eoa i>%a .. ·.

developments in clinical biochemistry

volume 2

G. S iest, editor

DRUG EFFECTS ON
LABORATORY
TEST RESULTS

Martinus Nijhoff
for the Commission of the European Communities
DRUG EFFECTS ON LABORATORY TEST RESULTS
DEVELOPMENTS
IN CLINICAL BIOCHEMISTRY

VOLUME 2

Other titles in this series

1. R. Malvano, ed., Immunoenzymatic Assay Techniques. 1980

ISBN 90-247-2314-0

Series ISBN: 90-247-2335-3

DRUG EFFECTS ON
LABORATORY TEST RESULTS
Proceedings of a Workshop on 'The Use of Laboratory Test Results. Variations due to
Drug Intake' sponsored by the Commission of the European Communities, as advised
by the Committee on Medical and Public Health Research and held
at the Abbaye des Prémontrés, Pont-à-Mousson (France), December 17-19, 1979

editor
G. SIEST, Nancy, France

co-editors
J.G. SALWAY, Guildford, United Kingdom
D. REEDER, Washington, USA
Ν. TRYDING, K ristianstad, Sweden
CA. DUJOVNE, K ansas City, USA
F.W. SUNDERMAN, Farmington, USA

executive editor
D. NOTTER, Nancy, France

PARL EUROP. Biblioth.

Κ
Com. 12.8*0 J

1980
MARTINUS NIJHOFF PUBLISHERS
THE HAGUE / BOSTON / LONDON
for
THE COMMISSION OF THE EUROPEAN COMMUNITIES
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Library of Congress Cataloging in Publication Data OH

Main entry under title:

Drug effects on laboratory test results.
(Developments in clinical biochemistry; v. 2)
1. Chemistry, clinical­Congresses. 2. Drugs­Physiological effect­Congresses. 3. Drugs­Side
effects­Congresses. I. Siest, G. II. Commission of the European Communities. III. Commission of
the European Communities. Committee on Medical and Public Health Research. IV. Series [DNLM:
1. Drugs­Analysis­Congresses. 2. Drugs­Metabolism­Congresses. 3. Chemistry, Clinical­Congresses.
Wl DE997VR v. 2 / QY90 D794 1979]
RB49.D78 616.07'56 80­24263

ISBN 90­247­2419­8 (this volume)

ISBN 90­247­2335­3 (series)

Publication arranged by:

Commission of the European Communities,
Directorate­General Information Market and Innovation,
Luxembourg

EUR 6860 EN
Copyright © ECSC, EEC, EAEC, Brussels-Luxembourg, 1980
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system,
or transmitted in any form or by any means, mechanical, photocopying, recording, or otherwise,
without the prior written permission of the publisher,
Martinus Nijhoff Publishers bv, P.O.Box 566, 2501 CN The Hague, The Netherlands.

LEGAL NOTICE
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PRINTED IN THE NETHERLANDS

CONTENTS

Preface
G. Siest IX

List of participants XI

BASIC PROBLEMS

Introductory considerations on drug effects in clinical biochemistry 3

G Siest, MM Galteau and D. Notter

Clinico-pharmacological problems in the evaluation of side effects of drugs 14

F. Colombo and G. Tognoni

Data banks and other documents on drug effects in clinical chemistry.

A new file for clinical use in the computerized Swedish drug information system 27
JV. Tryding, S.G. Johansson and P. Manell

ANALYTICAL INTERFERENCES

Analytical interferences of drugs 39

A. Galli

Place of reference materials and reference methods in the evaluation of drug effects 49
D.J. Reeder

Detection of drug interferences in method evaluation programs 58

R. Haeckel and 0. Sonntag

Analytical interferences due to antiinflammatory drugs 74

R. Galimany
VI
DRUG EFFECTS ON LABORATORY TESTS

Drug effects on laboratory tests: uric acid 85

J. G Salway

Effects of drugs on total serum cholesterol 91

J. Delattre and P. Bastide

Drug effects on cholesterol plasma lipoproteins fractions 99

P. Braquet and M. Braquet

Effects of drugs and physiological substances on serum and urine creatinine 134
J. Rogulski and A. Pacanis

Drug effects on hematological tests 146

J.F. Guelfi

PHYSIOLOGIC OR TOXIC EFFECTS

. Interpretation of laboratory tests: the example of drug effects on aminotransferases 159

MM. Galteau, C. Morin and G. Siest

On the influence of Phenylhydrazine and related compounds on the activity of enzymes 170
D. W. Scheuch

Laboratory tests as indirect indicators of the activity of drug metabolizing enzymes:

use of glucaric acid and gamma-glutamyltransferase 178
A.M. Batt and G. Siest

Drug effect on vitamin levels 193

J.-J. Himberg

The interaction between antiepileptic therapy and folate absorption and distribution.
A kinetic approach to obtain more meaningful laboratory data 206
J. Hendel, P. Winkel and M. Dam

Differentiation of toxic from spurious abnormalities after drug administration in man 214
CA. Dujovne

Effects of xenobiotics on heme oxygenase activity 221

F. W. Sunderman
 VII
Use of laboratory tests for estimation of patients' alcoholic intake 236
A. Bagrel, B. Herbeth and D. Barrucand

Effects of drugs on thyroid function tests 245

K. Liewendahl

PHARMACOLOGICAL EFFECTS

Effects of antiepileptic drugs on laboratory tests 261

D. Bagrel, A. Bagrel, B. Le Perron and G. Siest

Pharmacological effects due to oral contraceptives on laboratory tests 274

F. Schiele and G. Siest

Pharmacological effects due to hypolipidemic drugs 295

J. Steinmetz, E. Gaspart and D. Not ter

Pharmacological effects of tranquilizers and laboratory tests interferences 304

F. Trivin and M.F. Gerhardt

Effect of Imipramine on laboratory tests 312

A. Tazi, M.M. Galteau and G. Siest
 PREFACE

This book constitutes the Proceedings of a Workshop held at

Pont-à-Mousson under the aegis of the Committee of Medical Research and
Public Health (CRM) of the Commission of European Communities.

After some introductory presentations, the different papers

on drug effects concern four main themes:

- analytical interferences
- drug effect on specific laboratory tests
- physiological or toxical effects
- general pharmacological effects

Drugs and xenobiotics can increase or decrease the level of

certain blood constituents. This effect may be desirable or not. It can be
due to an analytical or to a pharmacological effect. It is necessary after
such workshop to prepare detailed technical protocols for the two types of
effects on laboratory tests.

The "in vitro" effects, called analytical interferences, have

to be systematically checked, especially for chemical tests. There are
likely to disappear with further progress in analytical methods towards
specificity.

The "in vivo" pharmacological effects of drugs must be measu-

red and calculated for every new drug or new laboratory method. The know-
ledge of these effects in terms of percentages will permit a better inter-
pretation of laboratory results by physicians and clinical biochemists.

A consensus was established between the different participants

of the workshop to emphasize the necessity of having a data bank on drug
interferences and drug effects in clinical chemistry. This bank should also
enclose information concerning the influence of other biological variation
factors. Contrarily to the existing data banks, the new one should be fil-
led up with validated information from the literature. It would be open
for general practitioners, clinical chemists, pathologists, pharmaceutical
industries... to better interpret laboratory results and diminish the in-
correct diagnoses and mistreatment of patients.

We thank all those individuals who contributed to the effi-

ciency and success of the meeting, as chairmen or secretaries of the scien-
tific sessions. We wish also to thank Chantal Thirion for the general
secretariat organisation of the meeting and the proceedings.

Gérard SIEST
 LIST OF PARTICIPANTS

BACHMANN, C. Insclspital Bern, Chemisches Zentrallabor der Universitäts­

kliniken, CH­3010 BERN (Switzerland)

BAGREL, A. Laboratoire d'Hygiène, Faculté des Sciences Pharmaceutiques

et Biologiques, 7 rue Albert Lebrun 54000 NANCY (France)

BAGREL, D. Laboratoire de Biochimie Pharmacologique, Faculté des

Sciences Pharmaceutiques et Biologiques, 7 rue Albert Le­
brun 54000 NANCY (France)

ΒATT, A.M. Laboratoire de Biochimie Pharmacologique, Faculté des

Sciences Pharmaceutiques et Biologiques, 7 rue Albert Le­
brun 54000 NANCY (France)

BRAQUET, P. Laboratoires FOURNIER, B.P.130, 21004 DIJON Cedex (France)

CASTILLO DE Torres Adaid j* 508, Col Del Valle, MEXICO D.F., Mexico
SANCHEZ M.L. ZP 12 (Mexico)

DELATTRE, J. Faculté de Pharmacie, 28 Place Henri Dunant, B.P. 38

63001 CLERMONT FERRAND Cedex (France)

DELWAIDE, P. Université de Liège, Laboratoire de chimie médicale, rue

des Bonnes Villes, Β 4020 LIEGE (Belgium)

DUJOVNE, C.A. University of Kansas Medical Center, College of Health

Sciences and Hospital,
Clinical Pharmacology Toxicology Center,
Rainbow Bd AT 39TH, KANSAS CITY, KANSAS 66103 (USA )

FERRAND, C. Laboratoire PARCOR, Département de Biologie Clinique, 195

Route d'Espagne, 31300 TOULOUSE (France)

GAL IMANY, R. Departemento de analisis clínicos, Hospitalet de Llobregat,

BARCELONE (Spain)

GALLI, A. Service de Biochimie, Hôpital de la Salpètrière, 47 Bd de

l'Hôpital, 75634 PARIS Cedex 13 (France)

GALTEAU, M.M. Laboratoire du Centre de Médecine Préventive, 2 Avenue du

Doyen Jacques Parisot, 54500 VANDOEUVRE LES NANCY (France)

GASPART, E. 48 Bd Docteur E.Feltgen, LUXEMBOURG (Grand Duché)

GILLET, M. Communauté Européenne, 200 rue de la Loi, 1040 BRUXELLES

(Belgium)

GUELFI, J.F. Ecole Nationale Vétérinaire, 23 Chemin des Capelles,

31076 TOULOUSE Cedex (France)
χπ

HAECKEL, R. Klinische Hochschule Hannover, Karl Wiechert Allee 9,

3 HANNOVER 61 (German Federal Republic)

HENDEL, J. Finseinstitutet, Strandboulevarden 49, 21OO COPENHAGUE 0

(Denmark)

HENNY, J. Laboratoire du Centre de Médecine Préventive, 2 Avenue du

Doyen Jacques Parisot, 54500 VANDOEUVRE LES NANCY (France)

HIMBERG, J.J. Department of Clinical Pharmacology, University of Helsinki,

Haartmaninkatu 4, 00290 HELSINKI 29 (Finland)

HITZ, J. Laboratoire de Biochimie Pharmacologique, Faculté des

Sciences Pharmaceutiques, 7 rue Albert Lebrun 54000 NANCY
(France)
HOUOT, O. Laboratoire du Centre de Médecine Préventive, 2 Avenue du
Doyen Jacques Parisot, 54500 VANDOEUVRE LES NANCY (France)

HÜBSCH, G. Laboratoire du Centre de Médecine Préventive, 2 Avenue du

Doyen Jacques Parisot, 54500 VANDOEUVRE LES NANCY (France)

LEVI, E. Commission of European Communities, Biology Division D.G.

XII 21020 ISPRA (Italy)

LIEWENDAHL, Κ. Department of Clinical Chemistry, University Central Hos­

pital, HELSINKI (Finland)

LOVE, C. Department of Biochemistry, Trinity College, Dublin Univer­

sity, DUBLIN 2 (Ireland)

MAHASSEN, Α. Laboratoire du Centre de Médecine Préventive, 2 Avenue du

Doyen Jacques Parisot, 54500 VANDOEUVRE LES NANCY (France)

MORIN, C. 13 Avenue Foch, 54000 NANCY (France)

NÖTTER, D. Laboratoire de Pharmacodynamie, Faculté des Sciences Phar­

maceutiques et Biologiques, 7 rue Albert Lebrun 54C OO
NANCY (France)
RATANASAVANH,D. Recherche Biologique et Pharmaceutique Lorraine, 7 rue
Albert Lebrun 54000 NANCY (France)

REEDER, D. Chemistry Building, National Bureau of Standards,

WASHINGTON, DC 20234 (USA)

REGNIER, F. Laboratoire SYNTHELABO, 6 Boulevard Emile Zola

54520 LAXOU (France)

ROGULSKI, J. Institute of Pathology, Department of Clinical Biochemis­

try, Medical Academy, Debinki 7, GDANSK (Poland)

SALWAY, J. University of Surrey, Department of Biochemistry, GUILDFORD,

SURREY GU2 5XH (England)
 ΧΙΠ

SARHAN, Ν. Laboratoire de Biochimie Pharmacologique, Faculté des

Sciences Pharmaceutiques et Biologiques, 7 rue Albert
Lebrun 540CO NANCY (France)

SCHEUCH, D.W. Medizinische Akademie "Carl Gustav Carus", Fetscherstrasse

74, 8019 DRESDEN (German Democratic Republic)

SCHIELE, F. Laboratoire du Centre de Médecine Préventive, 2 Avenue du

Doyen Jacques Parisot 545CO VANDOEUVRE LES NANCY (France)

SIEST, G. Laboratoire du Centre de Médecine Préventive, 2 Avenue du

Doyen Jacques Parisot 54500 VANDOEUVRE LES NANCY et
Laboratoire de Biochimie Pharmacologique, Faculté des
Sciences Pharmaceutiques et Biologiques, 7 rue Albert
Lebrun 540CO NANCY (France)

STEINMETZ, J. Laboratoire du Centre de Médecine Préventive, 2 Avenue du

Doyen Jacques Parisot 54500 VANDOEUVRE LES NANCY (France)

SUNDERMAN, F.W. Department of Laboratory Medicine, University of Connec­

ticut, School of Medicine, Box G, FARMINGTON, CONN.06032
(USA)
TAZI, A. 26 rue 251 bis, AIN­KADDOUS, FES (Marocco)

TOGNONI, G. Istituto di Ricerche Farmacologiche, "Mario Negri", Via

Eritrea, 62 ­ 20157 MILANO (Italy)

TRIVIN, F. Service de Biochimie, Hôpital Saint Joseph, 7 rue Pierre

Larousse, 75674 PARIS Cedex 14 (France)

TRYDING, N. Centralsjukhuset, 291 85 KRISTIANSTAD (Sweden)

VASSEUR, P. Institut Européen d'Ecologie, 570O0 METZ (France)

WINKEL, P. Finseninstitutet, Strandboulevarden 49, 2100 COPENHAGUE

0 (Denmark)
BASIC PROBLEMS
 INTRODUCTORY CONSIDERATIONS ON DRUG EFFECTS IN CLINICAL BIOCHEMISTRY

G. Siest, M.M. Galteau and D. Notter

Laboratoire de Biochimie Pharmacologique ERA CNRS 698
Faculté des Sciences Pharmaceutiques et Biologiques
7, rue Albert Lebrun 54000 NANCY France

ABSTRACT
Drugs and xenobiotics can increase or decrease the level of certain
blood constituents. This effect may be desirable or not. It can be due to
an analytical or to a pharmacological effect.

The "in vitro" effect, called analytical interference, has to be sys-

tematically checked, especially for chemical tests. It is likely to disap-
pear with further progress in analytical methods towards specificity.
The "in vivo" pharmacological effects of drugs must be measured and
calculated for every new drug or new laboratory method. The knowledge of
these effects in terms of percentages will permit a better interpretation
of laboratory results by clinical chemists and physicians.
Laboratory tests can be used, in addition, to monitor drug effects.
The authors rapidly describe the usefulness of laboratory tests in this
field. The general introduction clearly shows that it is now necessary to
prepare technical protocols for analytical interferences and pharmacologi-
cal effects. It is obvious that international recommendations must be wri-
ter! in order to collect information. All this work will be grouped in the
next future into a data .bank on drug effects. Clinical chemists, physicians,
will improve the interpretation of laboratory test results in treated peo-
ple by consulting this bank.

INTRODUCTION
Clinical chemists, clinicians and drug manufacturers are increasingly
aware of the problems related to the effects of drugs on laboratory tests
(Siest et al., 1980). These effects can assume several aspects :
- a purely analytical aspect, in which the drug and/or its meta-
bolites can perturb the assay of a constituent at any stage ; in clinical
chemistry, the term "interference" can be reserved for this "in vitro"
effect ;
- a biological aspect, in which the drug provokes a change in
a biological parameter by a physiological, pharmacological or toxicological
mechanism. This second aspect constitutes what can be called the unexpected
secondary effects of drug, desirable or not.
Laboratory tests exploring the liver, the kidney, etc, may be carried
out for a person who, without the knowledge of the physician ordering the
tests, takes tranquillizers, hypnotics, or oral contraceptives... So it is
very important to know exactly the sort of drug taken by a patient and the
exact conditions of administration. It is hopeful to get a precise question­
naire which can be filled at the same time the blood is drawn. The figure 1
presents an example of sampling form.

ORGANISATION RE GIONALE DES E XAME NS DE SANTE

Surname . ■ c.c. 5 9 9
First name _ Lab. N"
Age Individual N°
Months

Sex :
N° Nurse :
Time of blood sampling
Are you fasting ?

1 If yes If no 2 ­ breakfast
3 ­ meal at center (700 cal.)
A ­ others

Gynaecology :

Cycle 1 Pregnancy 2 Menopause 3 Other cases 4

Date of first day of last period : Day »
Since when do you smoke (in number years) ?
Have you smoked within the last two hours"?
Are you taking the "pill"" ?
Have you taken aedicines 0 ?
Sampling not possible"
Weight :

Laboratory :

Serum"
Blood hemolyzed"
Plasma a p p e a r a n c e
Normal 0 Lactescent k Icteric and opalescent 8
Opalescent 2 Icteric 5 Various (carotenolds) 9
*1 if yes

Fig. 1 : Sampling sheet

The drug intake questionnaire (Fig. 2) is used only if the patient

has­answered "yes" to one of the questions about drugs on the sample sheet.
Laboratory tests are also playing an increasingly important part in
the monitoring of drug treatments. So far such tests have mainly been used
only to evaluate the efficiency of treatments (with hypoglycemic agents,
hypolipemic agents, anticoagulants, etc.). More recently, biological tests
for such substances as triacylglycerols (triglycerides) have been more and
more commonly used in the surveillance of patient taking oral contracepti­
ves. Now a new chapter is beginning i.e. laboratory tests are used as indi­
ces of the activity of drug­metabolizing enzymes, particularly for their
 ORGANISATION RE GIONALE DE S EXAMENS DE SANTE

Sumane
F i n t nai _ Lab. Ν"
Individual Ν*

I ­ Ar« you taking médecine» a · part of a long­term

treatnent E I:Y S 2:N0
If y e s , which ?

Nan· a Nanea

II ­ Have you taken n e d i c i n e i in e x c e p t i o n a l

circunstancaa (during the l a s t 48 hours)
I:YES 2:N0
If yea, which 7

N Nanea
—

Have you undergone a l o n g ­ t e r n treatnent (more

than 3 month·) which you have t i n c e stopped ?
1 ­ l e s s than I nonth
2 ­ I week to 1 month
3 ­ I t o 6 nonthe
4 ­ nore than 6 nonthe
Have you already taken the " p i l l " ?
I:YES 2:NO
If no, you are not concerned by the f o l l o w i n g
You take contraceptive p i l l s :
. Since when ?
. Which p i l l are you using now ?
01 Anoviar 10 Ovariostat 24 Trentovlane
02 Gynophase 11 Ovulene SO 31 Norquentiel
03 Cynostat 12 Ov 28 32 Ovanon
04 Cynovlane 13 Planor 33 Physioatat
05 Lyndiol 14 Re lovii 41 Exluton
06 Metrulene IS Stediril 42 Microval
07 Hillianovlar 21 Adepal 43 Hilligynon
08 Noracycline 22 Hinidril
09 Nov SO 23 Hinipaese
. Have you changed the kind of pill ? I:YES 2:NO
­ Old pill :
Length of t i n e taken ( i n nonths) :
­ New p i l l :
Length of t i n e taken ( i n nonths) :
VI ­ Have you stopped taking the p i l l ? EI:Y S 2:N0
If y e s , s i n c e when ( i n nonths) :
Nan· of p i l l which you took :

Fig. 2 : Questionnaire on the intake of drugs

induction (Siest et al., 1977).
Therefore four groups of problems and recommendations seem to be im-
portant :
- Some molecules perturb the analytical procedure, because of the re-
activity of their functional group.
- The biological activity of other molecules produces real in vivo
changes in one or several biological parameters.
- Laboratory tests can be useful to improve the monitoring of treat-
ment.
- Finally, it would be useful to make particular recommendations con-
cerning drugs as factors of exclusion when reference values are being pro-
duced. (Metais, 1980).
I - ANALYTICAL INTERFERENCE
Interferences with the analysis constitute a part of the total drug
effect, but account for only about 20 % of the cases summarized in Young's
listing (Young et al., 1975).
They can be due to the drug itself or to its metabolites. Drugs can
interfere because of their physical properties : inherent coloration or
fluorescence. This type of interference is very important when the reaction
is performed in a very acid or alkaline medium.
Interferences can also be due to the chemical properties of drugs, pre-
sent usually in rather large amounts. The reducing power of a drug or its
metabolites is often the main factor ; for example, ascorbic acid interfe-
res with glucose or uric acid test because of its marked reducing properties
(Fig. 3 and 4 ) .
Of course, other mechanisms such as the formation of complexes and
the development of precipitates can also contribute. And drugs can act more
especially on the enzymes and proteins, blocking or changing active sites
used to reveal them, either by their catalytic properties or by immunology.
This" type of interference leads usually to inhibition, more rarely to
activation of plasma enzymes.
These several examples could lead to a fear that analytical interfe-
rences pose important problems for analyses carried out in the clinical
chemical laboratory.
Very fortunately, methods are gradually becoming more specific and
more accurate. However, every new suggested method and every new drug has
to be tested for this type of interference. Work is needed to define a ge-
neral protocol applicable to this type of study.
 /'
NO ­

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=600 I
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Attor Mc aed IniaoMI Mcorbc Ο Η Ι Ι Ι Μ Ί

2. 4. S. t Neocuproln SMA 12/60 hydroxylamuie

1 CIUCOM oxidase Vllatron UC 200 S ♦ 1 Enzymatic color test EppencVirfllOI
Pend method Dsjilot DRf 200 Uncue. Boehringer
Boehnrajei irf 1J7S4 reí. 15865
CIUCOM oxldase­pctkl Ttchnlcon 7 UV test LKB 2071­
Glucose oxidase­ LKB 7400 Unease­ Boehruifer LKBS6O0
peroxidase ref 15986
TXJNfVr method. Gilford β Urtcase­Ca**­neo­ Auto­
glUOOM OXldlM cuproin chemist
9 Urkasc­hydraxone

Fip Interference by ascorbic Fis, Interference by ascorbic

acid in glucose teste, acid in uric acid tests,
(from Sies t et al., 1978 a) (from Siest et al., 1978 a)

II ­ BIOLOGICAL EFFECTS IN VIVO

Biological effects in vivo are harder to demonstrate, to classify, and
what is more, to overcome. We will be content with presenting only few cau­
ses of this type of interference :
­ Drugs can bind competitively to proteins : phenylbutazone, for
example, by displacing protein ­bound anticoagulants, potentiates their
action and thus lowers the prothrombin level.
­ Secondly, the synthesis of a substance can be inhibited : certain
antihypertensive drugs decrease the synthesis of catecholamines metabolites,
thus lessening their excretion and that of their metabolites and making
false their levels in the urine.
­ Thirdly, synthesis of drug­metabolizing enzymes can increase.
­ And fourthly, the secretory mechanisms can be affected (elimination
8

of enzymes in bile, contraction of Oddi's sphincter, or changes in membrane

structure that provoke the release of enzymes).
The effects of drug on biological parameters can therefore be studied
in various ways :
- through their effect on a metabolite, a protein or an enzyme ;
- on a functionnal test (the effect of oral contraceptives on oral
glucose tolerance test) ;
- on a metabolism (the effect of hypolipemic agents) ;
- on an endocrine function (the impact on the thyroid gland) ;
- on an organ (usually the liver) ;
- on protein binding.
The drug can act on certain physiological parameters (shifts in bio-
logical rhythms for example).
Ill - LABORATORY TESTS IN MONITORING TREATMENT
3.1. Laboratory tests during drug trials
First, laboratory tests are very widely used in the monitoring of
drug trials. Some countries have already organized working groups and these
groups have even recommended a standard program of assays (Breuer et al.,
1976 ; Breuer, 1977) to be carried out during trials in animals. Such a
program would comprise : I
- hematological examinations, particularly the observation of blood
cells,
- a series of enzymes and main chemical constituents measurements,
- a simple urine analysis,
- and creatinine clearance.
These working groups have also specified methods to be used to avoid
inaccuracy. They recommend statistical analysis of the results. They invite
laboratories to preserve the originals, particularly the electrophoresis
diagrams and the cytological preparations, for 10 years.
3.2. Iaboraţory_ţesţs_in_moniţoring_ţhe efficiency of drugs
The effect of hypoglycemic agents is monitored by the measurements of
glucose, that of hypolipemic agents by the measurement of cholesterol and
triglycerides, that of agents acting on uric acid levels by the measurements
of uric acid, that of coumarin­type anticoagulants by prothrombin levels,
that of heparin by Howell's time...
The effects of drugs have' nhus long been monitored directly by follo­
wing changes in certain constituents of plasma.
Some drugs are also used specifically to lower the concentration of
troublesome constituents the levels of which are too high : for example,
that of bilirubin with phénobarbital, and that of iron with chelating
agents.
It is also useful to know the associated drugs. Thus, the association
of various drugs with a hypoglycemic agent can have undesirable effects.
Changes seen during the administration of these drugs, especially
those intended to correct risk factors, are usually small ; a 10 to 20 Ζ
reduction is generally settled. To interpret such a change, of course, one
must be familiar with the other biological variation factors (Siest et al.,
1979).
Drugs thus provide good examples of the need for clinicians to know
and take into account the factors of biological variations in order to im­
prove the interpretation of laboratory tests.
Laboratory tests are used to follow the effects of drugs on damaged
organs ; for example, the lowering of hepatic enzymes blood levels is a
good surveillance for the treatment and evolution of hepatitis.
3.3. Laboratory__examinations in monitoring the secondary effects of
treatment
Some drugs cause variations in biological parameters that must be
monitored because the changes can provoke disorders and iatrogenic accideits.
This is the case with changes in, for example, the concentration of potas­
sium under the influence of diuretics, of uric acid after the intake of
cytostatics, and of xanthine and hypoxanthine under the influence of cer­
tain agents used to lower the concentration of uric acid in the blood.
3.A. Laboraţqry_ţesţs_in_moniţorÎng_for therapeutic risk or in choo­
2 Í.ÜS_â1!°ÜS_Íiy S2
Before certain drugs are ordered, laboratory tests are needed to ve­
rify the function of certain important organs such as the kidney or the
liver. For the kidney, creatinine is usually assayed for adjustment of the
drug dosage. Similarly, strict monitoring of the blood cells is necessary
when cytostatic drugs are used. And in recent years it has become usual to
monitor oral contraceptives intake through the plasma triglyceride concen­
tration and sometimes through coagulation tests, as anti thrombin III ; but
in this cases, also the physiological range of biological variations and
the decision limits should be better defined (Fig. 5 ) .
10

r~!
15 — Controls
Orai
contraceptives
IO

2.5 mmol/I

Fig. 5 : Effect of taking oral contraceptives (OC) on plasma

triglycerides. Women 20 to 40 years old and not overweight by
more than 15 %. (From Siest et al., 1978 b ) .

3.5. Laboratory tests to detect threshold toxicity

Drugs affect mainly liver function. Therefore the laboratory test
results for hepatic enzymes and bilirubin are among those most often affec-
ted by drugs. The same is true for kidney, and certain urinary enzymes are
used for the surveillance of potentially nephrotoxic products. N-acetyl-ß-
D-glucosaminidase has even been proposed as a very sensitive and specific
test.
Sometimes the constituents of erythrocytes can give interesting in-
formation : incipient intoxication by digitalin can. be monitored through
the lowering of potassium and the increase of sodium in the erythrocytes.
3.6. Laboratory tests in defining genetic constitution
Under this heading can be cited the measurement of glucose-6-phospha-
te dehydrogenase in the erythrocytes, the study of serum Cholinesterase
anomalies, and the capability measurement of Phenytoine hydroxylation. But
the determination of acetylator phenotypes is the best known example, and
will probably become more common in the next few years.
3.7. Laboratory tests as indicators of the activity of drug-metaboli-
zing_enzymes
Among the enzymes that transform drugs, it is most important to fol-
low and to measure those distributed in the endoplasmic reticulum and the
 11

activity of which is reduced or, especially, increased by certain drugs

(or xenobiotics).
While the direct measurement of tissue enzymes remains difficult in
current practice in human indirect methods, based either on the study of
endogenous or exogenous metabolites in plasma or urine, or on the measure-
ment of macromolecules in plasma or blood cells are useful.
Six different approaches can be used:
. Antipyrine half-time
. Measurement of radioactive CO2 after the administration of label-
led aminopyrine
. Measurement of changes in substrates or in endogenous metabolites
. Measurement of drug metabolites in urine or plasma
. Measurement of the enzymes in blood cells
. Measurement of the plasma concentrations of proteins and specific
enzymes
(Batt et al., in this book)

IV - DRUGS AND REFERENCE VALUES

Since intra- and inter-individual variations in the effects of drug
on biological parameters are very great, it is not possible at the moment
to control this variation factor. Therefore, drugs must be generally con-
sidered as exclusion factors (Siest et all., 1978c).However, for certain
classes of drugs and for limited aims (with oral contraceptives, for exem-
ple) it is worthwhile to know their effect on a well-defined population.

CONCLUSION
Laboratory tests are very useful tools for monitoring the effects of
drugs that provoke changes at the same time desirable and unexpected. Becau-
se these variations are usually small, all other variation factors must be
rigorously controlled. It is essential that clinical chemists get into the
habit of not carrying out laboratory tests on patients who take drugs
unless the person doing the tests knows exactly what substances are being
given, as well as the dosages. This is absolutely necessary for improve-
ment in the interpretation of laboratory tests. The effects of drugs can
lead to false diagnoses if the clinician does not realize the relation
with the drug. In order to better interpret laboratory tests, a list of
interfering drugs must establish for every assay method. But the drugs
12

which do not interfere must be also pointed out. Correspondingly, when a

new drug is offered for sale, it should ideally be accompanied by a list of
the main tests which vary.
But it should not be forgotten that most of drug effects on laborato-
ry tests are useful, particularly for following a treatment.
We will try in the future . to measure drug effects on different clini-
cal chemistry tests : uric acid, transaminases (Galteau et al., in this book)
. to make some practical recommendations, mi-
nimal information to collect for description of a drug effect (recommenda-
tion to scientific journals).
The effects of drugs, which in recent years have been particularly
preoccupying for clinical chemists, should become a subject of study and
reflection for all members of the health professions, especially clinicians
(Siest et al.. 1980). Patients, too, should be informed.
Clinical chemists must participate in this desirable development by :
- gathering the information about drugs ;
- defining particular drug-related risks through comparison and know-
ledge of biological variations ;
- eliminating persons taking drugs from reference samples ;
- using the accumulated data about drug effects, they can help to in-
form clinicians and to educate patients.
A just appreciation of these effects which have too long been consi-
dered undesirable, should be restored and showed that, on the contrary,
some of them, at least, are interesting or are even useful.
All this work will be included in a data bank useful for clinical
chemists and physicians in order to better interpret laboratory test results
in treated people.

REFERENCES
Batt, A.M. and Siest, G. : Laboratory tests as indirect indicators of the
activity of drug metabolizing enzymes: use of glucaric acid and
gamma-glutamyltransferase. In this book.

Breuer, J., Gerhards, P., Hajdu, P., Rick, W., Roka, L., Stamm, D. and
Wolf, H.P.: Empfehlungen der Deutschen Gesellschaft für Klinische
Chemie zur Durchführung Klinisch-chemischer Untersuchungen bei der
Prüfung von Arzneimitteln. J. Clin. Chem. Biochem., 14: 161-164, 1976.
Breuer, J.: Empfehlungen zur Durchführung von Klinisch - chemischen Unter-
suchungen bei der Prüfung von Arzneimitteln. Dt. Ges. f. Klin. Che-
mie e.V.- Mitteilungen, 5: 146-147, 1977.
 13

Galceau, M.M., Morin, C. and SiesC, G.: Interpretation of laboratory test:

the example of drug effects on aminotransferases in human. In this
book
Métais, P.: Biochimie Clinique, Simep, Villeurbanne Pubi., 2: 25­40, 1980
Siest, G., Batt, A.M. and Galteau, M.M.: Les examens biologiques témoins
indirects de l'activité des enzymes du métabolisme des médicaments.
Place de l'acide glucarique et de la gamma­glutamyltransferase. A nn.
Biol. clin., 35: 425­432, 1977
Siest, G., Appel, W., Blijenberg, G.B., Capolaghi, Β., Galteau, M.M.,
Heusghem, C , Hjelm, M., Lauer, K.L., Le Perron, B., Lopp inet, V.,
Love, C , Royer, R.J., Tognoni, C. and Wilding, P.: Drug interference
in clinical chemistry: studies on ascorbic acid. J.Clin.Chem.Biochem.
16: 103­110, 1978a.
Siest, G., Galteau, M.M., Bagrel, Α., Panek, E. and Schiele, F.: A lteration
of the biochemical profile by drugs, alcohol and xenobiotics. Proc.
8th Technicon International Congress, London, 1978b.
Siest, G. and Petitclerc, C : Selection of people for the production of po­
pulation reference values. Ann. Biol. clin., 36: 217­220, 1978c.
Siest, G., Bretaudière, J.P., Buret, J., Favre, R., Gueguen, R., Petitclerc,
C , Sachs, C., Vernet, M. and Zender, R. : Les variations biologiques
des examens de laboratoire. Ann. Biol. clin. 37: 229­239, 1979.
Siest, G., Galteau, M.M. and Notter, D.: Notions générales concernant l'ef­
fet des médicaments sur les examens de laboratoire. Société Française
de Biologie Clinique. Commission "Effets des Médicaments sur les
Examens de Laboratoire". Information Scientifique du Biologiste, I:
4­9, 1980.
Young, D.S., Pestaner, L.C. and Gibberman, V.: Effects of drugs on clinical
laboratory tests. Clin. Chem. 21: 1D­432D, 1975.

Footnote: Thia paper hae been prepared for the Expert Panel on "Drug Effecte
in Clinical Chemistry" of the I nternational Federation of Clinical
Chemistry.
CLINICO-PHARMACOLOGICAL PROBLEMS IN THE EVALUATION OF SIDE EFFECTS OF DRUGS

F. Colombo and G. Tognoni

Istituto di Ricerche Farmacologiche "Mario Negri"
Via Eritrea, 62 - Milan, Italy

ABSTRACT
The relatively new field of interactions between therapeutic drug use
and biochemical tests is a challenging occasion for interactive work between
two disciplines which have grown somewhat apart both in research and in
clinical practice.
To optimize the gains likely to be obtained by this approach, a solid
knowledge and sound understanding of what has been achieved in the field
of surveillance of untoward and unexpected drug effects seem desirable.
The methodology of drug monitoring, its main achievements, together with its
limitations and needs, are briefly outlined with the main purpose of offer-
ing a working frame or reference for clinical pharmacologists and clinical
chemists who are active in producing and-more often-in utilizing data on
drug-laboratory test interactions.
Examples taken from pharmacokinetic investigations and clinical trials
are provided to support the thesis that priority should be given to creating
true interdisciplinary teams where real clinical situations of drug use and
biochemical testing are investigated and planned. Much attention and effort
should be given to closing the gap between what is shown in in vitro and
in vivo situations, and to minimizing the risk of unjustified extrapolation
from extreme clinical situations and case reports to general clinical prac-
tice. It is suggested that the experience acquired in the field mainly of
drug kinetics and drug interactions should be carefully assessed. Further-
more the role of a well-oriented policy of transfer of information from
research to routine conditions cannot be overemphasized.

INTRODUCTION
For a clinical pharmacologist, it is interesting to recall a contribu-
tion to the issue being discussed here today, which was delivered during a
seminar on drug interactions held back in 1973 in this same Institution
where the work on which we have been asked to report is in full development
(Young et al., 1974). A clinical chemist was then the last speaker among
a host of pharmacologists; a clinical pharmacological perspective is propo-
sed today to clinical chemists.
 15

The reason for the citation is not to plead historical backing, but
to provide a context to the discussion which has continued since, as a
major topic of debate in the field: how can one confidently ensure transla-
tion of tiie results of tube and bench work to clinical interpretation and use?
The question has been·and still is much the sane for drug interactions-
so many reports, so much fine investigation to filter out what is worthy
of consideration for understanding basic mechanisms of inter(action) and
for clinical use.
In this framework, the topic assigned to this paper is not outside
the terms of references of the present Workshop, as it conments
briefly on why and how a clinical pharmacological perspective on (side-)
effects of drugs could be of interest for clinical chemists involved in
assessing the influence of drug Intake and therapy on the use of laboratory
tests. Three main reasons can be found which illustrate the connection
between the two lines of research:
- surveillance of drug effects is a comprehensive task, which requires
intelligent participation and the application of common methodology by
all those in charge of patient care.
- Understanding the possibilities and limits of detecting a causal relation-
ship between drug-exposure and clinical (including biochemical, and more
broadly, laboratory) findings could be facilitated by consideration of
experience and results acquired in the field of monitoring drug side-
effects, which can now be considered a sufficiently established branch of
clinical pharmacology and general medicine.
- A major problem facing clinicians and the man in the laboratory in
evaluating drug effects (especially those outside what is specifically
intended or expected following a specific treatment) is how to distinguish
between a purely casual finding and a result pointing to an event which
could occur with a certain frequency (as yet to be defined or confirmed)
1n the course of identical or similar therapy in the future.
The last point is especially relevant to our topic, and merits more
comment. Frequent discrepancies are known to occur between results obtained
1n experimental settings where predefined conditions are the rule, and data
observed in real life situations where many confounding factors (e.g. multi-
drug therapy, different baseline values because of underlying and evolving
pathology, not to mention interindividual variability) must be taken into
account when looking for grounds for clinical decision. Another well-known
16
distinction should be recalled between anecdoctal vs systematic observation.
The former is worth consideration and reporting as it arouses awareness of
the possibility of the event which can be helpfully recalled in interpreting
individual cases. Only the latter type of observation, however, can be
considered a reliable source of information to be used as an instrument in
clinical surveillance. The relevance of this apparently obvious word of
caution cannot be overestimated. It is just as common an occurrence in
clinical practice to encounter an inappropriate inference from an (un-
expected) occasional finding to a clinical decision as it is to see an
(equally unexpected) result being disregarded as originating from some
source of error, while in fact it perfectly fits a not "normal" distribution.
To remain in the field of clinical pharmacology (where much less work
has been done in this area than in the evaluation of reference values in
biochemical and functional testing) it is worth recalling the still very
much pending and challenging debate on the clinical relevance of so-called
therapeutic drug levels and ranges. Responders to subtherapeutic concentra-
tions, non-responders to drug concentrations which borderline toxic levels
and toxic effects-free patients with toxic levels do not merely represent
"exceptions" to the rule. On the contrary they represent a specific sub-
population within the overall "universe" of treated patients; they should
therefore be considered on their own, and could suggest different causal
links between the underlying pathology and the chosen drug regimen.
According to the basic attitude adopted towards the above observations,
we could expect rather conflicting decision-making practices both with
regard to appreciation of(side-) effects and to the importance attributed
to laboratory data. Recent reviews on the question of blood level monitor-
ing (Pippinger, 1979; Richens, 1979; Sjöqvist et al., 1979; Tognoni et al.,
1980a; Tognoni et al., 1980b) and discussion on the clinical relevance of
side-effect reporting systems (Colombo et al., 1977; Gross and Inman, 1977;
Inman, 1980; Ricadi s, 1980) offer an interesting methodological background
and a rich inventory of examples pertinent to the development of programs
aiming at detecting and using data on laboratory test interferences by. drugs.
OBJECTIVES AND GENERAL STRUCTURE
An exhaustive review of the topic is clearly beyond the aim of this
paper, which therefore will concentrate on quoting key-works and methodo-
logical problems,and on supplying major reference and descriptive sources
for consultation (Table 1 ) . Some examples from representative drug classes
and treatments will also be provided.
TABLE 1 SYSTEMATIC PUBLICATIONS ON ADVERSE DRUG REACTIONS

I. Textbooks
Meyler and Peck, 1962 et Seq. D'Arcy and Griffin,1972 Davies, 1977 Dukes, 1957 et Seq.
- Systematic introductory - Brief introductory - Systematic introductory
chapters remarks chapters
- Disease-oriented - Disease - oriented - Disease-oriented - Drug-oriented
.Inducing drugs .Inducing drugs .Inducing drugs .Induced diseases
- Subject index - Subject index - Subject index - Index of drugs
Cross index of generic Index of generic names Index of side-effects
and proprietary names Drug laboratory tests Index of synonyms
interferences
- References in alphabetical -References in aphabetical - References in alphabetical - References in numerical
order at the end of each order at the end of each order at the end of each order at the end of each
chapter chapter with some further chapter chapter
indications

II. Sample periodical publications

Adverse Drug Reaction Bulletin
- Fortnightly review on specific
drug(s) and/or topic(s)
- References in numerical
order at the end of the review
- Biannual and annual cumulated
subject and drug index
CI in-Alert
- Fortnightly comments on single
reports or articles with specific
bibliographic information
- Quarterly and annual cumulated
subject index
Pediatric Alert
- Fortnightly comments on single
reports or articles reported in
pediatric international literature
with specific bibliographic
information
- Quarterly and annual cumulated
subject index
18
GENERAL FRAMEWORK
Side-effects as defined by WHO (Royall and Venulet, 1972) can be
expected to occur at any step during the development phases and therapeutic
use of a drug or a drug treatment. Accordingly, all techniques adopted to
define the drug profile can be a source of information and a tool for
describing the type, incidence, and prevalence of side effects. An in-depth
search for mechanisms underlying such side-effects must obviously rely on
techniques that are specific for the type of effect which has become
evident (e.g. allergy, hypersensitivity, functional impairment, biochemical
changes).
Table 2 is an attempt to give an overview of the field, which
can be comprehensively defined as "clinical pharmacology and drug epidemio-
logy". The complementary role of these two approaches will become evident
from the examples given below. It is all the more regrettable that the
two approaches have often developed along rather separate lines and only
recently has more systematic attention been given to their mutual integration.
TABLE 2 - SIDE-EFFECTS OF DRUGS: DETECTION AND EVALUATION

Definition How and when Examples of information obtained

Any response to drug Clinical trials (Early) risk/benefit evaluation in
which occurs at doses selected populations
used in man for Surveillance Detection of new and prevalence
programs estimation of old and new side
prophylaxis, effects occurring in routine
practice
diagnosis and therapy
and which is Morbidity and Variations in incidence and pre-
mortality valence of diseases (e.g. mal-
noxious and unintended. indexes from formations, M.I.and oral contra-
vital statistics ceptives, bone marrow depression
and marketed drugs)
This definition would
not include Ad hoc epidemio- Risk/benefit ratios and causal
logical studies relationship can be established
expected responses
Kinetic studies Toxic drug accumulation in organ
(e.g. cancer chemo- and monitoring failure; dose-dependent kinetics;
therapy) programs drug interactions; genetically
determined metabolic variations

Merits and pitfalls of the main types of surveillance and detection systems
are recalled in Table 3. Intensive in-hospital monitoring for acute drug-
effects represented an important contribution to this field more because
they raised a more generalized alertness to the problem and gave incidence
TABLE 3 ­ A D HOC METHODS FOR MONITORING SIDE­EFFECTS
Methods Areas covered and main advantages
(Intensive) in­hospital All drugs:acute and subacute effects;
possible documentation with ad hoc
monitoring testing; educational role for house
staff
Voluntary reporting All drugs
All consumers
system All types of ADRs
Inexpensive
Post­marketing Timely detection of type and inci­
dence of side effects of a (new)
surveillance drug over large populations
Record­linkage Surveillance of all drugs and
medical events in a defined
population
Cohort studies Complete and reliable records
(Including long­term covering all medical events
clinical trials) Incidence rates and benefit/risk
ratios can be established
Ad hoc investigations on specific
parameters can be incorporated
Case­control studies Rapid and relatively inexpensive
Benefit/risk ratios can be
Case­control established
surveillance Rare, new and delayed effects
can be detected
Drawbacks Examples
Limited information yield B C D S Ρ
Aberdeen
Underreporting WHO
Lack of incidence figures Committee on Safety of
Causal relationship dif­ Medicine
ficult to establish Swedish Commi t tee on ADRs
Finnish Committee
Any advantage over Few examples
clinical trials? reported so far
Cost/effectiveness?
Cost/effectiveness heavily
dependent on the health Oxford Record Linkage
care system
Expensive; limited to a RCGP and Oxford Family
specific drug or treatment Planning studies on
Chances of detecting rare oral contraceptives;
and delayed effects depend UDGP; Clofibrate study
on duration and population
size
Strict methodology needed Many ad hoc studies
to avoid bias, to ascertain
degree of exposure, to Boston Drug Epidemio­
correct for confounding logy Unit
factors
20
figures than because of the new information they were able to produce in
terms of specific side­effects not already detected or detectable mainly
in phases III and IV of drug development. Some typical studies reported
in Table 4 give evidence of a noteworthy phenomenon. Incidence figures
vary widely pointing to a now acknowledged fact: "spontaneous" reporting
is a measure of doctors' attention, and is certainly well below the true
rate of occurrence, which is reflected best in systematic surveillance
studies relying on ad hoc full­time nurses. It is also an underestimated
measure of what can be picked up in centres where ad hoc programs for
side­effects surveillance are run by house staff.

TABLE 4 ­ FREQUENCY OF INCIDENCE OF ADRs IN HOSPITALIZED PA TIENTS

AUTHOR/S, year SERVICE No. of Incidence

PATIENTS of ADRs(%)
A ­ Ad hoc surveillance program
(based on house staff)
Schimmel, 1964 Medicai 1.014 10.0
Seidl et al., 1966 Medical 714 13.6
Smith et al., 1966 Medicai (semiprivate' 900 10.8
Ogilvie and Ruedy, 1967 Medicai 731 18.0
Hoddinott et al., 1967 Medicai 104 15.0
Sidei et al., 1967 Medicai 255 14.8
Β ­ Boston Collaborative Drug
Surveillance Program
(based on specialized full­
time nurse monitors)
Borda et al«., 1968 Medical 830 35.0
Miller, 1973 Medicai 11.526 28.1
C ­ Others
.With ad hoc personnel
Mitchell et al., 1977 Pediatrie 1.669 16.8
.Semispontaneous reporting
Hurwitz and Wade, 1969 Medical, Surgical 1.600 10.2
Psychiatric
Schmidt and McQueen, 1972 All 900 3.0
.Spontaneous reporting
Bellantuono et al., 1979 Psychiatric 930 2.6
Beghi et al., 1979 Neurological 370 3.5
 21

The last two studies mentioned in Table 4 report data obtained from retro-
spective surveys of medical records 1n specialized wards where a much higher
rate of side-effects 1s certainly to be expected and can easily be docunent-
ed. They are quoted here as an example of the use of data for educational
purposes to sensitize doctors and nurses to the problem, through simple
and periodical programs of drug utilization review (Bergman et al., 1979;
Tognonl et al., 1978; Wardell, 1978). For the topic discussed in this
Workshop a specific message can be derived from Table4; doctors are not
naturally inclined to attribute noxious events to drug action. Any un-
expected finding tends in most cases to be referred to or understood as
part of the disease (this is a real possibility, and its implications for
the reliability of this methodology have been discussed widely, Finney,1955;
J1ck, 1977; Karch and Lasagna, 1975). The same (reinforced) attitude, how-
ever, can be expected when confronted with laboratory findings where drug
interference could be considered a causal factor.
What happens in a hospital setting is all the more valid outside
hospitals. Underreporting is a well-documented fact in all countries,
with however a better degree of compliance from doctors when and where drug
surveillance has become a regular component of drug policy and educational
campaigns and where good feedback 1s sought between public authorities and
doctors (Böttiger et al., 1979; Inman 1980; McQueen, 1974). This is vital
to assure not only the survival but also the development of this approach,
which has in-built shortcomings; but it is likely nonetheless to remain a
substantial component of the surveillance system (Inman, 1977; Skegg and
Doll, 1977). The future of post-marketing surveillance programs is still
under evaluation. More data and experience, besides those produced so far,
are needed to decide whether a favourable, cost-benefit ratio is to be ex-
pected (Harcus et al., 1979; Lawson, 1979; SCRIP, 1979). The expansion of
such systems as the Oxford record-linkage depends by definition on the
structure of various health care systems (Skegg, 1979). The role of short-
and long-term clinical trials, the potentiality of cohort and of case-
control studies, and in particular the advantage of a case-control surveil-
lance system have been the subject of so many publications and debates ·
that we shall not take them up again here (Colombo et al., 1977; Gross and
Inman, 1977; Holland, 1973; Peto et al., 1976; Peto et al., 1977; Roth and
Gordon, 1979).
22
CLINICAL PHA RMA COLOGY A ND DRUG EPIDEMIOLOGY: SOME EXA MPLES
Tables 5 and 6 list model cases selected according to two main criteria:
­ more direct potential interest for those working in a clinical chemistry
laboratory and looking for a role in drug surveillance;
­ situations where the different aspects of surveillance are most evident.

TABLE 5 ­ KINETICS AND SIDE­EFFECTS

Problem drugs Evidence Open questions

Phenytoi η Dose­dependent kinetics Incidence of the phenomenon
Salicylates well established
Prediction impossible
Toxic effects not always cor­
related with toxic levels
Aminoglycosides Hal f­Life prolongation No clear relationship between
proportional to renal epidemiological data suggesting
failure blood levels related toxicity,
Accumulation in a deep and kinetic and clinical studies
compartment (kidney suggesting relation of toxicity
to estimated accumulation in
tissue) of gentamicin kidney
(only?)
Clinical relevance? Change the
drug?
Salicylates Kinetic patterns well Largely discrepant data on side­
described effects incidence and their rela·
Therapeutic ranges tionship to blood levels
checked with blood Dosage recommendations still
level monitoring based on inadequate clinical
data despite use for many years
and in large populations

The problems used for this purpose are well­known and will not be described
or discussed in detail. Their implications for the present discussion
should already be evident from the analysis proposed in the Tables. It is
further suggested that a drug surveillance approach should not be restricted
only to side­effect spotting but should aim at comprehensive evaluation
of positive interaction between drug therapies too.
 23
TABLE 6 - DRUG EPIDEMIOLOGY

Problem drugs Evidence Open questions

Oral contraceptives Established relationship Causal mechanisms unknown

with increased risk of
thromboembolic events Predictive tests not
and M.I.; smoking habits available
and age are main (syn-
ergistic) cofactors

L1p1d-lowering drugs Pharmacological and Benefit/risk ratio un-

biochemical effects settled with regard to
Oral hypoglycemic drugs established the main disease to be
treated/prevented and to
induced morbidity and
mortality with long-term
treatment

Anti-pi atei et drugs Suggestive contradictory Level(s) and mechanism(s)

results from large eli- of action:dose dependence?
cei trials and from Organs, district, sex
experimental and clinic- specificity?
al models Biochemical markers?

CONCLUSIONS
The problem of comprehensive monitoring of drug-effects has become an
Important part of clinical pharmacology, as consideration of the benefit-to-
risk ratio 1s a major parameter in drug selection and prescription (WHO,197?.
Kinetics, clinical trial methodology and epidemiology have shown their
potential 1n defining a drug profile where the probability of side-effects
should be predictable or at least spotted in good time, and evaluated
against the relevance of positive effects. Though still from being solved,
the problem 1s however at least being tackled with a greater degree of
awareness than 1t was even a few years ago. Looking for factors which could
condition successful progress in this field and therefore for tasks to be
given priority, the following indications seem appropriate and could be of
Interest for all those, Including clinical chemists, who are willing to
contribute to an Intelligent drug policy and to the proper use of drugs in
and outside hospitals:
- simplification and rationalization of drug therapy is the single most
favourable condition to be achieved, in order to obtain optimal surveillance
strategies and acceptable benefit/risk ratios.
24

­ During their curriculum and as part of their continuous education physi­

cians and nurses should receive specific training on matters related to drug
safety and on how to deal with drugs which are active and (because ofthat?)
can borderline toxicity: an all­or­none attitude is not likely to benefit
patients or therapeutic practice.
­ Understanding of drug kinetics (even more than monitoring programs) can
provide substantial support to evaluating causal relationships between
clinical findings and drug action. The contribution of kinetic knowledge
can already be seen in the one hand in the detailed description of drug
distribution patterns in various conditions and diseases, on the other in
interpreting individual cases. Its application to clinical trial planning
and evaluation and to interpretation of large­scale monitoring programs is
still in a preliminary stage.
­ Centrally coordinated national and international programs for data col­
lection and evaluation remain a backbone of drug surveillance. It is
likely however that they could benefit from enrichment and diversification
of their approach by adding to the task of data base construction an active
policy of coordination of ongoing projects for controlled trials, drug
epidemiology, post­marketing surveillance and education. The timely and
critically reviewed information originating from these sources is likely to
prove a most effective educational tool for both the medical community and
public opinion at large and both these partners are needed to create
conditions for a satisfactory feedback.
REFERENCES
Adverse Drug Reaction Bulletin, Shotley Bridge General Hospital, Consett,
Co. Durham.
Beghi, E., Sasanelli, F., Spagnoli, A. and Tognoni, G., 1979. Diagnosis and
treatment of patients with epilepsy in hospital practice in Lombardy.
Presented at: 11th Epilepsy International Symposium, Florence,
1­3 October.
Bellantuono, C , Reggi, V. and Tognoni, G., 1979. Drug monitoring expensive
­ toy or useful tool? The contribution of an epidemiological approach.
Presented at: World Psychiatric Association Symposium, London,
12­14 November.
Bergman, U., Grimsson, Α., Wahba, A.H.W. and Westerholm, B. (Editors), 1979.
Studies in drug utilization. Methods and applications. WHO Regional
Publications European Series No. 8, Copenhagen.
Borda, I.T., Sione, D. and Jick, H., 1968. Assessment of adverse reactions
within a drug surveillance program. JA MA , 205: 645­647.
Böttiger, L.E., Furhoff, Α.Κ. and Holmberg, L., 1979. Fatal reactions to
drugs. A 10­year material from the Swedish Adverse Reaction Committee.
Acta Med. Scand., 205: 451­456.
Clin­Alert, Science Editors, Inc., P.O.Box 7185, Louisville, Ky. 40207.
 25
Colombo, F., Shapiro, S., Sione, D. and Tognoni, G. (Editors), 1977.
Epidemiological Evaluation- of Drugs. El sevi er/North-Hol land,Amsterdam.
D'Arcy, P.F. and Griffin, J.P., 1972. Iatrogenic Diseases. Oxford University
Press, Oxford.
Davies, M.D., 1977. Textbook of Adverse Drug Reactions. Oxford University
Press, Oxford.
Dukes, M.N.G. (Editor), Series 1957 et seq. Meyler's side effects of drugs.
Excerpta Medica, Amsterdam.
Finney, D.J., 1965. The design and logic of a monitor of drug use.
J. Chronic Dis., 18: 77-98.
Gross, F.H. and Inman, W.H.W. (Editors), 1977. Drug Monitoring.
Academic Press, London.
Harcus, A.W., Ward, A.E. and Smith, D.W., 1979. Methodology of monitored
release of a new preparation: buprenorphine. Br. Med. J., 2: 163-165.
Hoddinott, B.C., Gowdey, C.W. and Coulter, W.K., 1967. Drug reactions and
errors in administration on a medical ward. Can. Med. Assoc. J.,
97: 1001-1006.
Holland, W.W. and Karhausen, L. (Editors), 1978. Health Care and Epidemio-
logy. Henry Kimpton Publisher, London.
Hurwitz, N. and Wade, O.L., 1969. Intensive hospital monitoring of adverse
reactions to drugs. Br. Med. J., 1: 531-536.
Inman, W.H.W., 1977. Thalidomide, Practolol - What next? In: F. Colombo,
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tion of Drugs. Elsevier / North-Holland,'Amsterdam, pp. 231-240.
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J1ck, H., 1977. The discovery of drug-induced illness. N. Engl. J. Med.,
296: 481-485.
Karch, F.E. and Lasagna, L., 1975. Adverse drug reactions: a critical review.
JAMA, 234: 1236-1241.
KICADIS, 1980. Proceedings of the Kyoto International Conference Against
Drug Induced Sufferings. Elsevier/North-Holland, Amsterdam.
Lawson, D.H., 1979. Detection of drug-induced disease. Br. J. Clin.
Pharmacol., 7: 13-18.
McQueen, E.G., 1974. New Zealand Committee on Adverse Drug Reactions: Ninth
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Mitchell, A.A., Goldman, P., Shapiro, S. and Slone, D., 1979. Drug
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26
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This work was supported by C Ν R ­ Convention Clinical Pharmacology,

27 March 1979.
 27

DATA BANKS AND OTHER DXUMENTS ON DRUG EFFECTS IN CLINICAL CHEMISTRY.

A NEW FILE FOR CLINICAL USE IN THE COMPUTERIZED SWEDISH DRUG IN-
FORMATION SYSTEM.

1 2 3
N. Tryding , S.G. Johansson and P. Manell
Department of Clinical Chemistry, Central Hospital,
S 29185 Kristianstad, Sweden
2
Uppsola University Data Center P.O. Box 2103, S 75002 Uppsala,
Sweden
Department of Drugs, National Board of Health and Welfare,
P.O. Box 607, S 75125 Uppsala, Sweden

ABSTRACT
A new file containing information on well documented drug
effects on laboratory investigations has been added to the compu-
terized Swedish drug information system. This includes all drugs on
sale as well as adverse drug reactions reported by Swedish clini-
cians since 1965. The information will be published for clinical
use as a new edition of our booklet on drug effects in clinical
chemistry. The content will be continuously updated and brought to
the attention of the clinicians. Local data terminals may be used
to get direct access to the information.

INTRODUCTION
The number of documents useful for the knowledge of drug
effects in clinical chemistry is enormous (cf Young, et al. 1975)
and grows rapidly. However, many of the publications have no signi-
ficance for clinical decisions or may even be misleading. It is
necessary to read the information critically. - A data bank on
clinically significant drug effects is needed. In 1977 the Swedish
Society for Clinical Chemistry and the National Corporation of
Swedish Pharmacies (Apoteksbolaget) published a booklet "Drug
effects on laboratory investigations". The starting point was the
publication by Young et al 1975. The material was reduced very '
extensively by excluding :
- All drugs not registered in Sweden.
- Methods not in routine use in Sweden.
- Reports regarding animals.
28

- Interactions between drugs except for serum concentrations of

sulfonamides, phenytoin, barbiturates and digoxin.
- Overdoses.
- Wanted therapeutic results.
Thus, only well documented effects of relevance to Swedish doctors
in clinical practice were included. - In 1979 the Department of
Drugs, Swedish National Board of Health and Welfare, joined the
project. The information on clinically relevant effects on labora-
tory results was then added to the reporting system for adverse
drug reactions and the computerized drug information system, SWEDIS.
REPORTS ON ADVERSE DRUG REACTIONS
It is essential that the relationship between the suspected
drug and the adverse reaction is well documented. Adverse drug
reactions are critically reviewed and excellently summarized in
"Meyler's Side Effects of Drugs" 1975 and the uniform Annuals of it.
In Sweden we have a reporting system for adverse drug reactions to
the Department of Drugs. The recording started in 1965 and is based
on reports sent in by physicians and dentists. The department
receives an increasing number of reports at present almost 3 000
per year. At the Adverse Drug Reaction Section of the Department
the relationship between the drug and the reported reaction is
evaluated. The final assessment is made by the Adverse Reaction
Committee (Boman, 1978). The chief contents of the Adverse Reaction
Register are as follows:
- report number
- patient's age and sex
- recorded date of adverse reaction
- code of reporting physician/dentist/hospital
- Adverse Reaction Committee's assessment of possible connection
between reported adverse reaction and suspected drug
- outcome of adverse reaction
- details regarding suspected drug, e.g. mode of administration,
daily dosage, duration of medication, indications for the use of
the actual drug
- adverse reactions reported. Swedish and WHO nomenclature is used.
A lot of adverse drug reactions are detected by laboratory tests.
There are more than one hundred different examples of clinically
 29

observed adverse drug reactions that correspond to drug effects on

laboratory tests i.e. Agranulocytosis (B-Leukocytes), Anemia
(B-Hemoglobin), Bleedings (B-Thrombocytes, P-Coagulation factors,
U-Hemoglobin etc), Diabetes (B-Glucose), Electrolyte disturbances
(S-Ca, S-K, S-Na, B-pH etc), Gout (S-Uric acid), Hemolytic diseases
(S-Bilirubin, S-Maptoglobin, Coombs test etc), Hepatic reaction and
Icterus (S-Bilirubin, S-Transaminases, S-Alkaline phosphatases etc),
Thyroid reaction (S-TSH, S-Thyroxine etc), Renal reaction and
Uremia (S-Creatinine, U-Osmolality etc). An inclusion of these
adverse reaction reports in our booklet "Drug effects on labora-
tory investigations" would increase 1) the value of the information
as well as 2) the doctors' knowledge of drug effects on laboratory
results. The results from the adverse drug reaction reports will be
presented in a new edition of the booklet together with the number
of reports for each drug reaction.
SWEDIS. A COMPUTORIZED DRUG INFORMATION SYSTEM
In close collaboration the Department of Drugs and the Uppsala
University Data Center have developed a computerized drug informa-
tion system, SWEDIS (Dagerus, Johansson and Manell 1978, Johansson
and Manell 1978). The overall logical structure of the corresponding
data base is presented in figure 1. The abbreviations used and the
contents of the different data base sections are listed below:
AD Adverse Drug Reactions, ADR. This register contains at present
about 17 000 reports on ADR notified since the compulsory
registration of side effects was introduced in Sweden 1965.
AF Administrative File contains administrative information on
pharmaceutical specialities, including details of the latest
investigations into applications for approval.
CC Chemical Classification including molecular formula and Wis-
wesser Line Notation for constituents of pharmaceutical specia-
lities.
CF Control File contains detailed descriptions of checks carried
out by the Pharmaceutical Division at the Department of Drugs.
CL Drug effects on Laboratory investigations.
CO Register of Companies. Names and addresses of all firms which
sell, import or manufacture drugs, herbal remedies and sterile
disposable articles are included.
30

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TlòURB I'STKUCWRB OF SWE DÌS
 31

CT Clinical Trials. This section contains information on clinical

trials.
DI Drug Ingredients. This section contains the complete composi-
tion of the pharmaceutical specialities stored in the drug
register, DR.
DO Document and literature section contains indexes and summaries
of literature and documentary references.
DR Drug Register contains the names of all currently approved
pharmaceutical specialities in Sweden. It also contains all
specialities for which application for approval has been made
and those withdrawn or rejected from the market. The total
number of specialities included in the register is about 6 000
at present.
DP Drug Packs. This register contains description of all packs of
pharmaceutical specialities which have appeared on the Swedish
market since 1971.
DS Drug Sales. The register contains details of sales of individual
drug packaging type marketing since 1971.
FC Drug File Circulation Register includes administrative routines
for files concerning pharmaceutical specialities.
FF Fees. The register contains information for the invoicing of
annual charges,
hü Herbal Remedies. This section contains all (about 600) herbal
remedies allowed for sale in Sweden.
ID International classification of Diseases. This section contains
ICD codes and their corresponding disease designations.
IN Register of Indications contains all the approved indications
for the use of pharmaceutical specialities.
LI Licenses. This section contains details of all applications for
licenced rending of non-approved pharmaceutical specialities.
SN Substance Names. This register contains about 18 000 names of
substances used in drugs or herbal remedies. The corresponding
CAS-number is also stored.
ST Register of Sterile Disposable Articles. This is a list of all
sterile disposable articles for use in health care.
32

TI Tablet Identification. This section contains description of

the appearance (size, colour, form, marking) of all registered
tablets, capsules, pills etc. Tablet identification can be
carried out directly from data terminals.
INTDIS World Health Organization (WHO) Computerized Drug Informa-
tion System, WHO Collaborative Center for International Drug
Monitoring, Uppsala, Sweden.
GENERAL USER REQUIREMENTS OF SWEDIS
In order to fulfill the present and future requirements from
different users, SWEDIS contains several general qualities:
- Expansion and modification of the data base without altering
existing programs.
- Existing data can bè used for new purposes and different users
can use the same data in different ways.
- Access to data is simple.
- Spontaneous questions can be answered without any programming by
means of a query language.
- New application needs may be met with existing data rather than
creating new files.
- Users can easily understand which data are available.
- Unauthorized access to the data will be prevented. The same data
may be restricted in different ways from different users.
- Data are quickly available to users at almost all times when they
need them.
THE SOFTWARE OF THE SYSTEM
The realization of the above mentioned requirements is done
by using a generalized data base system, MIMER. A MIMER data base
is a number of interrelated and independent files of single record
type (normalized data structures). Associative addressing is im-
plemented by partial inverted file technique. A logical data base
structure is established by dynamic networking in the application
program. The access path between two files contains no other logical
information than that derived from the data (cf. the relational
data model). By inverted list technique MIMER may implement any
data structure: relational, hierarchical or network.
 33

MIMER comprise· α
­ data definition facility
­ data manipulation language via a host programming language
­ query language, MIMAN
­ data dictionary
­ program generation facility, LIDAM (Rish, 1978).

As shown in figure 1 the adverse drug reaction reports are stored

in SWEDIS. The drug register contains the names of all pharma­
ceutical specialities in Sweden and their complete composition«
Files with chemical classification of the substances are also in­
cluded. Due to the flexibility of the system it was simple to add
a new file containing information on drug effects on laboratory
investigations. In this file the substance will be identified from
the Chemical Abstracts Service (CAS) registry number. The CAS
number will automatically give possibilities to use information
stored in the other files within SWEDIS. For example, information
on new drugs on the market will be available automatically. Reports
on new adverse drug reactions will also be added continuously.
SWEDIS contains a modern updating program and the updating of the
laboratory test file is simple. Computerized photocomposition will
be used for the printing of the new edition of our booklet on drug
effects on laboratory investigations.
It is possible to use information in SWEDIS interactively via data
terminals. Almost any terminal anywhere may be used. This gives
possibilities to include information from the system into local
computerized systems and into the routine work in local hospitals.
In the future information from INIDIS, the international drug in­
formation system of the WHO Collaborative Centre for International
Drug Monitoring, Uppsala, Sweden can be included. This system in­
cludes at present 170 000 reports on adverse drug reactions from
23 countries. However, a permission from the WHO in Geneva,
Switzerland is needed before this information can be used in our'
project.
34

CONCLUSIONS
Clinically relevant information on drug effects on laboratory
results has recently been collected from the literature. In 1977 it
was published in a booklet for clinical use by Swedish doctors.
SWEDIS, the computerized Swedish drug information system, contains
the names and complete composition of all drugs for sale in Sweden
as well as all adverse drug reactions reported. The data base system
used was well suited for the addition of a new file containing in-
formation on drug effects in clinical chemistry.
The main advantages with our new information system are:
- Reports from clinicians on drug effects on laboratory results are
included as well as documentation found in the literature.
- The information included is well documented.
- The case reports contain information about the dose and duration
of the drug administration.
- A new edition of our booklet on drug effects on laboratory in-
vestigations will be prepared for clinical use by computerized
photocomposition.
- New drugs on the market will be included automatically.
- New drug effects on laboratory test results will be added
continuously.
- The information will be available via data terminals.

One limitation is that at present only a small percentage of the

clinical cases of side effects of drugs are reported. After in-
formation to doctors and education of medical students we hope to
get a higher frequency of reports.
 35

REFERENCES
Apoteksbolaget: Läkemedel» påverkan pâ laboratorieundersökningar
(Drug effects on laboratory tests). National Corporation of
Swedish Pharmacies (Stockholm 1977).
Boman, G.: The Swedish Reporting System for Adverse Drug Reactions
1965-1977. Conference on Computer Aid to Drug Therapy and to
Drug Monitoring. Bern 1978. North-Holland Publishing Company
1978.
Dagerus, B., Johansson, S.G. and Manell, P.: SWEDIS, a Drug Informa-
tion System at the Department of Drugs, National Board of
Health and Welfare, Sweden. Conference on Computer aid to
Drug Therapy and to Drug Monitoring, Bern 1978. North-Holland
Publishing Company 1978.
Johansson, S.G. and Manell, P.: The Drug Information System SWEDIS
and its users. Proceedings of MEDIS' 78, Osaka 19/8.
Manell, P.: Computer aid to the analysis of adverse drug reaction
reports. Proceedings of XIX Congress of the Italian Pharmaco-
logical Society, Ancona 1978.
Meyler's Side Effects of Drugs Volume VIII. Ed. M.N.G. Dukes
(Excerpta Medica, Amsterdam-Oxford 1975).
MIMER. A general purpose data base management system. Report no
M78001. Uppsala University Data Center, P.O. Box 2103,
S 75002 Uppsala, Sweden.
Rish, T.: Compilation of multiple file queries in a meta-data base
system. Linköping studies in Science and Technology Disserta-
tions, 1978.
Young, D.S., Pestaner, L.C. and Gibberman, V.: Effects of Drugs on
Clinical Laboratory Tests. Clinical Chemistry 21: 1-432D, 1975.

A list of references to reviews and books on drug effects in

clinical chemistry is available on request from N. Tryding
ANALYTICAL INTERFERENCES
 ANALYTICAL INTERFERENCES OF DRUGS

A. GALLI
Hôpital de la Salpétrière (i)
75013 Paris

INTRODUCTION
The action or effect of drugs upon laboratory tests may present
two aspects :
- an analytical aspect. The drug and/or its metabolites can affect -
interfere with - the mechanism of the determination. The result thus
obtained is different from the normal or true value but in a totally
artifactitious way, the true value remaining unchanged.
- a biological aspect. Under the action of a drug and/or its metabolites,
some of the constituents measured in the body fluids are submitted to
significant variations by toxicology or primary or secondary mechanisms.
The biochemical aspect is often taken into consideration but the pro-
blems related to haematology and especially to haemostasis are also very
important and will be the topic of a separate study.
Such considerations apply also to a number of immunological determinat-
ions.
It is also important to be aware of the problem of physiological cons-
tituents present in abnormally high concentrations (e.g. bilirubin)
likely to disturb the measurement of other parameters, but this can be
considered as a different problem.
The knowledge of the analytical interferences produced by a drug can
only be acquired by means of a thorough study. This study must be car-
ried out :
1- When studying a drug.
2- When developing an analytical method.
THE INTERFERENCE DRUSS
Most often, the interfering drug is a molecule foreign to the body
and administrated for therapy, or possibly, diagnostic purposes.

(l) This paper is based on the work of the commission "Effet des Medica-
ments sur les Examens de Laboratoire" of the "Société Française de Biolo-
gie Clinique".
40

In the hospital, the interfering part played by biochemical or physiol-

ogical materials should not be overlooked, particularly when there is a
massive uptake of the parameter sought for : perfusion of sodium chlo-
ride solutions, bicarbonates, glucose, potassium, etc... The aminoacids
may be introduced as protein hydrolysates, or as artificial mixtures -
or separately (cystine; glutamic, aspartic acids) or bound to a thera-
peutic molecule : lysine - aspirin, arginine - aspirin.
Glycerol may interfere with the determination of triglycerides. Intra-
venous administration of lipids may also be a source of error.
Heparin, a drug administrated to a number of patients, delays the coa-
gulation in vitro with various results : formation of fibrin filaments
liable to block some instruments, and particularly the continuous-
flow systems.
The maintenance of a quantity of fibrinogen will vitiate the sedimenta-
tion rate determination, the floculation reactions and will result in an
additional peak on the protein profile recording.
The plasma substitutes, as polyvinylpyrolidone (P.V.P.), gelatines or
dextran may induce :
- a change in the total or ionized Ca content by lowering the albumin
fraction;
- a modification of the peptide or lipid determination by formation of
lipids-dextran, lipids-P.V.P. complexes,
- a perturbation of the inorganic phosphate determination via the molyb-
dic method by causing turbidity,
- a perturbation of some haematology tests : sedimentation rate, bleed-
ing time, hematocrit.
Preserved blood and blood products may also be the source of a number
of errors, transfusion of packed cells particularly are liable to in-
troduce very large quantities of potassium.
•Strictly speaking, the products used for organ function tests are not
drugs, but they can also lead to problems, and thus, it is not advis-
able to carry out biochemical determinations during a function test
requiring the administration of foreign material : for instance, the
B.S.P. affects various colorimetrie reactions (alkaline phosphatase
determination by the paranitrophenylphosphate method or creatinine
determination by the Jaffa reaction).
The problem of the use of skin disinfectants before taking a microsample
 41

of blood has been considered. The analytical impact appears to be of

small importance. However, when in doubt, volatile solvents : alcohol,
ether... should be used.
Very abundant literature states the unwanted effects of drugs upon labo-
ratory tests. However, if purely analytical disturbances are considered,
the number of active constituents is not as high as one would think.
The following drugs are among those proven to be sources of disturbance :
- DOPA and methylDOPA
- amidopyrine and noramidopyrine
- hydralazine
- isoniazide
- salicylates
- antimicrobial and hypoglycemic sulphamides
- vitamin C
- penicillamine
- various antibiotics.
The disturbing mechanisms can be of various types :
- physical
- chemical
- biochemical.
a) Physical
The perturbations are particularly significant in colorimetry and
fluorometry, however they are reduced by preliminary extraction or by
the specificity of the wave length.
Quinine is known to interfere with the Cortisol and catecholamine
determinations, B.S.P. with bilirubin measurement, and conversely,
phenazopyridine increases the B.S.P. value.
Some techniques such as gas- or liquid-chromatography can be disturbed
by the addition or superposition of a supplementary peak corresponding
to the drug or to one of its metabolites. An ammonium chloride load can
interfere with potassium determination by selective electrode.
Some drugs, by changing the physical conditions of the reaction mixture,
particularly the pH, can lead to erroneous results. For instance, ascor-
bic acid, by lowering the pH, interferes with the creatinine determinat-
ion by the Jaffa reaction.
b) Chemical
1- Action on the determination in the same direction as the biol-
42

ogical constituent sought for.

In this way, ascorbic acid and penicillamine, because of their reducing
properties, increase the apparent concentration of blood glucose when
measured by methods based on reduction.
2- Interferences with the mechanism of the reaction.
Conversely, ascorbic acid and penicillamine lower the apparent concen-
tration of glucose when measured by the glucose oxidase method. As a
matter of fact, this determination is based on the measurement of the
generated peroxide of hydrogen. How, hydrogen peroxide is partially
destroyed by reducing agents.
In presence of penicillamine, the apparent concentration of bilirubin
is dramatically lowered : it has been shown that the diazosulfanilic
acid is destroyed by the thiol group with release of nitrogen.
Some drugs interfere directly with the component to be measured : in
this way, the reducing penicillamine which should give a uric acid con-
centration higher than the true value when measured by the phosphotungst-
ic acid method, gives false negative results because of the formation of
a penicillamine - uric acid complex, with less reducing power than its
two constituents.
Some materials are liable to develop color reactions in the same direct-
ion as the measured biological parameter.
Thus, the ninhydrin-positive materials interfere with the aminoacid
determination; and products, such as dextran, interfere with the protein
determination by the biuret method,
c) Biochemical
A large number of effectors are liable to disturb the enzyme react-
ions where the chelators also play an important part by the sequestrat-
ion of enzyme activator metals (e.g. : decrease of the alkaline phosphat-
ase activity by formation of a zinc-penicillamine complex).
The metabolism of the drug itself may be a source of perturbation : for
instance, some material involving the formation of large quantities of
glucuronides can lead to a falsely elevated value in urine reducing-
sugars.

The problem of interferences in the urinary media is often more acute

than in blood; as a matter of fact, the measured constituent may be
present only in traces, even though the drug or its metabolites are in
high concentrations. The physico-chemical interferences are virtually
 43

the same as in plasma, however the proble« of urines has drawn the at-
tention of biochemists for a long time because of the various colors
the drugs are liable to generate either spontaneously or subsequently
to the action of strong bases or acids. However it is relatively easy
to find the origins of these various colorations; the case of dark
urines is more difficult and is highly significant from a diagnostic
point of view (melanoma, alkaptonuria, porphyria, hemoglobinuria).
The study of the physico-chemical disturbing mechanisms of drugs is of
great interest in its previsional aspect. Actually, it should allow us,
as our knowledge in this field will increase, to classify the interfe-
rences according to the structure and the chemical properties of the
evaluated drug. We saw previously that the most interfering drugs have
reducing functions : thiols, phenols or are chelators...
The structural analogy can also be a good guideline : sulfonyl urea
and urea, xanthic bases and uric acid.
I- EVALUATION OF A DRUS
The in vitro study appears as an essential prerequisite before
any animal or human experimentation in order to be sure of the validity
of in vivo result, in order to be able to evaluate with fidelity the
pharmacological or toxicological effects and thus to eliminate any risk
of false therapeutic results. Actually, it would be regrettable that such
a drug should only have an action on the determination itself and that
normal biological results should hide a possible toxicity.
1.1. Product to study
1.1.1. Raw material
Generally, it consists in the raw material used by the manufacturer
or by the Codex variety if the product is registered in the pharmacopeia.
1.1.2. Knowledge of the product ; physicochemical characteristics
1.1.2.1. It is a must to have the analytical file of the manufac-
turer, to know the formula of the product and its main physicochemical
properties.
- Physical properties : crystalline, amorphous, micronized form; solubi-
lity; pH of the solution, etc...
- Chemical properties : knowledge of the chemical functions and of their
degree of reactivity.
1.1.2.2. It is necessary to know the product stability, when solid
and particularly when in solution.
44

1.1.2.3· Metabolism : the metabolism strictly speaking may result

in a decreased chemical reactivity and an attenuated disturbing effect.
However, it is useful to know it as well as :
- the blood concentration after a single dose uptake;
- the blood concentration during treatment with therapy doses;
- the urinary elimination rate of the drug and/or its metabolites;
- the protein binding degree.
1.1.2.4. Finished product : one should be aware that the formulat-
ion may introduce additional components likely to affect the analysis :
- dies
- metals (titan)
- preservatives : reducing or antioxygen agents, ascorbic acid, sulfite,
nordihydroguaiaretic acid;
- antibacterial or fungicidal agents : phenolmethylparahydroxybenzoate,
propyl parahydroxybenzoate.
1.2. Parameters to study
1.2.1. A number of laboratory tests considered as the most often
requested might be determined systematically for each drug.
1.2.2. Tests aimed at revealing a possible toxicity.
1.2.3· Tests aimed at demonstrating the pharmacological action of
a drug.
1.2.4. Tests used during the animal toxicopharmacological experi-
ments.
1.2.5. Tests to be used during the clinical study.
1.2.6. Tests whose mechanisms are known to be likely affected be-
cause of the structure and physicochemical properties of the drug.
1.3· Selection of the analytical techniques
1.3.1· Check among the recommended or selected techniques.
1.3.2. The most widely used.
1.3.3. If there are several methods based on different principles,
select at least one in each category with consideration to the various
modes of automation.
1.3.4. The methods used in the toxicopharmacological and clinical
evaluations.
II- EVALUATION STUDY OF A METHOD
This is a proceeding opposite to the previous one. The various
tests listed may be applied in this case.
 45

Selection of druga to evaluate : as many as possible.

2.1. The drugs the most often given in the diseases for which the
parameter is tested.
2.2. Drugs which, because of their chemical properties, are likely
to interfere with the method.
2.3. Drugs most often prescribed (medical products).
2.4. Drugs most often taken (over the counter products).
PROPOSED /.KTHODOLOCY
1- Dissolution of the product
A stock solution is prepared. It will be used to prepare diluted solut-
ions in physiological media.
- If the product is water-soluble, bidistilled water is used.
- If the product is not water-soluble, the study will be different.
If it is soluble in other solvents (alcohol, acetone, dimethylsulfoxide,
dimethylformamide,...), an aqueous solution will be prepared with the
smallest possible quantity of solvent. Then, one must check that there
is neither denaturation of the proteins nor analytical interferences due
to the solvent.
In case of failure, it is advised to try with the metabolites.
2- Concentration of the drug. Preparation of the solutions
2.1. Liquid human plasma or serum
For products whose metabolism is known, choose a drug level equal to
the concentration of the therapy dose multiplied by 5.
For products the blood level of which are not known the dose should be
equivalent to 5 therapy doses diluted in 5 to 15 liters if the product
remains in the vascular bed or if it diffuses in the extracellular
volume.
Prepare a stock solution with a concentration 10 times higher than the
one of the working solution, then add 1 ml of this solution to 9 ml of
the sample for analysis.
We prepared a pool of serum or plasma coming from patients taking no
drugs, with 3 different levels for the evaluated parameters.
- 1 ml of the stock solution is added to 9 ml of the pool. The concen-
tration of this sample is five times the maximum therapy dose.
- 1 ml of the solvent used to prepare the product dissolution is added
to another 9 ml volume of the pool. This sample (reference solution)
has the same dilution as the test sample and is submitted to the same
46

effects from the solvent.

2.2. It is possible to use lyophilised human or animal plasma or serum.
The stock solution is added to the reconstituted sera according to the
proportions described above, with preparation of reference sample for
each serum.
The studied product may also be dissolved in the liquid or solvent
used for reconstitution of the serum. Animal sera are not to be used
for enzyme determinations.
2.3. Whole blood
Proceed as for serum, but check beforehand that the stock solution is
isotonic and does not induce hemolysis.
The stock solution must be prepared especially for whole blood, with
consideration to the possible anticoagulants.
2.4·. Urine
If the urinary elimination rate is known, take the maximum dose elimi-
nated in 24 hours, considering the possible metabolites.
If no information is available about the elimination, take the maximum
therapy dose diluted in two liters of water. As for serum, proceed with
a stock solution diluted to the l/lOth with urine.
Samples must be constituted by fresh urine from patients taking no
drugs.
3- Procedure
This procedure is applicable to the 4 media above described.
3.1. Each test and reference sample pair is analyzed simultaneously in
the same conditions, until 10 results are obtained, if the repeatability
of the method is good. If there is a significant scattering of the re-
sults a higher number may be selected.
If there is no variation with addition of a quantity of the product five
time higher as the maximum therapy dose, we consider the study as com-
pleted. If the variation between the test and the reference samples are
higher to 2 - 5 $> according to the parameter, we proceed to the next
step.
3.2. We prepare dilutions in series of the stock solution of the product
with the appropriate solvent.
For each type of sample and the corresponding series of.dilution samples
are analyzed simultaneously in the same conditions until we find a level
producing no variation compared to the reference sample.
 47

3.3· The studied parameter value is plotted against the therapy dose.
The value of the therapy dose retained for further study is taken where
the slope of the curve is maximum.
It would also be possible to work on a very high dose (20 g/l for ins-
tance) then on successive dilutions and then to mathematically or graph-
ically determinate a minimum active dose.
In this way, it would be possible to determine the smallest possible
dose, expressed in mg or ug/l able to induce a perturbation.
CONCLUSION
The analytical interference of drugs upon laboratory tests is ex-
tremely important if one wants to appreciate with accuracy the desired
or unwanted pharmacological effects as well as the possible toxicologica!
action. Existing literature is very abundant but it is often difficult
to make out the contribution of the analytical or toxicopharmacological
perturbations as well as the actual impact of the interferences. There-
fore, studies should be undertaken, according to perfectly defined
procedures, in order to evaluate the disturbing effects of the most
often used drugs and the reliability of the main laboratory tests in
presence of possible administration of drupa. A perfect knowledge of
the action mechanism is of high interest froa the prospective point of
view. If the new drugs and new methods are systematically evaluated in
this direction, we will have in the future a complete and updated
knowledge of this problem.
48

REFERENCES
Bailly M. : Perturbations des résultats d'analyses biochimiques provo­
quées par la thérapeutique. C.R. Journées pharmaceutiques interna­
tionales, Paris, 1972, 127.
Baselt R.C., Wright J.A., Cravey R.H. : Therapeutic and toxic concentra­
tions of more than 100 toxicologically significant drugs in blood,
plasma or serum : a tabulation. Clin. Chem., 1975, 21_, 44­62.
Capolaghi B. : Interférences analytiques des médicaments sur les paramè­
tres biologiques. Nancy, 1976 ­ Communication par G. Siest.
Caraway V.T. : Accuracy in clinical chemistry. Clin. Chem., 1971, H ,
63­71.
Caraway W.T., Kammeryer C.W. : Chemical interference by drugs and other
substances with clinical laboratory test procedures. Clin. Chim.
Acta, 1972, 41, 395­434.
Constantino N.V., Kabat H.P. : Drug induced modifications of laboratory
test values­revised 1973· Amer. H. Hosp. Pharmacy, 1973, ¿0, 24­71.
Delwaide P.A., Jadin Α., Heusghem C. : Interferences des médicaments sur
les déterminations effectuées en chimie clinique. In : Les effets
indésirables des médicaments. Eeusghem C, Le chat P. Ed., Masson
Pubi., 1973, Paris, 698­710.
Elking M.P., Kabat H.P. : Drug induced modifications of laboratory test
values. Amer. J. Hosp. Pharmacy, 1968, 25., 485­519.
Letellier G., Vinet B. : Etude in vitro de l'influence des drogues sur
les analyses : sérum de contrôle VS sérum frais. 5emes journées
internationales de Biologie Clinique, Tours, sept. 1976.
Marzin D. : Etude de l'interférence de la D penicillamine sur différents
dosages biologiques. Doctorat es sciences pharmaceutiques. Univer­
sité Paris Sud, 1979.
Reiss B.S. : Interactions of drugs and laboratory tests. J. Clin.
Pharmacol., 1975, feb­mar, 135­138.
Siest G., Bretaudière J.P., Buret J., Favre R., Gueguen R., Petitclerc
C , Sachs C, Vernet Η., Zender M. : Les variations biologiques
des examens de laboratoire. Inform, sci. Biol., 1978a, 4_, 3­14.
Siest G., Appel W., Blijenberg G.B., Capolaghi B., Galteau M.M.,
Heusghem C, Hjelm M., Lauer K.L., Le Perron, Loppinet V., Love C ,
Royer R.J., Tognoni C, Wilding P. : Drug interference in clinical
chemistry : studies on ascorbic acid. J. Clin. Chem. Clin. Biochem.,
1978b, 16, 103­110.
Siest G., Buret J., Gueguen R., Petitclerc C, Sachs C, Vernet Μ.,
Zender R., Panek E., Drosdowsky M., Guize L. : Modalités pratiques
de production des valeurs de référence. Inform, sci. Biol., 1979a,
í, 2.
Siest G., Bretaudière J.P., Buret J., Gueguen R., Petitclerc C, Sachs C ,
Vernet Μ., Zender R. : Influence des facteurs analytiques sur les
valeurs de référence. Ann. Biol. clin., 1979b, J7, 125­126.
Singh H.P., Hebert Μ.Α., Gault M.H. : Effect of some drugs on clinical
laboratory values as determined by the Technicon SMA 12/60. Clin.
Chem., 1972, 18, 137­144.
Sunderman F.W. : Drug interference in clinical biochemistry. Crit. Rev.
Clin. Lab. Sci., 1970, 1, 427­449.
Wirth W.A., Thompson R.L. : The effect of various conditions and subs­
tances on the results of laboratory procedures. Am. J. Clin. Pathol«,
1965, 21, 579­590.
PLACE OF REFERENCE MATERIALS AND REFERENCE METHODS IN THE EVALUATION OF
DRUG EFFECTS
Dennis J. Reeder
Organic Analytical Research Division, Center for Analytical Chemistry
National Measurement Laboratory, National Bureau of Standards
Washington, D.C. U.S.A.

ABSTRACT
The presence of analytical errors in clinical laboratory testing may
be the result of many factors. Even though most clinical tests are becom-
ing very precise, reliance on precise tests for accurate analytical values
is not sufficient because of potential bias caused by drug effects. Proper
analytical techniques can minimize the sources of inaccuracies caused by
variabilities and interferences but a true understanding of drug effects
can only be obtained when measurements are placed on an accuracy base.
Assurance that measurements are accurate can best be accomplished by use of
reference materials and reference methods that are free of interferences
and that can establish the accuracy of an analytical value. Development of
new reference materials and reference methods will become increasingly
important for proper health care.

INTRODUCTION
While this workshop is directed towards variations in results of
laboratory tests due to drug intake, we must keep in mind that it is the
Interpretation of test results by the physician that determines the resul-
tant treatment of a patient. If a physician places too much dependence on
a single clinical laboratory result, patients may be given incorrect quan-
tities or types of medication, although many physicians repeat tests to
confirm suspected out-of-normal values. Because of many complex chemical
reactions that are the basis of today's mass-produced, multiphasic, com-
puterized and automated test procedures, it is difficult for a physician to
anticipate aberrent test results that may occur as a result of the influ-
ences of medications on those tests. Furthermore, instances have been
reported in the literature where identical samples were sent to a number of
laboratories, simultaneously, and the ranges of reported values were both
alarming and disheartening. (Pippenger, 1976)
Numerous questions can be raised. How much confidence can be placed
on the analytical results from any single laboratory? Do the more auto-
mated and precise tests give accurate values for comparison? What kind of
Interferences are likely to influence a laboratory test and cause inaccu-
racy in the reported data? In essence, how specific are the analytical
values? Which medications definitely affect results of a clinical
50
laboratory test?· Are all tests affected? And to what extent? What place
does the use of reference materials and reference methods have In the
evaluation of drug effects?
The purpose of this paper is to review some of the factors that can
cause analytical errors in clinical laboratory testing and to suggest
current and expanding_roles for reference materials and reference methods
in making meaningful measurements for health care.
PRECISION OF ANALYTICAL TESTS
In any measurement procedure, precision is demonstrated when the same
numerical value is obtained repeatedly. This measure of precision is
termed repeatability within a single laboratory and is called reproduci-
bility between different laboratories. With the advent of highly sophis-
ticated, automated instruments that sample and pipette reagents and patient
fluids, precision of a given test within a single day has improved consid-
erably. Laboratories that report a high degree of precision in their test
results are often considered to be highly accurate. Often there is a high
and positive correlation between precision and accuracy. However, in
practice, some highly precise systems are sometimes found to be wildly
inaccurate. The important point is that reliance on precise tests for
accurate analytical values must be carefully considered. Drug interfer-
ences on a laboratory test may not alter the precision of the test, but may
grossly bias the reported values.
ACCURACY AND SOURCES OF INACCURACY

Accuracy of a chemical method has been defined by Anastassiadis and

Common (1975) as follows: "The accuracy of a chemical analytical method
for the determination of substance A may be defined as the degree to which
the mean measurement, and therefore the mean estimate, obtained by the
method approaches the true measurement, and therefore the true value, for
substance A after the effects of other substances giving the same measure-
ment as substance A have been eliminated (physically or mathematically)."
Analytical accuracy is important in every measurement from a scien-
tific point of view and also from the point of view of medical practice as
it relates to patient care. If methods are not accurate, it is impossible
to obtain meaningful baseline data on an individual's biochemical profile
and then later compare measurements performed on individuals after inter-
vals of several years. Biochemical changes within an individual may be
more important than previously realized. Accuracy must be assured over
many years (Harris, et al., 1970).
 51
Because laboratory procedures have limitations of accuracy, faulty
values are sometimes reported. Wrong conclusions, based on these spurious
values, can lead to incorrect diagnosis and, consequently, irrational
therapy resulting from faulty prescribing. Although this paper focuses on
the proper use of reference materials and methods in minimizing analytical
errors, it is prudent to be aware that serious inaccuracies may be gener-
ated by a host of other factors.
Variability Factors
Of the multitide of factors that may affect the accuracy of a reported
analytical test value, three categories emerge.
1. Biological variabilities - The concept of biochemical individu-
ality has long been mentioned in the literature (Williams, 1967). It is
often forgotten in laboratory interpretation of test data, but strong
evidence exists that variations between patients are often due to genetic
differences. Diurnal and cyclical changes within the same individual will
also influence the specimen given for analysis at periodic intervals
(Krieger, 1970).
2. Handling variabilities - Regardless of how sophisticated the
equipment and methodology spurious results may be the result of inadequate
handling. Adjusting pH, blood drawing, chromatographing, cooling, distill-
ing, extracting, filtering, heating, measuring, mixing, pouring, sampling,
separating, transferring, weighing, and performing other critical opera-
tions must be carried out in a skillful manner to avoid losses, contamina-
tion, or other effects that can adversely affect the ultimate value.
3. Laboratory variabilities - Laboratory errors often are a result
of failure to pay attention to details. Accurate timing, frequent checking
of instrument parameters, temperature control, cleanliness, use of control
sera, use of control charts, use of fresh reagents, and other controls
tend to minimize laboratory errors. Without strict adherence to set pro-
cedures, both accuracy and precision can be degraded to the point where
meaningful measurements are not feasible.
Interferences
In addition to the above mentioned sources of error there are a
variety of interferences to be considered. Dr. A. Galli, in this workshop,
will elaborate more on this subject.
1. Biological Interferences - Biological interferences may be the
result of pharmacologic, immunologic, or toxic actions of medications or
environmental stress situations. For example, elevated steroid levels due
52

to medication or· stress may alter the concentration of electrolytes in'the

urine and blood (Azarnoff, 1975).
2. Physical Interferences - Many physical interferences are mani-
fested in analytical tests that are based on colorimetrie or spectrophoto-
m e t r y determinations. For example, selection of the proper wavelength
(460 nm) for the analysis of bilirubin in serum rather than the 420 nm
wavelength which is sometimes specified, reduces the elevation of the
reading resulting from traces of hemoglobin (Caraway, 1962).
3. Chemical Interferences - Interfering chemicals are the most
frequently encountered sources of error in clinical laboratory testing.
The literature contains many hundreds of examples of values reported to be
abnormally high or abnormally low as a result of chemical interactions. In
some cases the effects are very subtle and unsuspected. For example, many
drug products employ chelating agents such as ethylenediaminetetraacetic
acid (EDTA) or similar derivatives to complex trace metals, thereby pre-
venting discoloration or oxidation. Trace amounts of these chelating
agents are sufficient to decrease values for serum calcium (Elking and
Rabat, 1968).

Some drugs interfere with oxidation or reduction reactions that are

the basis for in vitro analytical tests. Other drugs contribute elements
such as calcium, iodine, or sodium that would produce abnormally high serum
levels. Some drugs produce metabolites that interfere with certain tests.
For example, penicillin yields metabolites that produce false positive
values for protein in urine. Tetracyclines may interfere with laboratory
values for albumin, bilirubin, glucose, and catecholamines, as well as
alkaline phosphatase, aspartate aminotransferase, cholesterol and potassium
(Martin, 1971).
For some tests the list of interferences is extensive. Interferences
with determinations of glucose are most often reported (Elking and Kabat,
1968). Likewise, albumin in urine is widely modified by a variety of
drugs. Reports of elevated albumin in the urine can be dangerously mis-
leading since this condition occurs in congestive heart failure and renal
disease, as well as in many infectious diseases.
When multiple drugs are given, which is more likely the case in
today's therapeutics, the difficulty of correctly interpreting test results
is increased substantially.
ROLE OF REFERENCE MATERIALS AND REFERENCE METHODS
It is conceivable that many clinical tests are biased by drug effects
 53
and that these biases are never seen or recorded, either because the test
is Insufficiently precise so that variabilities are lost in the "noise" or
because there is no existing standard by which changes can be measured and
compared. Improvement of the quality of laboratory performance worldwide
is the goal of many international federations such as WHO or IFCC. One
common goal that has been stated repeatedly by these organizations is the
need to develop certified standardized reagent materials and to establish
standardized international methods. It has long been a position at the
National Bureau of Standards (NBS) that a hierarchy of reference materials
and measurement methods is necessary to establish a compatible clinical
measurement system on a national or international basis.
Understanding and evaluating drug effects on a myriad of clinical test
procedures can only be accomplished when suitable materials and methods are
developed for attaining accuracy. After all the sources of inaccuracy
which were mentioned in the section above have been minimized, there
remains the task of ensuring that the test method used will attain a value
that is free of bias or systematic error and that is specific for the
analyte being measured. Validation of the analyses and transfer of accu-
racy is best accomplished with reference materials in conjunction with
reference methods.
Reference Materials
The term "reference materials" is used to describe a generic class of
stable, homogeneous materials that are well-characterized and produced in
quantity and having one or more chemical or physical properties determined
within stated measurement uncertainties. The reference material is an
integral part in assuring accuracy because it provides a material with a
known answer.
Reference materials are generally used to either calibrate an instru-
ment or assess an analytical method. Reference methods also serve as
transfer devices to achieve measurement compatibility between laboratories.
If a reference material is used in a measurement system for a particular
analyte, and the certified answer is obtained, then one may with some
assurance expect that the measurement will yield the correct value for.the
unknown material (of similar nature to the reference material). If drug
effects or other interferences are present, correct interpretation of the
test result may be clouded unless a reference material is used for cali-
bration of the test method. The best reference material, of course, would
be a matrix-type reference material with certified levels of each desired
54
analyte. More likely one would use pure reference materials added to a
patient's serum for a standard addition type of analysis.
At the Conference held in Atlanta, GA in 1977, entitled "A National
Understanding for the Development of Reference Materials and Methods for
Clinical Chemistry", sessions were devoted to the specification of criteria
for selecting, validating, characterizing, and labeling standard materials.
From those sessions came twelve recommendations that cover the above points
and the reader is directed to the publication of those proceedings for full
details (Boutwell, 1978). It was recommended that two materials should be
considered as clinical chemistry "standards", and that these materials
should be defined in terms of their degree of characterization. The most
highly characterized material is termed a Certified Reference Material
(CRM) such as those issued by NBS as Standard Reference Materials, and is
characterized by definitive methods (where available) and/or exhaustive
characterization by independent reliable techniques. A second class of
standard is the Calibration and Test Material (CTM) that must be traceable
to a CRM (if available) by a reference method or well-accepted method(s).

Recommendations were also made that the following general criteria

should be specified for CRMs: (1) specific composition; (2) homogeneity;
(3) physical characteristics; (4) other chemical characteristics (e.g.
reactivity); (5) biological characteristics (e.g., biological hazards,
sterility); (6) stability; and (7) preservatives, if any.
Example of Reference Material for Drug Testing
A recently certified reference material for the standardization of
antiepilepsy drugs in serum (Standard Reference Material 900) was under
development for several years at NBS. Initially, a large amount of human
serum was processed to remove, chemical compounds that would interfere with
the analytical methods used in certifying the amount of drugs added to the
processed serum. A combination of dialysis, delipidation, and charcoal
treatments served to reduce a number of components as monitored at 254 nm
in a liquid chromatography separation procedure. Upon analysis at NBS,
analytical values obtained by both liquid chromatography and gas chroma-
tography on underivatized extracts of the serum proved to be consistent
with each other and close to the amount of drug added to the serum. How-
ever, when these prototype SRM's were sent to selected clinical labora-
tories, some concentrations of phénobarbital were overestimated by ten to
twenty percent. Furthermore, only those laboratories that employed a
 55
particular methylatlon procedure pri,or to the GC analysis for phénobarbital
experienced difficulties.
Investigation of the problem revealed the presence of a small amount
of a substance in the prototype serum reference material that co-eluted with
the methylated phénobarbital in GC analysis. Further separation and resolution
of the substance by GC and identification by maäs spectrometry showed that
the substance was phthalic anhydride (mw 148.1), derived from a common
plasticizer. The source of this compound was considered to be from the
original plastic bags used for storing the serum. Subsequent production
lots of serum were more thoroughly treated with charcoal and monitored more
stringently for interferences.
This type of control and monitoring is essential for reference mate-
rials that will be used to calibrate instrumentation used in trace organic
analysis of clinical specimens.
Reference Methods
At the Atlanta Conference mentioned above, the following definition
for a reference method was developed: "A reference method is an analytical
method with thoroughly documented accuracy, precision, and low susceptibil-
ity to interferences. The accuracy and precision shall be demonstrated by
direct comparison with the definitive method and SRM or, where not avail-
able, with other well-characterized and documented analytical approach(es)."
Reference methods are one of the key components of a chemical measure-
ment system and represent the primary mechanism for transferring the accu-
racy of a reference material or a definitive method into widespread use in
the field. Definitive methods are the ultimate methods of chemical analy-
sis that have a valid and well-described theoretical foundation, yield high
levels of precision, have negligible systematic errors, and with high
reliability give "true values". They are the most accurate methods avail-
able to measure a given chemical property and, in general, are uneconomical
for all but the most demanding uses. Their place is in the certification
of primary standard reference materials and in the development of reference
methodology. For amplification of the relationships between these methods
and reference materials, the reader is directed to the publication by
Urlano and Gravati (1977).
Reference methods are used for the following purposes: 1) to deter-
mine analyte concentration in calibrators; 2) to evaluate bias in routine
methods; 3) to test for interferences in routine methods; 4) to assess
56
product compliance; 5) To analyze patient samples in specific clinical
studies.
When inaccuracies in methodology have been shown to exist or if such
inaccuracies may impair the delivery of optimum medical care, then develop-
ment of a reference method is needed. When reference methods are applied
to the evaluation of drug- effects on laboratory tests, it is particularly
important that the methods be validated for their immunity to interfer-
ences. As described by Dr. Neil Lawson of the College of American Pathol-
ogists (Boutwell, 1978), the following problem areas need to be considered:
1) common drugs (such as aspirin and.caffeine, and their effect on the pro-
posed reference method); 2) drugs likely to be used in conjunction with the
disease state for which the test is ordered; 3) drugs known to interfere
with an already utilized method; 4) analytic properties of the method
itself which lend themselves to interference; 5) intrinsic chemical ana-
lytes in the matrix; 6) if the analyte is a drug, (identification of) other
drugs and metabolites of drugs which may be used in treatment along with
the analyte drug.
DIRECTIONS FOR THE FUTURE

The isolation, identification and quantitation of trace amounts of

organic compounds from the complex matrices found in clinical samples is
becoming increasingly important in patient care. While new instruments,
devices, and detectors are becoming increasingly sensitive, there exists an
even greater need to assure that accuracy is the basis of the measurements
obtained. As new field methods evolve for analysis of clinical specimens,
drug effects may be exerted in ways that are unexpected. Confusion will be
amplified as conflicting reports continue to be generated. It is primarily
through careful and accurate measurements based on reference methods and
materials that many of these problems will be solved. The present problem
is that there exist only a few definitive methods for clinical chemistry,
a few accepted reference methods, a paucity of matrixed certified reference
materials, and approximately twenty pure CRMs for clinical chemistry.

Clearly, there are many needs for the development of Certified Refer-
ence Materials and reference methods, particularly in those cases where
serious drug effects have been observed and patient care has suffered as a
result of laboratory analytical errors. Partial resolution of some of
these needs is currently underway at NBS with the certification of electro-
lytes and some organic constituents (such as cholesterol) in a large homo-
geneous batch of freeze-dried human serum. This proposed SRM (No. 909)
 57
will have analyte values certified by definitive methods. In addition,
informational values for other analytes will be provided as they are
measured by the best currently available methods. Calibration of instru­
mentation with this material may be very useful in elucidating drug effects
and interferences on clinical laboratory tests.
REFERENCES
Anastassiadis, P. Α., and Common, R. H.: Some Aspects of the Reliability of
Chemical Analyses. A nalyt. Biochem. 22: 409­423, 1968.

Azarnoff, D. L.: Steroid Therapy, p. 9, (W. B. Saunders Co., Philadelphia

1975).

Boutwell, J. H. (ed): A National Understanding for the Development of

Reference Materials and Methods for Clinical Chemistry. Proceedings
of a Conference. (A merican Association for Clinical Chemistry,
Washington, D.C. 1978).

Caraway, W. T.: Chemical and Diagnostic Specificity of Laboratory Tests.

Am. J. Clin. Path. 37: 445­464, 1962.

Elking, M. P., and Kabat, H. F.: Drug Induced Modification of Laboratory

Test Values. A mer. J. Hosp. Pharm. 25: 485­519, 1968.

Harris, E. K., Kanofsky, P., Shakarji, G., Cotlove, E.: Biological and
Analytical Components of Variation in Long­term Studies of Serum
Constituents in Normal Subjects. II. Estimating Biological Components
of Variation. Clin. Chem 16:11 1022­1027, 1970.

Krieger, D. T.: Factors Influencing the Circadian Periodicity of Adrenal

Steroid Levels. Trans N.Y. Acad Sci. 32: 316­329, 1970.

Martin, E. W.: Hazards of Medication, A Manual on Drug Interactions,

Incompatibilities, Contraindications, and Adverse Effects. Chapter 7,
156­215, (J. B. Lippincott Co., Philadelphia 1971).

Pippenger, C. E., Penry, J. K., White, B. G., Daly, D. D., and Buddington,
R.: Interlaboratory Variability in Determination of Plasma Anti­
epileptic Drug Concentrations. A rch. Neurol. 33: 351­355, 1976.

Urlano, G. Α., and Gravatt, C. C : The Role of Reference Materials and

Reference Methods in Chemical Analysis. CRC Critical Reviews in
Analytical Chemistry 6: 361­412, 1977.

Williams, G. Z.: Individuality of Clinical Biochemical Patterns in

Preventive Health Maintenance. J. Occup. Med. 9: 567­576, 1967.
DETECTION OF DRUG INTERFERENCES IN METHOD EVALUATION PROGRAMS

R. Haeckel and O. Sonntag

Medical School Hannover, Institute for Clinical Chemistry
Hannover, West-Germany

ABSTRACT
Drug effects are differentiated into interaction and in-
terference effects. Interference is defined as the effect of a
sample component other than the analyte on the determination
of the concentration or catalytic activity of the analyte.
Three various mechanisms are described.
A screening list of about 60 drugs representing the most rele-
vant preparations presently used are proposed for a scheme to
detect possible drug·interference in method evaluation programs,

Drug effects are usually differentiated into interaction

and interference effects. Drug interaction is the influence on
the concentration or catalytic activity itself of an analyte
("Einflußgröße" according to Büttner et al., 1978). This effect
is caused in vivo. Drug interference solely occurs in vitro and
is related to the analytical determination of an analyte ("Stör-
faktor" according to Büttner et al., 1978). The definition of
the term interference, however, is not unanimous in the lite-
rature .
In the following an interference is considered as the effect
of a sample component other than the analyte on the determi-
nation of the concentration or catalytic activity of the ana-
lyte. This influence leads to a difference between the value
measured and an assumed result which would have been obtained
in the absence of this component. This definition is close to
that proposed by the National Committee for Clinical Labora-
tory Standards. (Proposed Standard: PSEP-2. Protocol for
establishing performance claims for clinical chemical methods,
1979, p. 1o).
An interference can be positive or negative (that means in-
crease or decrease the final result) and can be caused by
 59

components of the sample either derived from endogenous

sources under specific conditions (such as bilirubinemia, al-
captonuria, etc.) or administered (such as druqs, ascorbic
acid, poisons, etc.).
Using this definition 3 various mechanisms can lead to an in-
terference (Table 1):
1. The interférant component affects the concentration of the
analyte molecule as it is measured (e. g. depression of the
ionisation of potassium atoms by sodium ions).
2. The influence on the chemical reaction(s) involved in the
detecting procedure.
3. The component interferes with the physical measurement of
the analyte or indicator without any influence on the che-
mical reaction.
These 3 groups can be further subdivided (Table 1).
TABLE 1 - VARIOUS MECHANISMS OF INTERFERENCES

1. Direct effect on the concentration of the analyte molecule

as it is measured.
2. Influence on the chemical reaction(s) involved in the de-
tecting procedure.
2.1 Component reacts in the same way as analyte (identical re-
action sequence).
2.2 Component reacts only with the indicator (reaction)
2.3 Component inhibits or enhances the over-all reaction.
3. Component interferes with the physical measurement of the
indicator.
3.1 The comnonent itself yields an overlaoping signal.
3.2 Component undergoes another reaction producing an over-
lapping signal.

1) In the case that an indicator reaction is present

60

Recently the Expert Panel on Quality Control on the Internati­

onal Federation of Clinical Chemistry has recommended (Büttner
et al., 1976) that only mechanisms No. 1 and 3 should be con­
sidered as interference. According to the definition of these
authors an interference "is the effect of a component, which
does not by itself produce a reading, on the accuracy of mea­
surement of another component. It results in either low values
for the analyte (inhibition) or high values (enhancement)".
All other mechanisms listed in table 1 would than lead to non­
specificity. Non­specificity is obtained because sample compo­
nents other than the analyte contribute to the reading".
Following the further explanations of the authors specificity
is the capability of an analytical method to measure only the
analyte that it pretends to detect (Büttner et al., 1976,
Stamm, 1979). In the specific case that the analyte is not pre­
sent in the sample, any signal measured due to a component is
considered a non­specificity (Fig. 1 ) . In the presence of the
analyte any signal caused by a component can be either non­
specificity or interference or both.

T3
d)
3
(0
m
ω
E

II
α>
η

co
c
σι

M\
Present in a c+a c+a
the sample: analyte component

(positive)
non­specificity interference

Fig. Λ_\ Difference between non­specificity and interference

according to Büttner et al. (1976).
 61

The differentiation between "non-specificity" and "interfe-

rence" as proposed by the Expert Panel on Quality Control re-
quires more experiments than are usually performed. In many
cases the exact mechanisms cannot be identified. In addition
it is of low practical usefulness for the evaluation of ana-
lytical procedures.
For the evaluation of analytical procedures we, therefore,
prefer to use "interference" as a common term for all effects
listed in table 1.
In the last years several national (Broughton et al., 1974,
Stamm, 1979) and international programs (BUttner et al., 1976)
for the evaluation of analytical methods have been proposed
which require the study of interferences from endogenous and
exogenous components. The investigation of interferences from
endogenous substances is less problematic. No details have been
proposed so far in these recommendations how the influence of
exogenous substances, especially of drugs should be studied.
In method comparison studies as suggested in most evaluation
protocols (Barnett and Youden, 197o, Broughton et al., 1974,
BUttner et al., 1976, Haeckel, 1979) 5o - 1oo samples from
hospital patients are used. This sample size is unlikely to de-
tect all relevant drug interferences. Therefore a separate
check for interferences from exogenous components is required.
Mathies et al. (1974) first selected 4o various drugs repre-
senting the most relevant groups of drugs applied and some an-
ticoagulants. These substances were added in pure from to
pooled human serum. The concentrations used corresponded to
the maximum single dosage which can be applied per day, solved
in 5 1 serum. This volume was considered to approximate the
primary distribution volume of the body. The 95 % reference
ranqe was calculated from 2o values determined in one series
from untreated pool serum samples (mean value + 2 standard de-
viations).
We have found this procedure very useful in several evaluation
studies (Haeckel, 1976, Haeckel et al., 1979). However, the
concentrations used appeared too high. Interferences were ob-
served too often which were not relevant to clinical chemistry
62

(Haeckel eind Sonntag, 1979) .Therefore, another criterion is

now suggested: the maximal plasma concentration which is de-
fined as the highest concentration which can be obtained in
patients treated with the corresponding substance. This value
can be the lethal concentration.
Baselt et al. (1975),Winek (1976) and Pentz et al. (1979) have
recently published a list of therapeutic, toxic and lethal
drug concentrations in human plasma. From these lists a ratio
of about 5 can be derived for the average relation of lethal
to the upper therapeutic concentration (n = 69). This ratio
is applied in all cases where the lethal concentration is un-
known.
For the evaluation of new analytical procedures, we propose a
screening list of about 6o drugs (table 2 ) . In the Medical
School Hannover about 96o drugs are presently used, half of
which presumably penetrate into the blood stream and, there-
fore, can possibly cause an interference. Among these we have
selected the most relevant groups.
The analgestics/antiphlogistics are a very large group. About
4oo preparations are available on the German market, which re-
cently have been related to 5 basic chemical structures
(Schulz and Gross, 1979): Salicylic acid, pyrazolon, indol
acetic acid (arylacetic acid) , anthranilic acid and 2-phenyl-
propionic acid (arylpropionic acid). In table 2 all these
groups are represented.
This list is probably not complete and must be modified for
special purposes. Apparently it must be unsufficient to charac-
terize a new method, especially to identify interferences of
mechanism 1 in table 1 (study of the specificity).
Furthermore it would be recommendable to include those drug
metabolites with reactive groups which reach relevant blood
concentrations. Drugs as for instance novaminsulfone, which
are rapidly metabolized and, therefore, hardly detectable in
the blood, are not considered.
In the present procedure, a five litre pool of clear sera is
collected from persons not having taken drugs during the last
3 weeks. The pure powder of the drugs is then directly solved
 63

in 1o - 1oo ml portions of the homogenous serum oool. All oor-

tions are analyzed in one batch. Portions to which no substan-
ces are added, are included at the beginning and at the end of
the batch and after each fifth position. At least 15 of these
control samóles are used to determine a 99,7 % reference ranae
(mean value + 3 standard deviations). All values which are out-
side this reference range as susoect to an interference effect.
If the drug addition experiment has revealed an interference,
2 separate experiments should be performed:
1. addition in vitro with several concentrations of the drug,
and
2. application in vivo.
In the first experiment, the corresponding drug is added to
the same serum pool in several concentrations which also cover
the therapeutic range (usually from 1o - 1oo % of the lethal
concentration). The results are presented graphically, as
shown in Fig. 2. An effect observed is tentatively considered
to be a significant interference at the in-vitro concentration
where the regression line crosses the unper or lower border of
the reference range. The uoward or downward trend of this line
should continue beyond this cross-over point.
The in-vivo application can be performed as proposed by
Stahler et al. (1975) with 3 men and 3 women. Blood samples
are taken o, 1,7 and 24 hours after administration of a single
dosage of the corresponding drug. If possible a comparison with
another method which is not subject to the interference sus-
pected should be included in this study. It is also very help-
ful if the blood concentration of the drug can be measured
simultaneously (Haeckel and Sonntag, 1979).
At the present time, the clinical relevance of an interference
observed must be regarded individually. Recommendations con-
cerning the declaration of a clinical relevance should be
worked out.
In summary, the screening procedure presented is proposed to
check for interferences. It is sufficient for evaluating ana-
lytical procedures. In the event and interference is observed,
its mechanism may then be investigated by those who intend to
 64

improve this method. The procedure appears as a recommendable

compromise between what is required and what can be achieved
with acceptable effort and expenses.

i 1
o
E

§ °
o
2co -1
ω

υ
ι
-3

100 200 400 300 500 600

Bilirubin [u m o l / l ]

Fig. Interference of bilirubin. The cholesterol concentration

in various bilirubinemic sera was determina with gas
chromatography (GC) and a Liebermann-Burchard procedure
(SMA 12/6o). The difference ( Δ cholesterol) between
the GC and the other method is plotted on the ordinate.
The upper and lower borderline were calculated as the
threefold standard deviation firom the results ( Δ
cholesterol) obtained with clear (non-bilirubinemic)
sera. Taken from Haeckel et al. (1979).
 65

REFERENCES
Barnett, R.N. and Youden, W.J.: A revised scheme for the com-
Darison of quantitative methods. Amer. J. Clin. Pathol.
'54: 454-462, 197o.
Baselt, R.C., Wright, J.A. and Carvey, R.H.: Therapeutic and
toxic concentrations of more than 1oo toxicologically
significant drugs in blood, plasma or serum: a tabulation.
Clin. Chem. 21: 46-62, 1975.
Broughton, P.M.C., Gowenlock, A.H., Mac Cormack, J.J. and
Neill, D.W.: A revised scheme for the evaluation of auto-
matic instruments for use in clinical chemistry. Ann.
clin. Biochem. 11: 2o7-218, 1974.
Büttner, J., Borth, R., Boutwell, J.H., Broughton, P.M.G. and
Bowyer, R.C.: Provisional recommendation on quality con-
trol in clinical chemistry. Part 2. Assessment of ana-
lytical methods for routine use. J. Clin. Chem. Clin.
Biochem. 14: 265-275, 1976.
BUttner, J., Rommel, K., Stamm, D. und Wisser, H.: Charakteri-
sierung der fachgerechten klinisch-chemischen Unter-
suchung. Dt. Ges. f. Klin. Chem. e. V. - Mitteilungen 9:
244-248, 1978.
Haeckel, R.: The use of aldehyde dehydrogenase to determine
H_0--producing reactions. J. Clin. Chem. Clin. Biochem.
14: 1o1-1o7, 1976.
Haeckel, R., Sonntag, 0., KUlpmann, W.R. and Feldmann, U.:
Comparison of 9 methods for the determination of cho-
lesterol. J. Clin. Chem. Clin. Biochem. 17: 553-563, 1979.
Haeckel, R. und Sonntag, 0.: Ektachem, ein neues Analvsen-
system. GIT Labor-Medizin 2: 317-327, 1979.
Haeckel, R. : Statistische Probleme beim Vergleich von klinisch-
chemischen Analysenverfahren. J. Clin. Chem. Clin. Bio-
chem, 198o, in press.
Mathies, H., Stähler, F., Wittner, H. und Vollmar, H.: Beein-
flussung eines neuen enzymatischen Harnsäure-Farbtestes
durch Pharmaka in vitro und in vivo. Med. Klin. 69: 6o7-
612; 1974.
Pentz, R., Strubelt, 0. und Gehlhoff, C : Theraoeutische,
toxische und lethale Arzneimittelkonzentrationen im
menschlichen Plasma. Dt. Ärzteblatt 43: 2815-282o, 1979.
Schulz, V. und Gross, R.: Therapie mit nichtsteroidalen Anti-
phlogista. Dt. Xrzteblatt 43: 2821-2828, 1979.
Stamm, D.: Recommendations for the description of a Selected
Method. J. Clin. Chem. Clin. Biochem. 17: 28o-282, 1979.
Stähler, F., Münz, E. und Kattermann, R.: Enzymatische Be-
stimmung von Gesamt-Cholesterin im Serum. Dt. Med. Wschr.
1oo: 876-887, 1975.
Winek, Ch.L.: Tabulation of therapeutic, toxic and lethal con-
centrations of drugs and chemicals in blood. Clin. Chem.
22: 832-836, 1976.
66

Table 2: SUBSTANCES PROPOSED FOR THE INVESTIGATION OF POSSIBLE

INTERFERENCES IN CLINICAL CHEMICAL METHODS

I. N. N. example for maximal reference

trade pre­ plasma
parations concen­
tration
mg/1

Aldosteron­antagonists
spironolactonum Aldaktone 5o 1» 2

Analgestics/antirheumatics
pethidinum DoIantin 3ο 3, 4
levomethadonum L­Polamidon­ 16,7 3, 5
Hoechst
phenylbutazonum Butazolidin 4οο 3, 6
oxyphenbutazonum Tanderil 6οο 7
acidum acetylo­ Aspirin 5οο 3, 8
salicylicum Colfarit
acidum niflumi­ Actol 45 9
nicum
Indometacinum Amuno 4ο 3, 1ο
naproxenum Proxen 2ο7ο 11, 12
D­penicillaminum Metalcaptase 55 13
paracetamolum Ben­u­ron 15ο 3,14,15
glafeninum Glifanan 3οο 16
azapropazon­di­ Prolixan 625 17,18
hydrat

Antiallergics
antazolinum Antistin 25 19

Antibiotics
gentamycinum Refobacin 11ο 2ο,21
cephalexinum Ceporexin 3οο 22,23,24
cefazolinum Gramaxiη 148ο 25,26
chloramphenicolum Paraxin 1οο 3,27,28
 67

Ι. Ν. Ν. example for maximal reference

trade pre­ plasma
parations concen­
tration
mg/1

erythromycinum Erycinum 1639 29

aminobenzylpeni­ Binotal 5oo 16oo 3o,31
cillinum
tetracyclinum Hostacyclin 4o 31,32
nitrofurantoinum Furadantin 5o 33,34

Antidiabetics
tolbutamidum Rastinon 5oo 3,35,36

Antiepileptics
acidum phenyl­ Luminal 60 3,37,38
aethyl­barbi­
turicum
acidum valpro­ Orfiril 5oo 3,39,4o,41
inicum
phenytoinum Phenhydan 5o 3,42,43,44
Carbamazepinum Tegretal 25 3,45

Antihypertonics
dihydralazinum Nepresol 11,5 46,47,48
methyldopa Sembrina 1o9 49,5o

Anticoagulantics
phenprocoumonum Marcumar 26 51,52
Na­citrabe Na­citrate 5 000 53
Na­heparinat Liquemin 75o 53
Na­fluoride Na­fluoride 2 000 53
Na­oxalate Na­oxalate 3ooo 53
EDTA Titriplex III I000 53

BronchosDasmolytics
theophyllin­ Euphyllin loo 3,54
monohydrat Solosin
68

Ι. Ν. Ν. example for maximal reference

trade pre­ plasma
parations concen­
tration
mg/1

Cortocoides
predni solonum Solu­Decortin 36 55

Diagnostics
acidum triiod­ Angiografin 6II00 56,57
benzoicum
adipinyltriiod­ Biligrafin 6000 58
anilidum

Diuretics
furosemidum Lasix 3o 59

Hypouricemic effective agents

probenecidum Benemid 1000 3,6o
benzobromaronum Uricovac 47 61,62
allopurinolum Zyloric 1o 63,64

Immun suppressiva
azathioprinum Imurek 1o 65

Plasmaexpander
dextranum 6 % Macrodex 6 % 8125o 66,67,68

Lipidlowering agents
clofibratum C
Dura­ lofibrat 378,5 69

Psychopharmaca
doxepinum Aponal 1o 3,7o
chlordiazepoxidum Librium 2o 3,71,72
 69
Ι. Ν. Ν. example for maximal reference
trade pre­ plasma
parations concen­
tration
mg/1

Chemotherapeutics

sulfanilamido­ Durenat 5oo 73

pyrimidinum
salicylazosulpha­ Azulfidine 225 74
pyridlnum

Antituberculostacis
isoniazidum Neoteben 1oo 75

Vitamins
Vitamin Β complex Polybion 8oo 76
acidum ascorbicum Cebion 235 77,78

Cytostatics

eyelophosphamidum En do χ an 452 79
acidum methyl­ Methotrexat 5oo 3,8o,
pteroyl­gluta­
minicum

The following substances were excluded from the list, because

the presumed lethal plasma concentration is 5 mg/1 or less:
chloroquinum (82, 83), glibenclamidum (84), reserpinum (85),
norfenefrinum (86), propranololum (3, 87, 88), digoxinum (3,
89 ­ 91), carbocromenum (92), promethazinum (93), chlorproma­
zinum (94).
70

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64. Rodnan, O.P., Robin, S.F. and Elion, G.B.: JAMA 231: 1143,
1975.
65. Elion. G.B. and Hitchings, G.H., Handbook of Experimental
Pharmacology 38, 2, Springer Verlag, Berlin, 1975.
66. Strey, W. , Schwartzkopff, W., Wurm, W. and Pedersen, O.M.:
Anaesthesist 26: 295­3o6, 1977.
67. Lüders, Κ., Kanold, P., Otten, G., Koslowski, L. und
Eichenseher, Ν.: Der Chirurg 44: 563­569, 1973.
68. Emmrich, P., Baumann, W. und Stechle, U.: Euroo. J. Pe­
diat. 125: 181­19o, 1977.
69. Persönliche Mitteilungen der Fa. Durachemie, 1979.
70. Ziegler, V.E., Biggs, J.T., Wylie, T., Rosen, S.H., Hawf,
D.J. and Coryell, W.H.: Clin. Pharmacol. Therap. 23:
573­579, 1978.
71. Randall, L.O.: Diseases of Nervous System Supplement 22:
1 ­ 9, 1961.
72. Smyth, D. and Pennington, G.W.: Arch. int. Pharmacodyn.
145: 154­165, 1963.
73. Wöhler, F., Otte, W. und Jung, H.: Arzneim. Forsch. (Drug
Res.) 11: 736, 1961.
74. Schröder, H. and Campbell, E.S.: Clin. Pharmacol. Ther. 13:
539­551, 1972.
75. Bartmann: Beitr. Klin. Tuberk. 122: 239, 196o.
76. Ammon, R. und Dirscherl, W.: Fermente, Hormone, Vitamine
III, 3. Aufl. 598, 1974.
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43. 2o1­211, 1973.
78. Gehler, J. und Kubier, W.: Int. Z. Vit. Forsch. 4o: 454­
464, 197o.
79. Schaumlöffel, E., Clausnitzer, M., Heß, F., Röttger, U.
und Thome, R.; Therapiewo. 27: 2953­2987, 1977.
80. Isacoff, W.H., Morrison, P.F., Aroesty, J., Willis, K.L.,
Block, J.B. and Lincoln, T.L.: Cancer Treat. Rep. 61:
1665­1674, 1977.
81. Shen, D.D. and Azarnoff, D.L.: Clin. Pharm. 3: 1­13, 1978.
82. Mc Chesney, E.W., Banks, W.F. and Mc Auliff, J.P.: Anti­
biotics and Chemotherapy: 583­594, 1962.
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Medicine and Hygiene 6o: 75­78, 1966.
84. Rupp, W., Christ, O. und Heptner, W. : Arzneim. Forsch.
(Drug Res.) 19: 1428­1434, 1969.
85. Maass, A.R., Jenkins, B., Shen, Y. and Tannenbaum, P.: Clin.
Pharmacol. Ther. 1o: 366­371, 1969.
86. Hengstmann, J.H. , Konen, C, Eichelbaum, M. and Dengier, H.
J. : Europ. J. Clin. Pharmacol. 8: 33­39, 1975.
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Merkus, F.W.H.M.: Clin. Pharm. Ther. 22: 853­857, 1977.
 73

88. Coltart, D.J. and Shand, D.G.: Brit. Med. J. 3: 731-734,

197o.
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Wschr. 1o4: 543-546, 1979.
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2o3, 1973.
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Kreisl. 11: 12-14, 1979.
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(Drug Res.) 2o: 19o5-1911, 197o.
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468, 1974.
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Leverkusen.
 ANALYTICAL INTERFERENCES DUE TO ANTIINFLAMMATORY DRUGS

Roman Gallmany
Clinical Chemistry Department, C S . Principes España
Hospitalet de Llobregat, Barcelona, SPAIN

ABSTRACT
The effect of a group of anti-inflamatory drugs of anti-rheumatic
action is studied upon the twenty analytic tests most frequently requested
in a laboratory. In the in vitro study, significant interferences are
appreciated, produced by therapeutic doses of D-Penicillamine (Alkaline
Phosphatase and Urate), Chloroquine (Phosphate and LDH), Dexamethasone
(Creatinine) and Methylprednisolone (Phosphate); there are no significant
interferences produced by Phenylbutazone, Oxyphenbutazone, Indomethacine
and Sodium Aurothiomalate in therapeutic doses.

INTRODUCTION
In spite of the disparity in the chemical structure of the antiinfla-
matory non steroid drugs, they all have a series of common characteristics
based on the similarity of their pharmacokinetics properties. In Table I
the values of absoption, metabolism and renal elimination of these drugs
are shown. The most important common characteristics of this group are :
1) high oral absorption 2) high degree of binding to plasmatic proteins
and 3) preferent hepatic metabolism. Due to this, the basic form of admi -
nistration is oral, limited only by the digestive toxicity inherent to these
compoounds.(Champion, et al., 1978)
The binding to plasmatic proteins is between 50 and 99 %. These are
capable of displacing other pharmacos from the places of binding to the
albumin, increasing in this way their free from and their pharmacological
activity. The drugs that are the most frequently displaced are the dicou -
marinics, sulfonylureas, glucocorticoids, diphenilhydantoine, sulfonamides
and tyroxine. There is also competition between themselves, which would
explain that the joint administration of aspirin with indomethacine or
phenoprophen reduces the half-life of these and increases the elimination
of their metabolites.

The metabolism of this group of drugs is basically undertaken in the

hepatic microsomal fraction, thus being able to be induced by the action
of barbiturates with the consequent reduction in the half-life and activity
 TABLE I - DIFFERENT GROUPS OF ANTI - INFLAMATORY DRUGS

Drugs Oral Binding to Half -life Hepatic Urinary excretion

absortion % proteins % (hours) metabolism % unmodified %

Acetylsal ic yl ic
acid derivatives 70 40-70 0.2-0.3 97 1-3
Phenylbutazone
derivatives 70 - 80 98-99 48-72 94 1-3

Indoles
derivatives 80 90-99 6-10 97 10-20

Phenylacetic
acid derivatives 85 95-99 8-10 97 2-10

Arylalcanoic
acid derivatives 70 99-100 3 90 1-2

Antranilic
acid derivatives 70 90 4 90 10
76
of the antiinflamatory drugs.
As a concequence of the metabolism of these compounds by the liver,
the amount of unmodified drug eliminated through the urine is less than
15 %, except for high dosages of salicylates and sulphide of sulindac. The
biliary excretion of the metabolites varies between 2 and 50 %, being spe -
cially pronounced for phenylbutazone, indomethacine, Ibuprofen, flutenamic
acid, sulindac, dyclophenac and alclophenac.
The majority of corticosteroids are used systematically as antiinfla -
matory agents (cortisone, hydrocortisone, prednisone, prednisolone, dexame-
thasone and triancinolone) and rapidly absorbed by gastric or cutaneous
routes; ordinarily they have a circulating half-life of 1 to 3 hours and
the maximal biological effect is reached between 2 and 8 hours after admi -
nistration. These compounds are metabolized and conjugated by the liver
before being eliminated.
The increased use of these drugs in clinical therapeutics, their ele-
vated toxicity and secondary effects, with serious pharmacological altera -
tions, confront us with a serious problem : to what an extent do they in -
terfere with our analytic methods and cause an alteration of their results?
In this work we study the effects of these drugs over the twenty test most
frequently requested in our laboratory. We present an study "in vitro"
with the intention of discovering the chemical interferences that the drugs
can provoque on the analytic method we use.
MATERIALS AND METHODS

Within the group of antiinflamatory drugs we selected the antirheu-

matic action drugs, nomally prescribed in the Rheumatology Service of our
hospital, and between these we chose some that were representative of each
chemical group, as we show on Tables II and III. For the "in vitro" study,
we used the pure active principle.
The analytical determinations were performed using a sequential auto-
analyzer of twenty channels SMAC. The channels included glucose, urea, iron,
creatinine, total protein, albumin, calcium, sodium, potassium, chloride,
phosphate, cholesterol, total and direct bilirubin, urate, alkaline phos -
phatase (EC 3.1.3.1.) , creatine kinase (EC 2.7.3.2.), lactate dehydrogenase
(EC 1.1.1.27) and aspartate and alanine aminotransferases (EC 2.6.1.1 and
EC 2.6.1.2). The chemical methods have been described by Schwartz et al.,
(1974) and westgard et al.,(197S) whose basis is shown on Table IV.
Pooled sera were used. For each perameter to be studied three diffe -
rent pools were prepared giving low, medium and high levels.
TABLES II - III . - GROUPS OF DRUGS AND DOSES STUDIED.

Chemical Pure active D a i l y Dose Plasma Comercial

group principle (DCI) ( mg ) concentration name
(ug/ml)
1 . - Non s t e r o i d s
Pirazolones Phenylbutazone 100 - 800 50 - 150 Butalgina
Butazolidina
Artrodesmol Extra
Oxyphenbutazone 100 - 800 0.3 - 3 Tanderil
Indoles Indométacine 50 - 200 0.3 - 3 Indometacina
Aminoquinolines Chloroquine 100 - 310 75 - 125 Resochin
Gold Salts Aurothiomalate 1 0 0 0 - 2 000 Myochrisina
de Sodium (weekly)
R
Aminoacids D - Penicillamine 250 - 2 000 40 - 320 Cupripen

2.- Corticosteroids Dexaméthasone 0.5 - 200 0.08 - 34 Forte-Cortin

Decadran

Méthylprednisolone 40 - 1 500 6 - 210 Urbason

78

TABLE IV - ANALYTICAL METHODS STUDIED

Glucose Glucose/oxidase. Peroxidase. MBTH-DMA.

Cholesterol Lieberman-Burchard.

Urea Diacetyl monoxime - thiosemicarbacide.

Creatinine Satured picric acid. Sodium hydroxide.

Urate Sodium tungstate. Hydroxylamine.

Calcium Cresolphtalein complexone. 8-Hydroxyquinoleine. Dime-

thylamine.

Phosphate Ammonium molybdate.

Iron Ferrozine. Ascorbic acid. Sodium acetate.

Total Protein Biuret

Albumin Bromcresol green.

Bilirubin total Diazo. Caffeine. Sodium Potassium Tartrate.

Bilirubin direc Diazo. Ascorbic acid. Sodium Potassium Tartrate.

Alkaline Phosph. p-Nitrophenol phosphate. AMP Buffer ph 10.3

A S T Malic dehydrogenase. NADH. Ketoglutaric acid.

A L T LDH. NADH. Ketoglutaric acid.

LDH NAD. Aminomethylpropanol buffer, pH 9.0

Creatin Kinase Creatine phosphate. Cysteine. Adenosine diphosphate.

Sodium Tris buffer, pH 8.0 Ion-selective electrode

Potassium Tris buffer, pH 7.5 Ion-selective electrode

Chloride Mercuric thiocyanate. Ferric nitrate.

 79

The initial concentration of the product to be studied is five times

the maximum therapeutic concentration. If we found a great variation among
the values obtained with this concentration and the sera withouth drug, a
series of dilutions of the initial concentration of the product are effected
until the interferences disappear. From each sample, and its corresponding
control (no drug added) sample,we made five more seried determinations in
order to guarantee the repeatability of the method. All the corresponding
determinations of seried dilutions of a drug are performed under the same
calibration, in order to avoid the small variations that can appear due to
other calibrations in the same series. The average of the results is cal ­
culated corresponding to each concentration of drug and it is compared with
the average of the control samples. For the test that present a variation
with the control sample, we calculate the percentage of this variation. In
order to do this we consider the analytic error due to the method, taking
into account the study of the repeatability and the reproducibility under ­
taken upon our automatic analytic system. (Galimany, et al., 1978)

We control if there is a variation in the pH of the samples after the

drug has been added.
RESULTS
The first selective test that we submited this group of drugs to, in
order to detect a significant interference upon some of the proposed tests,
in the study of the effect produced on a human serum by the addition of a
quantity of drug equivalent to a concentration of five times the therapeu ­
tic dosis.
Once the serums had been processed through the autoanalyzer, we obser­
ved that in this concentration there were no interferences detected upon the
method, or they were practically insignificant, concerning the following
drugs ι Aurothiomalate de Sodium, Indometacine and Phenylbutazone; for this
drug we detected an inhibition of Glucose (11% over 6.1 mmol/1 and 9.9%
over 9.1 mmol/1) and an increase in Creatinine (7.5% over 80 umol/1). For
the Oxyphenbutazone we observed in a concentration of four times the thera ­
peutic dosis, an increase in Urate (15% over 312 umol/1 and 14% over 270
umol/1) and an inhibition in Glucose (8% over 9.7 mmol/1).

For this same concentration we observed a significant interference

concerning the following drugs :
a) D­Penicillamine : complete inhibition of values of Glucose and
Alkaline Phosphatase in the three plasmatic levels and an increase in va­
80

lues of Urate (137% over 295 umol/1 and 57% over 471 umol/1) and Creatine
Kinase (20% over 685 U/l and 15% over 77 Ü/1).
b) Chloroquine : increase the values of Phsophate, going beyond the
lineality of the analytic method in the three plasmatic levels studied, and
Urate (5% over 270 umol/1 and 2.25% over 487 umol/1) and an inhibition in
Lactate dehydrogenase (56% over 227 U/l and 33% over 431 U/l).
c) Dexamethasone : increase the values of Creatinine, going beyond the
linality of the analytic method, and Creatine Kinase (24% over 400 U/l and
19% over 685 U/l) and inhibition in Urate (9.5% over 294 umol/1 and 6.8%
over 471 umol/1).
d) Methylprednisolone (sodium succinate) : increase the values of
Phosphate in the two plasmatic levels studied (233% over 0.90 mmol/1 and
107% over 1.82 mmol/1).
The results obtained when adding decreasing concentrations of a drug
to a series of samples belonging to a same pool are presented in figures
1 to 4. Upon the vertical scale we represent the percent of variation
(increase or decrease) of a certain test, with relation to its real value,
prior to the addition of the drug, and the horizontal scale the concentration
of drug that produces this variation.
For the D-Penicillamine (fig. 1) we observed an increasing inhibition
of the Alkaline Phosphatase from a concentration of drug of 0.01 mg/ml to
0.5 mg/ml in which the inhibition became total. For the Glucose, the inter -
ference is accentuated starting at a concentration of 1 mg/ml. Urate values
increased after 0.5 mg/ml of drug concentration in serum.
With the addition of Chloroquine (fig. 2) we observe a lineal increase
of the measured levels of Phosphate for a concentration renging from 0.009
mg/ml to 0.30 mg/ml; after 0.45 mg/ml of drug by ml of serum, the increase
goes beyond the lineality of the method. The Lactate dehydrogenase suffers
lineal inhibition at concentration of 0.09 mg/ml or above.
' We observed that Dexamethasone (fig. 3) provoked an increase of the
measured levels of Creatinine above a drug concentration of 0.4 ug/ml
where it goes above the lineality of the method. The interference it pro -
duces in the measurement of Urate is insignificant.
Methylprednisolone produced a strong increase in the meesured levels
of Phosphate (fig. 4 ) , studied using sera containing Phosphate a two
different levels. The variation that corresponds to the lower level is
double of the one that corresponds to the higher level, for an equal amount
 81

Varia· Ion

°/o Variation
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¡t
* eoo­

Ι Craatln
•—»Ural·

./ DaXnMaTHASONB

♦ ÌOO­ /
* SO
O
­ΙΟ­

ri 1—ι­
o.« » «o l a »o so
f almi

Λ» variation
MBTHVLPaiONISOLONI
«οο­ι
·—a i . i l

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O.· l.tO 2.SO1 8
Do··
FIO. 8
82

of drug added; the increase produced is due exclusively to the reaction of

the drug with the analytical method, for the values of Phosphate obtained
for the two serum levels are practically identical; only differ their per-
centages .
In the study done upon the evaluation of the samples pH after adding
the drug (fig. 5), we observe that the variation, with relation to the pH
of the control sample, is only significant for the D-Penicillamin and the
Chloroquine; an increasing acidification is observed in both and it conti-
nues to grow as the drug concentration is increased.
CONCLUSIONS
Within the group of drugs studied, only four of them produced a sig-
nificant chemical interference over the analytical method. In a plasmatic
level of drug that corresponds to the therapeutic dosage, we have detected
the following interferences : D-Penicillamine inhibits the alkaline phos-
phatase values in a 6% respecting its real value; Chloroquine increases of
35% in the value of phosphate and an inhibition in the values of LDH of 5%;
Dexamethasone increases in the value of creatinine up to even 32%; Methyl-
prednisolone increases in the value of phosphate in 29%. This interference
is of additive character in relation to the real value of phosphate in se-
rum. We have also observed that in therapeutic levels the drugs do not
alter the pH of the serum.

REFERENCES
Champion, D.G. and Graham, G.G.: Pharmacokinatics of non-steroidal anti-
inflamatory agents. Aust. N. Z. J. Med. 8 (1): 94-103, 1978
Galimany, R., Alsina, M.J. y Fernandez Simo, E.: Two years of experience
and evaluation of the SMAC. International Congress of Clinical Che-
mistry. Mexico, 1978.
Schwartz, M.K., Bethune, V.G., Pennachia, G., Menendez-Botet,C.J., and
Lehman, D.: Chemical and Clinical evaluation of the SMAC. Clin. Chem.,
20 (8): 1062-1070, 1974.
Westgard, J.O., Carey, R.N., Feldbruegge, D.H., and Jenkins, L.M.: Perfor-
mance studies on the Technicon "SMAC" Analyzer: Precision and compa-
rison of values with methods in rutine laboratory service. Clin.
Chem., 22 (4) : 489-496, 1976.
DRUG EFFECTS ON LABORATORY TESTS
 DRUG EFFECTS O N LABORATORY TESTS: URIC ACID

J.G. Salway

Biochemistry Department, University of Surrey, Guildford, Surrey, England.

ABSTRACT

There is an enormous literature on the subject of drugs interfering with the

interpretation of the results of serum urate analyses. Some of these reports are
well-established; however, many are contradictory. Such discrepancies cause
confusion and incredulity thereby distracting attention from important drug
effects which might, If unheeded by clinicians, lead to an incorrect diagnosis and
mistreatment of the patient. Concerted efforts are needed to extract from the
literature, quantitative information describing clinically significant effects of
drugs on the serum urate concentration. Once this has been achieved, this
information must be communicated efficiently to the clinician so that it can be
applied to routine clinical practice.

ARE DRUG EFFECTS ON SERUM URATE INVESTIGATIONS A SERIOUS

PROBLEM?
There have been several substantial reviews which deal with the effects of
drugs on the interpretation of serum urate investigations, see for example:
Demartinl (1965), Gill (1966), Krakoff (1966), Kelley (1975), Munan, et al. (1976).
It might therefore be reasonable to expect that a definitive statement on this
subject should be available. Unfortunately, however, there is a paucity of
quantitative information and, more seriously, there are many conflicting reports.
The phenomena of drug interference with laboratory investigation in general
is a seriously neglected problem. The computer-generated list of Young, et al.
(1975) lists approximately 16,000 entries of which 223 refer to serum urate (Table
1). This important work is well-known amongst clinical biochemists but few
clinicians are aware of its content.

Table lz References to literature on factors affecting serum urate concentration

(D.S. Young, et al. 1975).

No. of references
SERUM URATE: Decrease 70
" Increase 153

TOTAL 223
86
Because successful clinicians practise medicine without knowledge of most
of this i n f o r m a t i o n , the opinion is sometimes stated that drugs do not seriously
i n t e r f e r e w i t h the interpretation of serum urate results. I t is very d i f f i c u l t to
argue w i t h this opinion. Examples of cases where drug interference w i t h a test
had not been recognised are usually anecdotal and, not surprisingly, are rarely
(and never to my knowledge overtly) published. Occasionally, however, cases are
published as illustrated by the following examples:
Case 1
Bailey, et a l . (1976) described a female patient w i t h polycystic kidneys,
hypertension and chronic renal failure who developed gout (plasma urate 0.B6
mmol/1). She was treated w i t h frusemide and alprenolol. When allopurinol and
colchicine were added she developed an a r t e r i t i s as an unusual side-effect of the
allopurinol; this important observation was the subject of their communication.
However, i f the hyperuricaemic e f f e c t of frusemide (Humphreys, 1966; McSherry,
1968; Keyes, et a l . , 1968; Muth, 1968) had been drawn to the attention of these
physicians, then the above sequence of events, and subsequent adverse reaction t o
allopurinol might have been avoided. Hyperuricaemia is frequently associated
w i t h renal failure, but these patients do not usually develop gout. However,
frusemide is a potent hyperuricaemic agent and long-term maintenance therapy
w i t h frusemide may increase the serum urate by as much as 0.25 mmol/1 and w i l l
precipitate gout. I t is possible t h a t the patient would have benefited had
frusemide been replaced w i t h a diuretic not having a hyperuricaemic effect.
Allopurinol might then have been unnecessary, and the a r t e r i t i s would have been
avoided, Salway (1976).

Case 2
Keyes, et a l . (1968) made a detailed study of the e f f e c t of frusemide on
serum urate concentrations. They recorded the f a c t that one of their patients
was also receiving anticoagulants and aminophylline. However, they failed to
acknowledge that Christeinsen (1964) had reported anticoagulants sometimes have
a uricosuric e f f e c t and could possibly have a f f e c t e d the interpretation of their
results. Moreover, shortly afterwards i t was reported that aminophylline can also
interfere w i t h the interpretation of serum urate results (Paulus, et a l . , 1970).
Subsequent work in many laboratories has confirmed the hyperuricaemic e f f e c t of
frusemide. Nevertheless, i t is paradoxical that a paper reporting a drug e f f e c t
was itself vulnerable to drug interference.
 87
Casual inspection of Table 1 indicates the extent of knowledge of drug
interference with serum urate investigations. Coverage of all of these drug-
effects Is beyond the scope of this review. Some idea of the complexity of the
subject is revealed by examining the literature on the apparently simple subject of
paracetamol interference with serum urate determinations.

EFFECT OF PARACETAM OL ON SERUM U R A T E M EASUREM ENTS

An examination of the effect of paracetamol (N-acetyl-p-aminophenol;

acetaminophen) on serum urate measurements illustrates some of the difficulties
encountered when studying drug interference with laboratory investigations.
Singh, et al. (1972) examined quantitatively the effects taken orally of 2 g
of paracetamol on the serum urate concentration as measured by the Technicon
12/60. They found that this caused a slight false elevation of plasma urate (or
possibly eerum urate; it is not clear whether serum or plasma were studied since
Singh, et al. In their Table 1 refer to both of these fluids). This increase was a
maximum of 0.065 mmol/1, 2 hours after dosage.
Paracetamol is a widely-used analgesic which may be taken by patients with
joint pain who might be suspected of a diagnosis of gout and accordingly
investigated by measuring their serum urate concentration. It was therefore of
considerable Importance when Wilding and Heath (1975) reported gross
interference with serum urate determinations in two patients admitted to hospital
suffering from overdosage with paracetamol. In one patient (Patient 1 as
described by Wilding, et ah) a plasma paracetamol concentration of 15 mg/100 ml
coincided with a grossly elevated serum urate of 1.84 mmol/1, Table 2. The serum
urate concentration in this patient fell to a base-line of 0.24 mmol/1 four days
after admission when the serum urate was undetectable.

Table 2. From Wilding, et al. (1975).

Urate Paracetamol
mmol/1 mg/100 ml
Day 1 1.84 15
0.43 Undetectable
" 3 0.30 -
•ι n 0.24 _

Patient took 20 Panadol tablets plus a few tablets of bromide and diazepam.
88

It is reasonable on this evidence to conclude that the difference between

these results, i.e. 1.60 mmol/1 is due to the presence of paracetamol.
On day 2 the serum urate was 0.43 mmol/1. When the base-line
concentration is deduced a difference of 0.19 mmol/1 results. However, on this
occasion the plasma paracetamol was undetectable and therefore the observed
hyperuricaemia cannot be interpreted as a spurious effect due to paracetamol
dosage. The authors did not comment on this apparently paradoxical point. They
did, however, extend their studies to a problem of great clinical significance.
They studied the effect of therapeutic doses of paracetamol on serum urate
concentrations. They found that a 2 g dose caused the apparent urate
concentration to be increased by 0.18 - 0.24 mmol/1 and by 0.12 mmol/1 after a l g
dose. I have frequently quoted this work, e.g. (Salway, 1978, 1979).
It was therefore perplexing when Smith and Payne (1979) reported no
detectable change in serum urate concentration, as measured by the
phosphotungstic acid-urea/cyanide method, after the ingestion of lg of
paracetamol. Moreover, these results were confirmed using the standard
Technicon phosphotungstic acid - hydroxylamine/tungstate method on a Technicon
SMA 12/60 analyser.
Smith and Payne also studied patients admitted to hospital following
overdosage with paracetamol. In 10 of these patients, sequential urate
determinations were made using either the phosphotungstic acid-
hydroxylamine/tungstate or phosphotungstic acid-urea/cyanide method. They
reported no change in the apparent serum urate greater than 0.12 mmol/1. As a
result of these studies they concluded that it would be unlikely that patients being
investigated for gout, who were taking paracetamol as an analgesic, and who were
having their serum urate concentration measured by a phosphotungstate reduction
method, would have clinically misleading values for serum urate.
Further work is needed to resolve the apparent discrepancies between these
papers. Smith and Payne conducted studies with paracetamol added in vitro to
serum which was subsequently analysed by 15 different laboratories employing
different methods. The greatest effect observed was an increase of 0.12 mmol/1
using a phosphotungstic acid-urea/cyanide method with Technicon Wetting Agent A.
It is interesting to note the extent of methodological variation in current
 89

practice in that five different wetting agents were used by these laboratories. It
is possible that such details as the nature of the wetting agent could occasionally
be responsible for some of the analytical discrepancies and such minutiae are
rarely mentioned in accounts of experimental details.
Clearly, the problem of drug interference with clinical laboratory
investigations in general is an enormous one. Collaboration between members of
the medical and paramedical professions in collaboration with the pharmaceutical
and diagnostic industries is needed. Our objective should be to identify the most
important effects caused by drugs on laboratory investigation and devise simple
and effective means of communicating this information to the clinician.

REFERENCES

Bailey, R.R., Neale, T.J. and Lynn, K.L., 1976. Allopurinol-associated Arteritis.
The Lancet, ii: 907.
Carey, M.A., Jones, J.D. and G ustineau, C F . , 1971. Effect of Moderate Alcohol
Intake on Blood Chemistry Values. JAMA, 216: 1766-1769.
Christensen, F., 1964. Uricosuric effect of Dicoumarol. Acta Medica. Scand.,
175: 461-468.
Demartini, F.E., 1965. Hyperuricemia Induced by Drugs. Arthritis and
Rheumatism, 8: 823-829.
Gill, C R . , 1966. Iatrogenic G out. J. Kentucky Med. Assoc, 64: 411-417.
Humphreys, D.M., 1966. Acute G out apparently precipitated by Frusemide. Brit.
Med. J., 1: 1024.
Kelley, W.N., 1975. Effects of Drugs on Uric Acid in Man. Ann. Rev.
Pharmacol., 15: 327-350.
Keyes, M.H. and Belle, M.S., 1968. Long-term Maintenance Therapy with a new
Diuretic Furosemide. J. Florida, M.A., 55: 524.
Krakoff, I.H., 1966. Clinical Pharmacology of Drugs which influence Uric Acid
Production and Excretion. Clin. Pharmacol. Ther., 8: 124-138.
Lieber, C S . , Jones, D.P., Losowsky, M.S. and Davidson, C S . , 1962. J. Clin.
Invest., 4 1 : 1B63.
McSherry, J . , 1968. Acute G out complicating Frusemide Therapy. Practitioner,
201: 809.
Munan, L., Kelly, A. and Petitclerc, C , 1976. Am. J. Epidemiol., 103: 369-382.
Muth, R.G ., 1968. Diuretic properties of Furosemide in Renal Disease. Ann. Int.
Med., 69: 249.
Paulus, H.E., Coutts, Α., Calabro, J.J. and Klinenberg, J.R., 1970. Clinical
Significance of Hyperuricemia in routinely screened hospitalized men.
JAMA, 211: 277-281.
Saker, B.M., Toiler, O.B., Burvill, M.J. and Reilly, K.A., 1967. Alcohol
Consumption and Gout. Med. J. Aust., 1:1213-1216.
Salway, J.G ., 1976. Allopurinol-associated arteritis. The Lancet, i i : 1255.
Salway, J.G ., 1978. Drug interference causing misinterpretation of laboratory
results: How to solve the problem? Ann. Clin. Biochem., 15: 44-48.
Salway, J.G ., 1979. Factors causing misinterpretation of laboratory
investigations. Prescriben' J . , 19: 23-28.
90

Singh, H.P ., Hebert, M.A. and Gau I t , M.Η., 1972. E f f e c t of Some Drugs on
C l i n i c a l Laboratory Values as Determined by the Technicon SMA 12/60.
C l i n . Chem., 18: 137-144.
Smith, M. and P ayne, R.B., 1979. Re-examination of e f f e c t of P aracetamol on
Serum U r i c A c i d Measured by P hosphontungstic A c i d Reduction. Ann. C l i n .
Biochem., 16: 96-99.
Wilding, P . and Heath, D.A., 1975. E f f e c t of P aracetamol on U r i c A c i d
D e t e r m i n a t i o n . Ann. c l i n . Biochem., 12: 142-144.
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C l i n i c a l Laboratory Tests. C l i n . Chem., 2 1 : 1D-432D.
 EFFECTS OF DRUGS ON TOTAL SERUM CHOLESTEROL

J. Delattre and P. Bastide

Laboratoire de Chimie Biologique et de Pharmacodynamic
U. E. R. de Pharmacie
28, place Henri-Dunant 63001 Clermont-Ferrand - Cedex

ABSTRACT
Both chemical and pharmacological effects of drugs on total serum cho-
lesterol are considered. In none of the determination methodologies used
(Liebermann-Burchard, iron salts and enzymatic) have more than a few drugs
been reported to interfere chemically in cholesterol determination. Never-
theless, investigations intended to detect and classify this type of inter-
ference are frequently incomplete, especially in so far as enzymatic metho-
delogies are concerned, and these will, in the future, progressively re-
place traditional colorimetrie procedures.
On the other hand, drugs likely to increase or decrease the serum cho-
lesterol level through a pharmacological or toxicological effect are nume-
rous and may be classified into various (chemical) categories. The inter-
pretation of these interferences is often difficult. For a large number of
drugs, further investigations are necessary.

INTRODUCTION
The effects of drugs on serum cholesterol have been classified in ma-
jor studies of the effects of drugs on clinical laboratory tests. (Caraway
and Kammeyer, 1972 ; Constantino and Kabat, 1973 ; Young et al., 1975 ;
Hansten, 1979). These effects are of two kinds : pharmacological and chemi-
cal (analytical interference). Both will be considered in turn. However,
where analytical interferences are concerned it is necessary to consider
the methodology used. A brief preliminary reminder of the most widely used
cholesterol determination methodologies is therefore in order.
I - PRINCIPAL METHODOLOGIES OF CHOLESTEROL DETERMINATION
A large number of methodologies for cholesterol determination have
been described (Peynet and Chappuis, 1976 ; Zak, 1977). All make use of an
oxidizing agent which may be either chemical or enzymatic. Three major groups
of methods may be distinguished according to the nature of the reagent.
Colorimetrie methodologies based on the Liebermann-Burchard reaction.
This reaction results in a green coloration, classically measured at 630 run.
This may be. reacted directly with serum, or after various preliminary pro-
cedures (eg. the extraction of lipids, the saponification of cholesterol
esters) designed to produce more specific results.
Colorimetrie methodologies based on the use of iron salts in acid me-
92

diurn (The Zlatkis, Zak and Boyle procedure). A violet coloration occurs
and is read at 550 am. As with the method above, this may, or may not, be
reacted directly with serum.
Enzymatic methodologies.
After denaturation of serum lipoproteins, cholesterol esters are hydro-
lised by a cholesterol hydrolase. In the presence of molecular oxygen the
non-esterified cholesterol is oxidized by the cholesterol oxidase into cho-
lest-4-en-3-one, with formation of hydrogen peroxide.
Several procedures have been proposed as final reactions : for example,
(1) amperometric measurement of oxygen consumed, or (2) measurement of the
hydrogen peroxide produced, by means of an enzymatic coupled reaction (pe-
roxidase or catalase) resulting in a colored or flourescent compound.
TABLE 1 - ENZYMATIC MEASUREMENT OF CHOLESTEROL. MEASUREMENT OF HYDROGEN
PEROXIDE.

Indicator HO H H H
2°2 2°2 2°2
reaction + + +
+
4 amino homovanillic methanol
reduced
antipyrine acid
A.B.T.S.
Phenol
„xx

POD' POD POD HCHO

Final Quinone Oxidised Flourescent

I
Substituted
product imine A.B.T.S. compound lutidine

POD : peroxidase C : catalase

A.B.T.S. : 2,2'-azino-di-3[ethyl-benzthiazolin sulfonic acidi

The indicator reactions are thus based on a variety of principles, and

this diversity must be emphasised. In fact, as we shall see, the same drug
may have diametrically opposite effects on the enzymatic estimation of cho-
lesterol depending on the indicator reaction used.
II - ANALYTICAL INTERFERENCE OF DRUGS ON CHOLESTEROL DETERMINATION
Interference due to biological substances (hemoglobin, bilirubin, glu-
cose, etc..) will not be considered here.
A - Collection and classification of information
 93
With the aim of collecting information on analytical interference of
drugs, a commission*, "Effets dee médicaments sur les examens de labora­
toire" (Effects of drugs on laboratory tests) de la Société Française de
Biologie Clinique (S. F. B. C.) has drawn up a classification index card.
(Table II).
TABLE II ­ ANALYTICAL INTERFERENCE OF DRUGS ON CHOLESTEROL DETERMINA TION.
CLASSIFICATION INDEX CARD.

Reference : Drug Drug concentration Test medium

tested :

Clin. Chem. Cortisone 63 mg/1 serum Serum pool

1974, 20, acetate.
1282 Estradiol 25 mg/1 serum Serum pool
valerate.

Method : Results Comments

Enzymatic No interference
Trinder's reaction
ABA. J00

Β ­ Results
Table III summarises analytical interferences recorded for the three
major groups of cholesterol determination methodologies.
The number of such drugs is relatively low.
Liebermann­Burchard reaction
In the basic procedures of this reaction only one analytical interfe­
rence has, as far as we know, been described : that of Amphotericin Β
(Singh et al., J972) at a concentration of J mmol/liter.
Systematic investigations intended to research the influence of drugs
in vitro on the most widely used biochemical tests, have in fact shown that
a large number of drugs had no effect on the Liebermann­Burchard reaction.
(Singh lit al., J972 ; O'Kell et al., 1972a, 1972b ; Young and Panek, Ί976).
Iron­salts acid
Here we encounter the greatest number of interferences. There has been

χ Siest (Chairman), Delattre, Galli, Guelfi, Azria, Bastide, Braquet,

Chauvel, Galteau, Marie, Mousson, Notter, Trebaul, Trivin.
94

TABLE III ­ ANALYTICAL INTERFERENC ES ON C HOLESTEROL DETERMINATION.

Method Drug : Cholesterol

Liebermann­Burchard Amphotericin Β Increased

Iron­Salts Acid Aminopyrine Increased

Bromides Increased
Chlorpromazine Increased
Corticosteroids Increased
Iodides Increased
Salicylates Increased
Thiouracil Decreased
Viomycin Increased
Vitamin A Increased
Vitamin D Increased

Enzymatic Ascorbic Acid Increased or Decreased

Bromides Decreased

no systematic investigation, and therefore the number of interferences may

well be higher. Nevertheless, in the majority of instances, the interferen­
ces mentioned can be reduced, provided there is previous extraction.
Enzymatic Procedures
The influence of ascorbic acid has been the center of investigation
because of its reducing properties. As Table IV shows, the effect of ascor­
bic acid on the enzymatic determination of cholesterol depends on the indi­
cator reaction used. With Trinder's reaction (quinone imine) the level of
cholesterol remains unchanged (Meiattini et al., 1978) or is reduced
(Robinson et al., 1976 ; Pesce and Bodourian, 1977) ; on the other hand,
it increases with Hantzsch's reaction (substituted lutidine) (Pesce and
Bodourian, 1976). This shows that we must interpret all such analytical
interferences with prudence.
Enzymatic methods for cholesterol measurements are now used by 65 %
of U.S. Laboratories (Frost and Sullivan, 1979), and nearly 50 % in France
(Bailly, 1979). As enzymatic methods gradually replace the traditional co­
lorimetrie methods, the effect of drugs known for their reducing properties
(L. Dopa (Levodopa), Gentisic acid) will need to be studied carefully. In
 95
fact, these drugs are known to interfere with enzymatic determination of
glucose (Trinder's reaction) (Macart et al., 1978). The interference revea­
led would, in theory, apply also to the enzymatic determination of choles­
terol.
TABLE IV ­ ANALYTICAL INTERFERENCE OF ASCORBIC ACID ON THE ENZYMATIC DETER­
MINATION OF CHOLESTEROL.

Indicator reaction Trinder Trinder Trinder Hantzsch

Measuring equipment Beckman A

A II Centrifugal Centrifugal
Analyzer A nalyzer

Ascorbic acid 50 40 100 100

concentrations
tng/1 serum

Cholesterol level unchanged reduced reduced increased

14 X 15 X 25 X

III ­ PHARMACOLOGICAL AND/OR TOXICOLOGIC^ EFFECTS OF DRUGS ON THE LEVEL

OF SERUM CHOLESTEROL
Drugs known for their hypolipidemic properties (Cholesterylamine,
Clofibrate, etc) are not considered here. In addition, only results obtai­
ned with human subjects have been taken into account.
A ­ Collection and classification of information
In order to better define the effects of drugs on cholesterol levels
the commission "Effects of Drugs on Laboratory Tests" de la S.F.B.C, has
drawn up a table which is designed to include the following items : (1) the
nature of the drug tested, (2) concentrations administered, (3) means of
administration, (4) duration of treatment, (5) subjects studied (age, sex,
general state of health), (6) diet, (7) number of control subjects, (8) me­
thod of cholesterol determination used, (9) type of statistical analysis,
(10) action mechanism of the drug, (II) and other important parameters.
Β ­ Results
The drugs can be classified into two main groups : those which lower
serum cholesterol (Table Va) and those which increase it (Table Vb). Such
drugs are extremely numerous, and can be classified in various chemical
categories.
96
TABLE Va - DRUGS WHICH LOWER SERUM CHOLESTEROL (PHARMACOLOGICALLY OR
TOXICOLOGICALLY)

Aminosalicylic acid, Ascorbic acid, Asparaginase, Aspirin, Bran, Cellulose,

Chlortetracycline, Colchicine, Erythromycine, Isoniazid, Kanamycin, Mety-
rapone, Neomycin, Paramomycin, Pectin, Pentylenetetrazol, Salicylates,
Vitamin A.

TABLE Vb - DRUGS WHICH INCREASE SERUM CHOLESTEROL (PHARMACOLOGICALLY OR

TOXICOLOGICALLY)

Allopurinol, Anabolic steroids, Androgens, Chlorpromazine, Cincophen, Cor-

ticosteroids, Cyclophosphamide, Disulfiram, Epinephrine, Furosemide, Imi-
pramine, Levodopa, Lithium carbonate, Meprobamate, Miconazole, Penicil-
lamine, Phenothiazines, Phenytoin, Sulfadiazine, Sulfonamides, Thiazides,
Trimethadione, Vitamin D.

In some cases, anomalies have only been described in single reports

(Cyclophosphamide, Lithium Carbonate, Penicillamine). Confirmation is thus
necessary. For the same drug, contradictory results have been reported, as
is the case with corticosteroids. For example in a study involving female
asthmatic patients undergoing long-term treatment with prednisone, no evi-
dence was shown for a significant increase in serum cholesterol (El
Shaboury and Hayes, 1973). On the other hand, Stern et al. (1973) observed
a definite increase in cholesterol in patients suffering from rheumatic
disease, undergoing prednisone treatment over a period of three months.
Nevertheless, in both cases the daily doses of prednisone were similar.
Such contradicotory results have also been observed with other drugs, such
as ascorbic acid, phenytoin, practolol (Beta-blocking agent ) , etc.
It must be emphasesed that during pharmacological studies the aspect
of analysis, although essential, is often overlooked. The description of
the method of cholesterol determination is often incomplete, and may not
aven be mentioned. It is unusual for a drug undergoing testing to be stu-
died in order to confirm that it does not interfere with cholesterol deter-
minations. This precaution would certainly eliminate the erroneous inter-
pretations that have occured in the case of other biological parameters
(glucose or uric acid, for example). Where cholesterol variations are minor,
 97
an analytical error due to the determination technique itself is never
considered when results are analysed.
Therefore, interpretation of results can be a delicate process for a
large number of drugs. There is a need for further experimentation.
CONCLUSION
Few drugs, in vitro, have been reported to interfere with serum cho­
lesterol determination. In most cases, interference only occurs with high
drug concentrations, and these concentrations will rarely be encountered
in the serum of patients who are receiving normal therapeutic doses. Thus,
in practice, analytical interferences that are capable of modifying cho­
lesterol determination are infrequent.
Nevertheless, since enzymatic methods will gradually replace tradi­
tional colorimetrie procedures in laboratories in the future, it would
appear indispensable to investigate more carefully the influence of drugs
known to lower cholesterol values.
On the other hand, the list of drugs which may pharmacologically
and/or toxicologically modify the level of serum cholesterol is a long
one. Here the results are more difficult to interpret. It is not unusual
for apparently contradictory results to be observed for the same drug.
The diversity of experimentation procedures in use may well be at the root
of such divergences. In addition, it would appear essential to develop a
strictly controlled experimentation procedure which, if properly used,
would allow in the future greater precision in the assessment of the ef­
fects of drugs on serum cholesterol.
REFERENCES

Bailly, Μ., Personal Communication

Caraway, W.T., Kammeyer, C.W., 1972. Chemical interference by drugs and
other substances with clinical laboratory test procedures. Clin.
Chim. Acta, 41: 395­434.
Constantino, N.V., Rabat, H.F., 1973. Drug ­ induced modifications of la­
boratory test values­revised J973. Amer. J. Hosp. Pharm., 30: 24­71.
El­Shaboury, A.H., Hayes, T.M., J973. Hyperlipidaemia in asthmatic pa­
tients receving long­term steroid therapy. Br. Med. J., 2: 85­86.
Frost and Sullivan, J979. Clinical diagnostic reagents and test kit mar­
kets. Frost and Sullivan inc.
Hansten, P.D., 1979. Drug interactions, 4nd (Lea and Febiger. Philadelphia)
Macart, M., Koffi, Α., Henocque, G., 1978. Adaptation au centrifichem du
dosage de la glycémie selon Trinder. Etude cinétique des principales
interférences médicamenteuses. Ann. Biol. Clin., 36: 85­90.
Meiattini, F., Prencipe, L., Bardelli, F., Gianini, G., Tarli, P., 1978.
The 4­hydroxybenzoate 4­aminophenazone chromogenic system used in the
enzymìc determination of serum cholesterol. Clin. Chem., 24i 2J6J­
2J65.
98

O'Kell, R.T., Mantzey, L., Knepper, D.F., Elliot, J.R., 1972a. Intravenous
vitamins and clinical laboratory tests. Clin. Chem., 18; 403­404.
O'Kell, R.T., Mantzey, L., Knepper, D.F., Elliot, J.R., 1972b. Effect of
drugs on results of laboratory tests. Clin. Chem., 18: 1039.
Pesce, Μ.Α., Bodourian, S.H., 1976. Enzymatic rate method for measuring
cholesterol in serum. Clin. Chem., 22: 2042­2044.
Pesce, Μ.Α., Bodourian, S.H., 1977. Interference with the enzymic measure­
ment of cholesterol in serum by use of five reagent kits. Clin. Chem,
23: 757­760.
Peynet, J., C happuis, P., 1976. Dosage du cholestérol : Problèmes actuels
de biochimie appliquée, 8ème série, lipides, lipoprotéines et athéro­
sclérose p. 47­60 (Hasson, Paris).
Robinson, CA . , Hall, Jr.L.M., Vasiliades, J., 1976. Evaluation of an enzy­
matic cholesterol method. Clin. Chem., 22: 1542­J543.
Singh, H.P., Hebert, M.A., Gault, M.H., 1972. Effect of some drugs on cli­
nical laboratory values as determined by the Technicon SMA J2/60.
Clin. Chem., 18: 137­144.
Stern, M.P., Kolterman, O.G., Fries, J.F., Mc Devitt, H.O., Reaven, G.M.,
Alto, P., 1973. Adrenocortical steroid treatment of rheumatic diseases.
Arch. Intern. Med., J32: 97­101.
Young, D.S., Pestaner, L.C. and Gibberman, V., 1975. Effects of drugs on
clinical laboratory tests. Clin. Chem., 2J: 1D­42JD.
Young, D.S., Panek, E., 1976. Effects of drugs on the analytical procedures
of a Multitest analyser. Drug interference and drug measurement in
clinical chemistry. Proc. 3rd Int. Cell, or Prospective Biology, Pont­
ã­Mousson, p. 10­20 (Karger, Basel).
Zak, B., 1977. Cholesterol methodologies. A review Clin. Chem., 23: 1201­
12J4.
 DRUG EFFECTS ON CHOLESTEROL PLA SMA LIPOPROTEINS FRA CTIONS

Pierre Braquet (*) and Monique Braquet (~)

(") Laboratoires Merck Sharp et Dohme Chlbret
3, Avenue Hoche ­ F 75008 Paris.
(!!îi) Centre de Recherches du Service de Santé des Armées
Ibis, rue R. Batany ­ F 92140 Clamart.

ABSTRACT
Only the phoAmacological erecţi o¿ dmigi on cholutenol pla&ma lipo-
pn.otíÀni we/ie investigated. I t iA eaential to moniton, in the coufi&e
0(J a tAeatment, the. ¿,vum lévelo o& ¿oui denbity lipopnotein-choluteAol
(LOL­C) in negând to the highly positive connelation demonitnated betaeen
thitt inaction and athenoiclenoiii, the. majon caiue oA ¿ie.ha.emie. heant
duea&e (ItfP). Similan attention ¿hould be paid to the variation* in
high density lipopnotein-choleitenol (HPL­ C ) known to be negatively conne-
lated with athenoiclenoiii.
The phanmacological e¿]ect¿ oi the fallowing dnugò wene canefally
studied : honmonei, lipid lowening agenti, anti-diabetici, anti-hypen-
tensivei and ml&cellaneoui compound* togethen with the fallowing toxic-
agenti : pesticide* and alcohol.

INTRODUCTION
The literature to date provides very l i t t l e data on analytical
Interferences that may cause disturbances in the separation of plasma
lipoproteins. In addition, a large number of methods are available
(ultracentr1fugat1on, electrophoresis, polyanion ­or concanavalin A­
precip1tat1on) with their Interferences adding to those encountered in
the determination of cholesterol. These methods are treated elsewhere
(Delattre et a l . , 1979).
Atherosclerosis is characterized histologically by the accumulation
of l i p i d s , predominantly cholesterol, in the arterial wall together
with a connective tissue reaction (Adams, 1973). A wide variety of
epidemiologic data indicates that individual lipoproteins or combinations
of lipids and apolipoprotelns are better predictors of coronary artery
disease (CAD ), a major cause of IHD , than are levels total cholesterol
(TC) or triglycerides (TG). Of the four basic classes of lipoproteins"
1n human serum, three promote and one retards the development of CAD :
low density lipoproteins (LD L) and low density lipoproteins cholesterol
(LDL­C) is a strongly positive risk factor, particularly at younger ages
100

(<60 yrs) (Fredrickson e t a l . , 1972 ; Camejo, 1976). Although very low

density lipoproteins (VLDL) and very low density lipoprotein-cholesterol
(VLDL-C) are more controversial (Kannel et a l . , 1971 ; Rhoads et a l . ,
1976) evidence exists to implicate them as w e l l , p a r t i c u l a r l y when
associated with elevations of LDL and LDL-C (Blankenhorn et a l . , 1968 ;
Carlson e t a l . , 1975). Chylomicrons could be atherogenic as well
( Z i l v e r s m i t , 1973).
Conversely, a large body of epidemiologic information indicates t h a t
elevations of high density lipoproteins (HDL) and high density l i p o p r o -
tein-cholesterol (HDL-C) are equally strong negative r i s k factors a t
a l l ages (even 70 yrs) (Glueck et a l . , 1975 ; M i l l e r et a l . , 1977).
In view of the known relationships between lipoproteins and CAD,
a logical framework f o r combating atherosclerosis i s to reduce LDL,
(VLDL especially) and to increase HDL.
I t i s therefore of utmost value to f o l l o w drug therapy that modify
l i p o p r o t e i n s , by determination of cholesterol l i p o p r o t e i n f r a c t i o n s ,
since i t permits one to monitor and to evaluate the increase or the
decrease of an eventual CAD r i s k f a c t o r .
This report w i l l review some of the recent advances made in the findings
of pharmacological effects of drugs on cholesterol l i p o p r o t e i n f r a c t i o n s .

1 - HORMONES

1.1. Oral contraceptives (OC)

The observation that women of a l l ages have higher HDL-C levels than
men dates back more than 25 years (Barr et a l . , 1951 ; N i k k i l a , 1953).
More recent data demonstrates that the difference in sexes begins early
in adolescence and i s maintained u n t i l e l d e r l y ages (Rifkind et a l . , 1978):
although women taking estrogen or certain OC have higher HDL-C levels
than age-matched non OC-users, the mean level of t h i s parameter in women
remains above that of men, even in non OC users as w e l l , into postmeno-
pausal years.
In f a c t , the influence of OC on HDL-C was controversial : Krauss et a l . ,
1977 and Wallace et a l . , 1979 , found s l i g h t l y but s i g n i f i c a n t l y higher
levels i n OC users. Arntzenius e t a l . , 1978 ; Rossner, 1978a ; Bierenbaum,
1979 and Larsson-Cohn, 1979 found s i g n i f i c a n t l y lower leyels among users
and Schade et a l . , 1978 ; Meerloo et a l . , 1979 and Larsson-Cohn, 1979,
found approximately similar levels before and a f t e r use.
 101

A first explanation of these Inconsistent results is given by Bradley

et al., 1978. These authors report various changes in HDL-C levels
according to the formulations of OC : HDL-C levels appeared to be directly
related to estrogen dose and inversely related to progestin dose. This
result was confirmed by Goldzieher et al., 1978 ; Shelton et al. , 1978 ;
Hennekens et al., 1979 ; Wallace et al., 1979, Krauss et al., 1979.
In fact, estrogens are known to increase HDL (Goldzieher et al., 1978)
and Krauss et al., 1977 , 1979 have shown that this increase was the fact
of less dense HDL-2 : the differences are shown by subtracting the curve
of OC non users from that of the users. The curve obtained, showing
three peaks (two positive and significant, one negative and not statis-
tically significant) is the same between men and women (non OC users) in
the same population. Krauss et al., 1979, attribute these three peaks
to the three fractions HDL2b, HDL2a and HDL3. The estrogen used appears
to be associated with higher levels of HDL-2b and HDL-2a but not HDL-3.
Furthermore, estrogen treatment was associated with a strong increase
of VLDL-TG (Schaefer et al., 1977) except in women with type III HLP,
who are treated with 1 ug/kg/day of ethinyl estradiol. In thise case,
VLDL-C and VLDL-TG return to normal. This result, in contrast to the
usual hypertriglycerldemic activity of ethinyl estradiol in normal sub-
jects, might be attributed to the potential increasing activity of estro-
gens on the clearance of plasma VLDL remnants (Kushwaha et al., 1977).
The same effects are observed in postmenopausal women treated with
estradiol valerianate : LDL-C decreased (with a significant positive
correlation between the size of the decrease and initial levels), VLDL-C
also decreased but HDL-C significantly increased (Tikkanen et al., 1978 ;
Wallace et al., 1979).
In combination with estrogen , progestins tend to shift the increase
of HDL towards the more dense HDL-3 subspecies (Krauss et al.,1977 ) and,
depending on the type of progestins , HDL-C may increase or decrease
(Bradley et al., 1978 ).
The estrogen effect on HDL may be due to alterations in the normal
pituitary-gonadal feedback relationships. In the case of progestins, the
situation 1s more complex due to a number of pharmacological unwanted
effects such as androgenic, estrogenic, anabolic and anti-estrogenic
(Bradley et al., 1978).
102

For example, norethynodrel, which has almost no antiestrogenic effect

is associated with high concentrations of HDL-C, whereas norethindrone
acetate and norgestrel, both having strong antiestrogenic effects, are
associated with the lowest levels. The complexity of the problem is
increased by the fact that these hormones have effects on HDL-components
other than cholesterol (Cheung et al., 1977 ; Krauss et al., 1977).
Another important finding was the highly significant quantitative
increase in ß-lipoproteins during treatment with synthetic estrogen
while a non significant decrease was observed following natural estrogen
therapy (Bostofte et al., 1978). In fact, OC raise TC and LDL-C, particu-
larly in young women. OC that contain estrogen may secondarily raise the
serum TC by elevating the VLDL-C (and the HDL-C) (Fallat et al., 1976).
On the other hand, an elevation of HDL-C is noted at the half-way
menstrual cycle (Oliver et al, 1953 ; Barclay et al., 1965 ; Gustafson
et al., 1974) but the hormones involved in this increase were not defined
(Krauss et al., 1979).
Furthermore, HDL-C levels change during the different stages of
pregnancy (Nikkilä, 1978a).
In terms of HDL alone estrogens would theoretically have an ameliorating
effect on coronary risk and the majority of progestins would have a
neutral or negative effect. However, several authors (Stamier et al.,1963;
Jick et al., 1978 ; Gordon et al, 1978) have shown that estrogen use
increases the incidence of CAD in either sex. Recently, Phillips (1977)
identified a high endogenous estradiol/Testosterone ratio as a coronary
risk factor.
Any putative "protective" role of enhanced HDL-C in OC- users might
well be reversed by other estrogen effects such as increases in VLDL :
thus, there is a direct correlation between increases of HDL-C and serum
TG and, presumably, VLDL-TG (possibly by direct effect of hormones on
synthesis and/or catabolism of these lipoproteins) (Rössner et al., 1971 ;
Hazzard et al., 1973 ; Al fol al i, 1975) (see fig.l).
Furthermore, there may be other significant mechanisms by which the
estrogen and progestin within OC can be related to increased risk of IHD.
Progestin has been shown to be dose-related to the development of hyper-
tension in OC-users. OC can alter glucose tolerance, platelet adhesiveness,
plasma Cortisol levels and also can raise renin substrate and aldosterone
levels.
 103

130
• ­
•
•
··
120 ^ ·· ­

• J ' s 0.76
Ρ < 0.001
•
110 ­

•
1
I
30 40 50 60 70 80
HDL­C (mg/dl)

¿­¿g. ι . : CowieZatùm between TG and HOL­C ¿n OC lU&u

ΙΙΟΐΜύύ eX al., 1979).

1.2. Androgens
Androgens lower plasma HDL­C (Solyom, 1972 ; Freeman et al . , 1977 ;
Masarel et a l . , 1977). In f a c t , women with idiopathic hirsutism and
elevated levels of free or total plasma testosterone have somewhat lower
plasma HDL­C than hirsute women with normal total or free plasma testos­
terone (Witztum et a l . , 1979b).
Furthermore, the treatment with oxandrolone in patients with type
IIA, IIb and IV HLP Induced a significant reduction in VLDL­TG and HDL­C :
hypo HDL­C occurred in 7/10 patients with type IV, 3/6 with type I I B ,
1/4 with type IIA. Reciprocal elevation of LDL­C in 8/10 patients with
type IV and 1n 2/5 with type IIB was observed (Tamai et a l . , 1979a).
On the other hand, in rats, oxandrolone induced a significant decrease
of both LDL­C and HDL­C ; VLDL­C was not affected while LDL­PL (phospho­
l i p i d s ) , HDL­PL, LDL­TG and HDL­TG were also altered by the drug ( Freeman
et a l . , 1977).
1.3. Thyroxine
In patients with primary hypothyroidism and treated with thyroxine,
LDL­C fell to normal but no significant change occurred in the mean
VLDL­C or HDL­C (Ballantyne et al., 1979 ) .
104

The same r e s u l t s were obtained in patients with hyper­cholesterol ernia

(Schneider et a l . , 1978 ).

2 ­ HYPOLIPIDEMIC A GENTS

In view of the known relationships between CAD and serum lipoproteins,

a logical framework for combating this disease is to reduce LDL­C and
VLDL­C and/or increase HDL­C. The actions of hypolipidemic drugs on
cholesterol lipoprotein fractions are very complex ¿because they are :

. specific of the lipid lowering drug used,

. specific for one lipid lowering drug, of each type of HLP and often,
correlations are noted between parameters as VLDL­TG and LDL­C, VLDL­TG
and HDL­C or between initial (i.) parameter concentrations and the change
in parameter levels after treatment (a.t.) as LDL­C (i.) and LDL­C (a.t.),
HDL­C (i.) and HDL­C (a.t.)» HDL­TG (i.) and HDL­C (a.t.), VLDL­TG (i.)
and VLDL­TG (a.t.).
Table I presents the general effects of five well­known drugs affecting
serum lipoprotein concentrations.
2.1. Phenoxy compounds
2 . 1 . 1 . Adamantyloxyphenyl compounds
M i l l e r et a l . , (1975) found markedly decreased LD L­C and VLD L­C while
simultaneously increased HD L­C were observed in experimental animals
treated with adamantyloxyphenylpiperidine.
A closely related analogue adamantyloxyaniline, considered less t o x i c ,
reduced LD L­C and VLD L­C and i n h i b i t e d the development of experimental
atheromas (D ay et a l . , 1977).
2.1.2. Bezafibraţe

2 ­ Í 4 ­ [ 2­(4­chlorobenzamido)­ethyl ] ­ phenoxy | ­2­methyl

propionic acid.
This recently developed analogue of C l o f i b r a t e affects serum l i p o p r o ­
teins in much the same way as Clofibrate but at a lower dose. Bezafibrate
reduced VLD L­C and VLD L­TG in both types I I and IV HLP ; LD L­C was
reduced only in type I I HLP (Olsson e t a l . , 1977a), these data were con­
firmed l a t e r by the same authors (Olsson et a l . , 1977b, 1977c, 1978a,
1978b): maximal effects on VLD L­C and VLD L­TG were obtained with 450 mg
d a i l y . The maximum e f f e c t on LD L­C and LD L­TG was obtained with 900 mg
d a i l y (Olsson et a l . , 1978a). I t was also noted that LD L­C decreased by
15 % in type I I HLP but increased by 16 % in type IV HLP (Olsson et a l . ,
 TABLE I ­ Hypo 11 ρ i demie drugs effect on cholesterol of lipoprotein fractions.
Variations with different types of hyper 1 ipoproteinemia.

PARAMETER HYPERLIPOPROTEIHEHIA ORUGS

Clofibrate Bezafibrate Fenofibrate Colestipol Probucol

HDL­C IIA = or ­ + + or = = or ­
(or ­ HS)
IIB = or ­ + or = + or ­ HS = or ­
III *
IV * * + or ­ HS
V + + +

LDL­C IIA ­ ­ ­ ­ ­
IIB " ~ ~ ~
III _
IV + + ­ ­
V + ­
VLDL­C IIA ­ ­ ♦ ­
IIB — ~ ♦ ~
III _
IV ­ ­ ­ +
~ ~ ~
'

8
106

1977a ; Arntz et al., 1979). In faet ,the effect on LDL-C was dependent
on the LDL-C (i.) levels. A decrease was noted if the LDL-C (i.) was
above approximately 4.00 mmole/1. At lower initial concentrations, LDL-C
(a.t.) most often increased (see fig.2). The question of a bezafibrate-
induced increment in HDL-C needs further study to resolve conflicting
reports : in thair first report, Olsson et al., 1977a, did not find in-
creases of HDL-C in type II or type IV HLP after Bezafibrate treatment.
Subsequent reports (Olsson et al., 1977b, 1977c, 1978a, 1978b) revealed
that Bezafibrate increased serum HDL-C levels in the range of 20-30 %.
But Wechsler et al., 1977a, 1977b, were not able to demonstrate such
effects.
HDL-C increase was independant of VLDL-TG decrease and there were no
correlations between HDL-C (i.) and its increase or between the decrease
of HDL-TG and increase of HDL-C ; however, a significant relationship
was found between the HDL-TG (i.) and HDL-C (a.t.) rise (Olsson et al.,
1978a). Furthermore, the increases of HDL-C in type IV or V HLP were
less pronounced after six months (+ 9,4 %) than after two months
(+ 15,4 %) (Weisweiler et al., 1979).
2.1.3. Clofibrate (*) and related compounds (**)
(") éthyl ester of c l o f i b r i c acid ( 2 - ( 4 ' - chlorophenoxy)-2 methyl
propionic).
(-") c l o f i b r i d e , e t o f i b r a t e , aluminium or calcium or magnesium salts
of c l o f i b r i c acid.
Even though Clofibrate i s the most widely used ( f o r review see Witiak
et a l . , 1977) and one of the f i r s t developed l i p i d lowering drugs,
u r p r i s i n g l y few studies have appeared on the e f f e c t of t h i s compound on
cholesterol serum lipoproteins either i n experimental animals or in man.
In r a t s , Clofibrate at 200 mg/Kg/day reduced VLDL-C, LDL-C and HDL-C
by 20,22 and 41 %, respectively (Muller, 1977). In normal baboons LDL-C
i s increased by about 50 % at 50 mg/kg and HDL-C i s decreased by appro-
ximately 30 % at 200 mg/kg (Howard et a l . , 1977a).
In man, on type IIA HLP, t h i s drug led to a non-significant decrease
of LDL-C (Rössner et a l . , 1978b) on type I I B HLP to a s i g n i f i c a n t
decrease of VLDL-TG and to a non s i g n i f i c a n t decrease of LDL-C (- 9,6 %)
(Strisower et a l . , 1968 , Rose et a l . , 1976) but on type'lV HLP, i f
Clofibrate led to a highly s i g n i f i c a n t decrease of VLDL-TG, numerous
 107

authors (Strisower et a l . , 1968 ; Wilson et a l . , 1970, 1972 ; Rose et a l . ,

1975, 1976 ; Weisweiler et a l . , 1977 ; Carlson et a l . , 1974, 1977a ;
Ballantyne et a l . , 1978a ; Wallentin 1978) have observed a significant
increase of LDL­C and of HDL­C. Similar findings were obtained in type
V HLP (Carlson et a l . , 1972a).
The change in LDL­C ( a . t . ) was directly proportional to the LDL­C ( i . ) .
The regression Une showed that subjects with LDL­C ( i . ) above 3.7 mmole/1
would respond with a decrease while those with LDL­C ( i . ) below would
respond with a rise during treatment (see f i g . 2 ) (Carlson et a l . , 1974,
1977a, 1979). To clarify the mechanism by which LDL­C increases in patients
with type IV HLP* Carlson et a l . , 1977a, have studied the effects of
Clofibrate on serum cholesterol lipoprotein concentrations in 30 patients
(metabolic ward). The decrease of VLDL­C and increase of LDL­C levels
occurred simultaneously, indicating a close metabolic relationship
between both of them. One possible explanation for this phenomenon is
the increased transfer of apo Β from VLDL to LDL.

gemfibrozil ; r s ­ 0­51

·*bezafIbrat·¡ r a ­ 0 . 7 3

Fig. 2 : Regression lines for the changes in LDL-C [a.t.) against

LDL-C U.) [Otiion et al., 1977a).
.or V
108

The change i n VLDL-TG ( a . t . ) was already strongly related to the

VLDL-TG ( i . ) l e v e l . The decrease i n mean VLDL-TG levels was s i g n i f i c a n t l y
lower with Clofibrate than with Bezafibrate or Gemfibrozil.
In type I I I HLP, Clofibrate led to a s i g n i f i c a n t f a l l i n the VLDL-C
but i f the r a t i o of TC/TG decreased i t remained suggestive of type I I I
HLP. No change occurred i n LDL-C or HDL-C (Ballantyne et a l . , 1978b).
Calcium Clofibrate a c t i v i t y was studied by Lehtonen et a l . , 1979.
Compared with C l o f i b r a t e , the effects did not d i f f e r significantly.

2.1.4. Fenofibrate
Isopropyl ester of 2-[ 4-(4-chlorobenzoyl)phenoxy ] -2 methyl propionic
acid.
This new l i p o p r o t e i n modifying agent produces a more favorable e f f e c t
on serum lipoproteins i n man than any agent reported to date.
On d i e t a r y cholesterol r a t serum l i p o p r o t e i n s . Fenofibrate led to a
marked decrease of VLDL-C concomitantly with an increase of HDL-C to
a s i m i l a r degree (Wulfert, 1978).
Rössner e t a l . , 1977 a, 1977b, 1978b, compared Fenofibrate with
C l o f i b r a t e , Bezafibrate and Gemfibrozil in type I I and type IV patients
and found t h a t i t caused a 57 % decrease i n VLDL-TG in type IV, a 37 %
decrease i n LDL-C in type I I (27 % with Bezafibrate, 18 % with Gemfibrozil)
and a 14 % increase i n HDL-C i n type IV. These overall effects were better
than those of any of the other drugs that i t was compared w i t h . These
findings were confirmed l a t e r (Micheli et a l , 1979). In patients with
HLP type I I A , who are treated with Fenofibrate at a dosage of 100 mg,
the high reduction of plasma TC was due not only to a decrease in LDL-C
(-35 %), as expressed by a marked reduction of apo B, but also to a
decrease in VLDL-C, as reflected from a marked reduction i n plasma VLDL-TG
(-50 %). Furthermore, the same authors observed an increase of HDL-C, as
expressed by a marked increase (28 %) of apo A. The same results were
folind with type IIB (VLDL-TG : -40 %, LDL-C : - 32 %). A l l these variations
are highly s i g n i f i c a n t (p< 0.001) and they do not appear to change in
patients who were followed f o r 3 months (Lauwers, 1979). Furthermore,
Colestipol plus Fenofibrate, association used i n severe type I I HLP,
produced greater reductions i n cholesterol levels than Fenofibrate alone
(Sauvanet e t a l . , 1977)
 109

In patients with HLP, Stähelin et a l . , 1979, do not find HDL-C incre-

ases except in types I I I and V. In the meantime, Micheli et al.(1979)
found markedly decreased LDL-C and VLDL-C (in type Ha : -33 %, I I b :
- 32 %, I I I : - 49 %, IV : - 26 %, V : - 30 %).
Similar results were obtained by several authors (Drouin et a l . , 1979 ;
Sauvanet, 1979 ; Paoletti, 1979). As in the case of treatment with
Clofibrate, HDL-C ( a . t . ) concentrations were dependent on the VLDL-TG ( i . )
levels. Furthermore, there was a correlation between the change in
VLDL-TG ( a . t . ) and the VLDL-TG ( i . ) (Rössner et a l . , 1978b).

2.1.5. Gemfibrozil
2.2.-d1grethy1-5 (2.5.-xylyloxy)-valeric acid.
Although this drug was disclosed only recently, (Creger et a l , 1976 ;
Rodney et a l . , 1976) numerous studies have appeared concerning I t s effects
on serum lipoproteins 1n man (Howard et a l . , 1977b ; Olsson et a l . , 1976 ;
Vessby et a l . , 1976a, 1977a; Ei sal o et a l . , 1976a, 1976b ; NikkilS et a l . ,
1976 ; Janus et a l . , 1976 ; Bremner et a l , 1976 ; Howard et a l . , 1976 ;
Schwandt et a l . , 1979). Howard, 1977b, has summarized these studies.
Gemfibrozil led to a decrease of a l l lipoprotein TG fractions in a l l types
of HLP (VLDL-TG : - 29 t, ( H a ) , - 39 % ( I I b ) , - 57 % ( I I I ) , - 44 % (IV) ;
LDL-TG : - 23 %, - 37 %, - 38 %, - 22 % for the same types, respectively;
HDL-TG : - 25 % and - 23 9Í respectively in hypercholesterolemia (princi-
pally IIb) and hypertriglyceridaemia (especially I V ) . The most pronounced
decreases were found in conditions with VLDL elevation (types I I b , III
and IV). A significant correlation existed between the VLDL-TG ( i . ) levels
and the changes in VLDL-TG ( a . t . ) .
The decrease in serum TC was the net result of mean decreases in
VLDL-C (-69 % and - 49 % in hypercholesterolemia and hypertri glyceri-
daemi a respectively). The effect of Gemfibrozil on VLDL-C was essentially
similar to that on VLDL-TG except in type I I I where the VLDL-C decrease
was very pronounced.
LDL-C decreased - 22 % in type I I a and l i b or - 2 % (or slightly
increased) 1n type IV. In f a c t , in hypertri glyceridaemia, LDL-C levels did
not change significantly. HDL-C was always increased more strongly in
hypercholesterolemia ( + 21 t) than in hypertriglyceridaemia ( + 10 %).
For LDL-C, as for Clofibrate or Bezafibrate, there was a relationship
between LDL-C (1.) levels and the change in LDL-C ( a . t . ) concentrations.
110

The effect of Gemfibrozil was of the same order of magnitude as Beza-

fibrate (see fig. 2).
2.1.6. Şimfibraţe
1.3. -propanediyle ester of bis-2-(4'-chlorophenoxy)-2 methyl propionic
acid.
One would expect a p r i o r i t h a t t h i s closely related analogue of
C l o f i b r a t e , commercialized i n Japan, would have an identical e f f e c t on
serum l i p o p r o t e i n s . As expected, VLDL-TG and VLDL-C were substantially
reduced with t h i s drug, but s u r p r i s i n g l y , LDL-C was also reduced to a
s i m i l a r extent (Saba et a l . , 1977a, 1977b).
2.1.7. Cigrofibraţe
2- [4-(2.2.-dichlorocyclopropyl)-phenoxy ]-2-methyl propanoic a c i d .
This new l i p i d lowering drug induced a maximal decrease of LDL-C
(200 mg) while HDL-C increase was the best with lower doses (100 mg)
in patients with type I l a or I I b HLP . In type IV, VLDL-TG were decreased
while HDL-C increased (Olsson et a l . , 1979).
2.1.8. Ţerbufibrol
ρ ­ [3­(p­tertbutylphenoxy)­2­hydroxypropoxy] ­benzoic acid.
In experimental animals, Terbufibrol exerts a strong decrease on serum
l i p o p r o t e i n s , especially LD L­C ans VLD L­C (Howard et a l . , 1977a ; Enomoto
et a l . , 1976). In the normal r a t at a dosage of 120 mg/kg/day, t h i s drug
caused a 32 %, 83% and 40 % reduction in LD L­C, VLD L ­ C and HD L­C,
respectively. In the normal baboon, at a dosage of 200 mg/kg/day, Terbu­
f i b r o l led to a decrease of LD L­C and HD L­C by approximately 70 and
50 %, respectively.
2.2. Nicotinic acid and related compounds
Xantinol n i c o t i n a t e , ß­pyridyl c a r b i n o l , n i c e r i t r o l , n i c o f i b r a t e ,
nicoclonate, nicomol, i n o s i t o l n i c o t i n a t e .
Nicotinic acid is the most potent l i p i d ­ l o w e r i n g drug. In normal rats
n i c o t i n i c acid decreased LD L­C and VLD L­C but had no e f f e c t on HD L­C
(Muller, 1977). C l i n i c a l l y , n i c o t i n i c acid i s e f f e c t i v e in types I I A , I I B ,
III, IV and V HLP and normalizes raised levels of chylomicrons, VLD L­C,
LDL­C as well as decreased levels of HD L­C (Carlson, 1969). On VLDL­C
and VLD L­TG, n i c o t i n i c acid has been reported to behave l i k e Clofibrate ,
but on LD L­C the variations were dependent on LD L­C ( i . ) yalues (especially
in type IV) (Carlson et a l . , 1977a, 1978). HD L­C increased only i f the
i n i t i a l levels were low and the changes produced by t h i s drug were
 Ill

associated with marked alterations 1n the HDL-2 : HDL-3 ratio (sheperd,

1978a). Nicotinic a d d decreased LDL-TG, even 1n alcoholics (Wahlberg
et a l , 1979).
Xantlnol n1cot1nate has been reported to :
. decrease LDL-C and HDL-C (Haacke et a l . , 1977a ; Wechsler et a l . ,
1979) and not affect VLDL-C 1n type IIA HLP (Haacke et a l , 1977b).
. decrease slightly LDL-C and VLDL-C but increase HDL-C 1n type lib
(Haacke et a l , 1977b ; Hutt et a l . , 1979).
. decrease VLDL-C but Increase HDL-C and LDL-C in type IV (Hutt et a l . ,
1979).
. decrease chylomicrons and VLDL-C, increase LDL-C but not affect HDL-C
1n type V (Haacke et a l , 1976).
ß-pyr1dyl carbinol decreased LDL-C approximately in the range 25-30 %
1n type IIA HLP but had no effect on VLDL-C (Wechsler et a l . , 1977b,
1979) : HDL-C was either decreased (Wechsler et a l . , 1979) or not affected
(Wechsler et a l . . 1977c).
2.3. Bile acid séquestrants (Colestipol, Cholestyramine and Polidexide)
The effects of bile acid sequestering agents (ion exchange resins)
on serum lipoproteins appear to be approximately equivalent 1n their
biological effects (for review, see Day et a l . , 1976).
In type IIA HLP, Cholestyramine treatment led to a significant decrease
of LDL-C (-30 to - 40Ï) while the LDL-TG increased during the f i r s t day
of treatment and then stayed constant. VLDL-TG levels increased abruptly
after one day's treatment and then remained constant ; VLDL-C also
Increased i n i t i a l l y but decreased after the third day. HDL-C levels
decreased soon after cholestyramine while HDL-TG levels increased s i g n i f i -
cantly (Olsson et a l . , 1978c). Several authors had found the same results
(Levy et a l . , 1973 ; Mordas1n1, 1978). Consequently, Cholestyramine
should only be used when LDL-C 1s elevated and with some caution i f
VLDL-TG or VLDL-C are elevated (especially used in type I I ) .
Colestipol hydrochloride, while not affecting HDL-C levels (Witzum,
1979a ; Sauvanet, 1979) has been shown to result in a consistent increase
1n the Apo Al/Apo A2 ratio suggestive of HDL-2/HDL-3. Like cholestyramine,
Colestipol led to a significant decrease of LDL-C but i t also led to a
significant increase of VLDL-C (Rose et a l . , 1976 ; Witzum et a l . , 1976 ;
Kuo et a l . , 1979 ; Sauvanet et a l . , 1979). As a consequence, the lipopro-
tein profile determined as the ratio HDL-C/(VLDL-C+ LDL-C) was not
112

improved by Colestipol treatment. D uring treatment, VLD L­TG rose. Rose

et a l . , 1975, 1976 and Hunninghake et a l . , 1978, had observed the same
phenomenon when the elevation of LD L­C had been induced by C l o f i b r a t e
in type IIA HLP and in hypertriglyceridemia patients ( I I B and I V ) .

2.4. Miscellaneous compounds

2 . 4 . 1 . p­aminosalicylic acid
This well­known antituberculosis compound has been used on a very
l i m i t e d basis as a hypolipidemic drug since i t was f i r s t reported, i n
1954, to lower serum TC. Twenty two years l a t e r , Vessby et al . , 1976b,
published a detailed study of i t s e f f e c t on serum lipoproteins : in
patients who were mostly of type I I HLP, HD L­C was reduced ( ­ 14 %) as
were the atherogenic lipoproteins (LD L­C : ­ 8 % and VLD L­C : ­ 49 %).
Later, Vessby et a l . , 1977b, confirmed t h e i r f i r s t results on 30 patients
(type I I and IV HLP) but f a i l e d to detect any change in HD L­C. Kuo e ţ
al., 1976, found greater decrease than Vessby et a l . , i n LD L­C.

2.4.2. BR­931* and WY­ 14 643* *

»i: [ 4­chloro­6­(2.3 ­xylidino)­2­pyrimidinylthio ] acetic acid

These two compounds, potent inducers of hepatic peroxisomes, increased
HDL­C levels in both ethanol­induced and high fat diet­induced hyper­
lipidemic rats (Sirtori et al., 1977d) : HDL levels were increased by
53 % With BR­931.

2.4.3. Probucol

a,a ­dimethylmethane­4.4'­dithio­bis (2.6­ditertbutylphenol)

With t h i s new l i p i d lowering drug, in patients.with primary hypercho­
lesterolemia, LD L­C levels were reduced in parallel to the t o t a l choles­
t e r o l (­ 16 %) but a s l i g h t , though s t a t i s t i c a l l y n o n ­ s i g n i f i c a n t ,
decrease was also noted in the HD L­C concentrations. Under Probucol, no
s i g n i f i c a n t changes of VLD L­TG and LD L­TG were observed (Mordasini et a l . ,
1979a, 1979b). Rouffy et a l . , 1979a, found a same reduction of LD L­C
(10­15 %) but Le Lorier et a l . , 1977, observed a s i g n i f i c a n t decrease of
HDL­C. This change in LD L­C and HD L­C levels was associated with a reduc­
t i o n of Apo Β (­12 %) and Apo Al (­16 %), Apo A2 (­55%) l e v e l s , respec­
t i v e l y (Mordasini et a l . , 1979a, 1979b).

C") 4-chloro-6-('2. Z-xylidi.no)-2-pyrimidinylthio-(N-ß-hydroxy ethyl) -acetamide.

 113

2.4.4. Ţiadenol
b1s­1.10­(2­hydroxyethylthio)decane.
Ţiadenol treatment in patients with HLP (IIa) led to decreases of
both LDL­C and HDL­C by 23 % and 36 %, respectively, from basal concentra­
tions after 4 months of drug therapy.
No effect on VLDL­TG was found (Baggio et al, 1977).
2.4.5. β­Sitosterol
Recent studies demonstrated the efficacy of ß­Sitosterol, obtained
from t a l l o i l , in lowering plasma LDL­C in patients with type I I HLP
(Lees et a l . , 1977). However, in children with familial hypercholeste­
rolemia, ß­Sitosterol lowered LDL­C by only 6 % and HDL­C by 15 %. This
insufficient response for LDL­C and the marked f a l l of HDL­C appears to
advise against the use of this drug in juvenile type I I HLP (Schlierf
et a l , 1978).

2.4.6. Diosgenin
In cholesterol­fed r a t s , Diosgenin produced a significantly greater
decrease in LDL­C and increase in HDL­C than either ß­Sitosterol or
Cholestyramine. LDL­PL levels were also reduced, though to a lesser extent
than LDL­C. There was no effect on TG. This agent inhibited cholesterol
absorption without altering bile acid turnover (Cayen et a l . , 1979).
The combination of Clofibrate and Diosgenin produced greater decreases
in LDL­C than did either compound alone. Furthermore, the Diosgenin­
induced elevation in HDL­C was partially reversed by Clofibrate (Cayen
et a l , 1978).

2.4.7. Y:9738
Ethyl­2(4­chlorophenyl)­5­ethoxy­4 oxazoleacetate.
In rats with experimental hyper­ß­lipoproteinemia, this newly discovered
drug showed a dose­dependent lowering effect on LDL­C while an upward
trend was noted for HDL­C (Kobayakawa et a l . , 1978).

2.4.8. Surcose polyester (SPE )

SPE, a long chain fatty acid ester of sucrose, led to a significant
reduction in TC and LDL­C, in normal volunteers men, without altering
serum TG or HDL­C (Glueck et a! . , 1978).
114

3. ANTIDIABETICS
3.1. Insulin
Controversy exists about the e f f e c t of Insulin on HDL-C in man
(Nikkilä et a l . , 1977 ; Stalenhoef et a l . , 1978). However, several trends
are emerging. One i s that i n s u l i n - d e f i c i e n t diabetics have low HDL-C
levels ( N i k k i l ä , 1973, 1978a ; Schonfeld et a l , 1974 ; Bennion et a l , 1977)
and in large p a r t , the HDL-C returns towards normal with i n s u l i n . Such
patients have a s i g n i f i c a n t c o r r e l a t i o n between HDL-C and the i n i t i a l l y
subnormal levels of HDL-C ( N i k k i l ä , 1978a). Well-controlled I n s u l i n - r e q u i -
ring diabetics have elevated HDL-C levels and t h i s increase may represent
another argument f o r good diabetic control (Durrington, 1978 ; Nikkilä
et al , 1978b ; Calvert et a l . , 1978)
Furthermore, I n s u l i n treatment decreased LDL-C whereas HDL-C/LDL-C
r a t i o was increased. There was a negative c o r r e l a t i o n between i n i t i a l
VLDL-TG levels and changes of VLDL-TG with also a negative c o r r e l a t i o n
between VLDL-TG and HDL-C levels before treatment but not a f t e r treatment
(Tamai e t a l . , 1979b).
On other hand, i n maturity-onset diabetic patients several authors
(Lopes-Virelia e t a l . , 1977 ; N i k k i l ä , 1978a ; Kennedy et a l . , 1978 ;
El keles e t a l . , 1978) found decreases of HDL-C levels but only i f TG
or/and glucose concentrations were elevated. Furthermore, there was no
c o r r e l a t i o n between HDL-C and indices of diabetic c o n t r o l .

3.2. Other hypoglycemic agents

In t h i s area , the most studied hypoglycemic agent i s Metformine
(N-N dimethyl biguanid). Theoretically i t i s possible f o r a drug to a l t e r
the atherogenicity of serum lipoproteins without a l t e r i n g t h e i r absolute
concentrations. The best example of such drug i s Metformin wich i n h i b i t s
the developement of experimental atherosclerosis i n cholesterol fed rabbits
(Agid e t a l . , 1969) whereas a f a i l u r e i s noted with other hypoglycemic
agents (Sterne et a l . , 1973 ; Agid e t a l . , 1975). Metformin increased the
f r a c t i o n a l catabolic rate of VLDL i n hypercholesterolernie rabbits
(Rodriguez et a l . , 1976 ; S i r t o r i e t a l . , 1977a) and changed the apoprotein
composition of serum lipoproteins ( S i r t o r i e t a l . , 1977a, 1977b, 1977c).
In man, LDL-C and p r i n c i p a l l y VLDL-C were not s i g n i f i c a n t l y decreased
while HDL-C were not modified (Rouffy, 1979b). Furthermore, the concen-
t r a t i o n s of VLDL and IDL were reduced i n addition to the changes in
 115

apoprotein composition (B ar­On et a l . , 1977 ; Nikkilà" et a l . , 1977,

Stalenhoef et a l . , 1978). Glibenclamide, another hypoglycemic agent,
led to a reduction of LDL­C and HDL­C levels in diabetics and the HDL­C/
LDL­C ratio was decreased (Tamai et a l , 1979b).

4. MISCELLANEOUS COMPOUNDS
4 . 1 . Antihypertensive drugs
Thiazides and beta­blockers induce changes in serum l i p i d levels
(Helgeland et a l . , 1978) principally a decrease of HDL­C (Helgeland et a l . ,
1978b ; Streja et a l . , 1978).
Hydrochlorothiazide only resulted in a significant increase in mean TC
levels on the other hand HDL­C concentrations did not increase.
With propanol ol alone, no effect on TC was observed but LDL­C and
HDL­C decreased while VLDL­C increased (Tanaka et a l . , 1976). The
reciprocal changes in the lipoprotein composition might result from the
Inhibition of lipoprotein­lipase by propanolol.
4.2. Antihistamine drugs
Cinnarizine.an anti hi stami ni c and vasoactive drug has been used
for several years 1n the treatment of peripheral vascular disorders and
recently was tested for i t s effect on serum lipoproteins in hyperlipo­
proteinemic patients ( I I and IV HLP)(Saba et a l . , 1977c). After six
months of treatment with this drug, LDL­C and VLDL­C were reduced by
35 % and 78 %, respectively.
4.3. Phénobarbital
Phénobarbital is known to induce hepatic microsomal enzymes (Conney,
1967). I t has more recently been suggested that the induction of microsomal
enzymes might be an important factor determining plasma l i p i d concentra­
tions (Martin et a l . , 1975).
On the basis of this finding, certain authors (Miller et a l . , 1973 ;
Durrington et a l . , 1976 and Durrington, 1979) investigated the effect
of phénobarbital on plasma HDL levels. After phénobarbital administration,
several subjects showed an increase in both TC and LDL­C ( s t a t i s t i c a l l y
significant) while plasma VLDL­C levels did not change. LDL­TG and LDL­
protelns were significantly increased (Miller et a l . , 1973 ; Durrington
et a l . , 1976 ) . An Increase in serum HDL­C was also found. Relative
proportions of apoprotein Β and cholesterol in LDL were unchanged by
phénobarbital. Furthermore, the increase of LDL­proteins suggested that
116

there was an increase in another LDL-apoprotein, possibly apoprotein

C of the LDL-1 subclass containing more apoprotein C than LDL-2 (Durring-
ton, 1979).
Regarding a possible mechanism f o r these f i n d i n g s , increased hepatic
cholesterol synthesis has been demonstrated in phénobarbital-treated
hamsters (Jones et a l . , 1965) and rats (Middleton et a l . , 1969 ; Wada
e t a l . , 1967). This probably results from induction of the microsomal
enzyme 3-hydroxy-3-methyl-glutaryl-CoA-reductase which i s a r a t e l i m i t i n g
step in cholesterol biosynthesis (Durrington et a l . , 1976).
4.4. Phenytoin
Pel konen et a l . , 1975 and Nikkilä et a l . , 1978c, have found , in
e p i l e p t i c patients with Phenytoin treatment, a s i g n i f i c a n t increase
of HDL-C l e v e l s . Furthermore, high Apo Al values were found in patients
who had serum Phenytoin concentrations at therapeutic l e v e l s . HDL-C and
Apo Al levels showed a s i g n i f i c a n t correlation while the Apo A2 concentra-
t i o n was not s i g n i f i c a n t l y d i f f e r e n t from that of c o n t r o l s . This increase
of HDL-C levels would be able to explain Livingston's results (1976), who
noted an unusually low incidence of myocardial i n f a r c t i o n in e p i l e p t i c
patients.
4.5. Diazepam
In a preliminary r e p o r t , Wong et a l . , 1979 , indicated that HDL-C
of a group of roosters treated with valium (0,2 mg) was elevated as com-
pared to the c o n t r o l s .
4.6. Etidronate Di sodi urn
In patients with type IV HLP, Etidronate disodium, a diphosphonate
used c l i n i c a l l y to t r e a t Paget's bone disease induces a s i g n i f i c a n t
f a l l of LDL-C. However, t h i s drug had no s i g n i f i c a n t e f f e c t on HDL-C
and VLDL-C (Mellies et a l . , 1979).
4.7. Ascorbic acid
. Bates et a l . , 1977, found a strongly p o s i t i v e c o r r e l a t i o n between
plasma vitamin C and HDL-C in men but not in women treated with ascorbic
acid in an 18-month longitudinal study.
Additional studies are necessary to assess a r e l a t i o n between the
benefits of vitamin C on CAD and these hyper-HDL-C l e v e l s .
5 - TOXIC AGENTS
5 . 1 . Alcohol
Although alcohol has been known f o r a long time to a f f e c t l i p i d
 117

metabolism, Its HDL-C Increasing effect has been appreciated only In

the past few years.
The presence of disorders at the level of α-lipoproteins after alcohol
intoxication was demonstrated as early as 1969 by Johansson et a l .
Baraona et a l . ,(1970) report an elevation of HDL-C levels after
progressive alcohol Intake in animals.
In 1974, M ishkel noted an elevation in HDL-C levels in one alcoholic
woman at the time of peak ethanol Ingestion.On the other hand, HDL-C
levels dropped when the subject was sober again.
Johansson et a l . , (1976), have recently demonstrated that more than
80 % of chronic alcoholics showed increased HDL-C levels.
Previous studies have been confirmed and extended by Hulley et a l . ,
(1977) and Castelli et a l . , (1977), who also report an elevation in
HDL-C. Such association 1n strikingly related to the amount of alcohol
ingested : I . e . a subject consuming 5 to 6 oz alcohol per week exhibits
HDL-C levels 10 % higher than those determined in a non-alcoholic
subject.
This Increase 1n HDL-C 1s independant of age, weight and VLDL-TG
levels (Belfrage et a l . , 1977). In f a c t , moderate amounts of alcohol
can Increase HDL-C 1n most of the subjects even as VLDL-TG are rising,
though, 1n some Individuals, large amount of alcohol can suppress HDL-C.
This Increase of HDL-C could be ascribed to an increase in f r e e , as well
as ester1f1ed, cholesterol (Danielsson et a l . , 1978).
The Increase 1n HDL-C 1s most frequently correlated with the elevation
1n HDL-apoproteins found to be above normal values in 65 % of the cases.
However, correlation failed to be established 1n a few cases.
This last notion 1s consistent with the ultracentrifugai profiles
which showed no uniform pattern but revealed changes in HDL-2 as well
as in the HDL-3. The patients showing an increase mainly in the less dense
HDL-2 fraction had a much more pronounced increase in HDL-C than in HDL-
apoprotelns (Danielsson et a l . , 1978)
The mecanism whereby alcohol elevates HDL-C may be related in part
to I t s apparent a b i l i t y to increase adipose tissue lipoprotein lipase·
(Belfrage et al . , 1977) though i t probably acts to enhance lipoprotein
synthesis, as well (Baraona et a l . , 1977).
118

A reduction in LDL-C is observed concomitantly with the increase

in HDL-C. The intensity of this reduction varies according to the type
of subject considered. Certain patient populations studied by Castelli
et al., (1977), exhibited a reduction in LDL-C proportional to alcohol
intake and the negative correlation observed is subsequently less marked .
On the other hand, no significant correlation was found for VLDL-C. The
increase of HDL-C in case of moderate consumption of alcohol recently
led Kannel et al., (1979), to note that a moderate alcohol intake was
necessary to reduce the risk of coronary heart disease.
The authors quote the findings of St Leger et al. , (1979), recently
corroborated by those of Ricci et al.,(1979), who had demonstrated a
specific and negative correlation between wine intake and coronary death
in different countries, including France, and had, therefore, established
the benefits of wine.
Certain authors are, however, more prudent. Before including alcoholic
beverages among preventive therapeutic measures, consideration should
be given to the direct or indirect cardiotoxic effects of alcohol
(Barboriak,1977).
Statistically significant negative association between alcohol intake
and coronary occlusion remained even after adjustment for age and HDL-C
mediated effects of alcohol on coronary artery occlusion (Barboriak et al,
1979).
5.2. Pesticides
5.2.1. Chgl^ineşţerase_inhibiţgrş
In 1975, Kutty et al., observed in a 10-year-old boy who had been
hospitalized after ingestion of the Cholinesterase inhibitor parathion,
used as insecticide,a dramatic fall of LDL-C after only 6 hours from an
initial level of 240 mg/dl to 30 mg/dl. The drop of LDL levels was
attributed to the inhibition of serum Cholinesterase by the parathion
stnce this enzyme is intimately associated with LDL (Lawrence et al.,
1961). The same effects were reported with phospholine iodide, in guinea-
pigs (Kutty et al., 1975 ) and with neostigmine in rats (Kutty et al.,1977)
The decrease in serum LDL and LDL-C levels occurs simultaneously with
the reduction in serum Cholinesterase activity.
 119

5.2.2. Chlorinated hydrocarbons pesticides

Carlson e t a l . , 1972b reported that of 22 men exposed in t h e i r
a c t i v i t i e s to a mixture of chlorinated pesticides, mainly Lindane and
DDT, 40 % had hyper ­ α ­ lipoproteinaemia and serum hyper HD L­C l e v e l s .
Later, 8 of the 22 men who, in 1970, had been exposed to chlorinated
pesticides and who ceased to be exposed, showed a s i g n i f i c a n t decrease
of HD L­C l e v e l s , the average individual decrease being 12.5 ­ 2.1 mg/100ml
(p < 0.001) (Carlson et a l . , 1977b).
The cholesterol concentration of the other l i p o p r o t e i n classes
(LDL­C and VLD L­C) showed no s i g n i f i c a n t trend.
Ishikawa e t a l . , 1976, 1978 reported the same effects with several
chlorinated hydrocarbons, evaluated in rats : non toxic doses of
Aroclor, D ieldrin and Kepone elevated HD L­C ( 31 %, 26 % and 24 % respec­
t i v e l y ) . On these three compounds, only Aroclor maintained s i g n i f i c a n t
HDL­C elevations at 21 (28 %) and 60 (31 %) days p o s t ­ i n j e c t i o n .
Simultaneously, microsomal cytochrome P­450 content was s i g n i f i c a n t l y
Increased.
The HD L­C elevating e f f e c t does not appear to be a consequence
of h e p a t o t o x i c i t y but may represent some aspect of hepatic microsomal
enzyme Induction (Ishikawa et a l , 1978).
120

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 EFFECTS OF DRUGS AND PHYSIOLOGICAL SUBSTANCES ON SERUM AND
URINE CREATININE

Jerzy Rogulski and Anastasia Pacanis

Department of Clinical Biochemistry
Institute of pathology Medical Aoademy
Gdansk, POLAND

ABSTRACT
A major problem in serum creatinine measurement is in-
terference from noncreatinine chromogens. The most common
interferences include proteins, glucose, and ketoacids. Ele-
vated concentrations of glucose and ketone bodies in diabetic
patients may result in elevated spurious creatinine values.
Small differences in technics or reagents employed may cause
large discrepancies in the results obtained. The kinetic
method employed in centrifugal analyzers was found to be
especially sensitive to the interference by acetoacetate.
Bilirubin can negatively interfere with kinetic Jaffe creati-
nine determinations. All drugs potentially nephrotoxic should
be considered as interfering with serum and urine creatinine.
Gentamicin and co-trimoxazole are examples of drugs inducing
deterioration in kidney function, which is manifested by the
rise in serum creatinine concentration.

Serum creatinine may be determined by a number of

technics. They differ on the basis of method used, the manner
of eliminating interferences, the type of system (manual or
automated) employed, and the procedure (end-point or kinetic)
of deriving concentration. The most widely used methods
depend on the reaction of creatinine with alkaline picrate to
form a colored complex measured at 500-520 run. Despite
reports that other methods may have higher specificity, the
alkaline picrate method for the assay of creatinine appears
to be the most practical at this time. This method have been
adopted to a wide variety of automated instruments including
continous-flow, centrifugal and other discrete analyzers.
Manual as well as automated technics may employ end-point or
kinetic method of determination.
There are two chief sources of difficulty with the alka-
line picrate method; its lack of specificity and its sensiti-
 135
vity to certain variables, some of which have only recently
been reoognized. The color yield of creatinine, as well as
the time required to attain full oolor development depend
upon the concentration of both NaOH and picric acid. The rate
of oolor formation is decreased when pH of reaction mixture
is lowered. Different ways of eliminating of serum protein
yielding neutral, slightly or strongly acid creatinine solu­
tion affeot the rate of Jaffé reaction. The temperature and
duration of incubation time, and the presence of some other
constituents in the reaction medium are also of importance.
It is apparent that the color yield and the rate of reaction
of interfering substances are also sensitive to the above
variables, usually, hovever, in different ways. Although
these problems were not extensively studied, the existing
data allows one to predict that all published modifications
of Joffe reaction as well as all commercial test­kit proce­
dures differ in their sensitivity to various interferences.
Many procedures have been claimed to improve the specificity
of the reaction when applied to serum or urine. Modifications
to reduoe interferences employ preoipitation of proteins,
dialysis, kinetio determination of creatinine concentration,
Lloyds'β reagent, cation exchange resins, acidification of
the sample after color development and enzymatic degradation
of creatinine. In most oases increased specificity was intro­
duced, however, at the expense of expediency, requiring
extended handling of the specimen.
Clearly, the major problem in routine creatinine estima­
tion seems to be the elimination of interferences without
saorifice of effioiency, aocuracy and precision. Henry (196Ί)
listed several substances wbioh in addition to creatinine
reaot with alkaline plorate. Some of these substances are
normally present in blood or urine, others are found only in
disease states, while still others occur during drug therapy.
The most common interferences include proteins, glucose,
ketone bodies and keto aoids. All these substances may react
with alkaline plorate. Some drugs, including nitrofurans and
dihydroxybenzene derivatives, react directly with the Jaffe
136
reagent. Others, like ascorbic acid, levodopa and methyldopa,
are readily oxidized and affect the reaction. Still others,
like BSP and PSP, interfere physically due to their own
color in alkaline medium (Table 1 ).
TABLE 1 ­ SUBSTANCES INTERFERING IN JAFFE REACTION

Proteins, glucose, fructose Normally present

ketone bodies ­ acetoacetate, acetone in body fluids,
2­ketoacids ­ pyruvate,' diet constituents
ketoglutarate, metabolites
ketobutyrate,
ketocaproate,
■ ketovalerate

Ascorbic acid, diacetic acid, Drugs ­

aminohippurate, levodopa, methyldopa, therapeutics,
pyrocatechol, resorcinol, diagnostics,
hydroquinone, nitrofurans, BSP, PSP xenobiotics

Taken from the data of Henry (1964), Wirth and Thompson

(1965), Elking and Kabat (1968), Christian (1970), Young et
al (1972) .
E l i m i n a t i o n of serum p r o t e i n i n t e r f e r e n c e by p r e c i p i t a ­
t i o n with d i f f e r e n t a g e n t s i s an i n i t i a l s t e p i n the most
manual a l k a l i n e p i c r a t e t e c h n i c s . C are must be taken t h a t
serum f i l t r a t e or the u r i n e samples a r e completely p r o t e i n ­
f r e e . As was demonstrated by Heinegard and Tiderstrora (197J)»
serum albumin at concentration of 725 umoles/1 produces color equivalent
to about 177 ymoles/1 creatinine. C olor yield of protein depends largely
on the pH of the reaction mixture. The higher the pH, the higher is the
color obtained with protein.
As early as 195^, Haugen found that glucose and acetone
influence plasma creatinine determination. In diabetic and
especially in ketotic patients high level of "interference
can lead to erroneously low estimates of glomerular filtra­
 137

t i o n r a c e . I t was r e c o g n i z e d t h e r e a f t e r t h a t w i t h c r e a t i n i n e
l e v e l s w i t h i n t h e normal range serum g l u c o s e c o n c e n t r a t i o n s
of about 28-39 mmoles/1 could lead to an error of about 15-20%.
Heinegard and Tiderstrom (197U) reported that when
glucose was reacted with alkaline picrate, color formation
was lowest in the pH range between 11.65 and 12.20. Increase
or deorease of pH caused a gradual increase of glucose in-
terference. It is also known that interference by glucose,
as well as by fructose, at 37 is more pronounced than at
25 especially if the time of reaction is prolonged. The
absolute error in creatinine determination due to a given
concentration of"glucose appears similar whether the
solution is serum or urine. Since, however, the creatinine
concentration in urine may be 100 times that of serum, the
per cent error is usually much lower in urine and may be
considered negligible.
The major problem in serum creatinine measurement in
diabetio patients seems to be the interference of ketone
bodies. Watkins (I967) and Husdan and Rapoport ( 1968) showed
that acetoacetate present in serum of ketotic patients
caused a false elevation of the creatinine value, assayed
either with manual or with standard continous-flow technic.
TABLE 2 - KETONE BODIES INTERFERENCE IN CREATININE
MEASUREMENT

Serum creatinine
Diagnosis Ketostix ymoles/1
reaction Autoanalyzer Manual with
Lloyd's reagent

Diabetio
+++
ketoacidosis 442 133
Untreated 141 44
new diabetic
Untreated 80 62
new diabetic

(from Watkins I967) .

138
The above authors showed also that the error in creati-
nine is related linearly with servim acetoacetate concentra-
tion. The color yield of 2 inmoles acetoacetate per liter is
equivalent to 88,4 pmoles/1 creatinine. Kammeraat (1978) studied
the effect of acetoacetate and pyruvate on the determination
of creatinine by various methods. He found that acetoacetate
causes highest pseudocreatinine values in the test-kit pro-
cedure for Centrifichem. No effect of acetoacetate is seen
in the ACA procedure and the manual method with Lloyd's
reagent ( Table 3 ) .
TABLE 3 - EFFECT OF ACETOACETATE AND PYRUVATE ON THE
DETERMINATION OF CREATININE BY VARIOUS METHODS

Creatinine /umoles/1
Serum with 5.0 mM with 1.0 mM
acetoace tate pyruvate

Test-kit for U80

Centrifichem 100 150

ACA du Pont 100 100 149

Continuous-flow 100 203 137
SMA-C
Manual, test-kit
Boehringer 100 3W 150
Manual, with Lloyd's 100
reagent 100 95

(Kammeraat 1978) .
The kinetio method for creatinine determination, perhaps
most widely employed in centrifugal analyzers is based on
the direct relationship between reaction rate and creatinine
concentration. It greatly reduces the effects of those
interferences with reaotion rates much slower than crea-
tinine, e.g., proteins, glucose, asoorbic acid.
In oontrast, acetoacetate reaots much faster than crea-
tinine and interferes strongly in the centrifugal analyzer
in which the reaction is started without significant delay.
The presence of acetoacetate has no effect on the kinetio
 139
method with the discrete ACA analyzer because a delay time
of 29 sec at 37° issufficient for completing the reaction
between acetoacetate and picrate. Caraway and Kammeyer
(1972) also reported that by 1 min acetoacetate ceased to
bave any significant effect on creatinine measurement. This,
bowever, does not bold for otber ketoacids. It was also
found by Kammeraat (1978) that Increasing concentrations of
NaOH causes a more intense reaction for acetoacetate.
The effeot of this serious interference in manual methods
and in continous­flow methods can be reduced with appro­
priate reaotion conditions but not eliminated. Pyruvate
interferes in all direct methods although less strongly than
aoetoacetate.
It should be pointed out that high levels of inter­
ference in serum can lead to erroneously low estimates of
glomerular filtration rate.
It is also apparent that the treatment of diabetic
ketoacidosis with insulin would affect creatinine measurement
due to obanges in the level of interferences. This might be
responsible for large discrepancies even between within­a­
day creatinine determinations.
Considering the consumption of ascorbic acid as vitamin
tablets, juices, vegetables and fruits one may expect
ascorbio aoid interference in creatinine analysis to be re­
latively important. Caraway and Kammeyer (1972) found that
14,2 mmoles/1 of ascorbic acid produces color equivalent to 168 μnoles/l
of creatinine. Siest et al (1978) found that at therapeutic concentrations,
ascorbic acid distinctly interferes with the analysis of creatinine. The
extent of interference vary, depending on the technic, kit and apparatus
used.

Unlike the end­point Jaffa method, the kinetio methods

do not inolude protein separation, which also removes bili­
rubin. Thus when protein separation is omitted, bilirubin is
present during the assay of creatinine. Several authors have
reported recently that bilirubin interferes witb the kinetio
Jaffe method resulting in falsely low creatinine values
Daugherty et al( 1978),0sberg and Hammond (1978),Dorwart (1979) .
140

TABLE k ­ EFFECT OF BILIRUBIN ON RESULTS BY ΤΠΕ KINETIC

JAFFE METHOD

Bilirubin C reatinine Apparent error

ymoles/l pmoles creatinine/1

Control serum 3,4 81

no bilirubin 87 60 21
added 173 51 31
313 25 57

(According to Daugherty et al, 1978)

The most common explanation of this interference is that

bilirubin oxidizes in the alkaline medium with the concomitant
disappearance of its absorbance at about 500 run. The mecha­
nism of interference and its clinical significance are not
yet solved definitely. Other bile and heme pigments sometimes
associated with bilirubin are considered as a source of in­
terference. At this time the simplest treatment of icteric
sera would be to avoid the bilirubin interference using an
end­point method for creatinine determination.

TABLE 5 - C OMPARISON OK KINETIC AND END­POINT METHODS

(ICTERIC SERA)

Bilirubin Creatinine
End­point Kinetic
ymoles/1 jimoles/1

214 62 71
224 283 212
213 141 9
158 53 18
233 88 n e g a t i v e 'value

(According to Daugherty et a l , 1978)

 141
Wirth and Thompson in 1965 and Elking and Kabat in I968
published the comprehensive data dealing with causes of
aberrant laboratory values, including drugs. These valuable
contributions list the effects of drugs but do not distin-
guish between pharmalogical effects and chemical interferen-
ces. The works of Christian ( 1970) and that of Caraway and
Kammayer ( 1972) present discussion concerning the mechanism
of drug interferences. All these reviews list the drugs
having effect on creatinine measurement. The most complete
tabulation of drugs having pharmalogical effects on serum
creatinine concentration was, however, compiled by Young and
associates (1972).
TABLE 6 - DRUGS INCREASING SERUM CREATININE CONCENTRATION

Aoetaminophen, acetophenetidin,
amphotericin B, oapreomycin, carbutamid,
cephaloridine, chlorthalidone, Clonidine,
oolietiniettiate, Colistin, demeclocycline, ., . . .
* * ' Nephrotoxic -
deoxycyeline, gentamicine, kanamycine, . .. ,,
methioillin.* methoxyflurane,
' ' mithramycin.
' * . . .
nephrotoxic
mitomycin C, nalidixic acid, neomycin,
nitrofurantoin, oxacillin, phenacetin,
polimyxin B, streptokinase, tetracycline,
thiazides, triamterene
Arsenicale, mercurio salts, phosphorus, Nephrotoxic
paraldehyd
Mannitol due to dehydration
Clofibrate due to muscle damage

Most of the drugs listed affect serum creatinine due to

their nephrotoxicity. All drugs potentially nephrotoxic
should be considered as interfering with serum and urine
creatinine ( Curtis 1979 ). The clinical manifestations of drug-
induced renal disease may be missed in patient with initially
normal renal function. The only evidence may be a transitory
rise in the serum creatinine and urea concentrations. In
142

patienta with renal failure smaller doses may be nephrotoxic

The tabulation of potentially nephrotoxic drugs include
mercuric salts, sulphonamides, thiazides, cephalosporins,
aminoglycosides and many other antibacterial agents, as well
as barbiturates, arsenicale and phosphorus. In most cases no
detailed studies on the mechanism of interference were done.
Increase in serum creatinine or decrease of creatinine
clearance were reported among other clinical manifestations
of impared renal function. Nephrotoxicity is thought to be
present when the serum creatinine increase to a value 5O9Ó
greater than the pretreatment measurement. In patients with
marginal renal function, a smaller increment reflects nephro-
toxicity.
Presently, between 5 and W^l'o of all patients receiving
aminoglycosides display ototoxicity or nephrotoxicity. Amino-
glycoside nephrotoxicity is most frequently found with neo-
mycin and amikacin therapy, least common with kanamicin and
tobramicin. Aminoglycosides are not metabolized in the body
and are eliminated almost exclusively by renal excretion.
Pre-existing renal disease and concomitant administration of
other nephrotoxic drugs, particularly diuretics, increase
the risk of nephrotoxicity from aminoglycosides. Because of
the necessity of progressive cellular accumulation for the
induction of renal damage, abnormalities in kidney function
are not seen until 7-1^ days of therapy. Clinically this is
manifested by an increasing serum creatinine and urea con-
centrations.

TABLE 7 - CHANGES IN SERUM CREATININE ASSOCIATED WITH

GENTAMICIN THERAPY

Serum Creatinine
creatinine clearance
uraoles/l ml/min

Before t r e a t m e n t 106 65-88

Maximum d e t e r i o r a t i on 469 k-10

(According to Gary et al 1976),

 143

R e c e n t l y , m a n y a u t b o r e d r e w a t t e n t i o n to t b e d e t e r i o ­
r a t i o n i n r e n a l f u n c t i o n i n a s s o c i a t i o n with co­trimaxazole
therapy h a l o w s k i et a l ( l 9 7 3 Ì B e r g l u n d et βΐ(ΐ975λ Shouval
et a l (ΐ97β). T h i s d r u g c o n t a i n i n g t h r i m e t b o p r i m a n d s u l p b a ­
m e t h o x a z o l e b a s n o w r e p l a c e d tbe t e t r a c y c l i n e s a s t h e com­
m o n e s t d r u g c a u s i n g a s h a r p d e t e r i o r a t i o n of r e n a l f u n c t i o n .
T h e effect of o o ­ t r i m o x a z o l e w a s s t u d i e d i n p e o p l e w i t h
normal kidney function and rises in plasma creatinine by
about 25 , w i t h c o n c o m i t a n t d e c r e a s e s i n c r e a t i n i n e clearance
by about 30'/b w e r e f o u n d a f t e r treatment with therapeutic
d o s e s of the d r u g . I n p a t i e n t s w i t h a l t e r e d r e n a l f u n c t i o n ,
the d e t e r i o r a t i n g e f f e c t s of c o ­ t r i m o x a z o l e w e r e r e p o r t e d to
be m o r e s e v e r e .

T A B L E 8 ­ E F F E C T S OF C O ­ T R I M O X A Z O L E O N R E N AL F U N C T I O N

Creatinine
Creatinine Urea clearance
ymoles/1 nnr.oles/1 ml/min

Before treatment 265 16,3 39.7

Maximum deterioration 495 26,1 16.3

( According to Kalowski et al 1973 ).

It was found that the effect is secondary to the tri­
methoprim oomponent of tbe drug. It was also documented that
the addition of trimethoprim to serum in vitro does not af­
fect the measurement of creatinine by oontinous­flow method.
The overestimation by about 10^ of serum creatinine in the
presence of oo­trimoxazole was, however, reported. It seems
that the increase in serum chromogen during treatment with
other potentially nephrotoxic drugs should be also con­
sidered unless it is ruled out.
REFERENCES
Berglund, F., Killander, J. and Pompeius, R.: Effect of Tri­
me throprimsulphamethazole on the Renal Exoretion of
Creatinine in Man. J. Urology 11Ή 802-808, 1975.
Bye, A. and Fowle, A.S.A.: Co-Trimoxazole and Creatinine
Clearanoe. Lancet 1; 711-712, 1978.
144
Caraway, W.T. and Kammayer, C.W.: C hemical Interference by
Drugs and Other Substances with Clinical Laboratory
Test Procedures. Clin. Chim. Acta 41: 395­434, 1972.
Christian, D.G.: Drug Interference with Laboratory Blood
Chemistry Determinations. Am. J. Clin. Pathol. 54:
118­142, 1970.
Curtis, J.R. : Drug­Induced Renal Disease. Drugs 18: 377­391,
1979.
Dahlgren, J.G., Anderson, E.T. and Hewitt, W.: Gentamicin
Blood Levels; A Quide to Nephrotoxicity. Antimicrob.
Agents Chemotherap. 8: 58­6O, 1975.
Daugherty, N.A., Hammond, K.B. and Osberg, J.M.: Bilirubin
Interference Eith the Kinetic Jaffe Method for Serum
Creatinine. Clin. Chem. 24: 392­393, 197«.
Dorwart, W.V.: Bilirubin Interference in Kinetic Creatinine
Determinations. Clin. Chem. 25: 196­197, 1979.
Elking, M.P. and Kabat, H.F.: Drug Induced Modifications of
Laboratory Test Values. Am. J. Hosp. Pharm. 25:
485­519, 1968.
Gary, N.E. , Buzzeo, L., Salaki, J. and Eisinger, R.P.:
Gentamicinassociated Acute Renal Failure. Arch. Int.
Med. 136: 1101­1104, 1976.
Haugen, H.N.: Glucose and Aceton as Sources of Error in
Plasma "C reatinine" Determinations. Scand. J. Clin.
Lab. Invest. 6: 17­21, 1954.
Heinegard, D. and Tiderstrom, G.: Determination of Serum
Creatinine by a Direct Colorimetrie Method. Clin. Chim.
Acta 43: 305­310, 1973.
Henry, R.J.: Clinical Chemistry. New York; Paul B. Hoeber,
Inc. pp. 154­933, 1964.
Husdan, H. and Rapoport, Α.: Estimation of Creatinine by
the Jaffe Reaction. A Comparison of Three Methods.
Clin. Chem. 14: 222­238, 1968.
Kalowski, S., Naura, R.S., Mathew, T.H. and Kincaid­Sraith,P.:
Deterioration in Renal Function in Association with
Co­Trimoxazole Therapy. Lancet 1: 394­397, 1973.
Kammeraat, C: Modification of the Alkaline Picrate Assay
for Creatinine to Prevent Spuriously Elevated Values by
Keto Acids. Clin. Chim. Acta 84; 119­128, 1978.
Osberg, J.M. and Hammond, K.B.: A Solution to the Problem of
Bilirubin Interference with the Kinetic Jaffe Method
for Serum Creatinine. Clin. Chem. 24: 1196­1197, 1978.
Rosenthal, S.L.: Aminoglycoside Antibiotics. N.Y. State J.
Med. 75: 535­545, 1975.
Shouval, D., Ligumsky, M. and Ben­Ishay, D.: Effect of Co­
Trimoxazole on Normal Creatinine Clearance. Lancet 1 ;
244­245, 1978.
Siest, G. , Appel, W., Blijenberg, G.B., Capolaghi, Β.,
Galteau, M.M. , Heusghem, C, Hjelm, M., Lauer, K.L. ,
Le Perron, Β. , Loppinet, V., Love, C, Royer, R.J.,
Tognoni, C. and Wilding, P.: Drug Interference in
Clinical Chemistry; Studies on Ascorbic Acid. J. Clin.
Chem. Clin. Biochem. 16: 103­110, 1978.
Watkins, P.J.: The Effect of Ketone Bodies on the Determina­
tion of Creatinine. Clin. Chim. Aota 18: 191­196, I967.
 145
Wirth, tf.A. and Thompson, R.L.: The Effect of Various
Conditions and Substances on the Results of Laboratory
Procedures. Am. J. Clin. Pathol, k'3: 579-590, 1965.
Young, D.S., Thomas, D.W., Friedman, R.B. and Pestaner, L.C.:
Effects of Drugs on Clinical Laboratory Tests. Clin.
Chem. 18: IOUI-I30U, 1972.
 DRUG EFFECTS ON HEMATOLOGICAL TESTS

J.F. Guelfi
agrégé de pathologie médicale
Ecole Nationale Vétérinaire de Toulouse
Toulouse France

ABSTRACT
The author considers drug effects capable of modifying the hemogram
reference values. Variations due to drug accidents and to the looked-for
therapeutic effect are thus excluded. The drugs concerned interfere mostly
by modifying the cellular compartments. When variations manifest themselves
by a decrease in blood cells, they often imply a depressive effect (toxic,
in particular) that represents actually a first sign of intolerance.

INTRODUCTION
The bounds of our subject must first of all be clearly defined. We
have apOroached it in the Spirit of the "Société Française de Biologie cli-
nique" commission "Effects des médicaments sur les examens de laboratoire",
i.e. we have nrincipally searched for analytical and pharmacological effects
liable to modify unexpectedly the value of the blood parameters most asked
for and thus likely to deceive the practicioner.
Thus, our object is not to review the hematologic accidents due to drugs,
althou,sh their knowledge may help us ; neither is it to describe blood chan-
ges directly related to the therapeutic effect. On the other hand, we have
restricted our research to the blood cells, haemostatic troubles being the
object of a special study. Finally, we have not delt with antineoplastic
therapies.
Our bibliographic research has shown that a exeat number of articles have
been written concerning drug effects on blood tests. However, they often
deal with haematologic accidents ; furthermore, many studies are carried
out "in vitro" or on animals, or else relate to complicated tests. We have
assembled here the elements that seem adapted to our topic.
DROG EFFECTS ON WHITS BLOCH C3LL COUNT. PZATSLET COUNT AND WHITE BLOOD
PICTURE.
Analytical variations
Luke et al..(1971) achieved the comparison between Coulter S automatio
 147

leukocyte counts and hemocytometer counts. They observed a significantly

lower count with the first technique in patients receiving azathioprine and
prednisone, lymphocytes and granulocytes appeared to be involved, and dif-
ferences of up to 4500 cells/mnr vere observed, especially daring the first
1 or 2 weeks of azathioprine therapy. These differences may be due to the
destruction of abnormally fragile leukocytes as a result of the increased
mechanical and chemical trauma of the Coulter S counting system.
It is well known that error in platelet count may be observed after intra-
venous injection of lipid emulsions, due to formation of lipid micelles.
Fharmaoological effects
1- Granulocyte modifications
Their study is based on the knowledge of the different leukocyte seo—
tors. The kinetics of neutrophil Dlurinuclear cells may be summarized as
follows. The marrow comorises a multiplication sector a maturation sector
and a reserve sector. In the normal individual's blood vessels, the neutro-
phil cells are distributed between two compartments of comparable volume,
in oonstant interaction t the circulating compartment (typified by white
blood oell count) and the marginaied compartment (granulocytes, clinging to
the blood vessel walle). The granulocytes then proceed from the blood com-
partment into the tissue compartment. Thus a neutrophilia can be due to an
inorease in production of white blood cells, a liberation of neutrophil
cells into the circulating oompartment from marrow reserves or from the
marginate! compartment, a decrease of their departure into the tissues, or
an increase in their life-soan. On the contrary, a neutropenia will be due
to a decrease in production, a maturation problem, a peripheral destruction,
an increase in volume of the marginated compartment, or an egress of pluri-
nuclear cells into the tissues.
1.1 Neutrophilia
1.1.1 By liberation of marrow reserves and decrease in egress
towards tissues.
Cortisol produces a leukocytosis with neutrophilia ; we shall see
that it also brings about lymphopenia and eosinopenia. Let us mention here
that opposite modifications are observed in drug addicts (Cottrell, et al.
I972)· The mecanism of action of Cortisol seems to be a liberation of re-
serves from the marrow and a decrease of their erresc towards tissues. The
result is an inorease in volume of the vascular compartment (hence neutro-
philia) and of the marginated oompartment. Dale et al.. (1975) undertook a
148
comparative study of the neutrophilia provoked by hydrocortisone, predni­
sone, an endotoxin and etiocholanolone. SLood neutrophil count changes
were measured in eleven normal adult volunteers of either sex, receiving
single intravenous doses of 25, 50, 100, 200 and 400 mg of hydrocortisone
sodium succinate at 8.00 a.m., on separate days at least two days apart. Λ
second ,group of 15 volunteers received per os single doses of 51 10, 20,
40 and 80 mg of prednisone at 8.00 a.m. ; a third group of nine subjects
received no drug. For both groups, a blood neutrophil count was made before
the therapy and hourly for 6 hours after beginning the study. The maximum
increases in counts occurred within 4 ­ 6 hours. The three higher doses of
both steroids increased the blood neutrophil count approximately 4 000
cells/mm ·
On the other hand, the neutrophilia occur ing after administration of 200 mg
of hydrocortisone and 40 mg of prednisone was compared with that observed
after administration of an endotoxin (0,8 mg/kg) and etiocholanolone (0,1
mg/kg). The results were similar for all agents and it is concluded that
glucocorticoids can be used for measuring the neutrophil reserve response.
Mishler and Emerson (1977) also observe a maximum neutrophil count occuring
4 — 6 hours after oral or intravenous administration of dexamethasone ; a
second rise in the neutrophil count occurred 24 hours after oral ingestion
of dexamethasone.
It seemed interesting to us to mention the work of Breitenfield et al..
(I978). They observe the same variations as the other authors, but noticing
different results in each individual, they conclude that it is necessary to
take the blood sample, before the morning administration of corticoids, to
survey azathioprine treatment.
Some authors mention that prednisone and dexamethasone administrated before
leucopheresis, increase the number of granulocytes collected.
Finally, it would be useful to know the effects of long­acting corticoids.
1.1.2 By mobilization of the marginatedpool
Classically, adrenaline mobilizes neutronhils from the marginated
compartment, causing neutrophilia. Gader and Cash (1975), studied the ef­
fects of adrenalin, noradrenalin, isoprenalin (non sOecific ρ stimulant)
and salbutamol (p„ stimulant) by infusion of 7 uß/mn of these drugs during
50 minutes to 5 healthy volunteers. The number of white blood cells in­
creases with adrenalin and noradrenalin, this leukocytosis being due to an
afflux of neutrophils and lymphocytes. Isoprenalin and salbutamol only
 149
cauae an increase in the number of lymphocytes.
Atropine sulfate as well as nilocarpin may cause leucocitosis.
1.1.3 By increased production
λ PTinulocytosis Í3 ­leo observed in patients treated with lithium
carbonate for psychic disturbances ; some authors think that lithium atten­
uates neutropenia in patients undergoing cancer cherotherapy. Stein et al..
(I978) exnlain the mechanism of the rocess. Six healthy volunteers aged 24
to 31 years, received lithium carbonate, 3OO mg three times a day, orally,
durin/» 4 weeks. To measure marrow 'T­inulocyte reserve, hydrocortisone, 100
mg, wae injected intravenously. Aqueous epinephrine 1/1000, 0,4 ml/m t was
injected subcutaneoualy to easure marginited granulocytes. After the
therapy, the mean circulating granulocyte counts rose I24O cells/mm above
base line, an increase of 29 per cent. The granulocyte marrow reserve rose
significantly, 1 310 cells/mm above base line, an increase of 36 per cent ;
the marginated granulocytes dropped slightly, 314 cells/mm below the base
line, α decrease of I7 per cent, but this change was not significant stati­
stically. This study demonstrates that lithium induced granulocytes do not
merely arise from a redistribution of granulocytes that marginated or are
in the marrow reserves. It supports the hypothesis of increased granulocyte
production. The data are also compatible with increased survival of granu­
locytes after lithium administration.
1.2 Neutropenia
Among the many cuses of drug­induced neutropenia, medullar toxicity is
the foremost. Among the drugs responsible, let us mention chloramphenicol,
thianrph enicol, sulphametoxypyridazin, phenylbutazone, pol d salts, pheno­
thiazin and its derivatives. Other POssibie causes are a deficient medullar
maturation (dinhenylhydantoin . . . ) , a peripheral cell destruction by toxic­
ity (rifampicin, propranolol, ajmalin ...) or immunologic action (amidopyrin,
of methyl dopa . . . ) . A ctually, most cases described in literature refer to
drug­administration accidents, concerning one or more medullar cell­lines.
While immunologie phenomenous are not of concern here, the other causes
mentioned deserve our attention, for they are susceptible of causing, in
quite a number of subjects, blood modifications without clinical symptoms ;
these modifications must however be considered as a first sign of toxicity.
That is why we selected, instead of the articles refering to therapeutio
«ocidente, those that give a systematic study of the kinetics of blood
values in many patients receiving drugs reputed to be toxic. Pisciotta
150
(I969)f underwent a study of 6 200 patients treated by phenothiazine deri­
vatives. He observed 5 cases of reversible a.^ranulocytosis, but also, in a
third of the patients, a mild tenOorary leucopenia, ­it the beginning of the
treatment.
Several studies concern the association of sulfametoxazole and trimethoprim.
Lindahl et al., (l972) observed no .granulocyte modifications in 21 aşed
patients (mean ai^e of 78 years) with renal insufficiency, after several
months of treatment. However, Van Hove et al.. (1975) observed in 16 pa­
tients receiving Bactrim for a period of more than 6 weeks a hypersegmen­
tation of the nucleus of neutrophil cells and, in three patients, a mild
granulopenia. Maier and Heer (1972) administrated this drug to 78 patients
and observed, in one third of them, modifications in the granulocyte morpho­
logy and leuco­jenia.
Williams et al., (ΐ97β) treated 60 patients with levamisole ; 35 per cent
of them showed a persistent decrease in neutrophil cells and 10 per cent
(i.e. 6 patients) a marked neutropenia ; one\of these patients recuperated
before the end of the treatment and the other 5 after interruption of the
therapy.
2­ Eosinophil variations
Corticosteroids cause eosinopenia. Blockage of eosinophil release and
peripheral loss and migration of cells from bone marrow to other tissues
appear to be the major factors governing the response to corticosteroid
administration.
5­ Lymphocyte variations
Daring corticotherapy, all authors observed at the same time as neutro­
philia and eosinopenia, a lymphopenia, maximal after 4 to 6 hours. Counts
usually return to normal by­ 24 hours. Occasionally, after massive doses such
as 1 g of methyl prednisolone, suppression of counts may last up to 48 hours.
Mishler and Emerson (1977) report lymphopenia, 4 to 6 hours after oral ad­
ministration of dexamethasone and lymphocytosis 24 hours later.
It seems that the lymphopenia is due to a modification in lymphocyte circu­
lation. The equilibrium between the intravascular and extravascular portions
of the recirculating lymphocyte pool (spleen, lymph nodes, thoracic duct,
bone marrow) is affected by corticosteroid administration so that the
intravascular recirculating lymphocytes are depleted from the circulation
and accumulate in the extravascular compartments of the recirculating
lymphocyte pool (Fauci et al.. I976).
 151

Lymphopenia nay also be observed in subjects receiving phenytoin.

hac Kinney et al.. (1Ş72) observed in 66 patients a lymDhopenia all the
more marked as the serum level of the drug was high. However, no relation
was observed between this level and erythrocyte values. These results sug­
gested that the effect of phenytoin on circulating blood cells was specific
for lymphocytes and probably not rel ted to the decreased serum folate
levels associated with phenytoin therapy.
The observations that phenytoin de­pressed DNA synthesis in cultured lympho­
cytes as well as significantly lowering values of IgG in treated patients
led the authors to conclude that phenytoin causes immunodcpression. On the
oontrary, some drugs cause lymphocytosis t such is the case for isoprenalin
and salbutamol.
4­ Platelet count variations
The & ­ blockers (meteprolol) as well as propranolol (non specific B
blocker) increase the number of platelets.
Adrenalin, by stimulation of Preceptors, has the same effect, and is further
potentiated by propranolol.
On the contrary, specific or non specific Β stimulants decrease platelet
counts by increasing platelet captivation by the spleen.
Vincristine stimulate megacaryopoiesis.
DRUG EFFECTS ON RED BLOOD CELL COUNT. HE ATOCRIT ΑND HEMOGLOBINE IA
Analytical variations
Intravenous injections of lipid emulsions cause errors in the measure
of hemoglobinemia by spectrophotometry (Nicholls, 1573). The density of
methemoglobin is increased and so is hemoglobinemia. There thus ensues an
erroneous apparent increase of the mean cell hemoglobin concentration, that
may oatch the eye of the biologist when the values are aberrant.
Pharmacological effects
1­ Drupe causing hemodilution or hemoconcentration
Diuretics may cause hemoconcentration, thus increasing hematocrit.
Gavalaki et al.. (1973) noted that iodine­containing contrast products used
in angiography (Cardioconray) are responsible for hemodilution, appearing
very soon after the injection. These authors used Furosemide to speed up
the return to a normal hematocrit value.
Patients were treated by nandrolone decanoate (3 mg/kg/week) (Besa et al..
I972). They' showed a phase of hemoconcentration during the first 5 weeks.
There was no significant change in red cell mass but there was a 20 per
152

cent decrease in plasma volume and an increase of 15 per cent in hematocrit.

Sight to ten weeks later, the red cell mass increased 11 per cent with no
change in hematocrit.
Testosterone enan thate instead, increased plasma volume, presumably because
of its salt­retaining effects.
Larcan and Stoltz, (197&) strike the balance of effects of macro­molecules
(derivatives of polyvinylpyrrolidone, gelatins and dextrans) on blood
values (213 bibliographic references).
These macro­molecules cause hemodilution, with lowering of hematocrit. In
vivo, modifications are noted on red blood cell morphology, more distinct
with dextrans than with gelatins. No sign of hemolysis was found.
Some authors observed errors in blood group appreciation, but Larcan and
Stoltz do not mention any in their personal observations on 72 patients
receiving dextran.
In vitro, it is found that the sedimentation rate in Westergreen tubes is
accelerated by plasmagel, hemacel, subtosan and macrodex ; on the contrary,
dextrans of low molecular weight slow it down. In vivo, modifications ob­
tained vary according to different authors.
Adrenalin and JB stimulants may induce an increase of hematocrit by spleno­
contraction.
2­ Drugs with effects on circulating red blood cells
Much research has been done to test hemolysis, or on the contrary,
hemolysis­inhibiting effect of drugs. However certain studies were carried
out in vitro, and while they help to understand how drugs act, they do not
have a direct pragmatic interest.
Other articles relate to hemolytic accidents (toxic or immunologic) and
often take into account a special sensitiveness in subjects a G ­ 6 ­ PD
deficiency. Let us joint out the study of Halmekoski et al.. (1Ş78) on the
hemolytic effect of daosone on subjects treated for dermatitis herpetifor­
mis. Administration of 100 mg per day for 7 days is responsible for a
light hemolysis, detected by a decrease in seric haptoglobin.
3­ Drugs with effects on red blood cell production
3.1 Production decrease
3.1.1 Toxic medullar effect
The same remarks can here be made than in the case of white blood
cells. Many products provoke'accidents concerning one or more cell­lines.
A few articles study systematically the red blood cells of patients recei­
 153

ving druga considered to be toxic.

Kaltwasser et al.. (1973) treated 20 patients with thiamphenicol ; in most
of them was noted a hematocrit decrease, reticulocytopenia and an increase
of serum iron. Brythroblastopenia with cellular vacuolisation was noted in
the marrow. The disturbances arise much faster than with chloramphenicol,
but they are reversible.
3.1.2 Metabolic disturbances
By acting on folic acid metabolism, some drugs are responsible for
megaloblastic anemias. Among them are often mentioned anticonvnlsivant
agents. Systematic studies point out a relatively frequent maorocytosie
when hydantoin derivatives are administrated.
Strauss et al.. (ΐ97θ) do not note disturbances in 10 pregnant epileptic
women receiving phenytoin.
Fisch and Freedman, (1973) analysed red blood cell values in women taking
oral contraceptives. It was proven that these drugs cause an increase in
mean red blood cell volume, with a decrease in hemoglobin level, hematocrit
and red blood cell number, compatible with a folate and/or vitamine Β,, de­
ficiency. However, these authors add t "But the hazard of a clinically si­
gnificant macrocytic anemia in a healthy, well­nourished woman using an
oral contraceptive is probably minimal".
Trimethoprim­sulfamethoxazole association also has an impact. Haler and
Beer (1972) did not report anemia in 78 patients but Van Bove et al..
(1973) noted reticulocytopenia and an increase in free bilirubin in patients
treated for 6 weeks. Lindahl et al.« (1972) observed one case of reversible
megaloblastic anemia, after 2 months of treatment (on 21 aged patients).
3.1.3 Decrease in erythropoietic renal factor
Propranolol is responsible for such an effect, but only in mice.
Kryger et al.. (1978) evaluated the effect of medroxyprogesterone acetate
on 17 men with exoessive polycythemia at high altitude, the hematocrit fell
in all 17 patiente from 60.1 ­ 1.6 to 52.1 ­ 1.5 per cent. Concomitantly,
it was observed that there was a decrease in arterial PC0 2 and an increase
in arterial P0 9 · Medroxyprogesterone acts by its stimulating effect on
ventilation.
3.2 Production increase
According to certain observations, corticosteroids may stimulate
erythropoiesis.
154

Nortestosterone decanoate and oxymetholone cause proliferation of colony­

forming cells of the marrow.
It is proven that isoproterenol and salbutamol acting as B„ stimulants,
increase erythropoietic renal factor, by direct action on the Kidney.
CONCLUSION
At the end of this review, we find that studies carried out in the
spirit we defined in our introduction are relatively rare with regard to
the vast quantity of documents concerning hematological effects of drugs.
However, this first examination must nrompt us to continue bibliographic
research.lt would be interesting to study, following a protocol to be
precisely determinated, the analytical or pharmacological modifications
induced in reference values the following drugs frequently used, drugs
with a mechanism of action that allegedly have a hematological effect, and
new drugs.
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Bishop, C.R., Athens, J.W., Bogps, D.E., Warner, H.H., Cartwright, G.E.,
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Cader, A.M.Λ., and C ash, J . D . , 1975· The e f f e c t of a d r e n a l i n e , n o r a d r e ­
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PHYSIOLOGIC OR TOXIC EFFECTS
 INTERPRETATION OF LABORATORY TESTS :
THE EXAMPLE OF DRUG EFFECTS ON AMINOTRANSFERASES

K.M. Galteau, C. Morin and G. Siest

Laboratoire de Biochimie Pharmacologique, Faculté des Sciences Pharmaceu-
tiques et Biologiques, 7 rue Albert Lebrun 54000 NANCY (France) and Centre
de Médecine Préventive (dir. Pr. R. SENAULT), 2 avenue du Doyen Jacques
Parlsot 54500 VANDOEUVRE-LES-NANCY (France)

ABSTRACT
Plasma aspartate and alanine aminotransferases are very often used
to check the hepatic action of drugs. But the interpretation of an iso-
lated enzymatic activity really requires the control of the enzymatic
reactions and of the analytical variations, the cellular and tissue
localization and the physiological variations, the tissue (hepatic) level
in different pathological and experimental conditions, and the knowledge
of physiological plasmatic variations and the associated changes induced
by drugs in animal liver.

INTRODUCTION
The evaluation of drug toxicity is often based on the activity
measurement of plasma enzymes of hepatic origin. An increase in the
activities of these enzymes in the plasma of treated patients is usually
associated with a toxic effect of the drug on the liver due to necrosis
or cytolysis. These two notions of necrosis or cytolysis are now out of
date. Numerous tissues can release enzymes without damaging the vital
functions of the cells. In addition, physiological phenomena that can
change enzyme levels or activities are generally ignored. The interpre-
tation of an isolated enzymatic activity really requires the knowledge of
the different causes of variation.
We will exemplify these different points, as far as possible, in
humans. Some examples in animals are given only when no report for humans
is available (especially for tissue changes).
160

1. Control of the enzymatic reactions and of the analytical

variations
Aspartate aminotransferase (A ST : EC 2.6.1.1.) catalyzes rever­
sibly the following reaction :

COOH COOH COOH COOH

I I I
CH­NH2 CO CH­NH2 CO
ι
CH 2 CH 2 CH 2
I CH 2
COOH CH 2 CH 2
ι COOH
COOH COOH
Aspartic 2­0xoglutaric Glutamic Oxaloacetic
acid acid acid acid

and alanine aminotransferase (ALT : EC 2.6.1.2.), the following one :

COOH COOH COOH COOH

I I I I
CO CH _NH 2 CO
CH_NH2 ι
CH 3 CH 2 CH 3
CH 2 I
CH 2
CH 2 I
COOH
COOH
Alanine 2­Oxoglutaric Glutamic Pyruvic
acid acid acid

Both enzymes require pyridoxal phosphate as coenzyme (O'Kane and

Gunsalus, 1947). That is the reason why pyridoxal phosphate is now added
into the reaction mixture in order to measure AST and ALT total activi­
ties , even if the level of pyridoxal phosphate is diminished (Rej et al.,
1973; Ury and Chassy, 1973; Rosalki and Mbayoumi, 1975). Short and
long­term variations must be followed by means of rigorous quality
control for plasma as well as for tissue measurements. In the same way,
the analytical procedures must be adapted after kinetic studies to tissue
measurements. Finally, preanalytical steps, such as sampling, must be
controlled.
 161

2. Cellular and tissular localization and physiological variations

AST and ALT are distributed in many organs (table I)

Table I : Distribution of AST and ALT in human tissues

(results expressed in units / g of tissue)

TISSUE AST ALT

Heart 160 7
Liver 140 44
Skeletal muscle 100 5
Kidney 90 20
Pancreas 28 2
Spleen 14 1.2
Lung 10 0.7
Serum 0.02 0.02

In man, the activity of AST is high especially in heart and liver,

whereas the activity of ALT is high only in liver.
AST activity is found in the liver mitochondria and cytoplasm (Boyd,
1961). ALT is localized only in the liver cytoplasm (Wilkinson, 1976).
The physiological variations of tissue aminotransferases are not well-
known; even in animals. The studies are very rare in man, biopsies being
done only for pathology. However, in rats hepatic AST has been shown to
not vary with sex and to increase slowly from birth until puberty.
AST is an index of protein catabolism and neoglucogenesis. Its hepatic
activity increases after administration of a hyperprotidic diet (Borei
et al., 1958).

3. Tissue levels in different pathological and experimental

conditions
The AST and ALT activities vary in liver during different hepatic
disorders. For example, AST activity is diminished in rat liver 6 days
after bile duct ligation (Ideo et al., 1972).
In another experimental condition, i.e. 24 h after galactosamine
treatment, hepatic AST and ALT are decreased respectively from 15 Ζ and
40 Ζ (Galteau et al., in preparation). This is due to enzymes being
released in plasma.
It is important to know the stimulation factors and the physiological
limits of adaptation. Any interpretation of plasma AST or ALT activities
162

supposes that the causes of tissue variations are controlled.

The change of the enzyme amounts in the liver is followed by a change of
plasma enzyme activities.

4. Knowledge of physiological plasmatic variations

The two most important factors affecting transaminase activities
are age, sex and obesity.
In the human, the activities of AST and ALT are lower in women than in
men. In children, AST activity diminishes slightly until puberty but the
activities of both enzymes then increase to a peak at age about 40-50
years before decreasing again. Changes with age are different for men and
women, the decrease being delayed in women by one or two decades (Siest
et al., 1975). Weight and overweight greatly influence these enzymes,
especially ALT.
The table II summarizes these results.

TABLE II CLASSIFICATION OF THE DIFFERENT VARIABLES CALCULATED

WITH THE MEDIAN VALUES FOR AST AND ALT

Sex AST,% ALT,%

Age
Between children and adults females 45.3 9.8
males 22.5 31.5
20-30 to 40-50 years females 10.0 23.1
males 9.4 21.3
Sex
Between males and females (20-30 years) 21.0 37.5

Body weight
40-100 kg females 5.5 38.8
50-100 kg males 9.8 86.6
Meals
Before/after females 2.2 1.2
males 3.1 0.8
Oral contraceptives
Treated/control 12 32
Anticonvulsant drugs 36 36
Analytical variations
Day-to-day (coefficient of variation) 4.0 4.8
 163

A slight standardized exercise gives rise of AST (+ 4.7 Z) and ALT

(+ 6.1 Z) activities (Galteau, 1973). This increase is related to the
duration and the intensity of the effort (Fowler et al., 1968). For AST,
both isoenzymes (cytoplasmic and mitochondrial) are increased (Ohno
et al., 1978).

5. Changes induced by drugs in animal liver

Very few publications relate the variations of transaminases in
liver. The authors use plasma AST and ALT activities as indices of liver
diseases but the enzyme activities of the liver itself are very rarely
measured for this organ.
Dexamethasone significantly increases the activity of ALT but not that
of AST in the liver of treated rats. Cortisone (3 days, 120 mg/kg daily)
increases liver ALT by 59 Ζ and AST by 26 Z. Testosterone and thyroxine
have no effect on liver ALT (Knox and Greengard, 1965).
At the beginning of a long­term treatment with coumarin, liver transa­
minase activities are slightly decreased or not changed. After some days
of treatment the enzyme is released into plasma and the liver activity
decreases (Nievel et al., 1976). Treatment of rate with cefazolin signi­
ficantly supresses activity of alanine and aspartate aminotransferase in
serum and in the liver, brain, kidney and heart. Pyridoxal phosphate
partly reverses enzyme activity in the serum, liver and kidney, but does
not restore it to the amount observed in the control animals (Dhami
et al., 1979).
Phénobarbital and fluothane induces rat liver proteins and enzymes.
Hepatic AST and ALT are increased as other hepatic enzymes (Kuntz and
Schmieders, 1969).

6. Drug effect on the enzyme activities in the plasma

Countless numbers of drugs can produce variations of plasma
aminotransferases. In the listing published by Young et al., (1975), we
have found 318 different drugs producing an effect on plasma AST and
290 on plasma ALT.
Plasma activities can be altered by the presence of the drug in the ­
sample. For example, AST is diminished by clotiapine (9 mg/1) or by
pindolol (5 mg/1) (Jadin— Starodoubsky et al., 1973). But such "in
vitro" effects are not often observed, the technics being more and more
specific. More often, drugs produce pharmacological variations of plasma
164

AST and ALT.

During the three first months of treatment, isoniazid leads to a plasma
elevation of both transaminases in 7.5 % of treated children of one of
these enzymes in 9.6 % of treated children and has no effect in 83.0 %
of them (Spyridis et al., 1979).
The association of isoniazid, rifampicin and ethambutol increased trans-
aminase activities in 7 of 15 patients with active pulmonary tubercu-
losis and had no effect in 8 others (Knop et al., 1977). According to
Perry et al. (1978), a treatment with rifampicin and isoniazid does not
produce any change in ALT activity. Moreover, it has been demonstrated
that isoniazid does not interfere with colorimetrie or enzymatic deter-
mination of AST (Bailey et al., 1975).
In the mouse, morphine pellet implantation causes a 2 fold increase in
serum AST and ALT activities as compared with those of the placebo
control group. AST and ALT activities are returned to control levels
after 3 - 9 days of pellet morphine implantation and at 3 and 6 days
after the removal of the morphine pellet (Chang and Ho, 1979).
Patients who have no previous experience of chlorpromazine exhibit a
transient abnormal rise of AST after a single initial intramuscular
injection of the drug. A similar, though lesser reaction is observed
with ALT (Evans et al., 1976).
AST greatly increases in plasma after salicylates administration
(Saltzman et al., 1976). Marked transaminase abnormalities are transient
and appear unpredictably in individual patients (Miller and Weissman,
1976). Azathioprine, ajmalin and pentachlorphenol induce also an increase
of both transaminases (Schmidt, 1978).
Antibiotics increase AST and ALT activities. Table III summarizes these
effects.
 165

Table III : Pharmacological effects of antibiotics and β

lactamines on AST and ALT

­1
Antibiotics AST ALT ß lactamines AST ALT

Tetracyclines Pencillin G
/ / /
Tetracycline
Chlortetracycline / /

taninosides Penicillin A
Kanamycin
Gentamycin
/
/
Ampicillin
Carbenicillin
/
/ 4

iacrolides Penicillin M
Oleandomycin / /
Oxacillin / /

Chloramphenicol / / Cephalosporines
y y
Cephaloridine
Cephalothin y y

(Λ increase)

Many other examples may be found in the litterature. But the studies very
often describe isolated cases of overdosed people or patients not tolerant
to their treatment. Moreover, it is not enough to know that a drug
increases or decreases an enzymatic activity in the plasma. These varia­
tions must be calculated to be useful.
In our laboratory, we have done some studies by comparing treated and
control people using the method described elsewhere (Steinmetz et al.,
1980). The variation due to drugs for ALT are shown on the figure 1.
166

Vasodilator ι =b Vasodilator ! C ¡
1 Hypoliptmic ¡ ­^ι
Vasoconstrictor ι C Vasoconstrictor ι ^ '
Analgesic ■ Analgesic ι I !
Antidepressant ■ ; Antidepressant ¡ ­f ¡
1 Antianginal ιC ·
Tranquilizer ¡
Tranquilizer ¡C ¡
Antigout !
Antigout ¡ ιI
Anticoagulant ¡
Anticoagulant ¡ ] ¡
> 1 Antidiabetic ) ■ι
Antihypenention ι !
Cardiotonic ! • 1
Hypnotic ι ι ; Hypnotic ι
Anticonvulsant ι
72!
Anticonvulsant ¡
Oral contraceptive ι
¡
(20­30 years) J ! *
Agr (20­30 io 50­60 !
' ι
Se« Ittiwccn niales and ■
¡ femelei (20­30 vean) females (20­30 years) !

1 ι—'—
­20 ­10 0 Κ) ­20 ­10
ALT females ALT malei

Fig. 1 : Effects of long term drug intake on ALT activities

in males and females 40 ­ 60 years old as a percentage
of the median (From Schiele et al., 1980)

Thus, a lot of drugs can produce plasma enzyme variations.

But it is very difficult to interpret the data of the literature for
the following reasons :
. the activities are measured in animal or in man
. the number of subjects varies from 1 to more than 1500
. the studies are often performed with sick people who can receive many
drugs
. the statistical methods are not described and not often well adapted
to the problem
. the variations are indicated by increase or decrease and the percentages
of variations have not been calculated
Thus, after the pathological variations have been described and control­
led and the reference values defined, it will be possible to interpret
an isolated value for enzyme activity in the plasma.
In order to make such a work possible, it is necessary
. to examine the existing files and to reevaluate the described varia­
tions (Isolated variations observed on only few cases must be avoided)
 167

to describe and measure drug effects in clinical chemistry

to not systematically classify as toxic a plasma increase of AST or ALT
but to consider all other variations

Afterwards it will be possible to answer the questions :

1. Do the AST and ALT that are released in plasma really originate from
liver ?
2. Is the increase an inductive phenomenon ?
(Induction tests such as GGT, glucaric acid must be examined)
3. Are AST and ALT variations accompanied by other phenomena such as
steatosis ?
(Lipid metabolism must be explored)
4. Is the effect really cytotoxic ?
(Other parameters such as mitochondrial enzymes must be tested)

REFERENCES
Bailey W.C., De Rouen T.A., Ziskind M.M. and Greenberg H.B.: Autoanalytic
(colorimetrie) determinations of SGOT in isoniazid recipients are
reliable. Am. Rev. Respir, dis., Ill: 237-238, 1975.
Borei C.L., Ryser H. and Frei J.: L'élévation de deux transaminases hépa-
tiques considérée comme adaptation enzymatique du rat au régime
carné. Schweiz. Med. Wschr., 6: 135-137, 1958.
Boyd J.W.: The intracellular distribution, latency and electrophoretic
mobility of L-glutamate-oxaloacetate transaminase from rat liver.
Biochem. J., 81: 434-441, 1961.
Chang Y.Y.H. and Ho I.K.: Effects of acute and continuous morphine adminis-
tration on serum glutamate oxaloacetate transaminase and glutamate
pyruvate transaminase activities in the mouse. Biochem. Pharmacol.,
28: 1373-1377, 1979.
Dhami M.S.I., Drangova R., Farkas R., Balazs T. and Feuer G.: Decreased
aminotransferase activity of serum and various tissues in the rat
after cefazolin treatment. Clin. Chem., 25: 1263-1266, 1979.
Evans L., Hassanyeh F., Gallacher J. and Leitch J.M.: The effect of
intramuscular chlorpromazine on the serum transaminases (SGOT and
SGPT). Europ. J. clin. Pharmacol., 10: 289-292, 1976.
Fowler U.M., Gardner G.U., Kazerunian H.H. and Lawsted W.A.: The effect
of exercise on serum enzymes. Arch. Phys. Med. Rehabil., 49: 554-
565, 1968.
Galteau M.M.: Etude du phénomène de sortie d'enzymes musculaires. Relations
avec les variations des metabolites et des enzymes au cours de
l'exercice. Thèse de Pharmacie Nancy, 1973.
Galteau M.M., Ratanasavanh D. and Siest G.: Effect of galactosamine
treatments on hepatic and plasmatic enzymes in rats. In preparation.
Ideo G., Morganti A. and Dioguardi N.: Gamma-glutamyltranspeptidase: a
clinical and experimental study. Digestion, 5: 226-336, 1972.
168

Jadin­Starodoubsky Α., Delwaide P.A., Penders C, C ollard J. and

Heus ghem C: Psychotropic drug interferences with clinical chemistry
determinations, In : G. Siest Ed., Reference values in human chemis­
try, Karger Basel Pubi., pp 229­303, 1973.
Knop P., Kindler U. and Austerhoff Α.: Rifampicin und Isoniazid­plasma­
spiegel sowie Aminotransferasen im Serum bei Tuberkulostatischer
Kombinationstherapie. Dtsch. med. Wschr., 102: 1913­1915, 1977.
Knox W.E. and Greèngard 0.: The regulation of some enzymes of nitrogen
metabolism. An introduction to enzyme physiology. In : G. Weber Ed.
Advances in enzyme regulation, Pergamon Press Oxford, Publi.,
3 : pp 247­313, 1965.
Kuntz W. and Schnieders B.: RNA metabolism and induction of extramicrosomal
enzymes during liver enlargement due to drugs. In : R. Eigenmann Ed.
Proc. 4th Inter. Cong. Pharmacol., Schwabe and Co. Basel Pubi.,
pp 326­339, 1969.
Miller J.J. and Weissmann D.B.: Correlations between transaminase concen­
trations and serum salicylate concentration in juvenile rheumatoid
arthritis. Arthritis Rheum., 19: 115­118, 1976.
Nievel J.G., Anderson J. and Bray P.J.: Biochemical changes in liver and
blood underlying parenchymal damage of rat liver induced by a dose­
dependent effect of drugs before and after development of liver
enlargement and nodular hyperplasia. Bioch. Soc. Trans., 4: 932­933,
1976.
Ohno H., Watanabe H., Kishihara C, Taniguchi N. and Takakuwa E.: Effect
of physical exercise on the activity of GOT isozyme in human plasma.
Tokohu J. exp. Med., 126: 371­376, 1978.
O'Kane D.E. and Gunsalus I.C .: The resolution and purification of glutamic
aspartic transaminase. J. biol. Chem., 170: 425, 1947.
Perry W., Jenkins M.V., Erooga M.A. and Stamp T.C.B.: Elevation of plasma
levels of lysosomal enzymes during treatment with rifampicin and
isoniazid. Bioch. Med., 20: 153­159, 1978.
Rej R., Fasce C.F. and Vanderlinde R.E.: Increased aspartate aminotrans­
ferase activity of serum after in vitro supplementation with pyrido­
xal phosphate. Clin. Chem., 19: 92­98, 1973.
Rosalki S.B. and Mbayoumi R.: Activation by pyridoxal 5'phosphate of aspar­
tate transaminase in serum of patients with heart and liver diseases.
Clin. Chim. Acta, 59: 357­360, 1975.
Saltzman D.A., Gall E.P. and Robinson S.F.: Aspirin ­ induced hepatic
dysfunction in a patient with adult rheumatoid arthritis. Digestive
Dis., 21: 815­820, 1976.
Schiele F., Neuman J.L., Le Perron Β., Galteau M.M. and Siest G.: Plasma
enzyme reference intervals. Influence of medications taken on a long­
term basis. In : G. Siest and D.S. Young Eds. Drug measurement and
drug effects in laboratory health Science, Karger Basel Pubi.,
pp 185­193, 1980.
Schmidt F.W.: Use of enzyme determinations in liver disease diagnosis. In :
L.M. Demers and L.H. Shaw Eds., Evaluation of liver function, Urban
and Schwarzenberg, Baltimore, pp 51­77, 1978.
Siest G., Schiele F., Galteau M.M., Panek E., Steinmetz J., Fagnani F. and
Gueguen R.: Aspartate aminotransferase and alanine aminotransferase
activities in plasma statistical distributions, individual varia­
tions and reference values. Clin. Chem., 21: 1077­1087, 1975.
Spyridis P., Sinaniotis C .,· Papadea I., Oreopoulos L.,'Hadjiyiannis S. and
Papadatos C .: Isoniazid liver injury during chemoprophylaxis in
children. Arch. Dis. Childhood., 54: 65­67, 1979.
 169

Steinmetz J. and Gaspart E.: Pharmacological effects due to hypolipidemic

drugs. In : G. Siest Ed., The use of laboratory tests results.
Variations due to drug-intake, Nijhoff, The Hague Pubi., in press,
1980.
Ury A.C. and Chassy J.R.: Increased activity of serum aspartate amino-
transferase in the presence of added pyridoxal 5' phosphate. Clin.
Chem., 19: 140, 1973.
Wilkinson J.H.: The principles and practice of diagnostic enzymology.
E. Arnold Pubi., pp 87-95, 1976.
Young D.S., Pestaner L.C. and Gibberman V.: Effects of drugs on clinical
laboratory tests. Clin. Chem., 21: 1D-432D, 1975.
170
ON THE INFLUENCE OF PHENYLHYDRAZINE AND RELATED COMPOUNDS
ON THE ACTIVITY OF ENZYMES

D. W. Scheuch
Department of Pathological Biochemistry
Medical Academy Dresden, G. D. R.

ABSTRACT
In model experiments with erythrocytes, hemolysates,
crystalline enzymes, the mechanism of the hemolysing action
of Phenylhydrazine has been investigated and a new scheme of
the interaction of Phenylhydrazine with glutathione and SH-
enzymes in red blood cells has been developed.

INTRODUCTION
It is well known that injection of phenylhydrazinechlo-
ride (10 mg/kg body weight) causes within a week severe ane-
mia with nearly 100 % reticulocytes. Oxyhemoglobin is de-
stroyed by Phenylhydrazine and ultimately results in the de-
naturation of globin to a green pigment and hemin (Warburg,
0. et al. 1931; Beaven, G. H. et al. 1954). In this process,
glutathione is involved which protects hemoglobin and is
oxidized by Phenylhydrazine to form mixed disulfides with
hemoglobin socalled "Heinz Bodies" (Jandl, J. H. et al.1960;
Allen, D. M. et al. 1961). We were interested in obtaining
insight into the mechanism of Phenylhydrazine action on the
metabolism responsible for destroying the erythrocytes.
RESULTS
Incubation of washed rabbit blood cells with phenyl-
hydrazine (3 . 10 mol/1) for 10 hours, 37 °C in a Warburg
apparatus with air as the gas-phase, induced a pronounced
decrease in the activity of hexokinase (HK), glucose-6-phos-
phate dehydrogenase (G-6-PD), NADPH-glutathione oxidoreduc-
tase (GSSG-RI), and phosphoglycerinaldehyde-NAD-oxidoreduc-
tase (TPD) (Fig. 1 ) . We found parallel to the decrease of
enzyme activities a decrease of intracellular concentration
of glutathione (GSH). Addition of glucose (1,5 · 10" mol/1)
protected GSH partially as well ae the enzymes, while addi-
 171

Activity

¡.Ο­

ΙΟ

10

Z2 ^
^ ν^77λ
esH HK Θ­6Ρ0 6SS6­M TPD

Fig. 1 ­ Action Of Phenylhydrazine On Enzymes

Π Control %■ Phenylhydrazine

tion of lactate had no effect (Scheuch, D. et al. 1961)· In­

cubation of intact red blood cells with hydroxylamine (5 ·
10 rcol/1) or methyleneblue caused a decrease in GSK, as
well as in the activity of PP­ase and hexokinase. Glucose
protected GSH and the enzymes against hydroxylamine activity.
By addition of cysteine to the test­tube, a certain reactiva­
tion could be demonstrated. These experiments could be inter­
preted as inactivation of enzymes caused by oxidation of GSH
to GSSG with consecutive formation of mixed ESSG­disulfide.
With glucose as substrate GSSG could be partially reduced to
GSH by way of NADPH­GSSG­oxidoreductase. The NADPH necessary
is produced via the direct oxidative glucose shunt by G­6­PD
and 6­PGAD (Scheuch, D. 1963).
In the following experiments we investigated the direct
influence of GSSG on these enzymes. PP­ase was inactivated by
GSSG (5 . 10~ 3 mol/1) (Pig. 2 ) . There was no difference bet­
ween aerobic and anaerobic conditions. But in testing of the
reversibility with cysteine we found complete restoration of
activity that was lost under anaerobic incubation times up to
4 hours. After an incubation time of 12 hours under aerobic
conditions there was no detectable reactivation but under an­
172

Activity

• anaenò

Pig. 2 - Inactivation Of PP-ase By GSSG

Upper Linea: Reactivation By Cysteine
aerobic conditions we found a considerable reactivation even
up to 25 hours. The inactivation depends strongly on the
temperature. The inactivation begins above 35 C. There is a
significant decline of PP-ase activity even with GSSG-con-
centrations of 1 . 10 mol/1. Increased GSSG-concentrations
causes concomitant increases of inactivation. The strongest
inactivation could be seen above a pH-value of 8.0 (Pig. 3)·

3.0 pH

Pig. 3 - Influence Of pH On Inactivation Of Hexokinase And

TPD
 173
This finding suggests that the formation of mixed disulfide
is favoured in the existence of the anion­form of SH­group
(pK 8.4)· The inactivation is strongly depressed in the pre­
sence of substrates or co­substrates (co­enzymes). Further
TPD is significantly protected against GSSG in presence of
NAD and NADH (Pig. 4 ) . Inactivation of PP­ase by GSSG is pre­
2+
vented by Mg which forms with pyrophosphate the true sub­
strate of the enzyme. G­6­PD is also protected by co­substra­
tes against the action of GSSG (Scheuch, D. 1962).
Pig. 4 ­ Influence of GSSG on PP­ase and TPD of stromafree
Hemolysate (67 h)
Κ I II III IV V VI
Incubât ion
(M Õ 4 4 4 4 4 4
GSSG ­ ­ + + + + +
NADH ­ ­ ­ + ­ ­
NAD ­ ­ ­ ­ + ­
NADP ­ ­ ­ ­ ­ + +
G­6­P ­ ­ ­ ­ ­ +
PP­ase 221.2 23.1 0.3 0.3 0.3 0.3 23.5
TPD 31 .0 14.0 0.5 15.0 27.0 22.0 19.0
Finalconcentrations:
GSSG = 2.5 x 10"3mol/l; NADH = 2.5 x 10~ 4 mol/1;
NAD = 2.5 x 10"4mol/l; NADP = 2.2 χ 1 0 ­ 4 mol/1;
G­6­P = 5 x 10"3mol/l.

The following conclusions could be drawn: GSSG oxidized

from GSH by action of Phenylhydrazine inactivates SH­enzymes.
The extent of inactivation strongly depends on the existing
conditions such as concentration, temperature, pH and the
presence of substrates and co­substrates. The experiments
with crystallized enzynes shown in the following data demon­
strate that there is also a direct inactivation by phenylhy­
drazinu. This inactivation is more pronounced in presence of
O­, and at more alkaline pH values (Pig. 5) and with increa­
sing temperature. Inhibition could be reversed by addition
of cysteine which restored up to 50 % of the control tube
activity. After havinp proved a direct action of phenylhy­
174

Pig. 5 - Inactivation Of Crystalline TPD By Phenylhydrazine

drazine as well as action of intermediary-originated GSSG on

the SH-enzymes, we tried to test our hypothesis on a step-
wise completed system. We used the stroma-fixed ATP-ase of
erythrocytes. By washing the stroma 20 times with phosphate
buffer (pH 7.8), the supernatant which includes the hemoglo-
bin could be easily discarded. While the addition of phenyl-
hydrazine or Phenylhydrazine plus dialyzed stroma-free hemo-
lysate showed only a small inactivation, the action of Phe-
nylhydrazine in presence of GSH caused a decrease in ATP-ase
activity of about 50 %. Complementing the entire system with
SPH increased the inactivation up to 90 % (Fip.. 6 ) .
 175

PH PH PH
♦GSH ♦Hb
•GSH

t)­·

5­

1
Fip. 6 ­ Inact ivation Of ATP­aae By Phenylhydrazine With Or
Without Additions '777>{ N /C0
:^o„ ¡Π 2
CONCLUSIONS
Sumuarizing our findings we propose the following scheme
of the action of Phenylhydrazine on erythrocytes (Pig. 7 ) .

Hb denot.
Pig. 7 ­ Action Of Phenylhydrazine On Hemoglobin, GSH And
SH­Enzymes
176
By inactivation of the SH­enzytnes of the glycolytic pathway,
there must be, consequently, a decrease in intracellular ATP
concentration with consecutive hemolysis. This decrease of
ATP is shown in Pig. 8.

ATP

»without HA

■»with HA

Pig. 8 ­ Influence Of Hydroxylamine On ATP Concentration In

Erythrocytes

We think the self­regulating system of GSH and SH­enzymes is

most important in modifying the action of certain drugs and
­ toxic substances on the structure and function of red blood
cells (Scheuch, D. W. 198υ). This could be demonstrated in
^case of inborn errors of metabolism in the pathway of GSSG­
reduction (Scheuch, D. 1964).
 177
REFERENCES
Allen, D. \ . and Jandl, J. K.: Oxidative Hemolysis and Pre­
cipitation of hemoglobin. II. Role of Thiols in oxidant
Drug Action. J. Clin. Invest. 40: 454 ­ 475, 1y6l .
Beaven, G. H. and Wnite, J. C: Oxidation or Phenylhydra­
zines in the Presence of Oxynaemoglobin and the Origin
of Heinz Bodies in Erythrocytes, mature 17J: 38y ­ 3^1,
1954.
Jandl, J. H., Engle, L. K. and Allen, D. Vi.: Oxidative Hemo­
lysis and Precipitation of Hemoglobin. I. Heinz Body
Anemias as an Acceleration of Red C ell Aging. J. Clin.
Invest. 39: 1o13 ­ 1636, 1960.
Scheuch, D., Kahrig, C, Ockel, Ξ., Yagenknecnt, C. and
Rapoport, S. M.: The Role of Glutathione and of a Self­
stabilizing C hain of SH­Enzyr.es and Substrates in the
Metabolic Regulation of Erythrocytes. Nature 190:
631 ­ 632, 1961.
Scheuch, D. und S. Rapoport: Das Verhalten von anorganischer
Pyrophosphatase, Triosephosphatdehydrogenase und Glu­
kose­6­phosphatdehydrogenase bei Oxydation ihrer essen­
tiellen SH­Gruppen. Acta biol. med. germ. d: 31 ­ 4 1 ,
1962.
Scheuch, D.: Inaktivierung und Schutz von SH­Enzymen bei
Oxydation des Glutathion in intakten Zellen. Acta biol.
med. germ. Suppl. III, 294 ­ 295, 1963.
Scheuch, D.: Untersuchungen an Erythrozyten eines Glukose­
6­Phosphat­NADP­0xydoreduktase­Defektes. Folia hämatol.
82: 76 ­ 85, 1964.
Scheuch, D. V/.: Zur Durchführung von Modelluntersuchungen
über Seiteneffektwirkungen von Arzneimitteln oder ande­
ren biologisch aktiven Stoffen. Z. med. Labor­Diagn.
21: 1980 in the press.
Warburg, 0., Kubowitz, P. und Christian, W.: Über die Wir­
kung von Phenylhydrazin und Phenylhydroxylarcin auf den
Stoffwechsel der roten Blutzellen. Biochera. Zschr.
242: 170 ­ 205, 1931.
LABORATORY TESTS AS INDIRECT INDICATORS OF THE ACTIVITY OF DRUG METABOLIZING
ENZYMES : USE OF GLUCARIC ACID AND OF GAMMA-GLUTAMYLTRANSFERASE

A.M. Batt and G. Siest

Laboratoire de Biochimie Pharmacologique, ERA CNRS 698

Faculté des Sciences Pharmaceutiques et Biologiques

7, rue A. Lebrun Nancy FRANCE

ABSTRACT
Laboratory tests are increasingly important in the surveillance of
drug treatments. People in charge of clinical laboratory science must now
become aware of a new development,·the use of some tests as indicators of
the activity of drug-metabolizing enzymes, especially of the induction of
such enzymes. Ideally', the activity of such enzymes should be measured in
the tissues. In man, however, easier indirect methods are now most often
used : measurement of antipyrine half-life, urinary and plasma drug metabo-
lites, specific protein and enzyme levels in plasma, changes in endogenous
substrates and endogenous metabolites, and enzymes in circulating blood
cells. Of all these, two examinations are now widely performed, those for
urinary glucaric acid and plasma gamma-glutamyltransferase. The value and
limitations of these two tests are discussed.

INTRODUCTION
To improve drug efficacy and safety, it has been necessary to develop
clinical methods to establish the individual pattern of drug metabolizing
enzymes in patients and also their inducibility.
Let us first consider what the implications of induction are in clini-
cal medicine to day. There are examples where enzymes induction leads to
1. The loss of efficacy (detoxification)
2. The increase of toxicity (toxification)
3. Cancerogenicity
4. Substrate deficiency
5. Therapeutic mechanism
Many drugs and environmental xenobiotics can activate or inhibit en-
zymes, including those enzymes which convert them. These enzymes are dis-
tributed in various tissues (mainly the liver, kidney, intestine, lungs
and skin) and in various cell compartments. A large group being found in
the endoplasmic reticulum, but some of them belonging to the plasma membra-
nes, or to the soluble, cytoplasmic fraction of the cells.
 179

It is not yet possible to predict the behavior of a new molecule in

advance... The following general principlescan be accepted.
. Induction implies a real increase in the amounts of enzymatic proteins
. The amplitude of the induction effect depends on the solubility of the
inducer, the most liposoluble compounds being the most efficient inducers
. The amplitude of the induction also depends on the half-life of the sub-
stance in the organisms - the longer the half-life, the greater the ampli-
tude of the induction
. In animals three main groups of inducers have been described from their
analogy with typical inducers
- phénobarbital
- polycyclic hydrocarbons (3-methylcholanthrene)
- steroids
. Induction of drug metabolizing enzymes requires, relatively high doses of
a compound, and is, with a very few exceptions, correlated with other un-
wanted effects.
. The selection process designed to choose new drugs in human therapy try
to select drugs void of toxic reactions. Such a process removes most enzy-
me inducing drugs.
Therefore we should restrain us from over-emphazising enzyme induction du-
ring therapy. However, this should not prevent us to perform studies with
the aim to elucidate the impact of chemical inducers specially in human
beings. Two main ways of estimating the induction in human are proposed.
. Direct methods determining the activity of the enzymes involved in drug
metabolism in biopsies samples are relatively easy to apply to animals,
but practically and ethicaly extremely limited in humans. They are required
to assess the mechanism of a true induction.
. Indirect methods based on the study of endogenous or exogenous metaboli-
tes in plasma, urine and expired air or on changes in the levels of macro-
molecules in plasma are recommended. The significance of observed changes
must be interpreted with the knowledge of non-induction sources of varia-
tions.
1. Half-life of antipyrine
Determination of the decay rate in plasma or saliva of a single dose of
this substance which is easy to measure is a very useful test for evalua-
ting the hepatic conversion capacity in humans.
2. CO., expired in breath after N-demethylation of labelled C-aminopyriie
This allows the in vivo estimation of the maximal rate of demethylation
180

activity and gives results in a short period of time.

3. Determination of drug metabolites in urine or plasma
Modern chromatographic methods coupled with mass spectrometry allow one
to analyze the various stages of drug conversion and to assess the contri-
bution of the various enzymes responsible for the metabolism studied.
4. Determination of the levels of specific proteins and of enzymes in
blood plasma
The induction effect is more or less specific for protein synthesis.
In addition to the endoplasmic reticulum enzymes involved in drug metabo-
lism, there is often also an increase in a great number of other proteins,
some of which are normally found in the plasma—fcr example, ceruloplasmin,
gammaglutamyltransferase and lecithin-cholesterol-acyltransferase.
5. Determination of enzymes in circulating blood cells
Biochemists and pharmacologists do not make enough use of the circula-
ting blood cells, which can provide interesting information. Some of the
enzymes contained in them are subject to regulatoiy mechanisms similar to
those in the liver—for example, delta-amino levulinate synthetase in
erythrocytes and hydroxylases in lymphocytes.

6. Determination of changes in substrates or endogenous metabolite levels

The drug induced effects on enzymatic systems are reflected in the
levels of certain blood constituents, now routinely determined in the labo-
ratory, bilirubin, which is decreased by the induction of UDP-glucuronosyl-
transferase or ones that are more difficult to measure, ß-hydroxycortisol
or glucaric acid.
This paper will be restricted to two assays routinely performed in
pharmacological biochemistry laboratories : the estimation of glucaric
acid in urine and the measurement of gammaglutamyltransferase activity in
plasma.
The purposes of those two laboratory tests are :
- screening of new enzyme inducing drugs
- indicating use of drugs when not otherwise demonstrable
- detection of non compliant patients supposed to take knowA inducers
- indicators of absorption of enzyme inducing drugs into circulation
- monitoring individual variations (genetic) in the rate of drug metabolism
- detection of liver dysfunction
- detection of changes in glucuronidation or hydroxylation of drugs.
 181

GLUCARIC ACID ELIMINATION

Glucaric acid is produced in the liver during the metabolism of glucu-

ronic acid. The biosynthesis of glucaric acid (Burns and Connery 1966 ;

Aarts 1968 ; Hanninen 1968 ; Miettinen 1970 ; Marselos 1976 ; Khodjet El

D Glucose

Glucose hexoKlnase I G-6-Pase / PentosesV

I.' . . . ' PhosDhate »
^Galactose t» Glucose-fi-Phosphate
•Phosphate „ ». shunt
n-Hylulose-5-P

UDP-Galactose ^ - u c o s e — niycoqen

ϋΡΓ,-DH

UDP­GLUCURONIC ACID
Rillrubin
>^ Accepte
Acceptor ­ Steroids
h­Oh

UOP Ν.

G l u c u r o n i c a d d 1 P. B­Glucuronides
H
Phospha taseXy^O 2°v/flrl
^ ? \ \S B ­ G l u c u r o nl d a s e
^ >v Y \\cceptor
­ Mucopoly­ ^ GLUCURONIC
ΓΛ uriiDnMTQ ACID *
saccharides
Myoinositol 4
Uronolactonase ucuronate reductase

D­Glucuronolactone ulonlc add

GL DH

L­Gulonolactone L­Xylulose ' »­Xyl 1101—"

□ Glu aroM 4) L­Xylulose

tB 3) d l l a c t o n e reductase

Hydrolysis

D Glucaric a d d

Figure 1: D­glucuronic acid pathway in the human liver.

182

Khil 1976) includes two major steps, the synthesis of free glucuronic acid
followed by its conversion into terminal metabolites, especially glucaric
acid. Free glucuronic acid can be converted via three different metabolic
pathways, in which we are interested here, depends first on the action of
a D-glucurono-delta-lactone hydrolase. This enzyme is found both in micro-
somes and in the cytoplasm of hepatocytes and leads to the formation of
D-glucuronolactone. This compound in then oxidized into 1,4-saccharo-6,3-
dilactone by an irreversible process that requires the presence of NAD and
is catalyzed by a soluble enzyme already identified as D-glucuronolactone
NAD oxidoreductase. The dilactone produced hydrolyzes spontaneously into
two monolactones, 1,4-saccharo-lactone and 6,3-saccharolactone, which can
both convert into glucaric acid without the intervention of any enzyme.

All the methods for assaying glucaric acid are delicate. The techni-
ques most often used are based on the inhibition of beta-glucuronidase by
1,4-glucuronolactone obtained by heating glucaric acid in an acid medium.
Chemical and gas-chromatographic methods have also been developed.
Reference values obtained by these various methods are quite different
since besides the problems of accuracy and precision, physiological varia-
tions are not yet well understood.
TABLE 1 - GLUCARIC ACID ELIMINATION IN HEALTHY HUMANS (see Marselos 1976)

Inhibition methods (ymoles per 24 hours)

MARSH and CARR, 1965 53,0-10,0 men

4 1 , 0 - 8,0 women
AARTS, 1968 30,9-27,1
MOWAT, 1968 40,0-75,0
HUNTER et al., 1971 12,4
MARCH et al., 1974 98,0
SIMMONS et al., 1974 39,0-17,9 men
4 1 , 0 - 8,0 women
SMITH and RAWLINS, 1974 15,0- 6,0
SOTANIEMI et al., 1974 13,9- 4,9
HILDEBRANDT et al., 1975 32,5 - 4,9 men

Chromatographic method

GANGOLLI et al., 1974 33,4 - 13,0

 183
For example the amount of glucaric acid in urine varies with the person's
age and degree of fasting (see Marselos, 1976), increases during pregnancy
(Davis et al., 1973) and in pathological conditions such as diabetes, hy­
perthyroidism and liver diseases (Herzberg,1978) hepatites (Sorrell et al.,
1976 ; Cerella et al., 1978) and cirrhosis (Hezey, 1976 ; Escortin Morin
et al., 1976) and rheumatic diseases (also see Marselos, 1976). Only when
the physiological or pathological states are very well defined can the
effect of enzyme inducing drugs be assessed. Glucaric acid levels are usu­
ally high in man after administration of barbiturates and also after admi­
nistration of other drugs.
TABLE II ­ EFFECT OF DRUGS ON GLUCARIC ACID ELIMINATION

References Drugs

HUNTER et al., 1971 Anticonvulsants χ SO

HUNTER et al., 1971 Phetharbital χ 8,3
SOTANIEMI et al., 1974 Associated drugs χ 2
DAVIDSON et al., 1974 Anticonvulsants χ 8,6
CUNNINGHAM et PRICE­EVANS, 1974 Phenytoin χ 3,1
TALAFANT et al., 1975 Phénobarbital χ 1,7
HILDEBRANDT et al., Phénobarbital χ 6,0
Clemastine
LATHAM et al., 1976 Phénobarbital (240mg/j) χ 10
ROBERTS et al., 1976 Glutethinimide χ 5
FOLIOT, 1977 Clofibrate χ 3,1
HERZBERG et al., 1977 α­methyldopa χ 1,8

Among hospitalized patients (Sotaniemi et al., 1974) an increase was also

observed. Some unaltered results have been mentioned, specially after oral
contraceptives (Davis et al., 1973) or during pregnancy (Herzberg et al.,
1977) or tolbutamide or ethosuccimide (Gilbert et al., 1974), sodium valproa­
te among anticonvulsants and disopyramide (Rycroft, 1976). Decreases have
been observed in congestive heart failure (Tokola et al., 1975).
Applications to other series of drugs were developed in this laboratory in
order to better define the relation between the increase in glucaric acid
and the mechanism of induction of drug­metabolizing enzymes (Khodjet El
Khil, 1976). (Table III).
TABLE III : GLUCURONIC ACID PATHW AY ENZYMES A C T I V I T I E S I N RAT LIVER %

UDP glucose dehydrogenase UDP glucuronyltransferase UDPCA pyrophosphatase Glucuronolactone detydrogenase Glucaric Acid
TREATMENT timóles NAD X ran"' nmoles p.nilrophe ol χ rain-1 nmoles Pi χ min ' nmoles NAD X ran-' 'moles χ uH creatinine
-1
g" liver 7ig~l protein g"1 liver ι; protein g liver mg protein g liver mg protein

Control (8) o7 728.5 * 68.3 9.50 S 1.51 166.2 t 23.3 5.60 : 0.81 566.0 ; 46.1 14.07 ± 1 . 1 0 503 1 B9 6.55! I.I 21.6 t 2.2
Phénobarbital ( 8 ) 913.5 t 34.1 10.83 : 0.36 311.3 1 25.1 7.15 I 1.07 450.2 ! 30.8 8.19 1 1.03 1146 t 245 13.50 i 2.7 39.3 1 1.5
100 mg/kg 6/tt tt tt ttt ttt
i . p . 5 jours

Control ( 5 ) 480.0 ! 95.9 7.14 i 1.51 123.2 1 35.9 '.30 J 0.83 334.6 i 31 5.02! 0.6
Diphenylhydantoin (5) 544.0 : 48.8 7.86 ! 1.70 189.0 ! 38.4 -.58 ! 0.83 515.2 i 71 7.46! 1.06
100 mg/kg t tt tt
i . p . 5 'jours

Control ( 5 ) 508.0 2 163.2 6.58 t 2.44 154.1 t 62.3 ..13 s 1.26 442.3 : 140.4 11.13 ! 2.76 678.6 ± 163.4 7.94 1 23.5 15.6 t 3.5
C l o f i b r a t e (5) 596.0 ! 48.8 7.63 t 0.61 187.4 ! 21.5 '.82 ! 0.07 356.3 ! 99.6 9.26 t 1.92 1184.0 t 104.2 14.34 ! 1.26 34.7 ! 7.6

200 rag/kg ttt ttt

Control ( 5 ) 821.9 ! 72.9 12.96 í 2.43 219.1 t 31 .9 ..16 S 0.89 789.1 4 74.7 22.21 i 2.27 230.3: 94.9 3.71 ! 1.40 27.4 ! 10.9
Procetophene (5) 428.0 1 130.9 7.31 i 2.23 194.8 i 14.4 '.55 1 0.24 681.4 ! 46.7 19.48 t 1.90 369.7 t 132.9 6.21 1 1.85 31.7 ! 15.8
10 rog/kg
'tt tt t

Control ( 5 ) 587.5 i 156.6 7.64 i 2 . 1 3 94.2 S 17.3 ■40 1 0.95 385.4 ! 2.74 11.24 i 3.35 136.7! 24.8 1.77! 0.34 22.1 ! 11.2
Procetophene (5) 244.2 ! 68.4 2.74 t 0.49 72.5 i 32.6 ■29 i I.Ol 106.6 ! 3 3 . 1 4.95 ± 0.73 639.7 t 78.1 7.06 t 0.34 35.9 ! 13.5
100 mg/kg
tt tt t tt ttt ttt

o/ Number of animals i s given in parenthesis

b/ Significance was calculated with Student's t e s t
* : ρ < 0,05 ; ■" : ρ < 0,01 ; +++ : ρ < 0,001
 185

Diphenylhydantoin effect seems Co resemble phénobarbital, but with weaker

magnitude for the enzymes studied here. Procetophene is characterized by an
inhibiting effect on UDP glucose dehydrogenase, UDP glucuronyltransferase
and UDP glucuronic acid pyrophosphatase. Clofibrate at the dose studied
here is acting more like phénobarbital than like procetophene and may be
classified among inducers of microsomal enzymes.
The four drugs studied are acting the same way on UDPGA PPase which is de-
creased and on glucuronolactone dehydrogenase which is increased. Clofibra-
te and procetophene are both inducing the D-glucaric acid elimination.
From the decreases obtained with procetophene in UDP glucuronic acid pyro-
phosphatase and UDP glucose dehydrogenase it becomes obvious that these
enzymes are not involved in glucaric acid increased excretion. The only en-
zyme involved in this increase appears to be D-glucuronolactone dehydroge-
nase. This is in good agreement with reports by Marselos (1976).
The glucaric acid elimination seems to depend upon the regulation of that
enzyme, not involved in drug metabolism, rather than of enzymes involved in
drug metabolism.

Glucaric acid would depend on an enzyme present in the cytosolic fraction

and not in the endoplasmic reticulum. Since the specificity of this in-
duction in unknown at the moment, it is hazardeous to consider D-glucaric
acid alone as a direct index for induction of the whole glucuronic acid
pathway.
CAMMA-CLUTAMYLTRANSFERASE ACTIVITY
Gamma-glutamyltransferase (GGT) catalyzes the transfer of gamma- glu-
tamyl groups from gamma-glutamyl peptides to other peptides, L-amino acids
or water (Meister, 1978). In the liver, the enzyme has been found mainly
at the surface of the epithelial cells and also in bile ducts. Finally, it
has just recently been demonstrated in the rat that gamma-glutamyltransfe-
rase is much more abundant in nonparenchymatous cells (Kupffer cells) than
in hepatocytes. The subcellular location of this enzyme in the liver is
now established. Some is found in the soluble phase and some in the membra-
ne fraction (plasma membrane). The enzyme is also present in many human
physiological fluids, particularly seminal fluid, bile, urine and blood
plasma. In some animals, such as the rat, the plasma activity is very low
(Bagrel et al., 1978).

It must be remembered that the goal is to measure a plasma protein as

an indicator of a tissue modification, and that the enzyme activity is used
only to follow this protein. Furthermore, the enzyme exists in several mo-
186

lecular forre and in the tissues one part is bound to membrane structures.
The method that is currently most often used to measure this activity is
based on the transfer of a glutamyl residue from a synthetic substrate,
gamma-glutamylparanitroanilide, onto an acceptor, usually glycylglycine
(Szasz, 1976).
The GGT activity in human liver is to some extent elevated during va-
rious hepatic disorders (Schmidt, 1978). It seems that the fetal form is
often found in carcinomas (Sawabu et al., 1978). Experimentally, it is re-
latively easy to provoke an increase of GGT activity in rat liver by liga-
ture of the bile duct. Other types of cholestasis can also increase the ac-
tivity of this enzyme. It is important to recognize this effect before in-
terpreting a drug's enzyme inducing effect, since drugs often stimulate bile
secretion (Chivrac et al., 1978 ; Ohnhaus, 1979).
The activity in plasma is usually low and its distribution in an unse-
lected population is not Gaussian. In humans, the plasma GGT activity falls
from birth, until 3 years old, then rises slowly until forty or fifty years
of age. It is much lower in women than in men and depends greatly on the
amount of fat and of overweight (Schiele et al., 1977). Alcool consumption
is an important factor in increasing the activity.
When a population is selected to eliminate the main variation factors,
a much narrower distribution of the activity of this enzyme is obtained. It
is therefore necessary to choose carefully the individuals who constitute
the reference sample for plasma GGT, since in unselected population many
persons show high activities. Thus among men 40 to 60 years old, the 97.5
percentile is at 200 U/l, whereas it is at 65 U/l in the reference sample
of subjects of that age and sex (Figure 2 ) .

Phénobarbital or other drugs like oral contraceptives can act on the

concentration of the enzyme, or on the enzyme's cellular or subcellular
localization, or on the stability of membrane structures (Weinberg et al.,
1974 ; Siest et al., 1974).(See also Bagrel et al., 1978, for a review of
drug). Among this drugs, hypnotic, antianginal, antidepressive drugs have
been shown to increase the plasma GGT activity,at least the mean value,
and hypolipiemic drugs may decrease it (Schiele et al., 1979) (Figure 3 ) .

Unaltered results sometimes come from the fact that this increase is
small and therefore difficult to measure in liver homogenates or in micro-
somes from certain animal species, such as the rat. This is chiefly becau-
se GGT is located mainly in the cell membranes. The administration of phe-
 187

WOMEN

40­60
years
HL
♦
20­40
years
Efi réf
tol

MEN
♦
40­60 II 1 I I I ­
years 1 | tot
οι ι ι
4
20­40
years llll 1 1 1 ·*' | to.
M♦i l
0 50 100 150 200 °° T m / , )
Figure 2. D i s t r i b u t i o n of the plasma GGT a c t i v i t y i n an unselected popula­
t i o n in Nancy (France) ( t o t ) and i n a r e f e r e n c e population define by age
and sex ( r e f ) .

I
VModileior j Ζ Vnodiljior C
ι
Hypollptfmc ¡ I
VMocontttKlor
An*tgnic
I
¡ 1
3 VMoconunctor
AnaiotMC
C
] i
AWwtipflMêlH • Anttdapfttunt
Anilonçinol ι ι Antenori*! [
TrenquillMT · ι Tranqu*lu«r D;
Anttoout 1 I
Anticoagulant
AnlWUjtMtlC ¡
ι
AnilrtvotrttmJon ¡ , Ι
Cwdtolonie ¡ Cardiotonic

Hypnotic ι • Ι Hypnotic

Antfconvulunl ι ι i 200
Orii contraceptiv·
120­30 yMnl
¡
ι φ" ξ° Ao» 1 2 0 ­ 3 0 to
ι
• lima kt 120­30 v—ni ramam 120­30 rmnì

­» ­β η » ­20 ­ β Χ) 20
ΟΟΤΙι GGTnita

Figure 3. Effect of long term drug intake on GGT activities in males and
females 40­60 years old as a percentage of the median.
188

nobarbital increases the GGT activity much more in the plasma membranes
than in the microsomal fraction (Tazi et al., 1979).
The effect of inducers other than phénobarbital is less clearly esta-
blished. It seems that they operate not only in the liver, but also under
certain conditions in other organs, particularly the kidney and the intes-
tine. However, we have not found any significant changes in certain circu-
lating blood cells, such as granulocytes (Tarallo, 1978).
The GGT activity is present in hepatocytes and in non-parenchymal
cells isolated from control or phenobarbital-treated rats (Galteau et al.,
in press). The percentages of some other membrane enzymes in the cytoplas-
mic fraction were higher after the administration of inducers.
Chlorpromazine, like deoxycholate, can solubilize GGT from microsomes
and from plasma membranes ; the solubilization is easier when the membrane
preparations" are obtained from phenobarbital-treated animals. Thus phéno-
barbital does not act solely by increasing protein synthesis ; as is now
well known, it also alters the flow and secretion of bile and the lipid
composition of membranes. A more recent study of solubilization by litho-
cholate, deoxycholate, and cholate shows that the dihydroxy bile salts
solubilize membrane enzymes more easily than the trihydroxy salts do (Tazi
et al.,1979). It is important to be aware of this result, because the rela-
tive proportions of the various bile salts can also vary under the influ-
ence of inducing drugs.

In interpreting the value, it is necessary also to refer to various

pathological conditions. In an hospital department specialized in hepatolo-
gy GGT is the enzyme that is most often found to be perturbed. GGT is the-
refore not a good, candidate for detection of enzyme induction in such con-
ditions.
When the main factors of variation are eliminated from a population,the
distribution obtained for the activity of this enzyme is much narrower. In
a study on plasma GGT, it is therefore necessary to choose carefully the
individuals who are included in the reference sample.
In conclusion, a slight increase of plasma GGT can be interpreted as
a sign of an enzyme induction if any of the followings are true: An increa-
se greater than 20 % has been observed in any one subject or in an homoge-
neous group of subjects ; other causes of physiological, pharmacological
and pathological variation have been eliminated ; when other indirect in-
dices of induction such as urine glucaric acid, are also positive.
A summarizing table for the clinical chemist and the clinician is also
 189
useful in interpreting the results. (Figure A) .
W

Liver tumoo
SSO BUuy a n h o *
600 ObKractm janadfc·
500 Drag kteraa
400 Alcohol toxic drraoak
300 Alcohol toxic »epatiti·
Chronic K t m bepatitia
200 Vini hepatita
Myocardial Infarction dlabtta
150 Non apedfkreactivehepatite
Rhranataanal diaman
Infecţiei» mooooudeod·
120 Fatty Ihcr · Chronic pcnbtcal bepuiti·

105 Ownwifht \
80 Indnrnii ånt> " " * *» phninbareltal l ful
. 70 | Values
(peroraţiei 95) ♦ 40
f Reference ¡attrai (perccntika 95) 1
Maka 20­30 yean old ! . II 1 Femalei 20­30 yean old 1
7
4 Druga inch aa hypoiponka |

2
0 Deficiency of GGT activity (labora cuor of metaboasn)

Figure 4 : Physiological and pathological variations of plasma GGT.

CONCLUSION
In conclusion,we think that determining uric glucaric acid and plasma
ganrna­glutamyltransferase are potentially valuable for detecting the enzyme
inducing effects of new drugs for therapy and or experimentation on animals.
Furthermore, we believe that in the near future, these two tests in all cli­
nical chemistry laboratories will prove useful for following the course
of therapy with drug combinations that include at least one enzyme­inducing
drug (especially when the expected therapeutic results are not obtained).

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Cerella, M., D'Arienzo, Α., Manzillo, G. and De Ritis, F., 1976. An eleva­
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190

Chivrac, D., Dumont, M. and Erlinger, S., 1978. Lack of parallelism between
microsomal enzyme induction and phenobarbital­induced hypercholeresis
in the rat. Disgestion, 17 : 516.
Cunningham, J.L. and Price­Evans, D.A., 1974. Urinary D­glucaric acid ex­
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 DRUG EFFECT ON VITAMIN LEVELS

J.-J. Himberg
Department of Clinical Pharmacology, University of Helsinki
and Finnish Red Cross Blood Transfusion Service
Helsinki 29, Finland

ABSTRACT
Presently available data of drug effects on vitamin levels has been
reviewed. Antiepileptics, oral contraceptives and cortiocosteroids have a
well documented effect on vitamin concentrations.Antiepileptics increase
serum vitamin A concentration and decrease concentrations of 25-(OH)-chole-
calciferol in serum, vitamin B.. in cerebrospinal fluid and folic acid in
serum, erythrocytes and cerebrospinal fluid. Oral contraceptives increase
concentration of vitamin A in serum and decrease concentrations of toco-
pherol and vitamin B._ in serum, folic acid in serum and erythrocytes, and
ascorbic acid in leuKocytes. Prednisone decreases 1-alpha, 25-(0H). chole-
calciferol concentration in serum. Because the vitamin concentrations also
depend on other factors like 'diet, the lack of controlled studies with
modern analytical methods is evident.

The vitamins are essential exogenous micronutrients for the proper

physiological function of the body. Their mechanisms of action vary,
sometimes they are precursors of cofactors in enzymatic reactions, some-
times their metabolites function like hormones (for reviews see Kutsky,
1973; Körner and Völlm, 1976). Hypovitaminosis may develop because of
faults in absorption, transfer or utilization (Baker and Frank, 1968) and
hypervitaminosis because of excessive intake (DiPalma and Ritchie, 1977).
Drugs are foreign compounds often with unknown mechanism of action
and side effects. The body is trying to get rid of them and they are there-
fore burdens for the metabolism (for review see Goodman and Gilman, 1975).
The idea of drug-vitamin interactions has been used in the development
of antimetabolites. Most of the drug-vitamin interaction information on
other type8 of drugs comes usually first from the reports of effects .of a
drug on hematological or clinical chemistry parameters.
A systematic approach in the search of drug-vitamin interactions
mechanisms is first to collect the existing knowledge of drug effects on
vitamin levels. Already Young et al.(1975) in their extensive computer data
on drug effects on laboratory tests have some information which ends
194

October 1974. The purpose of this paper has been to collect the relevant
data and references presently available. The data has been organized ac-
cording to the vitamins by classical definitions (Kutsky, 1973) but some of
the water-soluble vitamins are not included because of lack of information
on interactions. The drugs which have by definition an effect on vitamins
(i.e. antimetabolites, anticoagulants) can be found in textbooks (Goodman
and Gilman, 1975) and the effects of ethanol or non-therapeutic drugs and
toxins are not included.
FAT-SOLUBLE VITAMINS
VITAMIN A
The compounds having vitamin A activity can be measured reliably with
chemical methods and they reflect the vitamin A status in man (Körner and
Völlm, 1976) . In the transportation of vitamin A in the blood the retinol
binding protein has an important function and some of the changes found
in vitamin A levels are correlated to the changes in the concentration of
the beta-lipoprotein (Smith and Goodman, 1976).
Curtis and Swicord (1976) surveyed one thousand mentally retarded
patients. They found that of the drugs used, only phénobarbital, diphenyl-
hydantoin and thioridazine as well as isosorbidedinitrate had a slight
increasing effect on plasma vitamin A levels.
The oral contraceptives were found to have a significant increasing
effect in three months on vitamin A plasma levels (Gal et al., 1971). In
the study the control group was matched by age and cyclus. The estrogens
were ethinylestradiol and mestranol, and the progestagenes were nore-
thisterone, lynesterol, ethynodioldiacetate and megestrolacetate. No toxic
vitamin A levels were found. Other studies confirm the results (Briggs and
Bennun, 1972) and the mechanism might be secondary to the increased beta-
lipoprotein levels in serum (Heilmann, 1979).
Perorally administered neomycin decreases serum vitamin A levels as
well as the levels of carotenes (Faloon, 1970) .
Cholestyramine decreases vitamin A levels in plasma at a dose of
0.6 g/kg/d during two years treatment in children with familial' hyper-
cholesterolemia (West et al., 1975).
VITAMIN D
During the last fifteen years the metabolism of vitamin D has been
largely discovered and the active metabolites have been found to be acting
like hormones (DeLuca, 1976). After having the methods for measuring the
 195

levels of active metabolites it has been possible to interpret the known

changes of calcium metabolism which have been found to occur during anti-
epileptic and corticosteroid therapy.
Since the first finding of bone changes during antiepileptic therapy
(Kruse, 1968) many reports have been published. However, the concentra-
tions of circulating forms of vitamin D depend also on many exogenous
factors like diet and intensity of exposure to sunlight. The radiological
findings in children are typical for rickets and in adult patients they
resemble osteoporosis. The decrease in 25-hydroxycholecalciferol (25-OHD.)
levels during anticonvulsant therapy has been reported for both adults and
children (Hahn et al., 1972; Hahn et al.,1975; Barragry et al., 1978;
Mosekilde et al., 1977; Stamp et al·., 1972; Tolman et al., 1975). These
changes were found with phénobarbital, diphenylhydantoin and primidone
alone or in combinations. Sumi et al.(1978) discovered that in children,
aged 10/12-16 years, taking phénobarbital 2-8 mg/kß/d, the 25-OHD.
levels in plasma increased significantly during the first month but in
six months they were on a lower level than before the treatment. When
phénobarbital or dipheylhydantoin were combined with other drugs no such
early increases were found and the levels were in few months significantly
lower than before the treatment. The Floridan children on antiepileptic
therapy and throughout the year exposure to sunlight had lower 25-OHD.
levels than the control group but higher levels than the normal values in
Pennsylvania (Weissman et al., 1979).
Corticosteroids inhibit the normal calcium absorption in intestinal
mucosa and they also effect the hormonal control of the absorption
(DeLuca, 1976; Lukert and Adams, 1976). One of the suggested mechanisms
has been the change in 25-OHD, production. Hahn et al. (1977) was not
able to demonstrate any change in 25-OHD. levels with a mean prednisone
dose 22 mg/d for five years compared to the control group, matched by
age, sex, race, vitamin D intake and sunlight exposure. Klein et al.
(1977) did not find a change in plasma 25-OHD. level with a low dose of
prednisone but higher doses decreased it significantly. 1-alpha,
25-Dihydroxycholecalciferol levels were decreased already with prednisone
doses of 0.2-2.0 mg/kg/d for at least a year in children between 5-16
years of age with glomerular disorder compared to matched control group
not receiving prednisone (Chesney et al., 1978).
Colestipol, a synthetic bile séquestrant polymer, decreased the
196

plasma 25­OHD, levels at doses 10­15 g/d during two years therapy of
children with familial hypercholesterolemia (Tsang et al., 1978).
VITAMIN Κ
The effect of drugs on vitamin Κ levels are seldom of clinical value
because the prothrombin status can be measured with other methods. Many
drugs change the prothrombin levels or interfere with the haemostasis in
other ways. For a review see standard textbooks (i.e. Goodman and Gilman,
1975).
VITAMIN E
The oral contraceptives decrease the alpha­tocopherol levels in serum
but it may be secondary to the changes of serum lipid or lipoprotein
levels (Aftergood et al., 1975).
Clofibrate at a dose of 400 mg/d for six months together with poly­
unsaturated fatty acid rich diet increased the plasma tocopherol levels
in hyperlipidemic patients compared to healthy controls (Vessby et al.,
1977).
Cholestyramine, at a dose of 0.6 g/kg/d, decreased serum tocopherol
levels during two years therapy but the levels remained in normal range
(West et al., 1975).
WATER­SOLUBLE VITAMINS
THIAMINE
Thiamine is assayable chemically from biological material but the
techniques have not been practical for clinical purpose. The erythrocyte
transketolase activity has been used for evaluation of vitamin B. status
(Brin, 1962; Dreyfus, 1962). The enzyme activity correlated well with the
thiamine levels in blood (Brubacher et al., 197.2).
Some antimetabolites are known to alter the thiamine levels in man
(Baker and Frank, 1968).
RIBOFLAVIN
The riboflavin blood levels do not correlate well with the vitamin B_
status. Usually the erythrocyte NAPDH_­glutathione reductase is used for
the evaluation (Baker and Frank, 1968; Glatzle et al., 1970; Körner and
Völlm, 1976; Kutsky, 1973) though it has to be pointed out that many drugs
and their metabolites also change the glutathione levels (C hasseaud, 1974).
Chlortetracycline at a dose of 1­3 g/d and Oxytetracycline 2.5 g/d
are found to increase the excretion of riboflavin in urine (cited in
Goodman and Gilman, 1975).
 197

NICOTINIC ACID
The evaluation of vitamin B. status on the basis of the nicotinic acid
levels has not been found to be clinically meaningful (Kutsky, 1973; Baker
and Frank, 1968; Körner and Völlm, 1976). One has to consider also that
tryptophan is a precursor of nicotinic acid and many drugs affecting
vitamin B, levels also affect the conversion of tryptophan to nicotinic
acid. In some cases the metabolites have been measured (Körner and Völlm,
1976).
Chlortetracycline increases the urinary excretion of N-methyl-
nicotinamide (Goodman and Gilman, 1975).
VITAMIN B 6
The vitamin B, blood levels correlate well with the vitamin B, status
in man (Körner and Völlm, 1976). In addition to the classical tryptophan
loading test (Wachstein and Gudaitis, 1952) some plasma or erythrocyte
enzymes like aspartate aminotransferase have also been used in the B,
status evaluation (Hamfelt, 1967; Stanulovic et al., 1967).
Antiepileptic drugs hydantoin and succinimide were found to lower
serum pyridoxal levels in four weeks in a prospective study (Reinken,
1973). This confirms the earlier results based on the tryptophan loading
test (Woodbury et at., 1972). The author refer apparently to diphenyl-
hydantoin and ethosuximide by generic names.
The oral contraceptives have a well documented effect on vitamin B,
status (Heilmann, 1979) which was also found with the tryptophan loading
test (Rose, 1966). Norethisterone alone does not change the pyridoxal
metabolism (Rose-Adams and Strong, 1973).
Ieoniazide and cycloserine as well as penicillamine and many natural
compounds are known as vitamin B, antagonists (Brin, 1978).
FOLIC ACID
The effect of drugs on folic acid status are reflected both in haema-
tological parameters and the folate levels. The effects of drugs on
folates have been reviewed by Waxman et al. (1970) and Blakley (1969).
The first megaloblastic anemia induced with antiepileptic drugs was
rapported in 1952, decades after the introduction of the classical anti-
epileptics. Phénobarbital, diphenylhydantoin and primidone decrease
folate concentration in serum, erythrocytes and cerebrospinal fluid in a
high proportion of epileptic patients but macrocytosis develops only in
few patients. The effect is well documented but the mechanism is still
198

disputable (Blakley, 1969; Frenkel et al., 1973; Reynolds, 1973; Waxman

et al., 1970; Woodbury et al., 1972). The possible mechanisms are the
interaction with folate absorption, the induction of microsomal enzymes
and the urinary excretion of folate (Labadarios et al., 1978; Latham et
al., 1973; Maxwell et al., 1972; Reynolds, 1973).
It has been considered also­that decreased folate values and megalo­
blastosis are simply reflections of inadequate nutrition of antiepileptics
(Rose and Johnson, 1978). The negative result in the deoxyuridine suppres­
sion test shows that the synthesis of thymidylate is not affected by anti­
convulsants (Wickramasinghe et al., 1975). On the other hand the macro­
cytosis disappears on withdrawal of the anticonvulsant (Blakley, 1969).
The following presentation of this workshop also deals with this problem.
■ Phenothiazines decrease the serum and red blood cell folate levels
in psychiatric patients (Labadarios et al., 1978) .
The oral contraceptives have been shown to decrease both serum and
erythrocyte folate levels in well controlled studies and there is no
additional decrease after two years (Heilmann, 1979; Shojania, 1975;
Shojania et al., 1971; Waxman et al., 1970). In some studies the investi­
gators were not able to find this effect (Heilmann, 1979; Karlin et al.,
1977).
Folate antagonists, methotrexate, pyrimethamine, trimethoprim and
Pentamidin decrease folate levels as expected (Blakley, 1969; Herbert,
1973; Waxman et al., 1970). In other studies no effect of trimethoprim
on folate levels was found (Bateson et al., 1976; Borkenstein et al.,
1979; Hjärtshöj et al., 1978).
Triamterene, a potassium sparing pteridine diuretic, has been shown
to decrease serum folate levels in some special cases (Waxman et al.,
1970; Wilms et al·., 1979).
Metformin significantly decreases both serum and blood folate levels
­ in patients using 3 g/d (Berchtold et al., 1971).
In the therapy of tuberculosis isoniazide and cycloserine have been
found to change folate metabolism (Blakley, k969).
Cholestyramine decreases erythrocyte and serum folate levels during
long­term therapy of children with familial hypercholesterolemia (West
et al., 1975).
Methionine in intravenous infusions of amino acid mixtures is sug­
gested to lower serum folate concentrations (C onnor et al., 1978).
 199

VITAMIN B.­
The earlier findings can be found in reviews (Corcino et al., 1970;
Waxman et al., 1970).
Long­term anticonvulsant therapy lowers the cerebrospinal fluid
vitamin B.. levels without effect on the serum B. ? and with no sign of
megaloblaetosis (Frenkel et al., 1973).
The oral contraceptives have been found to lower serum vitamin Β η _
levels (Wertalik et al., 1971; Heilmann, 1979) and after prednisone treat­
ment the unsaturated B..­binding capacity is significantly decreased in
pancytopenic patients (Wysocki et al., 1978).
The treatment of gout with colchisin, tuberculosis with para­amino­
salicylic acid and intestinal infections with neomycin lowers the absorp­
tion of vitamin B._ measured with Schilling­test (Corcino et al.,1970;
Faloon, 1970).
Both melphalan and busulfan normalize the increased unsaturated B.»­
binding capacity in the therapy of polycytemia vera (Rachmilewitz et al.,
1977).
Metformin decreases the absorption of vitamin B... and some diabetics
have decreased serum vitamin Β , levels (Berchtold et al., 1971; Tomkin
et al., 1971). The effect has been confirmed also in volunteers with a
dose of 1.5 g/d for two months (Berchtold et al., 1971).
ABCORBIC A CID
The oral contraceptives have effect on ascorbic acid levels in serum,
leukocytes and thrombocytes as well as on the urinary excretion (Heilmann,
1979). In a study of healthy women using oral contraceptives, sequential or
combined, for at least a year and comparing them to the control group
matched by age, weight, parity and educational level, McLeroy and Schendel
(1973) showed significant lowering of leukocyte ascorbate level of the
group on the pill, regardless of whether or not they took ascorbic acid
supplement. Harris et al. (1973) have found the excretion of ascorbic acid
in urine to be halved in women using oral contraceptives. The mechanism of
the effect of oral contraceptives on ascorbic acid levels is not known, but
it might be secondary to the increased ce nilopläsmin level which activates
ascorbate oxidase (Heilmann, 1979).
Acetosalicylic acid increases the plasma ascorbate levels and the
excretion in the urine but lowers the leukocyte levels at dosages 600 mg/
g.i.d. in four days (Loh et al., 1973).
200

Tetracycline at a dose of 1 g/d, lowers the leukocyte ascorbate

levels and increases the urinary excretion in five days in old men.
Similar changes were not found in the control group receiving one of the
following drugs, phénobarbital, phenylbutazone or aloxiprin (Windsor e_t
al., 1972).
SUMMARY
The drug-vitamin interaction is often preceeding the untoward effect
of a drug. Sometimes the first sign of an interaction is the effect on
vitamin levels in body fluids. Often the levels are changed but still
in normal limits. The lack of well controlled studies is apparent and
modern analytical methods should be used.
The antiepileptics, oral contraceptives and corticosteroids have
reliably documented effects on some vitamin levels. These and other effects
are summarized in the following table.

DRUG EFFECTS ON SOME VITAMIN LEVELS

Vitamin Increase Decrease

VITAMIN A
serum vitamin A phénobarbital cholestyramine
diphenylhydantoin neomycin
thioridazine
isosorbidedinitrate
oral contraceptives

VITAMIN D
serum 25-(0H)D, phénobarbital
diphenylhydanto in
primidone
colestipol
serum 1 alpha, 25-(0H)„D3 prednisone

VITAMIN E
serum tocopherol Clofibrate oral contraceptives
cholestyramine

RIBOFLAVIN
urine riboflavin Chlortetracycline
Oxytetracycline

cont.
 201

VITAMIN B6
serum p y n d o x a l p h o s p h a t e diphenylhydantoin
ethosuximide

VITAMIN B­ 2
serum vitamin Β 12 oral contraceptives
metformin
cerebrospinal fluid B._ anticonvulsants
Schilling­test/absorpfion
/absorption colchisin
para­aminosalicylic acid
neomycin
metformin

FOLIC ACID
serum, erythrocyte, phénobarbital
cerebrospinal fluid diphenylhydantoin
primidone
serum, erythrocyte phenothiazines
oral contraceptives
methotrexate
pyrimethamine
trimethoprim
Pentamidin
triamteren
metformin
isoniazide
cycloserine
cholestyramine
methionine

ASCORBIC ACID
serum/plasma acetosalicylic acid
leukocytes oral contraceptives
acetosalicylic acid
tetracycline
urine oral contraceptives
tetracycline

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 205

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THE INTERACTION BETWEEN ANTIEPILEPTIC THERAPY AND FOLATE AB-
SORPTION AND DISTRIBUTION. A KINETIC APPROACH TO OBTAIN MORE
MEANINGFUL LABORATORY DATA.

Jørn Hendel, Per Winkel and Mogens Dam

Departments of Clinical Chemistry,

Frederiksberg Hospital and Finsen Institute,
and Department of Neurology, Hvidovre Hospital, Copenhagen,
Denmark

Phenytoin, and to a lesser degree phénobarbital, primi-

done and carbamazepine have been reported to interfere with
the metabolism of folates in humans (Webster, 195**, Nelson,
et al., 1956, Gydell, 1957, Klipstein, 1964). Furthermore re-
cent studies have shown a correlation between folate deple-
tion and the incidence of neuropsychiatrie disorders in pa-
tients treated with antiepileptic drugs (Reynolds, 1972).
These findings should be related to our present knowledge of
folate metabolism in man.
All the folates are reduced and methylated derivatives
of folic acid (pteroylmonoglutamic acid, abbreviated PGA)
linked together in a rather complicated metabolic pattern
(Fig. 1 ) . Their role is to serve as coenzymes in metabolic
reactions where transfer of C-l fragments, like methyl, for-
myl and methylene, takes place. Intracellularily the differ-
ent folates are conjugated with glumatic acid to yield pen-
ta-, hexa- and heptaglutamates. Folatepolyglutamates are un-
able to cross cellmembranes, which enables the cell to main-
tain a much higher folate concentration than that extracellu-
rily. Furthermore, there is increasing evidence that these
polyglutamate forms are the active coenzymes., and not only a
storage form, as it was believed earlier.
 5-CHj-THF.· -6-CH3-TMF

POA- PGA

Figure 1.
Metabolic
scheme of intra-
DHF· cellular folate
pathways
Ihymtdl
datoiyuridln·

-
e.10-CH,-THF
5-CHO-THF -5-CHO-THF

For all practical purposes folate in food is exclusively

present as reduced folate polyglutaraates. Contrary to the mo-
noglutamates these folate polyglutamates cannot pass the in-
testinal epithelium. Therefore they first have to be deconju-
gated by glutamyldeconjugase. This enzyme is present in the
Jejunal brushborder. The resulting reduced folate monogluta-
mates are then absorbed into the portal blood stream. Once
they have entered the blood stream they are taken up by the
liver cells and reconjugated, or converted to 5-methyltetra-
hydrofolic acid, which is the predominant monoglutamate in
the organism, and by far constitutes the main part of the
plasma folates.
In this complicated metabolic pattern there are obvious-
ly many steps where the antiepileptic drugs could possibly
interfere. Many such interferences have also been suggested.
However, the hypothesis most consistent with clinical and ex-
perimental observations is that the use of the drugs results
in a malabsorption of folate. Thus in in vitro experiments
Phenytoin has been shown to be a potent inhibitor of the glu-
tamate deconjugase (Hoffbrand, et al, 1968 and Rosenberg, et
al, 1968), which is consistent with the clinical finding that
patients suffering from tropical- and nontropical sprue, show
a decreased glutamated^conjugase activity in their jejunal
brushborder, leading to severe folate malabsorption (Halsted,
et al, 1977).
208

A more specific investigation of the in vivo interfer-

ence of various antiepileptic drugs with folate absorption
therefore seems appropriate. Three conditions have to be ful-
filled before this problem can be approached in a rational
and quantitative way:
1) A well defined compound which is compatible to compounds
found in ordinary foodstuffs has to be available.
2) A specific and reasonably precise analytical method for
measuring the compound entering the organism from the
intestine must be available.
3) A model which will allow a reasonably realistic predic-
tion of the fate of the compound once it has been ab-
sorbed from the intestine should have been specified.
In the following we will address ourselves to these pro-
blems and present an experimental approach.
A number of experiments have earlier been performed
where monoglutamates have been given orally. It is unlikely
that the intestinal capacity for the absorption of folates
can be assessed on the basis of results obtained from such
experiments. Practically all folates in food are present as
polyglutamates and the rate limiting step in the absorption
is probably the breakdown of polyglutamates to monoglutamates
at the intestinal brushborder.
Therefore to study this important aspect of the intesti-
nal absorption without loosing the advantage of working with
a welldefined pure compound we decided to use a synthetic
triglutamate (pteroyi-àr* -L-glutamyl- à^-Lglutamyl-L-glutamic
acid (P-triGA). In the intestine it is degraded to PGA by
the action of glutamyldeconjugase. PGA was chosen because
•1) its conversion to reduced folates in the human organism
for practical purposes is irreversibel, 2) its concentration
in blood is usually very low, 3) it is chemically the most
stable of the folates, and 4) when given as a monoglutamate
the absorption from the intestine is very fast and complete.
So far, however, experimental work using PGA has been
hampered by the lack of a specific and precise analytical
technique for the measurement of its concentration in biolo-
 209

gical fluids, in that only nonspecific binding methods or

crude biological assays have been available.
To overcome this difficulty a more specific and precise
RIA­method was developed by one of us (Hendel, I98O). Once
the analytical problems were solved preliminary experiments
could be performed to obtain the building blocks necessary
for the construction of the kinetic model.
Four healthy male volunteers have been used for these
experiments. Figure 2 depicts the plasma PGA concentration
versus time curve based on data obtained from one of the vol­
unteers given an intravenous bolus injection of 3 mg PGA.
The best fitting curve based on a linear two compartment mo­
del is also shown in the figure. It appears that this simple
model provides a reasonable approximation to the observati­
ons. However, only four such experiments have been performed
so far.
Looking at the findings of other investigators concer­
ning the kinetics of reduced folate monoglutamates, it ap­
pears that these reduced folate forms after oral administra­
tion are cleared very rapidly from the portal stream by the
liver cells, i.e. exhibiting a pronounced so called first
pass effect.
plasma POA^jg/l

c »·.­<" ,,..­P>
200­
Figure 2.
β . ­oco» mm­' Plasma concentration curve
(t ­ ­00047 m»!­' after an intravenous bolus
injection of 3 mg PGA.
100­

20 40 60 80 100 120 minutas

Concerning the renal clearance of reduced folate, some

kind of renal threshold mechanism seems to exist. According
to the linear two compartment model the renal elimination of
PGA should be a first order process. This may be demonstrated
more directly if a fractionated collection of the urine is
210
performed following an intravenous load of PGA. Assuming
that the renal elimination is a first order process it may
be shown that a linear relationship exists between the a-
mount of PGA eliminated in the urine and the area under the
plasma concentration curve corresponding to the same time
interval during which the PGA was eliminated in the urine.
Figure 3 and H show such data obtained from an experiment
where PGA was infused intravenously at a constant rate of
1 mg/h. It appears that the relationship is reasonably li-
near. Since the plasma PGA concentration showed considerable
variation during the experiment these data support the hypo-
thesis that the renal elimination of PGA is a first order
process.
infusion rate 1 mg/60 min
Figure 3·
Plasma concentration
curve during i.v. in-
fusion of PGA. Timespan
of fractionated urine
collection indicated.

300 minutes

/jg PGA
excreted F i g u r e H.
Amount of PGA eliminated
in urine versus corres-
400 ponding area under curve
of PGA plasma concentra-
tion.

200 -

SO 100 A U C arb. units

 211
Assuming that the system is linear and the intestinal
absorption is complete it may be shown that if there is a
firstpajá effect, the area under the PGA concentration in
plasma versus time curve after oral administration should be
less than that observed after intravenous administration of
the same dose of PGA.
Figure 5 shows the PGA concentration in plasma versus
time curve after oral as well as after intravenous admini­
stration of 3 mg of PGA to the same healthy volonteer. Ac­
tually the area after oral administration is slightly larger
than that obtained after i.v. administration. A second expe­
riment involving another volunteer showed similar results.
Therefore in this limited series of experiments and in this
particular concentration range a first pass effect could not
be demonstrated.

plum« PQA^ig/l
Figure 5 .
200­
PGA concentration curves
from the same volunteer
after oral as well as after
i.v. administration of 3 mg
PGA.
100­

I I I I I I
20 40 Μ M 100 120 minut·«

Thus a very simple kinetic model evolves from these pre­

liminary experiments. A linear two compartment model which
seems to offer a reasonable approximation to the observations
when PGA is given intravenously as well as orally (i.e. no
first pass effect). To reach more definite conclusions more
experiments are of course needed.
However, even if the model should prove to be valid it
is still incomplete, since it does not include the important
first step in the intestinal absorption of genuine folates,
i.e. the breakdown of polyglutamates to monoglutamates at the
intestinal brushborder. To incorporate this process in the
212
model' it is necessary to obtain data from experiments where
a known amount of a polyglutamate is administered orally. So
far we have only performed one such experiment since the syn-
thesis of P-triGA has been very time consuming.
Figure 6 shows the plasma PGA concentration versus time
curve after oral administration of 2.5 mg of P-triGA (corres-
ponding to approximately 2 mg of PGA) to a healthy volunteer.
It is seen that compared to the type of curve obtained after
oral administration of PGA this curve is much lower and reach-
ing its maximum considerably later. This supports the conten-
tion that the breakdown of polyglutamates at the intestinal
brushborder is an important rate limiting process in the in-
testinal absorption.

plasma PGA jig/I

Figure 6.
PGA concentra-
tion curve after
oral administra-
10 -
tion of P-triGA
corresponding to
5 - 2 mg of PGA

At present a clinical study including patients with a

verified diagnosis of epilepsia, who have not received any
antiepileptic treatment, is in progress. Depending on the
type of seizures these patients will receive one of the fol-
lowing 4 antiepileptic drugs:
Phenytoin, phénobarbital, carbamazepine or valproic acid.
The absorption of orally administered P-triGA and the
distribution of an i.v. dose of PGA are followed in every
patient before the start of medication as well as subsequent
to three months of medication.
The knowledged gained in the present study of PGA kine-
tics has been essential in order to design the above mention-
ed clinical study.
 213
REFERENCES
Gydell, Κ.: Megaloblastic Anaemia in Patients Treated with
Diphenylhydantoin and Primidone. Acta Haemat. 17: 1­15,
1957.
Halsted, C.H. , Reisenauer,A.M., Romero, J.J., C antor, D.S.
and Ruebner B.: Jejunal Perfusion of Simple and Conju­
gated Folates in Celiac Sprue. J. Clin. Invest. 59:
933­9^0, 1977.
Hendel, J.: Radioimmunoassay of Pteroylglutamic Acid. Manu­
script submitted.
Hoffbrand, A.V. and Necheles, T.F.: Mechanism of Folate
Deficiency in Patients receiving Phenytoin. Lancet ii:
528­530, 1968.
Klipstein, F.Α.: Subnormal Serum Folate and Macrocytosis
Associated with Anticonvulsant Drug Therapy. Blood 23:
68­86, 1964.
Nelson, N.A. and Bishop, R.C.: Nonaddisonial Megaloblastic
Anemia. Clin. Res. Proc. Η: 231*, 1956.
Reynold E.H., Gallagher B.B., Mattson, R.H. Bowers, M. and
Johnson, A.L.: Relationship between Serum and Cerebro­
spinal Fluid Folate. Nature 2Uo: 155­157, 1972.
Rosenberg, I.H., Godwin, H.A., Streiff, R.R., and Castle W.B.:
Impairment of Intestinal Deconjugation of Dietary Folate.
Lancet ii: 530­532, 1968.
 DIFFERENTIATION OF TOXIC FROM SPURIOUS ABNORMALITIES
AFTER DRUG ADMINISTRATION IN MAN

Carlos A. Dujovne
Department of Clinical Pharmacology
University of Kansas Medical Center
College of Health Sciences and Hospital
Kansas City, KS 66103 U.S.A.

ABSTRACT
My experience in selecting tests and interpreting clinical laboratory
results during clinical trials is described. Examples of possible problems
in interpreting the abnormalities in commonly performed tests are given.
By keeping in mind the possible mechanism of alterations of test results
following drug administration one may learn to choose tests adequately
and/or to interpret correctly the results of tests used to monitor drug
effects.

INTRODUCTION
While taking care of patients and performing clinical trials with new
and old drugs over the last ten years, I have become increasingly aware of
some of the pitfalls in the interpretation of various clinical laboratory
tests performed to monitor drug toxicity during therapeutic trials or
disease treatments in man (Dujovne et_ aL· , 1971a,b; 1974; 1976a,b; Dujovne,
1977; Dujovne et_ al_., 1979). To evaluate toxic effects by means of the
change in the normal value of a clinical test after administration of a
drug, one must be aware that many causes other than toxic effects of the
drug itself could account for that change. Table 1 includes some of these
possible causes.

TABLE 1 - SOME CONDITIONS ASSOCIATED WITH SPURIOUS ABNORMALITIES OF

CLINICAL LABORATORY TESTS
Serum Bilirubin (S-Bilirubin)
Gilbert's syndrome
Rifampin
Clofibrate

Serum Alkaline Phosphatase (S-ALP)

Clofibrate
Albumin prepared from placental blood
Phenytoin
 215

Serum Gamma Glutamyl Transpeptidase (S-GT)

Microsomal enzyme inducers
Ethanol-phenytoin-phenobarbital-rifampin, etc.
Serum Creatine Phosphokinase (S-CK)
Intramuscular injection
Clofibrate
Hyperlipoproteinemia
Hypothyroidism
Serum Uric Acid (S-URAT)
Therapeutic cytolysis
Clofibrate
Serum Aspartate Aminotransferase (S-ASAT)
Isoniazid
Erythromycin
Intramuscular injections
Azotemia
Serum Alanine Aminotransferase (S-ALAT)
Vitamin B, deficiency
Knowledge of the various mechanisms by which the test
abnormality may occur helps in making an adequate interpretation of the
results ae well as in choosing the proper tests to be monitored.
The following two examples should serve to illustrate the message of
this presentation:
1. When a drug such as Clofibrate is being given to treat hyperlipi-
demia, serum creatine Phosphokinase (S-CK) levels are used to
monitor possible toxicity to skeletal muscle. We have learned of
the possibility of elevated S-CK secondary to the hyperlipidemia
itself (Dujovne et a l . , 1976a). This knowledge helped us to
exclude the possibility that the S-CK elevation may not have been
from drug toxicity to the muscle but rather from the disease
state itself.
2. A drug is being tested in man and serum gamma glutamyl transferase
(S-GT) is chosen to monitor possible hepatotoxic effects. If the
drug being tested is known or suspected of having a microsomal
induction effect (e.g. ethanol, phenytoin, etc.), the levels of
S-GT may rise due to enzyme induction independent of a hepatotoxic
effect. Consequently, tests other than S-GT should be monitored
for the purpose cited.
216

The results of many clinical tests can be modified by interactions

between the drug and the substance tested or by interference with the assay
methods. Long comprehensive lists of such effects have been published
(Hansten, 1979) and a complete review of this topic is not within the scope
of this paper. Rather, I will focus on the presentation of relatively un-
known situations involving the clinical tests cited in Table 1 which I have
encountered in clinical pharmacological practice.
DISCUSSION
1. Serum Bilirubin (S-Bilirubin)
a. Gilbert's syndrome is a genetically acquired deficiency of trans-
port and/or conjugation of bilirubin with no known pathological conse-
quences in man. Moderate elevations of 20 to 100SÓ of a baseline level of
S-Bilirubin can be found after fasting in subjects with this entity
(Felsher et aj_., 1970). During clinical drug testing it is not uncommon
to obtain a baseline S-Bilirubin under a non fasting state and to repeat
the level for monitoring toxicity after drug administration. If the repeat
level is obtained after 12 hrs of fasting or longer in an individual who
happens to have Gilbert's syndrome, the level will probably be abnormally
elevated. This elevation may be completely unrelated to the drug adminis-
tered, however some drugs, e.g. contraceptive steroids, are known to aggra-
vate the abnormality of increased S-Bilirubin in this syndrome.
b. It has been shown by Acocella et_ al_., (1965) that up to 80SÍ of
subjects developed transient elevation of S-Bilirubin following administra-
tion of rifampin. This drug competes with bilirubin for transport in the
liver resulting in slight to moderate elevation of S-Bilirubin level; after
a few days or weeks a new equilibrium takes place and the S-Bilirubin level
returns to normal. The fact that rifampin can also cause hyperbilirubinemia
by an hepatotoxic effect makes the differential diagnosis and interpreta-
tion of this change particularly important. Other drugs such as the
•antibiotic novobiocin can exert a similar effect.
c. A significant reduction in S-Bilirubin levels takes place after
administration of Clofibrate to hyperlipidemic patients. The mechanism is
unknown but it may be related to the subsequent discovery that Clofibrate
can also normalize elevated serum bilirubin in non hyperlipidemic subjects
who have hyperbilirubinemia of fasting due to Gilbert.'s syndrome. The
effect may be related to competition between bilirubin and lipids for
binding to serum proteins. This may be associated with, or be independent
 217

of, an effect of the drug on excretion of bilirubin by the liver or the

kidneys. This is an example of mechanism #2 in Table 2.

TABLE 2 - MECHANISMS OF CHANGES IN CLINICAL TESTS AFTER DRUG

ADMINISTRATION, NOT NECESSARILY FROM DRUG TOXICITY
1. Drug interaction with test assay methods or conditions.
2. Drug producing a change in physiological parameters with or without
toxicity.
3. Drug interaction with preexisting or coexisting pathology.
4. Drug affecting the amount of tested substance independent of a toxic
mechanism.

2. Serum Alkaline Phosphatase (S-ALP)

a. I have reported a significant reduction in S-ALP following
administration of Clofibrate (Dujovne eţ^ a_K, 1971b; Dujovne et al.,
1976a,b). Due to the ubiquitous origin of this enzyme (liver, bone, white
blood cells, intestinal mucosa, placenta, kidney) it becomes difficult to
elucidate the mechanism of this effect. Recent studies (Whitaker et al.,
1979) seem to indicate that the reduction is in the hepatic origin of
S-ALP.
b. Artificially elevated S-ALP has been reported in patients who have
received human serum albumin supplements manufactured from placental tissue
(Bark, 1969). This is an example of mechanism #4 in Table 2.
c. Phenytoin administration may lead to increased S-ALP resulting
from increased celcium turnover due to the effect of the drug on 25-hydroxy
califerol metabolism in the liver (Tolman eţ^ al_., 1975).
3. Serum Gamma Glutamyl Transpeptidase (S-GT)
The measurement of this enzyme in blood for detection of pathology
of the biliary tree has gained wide popularity in recent years. This is
because S-GT is absent from bone and placental tissues. H owever, the
findings of S-GT elevation after microsomal enzyme induction following
administration of drugs with unlikely effects on the biliary tree (e.g.
phénobarbital, ethanol, etc.) reveals the non specificity of this test to
detect drug induced hepatic injury in man (Rosalki et al., 1971); it is
particularly relevant that elevation of S-GT produced by alcohol without
evidence of hepatotoxicity may be related to this mechanism (Rosalki et
al., 1972). This is an example of mechanism #4 in Table 2.
218

4. Serum Creatine Phosphokinase (S­CK)

The blood levels of this enzyme could be markedly elevated by local
trauma or irritating effects of drugs on muscle tissue when the drug is
given intramuscularly. It is also known that some drugs can have systemic
toxic effects on skeletal muscle (drug­induced myositis) and this effect
may result in S­CK elevation; Clofibrate has been reported to produce such
an effect. However, I have found that the elevation of S­CK following
Clofibrate administration may also be related to the effects of the
hyperlipidemic condition itself on the skeletal muscle. In our clinical
trial, patients were found to have abnormal S­CK as often during the
placebo regimen as during Clofibrate treatment. In some of the patients
who had elevated S­CK during placebo treatment the subsequent administra­
tion of Clofibrate resulted in enhancement of the S­CK elevation (Dujovne
et_ al^., 1976a,b). This constitutes an example of mechanism #3 in Table 2.
5. Serum Uric Acid (S­URA T)
A drug can affect S­URAT levels by changing the rate of uric acid
production or elimination by direct or indirect mechanisms. A chemothera­
peutic agent against certain types of cancer may increase S­URAT levels by
means of destruction of large numbers of neoplastic cells. A hypolipi­
demic drug like Clofibrate or halofenate may decrease S­URAT levels by its
effect on serum lipoproteins, possibly by indirectly affecting uric acid
binding and excretion (Dujovne et al., 1976a,b).
People with hyperuricemia have been shown to have increased sulphobro­
mophthalein retention without detectable liver disease (Granarne et_ al.,
1968).
6. Serum Aspartate Aminotransferase (S­A SA T)
The activity of this enzyme may be spuriously elevated after isoni­
azid or erythromycin administration. This is due to an interference by
these drugs with some of the spectrophotometry assays of the enzyme, an
■example of mechanism #1 in Table 2. This effect needs to be ruled out
before assuming an hepatotoxic effect from these drugs. A lso, the intra­
muscular injections of some drugs can result in local skeletal muscle
injury from the needle or the drug sufficient to produce leakage of muscle
S­ASAT resulting in subsequent rise in S­ASAT levels.
Artificially decreased S­ASAT has been observed in azotemic patients
(Cohen et al., 1976).
 219
7. Serum Alanine Aminotransferase (S­A LA T)
S­ALAT ia one of the most specific enzymes measured to indicate
hepatocellular damage. Leakage from hepatic cells serves as an indicator
of liver injury of any type. Most patients with alcoholic liver injury are
found to have lower than expected rise or no rise at all of S­ALAT. This
19 most probably due to the presence of a significant deficiency in vitamin
B,, in such patients (Ning et_ al_., 1966). Since B, is a necessary cofactor
of alanine transamination, its deficiency results in lower measurable
levels of the enzyme in blood even when a large amount of the precursor is
leaked from injured cells. This explanation is supported by (a) a rise in
S­ALAT after intravenous B, infusion in such patients and (b) a rise in
measurable S­A LA T when vitamin B, is added in vitro to the serum specimen
of such a patient (Rej eţ^ aL·, 1973). The above is an example of a drug
(alcohol) coexisting with other pathology (nutritional deficiency), confus­
ing the interpretation of the drug effect. This is an example of mechan­
ism #3 of Table 1.

REFERENCES

Acocella, G., Nicolis, F.B. and Tenconi, L.T.: The effect of an intraven­
ous infusion of rifamycin SV on the excretion of bilirubin, bromosul­
phalein, and indocyanine green in man. Gastroent. 49:521­525, 1975a.
Bark, C.3.: A rtificial elevation of serum alkaline phasphatase following
albumin infusions. A m. J. Clin. Path. 52:466­467, 1969.
Cohen, G.A ., Goffinet, J.Α., Donabedian, R.K. and Coun, H.O.: Observations
on decreased serum glutamic oxalacetic transaminase. (SGOT) activity
in azotemic patients. A nn. Int. Med. 84:275­280, 1976.
Dujovne, C.A. and Strauss, H.W.: Changes in liver and spleen scans on pa­
tients during treatment with two hypolipidemic drugs. Radiology 98:
682­684, 1971a.
Dujovne, C A. , Weiss, P. and Bianchine, 3.R.: Comparative clinical thera­
peutic trial with two hypolipidemic drugs, Clofibrate and nafenopin
(SU­13437). Clin. Pharmacol. Ther. 12:117­125, 1971b.
Dujovne, C A. , Hurwitz, Α., Kauffman, R.E. and Azarnoff, D.L.: Colestipol
and Clofibrate in hypercholesterolemia. Clin. Pharmacol. Ther. 16:
291­296, 1974.
Dujovne, C A. , A zarnoff, D.L., Huffman, D.H., Pentikainen, P., Hurwitz, A.
and Shoeman, D.W.: One­year trials with halofenate, Clofibrate, and
placebo. Clin. Pharmacol. Ther. 19:352­359, 1976a.
Dujovne, C A. , A zarnoff, D.L., Pentikainen, P., Manion, C., Hurwitz, A.
and Hassanein, K.: A two­year crossover therapeutic trial with halo­
fenate and Clofibrate. A m. J. Med. Sc. 272:277­284, 1976b.
Dujovne, C A. : A clinical pharmacologist's view of drug hepatotoxicity.
Pharmacol. Res. Comm. 9:1­15, 1977.
Dujovne, C A. , A zarnoff, D.L., Pentikainen, P., Manion, C and Hurwitz, Α.:
Clofibrate and nicotinyl alcohol tartrate in hyperlipoproteinemic
patients. A m. J. Med. Sc. 277:255­261, 1979.
220

Felsher, B.F., Richard, D. and Redeker, A.G.: The reciprocal relation

between caloric intake and the degree of hyperbilirubinemia in Gil-
bert's syndrome. N.E.J.M. 283:170, 1970.
Granarne, R., Haslem, R.M. and Scott, 3.T.: Sulphobromphthalein retention
in gout and asymptomatic hyperuricaemia. Ann. Rheum. Dis. 27:19-26,
1968.
Hansten, P.D.: Drug effects on clinical laboratory test results. In:
Drug Interactions, pp. 303-485, 1979.
Ning, M., Baker, H. and Leevy, C M . : Reduction of glutamic pyruvic trans-
aminase in pyridoxine deficiency in liver disease. Soc. Exp. Biol.
Med. 21:27-30, 1966.
Rej, R., Fasce, C.F. Jr. and Vanderlinde, R.E.: Increased aspartate amino-
transferase activity of serum after in_ vitro supplementation with
pyridoxal phosphate. Clin. Chem. 19:92-98, 1973.
Rosalki, S.B. and Rau, D.: Serum alpha-glutamyl transpeptidase activity
in alcoholism. Clin. Chem. Acta. 39:41-47, 1972.
Rosalki, S.B., Tarlow, D. and Rau, D.: Plasma gamma-glutamyl transpepti-
dase elevation in patients receiving enzyme-inducing drugs. The
Lancet, Aug. 14, 1971.
Tolman, K.G. r Jubiz, W., Sanella, 3., Madsen, J., Belsey, R.E., Goldsmith,
R. and Freston, J.W.: Osteomalacia secondary to anticonvulsant drugs
in mentally retarded children. Pediatrics 56:45, 1975.
Whitaker, K.B., Costa, D. and Moss, D.W.: Selective effects of Clofibrate
on alkaline phosphatase isoenzymes in serum. Clin. Chem. Acta 94:
191-196, 1979.
 EFFECTS OF XENOBIOTICS ON HEME OXYGENASE ACTIVITY*

F. William Sunderman, Jr.

Departments of Laboratory Medicine and Pharmacology
University of Connecticut School of Medicine
Farmington, CT 06032 U.S.A.

ABSTRACT
Heme oxygenase (PO) is a microsomal enzyme in spleen, bone marrow,
liver, kidney, brain and other organs which cleaves the a-methene bridge of
heme to liberate blllverdln, iron and carbon monoxide. Hepatic HO activity
is induced by (a) methemalbuminemia and hemoglobinemia, (b) hypoglycemia and
fasting, (c) hormones (e.g., Insulin, glucagon and epinephrine), (d) organ-
ic xenoblotlcs (e.g., zymosan and bromobenzene), and (e) metal compounds
(e.g., cobalt chloride and gold sodium thlomalate). Induction of HO acti-
vity in liver and other organs may possibly result in increased serum
concentrations of bilirubin and iron, and increased rate of endogenous pro-
duction of carbon monoxide.

INTRODUCTION
During the past decade, research on heme oxygenase (EC 1.14.99.3;
abbreviated HO) has provided Insight into a previously unrecognized mechan-
ism whereby certain xenoblotlcs Influence the metabolism of bilirubin, iron
and carbon monoxide (Bonkowsky et al, 1979). As a consequence, HO has
become a focus for current investigations in laboratories of pharmacology,
toxicology, and clinical biochemistry. The objectives of this manuscript
are: first, to summarize the biochemistry of HO; second, to list the tech-
niques for HO assay; third, to discuss the pathophysiology of HO; fourth,
to tabulate xenoblotlcs which induce HO activity in hepatic microsomes;
fifth, to consider measurements of HO activity In tissue biopsies from
patients; and finally, to suggest that stimulation of HO activity by
hormones, metabolites, and xenoblotlcs may result in increased serum
concentrations of hillrubin and iron, and increased rate of endogenous
production of CO.
BIOCHFMISTRY OF HEME OXYGENASE
Tenhunen et al (1968,1969) showed that microsomes in rat liver and
spleen possess a mixed-function oxidase enzyme (HO), which cleaves the a-
methene bridge of heme. As Indicated in Flg.l, HO has requirements for
NADPH and O2. The products of the HO reaction are CO, Fe and biliverdin-
IXa, which subsequently is reduced by biliverdin reductase to yield bill-
ruhin-IXa. A flavoproteln, NADPH-cytochrome P-450 reductase, is a cofactor

* Supported by research grants from the U.S. Department of Energy (EY-3140)

and the National Institute of Environmental Health Sciences (ES-01337).
222

M ν

HC=T^J=CH NADPH

Ρ M

HEME
M V M P P M M V

BILIVERDIN K a

M V M Ρ Ρ M M V

"UUO­Π.­
Ν H Ν ^ Ν H V
NADP T

Fig. 1
BILIRUBIN IXa (See legend on
next page)

UROPORPHYRINOGEN HI

ILIVERÛIN H »

Fig. 2
(See legend on
next page)
 223

ENDOPLASMIC RETICULUM

■NADP*

(7NADPH)

HO)­HVDROXYHEME

©
­&■ CO

-►Fa'·
|(?Branch Pathway)
I
,,
BILVERDIN ΓΧ·

► NADP*
FÎŞ. 3
BILIRUBIN LX«
(See legend below)

Fig. 1 ­ Catabollc pathway of heme.

Fig. 2 Relationships of heme blosynthetlc and catabollc pathways.
(1) A LA ­synthetase; (2) A LA ­dehydratase; (3) PBG­deaminase; (A )
UPG­synthetase; (5) UPG­III­cosynthetase; (fi) UPG­decarboxylase;
(7) CPG­oxldase; («) PPG­oxldase; (9) ferrochelatase ; (10) heme
oxygenase; (11) billverdln reductase.
Fig. 3 Probable reaction sequence In heme catabollsm. The numbered steps
are explained In the text.
224

Table 1 ­ PROPERTIES OF MAMMALIAN HEME OXYGENASE

Physiological Role; Heme degradation by a­methene cleavage (Tenhunen et al,

1969; Tenhunen, 1975; Maines & Kappas, 1976b).
Organs: Spleen, liver, bone marrow, brain, kidney, lung & duodenum (Tenhu­
nen et al, 1970).
Localization: Smooth & rough endoplasmic recticulum & Golgi apparatus
(Krasny & Holbrook, 1<>78; Hino et al, 1979).
Molecular Weight: =150,000­200,000 daltons (subunits of =32,000 or 68,000)
(Yoshida et al, 1974; Maines et al, 1977; Yoshida & Kikuchi, 1978a).
Substrates: Specific for Fe­hemins (Schacter & Waterman, 1974; King &
Brown, 1978; Yoshida & Kikuchi, 1978c; Frydman et al, 1979).
Inducers : Heme, hemoglobin, bromobenzene, endotoxin, diethyl maléate, Fe­
dextran, Co, Ni, Au and other metals (see Tables 4 & 5 ) .

for HO activity (Schacter et al, 1972; Hino & Minakami, 1979). Some prop­
erties of microsomal HO are listed in Table 1.
The relationships between the heme biosynthetic pathway, microsomal
hemoproteins, and the heme degradative· pathway are diagramed in Fig.2.
Mitochondrial δ­ALA synthetase is the initial and rate­limiting enzyme in
heme synthesis. According to a general principle of molecular biology, the
flux of substrate down an unbranched metabolic pathway is often regulated
by end­product repression of the gating enzyme. C onsonant with this prin­
ciple, 6­ALA synthetase is repressed by heme. The inhibitory effect of
heme on δ­ALA synthetase activity is responsible for the therapeutic effi­
cacy of hematin in acute porphyria (Lamon et al, 1979). The intracellular
heme pool also regulates microsomal HO activity, which is the initial and
rate­limiting enzyme in the pathway of heme catabolism. Thus, the Intra­
cellular heme pool exerts a negative feed­back effect on heme synthesis by
repression of δ­ALA synthetase activity, and positive feed­back effect on
heme degradation by induction of HO activity. The probable reaction
sequence in heme catabolism is shown in Fig.3 (Yoshida & Kikuchi, 1978b).
In step #1, HO binds with ferriherae in a specific configuration so that
only the a­methene bridge is accessible for attack by molecular O2· In
•step #2, ferrlheme is reduced to ferroheme by NADPH­cytochrome P­450 reduc­
tase, with consumption of NADPH. In steps #3 and #4, activated oxygen
attacks the a­methene bridge of ferroheme to form a­hydroxyheme, which is
further oxidized by 2 molecules of O2 to form ferrlbilirubin­IXa complex,
with evolution of C O. The requirement for molecular O2 was proven by
18
experiments with 0 2 and H 2 1 8 0 , which showed that the terminal lactam
oxygens of biliverdin­IXa and the C O oxygen were derived from molecular O2
and not from water (Tenhunen et al, 1972). Double­labelling experiments
revealed that the terminal oxygens of the bile pigment were derived from
 225

two different 0 2 molecules (King & Brown, 1978). The postulatlon that <*-
hydroxyheme is an intermediate Is supported by an observation that a-oxy-
mesoferrlheme-3H yields mesoblllverdln-3H (Kondo et al, 1971). Epoxides
have been suggested as intermediates in the oxidative attack on ferroheme
(step #3) (Hamilton & Dolphin, 1977). According to Yoshlda and Kikuchi
(1978b), the Fe[III]-blllverdln-IXa complex is reduced by NADPH-cytochrome
P-450 reductase in step #5, with release of biliverdin-IXa and iron.
Yoshlda and Klkuchl (1978b) inferred that step #5 may be the rate-limiting
stage of the over-all HO reaction.
In Pig.3, a dashed arrow has been inserted in the reaction sequence
after step #5 to indicate the possible existence of a branch pathway in
heme degradation. Landaw et al (1970) administered heme- C by iv injec-
tion to rats that had been treated with allylisopropylacetamide (AIA), and
observed that the molar ratio of '''CO production to bilirubin-ll4C produc-
55
tion was significantly >1.0. Raffin et al (1974) measured release of Fe
55
and production of bilirubin from heme- Fe by post-mitochondrial superna-
55
tant of rat intestinal mucosa. On a molar basis, the rate of Fe-release
significantly exceeded the rate of bilirubin formation. Pimstone et al
(1971b) noted that in assays of HO activity in rat and rabbit macrophages,
the rates of heme consumption consistently exceeded the rates of bilirubin
production. Lodola et al (1979) also observed that the rate of heme disap-
pearance exceeded the rate of bilirubin appearance during heme cataholism
by rat hepatic post-mitochondrial supernatant. From these studies, it
appears that biliverdin and bilirubin, albeit predominant, may not be the
only pyrrole-containing products of heme degradation.

Table 2 - ASSAYS OF HEME OXYGENASE ACTIVITY

Heme consumption, by spectrophotometry of pyridine hemochromogen at 557 nm

(Yoshlda & Kikuchi, 1978b).
Biliverdin production, by spectrophotometry, of reaction mixture at 690 nm
(Hino & Minakame, 1979).
Bilirubin production, by coupled reaction with biliverdin reductase & spec-
trophotometry of reaction mixture at 468 nm, or of CCli, extract at 455
nm (Maines et al, 1977; Schacter, 1978; Yoshlda & Kikuchi, 1978a).
^C-Bilirubin release, from heme-^C derived from ALA-a.A-^C. CC1 4 extrac-
tion is monitored by addition of bilirubin-3H (Tenhunen, 1972).
Fe release, from protoporphyrin-IX-55Fe (Raffin et al, 1974; Sassa et al,
1979).
1 >
* C0 release, from heme-1''C derived from g l y ^ - ^ C (Pimstone et al, 1971b).
CO release, from heme by gas chromatography (Cavallin-Stahl et al, 1978;
Sunderman & Downs, 1979).
226

ASSAYS OF HEME OXYGENASE A CTIVITY

Seven procedures for assay of HO activity are summarized in Table 2.
Spectrophotometry assays involve either measurements of heme consumption
(based upon spectrophotometry of pyridine hemochromogen) or measurements of
biliverdin or bilirubin production. These spectrophotometric techniques
are relatively insensitive. Radiometric assays involve (a) measurement of
55
Fe liberated from heme that has been labelled with radioactive iron; (b)
measurement of bilirubin­*N? produced from heme which has been labelled
1 11+
with '*C in the pyrrole rings; or (c) measurement of C0 released from
heme which has been labelled with *^C in the methene bridges. These radio­
metric techniques are sensitive and specific, but the labelled substrates
are difficult to prepare and are not commercially available. In the
author's experience, the most sensitive, convenient and practical approach
to HO assay involves gas chromatography of CO released from heme, as first

Table 3 ­ ASSAY OF HO ACTIVITY BY CO RELEASE (Sundlerman & Downs, 1979)

Procedure: Microsomes are incubated in the reac tion mixture at 37°C. A

blank contains the same ingredients, but HO is inhibited with p­
hydroxymercuribenzoate.
Reaction Mixture Conc/2.0 ml Purpose
Fe­heme 30 nmol Substrate
Bovine albumin 0. 5 mg To bind heme
Desferoxamine 10 uraol To bind Fe
Oxyhemoglobin 0.1 mg To bind CO
Glucose­5­PO^ 1.7 umol • NADPH production
NADP 1.6 ymol ··
Mg[II] 4 μιηοΐ ·* »«
Glucose­6­PO|4­dehydrogenase 1 IH *· It

ΡΟΐψ buffer 200 yraol pH 7.A

Microsomes 0. 5 mg of Source of HO & NADPH­
protein cyt. P­450 reductase
Release of CO: A fter 10 min incubation, the reaction is stopped with p­
hydroxymercuribenzoate (2 pmol/O.l m l ) . Aliquots (0.5 ml) of reaction
and blank tubes are added to septum vials which contain 0.2 ml of
K3Fe(CN)g­sterox­citrate reagent, and incubated for 4 min with stir­
ring to release CO into head­space.
Gas Chromatography: The head­space is swept onto a train of 3 columns: (a)
drierite (25°C) to remove H 2 0; (b) molecular sieve (25°C) to separate
CO from CO2; and (e) Ni catalyst (325°C) to reduce CO to CHi*. The
carrier gas is H 2 /N 2 (2:3, v / v ) . CH^ is detected with an H2~flame ion­
ization detector, and the area under the CH,, peak is measured.
Computations : Heme oxygenase activity is expressed as pmol of CO formed/
sec/mg of microsoraal protein (pkat/mg).
 227

described by Cavaliin-Stahl et al (1978). The gas chromatographic proce-

dure of Sunderman and Dovne (1979) Is outlined In Table 3. Based upon the
present author's unpublished observations, renal HO activity In male Fis-
cher rats averages 0.87 pmol/sec/mg of microsomal protein (range - 0.25 to
2.3 pmol/sec/mg, N-12). This low HO activity can readily be measured by
the gas chromatographic procedure. The coefficient of variation of HO
assays In renal microsomes of untreated rats Is ±8% (within-run), and the
detection limit Is -0.05 pmol/sec/mg, expressed as HO activity that yields
a CO peak which Is 2X the noise-level of the recorder tracing of the gas
Chromatograph. Because gas chromatographic HO assays are a recent innova-
tion, published data on the effects of hormones, metabolites, and xenobi-
otlcs on HO activity are derived primarily from HO assays by spectrophoto-
metric or radiometric procedures.
PATHOPHYSIOLOGY OF HEME OXYGENASE
Tenhunen et al (1970) showed that HO activity in rat liver microsomes
is Increased 2-3X following splenectomy, increased 2-7X following iv Injec-
tion of hemoglobin or methemalbumin, and increased 3-5X during hemolytic
anemia Induced by erythrocyte antibodies or Phenylhydrazine. In brain, HO
activity Is responsible for xanthochromia that develops following cerebral
hemorrhage (Roost et al, 1972). HO in renal tubular cells provides a
defense against kidney damage by hemolysis (Pimstone et al, 1971a). Hemo-
globin In the glomerular filtrate is absorbed by renal tubular cells and
heme is degraded to bilirubin, so that hemoglobinuria does not occur until
the catabolic capacity of renal HO has been exceeded. Pimstone et al
(1971a) found that renal HO activity in rats increased 30-100X following
hemogloblnemla. Administration of cycloheximide, puromycln or actinomycin
D prior to iv injection of hemoglobin prevented the increase of renal HO
activity. HO In duodenal mucosa liberates iron from heme that is derived
from myoglobin and other hemoproteins in the diet, and therefore HO may
play a critical role in iron nutrition (Raffin et al, 1974).

Bakken et al (1972) studled endocrine and metabolic Influences on HO

activity in post-mitochondrial supernatants of rat liver and spleen. Fast-
ing for 3 days Increased hepatic HO activity 3X, while starvation beyond
this period led to gradual decline in HO activity. Hypoglycemia induced by
Injections of insulin or mannose increased hepatic HO activity 7X. The
stimulatory effect of Insulin on hepatic HO activity was abolished by con-
comitant administration of glucose. Parenteral treatments with glucagon,
dlbutyryl cycllc-AMP, arginine or epinephrine Increased hepatic HO activity
2X, 3X, 5X, and 6X, respectively, while thyroxine and Cortisol were inef-
fective. HO activity in spleen was not affected by starvation or by admin-
istration of any of the hormones.
228

Thaler et al (1972) measured HO activities in liver and spleen of

fetal, neonatal and adult rats. In fetuses from 15 to 21 days of gesta-
tion, hepatic HO activity averaged 2X mean adult activity. Hepatic HO
activity increased rapidly during the first postnatal day and reached 4X
mean adult activity by 7 days of age. Thereafter, hepatic HO activity
declined to adult levels by weaning. Splenic HO activity was relatively
low during the fetal and postnatal period, compared to HO activity in adult
spleen. However, in adult animals, splenic HO activity averaged 11X the
hepatic activity. In newborn rats which were fasted for 3 or 6 hours,
hepatic HO activity Increased 2X and 3X, respectively, compared to fed
controls. Splenic HO activity was not affected by fasting.
INDUCTION OF HEME OXYGENASE ACTIVITY BY XENOBIOTICS
Xenobiotic stimulation of HO activity was first reported by Tenhunen
et al (1970). They found that administration of zymosan (a cell-wall prep-
aration from S. cereviseae yeast) to rats by 4 iv injections at 48 hour
intervals caused hepatic HO activity to increase 10X. The stimulatory
effect of zymosan was not solely attributable to adrenal secretion of epi-
nephrine or glucocorticoids, since adrenalectomy had little effect on
zymosan-mediated induction of hepatic HO activity. Schacter and Mason

Table 4 - EFFECTS OF ORGANIC XENOBIOTICS ON LIVER HEME OXYGENASE ACTIVITY

Investigators Species Compound, Route & Dose* Heme Oxy-

(mg/kg) & no. of doses genaset

Tenhunen et al (1970) Rat zymosan iv 30 x4 10.4

Schacter & Mason (1974) Rat phénobarbital ip 40 x4 0.9
benzpyrene ip 20 x4 1.0
methylchol- ip 13 x4 0.6
anthrene
pregnenolone- po 50 x3 0.7
carbonitrile
Gemsa et al (1974) Rat endotoxin ip 0.5 xl 3.9
De Matteis (1976) Rat iron-dextran ip 50 8.0
Maines & Kappas (1976c) Rat dimethyl- ip ? x3 3.0
sulfoxide
Yoshida et al (1978) Mouse OK-432 ip 100 5 l.R
PSK ip 100 1.6
Eiseman & Alvares (1978) Rat gold sodium ip 755 1.5
thiomalate
Burk & Correia (1<>79) Rat diethyl maléate IP 517 3.0
Guzelian & Elshourbagy (1979) Rat bromobenzene ip 785 10-15

*16 to 24 hr from last injection to death (except as indicated).

^Multiple of mean activity in controls.
§4 hr from injection to death.
 229

(1974) reported that Inducers of hepatic cytochrome P­450 (including phéno­

barbital, 3­methylcholanthrene, 3,4­benzpyrene and pregnenolone­16a­carbo­
nitrile) did not increase hepatic HO activity in rats. Instead, 3­methyl­
cholanthrene and pregnenolone­lfia­carbonitrile caused modest reductions of
hepatic HO activity. Several organic xenoblotics have been found to in­
crease HO activity in livers of rodents (Table 4 ) , including endotoxin,
iron­dextran complex ("Imferon"), dimethylsulfoxide, two anti­tumor drugs
derived from bacterial polysaccharides ("OK­432" and " P S K " ) , gold sodium
thiomalate, diethyl maléate, and bromobenzene. Bromobenzene is the most
potent organic compound for induction of hepatic HO activity that has been
identified. Guzellan and Elshourbagy (1979) found that ip administration
of bromobenzene (5 mg/kg) to rats increased hepatic HO activity 15X after
20 hours, but did not affect HO activity in kidney or spleen. The increas­
ed hepatic HO activity was accompanied by diminution of hepatic cytochrome
P­450 concentration and Increased rate of hepatic degradation of h e m e ­ ^ C
to ''♦CO. Bromobenzene stimulation of hepatic HO activity was blocked by
cyclohexlmide hut not by actinomycin D, suggesting that bromobenzene stimu­
lates _de_novo HO synthesis.at the step of translation. Furthermore, induc­
tion of hepatic HO activity was unaffected when bromobenzene hepatotoxicity
was blocked by pre­treatment rats with ß­diethylaminoethyldiphenylpropyl­
acetate ("SKF­525A ") or when hepatic eulfhydryl content was supplemented by
administration of cysteine. Guzelian and Elshourbagy (1979) concluded that

Table 5 ­ EFFECTS OF METAL COMPOUNDS ON LIVER: HEME OXYGENAS*: ACTIVITY

Investigators Species Compound, Route & Dose Heme Oxygenase^

(praol / k g ) *

Maines & Kappas (1976a) Kat Coni] sc 250 8.0

Cd[II] 8C 2.5 6.7
Cu[II] SC 12.5 5.1
Hg[II] 8C 250 4.5
Zn[II] 8C 100 4.3
Fe[II] se 250 3.6
Mn[II] se 250 3.1
Pb[II] IP 250 2.9
Ni[II] se 250 2.8
Cr[IIl se 250 2.5
Maines & Kappas (1977b) Rat Sn[II] se 250 5.5
Pt[II] se 250 6.0
Maines (1979) Rat Au[IV] 8C 125 2.3
Woods et al (1979) Rat Infili] ip 180 4.7
Yoshida et al (1979) Mouse Cd[II] IP 13.7 31.
*lfi to 24 hr from injection to death.
"^Multiple of mean activity in controls.
230

bromobenzene ­ is a novel inducer of hepatic HO activity, differing from

other non­heme substances in potency and specificity for the liver, and in
utilizing mechanisms which require neither production of hepatotoxicity,
depletion of hepatic glutathione, or sensitivity to actinomycin D. Hepatic
HO activity is induced following parenteral administration of various toxic
metals to rodents as listed in Table 5. Maines and Kappas (1977b) reported
substantial differences in the relative effects of these metals in various
organs. A s an example, Ni[II] is of the most potent inducers of renal HO
activity, but is one of the least potent inducers of cardiac HO activity
(Maines & Kappas, 1977a). A s another example, Hg[II] is the most potent
inducer of cardiac HO activity, but is considerably less potent in kidney
(Maines & Kappas, 1977a). Co[II] is the most potent metal for induction of
hepatic HO activity in rats (Maines & Kappas, 1177b), but has no effect on
HO activity in pig alveolar macrophages (Shibahara et al, 1978). Maines
and Kappas (1975a,1976b) showed that CoTIIJ­induction of hepatic HO acti­
vity in rats is (a) attended by diminution in hepatic concentration of
microsomal cytochrome P­450, (b) inhibited by prior administration of acti­
nomycin D or puromycin, consistent with· an effect at the level of tran­
scription, and (c) potentiated by prior administration of A IA . Maines and
Kappas (1975b) observed that administration of Co[II] to pregnant or
nursing dams did not cause any increase of HO activity in fetuses or neo­
nates. Yoshida (1979) reported that pre­treatment of mice with cyclohexi­
mide almost completely blocked Cd[II]­mediated increase of hepatic HO acti­
vity, whereas pre­treatment with actinomycin D was less effective, suggest­
ing that HO induction by Cd[II] may occur at a post­transcriptional step.
Hino et al (1979) deduced that the active site of HO is exposed on the
cytoplasmic surface of smooth­surfaced microsomal membranes in liver of
Co[II]­treated rats.
HEME OXYGENASE ACTIVITY IN HTIMAN TISSUES

Tenhunen and Vuopio (1971) were the first investigators to demonstrate

HO activity in human liver and spleen. Only a few measurements of HO acti­
vity in biopsy specimens of human tissues have been reported (Table 6 ) .
■Mahonen et al (1976) and Schacter et al (1979) observed increased HO acti­
vity in bone marrow and spleen of patients with certain hematological
diseases, including hemolytic anemias and lymphoproliferatlve disorders.
Pasternak and Tenhunen (1976) and Lahdevirta and Tenhunen (1977) measured
HO activity in splenic and renal biopsies from patients with chronic renal
insufficiency to ascertain if diminished concentrations of serum bilirubin
in these patients might be 'a manifestation of diminished HO activity. The
experimental results did not support this hypothesis, since mean HO activi­
ties in spleen and kidney of patients with renal diseases did not differ
 231
Table 6 - ASSAYS OF HEME OXYGENASE IN HUMAN TISSUES

Investigators Tissues Pathological Observations

Conditions

White et al (1972) Spleen Splenic PO has major role

In heme catabolismi
Mahonen et al (1076) Bone Hematological Increased HO activity in
narrow disorders hemolytic & secondary
anemias ; chronic leukeralas
Pasternak & Spleen Chronic renal Normal HO activity
Tenhunen (1976) insufficiency
Lahdevlrta & Kidney Various Normal HO activity
Tenhunen (1977) nephritldes
Schacter et al (197°) Spleen Hematological Increased HO activity in
disorders hemolytic anemias, chronic
lymphatic leukemia, &
Hodgkin's disease

significantly from corresponding values for HO activities In spleen and

kidney from control subjects·
DISCUSSION
Since HO is the rate-limiting enzyme in the pathway of heme degrada-
tion, HO activity regulates the formation of bilirubin, CO, and Fe from
heme. Coordinated release of these 3 products of the HO reaction may
explain the significant correlations observed In healthy adults by Lynch
and Moede (1972) between (a) serum unconjugated bilirubin concentrations,
(b) endogenous CO production rates, and (c) serum iron concentrations.
Significant correlation between serum total bilirubin concentrations and
endogenous CO production rates In healthy adults was noted by Lundh et al
(1972), and significant correlation between serum total bilirubin concen-
trations and eerum iron concentrations In healthy adults was reported by
Morrison et al (1979).
Bakken et al (1972) suggested that increased hepatic HO activity may
be responsible for increased concentrations of serum bilirubin which occur
In human subjects during fasting or hypoglycemia. Diminished rates of
glucuronide conjugation and impaired biliary excretion of bilirubin also
occur during fasting and hypoglycemia, and may contribute to hyperbilirubi-
nemia (Barrett, 1971; Bloomer, 1971; Bensinger et al, 1973). However,
Impaired glucuronide conjugation could not account for the enhancement of
endogenous CO production which occurs in healthy adults during caloric
restriction (Lynch et al, 1972). The present author speculates that induc-
tion of HO activity by progesterone may account for cyclical changes in
rates of endogenous CO production in healthy young women during the men-
strual cycle (Lynch & Moede, 1972; DelIvoria-Papadopouloe et al, 1974).
232

Maines and Kappas (1975b) proposed that certain instances of human

neonatal jaundice may result from excessive production of bilirubin by the
HO system, in addition to impairment of bilirubin conjugation by the hepa­
tic glucuronyl transferase system. Bartoletti et al (1979) reported signi­
ficant increase in mean C O production in 6 neonatal infants who developed
peak concentrations of serum bilirubin in excess of 200 ţnnol/1. In these
infants, polycythemia, starvation, and Rh or ABO hemolytic disease were not
present, and the reticulocyte counts were normal. Bartoletti et al (1979)
considered that mild hemolysis, ineffective erythropoiesis, or increased
catabolism of hepatic heme might be pathogenetic mechanisms in these
infants with idiopathic neonatal hyperbilirubinemia. Necheles et al (1976)
found significant correlation between blood carboxyhemoglobin concentra­
tions and serum bilirubin concentrations on the third postnatal day in 250
consecutive full­term, normal infants. Necheles et al (1976) inferred that
increased rate of heme catabolism plays an important role in "physiologic"
neonatal jaundice.
Drugs (such as Imferon and gold sodium thiomalate) and toxic suhstan­
ces (such as bromobenzene and cobalt chloride) which cause pronounced
induction of hepatic HO activity in rodents may be predicted to increase
the rates of endogenous C O production and bilirubin formation in human
subjects. The present author speculates that induction of hepatic HO acti­
vity might account for increased carboxyhemoglobin concentrations in blood
samples from men exposed to inhalation of dichloromethane ( Stewart et al,
1972). Insofar as the author can ascertain , HO activity has not yet been
detected in human blood cells or plasma. Such measurements may soon become
feasible, owing to improved sensitivity of HO assays.
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234
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 235

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236

USE OF LABORATORY TESTS FOR ESTIMATION OF PATIENTS'ALCOHOLIC INTAKE

A.Bagrel 0 , B. Herbeth" and D. Barrucand00

0
Laboratoire du Centre de Médecine Préventive (Dir.Pr G.Siest), 2 Avenue
du Doyen Jacques Parisot 54500 Vandoeuvre-lès-Nancy, France
00
Chef du Service de Médecine Interne orientée vers l'alccologie
Centre Hospitalier Régional de Nancy, 29 Avenue du Maréchal de Lattre de
Tassigny, Case Officielle n°34, 54037 Nancy Cedex, France

ABSTRACT
After having received the principle mechanism of alcohol metabolism,
the authors reaffirmed the high correlation between GGT and an increasing
consumption of alcohol. Moreover the variations of this parameter before
and after the detoxication cure, reconfirm the effect of alcoholic con-
sumption on this enzyme. The authors demonstrate a significant decrease
in the levels of AlAT in males and in females, when the declared consump-
tion of alcohol is higher than class 22-44 g of alcohol daily. In the
males, the mean comparison showed a significant decrease in the level of
ceruloplasmine according to increased alcohol consumption.
However, if it is necessary to have as exact as possible an estima-
tion of a patient's alcoholic intake, still more discriminative biolo-
gical indices of alcohol consumption must be sought.

INTRODUCTION
It has been shown that habitual ethanol exposure results in profound
biochemical and morphologic changes (Lieber, 1975). Ethanol can be synthe-
tized endogenously in trace amounts but it is primarily an exogenous
compound that is readily absorbed from the gastro-intestinal tract. Only
2 to 10 % of the amount absorbed is eliminated through the kidneys and
lungs. The rest must be oxidized in the body, principally in the liver.
The main hepatic pathway for ethanol disposition involves alcohol
deshydrogenase, an enzyme that catalyses the conversion of ethanol to
acetaldehyde. Hydrogen is transfered from ethanol to the cofactor
nicotinamide adenine dinucleotide (NAD), which is converted to its
reduced form (NADH). The acetaldehyde which is newly produced loses
hydrogen and is converted to acetate. In addition, ethanol can also be
metabolized by an accessory pathway that requires NADPH as a cofactor and
is localized in the endoplasmic reticulum (figure 1). The rate of the
non ADH mediated oxidation varied depending on the concentrations of
ethanol used, from 20 to 25 %, to half or more of the total ethanol
metabolism.
 237

HYPERURICEMIA ■ HYTERLACTACIDEMIA E
MUCOS

c
'­fc
%
^ROCHOWMQ,
PYRUV»tf · * ­ '

POI»« METuarrts HYPERLIPEMIA

Figure 1: Metabolism of ethanol in the hepatocyte (Lieber, 1977)

The various metabolic effect of ethanol can be attributed to the

NADPH generation by the ADH pathway or to the interaction with other mi­
crosomal functions associated with metabolism by the microsomal ethanol
oxidizing system (MEOS). Some of the effects are also due to the metabo­
lites of ethanol, acetaldehyde and acetate.
It was therefore proposed that the altered NADH­NAD ratio is respon­
sible for a number of metabolic abnormalities associated with alcohol
abuse. The enhanced NADH­NAD ratio reflects itself in a increased lactate­
pyruvate ratio that results in hyperlactacidemia because of both decreased
utilization and enhanced production of lactate by the liver. The hyper­
lactacidemia contributes to acidosis and also reduces the capacity of the
kidney to excrete uric acid, to secondary hyperuricemia. The increased
NADH­NAD ratio also raises the concentration of α­glycerophosphate that
favors hepatic triglycerides accumulation by trapping fatty acids. À
dramatic but uncommun complication of acute alcohol abuse is severe hypo­
glycemia, due in part to the blockage of hepatic glucuneogenesis by
ethanol.
238

After chronic alcohol consumption the membranes of the rough endo­

plasmic reticulum appear decreased in electron microscopy. One of the main
function of the rough endoplasmic reticulum is protein synthesis (Lieber,
1977).
In view of the numerous metabolic disturbances caused by an excessive
intake of alcoholic beverages, it seemed necessary to investigate the most
sensitive and discriminating indicators that are directly related to a
habitual intake of alcohol. Accordingly, we paid particular attention to
GGT variations and to certain plasmatic proteins, such as cerulopläsmin
and α 1 antitrypsine.
MATERIAL AND METHODS
The population sample consisted of people examined at the Center for
Preventive Medicine (Vandoeuvre­Les­Nancy). The individuals included in
the sample were not medically selected, but constituted the "unselected"
clientele of a health examination Center. We also studied subpopulation
of males between the ages of 19 and 59, all of whom were patients in
Professor Barrucand's clinic for alcoholic treatment. They were medically
examined upon admission and again upon discharge following a three weeks
hospitalization period. For the study carried out at the CMP, GGT activity
was measured on a Greiner GSA II by colorimetrie methods at 37°C. For the
other results determined at the hospital a method adapted on an auto­
analyzer centrifuge GEA MSEC was used.

In order to determine the levels of the specific proteins which

were studied, a laser nephelometer (Behring) was used for ceruloplasmine
and nephelemeter ICS Beckman for the o 1 antitrypsine.
RESULTS
The alcohol consumption of the patients was based on a questionnaire.
Their answers are arranged in a classification of 6 increasing levels
(table I ) .
TABLE I ­ ALCOHOL CONSUMPTION

1. Little or none
2. Less than 1/4 liter per day
3. Between 1/4 and 1/2 liter per day
4. Between 1/2 and 1 liter per day
5. Between 1 and 2 liters per day
6. More than 2 liters per day
 239

GGT activity in all of the subjects confirms that the median and dis-
persion of the values increase as the avowed consumption of wine increases
(figure 2 ) . This variations was also observed in subjects aged between
20 to 30 years old, who where selected from a male population ranging from
20 to 100 years. The study of the 5, 50 and 95 percentiles confirm this
increase in GGT activity parallel with an avowed increase of wine
consumption (table II) .

GOT (D I )

MEN
A(* a o . l o o

N. 3787 SK» 4417 »413 «»I

ol&aa of ooMumptlon

Figure 2: Effect of alcohol consumption on GGT activity

240

TABLE II - CHANGES IN THE PERCENTILES 5, 50 AND 95 OF GGT AS A FUNCTION

OF REPORTED CONSUMPTION OF WINE AMONG MEN AGED 20 TO 29 YEARS

Percentiles
Reported consumption Ν 5 50 95
(wine)

1 1 446 9.41 19.68 51.94

2 872 11.33 20.97 67.71
3 740 12.04 25.70 80.29
4 203 12.74 36.68 170.50
5 11 13.75 46.00 115.50

It is equally interesting to study the variations of GGT values

higher than a given value, as for example 61 UI in the males between
20 to 30 years old as a function of the declared consumption of wine
(table III).
TABLE III - FREQUENCE OF~~SUBJECTS HAVING A GGT VALUE BELOW 61 UI

Class of consumption

0.961
0.937
0.901
0.700

We observe that 6 to 30 % of the individuals have a GGT higher than

61 UI when their declared consumption of wine less than 1/4 liter opposed
to people who drink between 1/2 and 1 liter per. day. We have not spoken
of class 5 due to a very low number (N = 11).
Histograms plotted for a sampling of 14 166 male patients at the
Center for Medicine Preventive are presented in figure 3.
It is interesting to note the values of GGT activity before and
after a desintoxication cure. At the time of admission to the clinic, a
histogram of the GGT activity of 86 males aged between 19 and 59 showed
a very high mean: 102.93 and a large dispersion of values: 10 subjects
showed GGT higher than 260 UI. However, about 50 % of the patients had a
GGT lower than 61 UI (figure 4 ) .
 241

■Un > ( · 1 0 . too

Ν . 14 I M
ft % . 11.»»
ao %. «β.βο (rn.44.it . » . » . M )
M t . 184.0

Figure 3: Histogram of CGT activity in an unselected population

OPT (bafora traalmaiU)

Man * ( * 19.68 (m.40.4. 4.8,8)

Ν .88
m . 108.83
• .181.M

­ ­ ­ ­ ­ β S ** S

­ s ; ; s § s s s s
Figure 4: Histogram of GGT activity before a desintoxication cure

After three weeks hospitalization (figure 5 ) , the mean of the dis­

tribution approaches that of our non selected population at the CMP. The
dispersion is different and we can point out that 19 individuals in 84
(12 Z) had a GGT higher than 61 UI (16.2 Ζ in the group at the CMP).
242

OOT (arter treatment 3 week*)

Men age 19-59 ( m . 4 0 , 4 - a . 8.Θ)

Ν.84
m . S3,27
a . 62.21

s
= . Ş ς ş 3 S S E ! ij Ι °° τ
3 ; S 5 S 3 Ï S S S H
Figure 5: Histogram of 66T a c t i v i t y afiter a d e s i n t o x i c a t i o n cure
As f a r as s p e c i f i c p r o t e i n s are concerned t h e l e v e l s of A.AT in a
subpopulation of 675 i n d i v i d u a l s o l d e r than 20 (319) males. The d a i l y
consumption of alcohol was c a l c u l a t e d i n grams according to the following
classification:
­ l e s s than 10 g
­ 10 t o 22 g
­ 22 t o 44 g
­ 44 t o 88 g
­ more than 88 g alcohol in a d a i l y b a s i s ( f i g u r e 6)
We were thereby able t o demonstrate a s i g n i f i c a n t e f f e c t of h a b i t u a l
alcohol consumption i n the l e v e l s of A.AT. Indeed, we confirmed a s i g n i f i ­
cant decrease i n the l e v e l s of A AT i n males ( t » 3 . 0 8 , ρ > 0.01) and in
females ( t = 3 . 9 3 , ρ > 0 . 0 0 1 ) , when the d e c l a r e d consumption of alcohol
i s h i g h e r than c l a s s 22­44 g of alcohol d a i l y . However, an alcohol i n t a k e
h i g h e r than 44 g leads t o an i n c r e a s e in the l e v e l s A.AT compared with
values observed i n t h e preceding c l a s s .
We were a l s o i n t e r e s t e d by another s p e c i f i c p r o t e i n namely c e r u l o ­
plasmine. This p r o t e i n was determined on a p o p u l a t i o n of 639 i n d i v i d u a l s
of both sex and o l d e r than 4 y e a r s . In the males, the mean comparison
(determined by s t u d e n t ' s t e s t ) showed a s i g n i f i c a n t decrease in the l e v e l
of ceruloplasmine according t o i n c r e a s e d alcohol consumption (a < 0 . 0 1 ) .
 243

In females, however, no s i g n i f i c a n t e f f e c t was observed (figure 7).

i/i

i.*o

i.T»

U t

t.M IA·

10 U 44 88 Aloohol oonaumptlon 10 88 44 88 Alcohol eanaumpUan

t/»«ar
Figure 6: Effect of alcohol consumption on A· Antitrypsin levels

f/l

01M
0,·!· ati·
o,*oa o.ao«

O.IU JIM* O.IW

0 10 44 88
Aloohol oooBUmaUon/dvy Alcohol oonaumptkio/dagr

Figure 7: Effect of alcohol consumption on ceruloplasmine levels

244

CONCLUSION
We have reaffirmed Che high correlation between GGT and an increasing
consumption of alcohol. Moreover the variations of this parameter before
and after the detoxication cure, reconfirm the effect of alcoholic con­
sumption on this enzyme. Nevertheless as a reported in an earlier article,
GGT, by itself, appears to have an insufficient discriminative power.
(Bagrel, 1979). However, after having tested different variables (iron,
GGT, mean erythrocyte volume (MCV), mean erythrocyte hemoglobin concentra­
tion, urea, uric acid and alanine aminotransferase). We showed that the
three variables that correlated most significantly with alcohol consum­
ption were GGT, MCV and the use of tobacco.
The work involving AjAT and ceruloplasmine permits us to confirm
that alcohol, as a result of its effect in the rough endoplasmic reticu­
lum, leads to a decrease in protein synthesis.
The variations in GGT and the decrease of certain specific proteins
may be investigated in a multiparametric analysis. However, critical
evaluation leads us to conclude that still more discriminative biological
indices of alcohol consumption must be sought.
We have two motivating reasons to establish the real alcoholic
consumption in our population:
1. Firstly, due to the risk which chronic alcoholism constitutes for the
individual himself, for his family and the immediate environment
2. Secondly, before interpretation of the results of laboratory tests,
it is necessary to have as exact as possible an estimation of a patient's
alcoholic intake.
REFERENCES
Bagrel, Α., D'Houtaud, Α., Gueguen, R. and Siest, G.: Relations between
reported alcohol consumption and certain biological variables in an
"unselected" population. Clin. Chem., 25: 1242­1246, 1979.
Lieber, C S . : Interference of ethanol in hepatic cellular metabolism.
Ann. N.Y. Acad. Sci., 252: 24­50, 1975.
Lieber, C S . : Metabolic aspects of alcoholism. MTF ed., 1977.
 EFFECTS OF DRUGS"ON THYROID FUNCTION TESTS

Kristian Lievendahl
Department of Clinical Chemistry, University of Helsinki,
and Minerva Foundation Institute, Helsinki 29, Finland

The list of pharmacological agents that interfere with laboratory

tests for the assessment of thyroid function is quite extensive. These
drug effects are usually mediated through pharmacological mechanisms but
there are also drugs that interfere with the analytical methods. Failure
to recognize drug interference can result in misinterpretations and even
the wrong treatment. This review will deal mainly with the effects of
various frequently used therapeutic and diagnostic agents, but information
on the effects of some important addictive and toxic substances has also
been included.
ANTITHYROID DRUGS
Antithyroid drugs interfere with thyroid hormone biosynthesis
thereby reducing the levels of circulating thyroxine (T.) and triiodo­
thyronine (Tß). This effect is utilized in the treatment of hyper­
thyroidism and can, when drug administration is inappropriate, result in
hypothyroidism. A ntithyroid drugs may act by blocking thyroidal uptake
of iodide (e.g. Perchlorate, fluoride, nitrate) or by inhibiting the
organic binding of iodide to thyroglobulin (e.g. thioureas, thiouracils,
mercaptoimidazoles, sulphonamides, sulphonylureas, phenylbutazone,
p­aminosalicylic acid). In addition to its intrathyroidal effect propyl­
thiouracil reduces the peripheral conversion of T, to T,, an effect
accompanied by an increased concentration of the metabolically inactive
reverse triiodothyronine (rT.) (Oppenheimer et al., 1972; Westgren et al.,
1977). The peripheral action of propylthiouracil appears advantageous in
the treatment of hyperthyroidism.
Whether naturally occurring goitrogenic substances such as thio­
oxazolidones (e.g. S­vinyl­2­thiooxazolidone), isothiocyanates and thio­
cyanate are of clinical importance is still an unresolved question
(Langer and Greer, 1977; Elfving and Peltola, 1978).
IODINE­CONTAINING DRUGS
Thyroxine forms a predominant fraction of the serum protein­bound
iodine (PBI). Normally, about 10 Ζ of the PBI is made up of iodothy­
246

ronines other than T., mono­ and diiodotyrosines and iodoproteins, the
proportion of which can increase in various thyroidal and non­thyroidal
diseases. Iodinated drugs that bind to serum proteins, spuriously elevate
the serum PBI. The number of such drugs is very large and includes, among
others, expectorants, local disinfectants, intestinal antiseptics and
radiographic contrast media. The effect on the PBI can last from a few
days to many years depending on the amount of drug ingested, the affinity
between drug and serum proteins, and the rate of drug elimination.
Urographie and other intravenous contrast media usually cause false PBI
values for a few weeks, whereas the effect of cholecystographic contrast
media and clioquinol may last for several months. After the use of oily
contrast media elevated PBI values can persist for many years (see
reviews by Liewendahl, 1968; Acland, 1971).

A falsely elevated serum PBI due to iodinated drugs is no longer a

diagnostic problem in laboratories where specific assays for thyroid
hormones have replaced the serum PBI. There are, however, iodinated drugs
influencing the serum thyroid hormones via effects on their peripheral
metabolism. Amiodarone, an antiarrythmic and antianginal drug, and the
oral cholecystographic contrast media iopanoate and ipodate were found to
lower T 3 and elevate rT, (Burger et al., 1976; Bürgi et al., 1976; Wu
et al., 1978). These effects are attributable to decreased phenolic ring
and increased or normal tyrosyl ring monodeiodination of T,. The small
increase in serum T, also observed after administration of these drugs,
could result from a decreased T, degradation rate or an increased thy­
roidal secretion of T, due to increase pituitary release of TSH. Since
amiodarone did not affect the free T. fraction the decrease in T, must be
accompanied by a decrease in free Τ of similar magnitude. There were
concomitant increases in free T, in patients with iopanoate­induced rises
in T 4 .

Deiodination of organic drugs in the organism increases the concen­

tration of serum inorganic iodide. The remarkable differences in the rate
of deiodination of the various iodinated drugs seem to be related to dif­
ferences in their serum and tissue binding (Liewendahl and Wägar, 1970).
A pharmacological concentration of iodide suppresses the thyroid accumula­
tion of a radioiodide tracer therefore invalidating the measurement of
thyroidal radioiodide uptake as a diagnostic procedure. However, the
diagnostic accuracy of the tracer test is already limited due to large
 247

interindividual variations in dietary iodide and the use of this test has
declined as a result of the introduction of specific hormone assays. It
must also be recognized that chronic administration of excess iodide
occasionally results in hypothyroidism (Wolff, 1969), particularly in
patients with an underlying thyroid abnormality such as autoimmune thy-
roiditis (Liewendahl and Turula, 1972), and that excess iodide can pre-
cipitate hyperthyroidism in susceptible individuals (Liewendahl and
Gordin, 1974).
ANTICONVULSANT DRUGS
In patients given diphenylhydantoin (DPH) the serum PBI is depressed
(Oppenheimer et al., 1961; Wolff et al., 1961). This observation has been
verified in subsequent studies using T, competitive protein-binding (CFB)
procedures (Stjernholm et al., 1975; Liewendahl and Majuri, 1976) and T^
radioimmunoassay (RIA) (Liewendahl et al., 1978). It is particularly
interesting that although these patients also have decreased serum free
T, levels they remain eumetabolic. The decrease in T. and free T_ was
found to be quite small, therefore providing an explanation, at least
partly, why clinical euthyroidism is maintained and why there is no signi-
ficant effect on serum TSH or the TSH response to TSH-releasing hormone
(TRH) (Liewendahl and Majuri, 1976; Peyma et al., 1977).
High concentrations of DPH displace T, from its binding sites on T,-
binding globulin (TBG) (Wolff et al., 1961; Oppenheimer and Tavernetti
1962). However, the in vitro displacing effect of therapeutic concentra-
tions of DPH is much too small to explain the average decrease in T, and
free T, of about 25 X seen in patients (Liewendahl et al., in press).
There is now, indeed, substantial evidence that the effect of DPH on
serum T, is mainly due to an accelerated T. metabolic clearance rate
(Larsen et al., 1970). DPH is a potent stimulator of hepatic microsomes
(Hvidberg and Dam, 1976) and the increased T, clearance rate is probably
due to induction of T, metabolizing enzymes. In rats DPH stimulates the
hepatic enzyme converting T, to T, (Hiifner and Knöpf le, 1976). Assuming
that DPH also increases the production of T. from T, in man via stimula-
tion of the converting enzyme the almost normal serum T, despite an -
accelerated T- metabolic clearance rate, becomes understandable.
Carbamazepins (CBZ) was recently found also to decrease serum total
and free T,, whereas the effect on total and free T 3 was small (Liewen-
dahl et al., 1978; Rootwelt et al., 1978). CBZ is a stimulator of hepatic
248

microsomal enzymes and therefore the effect of CBZ is presumably due to a

mechanism similar to that postulated for DFH.
Phénobarbital (PB), although a strong inducer of microsomal enzymes,
does not significantly affect serum thyroid hormones in euthyroid subjects
and neither does primidone (PD), the main metabolite of which is PB, in
spite of the fact that PB accelerates T, and T. metabolic clearance rate
(Cavalieri et al., 1973; Liewendahl et al., 1978). However, in hyper­
thyroid patients and in hypothyroid subjects on T, replacement therapy PB
decreases the concentration of T, (C avalieri et al., 1963). These obser­
vations show that the normal thyroid gland is able to compensate for the
PB­enhanced metabolism of hormones by increasing thyroidal secretion,
whereas in situations with a fixed supply of T, administration of PB or
PD will reduce the serum levels of thyroid hormones. It is also inter­
esting to note that 'PB and PD significantly depressed the serum levels of
rT. and 3,3'­diiodothyronine (3,3'­T„), probably because of an accelerated
metabolic clearance race that could not be compensated for by increased
thyroidal secretion due to the fact that rT_ and 3,3'­T9 derive almost
entirely from the peripheral metabolism of thyroid hormones with very
little coming from thyroidal secretion (Liewendahl et al., in press).
STEROIDS
Increased estrogenic activity, whether due to pregnancy or oral
contraception, is a well­established cause of increased serum TBG result­
ing in elevated concentrations of T,, T, and, to a lesser degree, rT_.
This effect is considered to be due to estrogen­stimulated hepatic syn­
thesis of TBG. Medication with androgens or anabolic steroids can moder­
ately reduce the levels of serum thyroid hormones, but whether this is
the result of decreased synthesis or increased catabolism of TBG and/or
Τ.­binding prealbumin (TBPA) appears to be unclear. C linical euthyroidism
is maintained in patients administered excess sex steroids because,
­according to the free T, (T,) hypothesis, under steady­state conditions
the concentration of free thyroid hormone is virtually uninfluenced by
alterations in plasma protein binding.
Administration of pharmacological doses of glucocorticoids reduces
serum T_ and increases serum rT_ (Duick et al., 1974; Chopra et al., 1975).
This effect is due to a decreased peripheral conversion of T, to T. and
an increased production of rT_ from T,. Glucocorticoids also induce a
decrease in serum Τ,, at least partly due to a decrease in serum TBG
 249

(Gamstedt et al., 1977). Low serum T appears to result also from a

suppressive effect of glucocorticoids on the pituitary secretion of TSH
and therefore a suppression of the thyroidal secretion of T, (Wilber and
Utiger, 1969; Faglia et al., 1973). Glucocorticoids also decrease free
T. but has little or no effect on free T,. Mineralocorticoids do not
alter the levels of serum iodothyronines (Westgren et al., 1977).
ANTICOAGULANTS
When therapeutic doses of heparin are added to serum in vitro there
is no effect on the thyroid hormone assays, but, when heparin is admin-
istered in vivo there is an increase in the free T, and free T, concen-
trations accompanied by a reciprocal decrease in serum TSH. This effect
on the free hormone levels appears to result from the displacement of
thyroid hormones from their plasma binding sites due to a rise in free
fatty acids (FFA) (Hollander et al., 1967). An increase in free T, was
also observed when the heparin-induced stimulation of lipoprotein lipase
causing the rise in FFA was blocked by prior injection of protamine,
indicating that heparin might stimulate some other factor(s) decreasing
plasma binding of thyroid hormones (Schatz et al., 1969). Elevated FFA,
whether heparin-induced or due to storage of serum at ambient temperature,
spuriously increase serum T, measured with the CPB assay but not with RIA
(Rootwelt, 1975; Liewendahl et al., 1976). Increased FFA also affect
other tests based on protein binding of thyroid hormones such as the T,
uptake test (T.U; a test for estimation of the number of unsaturated
thyroid hormone binding sites). The increase in T.U is small when anion
resin is used as the adsorbent of radiolabelled T. and not dextran par-
ticles (Sephadex), probably because of the higher capacity of the resin
than the Sephadex for trapping FFA (Liewendahl and Helenius, 1976). We
were also able to show that unsaturable FFA are much more effective in
raising T^ (CPB) and T.U than saturated FFA.
The T.U results when red cells are utilized as binders of radio-
labelled T- art increased following administration of dicoumarol (Hamolsky
et al., 1957), but results from subsequent in vivo and in vitro studies
on the effect of oral anticoagulants on the T.U resin and Sephadex tests
are conflicting. It appears, however, that the coumarin derivatives do
not usually significantly interfere with the T.U resin test (Quaife and
Matoole, 1967).
250

PSYCHOTROPIC DRUGS
Studies on the in vitro effects of benzodiazepine derivatives have
shown that diazepam and, to a lesser extent, chlordiazepoxide, by com­
peting with thyroid hormones for binding sites on TBG, raise the T, (C PB)
and T U tests (Schussler, 1971; El­Hazmi, 1975). Although the results of
in vivo studies are controversial the main bulk of evidence favours the
conclusion that therapeutic doses of these drugs have no significant
effect on thyroid function.
The tricyclic and tetracyclic antidepressant drugs amitriptyline and
mianserin did not affect serum total or free thyroid hormones (Liewendahl
et al., in press). Neither did treatment with the tricyclic antidepres­
sants chlorimipramine and nortriptyline influence the basal or TRH­stimu­
lated level of TSH (Widerlöv et al., 1978).
Lithium therapy can cause goitre and hypothyroidism but the exact
mechanism by which the lithium ion inhibits thyroid hormone biosynthesis
is still a matter of dispute (Blomqvist et al., 1977) . Patients with an
underlying thyroidal abnormality appear to be particularly susceptible to
lithium and in this respect the effect of lithium resembles that of excess
iodide. There is usually reversal of hypothyroidism after withdrawal of
lithium and iodide.
Raised serum PBI levels during treatment with perphenazine have not
been confirmed with methods specific for Τ,, and appear to have been caused
by iodine contaminants in some drug formulations (Mølholm Hansen and
Siersbaek­Nielsen, 1967). The phenothiazine drugs chlorpromazine and
thioridazine suppress the serum TSH response to TRH but do not affect
thyroid function (Lamberg et al., 1977). This effect might be due to the
alfa­adrenoreceptor blocking property of these drugs since phentolamine,
a specific alfa­adrenergic antagonist, also inhibits the TSH response to
TRH (Nilsson et al., 1974). Administration of L­dopa, or its metabolite
­dopamine, also inhibit the TSH response to TRH without affecting the serum
thyroid hormones (Spaulding et al., 1972). These and other studies with
central neurotransmitter agonists and antagonists indicate the existence
of stimulatory alfa­adrenergic and inhibitory dopaminergic physiological
mechanisms regulating the pituitary secretion of TSH (Scanlon et al.,1978).
Patients with alcoholic liver disease may have elevated serum free T,
and TSH, and low serum T, and free T, (C hopra et al., 1974) , but no acute
effects of ethanol on serum thyroid hormones and basal or TRH­stimulated
 251

serum TSH have been observed (Ylikahri et al., 1978). During the hangover
period, the prolactin response to TRH was totally blocked indicating that
withdrawal symptoms are associated with increased dopaminergic activity in
the hypothalamus (Ylikahri et al., 1978).
ADDICTIVE DRUGS
The use of heroin, its active metabolite morphine, or methadone can
result in increased serum T, and T. due, at least partly, to an increase
in TBG, whereas the free hormone concentrations remain essentially normal
(Webster et al., 1973; Bastomsky et al., 1977; Chan et al., 1979). The
increase in TBG is accompanied by a retarded T, metabolic clearance rate,
but the T, degradation rate is normal in accordance with the clinical
euthyroidism in narcotic addicts (Azzizi et al., 1974). Since hepatic
dysfunction in drug addicts was usually mild other pathogenetic mechanisms
responsible for the observed abnormalities in thyroid hormone metabolism
probably exist.
ENVIRONMENTAL TOXIC SUBSTANCES
The highly toxic tetrachlorodibenzo-p-dioxan (TCDD), a contaminant
in herbicides and pesticides, also seems to be an environmental risk
factor from the point of view of thyroid function: in the rat TCDD, a
potent inducer of hepatic microsomal enzymes, was found to increase
biliary excretion of T,, but not of T_, resulting in decreased serum T,,
increased TSH and goitre (Bastomsky, 1977).
Carbon disulphide (CS~) seems to be another environmental factor with
adverse effects on thyroid function since a significant decrease in serum
T, with increased duration of exposure to this compound in viscose factory
workers has been reported (Cavalieri, 1975). Whether this decrease in
thyroid function is due to a direct effect on the thyroid gland, altera-
tion of T> metabolism, or results from involvement of hypothalamic or
hypophyseal functions is unclear. Clinical hypothyroidism during exposure
to CS2 seems unlikely since a decrease in serum free T, or T- does not
occur (Wägar et al., personal communication).
MISCELLANEOUS
When added in vitro to serum salicylates displace T, and T, from
their binding to TBG and TBPA thereby increasing their free fractions
(Larsen, 1972). Administration of therapeutic amounts of salicylate
results in a decrease of about 25 X in serum T, and T., and also, due to
a higher percentage increase in the corresponding free hormone fractions,
252

in slightly elevated free hormone concentrations. It is, however, unclear

whether these small increases in free hormone concentrations persist or
whether, in the course of therapy, they will be normalized by compensatory
decreases in the total hormone concentrations. The salicylate-induced
decrease in thyroid hermone binding is also reflected in elevated T,U
values (Hvid Hansen, 1966).
Propranolol does not affect the secretion of thyroid hormones and
the symptomatic relief in hyperthyroid patients treated with this drug is
believed to be mediated through beta-adrenergic receptor blockade. More
recently propranolol was found to decrease serum T_ by suppressing the
peripheral conversion of T, to T_ (Murchinson et al., 1976; Wiersinga and
Touber, 1977). This decrease in T_ is associated with an increase in rT«
whereas T, remains essentially normal (Verhoeven et al., 1977). The
favourable effects of beta-adrenergic antagonists in hyperthyroidism
appear, therefore, to result partly from a diversion of T, metabolism
towards inactivation.

Theophylline enhances the serum TSH response to TRH (Faglia et al.,

1972) . This effect is probably related to an increase in pituitary cAMP
due to the theophylline-induced inhibition of phosphodiesterase.
The hypocholesterolaemic drug Clofibrate (ethyl-alfa-(p-chlorphenoxy)
isobutyrate) competes with T, for binding sites on TBPA and albumin in
vitro, but consistent changes in T, or free T, have not been observed in
patients administered therapeutic doses of this drug. Clofibrate inter-
feres with the hepatic metabolism of T, but the exact mechanism of its
effect on cholesterol metabolism is still unclear (Harland and Orr, 1975).
It is well-known that 2,4-dinitrophenol increases oxygen consumption
by uncoupling mitochondrial phosphorylation, but it is uncertain whether
the dinitrophenol-induced displacement of T, from its plasma binding sites
described by Korsgaard Christensen (1959) contributes to the calorigenic
.effect of this drug. Dinitrophenol inhibits binding to TBPA (Wolff et al.,
1961).
Penicillin displaces T, from TBPA when added in vitro and could,
therefore, induce a rise in free T, and a decrease in T, when administered
in sufficiently high doses (Surks and Oppenheimer, 1963).
Treatment with orphenadrine, a drug used in Parkinson's disease,
increases serum T, measured with the CPB but not the RIA technique
(Wiersinga et al., 1977). This effect was found to be due to an in vitro
 253

competition between Τ, and an orphenadrine metabolite for binding sites

on TBG. Other examples of interference with analytical methods are the
low PBI values caused by heavy metals in mercurial diuretics and therapeu­
tic gold salts: mercury and gold interfere with the catalytic eerie—
arsenite reaction for determination of iodine.

SUMMARY

Drugs affecting serum concentrations of iodothyronines

High T. High free T,:

estrogens salicylates
heparin (CPB assay) heparin
orphenadrine (CPB assay) amiodarone
propranolol iopanoate, ipodate
propylthiouracil penicillin
amiodarone dinitrophenol
iopanoate, iponate
heroin, morphine
methadone

Low T, Low free T.

antithyroid drugs antithyroid drugs
diphenylhydantoin diphenylhydantoin
carbamazepine carbamazepine
salicylates
glucocorticoids
androgens
anabolic steroids
penicillin
dinitrophenol
carbon disulphide
dioxan (TCDD)

High T. High free T_:

estrogens salicylates
heroin, morphine heparin
methadone

Low T. Low free T,

antithyroid drugs antithyroid drugs
salicylates glucocorticoids
glucocorticoids propranolol
propranolol propylthiouracil
propylthiouracil amiodarone
amiodarone iopanoate, ipodate
iopanoate, ipodate
254

High rT, Low rT

3" 3"
estrogens antithyroid drugs
glucocorticoids diphenylhydantoin
propranolol carbamazepine
propylthiouracil phénobarbital
amiodarone
iopanoate, ipodate

Drugs affecting the serum TSH­response to TRH

Suppression: Enhancement:
chlorpromazine theophylline
thioridazine amiodarone
phentolamine iopanoate, ipodate
L­dopa, dopamine estrogens (in men)
bromo cr i ptine
glucocorticoids,
salicylates

REFERENCES
Acland, J.D.: The interpretation of the serum protein­bound iodine.
A review. J. C lin. Path. 24: 187­218, 1971.
Azzizi, F., Vagenakis, A.6., Fortnay, G.I., Braverman, L.E. and Ingbar,
S.H.: Ann. Int. Med. 80: 194­199, 1974.
Bastomsky, C.H.: Enhanced thyroxine metabolism and high uptake goiters
in rats after a single dose of 2,3,7,8­tetrachlorodibenzo­p­dioxin.
Endocrinology 101: 292­296, 1977.
Bastomsky, C.H., Dent, R.R.M, and Tolis, G.: Elevated serum concentrations
of thyroxine­binding globulin and caeruloplasmin in methadone­main­
tained patients. C lin. Biochem. 10: 124­126, 1977.
Blomqvist, Ν., Lindstedt, G., Lundberg, P.­A. and Wålander, J.: No inhibi­
tion by L i + of thyroxine monodeiodination to 3,5,3'­triiodothyronine
and 3,3',5'­triiodothyronine (reverse triiodothyronine). C lin. Chim.
Acta 79: 457­464, 1977.
Burger, Α., Dinichert, D., Nicod, P., Jenny, M., Lemarchand­Béraud, T.
and Vallotton, M.B.: Effect of amiodarone on serum triiodothyronine,
reverse triiodothyronine, thyroxine and thyrotropin. J. Clin. Invest.
58: 255­259, 1976.
­Biirgi, H. , Wimpfheimer, C., Burger, Α., Zaunbauer, W., Rosier, Η. and
Lemarchand­Béraud, T.: Changes of circulating thyroxine, triiodo­
thyronine and reverse triiodothyronine after radiocontrast agents.
J. Clin. Endocr. 43: 1203­1210, 1976.
Cavalieri, R.R., Sung, L.C. and Becker, CE . : Effects of phénobarbital on
thyroxine and triiodothyronine kinetics in Graves' disease. J. Clin.
Endocr. 37: 308­316, 1973.
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 255

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PHARMACOLOGICAL EFFECTS
 EFFECTS OF ANTIE PILE PTIC DRUGS ON LABORATORY TE STS

D. Bagrel", A. Bagre1°, Β. Le Perron"", G. S i e s t *

* Faculte dee Sciences Pharmaceutiques e t Biologiques, 7 Rue Albert
Lebrun, 54001 Nancy Cedex, France
'" Centre de Médecine Préventive (Dir. Pr. R. SE NAULT), 2 Avenue du
Doyen Jacques P a r i s o t , 54500 Vandoeuvre­Les­Nancy, France

ABSTRACT
We studied the effect of some antiepileptic drugs on human laboratory
tests.
Among 150 epileptic subjects ("total population") we have singled
out 32 patients treated with phénobarbital (sub­population I) and 18
treated with phénobarbital + phenytoîn association (sub­population II)
without any other therapy.
These epileptic patients have been paired with a healthy population
according to their age, sex and overweight.
Some parameters such as urates, cholesterol, triglycerides and
B­lipoproteins undergo no significant variations under the influence of
these treatments.
Most of the other biochemical parameters decrease while enzyme and
erythrocyte cellular mean volume increase.
We can observe the most important variations for creatinine, bili­
rubin, alkaline phosphatase and gamma­glutamyltransferase.
Moreover, combination of phénobarbital with Phenytoin leads to a
more important effect than phénobarbital itself.

INTRODUCTION
Antiepileptic drugs forma large pharmacological group, the principal
drugs of which are composed of very distinct chemicals. According to the
classifications, they may be linked to 8 or 9 chemical structures with
the same heterocyclic nucleus which seems to be necessary for an anti­
epileptic activity.

Independent of their pharmacological activity, these molecules may

cause certain disturbances in metabolism as shown by different biological
parameters.
For many years, our laboratory has investigated this problem (Siest,
et al., 1974, Batt, et al., 1976).
We studied the effects of certain antiepileptic drugs or combinations
thereof, in presence and in absence of other medication. Epileptic
patients, however, are often polymedicamented patients. We wanted to
measure the effects of a treatment using several medications, where
262

a n t i e p i l e p t i c drugs are combined with o t h e r n o n ­ a n t i e p i l e p t i c drugs. We

compared the r e s u l t s of p a t i e n t s undergoing treatments t o the r e s u l t s of
a supposedly h e a l t h y p o p u l a t i o n .
MATERIAL AND METHODS
Choice of population samples
Ep¿ízp£¿c iubjzcti
Of 150 e p i l e p t i c s u b j e c t s ­ which we h e r e a f t e r r e f e r t o as the
" t o t a l p o p u l a t i o n " ­ examined between January 1st and J u l y 15th, 33 were
i n ­ p a t i e n t s and o u t ­ p a t i e n t s a t C entre H o s p i t a l i e r Regional de Nancy, i n
the n e u r o l o g i c a l ward of Professor M. Weber. The o t h e r s were o u t ­ p a t i e n t s
a t the C entre de Médecine Préventive (C MP) of Vandoeuvre­Les­Nancy,
undergoing a n t i e p i l e p t i c t r e a t m e n t .
Among 150 s u b j e c t s , 32 were t r e a t e d with p h é n o b a r b i t a l under the
t r a d e name of Gardenal, Aparoxal, Alepsal or O r t e n a l . This was in the
absence of any o t h e r therapy (sub­population I ) .
We s i n g l e d out 18 p a t i e n t s having received only the combined medi­
c a t i o n p h é n o b a r b i t a l + phenytoïn, ( t h i s l a s t was given under the form
of Solantyl or Dihydan). They formed the sub­population I I .
The o t h e r s u b j e c t s underwent very d i f f e r e n t t h e r a p i e s . We s i n g l e d out
28 d i f f e r e n t p r e s c r i p t i o n s a s s o c i a t i n g or not d i f f e r e n t antiepileptic
d r u g s . This was in absence of any o t h e r non a n t i e p i l e p t i c t r e a t m e n t .
Those e p i l e p t i c s who had consummed an excessive amount of alcohol
were e l i m i n a t e d from t h i s s t u d y .
CoYitAjol patiznti
The c o n t r o l p a t i e n t s were s e l e c t e d from an u n s e l e c t e d and supposedly
h e a l t h y p o p u l a t i o n at the C MP (Vandoeuvre­Les­Nancy). They were r e c e i v i n g
no medication.
The t r e a t e d p a t i e n t s and the c o n t r o l s u b j e c t s were p a i r e d according
t o t h e i r age, t h e i r sex and t h e i r overweight (according to Lorenz's
formula) .
At the time of p a i r i n g , no age d i f f e r e n c e were accepted among the
c h i l d r e n . A d i f f e r e n c e of more or l e s s than one y e a r was t o l e r a t e d for
a d u l t s o l d e r than 30 and d i f f i c u l t t o p a i r due t o t h e i r overweight. I t
should be added t h a t a maximum d i f f e r e n c e of 15 Ζ was t o l e r a t e d a t the
time of p a i r i n g according, to overweight (mean d i f f e r e n c e : 5 X) .
 263

Methods
Blood was taken with an anticoagulant from the fold of the arm and
by means of venepuncture. Immédiatly thereafter, plasma was separated
from blood cells.
Determination methods are summarized in table I.
TABLE I - METHODS OF BIOLOGICAL PARAMETERS MEASUREMENT

Biological parameter· Reagenta Apparatus

Total p r o t e i n Biuret SKA I I Technicon

Albumin Bromocresol green
Ura a Diacetylmonoxime
Ciuco·· Clucos*~oxydas«-para-aminoph«nazon«
Bilirubin S u l f u r i c acid - sodium n i t r i t «
Creatinin« Jaff«
Phosphotungstic acid -
Uric acid
sodium tungstat«
Ceruloplaaain Orthodianisidin« - chlorhydric acid CSA I I Gr«in«r
e-lipoproteina Heparin teat
Triglycerid·· Lipase - glycsrokinas«
ChoUatvrol C h o l e s t e r o l oxydase - Peroxydase SKA II Technicon
Calcium Complexon - cresolphtaléine
Phoiphat·· Ammonium molybdate - s i n e chlorhydrate
Alkaline phosphatases Paranitrophenylphosphate, aminomethyl
(AP) propanol 37*C
Camma-glutaayl- >-glutamyl-3-carboxy-4-nitroani l i d e
CSA II Greiner
t r a n s f e r a s e (CCT) g l y c y l g l y c i n a , 37*C
Alanine amino- L-alanina, l a c t a t e dashydrogenaae Automat Eppcndorf
t r a n s f e r a a · (ALT) 37'C 5010
Aspartat« amino- L-aapartate, malate deshydrogenase
t r a n s f e r a s e (AST) l a c t a t « dashydrogwnas· 37*C

S t a t i s t i c a l analysis
For each sub-population, the variations of the different blood
constituents have been shown :
- b y a Student's t e s t carried out on the differences obtained between
the treated group and the control
- by comparing the values obtained for the p e r c e n t i l e s 5, 50 and 95.
RESULTS
Many parameters have been disturbed under the influence of a n t i -
e p i l e p t i c drugs, as can be seen in table I I .
Most of the biochemical parameters under study decreased among
patients undergoing t h i s treatment when the enzymes and the mean corpus-
cular volume of red c e l l s increased.
Soma parameters such as u r a t e s , c h o l e s t e r o l , t r i g l y c e r i d e s and
264

B ­ l i p o p r o t e i n s do not seem t o undergo any change during a n t i e p i l e p t i c

t r e a t m e n t s . We d i d n ' t show them in t a b l e I I .
TABLE I I ­ EFFEC T OF ANTIEPILEPTIC DRUGS ON SOME BIOLOGIC AL PARAMETERS.
STUDENT TEST (Δ i n c r e a s e , T d e c r e a s e , ­ no s i g n i f i c a n t
v a r i a t i o n . Values given in p a r e n t h e s e s i n d i c a t e significance
level)

Sub­population I Sub­population II Population totale

(n ­ 32) (η ­ 18) (η ­ 150)

Total protein ­ ­ τ (0,01)

Albumin ▼ (0,01) Τ (0,01) τ (0.001)
Urea ­ Τ (0,05) τ (0,01)
Glucose ­ ­ ψ (0,02)
Bilirubin ­ τ (0,01) τ (0,001)
Creatinine ? (0,01) τ (0,001) τ (0,001)
Ceruloplasmin" Δ (0,01)
Calcium T (0,001) τ (0,01) » (0,001)
Phosphate ­ τ (0,02) ­
Alkaline
phosphatases ­ Δ (0,05) & (0,001)

Gamma­glutamy 1­
Δ (0,001) Δ (0,001) Δ (0,001)
traneferaae
Alanine amino­
transferase ­ ­ Δ (0,02)

Red cells count ­ Τ (0,02) Τ (0,001)

Hemoglobin ­ τ (0,02) Τ (0,01)
Mean corpuscular Δ
volume (0.01) ­ Δ (0,001)

κ
Evaluation of ceruloplaamin has been carried out on 80 aera only

S i g n i f i c a n c e of v a r i a t i o n s of parameters which underwent a change by

a n t i e p i l e p t i c drugs i s often h i g h e r at t h e l e v e l of the whole population
than t h a t of t h e two sub­populations I and I I . C onsequently, we have t r i e d
t o compare t h e s e sub­populations with the r e s t of the population (total
population ­ sub­population I or t o t a l population ­ sub­population I I ) .
Then, for each population and sub­population we compared the d i f f e r e n c e
of the mean value of each parameter between p a t i e n t s undergoing treatment
and c o n t r o l s u b j e c t s . The decrease of plasma c r e a t i n i n e and phosphate
in t r e a t e d p a t i e n t s of sub­population I I i s s i g n i f i c a n t l y more i n t e n s e
than in the r e s t of the p o p u l a t i o n . Moreover, t h i s phenobarbital­phenytoîn
a s s o c i a t i o n has a s i g n i f i c a n t l y more i n t e n s e e f f e c t than t h a t of phéno­
b a r b i t a l for GGT, b i l i r u b i n and c r e a t i n i n e .
 265

Sub-populations I and I I show a reduced number of subjects and a l l

t h e i r parameters do not present a gaussi an d i s t r i b u t i o n . That i s the
reason why we have exami ned the p e r c e n t i l e s 5, 50 and 95 for each
parameter undergoi ng a s i g n i f i c a n t vari ati on under treatment (Fi g. 1A-1L).

80 gi- 4 β mmol.I·1

m
Total protei n - Glucose

Q"·
mmol. I -1

I I 1 ,

Γ~Γ~Ί
Q
- Albumi n - Urea
266

μποΙ.Γ1 ' mmol.I' 1

E 60

Creatinine Calcium
)jmol.l _1 mmol.I" 1
p o 10 20 |-| 0.8 10 1. 2 1.4

PT

Ι Γ

ι ι
Bilirubin Phosphates
 26"!

200 4O0 Κ IO

E^
Π2-

- Alkaline phosphatases - H emoglobin

80 90 100
J °
S
uu. ^

□ZL
Gamma-glutamyltransferase
MCV

Figure 1: Comparison of the percentiles 5, 50 and 95 of different plasma

parameters.
PT : Total population I I Control
I : Sub-population I (= 1 Treated patients
II : Sub-population II
268

We observe that creatinine, bilirubin, gamma­glutamyltransferase and

alkaline phosphatase undergo the greatest variations (table III). (For
alkaline phosphatase, no discrimination of age has been made. This
explains variations of distributions of this enzyme between total popula­
tion and sub­population II which can be seen in figure 11).
TABLE III ­ VARIATIONS OF SOME BIOLOGICAL PARAMETERS CAUSED BY
ANTIEPILEPTIC DRUGS
Number indicate the value of the ratio — ­ — χ 100
t ­ percentile 50 of treated group
c « percentile 50 of control group

Sub­population I Sub­population 11 Total population

(n ­ 32) (n ­ 18) (n ­ 150)

Protein ­ 1.1 ­ 1.8 ­ 1.4

Albumin ­ «.5 ­ 4.7 ­ 4.8

Urea + 3.2 ­ 18 ­ 2.B

Glucose ­ 1 ­ 3.8 ­ 3.5

Bilirubin ­ 17 ­ 45 ­ 35
Creatinine ­ 5.1 ­ 18 ­ 12

Calcium ­ 4.7 ­ 3.1 ­ 3.7

Phosphatée ­ 0.2 ­ 11 ­ 3.6
Alkaline
phosphatases
­ 4.9 + 19 + 22

GGT + 101 + 245 + 185

Hemoglobin ­ 0.5 ­ 2.6 ­ 3.2

MCV + 2.9 + 3.7 + 3.5

DISCUSSION
Taking antiepileptic drugs modified some biological parameters.
Disturbances in the phosphocalcic metabolism, as we pointed out, have
been described by several authors (Greenlaw, et al., 1972, Gauchei,
et al., 1973, Siest, et al., 1974, Batt, et al., 1976, Fichsel, et al.,
1976, Young, et al., 1977, Pylypchuk, et al., 1978). They appear to be
the result of an osteomalacia which appears among epileptic patients
undergoing a long term treatment. The explanation of this complication is
a deficiency of vitamin D produced by an accelerated metabolism as a
consequence of an hepatic enzymatic induction. Thus, among some patients,
the decrease of serum calcium is correlated to the increase of glucaric
acid excretion.

On the other hand, the hyperparathyroïd type of reaction which is the

consequence of the calcium deficiency does not seem sufficiently compensa­
tive (Hahn, et al., 1972) or systematic.
 269

As a summary, we can conclude that anticonvulsant treatments tested

over a long period cause mild abnormalities of the calcic metabolism and
decrease the mineral mass among a good number of epileptic patients.
These anomalies probably predispose patients to an incidence of clinical
bone diseases (Pylypchuk, et al., 1978) which can be avoided by giving
patients vitamin D (Hahn, et al., 1972).
For the total population and sub­population II, we found a decrease
of plasmatic bilirubin. Phénobarbital affects bilirubin metabolism
according to two contradictory processes : its synthesis is accelerated
by an induction of oxygenase which converts haem into biliverdin which is
the precursor of bilirubin (Hunter, et al., 1976) ; its catabolism is
accelerated by an increased concentration of Y protein (one of the two
hepatic cytosolic proteins responsible for the take­up of bilirubin
(Meijer, et al., 1977) and its conjugation is increased by the inducing
action of phénobarbital on UDP­glucuronyltransferase. As a final result
of these two actions, there is a decrease in the plasmatic bilirubin.
The cause of the increase of bilirubin discovered by some authors
under the action of phénobarbital and Phenytoin is no doubt due
to an hepatic attack and not to the drug itself.
The main hematological disturbance discovered among epileptic
patients undergoing treatment was a decrease of blood cells. As for red
cells, we can observe a decrease of hemoglobin and hematocrit together
with an increase in the mean corpuscular volum (Young, et al., 1975).
This phenomenon results in I Ζ of reported cases in a megaloblastic anemia
which may be caused by a deficiency in folates (Reynolds, et al., 1978).
In fact, folic acid, in spite of its hydrosolubility could be a substrate
of the endothelial reticulum enzymes as is evident by its decrease during
inducing treatments (Hunter, et al·., 1976). Other explanations have been
put forward ; interferences of the drug in the biosynthesis of folic acid
or in its intestinal absorption, displacement of the folate from its
plasmatic carrier or inhibition of intestinal conjugases. At the present
time, the most convincing hypothesis is that of interference in its
intestinal absorption (Waxman, et al., 1970). Nevertheless, this explana­
tion has been vigorously criticized (Rose, et al., 1978, Strauss, et al.,
1978) and hematological disturbances that we have observed during this
study ara not very important.
270

We can note also a decrease in erythrocyte sedimentation rate caused

by sodium valproate which may lower the level of plasmatic fibrinogen
(Nutt, et al., 1978). However, not all the authors agree with this
explanation (Hutchinson, et al., 1978).
As for the variations in total proteins and albumin, they have
already been studied (Siest et al.,1974, Batt, et al., 1976). It is worth
noting the weak variations of total proteins when albumin decreases
significantly among our patients. As reported in the litterature, globu-
lins as well are disturbed by antiepileptic treatments. Although a defi-
ciency in IgA was discovered among patients treated with phenytoïn
(Smith, et al·., 1979), irregular variations were reported among patients
undergoing phenytoïn treatment wether or not in association with
other antiepileptic drugs (Grob, et al., 1972, Andersen, et al·., 1977).
IgG are also inhibited when IgM are increased (Andersen, et al., 1977)
or decreased (Sorrell, et al., 1975).

As for the immunoglobulins, the situation is not very clear therefore

it would be .of interest to study the variations in certain glycoproteins.
For our part, we observed variations of cerulopläsmin.
The study carried out on ceruloplasmin has been done on 80 subjects
of the total population. Our results confirm those of Seager's (Seager,
1977) who noted an increase of this parameter among a small population of
children undergoing phenytoïn treatment. This phenomenon depends
on the dosage and the duration the drug is taken (Cantu, et al., 1966)
such is not the case for the level of serum copper (Taylor, et al., 1974).
On another level, large decreases in plasma creatinine observed
during testing lead us to doubt other findings. Creatinine is essentially
an index of glomerular filtration. If this filtration is disturbed, there
is an accelerated movement of elements towards urine. That enables us to
put forward a new explanation for the decrease of some plasmatic parameter
. parameters such as phosphate and calcium.
Finally, for the 3 groups of treated patients, an obvious increase
of gamma-glutamyltransferase was observed. These results confirm data
found in the litterature (Gauchei, et al., 1973, Bartels, et al·., 1975,
Hildebrandt, et al., 1975, Acheampong-Mensah, 1976, Fichsel, 1976a,
Skillen, et al., 1976, Heipertz, et al., 1978) which suggest GGT as an
index of microsomal enzyme induction for humans. However, one must note
that GGT increases about three fold more among patients undergoing
 271

combined phénobarbital + Phenytoin treatment than among epileptic

patients taking only phénobarbital.
Furthermore, this increase in GGT activity is not concomitant with
a disturbance of triglycerides. But Nikkila, et al., (1978a, 1978b)
reported an increase of triglycerides under the influence of
Phenytoin. Martin, et al., (Martin, et al., 1975) pointed out a positive
correlation between GGT activity and plasmatic concentration of trigly­
cerides and pre­6­lipoproteins, which is due to an induction phenomenon.
Our results do not permit to confirm what these authors concluded.
CONCLUSION
Comparing three types of populations allowed us to point out how
important one antiepileptic treatment is vis­à­vis all others. Thus
combination of phénobarbital with phenytoln leads to a more important
effect than phénobarbital by itself, clearly showing an additional
inductive effect of these two drugs.
On the other hand, variations of biological parameters in sub­
population I do not appear different from those of the whole epileptic
population. The reason for this is that among 150 epileptic patients,
124 were undergoing treatment with phénobarbital alone or concomitantly,
with diverse other drugs. Variations of biological parameters observed
among the total population would seem to reflect the action of phénobar­
bital. That is why it would be of interest now to study variations of
biological parameters caused by other molecules such as carbamazepine or
valproate.
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272

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Hildebrandt, A.G., Roots, I., Speck, M., Saalfrank, K. and Kewitz, H.:
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in man after administration of clemastine, phénobarbital or placebo.
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Martin, P.J., Martin, J.V. and Goldberg D.M.: γ­glutamyltranspeptidase,
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Meijer, D.K.F., Vonk, R.J., Keulmans, Κ. and Weitering, J.G.: Hepatic
uptake and biliary excretion of dibromosulphtalein. Albumin depen­
dence, influence of phénobarbital and nafenopin pretreatment and the
role of Y and Ζ protein. J. Pharmacol. Exp. Ther., 202: 8­21, 1977.
Nikkila, E.A., Kaste, M., Ehnholm, C and Viikari, J. (a): Increase of
serum high density lipoprotein in phenytoin users. Br. Med. J., 2:
99­101, 1978.
Nikkila, E.A., Kaste, M., Ehnholm, C. and Viikari, J. (b): Elevation of
high density lipoprotein in epileptic patients treated with phenytoïn.
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K.G., Neema, H.E., Ogilvie, R., Sturtridge, W.C. and Murray, T.M.:
Calcium metabolism in adult outpatients with epilepsy receiving
long­term anticonvulsant therapy. CM A J . , 118: 635­638, 1978.
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treatment. Lancet, i: 682, 1978.
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Lancet, i: 994, 1978.
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Clin. Chim. Acta, 79: 603­606, 1977.
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ference des contraceptifs oraux et des antiépileptiques sur les
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 273

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alkaline phosphatase activities in epileptics receiving anticonvul-
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Smith, G.T., Hamilton, M.J., Biros, M.H. and Philstrom, B.: Salivary and
plasma IgA of serum subjects receiving phenytoin. Epilepsia, 20:
17-23, 1979.
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toln and carbamazepine. Clin. Exp. Immunol., 20: 273, 1975.
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effects of phenytoln therapy during pregnancy. Obstet. Gynecol., 51:
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diphenylhydantoîn. Amer. J. Clin. Pathol., 61: 577-578, 1974.
Waxman, S., Corcino, J.J. and Herbet, V.: Drugs, toxins and dietary amino
acids affecting vitamin BJ2 or folic absorption or utilization. Amer.
J. Med., 48: 599-608, 1970.
Young, D.S., Pestaner, L.C. and Bibberman, V.: Effect of drugs on
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 PHARMACOLOGICAL EFFECTS DUE TO ORAL CONTRACEPTIVES ON LABORATORY TESTS

F. Schiele and G. Siest

Centre de Médecine Préventive (Dir. Pr. R. Senault), 2 Avenue du
Doyen Jacques Parisot, 54500 Vandoeuvre-Les-Nancy, France

ABSTRACT
The use of oral contraceptives by women may be associated with labo-
ratory tests results. These effects have to be known to affect interpre-
tation of laboratory data, and permit to use them in evaluating therapeutic
risk or choice of pill. These effects are different according to the type
and dosage of pill and also to the physiological or other variables such
as age, weight, hormonal status and duration of the treatment. This work
attempt to demonstrate the influence of these numerous factors on the
interpretation of laboratory data.

INTRODUCTION
The use of oral contraceptives represents an important example of a
long term drug intake by presumably healthy subjects. At this time, about
fifty five percent of women twenty to forty four years old, have used oral
contraceptives and twenty eight percent take the "pill" regularly in
France (Marie, 1979).
The knowledge of the effect of oral contraceptives intake on labora-
tory tests permits to use these tests in survey of a therapeutic risk or
in choice of "pill".
These effects on biological parameters have been studied and well
described. All human physiological systems were studied and variations
were shown for carbohydrate, lipid, hepatic and hormonal metabolism.
From a general point of view, oral contraceptive use causes
variations in numerous biological parameters. These modification can be
classified according to metabolic risk, or to biochemical parameters
.group.
However, if the knowledge of drug effect on laboratory test is an
important thing, all unexpected results must not be explain by drug
interference.
EFFECT OF ORAL CONTRACEPTIVE USE ON LABORATORY TESTS
In a review, Miale, et al., (1974) tabulated one hundred laboratory
tests, which were affected significantly by oral contraceptive use. These
authors collected 120 references from 1963 to 1971 and nine in 1973.
 275

We attempted to perform a similar survey from 1972 to 1979 and to

calculate in percentage the effect of oral contraceptive intake on
laboratory tests.
The data are presented according to laboratory test, biological
fluid, type of pill and duration of the treatment. Only modified parameters
are listed.

Effects of oral contraceptive use on laboratory tests evaluating glucose

metabolism
In table I, we grouped glucidic parameters: glucose, insulin and
glucogen. Fasting glucose (Batt, et al., 1974) and insulin (Beck, et al.,
1975, Gershberg, et al., 1976, Rose, et al·., 1977, Spellacy, et al., 1978)
are not influenced by oral contraceptive intake.

TABLE I ­ EFFECTS OF ORAL CONTRACEPTIVES USE ON LABORATORY TESTS EVALUATING

GLUCOSE METABOLISM

lloloiical Typa of Duration of

fluid Authore Yeara
Inereaoe X Ho Chang« Ζ Decreeee Ζ pill troetaent

naiaatrol
Gloeooo 2 houra attar acetate
At leaat Spellacy et „
llucoM telatane« taat 0.5 a f
6 nontha el.

Oluceee 2 houra after northiadrone At laaat

■lucono tolerance teat 0 . » an 6 nonthe
Ko difference
Olucoae I hour after in veriouo Phillipe
■lucoeo tolerance teet pill
Qi lormedinone
ecetete
elucoe« 2 hour« after lynaacrol I to 6 Aner et e l . I97S
|lu«ooe tolerance teet aaeeetrol nonthe
ecetete

Glucoae 2 hour« after » 1} Ζ ι Séquentiel 3 to 6

■lucoeo tolerance teet ■lood ♦ 20 Ζ pill nonthe Asner et a l . 1*7}

Olueeee 2 houra after > 2123' Spellacy

■lucoeo tolerance teet ■ lood 5 ns weekly et e l .
Nageetrol
Inenlin 2 hour· after ecatate At leeet Spellecy
■lucono tolerence teat • Ut 6 nonthe et al.
0.5 as,

Inaulin 2 houra after Norethindrooe Spellacy

■ lucoeo tolerance teet • UZ 0.35 as at e l .
Ineulin i hour· after Conbined At leaat
e l e n i » hed 0.2 ■/■> • ι·.s : pill l o · · et e l . 1977
6 nonthe
«••tra o l
Incolte M un after 0.OS η« At leeet Spallecy. ,,„
1­aillaiae load 30 | Norethlndroae 6 nonthe
I m
HtatrtuM>l
Ο.Οβ ■«
■or«cbladroo« »•ek «t «1. 1975

Glue··«« W an mtft pi « a u
I­art·»­«· ­β·"" Χ) ι
276

E f f e c t s of o r a l c o n t r a c e p t i v e s use on l a b o r a t o r y t e s t s e v a l u a t i n g l i p i d
metabolism

In t a b l e I I , we p r e s e n t e d l i p i d i e parameters which change with o r a l

c o n t r a c e p t i v e u s e . The v a r i a t i o n s of serum or p l a s m a t i c c h o l e s t e r o l are
very d i s c u s s e d , and only Aftergood, e t a l . , (1974) found an augmentation
of 7 %. Gershberg, e t a l . , (1976) showed an i n c r e a s e of 6 % in obese
p a t i e n t s only and Wallace, e t a l . , (1977), an i n c r e a s e or a decrease of
5 Ζ according to age of women. Others s t u d i e s showed no d i f f e r e n c e s in
women t a k i n g o r a l c o n t r a c e p t i v e s (Smith, e t a l . , 1975, Pometta, et a l . ,
1978, S p e l l a c y , e t a l . , 1978, Larssan­C ohn, e t a l . , 1979).

TABLE II ­ EFFECTS OF ORAL CONTRACEPTIVES USE ON LABORATORY TESTS EVALUA­

TING LIPID METABOLISM

Biological Drug effect Type of Duration of

Author· Yeara
fluid pill treatment
Increue Ζ Ho change Ζ Decrease Ζ
3 month· to A ftergood
Choléaterol Plasma + 7 Ζ
6 yeara et al.

Heatranol
0.08 mg 2 years
Horethindrone
Ι ­β

Ethinyl­
estradiol
+ 6 Ζ in Ν.S. in
0.05 mg Gershberg
obeae normal
Ethynodiol et al.
patienta patienta
diacetate
1.0 mg

Meitrano1
0.05 mg
Horethindrone
1.0 mg
+ 5 1 ÍD ­ 5 Ζ in
Oral contra­
'women up women more Wallace
ceptives or
to 55 than 55 et al.
eatrogen
yeara old yeara old

Triglycerides Horethindrone Spellacy

0.35 mg et al.

Megeitrol
Spellacy
acetate
et al.
0.05 mg

Combined pill
Heade
estrogen
et al.
0.05 mg

Ethinyl­
f 23 Ζ in estradiol
obeae 0.05 mg Gershberg
women Ethynodiol et al.
♦ 21 Ζ in diacetate
normal 1.0 mg

Heatranol
0.05 mg
Horethindrone
1.0 mg
 277

Drag «ffect
■lelogicel Type of D o r a t i « of
»ill Aart tra Tears
fluid tn<BMt
lacrea·« Ζ Ho di—ga X Decrease Ζ

Triglycerid·· kaacraaol
0 . 0 · ■« •Utk
2 year·
■arackladroa M al.
■ ·«
• U I
• M a c «0
yaara eld
Oral cont r e ­
Plaaaui ai—a Vall·«
• SI «ora c e p t i v · or
ac a l .
eka· «0
yaara aided

• —ecke
•ara ♦ 71 I H77
ac laaat
»m
(anal ♦ 17 I Vario— ro—cea 117·
ac a l .

OB)" •pellacy |»7·

'lj ac a l .

■Ckleyl­
•acradiol
0.0S ag a cycl­ Laraaoa­
i c — r g e a c rel Coaa ac a l .
0.25 ag

•celayl­
eecradiol
0.0} at "
Levoaorgaecrel
0.15 a t

Iehinyl­
aetradiol
« Ι · Ζ co
0.05 eg "
♦ 42 X
Levooorgeacrel
0.12} a«

•cala·
Varloua
at a l .

T r i g l y c e r i d · · 3 hour·
Coakiaad • aoatka
• f e e r « alanine load Iena ♦ 900 Ζ ι ac a l . Ι·77
0.2/Ug pill ac laaac

a lipoprotein· Plata« ♦ 16 X 3 aoatka Afcergood

Varia
co · vaara ac a l .

Β lipoprotein· Plaeae 3 aoacka Aicargood

Vario
co 4 year· ac a l .

lacroaaad Decreaead Iffacc

HDL­chelaateroi Serva »radley
•y •y proeaa­ dapeade oa
ac a l .
o« atrofan caroaa eype of pili

Po—Ita
•e a l .

•ehlayl­
aacraaiol
l l u 0.05 eg • cyclaa
' 12 I ■orgaatrel
t ac a l .
0.2S a«

•tklayl­
aacradiol
0.03 a«
■orgaacral
0 . IS a«
278

Biological Type of Duration of

fluid pill tnimit Author· Years
Increate Ζ No change Ζ Decrease Ζ

Ethinyl­
estradiol
BDL­choleat«rol t cycles Lartson­
0.05 m Cohn «t al.
■org··tral
0.123 ■«
Uhimyl­
•stradiol
■PL­pfcoepholipides 0.03 ag
■org«itral
0.123 ■«
Ethinyl­
estradiol
0.03 ag
Morgestrei
0.123

Ethinyl­
estradiol
0.05 s«
Norgestrel
0.123 ag

♦ 31 Ζ
woeta 30 to
VLDL­triglycerides 39 year·
old

♦ 18 I
voaen 30 to
LDL­cholesterol 39 years
old
♦ 19 Ζ 20 to
29 years old
LDL­triglycerides ♦ 39 Ζ 30 to
39 years old

Effects of oral contraceptives use on hematological t e s t s

The effects of oral contraceptives use on hematological t e s t s and
coagulation factors were summarized on table I I I .
TABLE III ­ EFFECTS OF ORAL CONTRACEPTIVES USE ON HEMATOLOGICAL TESTS

Biological Type of Duration of

Authors Years
fluid pill treatment
Increase Ζ No change Ζ Decrease Ζ

Under or
Fish
Red blood cell count 3 Ζ more than S
• t al.
years

7 Ζ

Mestrano1
Blood + 5 Ζ Ο.Οβ mg
Norethindrone

Under or Fish
Hematocrit more than 5
et al.
years
Red blood cell indices Blood ♦ 3 Ζ
MCV Blood + 1.5 Ζ
Heade
Platelets count 1976
et al.
Ethinyl­ Lorrain
estradiol 1972
et al.

Dimetti i­
sterone 25 mg
 279

Drag efface
•lolotical Typ« af Duraci« of ._ .
aataore
fluid pill treataaac
Iacroaea Ζ Mo ebaate Ζ Oser·«·« Ζ

Carboalc aahydraae ■ Zrytbre­ ♦ »7 Ζ Coabiaad or

iaooacyae cytea aeaoeatial
piu
Itklayl
oacradiol
0.1 at
Diaetayl­
■.·. etaroaa 25 at
Coabiaad pill
oeattuaaa
0.05 at
■tkyayl­
oetradiol
0.1 a«
Diaatbyl­
aterone 25 at
Coabiaad
pill
■cada l*7t
Estrogen
0.05 a«

Coaalned
Pill
latrogeo
0.05 at

•tbiayl­
oetredici
0.1 at Lorraia
Maatkyl­ ac al.
atoroaa 25 ι S yaara

■tblayl­
­ 20 Ζ eetradiol
0.1 at Lorraia
ribrlaoeaa
U.S. Dieatbyl­ at al.
ataroaa 2) at S yeere

Coabiaad
pill Ha ala
Pibrlaogaa tarta ♦ 5» Ζ
Iatrogen at al.
0.05 at

Meetranol Craaff
Fibrinogen 0.1 at at ai.
« 10 Ζ Itbynodlol
♦ 17.« Ζ diacetate I ag

Coabiaad
pill Ma ado
Fibrinolytic activity iariai « 31 Ζ
getrogea at al.
0.05 at

Itbioyl­
aatradiol
Soluble flbrio aoaoaer At least Craaff
Plaaaa ♦ Μ Ζ 0.05 at
f aoatba ot al.
llorgaatrol
0.25 at

Ithlnyl­
aatradiol
3 to ·
0.03 at
aoatba
■orgoetrel
0.15 at

Naatraaol
0.1 at At laaat
Itboyodiol • aoatba
diacatat« I at

ttbiayl­
« t Ζ aatradiol
Lorraia
0.1 at
at a l .
Diaatbyl­
♦ 12 Ζ 5 yaara
■taroaa 25 ι

gtbioyl­
♦ 17 Ζ aatradiol I year
Fl eoa! nogen 0. I at
Oiaetbyl­
♦ «J Ζ 3 year
eteroae 25 at
280

Effects of oral contraceptives use on laboratory t e s t s evaluating hepatic

function
The liver synthesizes many proteins and aminoacids. The effects due
to oral contraceptives intake on specific or enzymatic proteins may be a
reflect of hepatic metabolism. In table IV, we grouped thus, t o t a l and
specific proteins, aminoacids and hepatic enzymes.

TABLE IV ­ EFFECTS OF ORAL CONTRACEPTIVES USE ON LABORATORY TESTS EVALUA­

TING HEPATIC FUNCTION

Drug e f f e c t
Biological Type of Duration of
fluid treatnent Authors Tears
pill
Increase Ζ No Change Ζ Decrease Ζ

Proteins 2 Ζ Combined pill Bett e t · 1 . 197t

Albumin 3 Ζ Combined pill Bett e t al. 197«

Chlormadinon·
Serua or I to 12 Bergsjö
Antithrombin III •cacata
plasma month· et e l .
0.5 a«

Combinad pill
«•erogan Me ede
0.03 ·| et · 1 .

Combined pill
Q 2 Microglobulin Serum Heade
eatrogan
et al.
0.05 mg

Combinad t
Serum or 0.05 mg Ondar Herbath
al antitrypsin + 36 Ζ
plasma estrogena 2 year· et e l .
0.03­0.0» ι

■· ♦ 22 Ζ Mora thai
5 years

Ceruloplasmio Serum ♦ 69 Ζ Horvitt

et e l . 1979

Ondar or
Begre1
Combined pill mora than 1979
et e l .
2 year·

Ethinyl­
estradiol
Total aminoacids 0.05 mg Nor« than Creft
Norethiataroi 3 montha et e l .
3.0 mg

Leukocytes ­ 57 Ζ Tarallo
Combined p i l l 1977
et al.

Fl u u Conminad p i l l Bo·· 1976

•t al.
Alanina . hour after
alanine load
Plina

Ithlnyl­
CIutamine and eatradiol
More then Craft
0 . 0 5 mg
glutamic acid 3 month· et a l .
Bjorethiaterooe
3­1
 281

• iolofUal Typ« of Duration of

fluía pill Autaora Taara
l a c r e a · · Ζ lio chana· Ζ Decreaae Ζ

Coatis·· pill Tarai lo

at al.
Ithiayl­
eetradiol
Nor« than Craft
O.Oi a«
3 aontba at al.
Norethisteron·
3.0 η«
At U u t i loa·
a.f. Diñad p i l l aontba *t al.
Glycine 1 hour afear
U.S.
glycine load
Ithinyl­
•atradiol
Hor· than Craft
0.05 u* 3 anntha mi
et a l .
aorethlatarooe
3.0 m
Methionine ( a f t e r 24 houra Hor· than Miler
aethiooine loaf 3 ■) Coahinad p i l l 117·
urina· t aontba et a l .

Leukocytae Coukioed p i l l ­ Tarallo

at a l .
1177

gthlayl­
aatradiol Mom than
Tyrosine Craft
0.03 a« 1*71
3 noothe et a l .
hor·tblaterone
3.0 η«
float
Alanina aninotranifar
Piaana ♦ MI ­ et a l .
197«

Calteau
Piaana ♦ 12 I Variou· 1977
at a l .

Si.it
Plaaaa ♦ 12 Ζ Variou· 197«
Aapartata aninotranafara·· et a l .
Caltaau
Plana Variou· 1977
et a l .

Craatine phoephohinaao riaaaa ♦ 2» Ζ Varioua ­ Siaat

•t a l .
197«

glut amp Itrenef a r a · ·

Plaaaa ♦ 21 Ζ Varloua ­ Sleet
at a l .
197«

Plaaaa ♦ 20 Ζ Various Calteau

1977
at a l .

Plaaaa Heet
Variou· 197«
et a l .
­ 1 X co
Lactata dehydrogenase
­ 13 Χ icblala
(according at a l .
Co at·)

Heet
­ 10 X Variou·
et a l .
Pheaphataaea a l c a l i n a · ­ 7 X to
ichiale
(accordio. *"lew· ­ •t a l .
to a«·)

Pyruvate At laaat
Hood ♦ 33 X èia·*) pill no··
t aontba et a l .
282

E f f e c t s of o r a l c o n t r a c e p t i v e s use on hormonal t e s t s
The i n t e r a c t i o n s of o e s t r o g e n s and progestogens with o t h e r s hormonal
parameters i s w e l l known. Many authors s t u d i e d the e f f e c t s of o r a l c o n t r a ­
c e p t i v e s use on hormones ( t a b l e V) .
TABLE V ­ EFFECTS OF ORAL CONTRACEPTIVES USE ON HORMONAL TESTS

Biological Drug effect

Type of Duration of
fluid pill treatment
Increase Ζ Ho change Ζ Decrease Ζ

Coafained to 6 Eats
Aldo·terone
ae·tranol on tbs at a l .

Angiotensin Morethindrone
2 ag, aestra­
nol 0.1 ag
Ethinyl­
estradiol 3 Months Beckerhoff
0.05 ag et a l .
Horeth indroae

2.S χ

Coaninad 1 to 6 Kats
Plasaa + 2S0 Ζ
aastranol lag months at a l .

Chloraadinone
Plasaa N.!
acetate

Ethinyl­
+ 250 Ζ estradiol
Pre« C o r t i s o l Sana aspi i tude I aonth at Van Cantar
0.05 ng
increase least et al.
Norgestrel
0.5 ­ g

Progestagen Durtor
only et al.

Combined
0 . 0 5 0 ag
Fourteen
estrogen
cycles
0.050 s«
progestagen

Cortisol binding capacity Plai Progestagen Durbar

lbo cycles
only et a l .

Coafained
0.050 s«
Plasma ♦ 240 Ζ estrogen Fourteen
0 . 0 5 0 ag cycles
progestagen

Ethinyl­
estradiol
Gonadotropin Hore than He Cuire
0.035 ­ g
18 aontha et e l .
Horethindrone
1.0 or 0 . 5 ag

Hegestrol
Grovthhoraone At laast Spellacy
acetate
6 aonth· et a l .
0 . 5 ag
 283

Drug « f f « c t
Iiolofical Typ· of Durst i o « of
Author· Tear·
fluid pill traatnaat
I n c r * · · · Ζ Mo chang* Ζ Dacraas« Ζ

Ovulatory
peak 0 . 0 5 ag
auppresMd ■oretbiaton
U.S. I ■*

Ko peak
I to 6 Vi l é ñ e l a
cycles «t a l .

Ithiayl­
0aetrof*na ι oaatrediol ♦ eitradiol
9 eyela« Joaaaaaoa 197)
Matron· 0.035 M
lyuastrol I ag
I to ft Vintola
Urin
cycl·· at a l .
ProfBMMdiol Urlo.

Ithinyl­
eatrediol
Hor· than Mc Cuir«
0.035 a t
18 n o o t h · «t · 1 .
Vor« t tu adroni
1.0 or 0 . 5 m
Ithinyl­
••tradiol
9 cycl·· Johaneson
0 . 0 3 5 η«
Lyn··trol la)

Conn load I t o ft Katt

riuu * iso ζ naat rano1 tas aontha •t al.
Chlormadinon·
Plata«
•catata

■oratili ndrona
2 >*
■•■tranol
0.1 a s ■ackarhoff
Ethinyl­
estradiol
50 ng
Nor«thindrona
•catata 2.5ag

Morothtndroo·
Fr·· UilMtiroM ueatranol1/
1/50 íríi* ""
■org··tral
•thinyl­
•atradio1

non­U SC bound Norothindrona

Troablay
tlllMUrOM •■•tranol
at a l .
1/50

Nora··tral
PIUM * IM Ζ «thinyl­
astradiol

Ethinyl­
estradiol
Thyrotropin I aonth Tan Cantar
Ι .ν · I 0 . 0 5 ng
at least at al.
♦ 212 Ζ t o Noraaatrsl
1000 Ζ 0.5 · «
284

Effects of oral contraceptives on oligoelements

Serum or plasmatic chloride, sodium, potassium are not influenced by
oral contraceptive intake (Batt, et a l . , 1974). Serum or plasmatic
magnesium l e v e l does not change also (Thin, 1971, Dale, et a l . , 1972,
Simpson, et a l . , 1972). The e f f e c t s found or other metals are summarized
i n table VI.
TABLE VI ­ EFFECTS OF ORAL CONTRACEPTIVES ON OLIGOELEMENTS

Biological Type of Duration of

fluid pill treataant
Increase X No change Ζ Decrease X

Ethinyl­
estradiol
Total electrocute· HagoujioD
Salivary 0.05 mg 2 month·
concentration· •t «1.
Norgeatrei
0.5 mg

Ethinyl­
estradiol
0.050 mg Four years Thin
No re thi s te rone
•cetate 3 mg

Hestranol
0.1 mg 1 to more
Siapion
Ethynodiol than 12
•t al.
diacetate months
1.0 mg

Batt
Plasma combined pill ­ •t ·1.
197«

Copper Serum Various Horvitt 1975

Hestranol
O.OB mg Smith
Serum 2 yaarf 1975
Norethindrone •t «1.

Total iron bonding

Serum * 10 X
capacity

Hestranol
0.1 mg 1 to more
Simp·on
Ethynodiol than 12
et al.
diacetate months
1.0 mg

Combined pill ­
Ethinyl­
estradiol
5 to 26 Hahn
0.05 mg
cycl·· at al.
Líneatrol
2.5 mg
 285

E f f e c t ! of oral contraceptives use on vitamins

In the l a s t t a b l e , we grouped the studies of the e f f e c t s upon
vitamins. Vitamin B , , tryptophan metabolites, vitamin Bj2 and f o l a t e s
were the most studied.
TABLE VII ­ E FFE CTS OF ORAL CONTRACE PTIVE S USE ON VITAMINS

■iol.tlc.l ""­ Trpa ol Doratio. of . . „ _ . ...„

fluid ~ ­ _ . Γ Τ " — pill liuont »·"»« T··*·
r
Imcraaaa Ζ Ho chamsa Ζ Daeraaaa Ζ

Coat, load p i l l ­ •riu·

ac a l .
Ithiirrl­
•atradlol
2 mootha Towns
0.05 am
to 2 jaara ac a l .
•ortaatral
O.S am or
northiodroma
ι m

Varioua ­ ■θ twiCC
at a l .

Hi· traici
0.08 a« Smith
Roratbiodroaa 2 year·
at a l .
1 ·*

Coafcload p i l l ­ ■riu·
at a l .

Haatranol
0.05 ms
•oralblod roo·

­»l ""Sloi »»·«· ■«­■

0.03 ms
Korgaatral
0 . ] ·>

bmmlmadpiU .„.„,,

­··»« 7ΖΤΆ «·'· ITS!

aodO.lm, ""■
Coafciaad p i l l
aaciotaaa 1 to mora
0.05­0.075 "*" "
.od 0.1 χ " O "

Saqoamcial
­ 15 t
pill

Soaliofh
Coatload p i l l Siam ink
at a l .

Srista
Comaload p i l l
at a l .

Maatraool
­ U Z 0 . 0 · aa 2 vaara Smith
Bora catadromo at a l .
1 «t
­ 22 Ζ At laaat Broa
Coahlaad p i l l
O.S.) t •ootoa at a l .
286

Drug effect ­_ * ­. .
Biological Type of Duración of
Authors Tun
fluid Increate Ζ Ho change Ζ Decreaie Ζ PiU treatment

­ 20 Ζ At leeit Leklem
Combined pill 1975
(H.S.) 6 months et al.

Schiele
Combined pill 1977
et al.

3 to I» Boss«
Verioui 1979
monthi et al.

Xanthurenic acid β hour·

after tryptophan load (2 g) Urine Ac least Leklem
♦ 131 Ζ Combined pill 1972
(2 g) 6 months et al.

Urine At leeit Uklem

♦ 139 Ζ Combined pill
6 months et al.

Coelinl
Urine ♦ «88 Ζ Combined pill Banning
et al.

Urine ♦ 450 Ζ Verloue Notvitt

ec al.

2 to 60
Combined pill
months

Briggs
Combined pill ec al.

Hestrenol
0.0B mg Smith
2 years
Norethindrone at al.

At leest Stephana
3 months et al.

Hestrenol
0.08 mg 3 to 6 Faine
1975
Norethindrone montha et al.
I mg

Urine
(12 hours) ­ 8,5 Ζ Combined pill Shogania I97S

Briggs
Combined pill 197«
et al.

Heetrenol
0.08 mg Smith
2 years 1975
Norethindrone et al.

Horwitt
Serum « 21 Ζ 1975
et al.

Combined pill Briggs

197«
et al.

3 to t Aftergood
197«
montha et al.

Hestrenol
0.08 mg Smith
■lood ♦ 28 Ζ 2 yeers
NorethindroM et al.

Horvitt
et al.

Hestraool
0.05 mg or
­ 21 Ζ to I yeer or Tengney
0.08 mg
­ 23 Ζ Norethindrone more et al.
I mg
 287

These effects are very important to know to interpret laboratory

tests results. But this interpretation has to take into account the
different factors which can influence the biological parameters. We will
now show the importance of this factors with several examples.

INFLUENCE OF OTHER VARIABLES ON ORAL CONTRACEPTIVES EFFECTS ON LA BORA TORY

TESTS
We studied the effects of age, hormonal status, overweight, duration
of the treatment and associations with other drug, upon variations due
to oral contraceptives use on laboratory tests. Our study population was
people coming for routine health examination at our Centre de Médecine
Preventive. About 20 Ζ of these were volunters ; the remaining 80 Ζ were
people summoned to the Centre after their names were drawn at random
from the files of Caisse Primaire d'A ssurances Maladie of Nancy.
About 20 000 subjects were examinated each year and more than 700
are collected per patient by questionnaires and by physiological and
biological tests. One of the original functions of the Centre was to give
such family­health examinations. These subjects who were presumed to be
healthy, range in age from 4 to 99 years.
During sampling, data concerning drug intake are collected with the
aid of special questionnaires and informations are memorised according
to therapeutic class and subclass (Steinmetz, et al., 1979).
Effect of age
For factors V, VII and VIII, fibrinogen, platelet count and adhesi­
veness and fibrinolytic activity, the differences between women using
and not using oral contraceptives were greater in younger than older
women (Meade, et al., 1976). These authors conclude that the differential
effects of oral contraceptives by age must be borne in mind in evaluating
the effects of these preparations on the hemostatic and lipid system.
An other example is the decrease of LDH and AP activities as a
function of age. These decreases are maximal for women aged more than
fourthy years (Schiele, et al., 1979).
Fish, et al., (1972) show a slight increase in blood pressure which
is related to age and weight.
Effect of hormonal status
The increasing effect of menopause on total alkaline phosphatase
activity is well known. In menopausal women total activity of AP is
increased. This phenomenon is observed for the two isoenzyme bone and
288

liver. The administration of oral contraceptive modify this physiological

increase after menopause. But only the liver alkaline phosphatase activity
is changed by oral contraceptive use and the activity decrease from
twenty four to twenty units per liter.
Effect of overweight
Many recent works deal with lipid metabolism. The effects of oral
contraceptive use on triglycerides levels is well known at present.
Steinmetz, et al·., (1979) show that in women, 20 to 39 years old, the use
of oral contraceptives was the major discriminating factor in the group
of women who were not taking oral contraceptives. In women, 40 to 49 years
old, overweight was the main dispersion factor. The use of oral contra­
ceptives was the second most discriminating factor in women who were less
than 30 % overweight. These authors conclude that women who take these
drugs reveal an indirect overall tendancy to latent hypertriglyceridemia
which is aggravated by overweight.
Effects of duration of treatment

This is an important parameter that can affect the action of oral

contraceptives. The action of oral contraceptives on enzyme activities
for women aged between 20 to 30 years old change with the length of time
on the pill. Enzyme activities are affected as early as the first month.
This effect reaches its maximum within a period which varies with each
enzyme, and then tend to normalize after two years (Galteau, et al·., 1977) .
Effect of type and dosage of pill
Larsson, et al., (1979) studied the effect of three types of pill on
lipid parameters. The ratio of ρrogestagen/estrogen seems to be an impor­
tant factor for triglyceride values. Progestational hormones cause a
decrease in triglycerides while estrogens increase them (Beck, 1975 ;
Wallace, et al., 1977).
HDL­cholesterol and HDL­phospholipid concentrations are reduced by
­progestagen and increased by estrogen (Bradley, et al., 1978 ; Manninen,
et al., 1978 ; Shelton, et al., 1978).
Associations with other drugs
It is well known that the drugs which induce drug metabolizing
enzyme, such as phénobarbital increase the catabolism of oral contracep­
tives. Many other drugs inţerfer with oral contraceptives. These effects
can be divided in two groups of antagonist effects and potentialization
or additionnai effects (Neuman, 1978).
 289

Antagonist effects can occure with oral anticoagulants, enzyme

inducers such as phénobarbital, hypolipernie agents, antihypertensives,
rifampicine, vitamins such as pyridoxal phosphate or folic acid, thyroxine,
ovulation inducers, antiestrogenic and androgenic drugs. Potentialisation
or additionnai effect are noted with antifibrinolytics with a thrombosis
risk, tricyclic antidepressant by cellular effect (Tazi, et al., 1979)
anti-inflamatory such as antipyrine, neuroleptics such as antipyrine,
neuroleptics such as phenothiazines, morphinic analgesics and vitamin D.
CONCLUSION

The effect of drug Intake on each laboratory test depends on:

- drugs
. type of medication
. duration of therapy
. dosage
- individual physiological factors
. sex
. age
. overweight
- association with other drugs
The knowledge of the effect of oral contraceptive tests leads to
three points.
First, intake of oral contraceptive is another factor of variation
taking on laboratory tests. So, women taking oral contraceptives must be
excluded from a reference population in order to define reference values.
Second, laboratory data must be used as indicators of therapeutic
risk or choice of pill. In this case, the interpretation of laboratory
results must take into account the physiological variations of biological
parameters. To use laboratory tests results, it is necessary to know
values before and during the administration according to physiological
status of women.
In the future, it would be possible to define reference values for the
particular group of women on oral contraceptives according to physiolo-
gical factors and perhaps type of pill.
290

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 291

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292

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 293

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294

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ACKNOWLEDGMENTS
We thank Anne-Rose MAIRE for her documentation assistance.
 PHARMACOLOGICAL EFFECTS DUE TO HYPOLIPIDEMIC DRUGS

J. Steinmetz", E. Gaspart", D. Notter'0*

* Laboratoire du Centre de Médecine Préventive (Dir. Pr. G.Siest)
2 , Avenue du Doyen Jacques P a r i s o t , 54500 Vandoeuvre-Les-Nancy, France
KX Faculté des Sciences Pharmaceutiques et Biologiques, 7 Rue Albert
Lebrun, 54017 Nancy Cedex, France

ABSTRACT
In order to know the unexpected biological effects of hypolipidemic
drugs, we performed different plasmatic tests on hyperlipidemic subjects
treated by Clofibrate and Fenofibrate and control group.
The hypolipidemic action of Fenofibrate and Clofibrate is evident.
The concentration of bilirubine, the enzymatic activities of alkaline
phosphatases and gamma-glutamyltransferase were decreased with the two
treatment.
In general, the transaminase activity (TGP) was not significantly
different between treated and control subjects. However, some treated
women had higher values than controls.

INTRODUCTION
Our aim was to study the effects of hypolipidemic drug intake on the
biological tests carried out in the laboratory, in order to elucidate
the shift in distribution of the biological values due to these drugs.
A research procedure in two parts was therefore devised. The first is
concerned with the biological variations which are observed in the entire
o
population of patients undergoing Fenofibrate or Clofibrate treatment,
regardless of whether or not other treatment is coadministered.
In the second part of the study, the possible effects of related
treatments upon the studied parameters were eliminated since we only
considered patients treated by one of the two hypolipidemic drugs. The
results obtained in these two phases are compared with those of the control
groups that are matched with the groups under treatment.
MATERIALS AND METHODS
The drug index
The evaluation of drug interference on laboratory test results is made
possible by our drug index.
Indeed, all the persons who are invited to Center for Preventive
Medicine (CPM) are questioned, during blood sampling, about a possible drug
intake. The name of the patent medicine is noted on a specially printed
Lipanthyl ® Laboratoires Fournier, Dijon, France
296

sheet, that allows us to distinguish between occasional drug intake and

long-term treatments. The dose administered is not recorded.
This information cannot be memorized in this form. So we have develo-
ped a codification system using six numbers :
- the first two numbers indicate the therapeutic class
- the two following refer to the sub-classes
- the last two numbers represent the patent medicine.
Example : DAONIL (120201)
12 02 01
Class : Subclass:: Patent medicine
Hypoglycemiant Sulfamide Daonil
The system is based on an index that is periodically reviewed and
comprises at present 42 therapeutic classes.
The subclasses are established by considering the analogies of
chemical structure or of biochemical types of activity.
Different patent medicines which contain the same active principle
are listed under the same numbers.
This index has a flexible structure that permits addition at any
given moment of a new medicine belonging to a defined class or sub-class.
It will then be given a new number. Likewise, new therapeutic classes
may be created or new sub-classes may be integrated into an already
existing class.
The drug codification is made by pharmacists in the laboratory. This
codification system also distinguishes between occasional drug intake
and long-term treatments by two different code numbers. Each index-card
can record up to twelve medications per person.
The preceding description explains the information item concerning
drug intake. This information is recorded at the same time as certain
other data obtained at the CPM such as various questionnaires, biological
- test results, clinical examinations...
All this information can later be drawn upon, permitting
- the study of drug effects on laboratory test results (aiming the
constitution of a data bank)
- the definition of "drug risk-group"
- a post-marketing surveyance.
 297
Frequency of drug intake by therapeutic classes
The information collection on drug intake has permitted to determine
in 1978 the drug intake frequency for each therapeutic class (table I and
II).
TABLE I - FREQUENCIES (NUMBERS AND PERCENTAGE) OF DRUG INTAKE BY
THERAPEUTIC CLASSES IN FEMALES DURING 1978
Number of treated
femalea Percentagea

Oral contraceptiv« 2631 41

Tranquiliier 852 13
Analgeaic 501
Vaaculotrop 486
Vasodilatator 475
Antiinflammatory 304
Paychotonic 294
C a a t r o - i n t a a t l n a l tharapautica 294
Antihypartenaiva 261
Hapatic therapeutic 222
Antiaathmatic 211
Antibiotic 209
Hypnot i c 196
Anti-angor 184
Progoaterone derivataa 178
Diuratic 172
Laxativa 152
Antiarythmic 143
Sedativa 140
Oestrogen 115
Hypolipidemic 115
Anttdepreaaant 110
Neuroleptic 97
Antiparkinaon 96
Hypoglycemias 86
Cardiotonic 75

TABLE I I - FREQUENCIES (NUMBERS AND PERCENTAGE) OF

THERAPEUTIC CLASSES IN MALES DURING 197

Number of treated
malea Percentagea

Tranquiliiera 389 13
Analgeaica 374 13
C a a t r o - i n t a a t i n a l therapeutcia 307 10
Antlaathmatic 283
Ant langor 280
Vaaodilatator 270
Paychotonic 231
Antihypartenaiva 185
Antibiotic 178
Hypolipidemic 163
Hypoglycemiant 149
Antiinflammatory 135
Hypouricemlc 119
Vaaculotrop 114
Antiarythmic 1 10
Anticoagulant 104
Diuratic 92
Hypnotic 91
Neuroleptic 88
Hepatic therapeutic 87
Antiepileptic 74
Antidepreaaant 58
Sedativa 54 1
Cardiotonic 53 1
Antiparkinaon 46 1
298

The population taking drugs included 9225 persons with 2849 men
(31 2) and 6376 women (69 Z ) . Women taking drugs are about two times more
numerous than men.
The oral contraceptives are at the first place (41 % of drugs intake),
The tranquilizers take the second place with 13 %, followed by
analgesics (7 Z) and vasodilatators (7 %). Only 1 % of females are
treated with hypolipidemic drugs.
In men, the most frequently used drugs are tranquilizers (13 %) and
analgesics (13 % ) . A higher percentage (5 %) of men than of women ingest
hypolipemics.
Description of population sample
411 patients treated with hypolipidemic drugs, were examined at the
Center for Preventive Medicine during 1976, 1977, 1978. Among these 411
patients, males were 262 (63,7 %) and females 149 (36,3 % ) . Their
repartition according to age, sex and hypolipidemic drug is summarized
in table III.
TABLE III - DISTRIBUTION OF SUBJECTS TREATED WITH HYPOLIPIDEMIC DRUGS

Men (n - 262) Women (n - U9)

Age - Years old 20 - 40 40-60 > 60 20 - 40 40 - 60 > 60

Fenofibrate 15 83 28 6 35 36
Clofibrate II 97 28 3 42 27

The biological results obtained were compared in this group with

those of the equivalent group who were assumed 'healthy subjects, under-
going no treatment.
The treated patients and the control subjects were paired according
to their age, their sex and to their degree of overweight.
Analytical methods
The plasma constituents were analysed on SMA 12/60 Technicon and
GSA II Greiner (Steinmetz J., et al., 1978). The TGP and LDH activity
was measured at 37°C on Automat Eppendorf 5010 with an optimized method.
Those of alkaline phosphatases were measured on SMA 12/60 Technicon and
those of GGT at 37°C on GSA II Greiner.
 299

Statistical analysis
The statistical analysis has been previously described (Steinmetz,J.,
et al., 1978).
RESULTS AND DISCUSSION
The hypolipidemic drugs have no effect upon concentrations of total
proteins, albumin, glucose, urea, creatinine, calcium and upon LDH
activity.
The hypolipidemic effect of Fenofibrate is confirmed in the two
groups of treated patients (first and second phases of the study). In the
treated male patients, the uric acid concentration, for the percentile
50 is 315 ptnol/l and 393 iimol/1 in the control group. In the female
group, the concentrations are respectively 280 Umol/l and 303 Umol/1.
These differences are statiscally significant (p < 1 2 ) . This uricemie
effect, already reported by Drouin P., et al. (1976) is a positive effect
of the drug since hyperuricemia is correlated with the increase of plasma
lipids. We did not find any significant decrease of uricemia during
Clofibrate treatment.
The decrease of bilirubin is established in the subjects treated
with Fenofibrate or Clofibrate. The difference between the treated and
control groups is slight (3 Vimol/1) , but statistically significant regard­
less of whether or not the subjects are treated by several drugs or only
by the hypolipidemic agent. This decrease seems to be related to an
inducing effect of the hypolipidemic drugs. In rats, the protein synthesis
in the liver increases and is accompanied by an increase in liver weight
(Batt, A.M., et al., 1978, Foliot, Α., et al., 1977, Mackerer, C R . , 1977).
This hepatomegaly is due principally to a proliferation of the smooth
endoplasmic reticulum and to an increase in the number and the size of
mitochondria and lysozomes (Salvador, R.A., et al., 1970, Taylor, K.G.,
et al., 1977) the increased urinary excretion of glucaric acid, of the
glucuronolactone deshydrogenase activity and of cytochrome F­450 confirms
the inducing effect of the drugs Fenofibrate and Clofibrate (Batt, A.M.,
et al., 1978).
The plasma concentration of inorganic phosphates seems to be parti­
cularly sensitive to Fenofibrate intake. The values in the treated
subjects (0.97 mmol/1) are statistically lower than those in the corres­
ponding controls (1.04 mmol/1).
300

In a group of hyperlipidemic children treated with Fenofibrate for

a three months period, we observed a 12 % decrease of the inorganic
phosphates values (Steinmetz, J., et al., 1978).
Other drug, namely oral contraceptives and anticonvulsivants, also
induce a decrease of phosphataemia (Batt, A.M., et al., 1974, Batt, A.M.,
et al., 1976). We have shown that Fenofibrate and Clofibrate lead to a
decrease of plasmic GGT activity. The differences of activity between the
control group and the group treated by Clofibrate are 10 U/l (percentile
50) and 33 to 100 U/l (percentile 95) (table IV).
TABLE IV - GGT, ALKALINE PHOSPHATASE AND TGP ACTIVITIES IN A GROUP OF
SUBJECTS TREATED BY FENOFIBRATE OR CLOFIBRATE AND CONTROL
GROUPS (Results in U/l)

Fenofibrate Controls Clofibrate Controls

η η
5 50 95 5 50 95 5 50 95 5 50 95

CGT
Mea 77 12 29 82 II 30 152 39 8 21 74 11 31 172
Uomen 50 7 17 87 9 22 63 25 6 12 35 9 22 57
Alkaline phosphates
Hen 126 21 42 76 33 51 82 136 23 41 70 31 51 91
Women 77 24 44 86 30 54 91 73 26 42 82 36 58 91
TGP
Men 109 15 26 64 15 29 66 109 16 27 51 15 27 69
Women 66 15 25 73 II 20 42 62 13 23 69 II 19 36

Under Fenofibrate treatment, the differences are less marked. The

decrease in GGT activity is surprising. Indeed, this enzyme is often
used as a plasma indicator of enzymatic induction. However, the decrease
in GGT activity may be directly related to the hypolipidemic action.
Indeed, the enzyme seems to be attached to lipoproteins (Simonelli, C ,
et Eaton, R.P., 1978). As a consequence, it's activity will be decreased
at the same time as it's ligand. The decrease of alkaline phosphatase
■activity during treatment with Fenofibrate and Clofibrate is evident
(table IV). This decrease appears to be a late reaction during treatment
(Fromentin, M., et al., 1978). In a group of 68 adults treated by
Fenofibrate, we showed that the decrease of alkaline phosphatase
activity is due to the hepatic isoenzyme and not to'the bone isoenzyme
(Fig. 1). The total alkaline phosphatase activity is 50 U/l for the
percentile 50 in the treated group and 68 U/l in the control group.
That is a difference of 26 %.
 301

Total alkaline phosphatase

50
Fenofibrate

68
Control

Hepatic isoenzyme

29 .
Fenofibrate

1=3 Control

Bone isoenzyme

28
Fenofibrate

29
Control

25 50 75 100 125 U/L

Figure 1 : Total alkaline phosphatase activity, hepatic and

bone isoenzyme activities in subjects treated by Fenofibrate
and controls (results for percentile 5, 50 and 95).

We find this same difference regarding the hepatic isoenzyme (20 U/l
in the treated group and 37,5 U/l in the control group). A similar effect
has been shown for Clofibrate (Shade, R., et al., 1978, Zunmoff, Β., 1978).
The TCP activity is the same in the treated and control groups
(table IV). However, the value for percentile 95 is 34 U/l higher in the
treated women compared with the controls, regardless of the hypolipidemic
agent used. An increase of transaminase activity in the Clofibrate treated
subjects has been pointed out (Delwaide, P.A., et al., 1973, Sundermann,
F.W., 1970). However the patients under Fenofibrate treatment did not
show this increase.
302

CONCLUSION
The intake of hypolipidemic drugs induces disturbances in the
biological test results of treated subjects, (decrease of the values of
uric acid, bilirubin, inorganic phosphates and of enzymatic activity of
GGT and alkaline phosphatase). These modifications are not necessarily of
a pathologic nature. However, subjects who show serious changes particu­
larly of enzyme activity should be submitted to more frequent biological
testing.

REFERENCES
Batt, A.M., Siest, G., Loppinet, V., Guerin, P., Gueguen, R., Floc'h, Α.:
Médicaments et valeurs de référence en biologie. I Contraceptifs
oraux. Ann. Biol. Clin., 32: 245­256, 1974.
Batt, A.M., Siest, G., Khodjet El Khil, R., Le Perron, B., Weber, M.,
Tridon, P.: Drugs and reference values in clinical chemistry. II
Anticonvulsivants. In: Drug interference and drug measurement in
clinical chemistry, Siest G. Ed., 33­41 (Karger, Basel), 1976.
Batt, A.M., Khodjet El Khil, R., Ν'Guyen, T., Siest, G.: Effet des
hypolipémiants sur l'hydroxylation et sur la synthèse de l'acide
glucarique. Soc. Chim. Thérap., 1978.
Delwaide, P.A., Jaclin, Α., He us ghem, C : Interference des medicaments sur
les déterminations effectuées en chimie clinique. In: Les effets
indésirables des médicaments. 698­709, 1973.
Drouin, P., Mejean, L., Sauvanet, J.P., Pointel, J.P., Gay, G., Debry, G.:
Etude de l'action hypolipidémiante du Procétofène chez des malades
porteurs d'une HLP du type IIa ou IIb. Gaz. Med. France, 83:
3848­3860, 1976.
Foliot, Α., Droucourt, J.C., Etienne, J.P., Housset, E., Fiessinger, J.N.,
Christoforov, B.: Increase in the hepatic glucuronidation and
clearance of bilirubine in Clofibrate treated rats. Biochem.
Pharmacol., 26: 547­549, 1977.
Fromentin, M., Drouin, P., Sauvanet, J.P., Rouffy, F.: Etude de la to1eran­
tolérance hépatique après quatre ans de traitement par le Procétofène.
Nouv. Presse. Med., 7: 938­940, 1978.
Mackerer, C.R.: Effect of'Clofibrate administration on several biochemical
parameters of normal and thyroidectomized rats. Biochem. Pharmacol.,
26: 301­306, 1977.
Salvador, R.A., Haber, S., Atkins, C , Gommi, B.W., Welch; R.M.: Effect of
Clofibrate and l­methyl­4­piperidyl bis (p­chlorophenoxy) acetate
(Sandoz 42­348) on steroid and drug metabolism by rat liver microsoms.
Life Sciences, 9: 397­407, 1970.
Shade, R.W.N., Demacker, P.N.M., Van't Laar, Α.: Clofibrate effect on
alkaline phosphatase. Bone or liver fraction. New Engl. J. Med.,
297: 669, 1978.
Simone H i , C., Eaton, R.P.: Effect of Clofibrate on in vivo triglyceride
production and clearance in genetically hyperlipemic rats.
Atherosclerosis., 29: 269­275, 1978.
Steinmetz, J., Gaspart, E., ?anek, E., Morin, C , Drouin, P.: Effect of
hypolipidemic drugs on ten blood constituents. In::Drug measurement
and drug effects in laboratory health science. The International
 303

Colloquium on Prospective Biology, Pont-A-Mousson, 1978. S i e s t , G.

and Young, D.S. Ed., 194-203 (Karger, B a s e l ) .
Sunderman, F.W.: Drug interference i n c l i n i c a l biochemistry. C r i t i c a l
reviews in Clinical Laboratory Sciences. 427-448, 1970.
Taylor, K.G., Holdsworth, G., Galton, D . J . : C l o f i b r a t e increases
l i p o p r o t e i n e - l i p a s e a c t i v i t y i n adipose t i s s u e of h y p e r t r i g l y c e r i -
daemic p a t i e n t s . Lancet, 2 : 1106-1107, 1977.
Zumoff, N.: Effect of Clofibrate on plasma l e v e l s of a l k a l i n e phos-
phatase. New Engl. J . Med., 297: 669, 1978.

ACKNOWLEDGMENTS

T h i s work was s u p p o r t e d by c o n t r a c t INSERM, CRL 7 7 - 1 - 1 9 2 - 8

 PHARMACOLOGICAL EFFECTS OF TRANQUILIZERS
AND LABORATORY TESTS INTERFERENCES

Francois TRIVIN and Marie Françoise GERHARDT

Laboratoire de Biochimie Hôpital Saint-Joseph F. 75674 PARIS Cedex 14
et Centre d'Etudes Pharmaceutiques Paris XI- 92270 CHATENAY MALABRY.

ABSTRACT
The effects of tranquilizers on laboratory tests may be related
to their pharmacological activity. The molecular basis of pharmacological
effects of major tranquilizers are dominated over by action on membranes,
and dopaminergic neurons receptors.
In the case of minor tranquilizers, basis of molecular action is
not clear, and cooperative effects on Gamma Aminobutyric Acid (GABA)
receptors is the usual hypothesis. Unfrequents effects on laboratory
tests are observed. However, some of them, specially on endocrine system
must be known.

INTRODUCTION
Tranquilizers are psychotropic drugs and represent a major milestone
in the history of psychopharmacology.
They are classified in two main categories :
- Major tranquilizers, or antipsychotics or neuroleptics drugs.
- Minor tranquilizers or anxiolytics drugs.
Each classes may be further divided as :
- Major tranquilizers :
. Phenothiazines (Chlo.rpromazine, Prochlorperazine, Thioridazine)
. Butyrophenones (Haloperidol, Dorperidol, Penfluridol, Lenperone)
. Thioxanthenes (Thiothixene, Chlorprothixene, Flupenthixol)
. Miscellaneous (Poxapine, Metrapine, Clorapine, Sulpiride).
- Minor tranquilizers :
. Benzodiazepines.
. Propanediol carbamate.
 305

The weak frequency of side effects of tranquilizers on laboratory

tests, as consequence of their pharmacological effects must be compared
to the large employement of these drugs.
In the first part we will mention the molecular and biochemical
underlying of the pharmacological effects.
In the second part we will report literature data of effects
on laboratory tests.
I - MAJOR TRANQUILIZERS
The fundamental action of neuroleptics on biological membranes help
us to understand the main pharmacological effects as well as side effects.
Experimental data demonstrate that biological membranes are
expanded by neuroleptics (SEEMAN, et al, 1974).
At the concentration of 2. 10" M, the membrane area of spherical
erythrocytes ghosts progressively expands by a maximum of 5 per cent.
For higher concentration, this effect was inhibited. These pheno-
menons may explain the anti-hemolytic role of neuroleptics. Low concentra-
tion of chlorpromazine are efficient as protecting agents of erythrocytes
from hypotonic hemolysis because the increase of area/volume ratio of
the erythrocytes (VAN STEVENINCK, et al, 1974). However LOPPINET, (et al,
1975), with human leucocytes as model, determined that phenothiazine
Induce protein and enzyme release. Interpretation of these phenomena
required consideration on structural, electronic and steric effects on
membrane.

Mechanism of membrane action may be explained from

a) membrane occupation by drug
b) membrane lipid fluidlzation
c) membrane protein expansion
2+
d) displacement of membrane Ca
a) The neurolept1c-1nduced membrane expansion may result in a
membrane-occupying volume of the neuroleptic molecules by insertion
Into constituents of membranes.

b) I t 1s thought thas wide variety of membranes are fluidized by

the neuroleptics (METCALFE, et a l , 1968) as shown with anesthetics
(VAN DEENEN, et a l , 1965) which expand and fluidize l i p i d films at a i r /
water interface.
306

c) Tranquilizers change the conformation of proteins following their

adsorption to membranes and alter the protein conformation. This
phenomenon may account for the observed membrane expansion. These
distorting facilitated diffusion channel (HUNTER, et al, 1965) and
specially the Na + conductance channel (GRUENER, et al, 1972).
2+
d) Neuroleptics readily displace membrane associated Ca .
Such displacement may reduce the contracted stade of postulated
contractile protein in membrane* or may reduce the distance between
2+
lipids molecules normally nonliked by Ca .
e) Membrane-membrane fusion.
Tranquilizers increase the frequency of miniature end plate
potentials ; this is thought to result from the drug's fluidizing action
on both the presynaptic membrane and the vesicle membrane.
Finally, neuroleptic drugs may be considered as potent releasing
drugs. Explanation for their pharmacological action are that neuroleptic
potentiate exocytose in the dopaminergic and cholinergic system. These
drugs are also known as membrane electrical stabilizing agents.(SEEMAN,
et al, 1972).
These data may explain various effects of neuroleptics and especially
the main side effects :
- the extrapyramidal syndrome (Parkinsonism and tardive dyskenesia).
- cardiac and pulmonary effects.
- laboratory tests interferences.
II - MINOR TRANQUILIZERS
The molecular mechanism of pharmacological effects of Benzodia-
zepine is not well-known.
These drugs do not act as competitor for a specific site in brain
receptor. It is postulated that there are specific receptors for
Benzodiazepine in human brain. These receptors have affinity for a
metabolite as physiologic ligand : the GABA. At the physiologic statement,
an hypothetical endogeneous protein, is fixed on these receptors, and
decrease their affinity for GABA. Increase of GABA affinity for receptor
results form the competitive effect between the endogeneous protein and
the Benzodiazepine drugs.
 307

Facilitation 1n fixation of GABA on the post synaptic membranes may

explain the side effects of these drugs :
- hypnotic, anticonvulsant, muscle relaxant.
Biogenic amine may be Involved in the biochemical mechanism of Benzodia-
zepine action.
Ill - MAJOR TRANQUILIZERS EFFECTS ON LABORATORY TESTS.
III.l. ENDOCRINE SYSTEMS
Binding of tranquilizers on biogenic amines receptors sweept
along an in vivo modification of hormones level.
111.1.1. Biogenic Amines
Modification of turnover of biogenic amine and more specially of
dopamine and It's metabolite homovanillic acid are consequence of
neuroleptic Intake (GARB, 1971).
Mechanism results of an decrease of dopamine captation by specific
receptor (blockade theory) and probably enlargement of secretion, which
may be an explanation for the dyskenesia syndrome (impulse blockade
theory). Perhaps this last mechanism may explain data on the rise of
cathecholamine level after administration of sulpiride in diagnostic
of latent phoechromocytosis.
111.1.2. Prolactin
It is well-known that serum prolactin level is increased after
chlorpromazine Intake. The galactorrhea induced in both male and female
1s a side effect of long-term treatment with phenothiazine (PARE,1976).
Serum prolactine level follows a circadian rythm, controlled by a protein,
the Prolactin Inhibitor Factor. Dopaminergic neurons are involved in the
control of this factor. Antagonists of Dopaminergic neurons, as
phenothiazine and butyrophenone drugs cause a dose - related rise in
prolactin levels, in a short time after intake.
111.1.3. Chlorpromazine, as Perphenazine and Spiroperidol may block
ovulation by an unhnown mechanism. (FUXE, 1975). Perhaps blockade of
norepinephrines receptors may induce this blockade. This effect is
different from the analytical effects observed in pregnancy tests
(SUNDERMAN, 1970).
308

111.1.4. Growth Hormone : neuroleptics increase level plasma of growth

hormone (G.H.), probably by blocking effect on hypothalamic dopaminergic
receptors which are mainly implicated in secretion of G.H. (SHERMA N, 1971).
111.1.5. Thyroid
Some reports indicated (A CLA ND, 1971) that modification of thyroid
function was induced by majors tranquilizers as Phenothiazine, Haloperidol
but mechanism was lacking in explanation. 1. level decrease, probably
by activation of microsome turnover, as result of metabolism and catabo­
lism of Phenothiazine.
111.1.6. Steroid
No modification of serum Cortisol level was reported until to day ;
however, ACTH secretion decrease, following lowering of hypothalimic
activity (GA UNT, R., 1968) may induce interferences.
Levels of urinary 17 KetosteroTds may be modified. But further
investigation will be necessary to elucidate these observations.
III.2. LIVER
Majors tranquilizers are organic molecules which implicate an
microsomal metabolism, with hydroxylation and conjugation with
glucuronic acid.
In a recent study, (GONÇA LVES, 1977) reported data on effects of
chronic administration of neuroleptic on hepatic functions.
ASAT and ALAT were increased as observed from γ GT. The mechanism
of these augmentations was unexplained, but may be explained by the
results of LOPPINET, et al, (1976), on the increase of membrane cell
permeability. On the other hand it is well known that chlorpromazine may
induce an intrahepatic cholestasis with jaundice and increase of total
and conjugated bilirubin level. BSP test retention rate and total alkaline
phosphatase may increase. Augmentation of cholesterol level may be related
to this cholestatis. This drug­linked cholestatis may disapear shortly
after the withdrawal.
Phenothiazines induce modifications in glucose tolerance.test.
Abnormal curve of glucose tolerance was observed in 30 p.cent of patient
intaking Phenothiazine. Plasmatic glucose level increase and may induce
glucosuria. Mechanism of this hyperglycemia is unknown (MEYLER, 1962).
 309

111.3. URIC ACID

Uric a d d metabolism is modified by Intake of Chlorpromazine
(SUNDERMAN, 1970). Serum uric adid decrease, and urinary uric acid
Increase. Tubular reabsorption of this molecule is lowered and allow
to conclude 1n favour of an uricosuric effect. The intensity of this pheno-
menon 1s related to drug concentration.
111.4. RED CELLS
Intake of Phenothiazine, Butyrophenone may cause an hemolytic
anemia with hypo hemogloblnemia and Increase of reticulocytosis
(COOPERBERG, et al, 1956). The mechanism of this effect is unclear , but
may be related to linkage of drug on membrane. The same phenomenon may
be noted with leucocytes.
IV - MINOR TRANQUILIZERS EFFECTS ON LABORATORY TESTS
There are many publications on the chemistry, pharmacology,
structure-activity, metabolism and clinical effects of minor tranquilizers
but very few effects on laboratory test are mentioned, in the literature.
Benzodiazepines, affect many of the neurotransmitters and neuromodulator
systems of the brain (catecholamine, serotonine.yaminobutyric acid,
glycine, acetylcholine). These biochemical effects appear to be relatively
Indirect probably by decrease of turnover of catecholamines and 5 hydroxy-
tryptamlne.
However no modification on level of plasma catecholamines was
observed (CRYER, et al, 1971) excepted unfrequent slight decrease of
HVA (PAPESCHI, et al, 1972). Effects on others endocrine system are not
described. A decrease of leucocytes rate had been reported.
CONCLUSION
Pharmacological effects of various tranquilizers may introduce
modifications on the level of biological parameters.
Main effects result from the membrane and neurotransmitter action
and perhaps all effects have not been investigated specially on
endocrine system and Intermediary metabolism.
On the other hand effects have not been correlated to the concen-
tration of plasma drugs ; moreover only some aspects of related structure
activity and interference on laboratory tests have been investigated
and must be rationally studied.
310

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58­67, Siest,G. and Young, D.S., Editors Karger 1976.
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presence of cyto membranes. Nature 220: 587­588, 1968.
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and depression. Pharmakopsychiatry, Neuro, Psychopharmakology,
9: 187­192, 1976.
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Pharmacol. Rev. 24: 583­655, 1972.
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Structurally related drugs, Forrest. I.S., Carr, C.J., Usdin, E.
Editors, pp. 137­148, Raven Press NY, 1974.
■Sherman, L., Kim,S., Benjamin, F., Kolodny, M.D., : Effects of chlorproma­
zine on serum growth hormone, New Eng. J. Med. 284 : 72­74, 1971.
Spano, P.F., Di Chiarra, G., Loddo, P., Tonon, G.C., and Trabbuchi, M., :
Dopamine stimulated adenylate cyclase in rat substancia nigra,
localisation and effects of neuroleptics in non striatal Dopaminer­
gic Neurons, Costa, E., Gessa, L., Edit. Advances in biochemical
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Rev. Clin. Lab. Sci.i 1 : 427­441, 1970.
 311

Van Deenen, L.L.M., Dehel, R.A. : Penetration of lipids monolayer by

psychoactive drugs, Biochim. Biophys. Acta, 94 : 314-316, 1965.
Van Steveninck, J. Gjosund, U.K., Booij, H.L. : The influence of
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Pharmacol. 16: 837,1967.
 EFFECT OF IMIPRAMINE ON LABORATORY TESTS

A. Tazi, M.M. Galteau and G. Siest

Centre de Médecine Preventive (Directeur : Pr. R. Senault)

2 avenue du Doyen Jacques Parisot, 54500 Vandoeuvre-les-Nancy, France

ABSTRACT
The mean value of gamma-glutamyltransferase activity was significantly
(+ 38%) increased (p < 0.01) in a population of 24 subjects treated over a
long term with Imipramine when compared to a corresponding control popula-
tion. This increase was especially high in women (+ 59% against 14% in
men). A slight elevation in the mean cholesterol values (+ 17%) was also
observed (p < 0.02) in the Imipramine treated population. Furthermore, an
increase of 140% in the gamma-glutamyltransferase was found in 5 women who
were simultaneously taking Imipramine and oral contraceptives.
The variations óf other studied parameters, such as alkaline phospha-
tase, lactate dehydrogenase, glutamate pyruvate transaminase bilirubin and
triglycerides were not significant either in the Imipramine treated popu-
lation, or in the case of the women who were treated with both Imipramine
and oral contraceptives. These data indicate that Imipramine increased
plasma ganrnia-glutamyltransferase activity, which could be related to the
hepatic membrane effect caused by this drug.

INTRODUCTION
The side effects of the treatment with antidepressants have been well
described in the literature. Most of the undesirable effects are minor, but
may, however, cause more serious troubles if their dose is not limited.
The hepatic toxicity of Imipramine, which is the most used tricyclic anti-
depressant drug, can lead to intrahepatic cholestasis (Hoaken, 1964;
Karkalas, et al., 1971) or necrosis (Powell, et al.,1968).
A relation between the metabolism of Imipramine and its side effects has
been discussed but never established. The effects of Imipramine on the
biological laboratory tests, and moreover, on the hepatic parameters were
-not even discussed. The pharmacological interaction of Imipramine with oral
contraceptives was extensively studied in several species (Meyerson, 1966;
Calhoun, et al., 1971; Bellward, et al., 1973; Fludder, and Tonge, 1977).
This interaction was often manifested by mental disturbances (Greengrass,
and Tonge, 1972; Khurana, 1972). This seems to be due to an interaction of
the hepatic metabolism of these two drugs. In fact, the oral contraceptives
are known to decrease the activities of drug metabolizing enzymes (O'Malley,
et al., 1972; Hertz, et al., 1978). It seems that patients who are
 313

simultaneously receiving high doses of oestrogen and Imipramine accumulate

in the brain and in other tissues a high concentration of Imipramine»

secondary to inhibition by oestrogen to the Imipramine metabolizing

enzymes and hence, leading to neurological and other symptômes described

earlier (Somani, and Khurana, 1973). Moreover, it is known that Imipramine

has a direct effect on the hepatic cell membranes (Kappus, and Remmer, 1975).

To clarify the hepatic impact of Imipramine in man, we studied the varia-

tions of certain biological parameters in a population under long term

treatment with this drug, and in another population of women treated with

this drug together with oral contraceptives. They were both compared to

supposed healthy control populations.

MATERIALS AND METHODS

1. Selection of the populations:

The subject of our study were all chosen among the individuals who

came to the Center for Preventive Medicine of Vandoeuvre-les-Nancy for a

routine health examination.

Among the total population we selected were:

. 24 subjects treated only with Imipramine (TOFRANIL^ or its

derivative, clomipramine (ANAFRANIL·0) for a period of more than two months.

This population was screened from the drug index (Steinmetz, et al.,

in press)

. 24 subjects (control population) for whose each subject was

chosen to correspond in sex, age, overweight and the date of analysis with

one of the subjects in the treated population (Table 1).

314

TABLE 1 ­ CHOICE OF IMIPRAMINE TREA TED POPULATION AND CORRESPONDING

CONTROLS

Patient Controls Treated

N° A ge Sex OW (%)« A ge Sex OW (Z)sc

1 12 M ­ 13 12 M ­ 16
2 10 M ­ 17 11 M ­ 14
3 36 F ­ 8 34 F ­ 8
4 12 M ­ 6 12 M ­ 7
5 36 M ­ .5 37 M ­ 7
6 17 M ­ 8 16 M ­ 6
7 52 M ­ 1 50 M ­ 5
8 27 F ­ 2 28 F ­ 4
9 8 M ­ 4 8 M ­ 4
10 8 F ­ 9 7 F ­ 4
11 5 M ­ 3 5 M ­ 3
12 5 F 0 5 F ­ 3
13 36 F ­ 3 36 F ­ 2
14 14 M + 2 14 M 0
15 11 F + 2 10 F + 4
16 47 M + 3 47 M + 7
17 35 M + 10 33 M + 12
18 39 F + 9 39 F + 12
19 40 M + 14 40 M + 13
20 45 F + 15 48 F + 17
21 36 F + 13 37 F + 19
22 44 M + 16 44 M + 18
23 55 F + 33 53 F + 42
24 55 F + 35 56 F + 44

" Degree of overweight (O.W) is calculated as:

actual weight ­ ideal weight
ideal weight χ 100
Ideal weight is calculated according to Lorenz formula:
ideal weight = (height ­ 100) ­ (height ­ 150)
4
where height is in centimeters and weight in kilograms.

Then we selected:
. 8 women who had been treated over a long term with Imipramine
and oral contraceptives. This was a heterogeneous population in that the
women selected were taking another drug in addition, which was in almost
all cases a tranquillizer (Table 2). For this population we present only the
preliminary results
. 8 women (controls) by the same criteria described above
 315
TABLE 2 - CHOICE OF TREATED WOMEN WITH IMIPRAMINE AND ORAL CONTRACEPTIVES
AND THE CORRESPONDING CONTROLS

Cone rols Treated

Age OW (Z) Age OW (Z)" Other drugs
taken

23 - 18 25 _ 18 1 Tranquillizer
19 - 3 19 - 6 1 Tranquillizer
31 + 1 32 + 3 2 Tranquillizers
34 + 5 35 + 5 None
30 + 8 30 + 11 1 Tranquillizer
44 + 17 43 + 20 1 Tranquillizer
39 + 45 40 + 38 None
31 + 41 30 + 40 1 Tranquillizer

,s
Overweight is calculated as in Table 1.

Alcoholics, subjects with clinical or biological abnormalities,

and those who had antecedent hepatic diseases were screened out.
2. Determination of the laboratory tests:
We chose seven parameters, all of which are indicative of the
hepatic effect of the studied drugs (Table 3 ) .
TABLE 3 - PARAMETERS STUDIED AND METHODS OF ANALYSIS

Parameter Method Analyzer

Gamma-glutamyItransferase
(GGT, E.2.3.2.1) Szasz, 1969 GSA II Greiner

Alkaline phosphatase
(ALP, EC.3.1.3.1) Morgenstern, et al.,1965 SMA 12/60 Technicon

Alanine aminotransferase Kinetic with reagent Automate Eppendorf

(ALT, EC 2.6.1.2) kit from Boehringer 5010
N° 15762

Lactate dehydrogenase CHU and Bishop, 1972 Automate Eppendorf

(LDH, EC 1.1.2.3) 5010

Total bilirubin Cambino and Schreiber, SMA 12/60 Technicon

1964

Total cholesterol Huang, 1961 SMA 12/60 Technicon

Triglycerides Bucolo and David,1973 GSA II Greiner

316

RESULTS
1. Variations due to the Imipramine:
. GGT:
The mean of the GGT activities in the treated population vas
38.5% higher than that of the corresponding control group, as shown in
table 4. This difference is a little higher in individuals over 14 years
(+ 37%) than in children (+ 30.6%); the variation in the adults is
emphatically larger in women (+ 59%), whereas only + 14% in men (Table 5 ) .
TABLE 4 ­ VARIATIONS OF LABORATORY TEST VALUES AFTER IMIPRAMINE TREA TMENT

Mean values Percents of

Controls Treated variation

GGT (U/l) 24 12.18 16.88 + 38.5 %"5!

ALP (U/l) 21 125.00 129.70 + 3.7 %
ALT (U/l) 19 20.60 24.48 + 18.8 %
LDH (U/l) 18 260.70 318.30 + 22.0 %
Bilirubin (umol/1) 19 9.58 11.12 + 16.0 %
Triglycerides (mmol/1) 23 0.86 0.73 ­ 14.0 %
Cholesterol (mmol/1) 21 5.23 6.16 + 17.8 %::

"" ρ < 0.01 " ρ < 0.02

percent of variation is calculated as C ­ Τ ιη.

— = — χ 100

(C = mean value of control group, Τ = mean value of treated group)

TABLE 5 ­ VARIATIONS OF GGT VALUES AFTER IMIPRAMINE TREA TMENT

Mean values (U/l) Percents of

Controls Treated variation

Children (age < 14 years) 9 t 7.77 10.15 + 30.6 %!!5S

Adults (age > 14 years) 15 15.07 20.70 + 37.4 %""

Men 7 16.80 19.12 + 14.0 %""

Women 8 13.78 21.95 + 59.3 %5S!Î

Percents of variation are calculated as in Table IV

"" ρ < 0.01

 317
Figure 1 shows that for all Che studied subjects the value of GGT was
higher in treated subjects rather than in controls, except in one case
(N* 17)
mu/ml

30

20

10

Patient No
-r-
10 15 20

Fig. _1_ - GGT ACTIVITIES IN IMIPRAMINE TREATED PATIENTS (o -o)

AND THOSE OF THE CORRESPONDING CONTROLS (· ·)

. Cholesterol:
There is a small (+ 17.8Z) but significant (p < 0.02) difference
between the mean of the values of cholesterol in the treated population
and that of the control population (Table 4 ) . This difference does not
occur in all studied cases (Fig. 2 ) .
318

mmol/l
10-

Patient No
if 15 20

F i g . _2 - CHOLESTEROL VALUES IN IMIPRAMINE TREATED PATIENTS (o- -o)

AND THOSE OF THE CORRESPONDING CONTROLS (· ·)

. Other parameters:
The variations of ALP, ALT, LDH, bilirubin and triglycerides
between treated and control populations are small and not significant
(Table 4 ) .
2. Variations due to treatment by the Imipramine associated with
-oral contraceptives.
The mean of the GGT values measured in 5 of the treated women was
higher (+ 140%) than in the corresponding controls. The variations found
for the other parameters are not significant.
 319

TABLE 6 ­ VARIATIONS OF LABORATORY TEST VALUES AFTER TREATMENT WITH

IMIPRAMINE A ND ORAL CONTRACEPTIVES IN WOMEN

Mean values Percents of

Controls Treated variation"

GGT (U/l) 5 9.60 23.28 + 140.0 X'""

ALP (U/l) 8 38.40 39.70 + 3.5 Ζ
ALT (U/l) 6 19.90 16.80 ­ 15.7 Ζ
LDH (U/l) 6 277.00 229.00 ­ 14.0 Ζ
Bilirubin (umol/1) 7' 10.49 8.79 ­ 16.0 Ζ
Cholesterol (mmol/1) 7 5.41 5.39 ­ 0.5 Ζ

" Percents of variation are calculated as in Table IV

SS!!
ρ < 0.01

DISCUSSION
Among the biological parameters studied, only the values for GGT
activity and cholesterol differed significantly in the Imipramine treated
population when compared to the corresponding control population.
GGT activity is known to be increased in plasma of subjects treated by
various drugs, especially those of the phénobarbital group (Rosalki, et aL,
1971; Hildelbrandt, et aL, 1975). Also, a 20Z increase is observed in women
receiving oral contraceptives (Weinberg, et aL, 1976). In experiments with
rabbits, we have recently shown that althrough pretreatment with phéno­
barbital induces the hepatic plasma membranes GGT, it does not lead to a
direct increase in plasma enzyme activity. This occurs only later and was
explained by a possible perturbation of the membranes structures caused by
phénobarbital treatment (Tazi, et al.,in press). On the other hand, we found
(not yet published results) an increase of plasma GGT in rats under Imipra­
mine treatment, while the hepatic activity was not induced.
The binding of Imipramine to hepatic membranes, as has been shown by
Kappus and Remmer (1975) leads to membranes disturbances (Romer, and
Bickel, 1979). The membrane effect of Imipramine is probably responsible
for the release of GGT into the plasma in the Imipramine treated subjects.
Furthermore, our present results demonstrate that the increase of GGT
activity in the Imipramine treated population is higher in women than in
320

men. In women,' metabolism of drugs is known to be lower than in men, hence

the drug effect is prolonged. This fact may explain the differences in GGT
values between men and women under Imipramine treatment. Young et al.,
(1975) report increase of ALP, ALT, LDH, cholesterol and bilirubin from
isolated cases of jaundice following therapy with Imipramine. The
variations we observed for these parameters were not significant except
for cholesterol and the subjects we selected have not any hepatic disease
established.
In women treated with Imipramine and with oral contraceptives, we observed
that the increase of GGT values was higher than that in women treated with
Imipramine alone. But those results must be supported by further studies
in larger and more homogeneous populations. Moreover, similar results were
obtained by our group in work done in rats (not yet published). The
increase in GGT activity in the plasma is higher in rats treated with both
Imipramine and contraceptives, than in rats treated with either Imipramine
or with contraceptives alone.

CONCLUSIONS
From this study of the effect of Imipramine on laboratory tests, the
important point to be retained is the increased GGT values in most of
subjects treated with this drug in comparison to those of the corresponding
controls. This phenomenon is of importance in clinical chemistry to avoid
misinterpretation of GGT high values in subjects under Imipramine treatment.
Moreover very high values of GGT in cases under treatment with this drug
can indicate a possible hepatic membrane damage and so the prescription or
the dose of Imipramine has to be modified.
This study must be extensived to other tricyclic antidepressants and to
their interaction with contraceptives towards usual laboratory tests.

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détermination de la gamma­glutamyltranspeptidase à la sëméiologie
hépatique (Boehringer Mannheim, Paris 1973).
Young, D.S., Pestaner, L.C. and Gibberman, V.: Effects of drugs on
clinical laboratory tests. Clin. Chem. 21: 140D, 1975.
This volume is an up-to-date survey of various drug effects on the analytical
procedures of clinical biochemistry. Drugs and xenobiotics can increase or
decrease the level of certain blood constituents. This can be due to an analytical
or to a pharmacological effect.
The 'in vitro' effect, called analytical interference, has to be systematically
checked, especially for chemical tests. It is likely to disappear with further progress
in analytical methods towards specificity.
The 'in vivo' pharmacological effects of drugs must be measured and calculated
for every new drug or new laboratory method. The knowledge of these effects in
terms of percentages will permit a better interpretation of laboratory results by
clinical chemists and physicians.
A data bank on drug interferences and drug effects in clinical chemistry is
needed in order to better interpret laboratory results and diminish the incorrect
diagnoses and mistreatment of patients.

ISBN 90-247-2419-8