Clinical Biochemistry, Vol. 31, No.
4, 195–220, 1998
PII S0009-9120(98)00015-0
Nitrite and Nitrate Analyses: A Clinical
Biochemistry Perspective
GRAHAM ELLIS,1 IAN ADATIA,2 MEHRDAD YAZDANPANAH,1,3 and SINIKKA K. MAKELA3
1
Department of Laboratory Medicine and Pathobiology, University of Toronto, 100 College Street,
Toronto, ON M5G 1L5, Canada, 2Divisions of Cardiology, Critical Care Medicine and Pediatrics,
University of Toronto, and 3Division of Clinical Biochemistry, The Hospital for Sick Children, 555
University Avenue, Toronto, ON M5G 1X8, Canada
Objective: To review the assays available for measurement of nitrite
and nitrate ions in body fluids and their clinical applications.
Design and Methods: Literature searches were done of Medline
and Current Contents to November 1997.
Results: The influence of dietary nitrite and nitrate on the concen-
trations of these ions in various body fluids is reviewed. An overview
is presented of the metabolism of nitric oxide (which is converted to
nitrite and nitrate). Methods for measurement of the ions are
reviewed. Reference values are summarized and the changes
reported in various clinical conditions. These include: infection,
gastroenterological conditions, hypertension, renal and cardiac
disease, inflammatory diseases, transplant rejection, diseases of
the central nervous system, and others. Possible effects of environ-
mental nitrite and nitrate on disease incidence are reviewed.
Conclusions: Most studies of changes in human disease have been
descriptive. Diagnostic utility is limited because the concentrations
in a significant proportion of affected individuals overlap with those
in controls. Changes in concentration may also be caused by diet,
outside the clinical investigational setting. The role of nitrite and
nitrate assays (alongside direct measurements of nitric oxide in
breath) may be restricted to the monitoring of disease progression,
or response to therapy in individual patients or subgroups. Associ-
ations between disease incidence and drinking water nitrate content
are controversial (except for methemoglobinemia in infants).
Copyright © 1998 The Canadian Society of Clinical Chemists
KEY WORDS: nitrates, nitrites, nitric oxide, nitric-
oxide synthase, arginine, human disease
Introduction
nterest in the measurement of nitrite and nitrate
I in various body fluids has increased in recent
years (1–5). These ions are ingested as part of the
diet and are also produced endogenously from nitric
oxide (NO). In this article, we shall review the
effects of dietary intake on nitrite and nitrate con-
centrations in plasma and urine. We shall discuss
Correspondence: Dr. Graham Ellis, Department of Clin-
ical Biochemistry, St. John’s Hospital at Howden, Howden
Road West, Livingston, West Lothian, EH54 6PP, United
Kingdom.
Manuscript received January 6, 1998; accepted March
24, 1998.
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
Copyright © 1998 The Canadian Society of Clinical Chemists
Printed in the USA. All rights reserved
0009-9120/98 $19.00 1 .00
the metabolism of NO to nitrite and nitrate and
summarize methods of measuring these ions in body
fluids. We shall comment on the clinical relevance of
the measurements. Finally, we shall discuss possi-
ble associations between dietary intake and disease
incidence.
Dietary intake of nitrite and nitrate
Nitrite and nitrate ions are both highly soluble in
water. With few exceptions, they form water-soluble
salts with almost all cations. When taken orally,
they are readily absorbed from the proximal small
intestine as reviewed by Walker (6). About 25% of
orally ingested available nitrate is actively secreted
into the saliva. This nitrate is then partially con-
verted to nitrite by oral bacteria (7) and to NO by
stomach acids, helping to reduce gastrointestinal
tract infection (6,8). The transport systems of ni-
trates and nitrites have only recently become better
understood in bacteria (9), in plants (10), and in
mammalian cells (11), where a nitrate and H1
cotransporter seems important. Nitrites and ni-
trates are added as preservatives and texture en-
hancers to various foods, such as meat products and
cheeses (12), but they are also a natural part of the
diet. The concentrations in drinking water are usu-
ally ,10 mg/L in the absence of bacterial contami-
nation (13). In areas where drinking water concen-
trations are high, steps are taken to lower them (14)
in order to avoid nitrate-induced methemoglobin-
emia in infants. Vegetables, especially beets, celery,
and leafy vegetables like lettuce and spinach are
rich in nitrates (6,15,16). Other vegetables contain
nitrate at lower concentrations, but because they are
consumed in greater quantity, they may contribute
more nitrate to the diet. Santillana et al. (17) sum-
marized findings from 710 commercial food samples
available in Spain. Cured meats had the highest
mean nitrite content (36 mg/g) and canned vegeta-
bles the highest mean nitrate content (88 mg/g).
195
 ELLIS
Improper storage and bacterial contamination may
increase nitrite (18). In Western diets, assessed by
dietary survey, the mean adult daily dietary intake
of nitrite (0.1 mg) is much lower than that of nitrate
(80 mg) (19). Others have found corresponding val-
ues of 1.4 mg and 54 mg in a 9 to 24-year-old Finnish
population (20). In some European countries, mean
daily intake may be as high as 8.7 mg of nitrite and
(in vegetarians) as high as 194 mg of nitrate (6).
Excessive nitrite or nitrate intake could potentially
generate N-nitroso compounds, that are carcino-
genic (21,22), but this is still controversial (6).
Nitrite and nitrate are excreted in the kidneys.
The nitrate is excreted in the urine as such or after
conversion to urea (23). Clearance of nitrate from
blood to urine approximates 20 mL/min in adults
(24), indicating considerable renal tubular reabsorp-
tion of this ion. Tubular reabsorption is also sup-
ported by studies of the urinary excretion of total
nitrate plus nitrite (NOx) concentrations in response
to diuretics (25). There is little detectable nitrite or
nitrate in feces (26). There is some loss of nitrate (40
mmol/L) or nitrite (3 mmol/L) in sweat, but this is not
a major route of excretion (27). (These values repre-
sent means for 6 subjects after a 40 min sauna.)
Patients on antibiotics had much lower sweat ni-
trite, implying a role for skin bacteria in nitrite
synthesis.
Variability in dietary intake (28) may contribute
greatly to the variability of plasma nitrate and to
urinary excretion. As would be expected, in con-
trolled studies, the daily urine output increased
with higher intake (26). Plasma nitrate increased
from about 30 mmol/L to about 200 mmol/L in re-
sponse to a high-nitrate diet and to about 300
mmol/L after 500 mg of oral potassium nitrate (29).
The ions disperse widely into body fluid spaces,
giving a large volume of distribution: 0.28 L/kg (30),
similar to that of the dog at 0.21 L/kg, equivalent to
the extracellular volume (31). A diet high in nitrate
may still elevate urinary excretion during the next
day (29,32). There was a wide variability in plasma
NOx over the course of 1 day in five individuals
taking their normal Japanese diet, as measured by 4
hourly samples (33). Between-day variation was also
observed by Wang et al. (34). Some authors have
recommended that random urines should be taken
immediately after getting up in the morning (35), as
the second fasting sample of the morning (36), or
just prior to dinner (32) to reduce this variability.
Excretion is then related to urine creatinine. One
group has recommended that there should be di-
etary restriction of nitrates and nitrites for at least
48 h prior to collection, if excretion is to be regarded
as a measure of NO metabolism (29). Others have
used a 15 h fast, with distilled water as the only
fluid permitted (37,38).
If a diet low in nitrite and nitrate is needed,
Westfelt et al. (39) recommends the exclusion of
alcoholic beverages, caviar, charcuteries (cooked or
cured delicatessen-style meats), cheese, herbs, pick-
led fish, roots and vegetables during the study and
196
ET AL.
for 2 days before the studies are undertaken. Viini-
kka (2) noted that many foodstuffs contain consid-
erable amounts of nitrate and listed meat, vegeta-
bles, fish, melon, herb and black teas, malt
beverages and wine as examples. Others have used
low-nitrate diets containing approximately 210
mmol/d (40) or ,180 mmol/d (41); the latter reference
lists both permitted and avoidance food groups. A
low nitrate diet for at least 3 to 4 days was found by
Wang et al. (34) to minimize the influences of the
Japanese diet on plasma NOx concentrations and
24 h urinary excretion, expressed in relation to
creatinine excretion. However, these authors col-
lected samples only between the third to the sixth
day of the low nitrite and nitrate diet. Plasma levels
taken at 0600 h were found to be consistent after 4
days of a well-balanced Japanese hospital diet (33).
In contrast, morning urines (the second fasting
sample of the morning in 12 healthy volunteers,
expressed in relation to creatinine) seemed little
affected by two days of low nitrate (,180 mmol/d)
diet, compared to the normal Scottish diet or a
nitrate-supplemented diet (36).
Nitrate-containing drugs have been used for many
years for the treatment of angina. They produce NO
(42,43) and give rise to urine nitrate. Because the
therapeutic doses are relatively small compared
with normal dietary intake, their contribution to
excretion is generally quite small.
Nitric oxide (NO)
In humans, NO is produced endogenously from
various sources, the most important of which is
L-arginine (4,5,29,39,44 – 48). Arginine is oxidized
by NO synthase(s) (NOS, EC 1.14.13.39) to produce
citrulline and NO. There are at least three isoen-
zymes of NOS (49 –53); one is located almost exclu-
sively in vascular endothelium. The NO that it
produces acts as a relaxing factor on smooth muscle
cells of the blood vessel wall, causing vasodilatation
(44,54). By so doing, it is an important determinant
of blood pressure (55). It is constitutively expressed.
A second (inducible) NOS is produced in macro-
phages and many other cell types in response to
inflammation or infection (52,56 – 63). The NO pro-
duced by the macrophage NOS not only damages the
agent under attack but also causes apoptosis of the
macrophage (64). NO may also induce apoptosis in
other cell types (65– 67). A third isoenzyme (neuro-
nal NOS) is found constitutively in brain, neuronal
tissue, neuroblastomas, skeletal muscle, b-cells of
the pancreatic islets, and also epithelial cells of
bronchioli uterus, and stomach (52). All three
isozymes are found in kidney (68). The activities of
the two constitutively expressed NOS isozymes are
not constant. They are calcium and calmodulin-
dependent enzymes. Transient increases in intracel-
lular calcium concentrations result in short-lived
bursts of activity. This enables NO to act as a
neurotransmitter/neuromodulator by diffusion (62,
69 –71) or by S-nitrosylation, nitrosothiol exchange,
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
 NITRITE AND NITRATE ANALYSES
and related mechanisms (72). Inducible NOS con-
tains tightly bound calcium and calmodulin and it is
generally insensitive to ambient calcium concentra-
tion (49,73) Hormones, such as estradiol, may up-
regulate NO synthesis (74). As much as 90% of
circulating nitrite is derived from the L-arginine:NO
pathway (38). On a low-nitrate controlled diet (210
mmol/d) about one-half the urine nitrate originated
from plasma arginine (40).
NO can also be produced from “recycled” nitrite or
nitrate in various ways. Acidification of nitrite may
release NO in the stomach (8). NO may be derived
from urine nitrate in infected urine (75). NO produc-
tion has been described in infarcted heart tissue
(76). The process is independent of NOS because it is
unaffected by specific NOS inhibitors. NO is also
produced in the mouth of both the rat (77) and
humans (78), or in human sweat (27). In these
instances, commensal bacteria may play a role in
synthesis and the NO that is produced may serve as
a defense mechanism against pathogens.
NO is used therapeutically in patients with pul-
monary hypertension and other conditions (79,80).
Serum nitrates and nitrites (measured as a com-
bined total) or plasma nitrate by high-performance
liquid chromatography (HPLC) increase with NO
inhalation (24,81). Most (90%) of the 15NO that is
inhaled is retained and at least 70% is recovered in
urine as 15N nitrate (39).
NO has an unpaired electron and this makes its
chemistry and biochemistry (82) and its pharmacol-
ogy (83) complex. Its biological half-life is 5 s or less
(84,85). Some of the NO produced by various tissues
rapidly binds to heme groups in enzymes such as
guanylyl cyclase, in effector cells such as the smooth
muscle cells of the blood vessel wall (86) or platelets
(87). This increases cGMP and in this way NO acts
as an intermediate or “second” messenger of hor-
mone action (88). In some cells, NO action is effected
through intracellular calcium cycling (89). Residual
amounts of NO react with water to form nitrite,
which in the presence of heme groups in proteins
such as myoglobin, hemoglobin, or enzymes, rapidly
oxidizes to nitrate and the corresponding met-heme
protein (85). For example, when NO diffuses into the
red cell, it reacts rapidly with oxyhemoglobin to form
methemoglobin and nitrite (90). In the presence of
oxygen and methemoglobin reductase, these are
rapidly converted back to hemoglobin and to nitrate.
NO also reacts with thiol groups to form nitrosothi-
ols, such as nitrosocysteine or nitrosoalbumin (91,
92) or nitrosohemoglobin (93,94). The association
between thiols and NO is relatively weak and nitro-
sothiols can exert long-acting “endothelium-derived-
releasing-factor-like” effects by slowly releasing NO.
As nitrosohemoglobin becomes deoxygenated, con-
formational changes take place and NO is released;
this may act on the endothelium to cause vasodila-
tation where it may be most needed (94). Because
albumin is so abundant and it has one free cysteine
residue, much of the protein-bound releasable NO
circulates as the albumin-adduct. Advanced glyca-
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
tion adducts may quench NO release from nitroso-
thiols, reducing their effectiveness as NO donors
(91). The exact role has not been determined for a
group of proteins described in insect salivary glands
that bind and readily release NO—“nitrophorins”—
(95), though they may act to increase blood supply to
the bite area. Their mammalian counterparts (ded-
icated high capacity NO carriers) have not been
described.
NO has important effects as a bactericidal agent
when it is released by macrophages during infection
(58,59,96). This defense mechanism seems to have
been conserved through evolution (97). Peroxyni-
trite (ONOO2) is formed from NO• and superoxide
(O2•2) (98) and may be responsible for some of the
cytotoxic effects of these molecules (57,99). When
protonated, ONOO2 gives rise to hydroxyl (OH•)
and nitrogen dioxide (NO2•) radicals, which can
induce tissue damage (100 –105). Nitrite may also
produce the nitrogen dioxide radical when oxidized
by hydrogen peroxide, catalyzed by heme peroxi-
dases (106). Tissue damage may result from tyrosine
nitration (107,108). In mammalian smooth muscle
cells, NO may inhibit proliferation by influencing
cyclin-dependent kinases that are critical to the cell
cycle (109).
Methods of measuring nitrite and nitrate in
plasma and urine
Methods for these analyses have been reviewed
(2,110,111). Nitrite is difficult to measure reliably in
blood because it is unstable, being rapidly oxidized
to nitrate. When added to blood, 16% of added nitrite
remained after 30 min at room temperature; at 60
min, the corresponding fraction was ,5%. At 4°C,
the percentages were 69% and 55%. Once plasma is
separated from blood (avoiding hemolysis), both ni-
trite and nitrate are stable at 220° C for at least 1
year (3). Urine nitrate and nitrite are stable in the
absence of microbial contamination. Acid pH de-
stroys nitrite, so acid preservatives should be
avoided. Samples should be collected on ice, possibly
with the addition of antibiotics or isopropanol to the
urine during the collection (4). After collection,
freezing is recommended for long-term storage.
Nitrite concentrations in plasma and urine are
usually only a small fraction (,5%) of the nitrate
concentrations, reflecting in part the dietary intakes
of these ions and also their bioreactivity. A combined
measurement of nitrate plus nitrite (termed NOx) is
often used with colorimetric assays because the
procedures can be simplified and the stringent sam-
ple preparation requirements for nitrite analysis
can be avoided. If investigators have no interest in
nitrite, serum can be used for NOx determinations.
Because nitrite is usually present at much lower
concentration than nitrate in body fluids, NOx is
almost synonymous with nitrate in most instances.
There is some confusion in the literature over no-
menclature. Some authors have used “nitrite” to
mean “NOx” because nitrite is the component mea-
197
 ELLIS
sured after reduction of nitrate (112–116). This error
has been noted by others (117). In this review, when
authors have used the abbreviation “NOx” to denote
something other than a combined total concentra-
tion of nitrite plus nitrate, e.g., “nitrite plus nitroso
compounds,” then we have explained their usage
and we have used quotes (“NOx”) to denote this.
Proposals to standardize nomenclature in NO re-
search have recently been published (118).
COLORIMETRIC METHODS
Nitrite is reactive and used extensively in dye
manufacturing. Over 50 colorimetric methods were
proposed for its determination; many based on the
formation of azo dyes (119). Currently, there are
occasional references to the use of the Brucine
method, in which nitrite is oxidized to nitrate by
permanganate and boiled with strychnine to pro-
duce a yellow color (35). However, most recent clin-
ical papers use colorimetric methods based on the
Griess reaction. Nitrite is reacted with sulfanil-
amide and N-(1-naphthyl)ethylenediamine (3) to
produce an azo dye. Nitrate may also be measured if
it is first converted to nitrite with the enzyme
nitrate reductase from bacteria (Escherichia coli,
Pseudomonas oleovorans) (111) or fungi (Aspergil-
lus) (120). Alternatively, metallic reduction over
copper-plated cadmium (121,122) or granulated cad-
mium (123) can be used. There are a number of
variants of the Griess reaction, designed to elimi-
nate interference from both reagents (120,124) and
the material assayed. Plasma assays require protein
removal, e.g., by precipitation with zinc hydroxide
(125), zinc sulfate (3), or by ultrafiltration (61).
When the methods are compared against gas chro-
matography-mass spectrometry, other sources of in-
terference become evident, such as free reduced
thiols and other poorly characterized plasma con-
stituents (126). Drugs, such as L-arginine analogs,
may interfere also (127,128). Recoveries have been
reported to be 91.5%, compared with copper-cad-
mium reduction (120) or 95% for nitrate and 100%
for nitrite in serum (124). However, when compared
with mass spectrometry, the variant of the Griess
reaction that was used for urine gave recoveries
varying between 30 – 80% (126). There are several
automated methods based on the Griess reaction
and flow injection analysis (121) or HPLC. Methods
based on HPLC are discussed subsequently. Vari-
ants of the method have used alternate coupling
reagents for nitrite determination (129).
ULTRAVIOLET SPECTROPHOTOMETRIC METHOD
A direct continuous kinetic spectrophotometric
method has been described for urine and serum
nitrate (130). The nitrate is reduced by NADPH
using nitrate reductase from Aspergillus. A similar
one-step assay used the method of standard addi-
tions to adjust for possible reaction interferents in
serum (131).
198
ET AL.
FLUORIMETRIC ASSAYS
Nitrite (and nitrate after reduction) have also
been measured fluorimetrically. In one method, ni-
trite enables the acid-catalyzed ring closure of 2,3-
diaminonaphthalene to form highly fluorescent 2,3-
diaminonaphthotriazole (132,133). The method is
50 –100 times more sensitive than the Griess reac-
tion and was used on serum or plasma and tissue
culture media. With minor adaptation, these meth-
ods may be used for determination of S-nitrosothiols
(134). A simple and highly selective fluorimetric
method for determining nitrite with Rhodamine 6G
has been described in which Rhodamine 6G is oxi-
dized in sulfuric acid medium. The assay has been
applied to the analysis of milk (135).
METHODS USING ELECTRODES OR BIOANALYTICAL
ELEMENTS
Several nitrate or nitrite-specific electrodes and
bioanalytical elements have been designed. They
have usually been shown to work with aqueous
samples (136 –143). One has been used with saliva
(144) and another with milk (145). Electrochemical
detection of nitrite has been used with HPLC (de-
scribed subsequently).
METHODS USING NO ANALYZERS
NO analyzers have been designed to measure NO
in air samples by chemiluminescence in the pres-
ence of ozone. The ozone is produced by an electrical
discharge acting on oxygen supplied from a cylinder.
The first product from NO is excited nitrogen diox-
ide, which emits chemiluminescence as it returns to
its ground state (146). The analyzers have been
refined and are used in clinical investigation to
monitor both NO and NO2 when NO is used in the
therapy of pulmonary vascular disease (79,80), and
NO production in exhaled breath in asthma and
other conditions (147–149). With this instrumenta-
tion, exhaled NO is easier to measure than nitrite
and nitrate. It has also been applied to the study of
NO metabolism in various conditions. However, the
apparent production rate of exhaled NO varies with
exhalation technique. Moreover, the site of origin of
the exhaled NO is unclear. Much of the measured
exhaled NO may be produced in the nasopharynx,
unless stringent precautions are used (147), or in
the airways, rather than at the level of the alveolus
(150).
In order to measure nitrite or nitrate, the NO
analyzers are coupled to an enclosed vessel through
which an inert gas, such as nitrogen or helium, is
purged (151). Then selective chemical reduction of
either nitrite or nitrate to nitric oxide can be moni-
tored directly as sequential samples are added to
excess of the appropriate reagent. Acetic acid/iodide
are often used for nitrite or vanadium III/NaOH for
nitrate. Various oxidants have been tested and com-
pared (152). It is subject to interference by a number
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
 NITRITE AND NITRATE ANALYSES
of arginine analogs currently investigated as poten-
tial pharmacological agents (127). The method has
also been applied to saliva (153). In a modification of
the method to include a photolysis step (154), or
thermolysis, followed by nitrate reductase (155),
S-nitrosothiols can be quantified.
CAPILLARY ELECTROPHORESIS
Capillary electrophoresis (CE) methods have been
applied to a wide range of analyses and they show
promise for the future (156). CE procedures have
been described for assay of nitrite and nitrate in
serum or plasma (37,157–160) and urine (158 –161).
Nitrite and nitrate show good resolution with a
short run-time of 4 –22 min. Both anions may be
quantified with a single injection after minimal
sample preparation, such as ultrafiltration. Capil-
lary isotachyphoresis methods permit assay of S-
nitroso compounds in addition to nitrite and nitrate
(162). Micellar electrokinetic capillary chromatogra-
phy has also been used for determination of nitrite
and nitrate in extracts prepared from milk and blood
(163). In this technique, migration of the ions is
modified by inclusion of a positively charged surfac-
tant, such as dodecyltrimethylammonium bromide.
CE is a microtechnique, suitable for small volumes
of fluid. It has been used experimentally for mea-
surement of airway surface fluid from rats (164) and
to assess nitrite and nitrate content of single neu-
rons (165). Despite the availability of alternative
approaches for nitrate and nitrite analysis using
capillary electrophoresis, CE equipment and exper-
tise are not yet widely available in clinical laborato-
ries.
GAS CHROMATOGRAPHY AND GAS CHROMATOGRAPHY-MASS
SPECTROMETRY
Gas chromatography has been used for the anal-
ysis of nitrite in water (166) and saliva (167) and for
nitrate analysis in rat urine (168). GC-MS methods
use 15N labeled internal standards or isotope dilu-
tion techniques and derivatization with pentafluoro-
benzyl bromide (169), benzene (168), or toluene (24).
They form the basis of reference methods for nitrite
and nitrate in plasma and urine (126) and water
(170). The specificity of a commonly used GC-MS
method for nitrite in plasma (171) has been ques-
tioned on the basis of 15N enrichment studies (38).
Nitrate recovery from rat serum was as low as 67%,
with one method (168).
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
High-performance liquid chromatography (HPLC)
is used for nitrite/nitrate measurement in environ-
mental studies and a direct UV spectrophotometric
method (at 215 nm) is approved by the US Environ-
mental Protection Agency for this purpose (172). A
number of similar methods are available for mea-
surements in foods (17,28), urine and serum (24,
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
173–176). The methods are generally based on sep-
aration by ion-exchange HPLC, although some have
been described for nonbiological matrices that use
reversed phase chromatography on C18 with ion-
pairing reagents such as cetyltrimethylammonium1
(177). The binding of ion-pairing reagents to C18
columns is often difficult to predict (178) and HPLC
columns with intrinsic ion-exchange functionality
are probably more reliable. Plasma assays require
ultrafiltration or protein precipitation and addi-
tional sample preparation by some form of chroma-
tography. Similar preparation (without protein pre-
cipitation) is used for urine (24,173). Not all methods
have been fully described and validated. Dedicated
ion chromatographs (with conductimetric or spectro-
photometric detection) can also be used for these
ions (179 –181). Chloride ion has to be removed to
prevent interference when conductimetric detection
is used because of its high concentration in biological
samples. Sensitive HPLC assays for nitrite in foods
(182) and tissues (183,184) have been developed
using electrochemical detection. A method has also
been described for whole blood, in which nitrite is
detected electrochemically and nitrate by spectro-
photometry (185). It is unclear whether there would
be significant interference from nitrosohemoglobin
when whole blood is analyzed. Ion-exchange HPLC
has also been used followed by copper-cadmium
reduction and the colorimetric Griess reaction to
measure plasma, urine and cell culture nitrite and
nitrate (173,186).
CONTAMINATION AVOIDANCE
It is important that clinicians and laboratory
workers be ever vigilant against possible contami-
nation of nitrite and nitrate assays (37). Glassware
is often contaminated with nitrate and sometimes
nitrite, unless it is sold for use in trace analysis (e.g.,
HPLC vials). Ultrafiltration filters are contami-
nated with nitrate (possibly from the azide, presum-
ably added to preserve the cellulose or other filter-
matrix) (174). We have generally found plastic
disposable wares clean. Disposable glass transfer
pipettes, gloves, and glove powder are potential
sources of contamination (187). We have found that
blood tubes containing anticoagulants such as
EDTA from some manufacturers contain nitrate.
Some heparinized syringes are contaminated and
rubber stoppers in vacutainer tubes may leach ni-
trate. Others have noted nitrite contamination of
blood tubes containing citrate or EDTA anticoagu-
lants (126). Type A water (18 MV) should be used for
reagent preparation and final rinsing of glassware.
Clinical correlates
REFERENCE VALUES
Sex differences
In one study (188), 22 men had mean plasma
nitrate (27 mmol/L) approximately twice that of 18
199
 ELLIS
women (14 mmol/L) and they exhaled more NO (34
vs. 20 ppb), even after correction for body weight.
Diet was uncontrolled in this study and no estimates
of intake were given, but samples were taken after a
12-h fast. Others have observed higher values for
plasma nitrate in men and have noted increasing
values with age and other factors associated with
atherogenesis, such as lipids, glucose, and blood
pressure (189). Giovannoni et al. (124) did not ob-
serve gender differences of this magnitude. Mean
serum NOx concentrations were 39 mmol/L in 12
men and 33 mmol/L in 12 women. These authors also
noted a two- to three-fold increase in values in
plasma, compared with serum. However, they did
not seem to exclude contamination from the antico-
agulant as a possible reason for the observed differ-
ences. No difference was found between serum and
plasma in another study (3). Men and postmeno-
pausal women had similar serum NOx concentra-
tions (190). Reference values have been summarized
in two reports (2,126); each contains information on
12 studies. Dietary restriction in 12 German sub-
jects reduced plasma concentrations and urinary
excretion by approximately one-third to one-half
(126,169).
Reports on the effects of the menstrual cycle on
values are conflicting. Plasma NOx values increase
in adult women in association with follicular devel-
opment (74). In this latter study, there was a posi-
tive correlation of plasma NOx with plasma estra-
diol in control women and in those undergoing
hormone therapy for in vitro fertilization. Diet was
uncontrolled, but most samples were fasting. Subse-
quent studies by these authors showed increased
plasma NOx in postmenopausal women when they
were treated with estrogens (191). In a similar
studies, serum NOx concentrations increased in
women (but not in men) following 3 days of trans-
dermal estradiol (190) or in women following 12
months of therapy (192). In contrast, Jilma et al.
(188) found no changes in breath NO or plasma
nitrate over the course of the menstrual cycle in 18
women and in a small group of 5 ovulating women,
Morris et al. (193) found no changes in exhaled NO
and early-morning urinary nitrite/creatinine ratios
over 30 days. However, amenorrheic women with
hyperprolactinemia had lower serum NOx than con-
trols and values increased after successful therapy
(194).
Values in children
In a group of 90 normal Japanese children, those
,3 years old had higher NOx/creatinine ratios than
older children, values showed no gender differences,
and were independent of age after about 12 years
(35,195). Plasma and urine NOx were also measured
in a small group of 16 healthy children (196). Values
for urine NOx (not “nitrite”) in 17 children have also
been presented (113). In groups of East Indian
children and adults, mean salivary nitrate concen-
trations (21–36 ppm) were approximately twice or
200
ET AL.
three times those of nitrite (12–17 ppm) (197). Ce-
rebrospinal fluid (CSF) NOx is age related in chil-
dren. In 35 British children with various neurologi-
cal conditions in whom no disturbances of
monoamine neurotransmitter metabolism or infec-
tion were suspected, values are highest in the first 2
years after birth and appear to decrease after this
time to 17 years (198).
GENERAL CONSIDERATIONS
Because NO is involved in so many diverse activ-
ities in the body, one might expect that its metabo-
lism would change in a wide variety of conditions,
leading to alterations in the concentrations of its
metabolites in blood and urine. While this is true to
some extent, many of the physiological activities
involve one of the two constitutive NOS isoenzymes
that change little in disease. However, other pro-
cesses are associated with the many tissues and
organs that have inducible NOS isozymes. These
enzymes can be massively induced by infection or
inflammation. The enzyme activity of a fully-in-
duced macrophage can be approximately 1000-fold
that of an endothelial cell (45).
URINE NITRITE AS A MARKER OF URINARY TRACT
INFECTION
Nitrite is produced from urine nitrate by bacterial
nitrate reductase (75) and small quantities may
perhaps arise by alternate pathways, involving NOS
(199). Some nitrite is converted to NO in acidified
urine (75), but much remains. Elevated urine nitrite
may assist in the diagnosis of urinary tract infec-
tions and test strips for this purpose have been
available for many years. Some have found that
strips for bacterial leukocyte esterase are more ef-
fective than those for urine nitrite (200). Nitrofuran-
toin may reduce the effectiveness of nitrite test
strips (201). The merits of using these test strips has
been the subject of much debate. Results should not
be used to direct antibiotic therapy (202), they had
low positive predictive value (203–205), but they
may be better than urinalysis (204,206) and they
have merit in certain specific situations (204,206 –
208). Others have reported that they perform well
(209).
NITRITE AND NITRATE IN OTHER INFECTIONS
In sick hospitalized patients, dietary intake, if
any, is less likely to contribute to plasma or urine
nitrate and nitrite. For this reason, increases in
these ions may become more reliable markers of
diseases, because of alterations in NO synthesis,
degradation or excretion. This may apply in septic
shock (210 –217), in patients with endotoxemia due
to severe cirrhosis (218,219), or in patients with
systemic inflammatory response syndrome (220,
221), and multiple organ failure (222). There were
significant differences between plasma NOx (by the
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
 NITRITE AND NITRATE ANALYSES

Figure 1 — (A) Urinary nitrate:creatinine molar ratios and (B) serum nitrate concentrations (mmol/L) in healthy volunteer
controls and in patients with rheumatoid arthritis or infectious gastroenteritis. Bars show the mean 6 SD. Broken line
shows mean 1 2SD for controls. Group means significantly different from controls are indicated by *p , 0.005 and **p ,
0.001. (Reproduced with permission from Lippincott-Raven Publishing from Grabowski PS, England AJ, Dykhuizen R, et
al. Elevated nitric oxide production in rheumatoid arthritis. Detection using the fasting urinary nitrate:creatinine ratio.
Arthritis Rheum 1996; 39: 643–7 [36].)

Griess reaction) in controls and infants with sepsis
and septic shock; mean values were: 35, 126, and
582 mmol/L for the respective groups (212). It is
unclear whether the assessment of efficacy of the
possible use of NOS inhibitors in septic shock (223)
would be assisted by measurements of plasma/urine
nitrite or nitrate. Plasma nitrite was increased in
AIDS with pulmonary involvement (224) and was
increased in other AIDS-related infection (225). Oth-
ers saw no change in NO production in AIDS pa-
tients without severe infection (41). Serum NOx was
increased in trypanosomiasis (226).
NITRITE AND NITRATE IN GASTROENTEROLOGY
Possible low vegetable consumption could en-
hance the utility and selectivity of plasma and urine
nitrate in patients with gastrointestinal diseases,
unmasking higher values in gastroenteritis (36,227–
229) (Figure 1). The precise role of NO and other free
radicals in secretory diarrhea is still to be deter-
mined (230). In inflammatory bowel disease, urine
nitrate/creatinine ratios were about four-fold those
of patients with non-inflammatory disorders (231).
However, others found no significant differences
between median serum nitrate in ulcerative colitis,
Crohn disease, and healthy controls; but patients
with active disease had higher values than those
with inactive disease (232). Greatly increased serum
NOx may help predict those patients with ulcerative
colitis who require a change in medical therapy or
surgery (233). Urine nitrate in 14 patients declined
in parallel with their recovery in response to therapy
(234). Plasma NOx was significantly increased in
small groups of patients with collagenous colitis and
also those with lymphocytic colitis; at colonoscopy,
NO sampled from the lumen of the colon, close to the
mucosal surface, was also increased in 4 of 5 pa-
tients with collagenous colitis compared with that in
10 controls (235).
Increased NO production might also be expected
in these conditions from other evidence. Colon mu-
cosa cultured from biopsy specimens showed 4 to
10-fold higher NOS activity and 4 to 8-fold higher
nitrite production in areas of active Crohn disease or
ulcerative colitis, compared to those from normal
controls, and values were lower when biopsies were
cultured in the presence of methylprednisolone or
ketotifen (236). Similar increases in mucosal NOS in
Crohn disease were seen by others (237). Also mu-
cosal L-citrulline (an indirect measure of NO syn-
thesis) is higher in biopsies from patients with
active colitis (238). Topical application of L-NAME
(N-omega-nitro-L-arginine methyl ester, a competi-
tive inhibitor of NOS) reduced colonic inflammation
in a rat model of ulcerative colitis, suggesting that
NO synthesis was associated with the inflammation
in this condition (239).
In mice, oral arginine supplementation protected
against bacterial translocation from the gut to other
organs after lipopolysaccharide-insult (240), sug-
gesting an important role for NO synthesis in host
defense from pathogen entry via the gut. The defen-
sive roles of NO production by the gut in gastroin-
testinal disease probably outweigh its potentially
harmful effects (241). Increased nitrite concentra-
tions were found in the gastric juice of patients with
chronic atrophic gastritis (242). The affected group
also had slightly higher gastric pH, which might be
expected to increase the stability of nitrite. Nitrite
production from nitrate by commensal bacteria in
the mouth may serve to protect against Candida and
other pathogens (78). The NO (nitrergic) nervous
system plays an important role in gut motility and
secretion and in other functions (243). Pyloric steno-
sis in infants appears linked to a neuronal NOS gene
polymorphism (244), and Hirshprung disease is as-
sociated with low neuronal NOS in intestinal nerve
fibers (245), but we are unaware of any studies on
NOx production in these conditions.

CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998 201

 ELLIS ET AL.

Mean serum NOx was 63 mmol/L in 24 patients

with chronic hepatitis C virus infection, compared
with 33 mmol/L in 8 patients with other liver disease
and 25 mmol/L in 12 healthy subjects. RNA tran-
scripts for inducible NOS in biopsy specimens were
also increased. It was unclear whether these effects
were caused directly by the virus or through inter-
feron-g (246). In contrast, in chronic hepatitis, from
various causes, serum nitrite was lower than in
controls, but many values in the groups overlapped
(247). Both exhaled NO and serum NOx were higher
in cirrhotic patients than controls (248). The differ-
ences were more marked for mean NO (252 vs. 75
nL/min/m2) than mean NOx (48 vs. 33 mmol/L) and
values did not correlate. Cirrhosis also increases
plasma NOx in rats (249) and in humans with
ascites (250). Nitrate concentrations in urine and
serum correlate with disease progression (251).
These increases seem to be related more to renal
impairment (250,252) than to endotoxin action on
hepatic or other macrophages as was first thought
(218). Plasma NOx was increased in patients with
hepatocellular carcinoma and other liver diseases;
values increased with increasing tumor size and
were higher in those patients who developed their
cancers from chronic hepatitis than from cirrhosis
(253). As judged from inhibitor studies, NO seems to
be involved in both exocrine and endocrine secretion
by the pancreas (254). Figure 2 — Nitrite concentrations in synovial fluid (SF)
and serum samples from patients with rheumatoid arthri-
NITRITE AND NITRATE AS MARKERS OF IMMUNE tis (RA), osteoarthritis (OA), and from controls matched
ACTIVATION for age and sex. Bars indicate group means. (Reproduced
with permission from BMJ Publishing Group, from Far-
Plasma nitrate has been proposed as an index of rell AJ, Blake DR, Palmer RM, Moncada S. Increased
immune system activation (58). During an immune concentrations of nitrite in synovial fluid and serum
samples suggest increased nitric oxide synthesis in rheu-
challenge; values increased 20-fold in experimental matic diseases. Ann Rheum Dis 1992; 51: 1219 –22 [261].)
animals after zymosan (59) or endotoxin (255). Li-
popolysaccharide increased NOx in the plasma of
rats and mice (61). Values peaked approximately in arthritic rats compared with controls (264). In 14
20 h after injection. Nitrite and nitrate in blood, patients with active spondyloarthropathy, mean se-
urine or culture fluids can also be used to investigate rum nitrate concentrations (taken 2– 4 h after
NO production and how it is affected by various breakfast, with no dietary control) were approxi-
drugs, cytokines (256,257) or hormones such as mately two-fold those of patients with inactive dis-
insulin (258), parathyroid hormone related protein ease or those of control subjects (265). Nitrate cor-
(259), or atrial natriuretic peptide (260). related with erythrocyte sedimentation rate and
Serum nitrite is increased in patients with rheu- C-reactive protein. Serum “NO” (nitrite, measured
matoid and osteoarthritis. Concentrations in syno- as NO after ascorbate reduction) has also been
vial fluid from affected joints are higher than corre- measured in rheumatoid arthritis and may be a
sponding serum concentrations, suggesting that more sensitive measure than NOx; mean values
much of the serum nitrite originates from the joints (293 nmol/L) were about 10-fold higher than those of
(261,262). The changes were more marked in rheu- controls (36 nmol/L) and patients with osteoarthritis
matoid arthritis (261) (Figure 2). Urine NOx (“ni- (33 nmol/L) (266).
trate”)/creatinine ratios but not serum NOx were Serum NOx may assist in monitoring the effects of
increased three-fold in rheumatoid arthritis and interleukin and interferon therapy of certain can-
provided a useful indicator of NO production in this cers (267). Measurements of exhaled NO have also
disease (36) (Figure 1). Urinary nitrate values fell by been proposed as marker of lung inflammation
28% in rheumatoid arthritis with prednisolone ther- (268). Mean serum NOx (not “nitrite,” as reported by
apy, but mean values remained elevated compared the authors) was increased in 46 patients with
with controls (263). There was extensive overlap systemic lupus erythematosis, compared to 5 control
between controls and patients, even before therapy, subjects. Overlap could not be assessed because of
and the test could not be used to support a diagnosis. the small number of controls. Endothelial cell and
Urine nitrate was approximately three-fold higher keratinocyte expression of inducible NOS protein

202 CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998

 NITRITE AND NITRATE ANALYSES
were also increased in skin biopsy specimens (112).
Yet in another study, plasma nitrate concentrations
were similar in controls, and patients with systemic
lupus erythematosis, thrombotic throbocytopenic
purpura and primary antiphospholipid syndrome
(269). In Kawasaki disease, a pediatric systemic
panvasculitis associated with immunological abnor-
malities, more urinary NOx and neopterin were
excreted at certain stages of the disease than in
controls (270). Mean early morning urine NOx/cre-
atinine was increased in eight patients with Ka-
wasaki disease; values rose with initial therapy and
declined to normal as the disease resolved (271).
NITRITE AND NITRATE IN HYPERTENSION AND IN RENAL
AND CARDIOVASCULAR DISEASE
The role of NO in the regulation of blood flow and
arterial pressure has been reviewed (272,273). Mice
lacking the gene for endothelial NOS are hyperten-
sive (274). The blood pressure of spontaneously
hypertensive rats can be lowered for several weeks
(relative to controls) by (non-genomic) delivery of the
gene for human NOS (275). This procedure in-
creased urine and serum NOx, along with urine and
aortic cGMP. The endothelium itself has a multi-
factorial role in blood pressure modulation (86), and
its dysfunction is important in atherogenesis (47,
276). The interaction of the NO system with aging
and atherosclerosis in general (277), and with homo-
cysteine metabolism in relation to atherothrombosis
are not fully understood (278,279).
NO has multiple effects within the kidney (5,280 –
283) and, as previously mentioned, renal tissue
contains all three isozymes of NOS and their precise
role in normal physiology, hypertension and diabe-
tes has not been fully elucidated (68). The use of
selective and nonselective NOS inhibitors and NO
donors may help elucidate the role of NO production
within the complex physiology of the renal tubule
(284,285) and its diuretic and natriuretic responses
(286). In rats, chronic specific inhibition of neuronal
NOS (found also in the macula densa cells of the
kidney) caused an increase in blood pressure, possi-
bly related to tubuloglomerular feedback and tran-
siently decreased GFR (287). Because nitrate and
nitrite are excreted by the kidney, renal function
and clearance have to be considered if they are
intended to be used as markers of NO production in
various conditions. In 71 patients in an intensive
therapy unit, there was a strong positive correlation
between NOx and creatinine concentrations in se-
rum (288). Mean plasma nitrate was approximately
three- to four-fold higher than normal in patients
with renal disease prior to dialysis and fell to below
normal following the procedure (289).
NO may have a role in the initiation of hypertension
(290). Dubey et al. (290) showed that in the one kidney,
one clip hypertensive rat model, increasing serum
NOx correlated with systolic blood pressure. Corre-
sponding human studies in renovascular hypertension
do not show significant changes. In 38 children with
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
Figure 3 — Urinary NOx (not “nitrite”) excretion
(nmol/mg creatinine) in healthy controls (CONT), children
with minimal change nephrotic syndrome (MCNS) in
remission (REM) or relapse (REL), with focal segmental
glomerulosclerosis (FSGS), or IgA nephropathy (IgAN).
Bars indicate group means; the asterisks indicate a p
value of ,0.025 versus other patient groups. (Reproduced
with permission from Mosby, Inc. from Trachtman H,
Gauthier B, Frank R, Futterweit S, Goldstein A, Tomczak
J. Increased urinary nitrite excretion in children with
minimal change nephrotic syndrome. J Pediatr 1996; 128:
173– 6 [113].)
hypertension, plasma NOx was increased compared
with 16 normal children. Values in all but one patient
with renovascular hypertension were normal, as were
about one-half the remaining patients with renal pa-
renchymal disease. Elevated plasma NOx was associ-
ated in part with the reduced GFR in the patients with
parenchymal disease (196). In nephrotic syndrome,
urine NOx (referred to as “nitrite”) was increased in
children with minimal change nephrotic syndrome
and normal in patients with focal segmental glomeru-
losclerosis or IgA nephropathy (113,114) (Figure 3). A
number of endogenous inhibitors of NOS have been
described: N(G)-monomethyl-L-arginine (L-NMMA),
asymmetrical dimethyl arginine (ADMA) and sym-
metric dimethyl arginine (SDMA). They are increased
in patients with hypertension, especially those with
low GFR and they may have physiological effects in
vivo, but it is uncertain whether they are a contribu-
tory cause of the hypertension or a consequence of it
and its related disease processes (291). In Bartter’s
syndrome, a normotensive, renal potassium-wasting
disease, mean NOx/creatinine ratios, in mmol/mmol
creatinine, (0.45) were approximately twice those of
controls (0.25) or patients with pseudo-Bartter syn-
drome (0.28), and urinary c-GMP excretion was also
increased (292).
The protective role of estrogens on the coronary
vasculature may in part be related to their complex
effects on NOS (293). Short-term oral high-dose
antioxidant therapy lowers blood pressure in normo-
tensives and hypertensives and increases nitrite
excretion, implying a role for NO in this effect (294).
Some of the deleterious effects of oxidized LDL also
relate to interactions with the NO system (295,296).
203
 ELLIS
Figure 4 — Serum nitrate concentration (mmol/L) for
women with preeclampsia, matched healthy pregnant and
nonpregnant women. Bars indicate group means. (Repro-
duced with permission from Blackwell Science Ltd., from
Smarason AK, Allman KG, Young D, Redman CW. Ele-
vated levels of serum nitrate, a stable end product of nitric
oxide, in women with pre-eclampsia. Br J Obstet Gynaecol
1997; 104: 538 – 43 [304].)
Plasma cholesterol and LDL were inversely corre-
lated with plasma NOx, taken at 0600 h on a
well-balanced hospital diet (33). The role of NO in
heart failure and cardiomyopathy is not known
(297), but cardiomyopathy is frequently associated
with disorders in which there is higher than normal
NO production. A high-capacity inducible NOS iso-
form is present in the myocardium of patients with
idiopathic dilated cardiomyopathy and plasma ni-
trate was significantly increased in 39 patients with
heart failure compared with 62 normal controls.
Mean concentrations were 51 and 25 mmol/L, but
values overlapped considerably between the groups
(298). Increases are also reported in chronic heart
disease (299) and in infants with heart failure due to
septal defects prior to cardiopulmonary bypass
(300). In ischemic heart tissue, NO can be produced
directly from nitrite and the significance of this
pathway has not been evaluated (76). Urine and
plasma nitrate measurements were unhelpful in
determining the cause of low systemic vascular
resistance syndrome after cardiopulmonary bypass
(301) or of primary Raynaud’s phenomenon (302).
After transesophageal variceal ligation in patients
with portal hypertension, there was a prompt fall in
serum nitrate (from 39 to 29 mmol/L) associated with
the changes in hemodynamics (303). However, there
was no indication that serum nitrate would serve to
predict a good long-term clinical outcome.
The literature on nitrate and nitrite in preeclamp-
sia is confusing. Mean serum nitrate concentrations
were higher in 20 women with preeclampsia (47
mmol/L) compared with 20 healthy pregnant (31
mmol/L) and 12 nonpregnant (32 mmol/L) controls.
There was no dietary restriction and nitrate would
be unhelpful in diagnosis because of extensive over-
204
ET AL.
Figure 5 — Serum NOx concentration (means and SEM)
for women with preeclampsia (PE), healthy normotensive
pregnant (NTP) and nonpregnant (NP) women, and
women with essential hypertension (EHT). Values repre-
sent means 6 SEM and n the number of samples (*p ,
0.0001; ‡p , 0.005; ANOVA with Fiser’s tests). (Repro-
duced with permission from S. Karger AG, Basel from
Nobunaga T, Tokugawa Y, Hashimoto K, et al. Plasma
nitric oxide levels in pregnant patients with preeclampsia
and essential hypertension. Gynecol Obstet Invest 1996;
41: 189 –93 [305].)
lap between the groups. It was not clear whether the
increases were due to increased production or de-
creased renal clearance (304) (Figure 4). Increases
were found in pregnant women throughout preg-
nancy (mean NOx 30 mmol/L), compared to nonpreg-
nant controls and values were even higher (mean
NOx 45 mmol/L) in preeclampsia (305) (Figure 5). In
this study, blood was taken after a 12–15 h fast.
Plasma NOx correlated positively with systolic blood
pressure in the preeclamptic patients. Previous au-
thors saw no change in NOx (not “nitrite,” as stated
by the authors) in maternal serum at 33–35 weeks
gestation; at delivery, fetal side umbilical venous
serum showed a slight increase over maternal (116).
Also both nitrate and nitrite were lower in pre-
eclampsia in the (non-fasting) group studied by
Seligman et al. (115) and serum NOx (not “nitrite”)
was inversely correlated with blood pressure in
preeclampsia. Davidge et al. (306) found that urine
NOx (not “nitrite”)/creatinine ratios were lower in 14
women with preeclampsia than in 20 normotensive
pregnant women (0.37 vs. 0.69 mmol NOx/mg creat-
inine), but mean plasma NOx was not significantly
different (33 vs. 26 mmol/L). Mean amniotic fluid
nitrite decreases in late pregnancy at 37– 41 weeks;
exhaled NO is unchanged (307).
Nitrate excretion and 15N arginine studies were
used to study newborns with persistent pulmonary
hypertension (308). The authors concluded that
there was decreased arginine utilization during the
acute vasoconstrictive stage of the disease, possibly
leading to insufficient endogenous NO production in
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
 NITRITE AND NITRATE ANALYSES

this condition. Others found that urinary NOx con-

centrations by the Griess reaction were lower in
newborns with persistent pulmonary hyperten-
sion than in controls (309). They also attributed
this to reduced pulmonary NO production in these
patients because of their lung disease. Values
increased transiently after the initiation of extra-
corporeal membrane oxygenation (ECMO). This was
believed to be due to enhanced lung production of
NO with the improved tissue oxygenation caused by
the ECMO. There was considerable overlap between
controls and patients. In newborns receiving blood
or indomethacin, NO metabolism (assessed by urine
nitrite excretion) was affected by these treatments
(310).

ARGININE SUPPLEMENTATION AND THE REGULATION OF

BLOOD PRESSURE AND BLOOD FLOW

Endothelial NO, synthesized from arginine, is an

important “second” messenger for blood pressure
control (53,311). Feeding arginine-deficient diets to
rats after small-intestine resection causes hyperten-
sion (312). An infant with arginosuccinate lyase
deficiency, who was unable to synthesize arginine,
was hypertensive prior to arginine replacement
(313).
Arginine infusion induces hypotension and diure-
sis/natriuresis in man (314). The blood-pressure-
lowering effects of arginine infusion are short term
in patients with essential hypertension, producing
significant change on only the first of four infusion
days (315). Both hypertension and angiotensin-con-
verting enzyme therapy affect the renal response to
L-arginine infusion (316). Arginine infusion in neo-
nates with possible endothelial dysfunction and de-
fective NO synthesis improved oxygenation (317). In
short-term experiments, arginine infusion improved
the circulation in affected ischemic limbs of patients
with atherosclerosis (318). In dogs undergoing car- Figure 6 — Plasma “NOx” concentration (nitrite plus
dioplegic arrest and coronary artery ligation and nitroso compounds, mmol/L) for patients after liver trans-
release, arginine supplementation of the cardiople- plant: (A) by clinical status, (B) by rejection grade on the
gia solution reduced postischemic injury (319). It day of biopsy confirmation. Bars show group means.
inhibits platelet aggregation in healthy subjects (Reproduced with permission from Williams and Wilken
(320). from Devlin J, Palmer RMJ, Gonde CE, et al. Nitric oxide
In partially nephrectomized rats, oral arginine generation a predictive parameter of acute allograft rejec-
gave some protection against renal failure (321). tion. Transplantation 1994; 58: 592–5 [333].)
Its chronic use in drinking water has also been
associated with attenuated cardiac hypertrophy result in part from its ability to act as a scavenger of
in spontaneously hypertensive rats (322). In superoxides as well as a substrate for NO synthesis
humans, oral L-arginine supplementation (0.1– (326,327). If L-arginine therapy becomes established
0.2 g/kg) may increase NO excretion and plasma practice for specific indications, plasma or urine
nitrate, without affecting blood pressure (323). nitrate or NOx measurements along with exhaled
Oral supplementation with lower doses may NO may be useful in the assessment of the treat-
slightly increase insulin without effect on nitrite ment regimes. However, there may be contraindica-
concentrations in plasma or nitrate excretion tions to arginine therapy. It enhances lipid peroxi-
(324). In men with coronary artery disease, oral dation in poorly perfused rat kidney (328). In mouse
L-arginine, taken over a 3-day period, improved malnutrition, supplementation with arginine alone
endothelium-dependent dilatation of the brachial may not be beneficial (329). Also some of the bene-
artery and reduced monocyte/endothelial cell ad- ficial effects of the low protein diet in renal disease
hesion (325). are believed to be a direct consequence of arginine
The proposed beneficial effects of L-arginine may restriction (330).

CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998 205

 ELLIS ET AL.

NITRITE AND NITRATE IN TRANSPLANTATION AND

REJECTION

NO is produced by various cells in the liver and

may serve as a “second” messenger and as a defense
against invading microorganisms, parasites and tu-
mor cells (331). Exhaled NO was increased in a
series of patients with hepatopulmonary syndrome
and values returned to normal in one patient after
transplantation (332). Plasma acid-labile “NOx” (ni-
trite and nitroso compounds, measured by chemilu-
minescence) was increased in liver transplant rejec-
tion in fifty consecutive patients. There was overlap
in values between the groups, but the highest values
were seen in Grade 2 and 3 rejection (333) (Figure
6). In this study, plasma nitrate did not correlate
with rejection. Samples, with no reported anticoag-
ulant, were separated within 20 min, to avoid the
reported rapid conversion of nitrite to nitrate in
whole blood (3). Others have criticized the use of
plasma nitrite as a marker of NO production, be-
cause of its instability (334). Plasma nitrate was
increased in patients with moderate to severe liver
transplant rejection, but not in those with mild
rejection. Mean values (mmol/L) were 50 in the
severe rejection group, compared with approxi-
mately 20 in groups with no or mild rejection at the
time of liver biopsy. Values fell to normal after
successful antirejection therapy with glucocorticoids
(335). Mild increases in plasma nitrate were found
in many patients in the postoperative period, per-
haps linked to temporarily ineffective anti-rejection
therapy (336). But rats show increased NO synthe-
sis following partial hepatectomy (337), so the rise in Figure 7 — NOx concentrations (mmol/L) in induced
nitrate following transplantation might be a nonspe- sputum in patients with asthma and normal controls.
cific response to injury. Bars represent mean values. (Reproduced with permis-
In rats, heart transplant rejection was associated sion from Mosby, Inc. from Kanazawa H, Shoji S, Yamada
with an eight-fold increase in urine nitrate excre- M, et al. Increased levels of nitric oxide derivatives in
tion, compared with controls. Unfortunately, rejec- induced sputum in patients with asthma. J Allergy Clin
tion in transplanted animals that had been treated Immunol 1997; 99: 624 –9 [347].)
with dexamethasone and cyclosporine was much
less dramatic, leading to only two- to three-fold
increases (338). These increases were probably due dipstick methods may not have sufficient sensitivity
to effects on the inducible NOS isoenzyme of the to detect bacteriuria (342).
myocardium as well as increased macrophage NOS
activity. Urine nitrate is increased in human heart NITRITE AND NITRATE IN DISEASES OF THE BLOOD SYSTEM
transplant patients during rejection. In a thorough
study of 86 patients, mean values (mmol/mmol cre- Plasma NOx concentrations were increased in
atinine) were 100 in patients with no evidence of acute painful crises in sickle cell disease when
rejection and 128 in those showing rejection (339). compared with healthy control subjects (90). The
The magnitude of these differences compared with reasons for this increase were not clear, but it is
intra- and interindividual differences was insuffi- unlikely that routine NOx measurements will find a
cient to make the test clinically useful for the diag- role in monitoring such patients because the in-
nosis of rejection. However, Benvenuti et al. (340) crease was quite modest (mean NOx 50 vs. 30
found that serum NOx correlated with the severity mmol/L) and sickle patients had elevated values
of heart transplant rejection, as assessed by histo- when not in crisis; their mean NOx was 42 mmol/L.
logical grade and a cutoff value of 20 mmol/L could be Cell-free hemoglobin can remove NO rapidly (343)
used to help detect Grade 2 rejection. and by doing so, its presence may lead to abnormal
Serum NOx has been shown to increase in exper- thromboregulation as summarized by Marcus et al.
imental small bowel rejection in the rat. Concentra- (344). This may be relevant in the development and
tions fell rapidly with the onset of anti-rejection possible introduction of hemoglobin-based blood
therapy (341). In renal transplantation, urine nitrite substitutes (343).

206 CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998

 NITRITE AND NITRATE ANALYSES
NITRITE AND NITRATE IN RESPIRATORY DISEASES AND IN
SMOKERS
The bronchiolar lavage fluid mean NOx concen-
tration was 20-fold increased in 20 lung cancer
patients compared with 8 smoker controls, presum-
ably due to cytokine activation (345). NOx in bron-
chio-alveolar lavage fluid was increased in 5 of 13
immunosuppressed children with pneumonia, com-
pared with 31 controls. The mean value (approxi-
mately 21 mmol/L) was about twice that of the
controls; serum NOx was similar in the two groups
(346). NOx by the Griess reaction is increased in
induced sputum from patients with asthma (347).
Mean concentrations (mmol/L) were 1086 in 18 asth-
matics and 577 in 10 controls. There was little
overlap in values (Figure 7). However, exhaled NO
(148,149,348,349) may give a more direct measure-
ment of NO production in lung than its metabolites,
nitrite or nitrate, and simple, sensitive NO analyz-
ers are increasingly used in clinical medicine.
The effects of smoking on NO metabolism are
complex. Cigarette smoke is an excellent source of
NO (up to 500 ppm), that can nitrate tyrosine (107).
Its NO concentration varies widely, depending upon
how the tobacco was grown and manufactured (350).
Smoking impairs endothelium-dependent vessel re-
laxation (350), but other effects are somewhat unex-
pected. Smoking, particularly heavy smoking, is
paradoxically associated with lower serum NOx
(351). Moreover, exhaled NO increases after quitting
smoking (352), perhaps implying better lung func-
tion.
CSF AND PLASMA NITRITE AND NITRATE IN NERVOUS
SYSTEM DISORDERS
In addition to acting as a neurotransmitter (353,
354), NO may cause damage to nerve cells, but its
role in various diseases is not well understood (51,
62,63,355,356). Whether it causes benefit or harm
may depend on the stage of the disease process
(357). Male mice deficient in neuronal NOS exhibit
bad behavior (358,359), but we know of no clinical
studies where nitrite or nitrate have been measured
in behavioral disorders.
Much of the clinical neurology literature relating
to changes in CSF and plasma nitrate and nitrite is
conflicting. In two infants with Hemophilus influen-
zae meningitis, CSF nitrite concentration was in-
creased. This was regarded as evidence for enhanced
nitric oxide production in this condition (360). CSF
nitrite was also increased in all six patients with
bacterial meningitis and declined with successful
therapy (361). In patients with recent stroke, argi-
nine and nitrite were both increased in CSF and
their concentrations correlated (362). The extent of
possible overlap in values between affected patients
and unaffected controls was not given, so it is
difficult to assess whether CSF nitrite measure-
ments could have a diagnostic role in this condition.
Minimal changes in CSF nitrite and nitrate oc-
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
curred during the treatment of adult head injury
patients with hypothermia, but values were slightly
higher in non-survivors than in those who survived
(363). CSF NOx was unchanged in Parkinson’s dis-
ease (364) and in aseptic meningitis, multiple scle-
rosis and Guillain-Barré syndrome (365), or in Par-
kinson’s disease, spinocerebellar ataxia and
amyotrophic lateral sclerosis (366). It was un-
changed in Huntington’s and Alzheimer’s disease,
amyotrophic lateral sclerosis, and HIV infection, but
values were significantly increased in a small group
of patients with bacterial and viral meningitis (367).
Others (224) described similar negative findings for
CSF, but noted some increases in serum nitrite in
generalized infections that may affect the brain,
such as cytomegalovirus in AIDS.
Lower than normal mean CSF nitrate was found
in Alzheimer’s and Parkinson’s diseases and in mul-
tiple system atrophy (368). But there were no
changes in fasting plasma NOx (mean concentration
33 mmol/L) or CSF NOx (mean 5 mmol/L) in the
group of patients with Alzheimer’s studied by Na-
varro et al. (369) and CSF nitrite was increased in
another Parkinson’s disease group (370). Plasma
nitrate was unchanged in Parkinson’s disease ac-
cording to another report (371). CSF nitrite and
nitrate concentrations were statistically signifi-
cantly higher in patients with lumbar spondylosis
compared with those of controls, and the authors felt
that NO may play a role in lumbar pain or nerve
damage in sciatica and that CSF nitrite/nitrate may
be used as a diagnostic parameter of spinal diseases
(372). Unfortunately, individual values for the 18
affected patients and 18 controls with other CNS
diseases are not shown, but there was significant
overlap between the two groups with mean (SEM)
values (mmol/L) of 4.48 (0.34) and 3.48 (0.22), respec-
tively. Serum NOx was increased in a group of
demyelinating diseases including multiple sclerosis,
inflammatory neurological diseases and AIDS pa-
tients studied by Giovannoni et al. (373). Mean NOx
(mmol/L) for the groups were 66, 56 and 58, com-
pared with noninflammatory neurological disease,
41, and normal controls, 33. There was significant
overlap in values between the groups.
On the basis of higher CSF NOx seen in younger
children (who were shorter in height and spinal
length than older children), Surtees et al. (374)
proposed that a rostrocaudal NOx gradient existed
in CSF and that most CSF NOx originated from the
brain.
NITRITE AND NITRATE MEASUREMENTS IN MISCELLANEOUS
CONDITIONS
Plasma nitrate concentrations were elevated in
patients after burn injury. The highest values were
in patients with 10 – 40% of the body involved (176).
Minor burns are not associated with significantly
increased plasma NOx; increased values were found
only when there was therapeutic cerium nitrate
contamination of the burn area, or generalized
207
 ELLIS
stress due to smoke inhalation injury (375). The
nitrate concentration in human tear fluid (like that
of the black vulture, Coragyps atratus) is lower than
that of serum (181), but there are no reports of its
use in the diagnosis of eye conditions (in either
species); although the NO system is very important
in many intraocular processes and in eye irritation
(376).
NO plays an important role in the initiation of
apoptosis in various cell types, such as neuronal
cells, pancreatic cells, chondrocytes and macro-
phages (64 – 67,377). NO interactions with the p53
tumor suppressor system are under intensive study
and NO may actually support tumor growth (378).
Plasma and urine NOx assays have not been used
extensively to monitor apoptosis or tumor progres-
sion clinically, except where they are associated
with inflammation. Plasma and CSF NOx were
generally unhelpful in malaria (379), but higher
plasma NOx was associated with deeper coma in
cerebral malaria (380). NO plays an important role
in intercellular communication in bone (381,382),
but its metabolites have not been studied in bone
and joint diseases, except in arthritis and the spon-
dyloarthropathies (382).
ENVIRONMENTAL NITRITE AND NITRATE: POSSIBLE EFFECTS
ON THE INCIDENCE OF DIABETES AND OTHER DISEASES
There are tenuous connections between the NO
system and several processes in diabetes (383). NO
and cytokines are associated with islet cell death in
diabetes (384 –386) and high glucose concentrations
induce NOS expression and superoxide anion in
human aortic endothelial cell culture, implying a
possible role for NO in the vascular complications of
diabetes (387). NO mediates increased blood flow to
pancreatic islets during hyperglycemia (388) and
experimental hyperinsulinemia increases urinary
NOx excretion (258). Type I diabetics have evidence
of arterial endothelial dysfunction (389) and specific
NOS inhibitors may influence urinary albumin loss
and the formation of advanced glycation end prod-
ucts in the diabetic rat kidney (390). Alternative
inhibitors may increase blood pressure and cause
proteinuria in other rat models (391). There are
early indications that tissue advanced glycation end
products may be important modulators of NO-medi-
ated responses (392).
However, because of the wide variability of di-
etary nitrate intake (393), it is difficult to under-
stand how disease incidence in childhood diabetes
could be causally associated with higher nitrate
concentrations in drinking water (394). The relative
risk was 1.27. Others have found no associations
between Type I diabetes and child or maternal
nitrate intake from food and water, but they found a
positive association with dietary nitrite (relative
risk 2.3 for the fourth quartile of intake) (395).
Drinking water (particularly well water) may be-
come contaminated by nitrate from fertilizer run-off
or from waste water contamination and this may
208
ET AL.
present an increasing problem (396). Associations
with the concentration of nitrate in drinking water
and disease incidence have also been described for
bladder cancer (397), non-Hodgkin’s lymphoma
(398,399), gastric and prostate cancer (400), sponta-
neous abortion in four women (401), chromatic/
chromosome breaks in children (402), and thyroid
enlargement (403).
In other studies, associations between environ-
mental contamination and cancer incidence were
deemed “hypothetical and unproven” (404). Bladder
cancer incidence showed no association with drink-
ing water nitrate, but it was linked to water chlori-
nation (405). No association was found with brain
tumors, but drinking water nitrate concentrations
(16 mg/L) were not exceptionally high (406). How-
ever, pediatric brain tumor incidence appeared as-
sociated with maternal cured meat consumption
assessed retrospectively (407). There was no associ-
ation between nitrate intake (median 99 mg from
foods and 4 mg from drinking water) and gastric
cancer in a recent Netherlands study of over 120,000
men and women with ages 55– 69 (408). In some
areas where drinking water was seriously contami-
nated (up to mean nitrate 135 mg/L), leukocyte
enzyme abnormalities were found and nitrosamines
were detected in urine; but some water samples
were mutagen-positive by the Ames test, so nitrates
may not have been the only contaminants (409). In
this study, salivary nitrite, salivary nitrate, and
urine nitrate showed rough correlations with drink-
ing water nitrate concentrations. If these associa-
tions with disease incidence are confirmed, it will be
important to show what components in vegetarian
diets protect against their high nitrate/nitrite con-
tent (6). Several have been suggested (407,410 –
413).
Methemoglobin reductase is less well developed in
infants and this may account for their well-recog-
nized greater susceptibility to methemoglobinemia
from drinking high concentrations of nitrate in
drinking water or other environmental sources
(414 – 417) or from therapeutic NO (418). Accidental
substitution of nitrite for nitrate as a food preserva-
tive attests to its potential for toxicity (419).
Conclusions
Assays for nitrite and nitrate in plasma and urine,
along with measurements of exhaled NO, have been
used to study various aspects of NO metabolism. In
most instances, because of its greater stability in
vivo, nitrate is the more predominant ion. Occasion-
ally, nitrite or “labile NOx” (nitrite and nitroso
compounds) may have higher predictive value than
nitrate. The involvement of NO in a wide range of
biochemical and physiological functions tends to
reduce the clinical utility of nitrite and nitrate
measurements for individual disease conditions or
pathological processes. Changes in metabolism that
may involve constitutive NOS isozymes, or slowly
developing, possibly chronic, conditions may not
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
 NITRITE AND NITRATE ANALYSES
register against the background “noise” of NO syn-
thesis or nitrate intake. Variable intake of nitrite
and nitrate from dietary sources, or less impor-
tantly, medications and NO intake from smoking
and air pollution additionally confound interpreta-
tion, outside the experimentally-controlled environ-
ment. Plasma and urine nitrite and nitrate mea-
surements may have a diagnostic or monitoring role
for individual patients with conditions in which
inducible NOS is massively up-regulated, such as
infection, rejection, and inflammation.
Acknowledgement
This work was supported by grants from The Physician
Services Incorporated Foundation.
References
1. Kostka P. Free radicals (nitric oxide). Anal Chem
1995; 67: 411R– 6R.
2. Viinikka L. Nitric oxide as a challenge for the clinical
chemistry laboratory. Scand J Clin Lab Invest 1996;
56: 577– 81.
3. Moshage H, Kok B, Huizenga JR, Jansen PL. Nitrite
and nitrate determinations in plasma: a critical
evaluation. Clin Chem 1995; 41: 892– 6.
4. Moshage H. Nitric oxide determinations: much ado
about NO•-thing. Clin Chem 1997; 43: 553– 6.
5. Kone BC. Nitric oxide in renal health and disease.
Am J Kidney Dis 1997; 30: 311–33.
6. Walker R. The metabolism of dietary nitrites and
nitrates. Biochem Soc Trans 1996; 24: 780 –5.
7. van Maanen JM, van Geel AA, Kleinjans JC. Modu-
lation of nitrate-nitrite conversion in the oral cavity.
Cancer Detect Prev 1996; 20: 590 – 6.
8. McKnight GM, Smith LM, Drummond RS, Duncan
CW, Golden M, Benjamin N. Chemical synthesis of
nitric oxide in the stomach from dietary nitrate in
humans. Gut 1997; 40: 211– 4.
9. Omata T. Structure, function and regulation of the
nitrate transport system of the cyanobacterium Syn-
echococcus sp. PCC7942. Plant Cell Physiol 1995; 36:
207–13.
10. Meharg AA, Blatt MR. NO3-transport across the
plasma membrane of Arabidopsis thaliana root
hairs: kinetic control by pH and membrane voltage. J
Membr Biol 1995; 145: 49 – 66.
11. Chow C-W, Kapas A, Romanek R, Grinstein S.
NO32-induced pH changes in mammalian cells. Ev-
idence for an NO32-H1 cotransporter. J Gen Physiol
1997; 110: 185–200.
12. Gloria MBA, Vale SR, Vargas OL, Barbour JF,
Scanlan RA. Influence of nitrate levels added to
cheesemilk on nitrate, nitrite, and volatile nitro-
samine contents in gruyere cheese. J Agricult Food
Chem 1997; 45: 3577–9.
13. Kross BC, Hallberg GR, Bruner DR, Cherryholmes
K, Johnson JK. The nitrate contamination of private
well water in Iowa. Am J Public Health 1993; 83:
270 –2.
14. Kapoor A, Viraraghavan T. Nitrate removal from
drinking water: review. J Environ Eng Assoc 1997;
123: 371– 80.
15. Meah MN, Harrison N, Davies A. Nitrate and nitrite
in foods and the diet. Food Addit Contam 1994; 11:
519 –32.
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
16. Vallance P. Dietary nitrate: poison or panacea. Gut
1997; 40: 288.
17. Santillana MI, Ruiz E, Nieto MT, de Alba M. High
performance ion chromatography determination of
nitrite and nitrate in foodstuffs. J Liqu Chromatogr
1993; 16: 1561–71.
18. Hunt J, Turner MK. A survey of nitrite concentra-
tions in retail fresh vegetables. Food Addit Contam
1994; 11: 327–32.
19. van Vliet JJ, Vaessen HA, van den Burg G, Scho-
thorst RC. Twenty-four-hour duplicate diet study
1994; nitrate and nitrite: method development and
intake per person per day. Cancer Lett 1997; 114:
305–7.
20. Laitinen S, Virtanen SM, Rasanen L, Penttila PL.
Calculated dietary intakes of nitrate and nitrite by
young Finns. Food Addit Contam 1993; 10: 469 –77.
21. Hecht SS. Approaches to cancer prevention based on
an understanding of N nitrosamine carcinogenesis.
Proc Soc Exp Biol Med 1997; 216: 181–91.
22. Hill MJ. Endogenous N-nitrosation. Eur J Cancer
Prev 1996; 5: 47–50.
23. Green LC, Ruiz de Luzuriaga K, Wagner D, et al.
Nitrate biosynthesis in man. Proc Natl Acad Sci
USA 1981; 78: 7764 – 8.
24. Wennmalm Å, Benthin G, Edlund A, et al. Metabo-
lism and excretion of nitric oxide in humans. An
experimental and clinical study. Circ Res 1993; 73:
1121–7.
25. Suto T, Losonczy G, Qiu C, et al. Acute changes in
urinary excretion of nitrite 1 nitrate do not neces-
sarily predict renal vascular NO production. Kidney
Int 1995; 48: 1272–7.
26. Bednar C, Kies C. Nitrate and vitamin C from fruits
and vegetables: impact of intake variations on ni-
trate and nitrite excretions of humans. Plant Foods
Hum Nutr 1994; 45: 71– 80.
27. Weller R, Pattullo S, Smith L, Golden M, Ormerod A,
Benjamin N. Nitric oxide is generated on the skin
surface by reduction of sweat nitrate. J Invest Der-
matol 1996; 107: 327–31.
28. van Vliet JJH, Vaessen H, Vandenburg G, Scho-
thorst RC. Twenty four hour duplicate diet study
1994: nitrate and nitrite: method development and
intake per person per day. Cancer Lett 1997; 114:
305–7.
29. Jungersten L, Edlund A, Petersson AS, Wennmalm
A. Plasma nitrate as an index of nitric oxide forma-
tion in man: analyses of kinetics and confounding
factors. Clin Physiol 1996; 16: 369 –79.
30. Jungersten L, Edlund A, Petersson AS, Wennmalm
A. Plasma nitrate as an index of endogenous nitric
oxide formation in man. Analysis of kinetics, con-
founding factors, and response to immunological
challenge. Circulation 1993; 88: I–369.
31. Zeballos GA, Bernstein RD, Thompson CI, et al.
Pharmacodynamics of plasma nitrate/nitrite as an
indication of nitric oxide formation in conscious dogs.
Circulation 1995; 91: 2982– 8.
32. Egberts J, Soederhuizen W. Urine samples before
dinner are preferable when studying changes in
endogenous nitrate production under uncontrolled
dietary conditions. Clin Chim Acta 1996; 254: 141– 8.
33. Tanaka S, Yashiro A, Nakashima Y, Nanri H, Ikeda
M, Kuroiwa A. Plasma nitrite/nitrate level is in-
versely correlated with plasma low-density lipopro-
tein cholesterol level. Clin Cardiol 1997; 20: 361–5.
34. Wang J, Brown MA, Tam SH, Chan MC, Whitworth
209
 ELLIS
JA. Effects of diet on measurement of nitric oxide
metabolites. Clin Exp Pharmacol Physiol 1997; 24:
418 –20.
35. Tsukahara H, Hiraoka M, Hori C, Miyanomae T,
Kikuchi K, Sudo M. Age-related changes of urinary
nitrite/nitrate excretion in normal children. Nephron
1997; 76: 307–9.
36. Grabowski PS, England AJ, Dykhuizen R, et al.
Elevated nitric oxide production in rheumatoid ar-
thritis. Detection using the fasting urinary nitrate:
creatinine ratio. Arthritis Rheum 1996; 39: 643–7.
37. Leone AM, Francis PL, Rhodes P, Moncada S. A
rapid and simple method for the measurement of
nitrite and nitrate in plasma by high performance
capillary electrophoresis. Biochem Biophys Res Com-
mun 1994; 200: 951–7.
38. Rhodes P, Leone AM, Francis PL, Struthers AD,
Moncada S, Rhodes PM. The L-arginine:nitric oxide
pathway is the major source of plasma nitrite in
fasted humans. Biochem Biophys Res Commun 1995;
209: 590 – 6.
39. Westfelt UN, Benthin G, Lundin S, Stenqvist O,
Wennmalm A. Conversion of inhaled nitric oxide to
nitrate in man. Br J Pharmacol 1995; 114: 1621– 4.
40. Castillo L, Beaumier L, Ajami AM, Young VR. Whole
body nitric oxide synthesis in healthy men deter-
mined from [15N]-arginine-to-[15N]citrulline label-
ing. Proc Natl Acad Sci USA 1996; 93: 11460 –5.
41. Evans T, Rasmussen K, Wiebke G, Hibbs JB. Nitric
oxide synthesis in patients with advanced HIV infec-
tion. Clin Exp Immunol 1994; 97: 83– 6.
42. Cederqvist B, Persson MG, Gustafsson LE. Direct
demonstration of NO formation in vivo from organic
nitrites and nitrates, and correlation to effects on
blood pressure and to in vitro effects. Biochem Phar-
macol 1994; 47: 1047–53.
43. Benedini F, Bertolini G, Gromo G, Mizrahi J, Sala A.
The discovery of a new organic nitrate: an overview.
J Cardiovasc Pharmacol 1995; 26(Suppl 4): S1–5.
44. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric
oxide—a physiologic messenger. Ann Intern Med
1994; 120: 227–37.
45. Anggard E. Nitric oxide—mediator, murderer, and
medicine. Lancet 1994; 343: 1199 –206.
46. Davies MG, Fulton GJ, Hagan P-O. Clinical biology
of nitric oxide. Br J Surg 1995; 82: 1598 – 610.
47. Dattilo JB, Makhoul RG. The role of nitric oxide in
vascular biology and pathobiology. Ann Vasc Surg
1997; 11: 307–14.
48. Farrell AJ, Blake DR. Nitric oxide. Ann Rheum Dis
1996; 55: 7–20.
49. Knowles RG, Moncada S. Nitric oxide syntheses in
mammals. Biochem J 1994; 298: 249 –58.
50. Schinikerth VB, Vanhoutte PM. Nitric oxide synthe-
ses in vascular cells. Exp Physiol 1995; 80: 885–905.
51. Sparrow JR. Inducible nitric oxide synthase in the
central nervous system. J Mol Neurosci 1994; 5:
219 –29.
52. Nathan C, Xie QW. Regulation of biosynthesis of
nitric oxide. J Biol Chem 1994; 269: 13725– 8.
53. Moncada S. Nitric oxide. J Hypertens 1994; 12:
S35–S9.
54. Magazine HI. Detection of endothelial cell-derived
nitric oxide: current trends and future directions.
Adv Neuroimmunol 1995; 5: 479 –90.
55. Cooper CJ, Landzberg MJ, Anderson TJ, et al. Role
of nitric oxide in the local regulation of pulmonary
210
ET AL.
vascular resistance in humans. Circulation 1996; 93:
266 –71.
56. MacMicking J, Xie QW, Nathan C. Nitric oxide and
macrophage function. Annu Rev Immunol 1997; 15:
323–50.
57. Miller RA, Britigan BE. The formation and biologic
significance of phagocyte-derived oxidants. J Inves-
tig Med 1995; 43: 39 – 49.
58. Miller MJS, Grisham MB. Nitric oxide as a mediator
of inflammation? You had better believe it. Media-
tors Inflamm 1995; 4: 387–96.
59. Jungersten L, Edlund A, Hafstrom LO, Karlsson L,
Petersson AS, Wennmalm A. Plasma nitrate as an
index of immune system activation in animals and
man. J Clin Lab Immunol 1993; 40: 1– 4.
60. Saleh D, Barnes PJ, Giaid A. Increased production of
the potent oxidant peroxynitrite in the lungs of
patients with idiopathic pulmonary fibrosis. Am J
Respir Crit Care Med 1997; 155: 1763–9.
61. Tracey WR, Tse J, Carter G. Lipopolysaccharide-
induced changes in plasma nitrite and nitrate concen-
trations in rats and mice: pharmacological evaluation
of nitric oxide synthase inhibitors. J Pharmacol Exp
Ther 1995; 272: 1011–5.
62. Dawson TM, Snyder SH. Feature article: gases as
biological messengers: nitric oxide and carbon mon-
oxide in the brain. J Neurosci 1994; 14: 5147–59.
63. Dawson VL, Dawson TM. Nitric oxide in neuronal
degeneration. Proc Soc Exp Biol Med 1996; 211:
33– 40.
64. Shimaoka M, Iida T, Ohara A, et al. NOC, a nitric-
oxide-releasing compound, induces dose dependent
apoptosis in macrophages. Biochem Biophys Res
Commun 1995; 209: 519 –26.
65. Blanco FJ, Ochs RL, Schwarz H, Lotz M. Chondro-
cyte apoptosis induced by nitric oxide. Am J Pathol
1995; 146: 75– 85.
66. Troy CM, Derossi D, Prochiantz A, Greene LA,
Shelanski ML. Downregulation of Cu/Zn superoxide
dismutase leads to cell death via the nitric oxide-
peroxynitrite pathway. J Neurosci 1996; 16: 253– 61.
67. Nicotera P, Bonfoco E, Brune B. Mechanisms for
nitric oxide-induced cell death: involvement of apo-
ptosis. Adv Neuroimmunol 1995; 5: 411–20.
68. Galle J, Wanner C. Impact of nitric oxide on renal
hemodynamics and glomerular function: modulation
by atherogenic lipoproteins? Kidney Blood Press Res
1996; 19: 2–15.
69. Snyder SH. Nitric oxide: first in a new class of
neurotransmitters? Science 1992; 257: 494 – 6.
70. Brann DW, Bhat GK, Lamar CA, Mahesh VB. Gas-
eous transmitters and neuroendocrine regulation.
Neuroendocrinology 1997; 65: 385–95.
71. Brenman JE, Bredt DS. Synaptic signaling by nitric
oxide. Curr Opin Neurobiol 1997; 7: 374 – 8.
72. Stamler JS, Toone EJ, Lipton SA, Sucher NJ. (S)NO
signals: translocation, regulation, and a consensus
motif. Neuron 1997; 18: 691– 6.
73. Griffith OW, Stuehr DJ. Nitric oxide synthases:
properties and catalytic mechanism. Annu Rev
Physiol 1995; 57: 707–36.
74. Rosselli M, Imthurm B, Macas E, Keller PJ, Dubey
RK. Circulating nitrite/nitrate levels increase with
follicular development: indirect evidence for estra-
diol mediated NO release. Biochem Biophys Res
Commun 1994; 202: 1543–52.
75. Lundberg JON, Carlsson S, Engstrand L, Morcos E,
Wiklund NP, Weitzberg E. Urinary nitrite: more
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
 NITRITE AND NITRATE ANALYSES
than a marker of infection. Urology 1997; 50: 189 –
91.
76. Zweier JL, Wang P, Samouilov A, Kuppusamy P.
Enzyme-independent formation of nitric oxide in
biological tissues. Nat Med 1995; 1: 804 –9.
77. Duncan C, Dougall H, Johnston P, et al. Chemical
generation of nitric oxide in the mouth from the
enterosalivary circulation of dietary nitrate. Nat
Med 1995; 1: 546 –51.
78. Dougall HT, Smith L, Duncan C, Benjamin N. The
effect of amoxycillin on salivary nitrite concentra-
tions: an important mechanism of adverse reactions?
Br J Clin Pharmacol 1995; 39: 460 –2.
79. Zapol WM, Bloch KD. Nitric oxide and the lung. In:
Lenfant C, Ed. Lung biology in health and disease;
vol. 98. Pp. 1– 469. New York: Marcel Dekker, Inc.,
1997.
80. Adatia I, Wessel DL. The use of inhaled nitric oxide
in congenital heart disease. In: Weir EK, Archer SL,
Reeves JT, Eds. Nitric oxide and radicals in the
pulmonary vasculature. Pp. 463–91. Armonk, NY:
Futura Publishing Company, Inc., 1996.
81. Valvini EM, Young JD. Serum nitrogen oxides dur-
ing nitric oxide inhalation. Br J Anaesth 1995; 74:
338 –9.
82. Stamler JS, Singel DJ, Loscalzo J. Biochemistry of
nitric oxide and is redox-activated forms. Science
1992; 258: 1898 –902.
83. Edwards AD. The pharmacology of inhaled nitric
oxide. Arch Dis Child 1995; 72: F127–F30.
84. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide:
physiology, pathophysiology, and pharmacology.
Pharmacol Rev 1991; 43: 109 – 42.
85. Ignarro LJ, Fukuto JM, Griscavage JM, Rogers NE,
Byrns RE. Oxidation of nitric oxide in aqueous solu-
tion to nitrite but not nitrate: comparison with enzy-
matically formed nitric oxide from L-arginine. Proc
Natl Acad Sci USA 1993; 90: 8103–7.
86. Luscher TF, Noll G. The pathogenesis of cardiovas-
cular disease: role of the endothelium as a target and
mediator. Atherosclerosis 1995; 118: S81–S90.
87. Rao GHR, Raij L, Lester BB, White JG. Inhibition of
agonist-induced human platelet activation by nitric
oxide. In: Moncada S, Higgs EH, Eds. Nitric oxide
from L-arginine: a bioregulatory system. Pp. 355– 63.
Amsterdam: Exerpta Medica, 1990.
88. Busse R, Fleming I. Regulation and functional con-
sequences of endothelial nitric oxide formation. Ann
Med 1995; 27: 331– 40.
89. Milbourne EA, Bygrave FL. Do nitric oxide and
cGMP play a role in calcium cycling? Short review.
Cell Calcium 1995; 18: 207–13.
90. Rees DC, Cervi P, Grimwade D, et al. The metabo-
lites of nitric oxide in sickle-cell disease. Br J
Haematol 1995; 91: 834 –7.
91. Farkas J, Menzel EJ. Proteins lose their nitric oxide
stabilizing function after advanced glycosylation.
Biochem Biophys Acta-Gen Subj 1995; 1245: 305–10.
92. Butler AR, Rhodes P. Chemistry, analysis, and bio-
logical roles of S-nitrosothiols. Anal Biochem 1997;
249: 1–9.
93. Jia L, Bonaventura J, Stamler JS. S-nitrosohaemo-
globin: a dynamic activity of blood involved in vas-
cular control. Nature 1996; 380: 221– 6.
94. Stamler JS, Jia L, Eu JP, et al. Blood flow regulation
by S-nitrosohemoglobin in the physiological oxygen
gradient. Science 1997; 276: 2034 –7.
95. Champagne DE, Nussenzveig RH, Ribeiro JM. Puri-
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
fication, partial characterization, and cloning of ni-
tric oxide-carrying heme proteins (nitrophorins)
from salivary glands of the blood-sucking insect
Rhodnius prolixus. J Biol Chem 1995; 270: 8691–5.
96. Lewis RS, Tamir S, Tannenbaum SR, Deen WM.
Kinetic analysis of the fate of nitric oxide synthe-
sized by macrophages in vitro. J Biol Chem 1995;
270: 29350 –5.
97. Franchini A, Conte A, Ottaviani E. Nitric oxide: an
ancestral immunocyte effector molecule. Adv Neuro-
immunol 1995; 5: 463–78.
98. Pfeiffer S, Gorren ACF, Schmidt K, et al. Metabolic
fate of peroxynitrite in aqueous solution: reaction
with nitric oxide and pH dependent decomposition to
nitrite and oxygen in a 2/1 stoichiometry. J Biol
Chem 1997; 272: 3465–70.
99. Zingarelli B, Day BJ, Crapo JD, Salzman AL, Szabo
C. The potential role of peroxynitrite in the vascular
contractile and cellular energetic failure in endotoxic
shock. Br J Pharmacol 1997; 120: 259 – 67.
100. Pryor WA, Squadrito GL. The chemistry of peroxyni-
trite: a product from the reaction of nitric oxide with
superoxide. Am J Physiol 1995; 268: L699 –722.
101. Moro MA, Darley-Usmar VM, Goodwin DA, et al.
Paradoxical fate and biological action of peroxyni-
trite on human platelets. Proc Natl Acad Sci USA
1994; 91: 6702– 6.
102. Muijsers RB, Folkerts G, Henricks PA, Sadeghi-
Hashjin G, Nijkamp FP. Peroxynitrite: a two-faced
metabolite of nitric oxide. Life Sci 1997; 60: 1833– 45.
103. Beckman JS, Koppenol WH. Nitric oxide, superox-
ide, and peroxynitrite: the good, the bad, and ugly.
Am J Physiol 1996; 271: C1424 –37.
104. Szabo C. The pathophysiological role of peroxynitrite
in shock, inflammation, and ischemia-reperfusion
injury. Shock 1996; 6: 79 – 88.
105. Gross SS, Wolins M. Nitric oxide: pathophysiological
mechanisms. Annu Rev Physiol 1995; 57: 737– 69.
106. van der Vliet A, Eiserich JP, Halliwell B, Cross CE.
Formation of reactive nitrogen species during perox-
idase-catalyzed oxidation of nitrite. A potential ad-
ditional mechanism of nitric oxide-dependent toxic-
ity. J Biol Chem 1997; 272: 7617–25.
107. Eiserich JP, Vossen V, O’Neill CA, Halliwell B, Cross
CE, van der Vliet A. Molecular mechanisms of dam-
age by excess nitrogen oxides: nitration of tyrosine
by gas-phase cigarette smoke. FEBS Lett 1994; 353:
53– 6.
108. Yi D, Smythe GA, Blount BC, Duncan MW. Per-
oxynitrite-mediated nitration of peptides: character-
ization of the products by electrospray and combined
gas chromatography-mass spectrometry. Arch Bio-
chem Biophys 1997; 344: 253–9.
109. Ishida A, Sasaguri T, Kosaka C, Nojima H, Ogata
J. Induction of the cyclin-dependent kinase inhibitor
p21(Sdi1/Cip1/Waf1) by nitric oxide-generating vaso-
dilator in vascular smooth muscle cells. J Biol Chem
1997; 272: 10050 –7.
110. Grisham MB, Johnson GG, Lancaster JR Jr. Quan-
titation of nitrate and nitrite in extracellular fluids.
Methods Enzymol 1996; 268: 237– 46.
111. Granger DL, Taintor RR, Boockvar KS, Hibbs JB Jr.
Measurement of nitrate and nitrite in biological
samples using nitrate reductase and Griess reaction.
Methods Enzymol 1996; 268: 142–51.
112. Belmont HM, Levartovsky D, Goel A, et al. Increased
nitric oxide production accompanied by the up regu-
lation of inducible nitric oxide synthase in vascular
211
 ELLIS
endothelium from patients with systemic lupus ery-
thematosus. Arthritis Rheum 1997; 40: 1810 – 6.
113. Trachtman H, Gauthier B, Frank R, Futterweit S,
Goldstein A, Tomczak J. Increased urinary nitrite
excretion in children with minimal change nephrotic
syndrome. J Pediatr 1996; 128: 173– 6.
114. Trachtman H. Urinary nitrite versus nitrate excre-
tion in children: reply. J Pediatr 1997; 130: 161.
115. Seligman SP, Buyon JP, Clancy RM, Young BK,
Abramson SB. The role of nitric oxide in the patho-
genesis of pre-eclampsia. Am J Obstet Gynecol 1994;
171: 944 – 6.
116. Lyall F, Young A, Greer IA. Nitric oxide concentra-
tions are increased in the fetoplacental circulation in
pre-eclampsia. Am J Obstet Gynecol 1995; 173:
714 – 8.
117. Tsukahara H, Hata I, Sudo M. Urinary nitrite versus
nitrate excretion in children. J Pediatr 1997; 130:
161.
118. Moncada S, Higgs A, Furchgott R. International
Union of Pharmacology Nomenclature in Nitric Ox-
ide Research. Pharmacol Rev 1997; 49: 137– 42.
119. Sawicki E, Stanley TW, Pfaff J, D’Amico A. Compar-
ison of fifty-two spectrophotometric methods for the
determination of nitrite. Tantala 1963; 10: 641–55.
120. Verdon CP, Burton BA, Prior RL. Sample pretreat-
ment with nitrate reductase and glucose-6-phos-
phate dehydrogenase quantitatively reduces nitrate
while avoiding interference by NADP1 when the
Griess reaction is used to assay for nitrite. Anal
Biochem 1995; 224: 502– 8.
121. Green LC, Wagner DA, Glogowski J, Skipper L,
Wishnok JS, Tannenbaum SR. Analysis of nitrate,
nitrite and [15N] nitrate in biological fluids. Anal
Biochem 1982; 126: 131– 8.
122. Wishnok JS, Glogowski JA, Tannenbaum SR. Quan-
titation of nitrate, nitrite, and nitrosating agents.
Methods Enzymol 1996; 268: 130 – 41.
123. Vodovotz Y. Modified microassay for serum nitrite
and nitrate. Biotechniques 1996; 20: 390 – 4.
124. Giovannoni G, Land JM, Keir G, Thompson EJ,
Heales SJ. Adaptation of the nitrate reductase and
Griess reaction methods for the measurement of
serum nitrate plus nitrite levels. Ann Clin Biochem
1997; 34: 193– 8.
125. Gutman SI, Hollywood CA. Simple, rapid method for
determining nitrates and nitrites in biological fluids.
Clin Chem 1992; 38: 2152 (letter).
126. Tsikas D, Gutzki FM, Rossa S, et al. Measurement of
nitrite and nitrate in biological fluids by gas chroma-
tography-mass spectrometry and by the Griess as-
say: problems with the Griess assay--solutions by gas
chromatography-mass spectrometry. Anal Biochem
1997; 244: 208 –20.
127. Greenberg SS, Xie J, Spitzer JJ, et al. Nitro contain-
ing L-arginine analogs interfere with assays for
nitrate and nitrite. Life Sci 1995; 57: 1949 – 61.
128. Nithipatikom K, Pratt PF, Campbell WB. Nitro-L-
arginine inteferes with the cadmium reduction of
nitrate/griess reaction method of measuring nitric
oxide production. Eur J Clin Chem Clin Biochem
1996; 34: 133–7.
129. Zhou JY, Dauphin C, Hamon M. [Use of 7-amino-4-
methylcoumarin for the spectrophotometric determi-
nation of nitrites]. Ann Pharm Fr 1993; 51: 94 –100.
130. Gilliam MB, Sherman MP, Griscavage JM, Ignarro
LJ. A spectrophotometric assay for nitrate using
212
ET AL.
NADPH oxidation by Aspergillus nitrate reductase.
Anal Biochem 1993; 212: 359 – 65.
131. Bories PN, Bories C. Nitrate determination in bio-
logical fluids by an enzymatic one-step assay with
nitrate reductase. Clin Chem 1995; 41: 904 –7.
132. Misko TP, Schilling RJ, Salvemini D, Moore WM,
Currie MG. A fluorometric assay for the measure-
ment of nitrite in biological samples. Anal Biochem
1993; 214: 11– 6.
133. Miles AM, Wink DA, Cook JC, Grinsham MB. Deter-
mination of nitric oxide using fluorescence spectros-
copy. Methods Enzymol 1996; 268: 105–20.
134. Park JK, Kostka P. Fluorometric detection of biolog-
ical S-nitrosothiols. Anal Biochem 1997; 249: 61– 6.
135. Jie NQ, Si ZK, Yang JH, et al. Fluorometric deter-
mination of traces of nitrite with rhodamine 6G.
Microchem J 1997; 55: 351– 6.
136. Perez MDA, Marin LP, Denis RD, Defuentes OA.
Development of a nitrate ion selective electrode.
Afinidad 1997; 54: 123–5.
137. Sun BT, Fitch PG. Nitrate ion selective sensor based
on electrochemically prepared conducting polypyr-
role films. Electroanalysis 1997; 9: 494 –7.
138. Mohr GJ, Lehmann F, Ostereich R, Murkovic I,
Wolfbeis OS. Investigation of potential sensitive flu-
orescent dyes for application in nitrate sensitive
polymer membranes. Fresenius J Anal Chem 1997;
357: 284 –91.
139. Aylott JW, Richardson DJ, Russell DA. Optical bio-
sensing of nitrate ions using a sol gel immobilized
nitrate reductase. Analyst 1997; 122: 77– 80.
140. Vanifatova NG, Isakova NV, Kolycheva NV, et al. Ion
selective field effect transistors with plasticized
membranes: nitrate ion sensor. J Anal Chem 1997;
52: 52–5.
141. Liu ZM, Xi XD, Dong SJ, Wang EK. Liquid chroma-
tography amperometric detection of nitrite using a
polypyrrole modified glassy carbon electrode doped
with tungstodiphosphate anion. Anal Chim Acta
1997; 345: 147–53.
142. Debeer D, Schramm A, Santegoeds CM, Kuhl M. A
nitrite microsensor for profiling environmental bio-
films. Appl Environ Microbiol 1997; 63: 973–7.
143. Hutchins RS, Bachas LG. Nitrate-selective electrode
developed by electrochemically mediated imprinting
doping of polypyrrole. Anal Chem 1995; 67: 1654 – 60.
144. Bertotti M, Pletcher D. Amperometric determination
of nitrite via reaction with iodide using microelec-
trodes. Anal Chim Acta 1997; 337: 49 –55.
145. Casella IG, Salvi AM. Voltammetric behaviour and
ion chromatographic detection of nitrite at a dis-
persed platinum glassy carbon electrode. Electro-
analysis 1997; 9: 596 – 601.
146. Fontijn A, Sabadell AJ, Ronco RJ. Homogeneous
chemiluminescent measurement of nitric oxide with
ozone. Anal Chem 1970; 42: 575–9.
147. Silkoff PE, McClean PA, Slutsky AS, et al. Marked
flow-dependence of exhaled nitric oxide using a new
technique to exclude nasal nitric oxide. Am J Respir
Crit Care Med 1997; 155: 260 –7.
148. Kharitonov SA, Yates D, Barnes PJ. Increased nitric
oxide in exhaled air of normal human subjects with
upper respiratory tract infections. Eur Respir J
1995; 8: 295–7.
149. Alving K, Weitzberg E, Lundberg JM. Increased
amount of nitric oxide in exhaled air of asthmatics.
Eur Respir J 1993; 6: 1368 –70.
150. Byrnes CA, Dinarevic S, Busst C, Bush A, Shine-
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
 NITRITE AND NITRATE ANALYSES
bourne EA. Is nitric oxide in exhaled air produced at
airway or alveolar level? Eur Respir J 1997; 10:
1021–5.
151. Cox RD. Determination of nitrate and nitrite at parts
per billion level by chemiluminescence. Anal Chem
1980; 5: 332–5.
152. Yang F, Troncy E, Francoeur M, et al. Effects of
reducing reagents and temperature on conversion of
nitrite and nitrate to nitric oxide and detection of NO
by chemiluminescence. Clin Chem 1997; 43: 657– 62.
153. Sen NP, Baddoo PA, Seaman SW. Rapid and sensi-
tive determination of nitrite in foods and biological
materials by flow injection or high-performance liq-
uid chromatography with chemiluminescence detec-
tion. J Chromatogr A 1994; 673: 77– 84.
154. Alpert C, Ramdev N, George D, Lorscalzo J. Detec-
tion of S-nitrosothiols and other nitric oxide deriva-
tives by photolysis-chemiluminescence spectrome-
try. Anal Biochem 1997; 245: 1–7.
155. Sonoda M, Kobayashi J, Takezawa M, et al. An assay
method for nitric oxide-related compounds in whole
blood. Anal Biochem 1997; 247: 417–27.
156. Landers JP. Clinical capillary electrophoresis. Clin
Chem 1995; 41: 495–509.
157. Ueda T, Maekawa T, Sadamitsu D, Oshita S, Ogino
K, Nakamura K. The determination of nitrite and
nitrate in human blood plasma by capillary zone
electrophoresis. Electrophoresis 1995; 16: 1002– 4.
158. Meulemans A, Delsenne F. Measurement of nitrite
and nitrate levels in biological samples by capillary
electrophoresis. J Chromatogr B 1994; 14: 401– 4.
159. Friedberg MA, Hinsdale ME, Shihabi ZK. Analysis of
nitrate in biological fluids by capillary electrophore-
sis. J Chromatogr A 1997; 781: 491– 6.
160. Rhemrevboom MM. Determination of anions with
capillary electrophoresis and indirect ultraviolet de-
tection. J Chromatogr A 1994; 680: 675– 84.
161. Janini GM, Chan KC, Muschik GM, Issaq HJ. Anal-
ysis of nitrate and nitrite in water and urine by
capillary zone electrophoresis. J Chromatogr B 1994;
657: 419 –23.
162. Tsikas D, Boger RH, Bodeboger SM, Brunner G,
Frolich JC. Formation of S-nitroso compounds from
sodium nitroprusside, nitric oxide or nitrite and
reduced thiols: analysis by capillary isotachophore-
sis. J Chromatogr A 1995; 699: 363–9.
163. Bjergegaard C, Moller P, Sorensen H. Determination
of thiocyanate, iodide, nitrate and nitrite in biologi-
cal samples by micellar electrokinetic capillary chro-
matography. J Chromatogr A 1995; 717: 409 –14.
164. Cowley EA, Govindaraju K, Lloyd DK, Eidelman
DH. Airway surface fluid composition in the rat
determined by capillary electrophoresis. Am J
Physiol Lung Cell Mol Physiol 1997; 17: L895–L9.
165. Cruz L, Moroz LL, Gillette R, Sweedler JV. Nitrite
and nitrate levels in individual molluscan neurons:
single-cell capillary electrophoresis analysis. J Neu-
rochem 1997; 69: 110 –5.
166. Jain A, Smith RM, Verma KK. Gas chromatographic
determination of nitrite in water by precolumn for-
mation of 2 phenylphenol with flame ionization de-
tection. J Chromatogr A 1997; 760: 319 –25.
167. Mitsuhashi T. Gas chromatographic determination
of trace nitrite after derivatization with ethyl
3-oxobutanoate. J Chromatogr 1993; 629: 339 – 43.
168. Johnson AA, Burleson DG. Nitrate analysis in bio-
logical fluids by gas chromatography-nitrogen-phos-
phorous detection. Anal Biochem 1996; 236: 331–7.
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
169. Tsikas D, Boger RH, Bode-Boger SM, Gutzki FM,
Frolich JC. Quantification of nitrite and nitrate in
human urine and plasma as pentafluorobenzyl de-
rivatives by gas chromatography-mass spectrometry
using their 15N-labelled analogs. J Chromatogr B
1994; 661: 185–91.
170. Wolff JC, Taylor DP, De Bievre P. Traceable values
for nitrate in water samples by isotope dilution
analysis using a small thermionic quadrupole mass
spectrometer. Anal Chem 1996; 68: 3231–7.
171. Tesch JW, Rehg WR, Sievers RE. Microdetermina-
tion of nitrates and nitrites in saliva, blood, water
and suspended particulates in air by gas chromatog-
raphy. J Chromatogr 1976; 126: 743–55.
172. Anonymous. United States of America Environmen-
tal Protection Agency EPA Test Method 300.0, The
determination of inoganic anions in water by ion
chromatography.
173. Muscara MN, de Nucci G. Simultaneous determina-
tion of nitrite and nitrate anions in plasma, urine
and cell culture supernatants by high-performance
liquid chromatography with post-column reactions.
J Chromatogr B 1996; 686: 157– 64.
174. Everett SA, Dennis MF, Tozer GM, Prise VE, Ward-
man P, Stratford MRL. Nitric oxide in biological
fluids: analysis of nitrite and nitrate by high perfor-
mance ion chromatography. J Chromatogr A 1995;
706: 437– 42.
175. Radisavljevic Z, George M, Dries DJ, Gamelli RL.
Determination of intracellular and extracellular ni-
trite and nitrate by anion chromatography. J Liqu
Chromatogr Relat Techn 1996; 19: 1061–79.
176. Gamelli RL, George M, Sharp-Pucci M, Dries DJ,
Radisavljevic J. Burn-induced nitric oxide release in
humans. J Trauma 1995; 39: 869 –77.
177. Ito K, Ariyoshi Y, Sunahara H. Determination of
inorganic anions in salt solutions by ion chromatog-
raphy using C18 reversed-phase columns coated
with cetyltrimethylammonium. J Chromatogr 1992;
598: 237– 41.
178. Canas-Montalvo B, Izqueierdo-Hornillos RC. Com-
parative behaviour of different reversed-phase pack-
ings when equilibrated with cetyltrimethylam-
monium bromide. J Liquid Chromatogr 1994; 17:
1461–78.
179. Monaghan JM, Cook K, Gara D, Crowther D. Deter-
mination of nitrite and nitrate in human serum.
J Chromatogr A 1997; 770: 143–9.
180. Stratford MRL, Dennis MF, Cochrane R, Parkins
CS, Everett SA. The role of nitric oxide in cancer:
improved methods for measurement of nitrite and
nitrate by high performance ion chromatography.
J Chromatogr A 1997; 770: 151–5.
181. Salas-Auvert R, Colmenarez J, deLedo H, et al.
Determination of anions in human and animal tear
fluid and blood serum by ion chromatography.
J Chromatogr A 1995; 706: 183–9.
182. Csiba A, Szentgyorgyi M, Juhasz S, Lombai G. A
high-performance liquid chromatographic (HPLC)
method for the simultaneous determination of nitrite
and formaldehyde from foods. Acta Vet Hung 1996;
44: 165–72.
183. Gorbunov N, Esposito E. Rapid and simple method
for the determination of nitrite in synaptosomal
superfusates by liquid chromatography with electro-
chemical detection. J Chromatogr 1993; 619: 133– 6.
184. Kaku S, Tanaka M, Muramatsu M, Otomo S. Deter-
mination of nitrite by high-performance liquid chro-
213
 ELLIS
matography system with electrochemical detector:
measurement of nitric oxide synthase activity in rat
cerebellum cytosol. Biomed Chromatogr 1994; 8:
14 – 8.
185. Preik-Steinhoff H, Kelm M. Determination of nitrite
in human blood by combination of a specific sample
preparation with high-performance anion-exchange
chromatography and electrochemical detection.
J Chromatogr B 1996; 685: 348 –52.
186. Pratt PF, Nithipatikom K, Campbell WB. Simulta-
neous determination of nitrate and nitrite in biolog-
ical samples by multichannel flow injection analysis.
Anal Biochem 1995; 231: 383– 6.
187. Makela SK, Yazdanpanah M, Adatia I, Ellis G.
Disposable gloves and Pasteur (transfer) pipettes as
potential sources of contamination in nitrite and
nitrate assays. Clin Chem 1997; 43: 2418 –9.
188. Jilma B, Kastner J, Mensik C, et al. Sex differences
in concentrations of exhaled nitric oxide and plasma
nitrate. Life Sci 1996; 58: 469 –76.
189. Takahashi H, Nakanishi T, Nishimura M, Tanaka
H. Measurements of serum levels of nitrate ions in
men and women: implications of endothetium-de-
rived relaxing factor in blood pressure regulation
and atherosclerosis. J Cardiovasc Pharmacol 1992;
20 (Suppl. 12): S214 – 6.
190. Kawano H, Motoyama T, Kugiyama K, et al. Gender
difference in improvement of endothelium dependent
vasodilation after estrogen supplementation. J Am
Coll Cardiol 1997; 30: 914 –9.
191. Rosselli M, Imthurn B, Keller PJ, Jackson EK,
Dubey RK. Circulating nitric oxide (nitrite/nitrate)
levels in postmenopasal women substituted with
17b-estradiol and norethistrone acetate. A two-year
follow-up study. Hypertension 1995; 25 [Pt. 2]: 848 –
53.
192. Imthurn B, Rosselli M, Jaeger AW, Keller PJ, Dubey
RK. Differential effects of hormone replacement
therapy on endogenous nitric oxide (nitrite/nitrate)
levels in postmenopausal women substituted with 17
beta estradiol valerate and cyproterone acetate or
medroxyprogesterone acetate. J Clin Endocrinol
Metab 1997; 82: 388 –94.
193. Morris NH, Sooranna SR, Steer PJ, Warren JB. The
effect of the menstrual cycle on exhaled nitric oxide
and urinary nitrate concentration. Eur J Clin Invest
1996; 26: 481– 4.
194. Shaarawy M, Nafei S, Abul Nasr A, el Sharkawy S,
Younis A. Circulating nitric oxide levels in galactor-
rheic, hyperprolactinemic, amenorrheic women. Fer-
til Steril 1997; 68: 454 –9.
195. Tsukahara H, Hiraoka M, Hori C, et al. Urinary
nitrite nitrate excretion in infancy: comparison be-
tween term and preterm infants. Early Hum Dev
1997; 47: 51– 6.
196. Goonasekera CD, Shah V, Rees DD, Dillon MJ.
Nitric oxide activity in childhood hypertension. Arch
Dis Child 1997; 77: 11– 6.
197. Siddiqi M, Kumar R, Kaul D, Spiegelhalder B,
Preussman R. Salivary nitrate and nitrite concentra-
tions from a sample population of children and
adults in high risk area for esophageal and gastric
cancers in Kashmir, India. Cancer Lett 1992; 64:
133– 6.
198. Clelland JD, Brand MP, Bolanos JP, Surtees RA,
Land JM, Heales SJ. Age dependent changes in the
cerebrospinal fluid concentration of nitrite and ni-
trate. Ann Clin Biochem 1996; 33: 71–2.
214
ET AL.
199. Smith SD, Wheeler MA, Weiss RM. Nitric oxide
synthase: an endogenous source of elevated nitrite in
infected urine. Kidney Int 1994; 45: 586 –91.
200. Hiraoka M, Hida Y, Hori C, Tuchida S, Kuroda M,
Sudo M. Rapid dipstick test for diagnosis of urinary
tract infection. Acta Paediatr Jpn 1994; 36: 379 – 82.
201. Anderson JD, Chambers GK, Johnson HW. Applica-
tion of a leukocyte and nitrite urine test strip to the
management of children with neurogenic bladder.
Diagn Microbiol Infect Dis 1993; 17: 29 –33.
202. Larson MJ, Brooks CB, Leary WC, Lewis LM. Can
urinary nitrite results be used to guide antimicrobial
choice for urinary tract infection? J Emerg Med 1997;
15: 435– 8.
203. Holland DJ, Bliss KJ, Allen CD, Gilbert GL. A
comparison of chemical dipsticks read visually or by
photometry in the routine screening of urine speci-
mens in the clinical microbiology laboratory. Pathol-
ogy 1995; 27: 91– 6.
204. Bachman JW, Heise RH, Naessens JM, Timmerman
MG. A study of various tests to detect asymptomatic
urinary tract infections in an obstetric population.
JAMA 1993; 270: 1971– 4.
205. Nauschuetz WF, Harrison LS, Trevino SB, Becker
GR, Benton J. Two rapid urine screens for detection
of bactiuria: an evaluation. Curr Microbiol 1993; 26:
43–5.
206. Liptak GS, Campbell J, Stewart R, Hulbert WCJ.
Screening for urinary tract infection in children with
neurogenic bladders. Am J Phys Med Rehabil 1993;
72: 122– 6.
207. Zainal D, Baba A. The value of positive nitrites in
screening asymptomatic bacteriuria amongst Malay-
sian school children. Southeast Asian J Trop Med
Public Health 1996; 27: 184 – 8.
208. Edwards A, Vandervoort J, Newcombe R, Thayer H,
Jones KV. A urine analysis method suitable for
childrens nappies. J Clin Pathol 1997; 50: 569 –72.
209. Lohr JA, Portilla MG, Geuder TG, Dunn ML, Dudley
SM. Making a presumptive diagnosis of urinary tract
infection by using urinalysis performed in an on-site
laboratory. J Pediatr 1993; 122: 22–5.
210. Nakae H, Endo S, Inada K, et al. Nitrite/nitrate
(NOX) and type II phospholipase A2, leukotriene B4,
and platelet-activating factor levels in patients with
septic shock. Res Commun Mol Pathol Pharmacol
1996; 92: 131–9.
211. Wong HR, Carcillo JA, Burckart G, Shah N, Janosky
JE. Increased serum nitrite and nitrate concentra-
tions in children with the sepsis syndrome. Crit Care
Med 1995; 23: 835– 42.
212. Shi Y, Li H-Q, Shen C-K, et al. Plasma nitric oxide
levels in newborn infants with sepsis. J Pediatr
1993; 123: 435– 8.
213. de Werra I, Jaccard C, Corradin SB, et al. Cytokines,
nitrite/nitrate, soluble tumor necrosis factor recep-
tors, and procalcitonin concentrations: comparisons
in patients with septic shock, cardiogenic shock, and
bacterial pneumonia. Crit Care Med 1997; 25: 607–
13.
214. Endo S, Inada K, Nakae H, et al. Nitrite/nitrate
oxide (NOx) and cytokine levels in patients with
septic shock. Res Commun Mol Pathol Pharmacol
1996; 91: 347–56.
215. Fukuyama N, Takebayashi Y, Hida M, Ishida H,
Ichimori K, Nakazawa H. Clinical evidence of per-
oxynitrite formation in chronic renal failure patients
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
 NITRITE AND NITRATE ANALYSES
with septic shock. Free Radic Biol Med 1997; 22:
771– 4.
216. Thiemermann C. Nitric oxide and septic shock. Gen
Pharmacol 1997; 29: 159 – 66.
217. Krafte Jacobs B, Brilli R, Szabo C, Denenberg A,
Moore L, Salzman AL. Circulating methemoglobin
and nitrite/nitrate concentrations as indicators of
nitric oxide overproduction in critically ill children
with septic shock. Crit Care Med 1997; 25: 1588 –93.
218. Guarner C, Soriano G, Tomas A, et al. Increased
serum nitrite and nitrate levels in patients with
cirrhosis: relationship to endotoxemia. Hepatology
1993; 18: 1139 – 43.
219. Bories PN, Campillo B, Azaou L, Scherman E. Long-
lasting NO overproduction in cirrhotic patients with
spontaneous bacterial peritonitis. Hepatology 1997;
25: 1328 –33.
220. Uzuner N, Islekel H, Ozkan H, Sen A, Yenice S,
Cevik N. Urinary nitrite excretion in low birth
weight neonates with systemic inflammatory re-
sponse syndrome. Biol Neonate 1997; 71: 362– 6.
221. Spack L, Havens PL, Griffith OW. Measurements of
total plasma nitrite and nitrate in pediatric patients
with the systemic inflammatory response syndrome.
Crit Care Med 1997; 25: 1071– 8.
222. Endo S, Inada K, Takakuwa T, et al. Nitrite/nitrate
(NOx) and sFas antigen levels in patients with
multiple organ failure. Res Commun Mol Pathol
Pharmacol 1996; 92: 253– 6.
223. Rees DD. Role of nitric oxide in the vascular dysfun-
tion of septic shock. Biochem Soc Trans 1995; 23:
1025–9.
224. Torre D, Ferrario G, Speranza F, Orani A, Fiori GP,
Zeroli C. Serum concentrations of nitrite in patients
with HIV-1 infection. J Clin Pathol 1996; 49: 574 – 6.
225. Zangerle R, Fuchs D, Reibnegger G, et al. Serum
nitrite plus nitrate in infection with human immu-
nodeficiency virus type-1. Immunobiol 1995; 193:
59 –70.
226. Sternberg JM. Elevated serum nitrate in Trypano-
soma brucei ‘rhodesiense’ infections: evidence for
inducible nitric oxide synthesis in trypanosomiasis.
Trans R Soc Trop Med Hyg 1996; 90: 395.
227. Oudenhoven IM, Klaasen HL, Lapre JA, Weerkamp
AH, Van der Meer R. Nitric oxide-derived urinary
nitrate as a marker of intestinal bacterial transloca-
tion in rats. Gastroenterology 1994; 107: 47–53.
228. Dykhuizen RS, Copland M, Smith CC, Douglas G,
Benjamin N. Plasma nitrate concentration and uri-
nary nitrate excretion in patients with gastroenter-
itis. J Infect 1995; 31: 73–5.
229. Dykhuizen RS, Masson J, McKnight G, et al. Plasma
nitrate concentration in infective gastroenteritis and
inflammatory bowel disease. Gut 1996; 39: 393–5.
230. Gaginella TS, Kachur JF, Tamai H, Keshavarzian.
Reactive oxygen and nitrogen metabolites as media-
tors of secretory diarrhea. Gastroenterology 1995;
109: 2019 –28.
231. Melichar B, Karlicek R, Tichy M. Increased urinary
nitrate excretion in inflammatory bowel disease. Eur
J Clin Chem Clin Biochem 1994; 32: 3– 4.
232. Oudkerk Pool M, Bouma G, Visser JJ, et al. Serum
nitrate levels in ulcerative colitis and Crohn’s dis-
ease. Scand J Gastroenterol 1995; 30: 784 – 8.
233. Rees DC, Satsangi J, Cornelissen PL, Travis SP,
White J, Jewel DP. Are serum concentrations of
nitric oxide metabolites useful for predicting the
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
clinical outcome of severe ulcerative colitis? Eur J
Gastroenterol Hepatol 1995; 7: 227–30.
234. Sasajima K, Yoshida Y, Yamakado S, et al. Changes
in urinary nitrate and nitrite during treatment of
ulcerative colitis. Digestion 1996; 57: 170 –3.
235. Lundberg JON, Herulf M, Olesen M, et al. Increased
nitric oxide production in collagenous and lympho-
cytic colitis. Eur J Clin Invest 1997; 27: 869 –71.
236. Rachmilewitz D, Stamler JS, Bachwich D, Karmeli
F, Ackerman Z, Podolsky DK. Enhanced colonic
nitric oxide generation and nitric oxide synthase
activity in ulcerative colitis and Crohn’s disease. Gut
1995; 36: 718 –23.
237. Kimura H, Miura S, Shigematsu T, et al. Increased
nitric oxide production and inducible nitric oxide
synthase activity in colonic mucosa of patients with
active ulcerative colitis and Crohn’s disease. Dig Dis
Sci 1997; 42: 1047–54.
238. Middleton SJ, Shorthouse M, Hunter JO. Increased
nitric oxide synthesis in ulcerative colitis. Lancet
1993; 341: 465– 6.
239. Neilly PJD, Kirk SJ, Gardiner KR, Anderson NH,
Rowlands BJ. Manipulation of the L-arginine nitric
oxide pathway in experimental colitis. Br J Surg
1995; 82: 1188 –91.
240. Adjei AA, Yamauchi K, Nakasone Y, Konishi M,
Yamamoto S. Arginine-supplemented diets inhibit
endotoxin-induced bacterial translocation in mice.
Nutrition 1995; 11: 371– 4.
241. Kubes P, Wallace JL. Nitric oxide as a mediator of
gastrointestinal injury? Say it ain’t so. Mediators
Inflamm 1995; 4: 397– 405.
242. Farinati F, Della Libera G, Cardin R, et al. Gastric
antioxidant, nitrites, and mucosal lipoperoxidation
in chronic gastritis and Helicobacter pylori infection.
J Clin Gastroenterol 1996; 22: 275– 81.
243. Konturek SK, Konturek PC. Role of nitric oxide in
the digestive system. Digestion 1995; 56: 1–13.
244. Chung E, Curtis D, Chen G, et al. Genetic evidence
for the neuronal nitric oxide synthase gene (NOS1)
as a susceptibility locus for infantile pyloric stenosis.
Am J Hum Genet 1996; 58: 363–70.
245. Larsson LT, Shen Z, Ekblad E, Sundler F, Alm P,
Andersson KE. Lack of neuronal nitric oxide syn-
thase in nerve fibers of aganglionic intestine: a clue
to hirschsprung’s disease. J Pediatr Gastroenterol
Nutr 1995; 20: 49 –53.
246. Mihm S, Fayyazi A, Ramadori G. Hepatic expression
of inducible nitric oxide synthase transcripts in
chronic hepatitis C virus infection: relation to he-
patic viral load and liver injury. Hepatology 1997; 26:
451– 8.
247. Amaro MJ, Bartolome J, Pardo M, Cotonat T, Lopez-
Farre A, Carreno V. Decreased nitric oxide produc-
tion in chronic viral hepatitis B and C. J Med Virol
1997; 51: 326 –31.
248. Rolla G, Brussino L, Colagrande P, et al. Exhaled
nitric oxide and oxygenation abnormalities in he-
patic cirrhosis. Hepatology 1997; 26: 842–7.
249. Chu CJ, Lee FY, Wang SS, et al. Hyperdynamic
circulation of cirrhotic rats: role of substance P and
its relationship to nitric oxide. Scand J Gastroenterol
1997; 32: 841– 6.
250. Campillo B, Bories PN, Benvenuti C, Dupeyron C.
Serum and urinary nitrate levels in liver cirrhosis:
endotoxemia, renal function and hyperdynamic cir-
culation. J Hepatol 1996; 25: 707–14.
251. Hori N, Okanoue T, Mori T, et al. Endogenous nitric
215
 ELLIS
oxide production is augmented as the severity ad-
vances in patients with liver cirrhosis. Clin Exp
Pharmacol Physiol 1996; 23: 30 –5.
252. Woitas RP, Heller J, Stoffel Wagner B, Spengler U,
Sauerbruch T. Renal functional reserve and nitric
oxide in patients with compensated liver cirrhosis.
Hepatology 1997; 26: 858 – 64.
253. Moriyama A, Masumoto A, Nanri H, et al. High
plasma concentrations of nitrite/nitrate in patients
with hepatocellular carcinoma. Am J Gastroenterol
1997; 92: 1520 –3.
254. Konturek JW, Hengst K, Kulesza E, Gabryelewicz A,
Konturek SJ, Domschke W. Role of endogenous ni-
tric oxide in the control of exocrine and endocrine
pancreatic secretion in humans. Gut 1997; 40: 86 –
91.
255. Deshmukh DR, Ghole VS, Marescau B, Dedeyn PP.
Effect of endotoxemia on plasma and tissue levels of
nitric oxide metabolites and guanidino compounds.
Arch Physiol Biochem 1997; 105: 32–7.
256. Misra A, Mukerjee R, Chaturvedi UC. Production of
nitrite by dengue virus-induced cytotoxic factor. Clin
Exp Immunol 1996; 104: 406 –11.
257. Ralston SH, Ho L-P, Helfrich MH, Grabowski PS,
Johnston PW, Benjamin N. Nitric oxide: a cytokine-
induced regulator of bone resorption. J Bone Miner
Res 1995; 10: 1040 –9.
258. Tsukahara H, Kikuchi K, Tsumura K, et al. Experi-
mentally induced acute hyperinsulinemia stimulates
endogenous nitric oxide production in humans: de-
tection using urinary NO22/NO32 excretion. Metab-
olism 1997; 46: 406 –9.
259. Jiang B, Morimoto S, Yang J, et al. Parathyroid
hormone-related protein upregulates interleukin-1b-
induced nitric oxide synthesis. Hypertension 1997;
30: 922–7.
260. Kiemer AK, Vollmar AM. Effects of different natri-
uretic peptides on nitric oxide synthesis in macro-
phages. Endocrinology 1997; 138: 4282–90.
261. Farrell AJ, Blake DR, Palmer RM, Moncada S.
Increased concentrations of nitrite in synovial fluid
and serum samples suggest increased nitric oxide
synthesis in rheumatic diseases. Ann Rheum Dis
1992; 51: 1219 –22.
262. Miyasaka N, Hirata Y. Nitric oxide and inflamma-
tory arthritides. Life Sci 1997; 61: 2073– 81.
263. Stichtenoth DO, Fauler J, Zeidler H, Frolich JC.
Urinary nitrate excretion is increased in patients
with rheumatoid arthritis and reduced by pred-
nisolone. Ann Rheum Dis 1995; 54: 820 – 4.
264. Stichtenoth DO, Gutzki FM, Tsikas D, et al. In-
creased urinary nitrate excretion in rats with adju-
vant arthritis. Ann Rheum Dis 1994; 53: 547–9.
265. Stichtenoth DO, Wollenhaupt J, Andersone D, Zei-
dler H, Frolich JC. Elevated serum nitrate concen-
trations in active spondyloarthropathies. Br J Rheu-
matol 1995; 34: 616 –9.
266. Ueki Y, Miyake S, Tominaga Y, Eguchi K. Increased
nitric oxide levels in patients with rheumatoid ar-
thritis. J Rheumatol 1996; 23: 230 – 6.
267. Citterio G, Pellegatta F, Lucca GD, et al. Plasma
nitrate plus nitrite changes during continuous intra-
venous infusion interleukin 2. Br J Cancer 1996; 74:
1297–301.
268. Stewart TE, Valenza F, Ribeiro SP, et al. Increased
nitric oxide in exhaled gas as an early marker of lung
inflammation in a model of sepsis. Am J Respir Crit
Care Med 1995; 151: 713– 8.
216
ET AL.
269. Porta C, Buggia I, Bonomi I, Caporali R, Scatola C,
Montecucco C. Nitrite and nitrate plasma levels, as
markers of nitric oxide synthesis, in antiphospho-
lipid antibodies-related conditions and in thrombotic
thrombocytopenic purpura. Thromb Haemost 1997;
78: 965–7.
270. Iizuka T, Oishi K, Sasaki M, et al. Nitric oxide and
aneurysm formation in Kawasaki disease. Acta Pae-
diatr 1997; 86: 470 –3.
271. Tsukahara H, Kikuchi K, Matsuda M, et al. Endog-
enous nitric oxide production in Kawasaki disease.
Scand J Clin Lab Invest 1997; 57: 43–7.
272. Umans JG, Levi R. Nitric oxide in the regulation of
blood flow and atrial pressure. Annu Rev Physiol
1995; 57: 771–90.
273. Nava E, Noll G, Luscher TF. Nitric oxide in cardio-
vascular diseases. Ann Med 1995; 27: 343–51.
274. Huang PL, Huang ZH, Mashimo H, et al. Hyperten-
sion in mice lacking the gene for endothelial nitric
oxide synthase. Nature 1995; 377: 239 – 42.
275. Lin KF, Chao L, Chao J. Prolonged reduction of high
blood pressure with human nitric oxide synthase
gene delivery. Hypertension 1997; 30: 307–13.
276. Celermajer DS. Endothelial dysfunction: does it mat-
ter? Is it reversible? J Am Coll Cardiol 1997; 30:
325–33.
277. Susic D. Hypertension, aging, and atherosclerosis.
The endothelial interface. Med Clin North Amer
1997; 81: 1231– 40.
278. Upchurch GR Jr., Welch GN, Fabian AJ, Pigazzi A,
Keaney JF Jr., Loscalzo J. Stimulation of endothelial
nitric oxide production by homocyst(e)ine. Athero-
sclerosis 1997; 132: 177– 85.
279. Upchurch GR, Jr., Welch GN, Fabian AJ, et al.
Homocyst(e)ine decreases bioavailable nitric oxide
by a mechanism involving glutathione peroxidase.
J Biol Chem 1997; 272: 17012–7.
280. Tikkanen I, Fyhrquist F. Nitric oxide in hyperten-
sion and renal diseases. Ann Med 1995; 27: 353–7.
281. Schnackenberg C, Patel AR, Kirchner KA, Granger
JP. Nitric oxide, the kidney and hypertension. Clin
Exp Pharmacol Physiol 1997; 24: 600 – 6.
282. Mattson DL, Lu S, Cowley AW Jr. Role of nitric oxide
in the control of the renal medullary circulation. Clin
Exp Pharmacol Physiol 1997; 24: 587–90.
283. Just A. Nitric oxide and renal autoregulation. Kidney
Blood Press Res 1997; 20: 201– 4.
284. Thorup C, Petersson AEG. Macula densa derived
nitric oxide in regulation of glomerular capillary
pressure. Kidney Int 1996; 49: 430 – 6.
285. Schnackenberg CG, Wilkins FC, Granger JP. Role of
nitric oxide in modulating the vasoconstrictor ac-
tions of angiotensin II in preglomerular and postglo-
merular vessels in dogs. Hypertension 1995; 26:
1024 –9.
286. Majid DSA, Navar LG. Nitric oxide in the mediation
of pressure natriuresis. Clin Exp Pharmacol Physiol
1997; 24: 595–9.
287. Ollerstam A, Pittner J, Persson AK, Thorup C.
Increased blood pressure in rats after long-term
inhibition of the neuronal isoform of nitric oxide
synthase. J Clin Invest 1997; 99: 2212– 8.
288. Mackenzie IMJ, Ekangaki A, Young JD, Garrod CS.
Effect of renal function on serum nitrogen oxide
concentrations. Clin Chem 1996; 42: 440 – 4.
289. Martensson L, Hegbrant J, Thysell H. Generation of
nitrate during dialysis as a measure of nitric oxide
synthesis. Artif Organs 1997; 21: 163–7.
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
 NITRITE AND NITRATE ANALYSES
290. Dubey RK, Boegehold MA, Gillespie DG, Rosselli M.
Increased nitric oxide activity in early renovascular
hypertension. Amer J Physiol-Regul Integr C 1996;
39: R118 –R24.
291. Goonasekera CD, Rees DD, Woolard P, Frend A,
Shah V, Dillon MJ. Nitric oxide synthase inhibitors
and hypertension in children and adolescents. J Hy-
pertens 1997; 15: 901–9.
292. Calo L, D’Angelo A, Cantaro S, et al. Increased
urinary NO22/NO32 and cyclic guanosine mono-
phosphate levels in patients with Bartter’s syn-
drome: relationship to vascular reactivity. Am J
Kidney Dis 1996; 27: 784 –9.
293. Darkow DJ, Lu L, White RE. Estrogen relaxation of
coronary artery smooth muscle is mediated by nitric
oxide and cGMP. Am J Physiol 1997; 272: H2765–73.
294. Galley HF, Thornton J, Howdle PD, Walker BE,
Webster NR. Combination oral antioxidant supple-
mentation reduces blood pressure. Clin Sci 1997; 92:
361–5.
295. Jay MT, Chirico S, Siow RC, et al. Modulation of
vascular tone by low density lipoproteins: effects on
L-arginine transport and nitric oxide synthesis. Exp
Physiol 1997; 82: 349 – 60.
296. Bruckdorfer KR, Naseem KM, Mohammadi B, et al.
The influence of oxidized lipoproteins, oxidation
products and antioxidants on the release of nitric
oxide from the endothelium and the response of
platelets to nitric oxide. Biofactors 1997; 6: 191–9.
297. Debelder A, Moncada S. Cardiomyopathy: a role for
nitric oxide? Int J Cardiol 1995; 50: 263– 8.
298. Winlaw DS, Smythe GA, Keogh AM, Schyvens CG,
Spratt PM, Macdonald PS. Increased nitric oxide
production in heart failure. Lancet 1994; 344: 373– 4.
299. Weiss G, Umlauft F, Grunewald K. Raised nitrate
concentrations in chronic heart disease. Lancet 1994;
344: 960 –1.
300. Seghaye MC, Duchateau J, Bruniaux J, et al. Endog-
enous nitric oxide production and atrial natriuretic
peptide biological activity in infants undergoing car-
diac operations. Crit Care Med 1997; 25: 1063–70.
301. Myles PS, Leong CK, Currey J. Endogenous nitric
oxide and low systemic vascular resistance after
cardiopulmonary bypass. J Cardiothorac Vasc
Anesth 1997; 11: 571– 4.
302. Ringqvist A, Leppert J, Myrdal U, Ahlner J,
Ringqvist I, Wennmalm A. Plasma nitric oxide me-
tabolite in women with primary Raynaud’s phenom-
enon and in healthy subjects. Clin Physiol 1997; 17:
269 –77.
303. Yokoyama M, Shijo H, Ota K, et al. Systemic hemo-
dynamics and serum nitrate levels in patients un-
dergoing endoscopic variceal ligation. Hepatology
1996; 24: 47–52.
304. Smarason AK, Allman KG, Young D, Redman CW.
Elevated levels of serum nitrate, a stable end prod-
uct of nitric oxide, in women with pre-eclampsia.
Br J Obstet Gynaecol 1997; 104: 538 – 43.
305. Nobunaga T, Tokugawa Y, Hashimoto K, et al.
Plasma nitric oxide levels in pregnant patients with
preeclampsia and essential hypertension. Gynecol
Obstet Invest 1996; 41: 189 –93.
306. Davidge ST, Stranko CP, Roberts JM. Urine but not
plasma nitric oxide metabolites are decreased in
women with preeclampsia. Am J Obstet Gynecol
1996; 174: 1008 –13.
307. Morris NH, Carroll S, Nicolaides KH, Steer PJ,
Warren JB. Exhaled nitric oxide concentration and
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
amniotic fluid nitrite concentration during preg-
nancy. Eur J Clin Invest 1995; 25: 138 – 41.
308. Castillo L, DeRojas-Walker T, Yu YM, et al. Whole
body arginine metabolism and nitric oxide synthesis
in newborns with persistent pulmonary hyperten-
sion. Pediatr Res 1995; 38: 17–24.
309. Dollberg S, Warner BW, Myatt L. Urinary nitrite
and nitrate concentrations in patients with idio-
pathic persistent pulmonary hypertension of the
newborn and effect of extracorporeal membrane ox-
ygenation. Pediatr Res 1995; 37: 31– 4.
310. Miller MJ, Eloby-Childress S, Snapp B, Chotinarue-
mol S, Steen VL, Clark DA. Urinary nitrite excretion
in premature infants: effects of transfusion or indo-
methacin. Acta Paediatr 1993; 82: 291–5.
311. Moncada S. Nitric oxide in the vasculature: physiol-
ogy and pathophysiology. Ann N Y Acad Sci 1997;
811: 60 –7.
312. Wakabayashi Y, Yamada E, Yoshida T, Takahashi
H. Deficiency of endogenous arginine synthesis pro-
vokes hypertension by exhausting substrate arginine
for nitric oxide synthesis. Biochem Biophys Res Com-
mun 1994; 205: 1391– 8.
313. Fakler CR, Kaftan HA, Nelin LD. Two cases suggest-
ing a role for the L-arginine nitric oxide pathway in
neonatal blood pressure regulation. Acta Paediatr
1995; 84: 460 –2.
314. Kanno K, Hirata Y, Emori T, et al. L-arginine infu-
sion induces hypotension and diuresis/natriuresis
with concomitant increased urinary excretion of ni-
trite/nitrate and cyclic GMP in humans. Clin Exp
Pharmacol Physiol 1992; 19: 619 –25.
315. Malczewska-Malec M, Goldsztajn P, Kawecka-Jas-
zcz K, et al. Effects of prolonged L-arginine admin-
istration on blood pressure in patients with essential
hypertension (EH). Agents Actions-Suppl 1995; 45:
157– 62.
316. Mimran A, Ribstein J, Ducailar G. Contrasting effect
of antihypertensive treatment on the renal response
to l-arginine. Hypertension 1995; 26: 937– 41.
317. Mccaffrey MJ, Bose CL, Reiter PD, Stiles AD. Effect
of L-arginine infusion on infants with persistent
pulmonary hypertension of the newborn. Biol Neo-
nate 1995; 67: 240 –3.
318. Bode-Boger SM, Boger RH, Alfke H, et al. L-arginine
induces nitric oxide-dependent vasodilation in pa-
tients with critical limb ischemia: a randomized,
controlled study. Circulation 1996; 93: 85–90.
319. Sato H, Zhao ZQ, McGee DS, Williams MW, Ham-
mon JW, Vintenjohansen J. Supplemental L-argi-
nine during cardioplegic arrest and reperfusion
avoids regional postischemic injury. J Thorac Car-
diovasc Surg 1995; 110: 302–14.
320. Bode-Boger SM, Boger RH, Creutzig A, et al. L-
Arginine infusion decreases peripheral arterial resis-
tance and inhibits platelet aggregation in healthy
subjects. Clin Sci 1994; 87: 303–10.
321. Ashab I, Peer G, Blum M, et al. Oral administration
of L-arginine and captopril in rats prevents chronic
renal failure by nitric oxide production. Kidney Int
1995; 47: 1515–21.
322. Matsuoka H, Nakata M, Kohno K, et al. Chronic
L-arginine administration attenuates cardiac hyper-
trophy in spontaneously hypertensive rats. Hyper-
tension 1996; 27: 14 – 8.
323. Kharitonov SA, Lubec G, Lubec B, Hjelm M, Barnes
PJ. L-arginine increases exhaled nitric oxide in nor-
mal human subjects. Clin Sci 1995; 88: 135–9.
217
 ELLIS
324. Beaumier L, Castillo L, Ajami AM, Young VR. Urea
cycle intermediate kinetics and nitrate excretion at
normal and “therapeutic” intakes of arginine in hu-
mans. Am J Physiol 1995; 269: E884 –96.
325. Adams MR, McCredie R, Jessup W, Robinson J,
Sullivan D, Celermajer DS. Oral L-arginine im-
proves endothelium-dependent dilatation and re-
duces monocyte adhesion to endothelial cells in
young men with coronary artery disease. Atheroscle-
rosis 1997; 129: 261–9.
326. Chowienczyk P, Ritter J. Arginine: NO more than a
simple aminoacid? Lancet 1997; 350: 901–2.
327. Wascher TC, Posch K, Wallner S, Hermetter A,
Kostner GM, Graier WF. Vascular effects of L-argi-
nine: anything beyond a substrate for the NO-syn-
thase? Biochem Biophys Res Commun 1997; 234:
35– 8.
328. Cristol JP, Thiemermann C, Guerin MC, Torreilles
J, Depaulet AC. L-arginine infusion after ischaemia-
reperfusion of rat kidney enhances lipid peroxida-
tion. J Lipid Mediat Cell Signal 1996; 13: 9 –17.
329. Peck MD, Babcock GF, Alexander JW, Billiar T,
Ochoa J. High doses of dietary arginine during
repletion impair weight gain and increase infectious
mortality in protein-malnourished mice. Br J Nutr
1995; 74: 787–95.
330. Narita I, Border WA, Ketteler M, Ruoslahti E, Noble
NA. L-arginine may mediate the therapeutic effects
of low protein diets. Proc Natl Acad Sci USA 1995;
92: 4552– 6.
331. Suzuki H, Menegazzi M, Deprati AC, Mariotto S,
Armato U. Nitric oxide in the liver: physiopatholog-
ical roles. Adv Neuroimmunol 1995; 5: 379 – 410.
332. Cremona G, Higenbottam TW, Mayoral V, et al.
Elevated exhaled nitric oxide in patients with hepa-
topulmonary syndrome. Eur Respir J 1995; 8:
1883–5.
333. Devlin J, Palmer RMJ, Gonde CE, et al. Nitric oxide
generation a predictive parameter of acute allograft
rejection. Transplantation 1994; 58: 592–5.
334. Benjamin N, Vallance P. Plasma nitrite as a marker
of nitric oxide production. Lancet 1994; 344: 960.
335. Fabrega E, Casafont F, de la Pena J, et al. Nitric
oxide production in hepatic cell rejection of liver
transplant patients. Transplant Proc 1997; 29:
505– 6.
336. Ioannidis I, Hellinger A, Dehmlow C, et al. Evidence
for increased nitric oxide production after liver
transplantation in humans. Transplantation 1995;
59: 1293–7.
337. Decker KF, Obolenskaya MY. Cytokines, nitric oxide
synthesis and liver regeneration. J Gastroenterol
Hepatol 1995; 10(Suppl 1): S12–S7.
338. Winlaw DS, Schyvens CG, Smythe GA, et al. Urinary
nitrate excretion is a noninvasive indicator of acute
cardiac allograft rejection and nitric oxide produc-
tion in the rat. Transplantation 1994; 58: 1031– 6.
339. Mugge A, Kurucay S, Boger RH, et al. Urinary
nitrate excretion is increased in cardiac transplanted
patients with acute graft rejection. Clin Transpl
1996; 10: 298 –305.
340. Benvenuti C, Bories PN, Loisance D. Increased se-
rum nitrate concentration in cardiac transplant pa-
tients. A marker for acute allograft cellular rejection.
Transplantation 1996; 61: 745–9.
341. Langrehr JM, Muller AR, Lee TK, Schhraut WH,
Simmons RL, Hoffman RA. Serum NO22/NO32 from
oxidative L-arginine metabolism: a possible marker
218
ET AL.
for small bowel allograft rejection. Transplant Proc
1992; 24: 1148.
342. Hashmi P, Ho C, Morgan S, Stephenson JR. Routine
urinanalysis in renal transplant patients. J Clin
Pathol 1995; 48: 383– 4.
343. Loscalzo J. Nitric oxide binding and the adverse
effects of cell-free hemoglobins: what makes us dif-
ferent from earthworms. J Lab Clin Med 1997; 129:
580 –3.
344. Marcus AJ, Broekman MJ. Cell-free hemoglobin as
an oxygen carrier removes nitric oxide, resulting in
defective thromboregulation. Circulation 1996; 93:
208 –9.
345. Arias-Diaz J, Vara E, Torres-Melero J, et al. Nitrite/
nitrate and cytokine levels in bronchoalveolar lavage
fluid of lung cancer patients. Cancer 1994; 74: 1546 –
51.
346. Grasemann H, Ioannidis I, de Groot H, Ratjen F.
Metabolites of nitric oxide in the lower respiratory
tract of children. Eur J Pediatr 1997; 156: 575– 8.
347. Kanazawa H, Shoji S, Yamada M, et al. Increased
levels of nitric oxide derivatives in induced sputum
in patients with asthma. J Allergy Clin Immunol
1997; 99: 624 –9.
348. Persson MG, Zetterstrom O, Agrenius V, Ihre E,
Gustafsson LE. Single-breath nitric oxide measure-
ments in asthmatic patients and smokers. Lancet
1994; 343: 146 –7.
349. Barnes PJ. Nitric oxide and airway disease. Ann Med
1995; 27: 389 –93.
350. Powell JT, Higman DJ. Smoking, nitric oxide and the
endothelium. Br J Surg 1994; 81: 785–7.
351. Node K, Kitakaze M, Yoshikawa H, Kosaka H, Hori
M. Reversible reduction in plasma concentration of
nitric oxide induced by cigarette smoking in young
adults. Am J Cardiol 1997; 79: 1538 – 41.
352. Robbins RA, Millatmal T, Lassi K, Rennard S,
Daughton D. Smoking cessation is associated with
an increase in exhaled nitric oxide. Chest 1997; 112:
313– 8.
353. Rand MJ, Li CG. Nitric oxide as a neurotransmitter
in peripheral nerves: nature of transmitter and
mechanism of transmission. Annu Rev Physiol 1995;
57: 659 – 82.
354. Garthwaite J, Boulton CL. Nitric oxide signaling in
the central nervous system. Annu Rev Physiol 1995;
57: 683–706.
355. Dawson VL. Nitric oxide: role in neurotoxicity. Clin
Exp Pharmacol Physiol 1995; 22: 305– 8.
356. Paakkari I, Lindsberg P. Nitric oxide in the central
nervous system. Ann Med 1995; 27: 369 –77.
357. Iadecola C. Bright and dark sides of nitric oxide in
ischemic brain injury. Trends Neurosci 1997; 20:
132–9.
358. Coles H. Neurobiology—nitric oxide and bad behav-
iour. Nature 1995; 378: 336.
359. Nelson RJ, Demas GE, Huang PL, et al. Behavioural
abnormalities in male mice lacking neuronal nitric
oxide synthase. Nature 1995; 378: 383– 6.
360. Tsukahara H, Hara Y, Tsuchida S, et al. Nitrite
concentration in cerebrospinal fluid of infants: evi-
dence for enhanced nitric oxide production in He-
mophilus influenzae meningitis. Acta Paediat Jpn
1996; 38: 420 –2.
361. Pfister HW, Bernatowicz A, Kodel U, Wick M. Nitric
oxide production in bacterial meningitis. J Neurol
Neurosurg Psychiatry 1995; 58: 384 –5.
362. Halawa A, Baig SM, Qureshi GA. Amino acids and
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
 NITRITE AND NITRATE ANALYSES
nitrite in ischemic brain stroke. Biogenic Amines
1996; 12: 27–36.
363. Clark RS, Kochanek PM, Obrist WD, et al. Cerebro-
spinal fluid and plasma nitrite and nitrate concen-
trations after head injury in humans. Crit Care Med
1996; 24: 1243–51.
364. Molina JA, Jimenez-Jimenez FJ, Navarro JA, et al.
Cerebrospinal fluid nitrate levels in patients with
Parkinson’s disease. Acta Neurol Scand 1996; 93:
123– 6.
365. Ikeda M, Sato I, Matsunaga T, Takahashi M, Yuasa
T, Murota S. Cyclic guanosine monophosphate
(cGMP), nitrite and nitrate in the cerebrospinal fluid
in meningitis, multiple sclerosis and Guillain-Barre
syndrome. Int Med 1995; 34: 734 –7.
366. Ikeda M, Sato I, Yuasa T, Miyatake T, Murota S.
Nitrite, nitrate and cGMP in the cerebrospinal fluid
in degenerative neurologic diseases. J Neural
Transm Gen Sect 1995; 100: 263–17.
367. Milstien S, Sakai N, Brew BJ, et al. Cerebrospinal
fluid nitrite/nitrate levels in neurologic diseases.
J Neurochem 1994; 63: 1178 – 80.
368. Kuiper MA, Visser JJ, Bergmans PL, Scheltens P,
Wolters EC. Decreased cerebrospinal fluid nitrate
levels in Parkinson’s disease, Alzheimer’s disease
and multiple system atrophy patients. J Neurol Sci
1994; 121: 46 –9.
369. Navarro JA, Molina JA, Jimenez-Jimenez FJ, et al.
Cerebrospinal fluid nitrate levels in patients with
Alzheimer’s disease. Acta Neurol Scand 1996; 94:
411– 4.
370. Qureshi GA, Baig S, Bednar I, Sodersten P, Forsberg
G, Siden A. Increased cerebrospinal fluid concentra-
tion of nitrite in Parkinson’s disease. Neuroreport
1995; 6: 1642– 4.
371. Molina JA, Jimenez-Jimenez FJ, Navarro JA, et al.
Plasma levels of nitrates in patients with Parkin-
son’s disease. J Neurol Sci 1994; 127: 87–9.
372. Asahara H, Yokoi I, Tamada T, et al. Increased
cerebrospinal fluid nitrite and nitrate levels in pa-
tients with lumbar spondylosis. Res Commun Mol
Pathol Pharmacol 1996; 91: 77– 83.
373. Giovannoni G, Heales SJR, Silver NC, et al. Raised
serum nitrate and nitrite levels in patients with
multiple sclerosis. J Neurol Sci 1997; 145: 77– 81.
374. Surtees R, Heales S. A rostrocaudal gradient of
nitrate plus nitrite concentrations in CSF. J Neurol
Neurosurg Psychiatry 1997; 62: 100 –1.
375. Harper R, Parkhouse N, Green C, Martin R. Nitric
oxide production in burns: plasma nitrate levels are
not increased in patients with minor thermal inju-
ries. J Trauma 1997; 43: 467–74.
376. Becquet F, Courtois Y, Goureau O. Nitric oxide in the
eye: multifaceted roles and diverse outcomes. Surv
Ophthalmol 1997; 42: 71– 82.
377. Nicotera P, Brune B, Bagetta G. Nitric oxide: inducer
or suppressor of apoptosis? Trends Pharmacol Sci
1997; 18: 189 –90.
378. Ambs S, Hussain SP, Harris CC. Interactive effects
of nitric oxide and the p53 tumor suppressor gene in
carcinogenesis and tumor progression. FASEB J
1997; 11: 443– 8.
379. Agbenyega T, Angus B, Bedu-Addo G, et al. Plasma
nitrogen oxides and blood lactate concentrations in
Ghanaian children with malaria. Trans R Soc Trop
Med Hyg 1997; 91: 298 –302.
380. Al Yaman FM, Mokela D, Genton B, Rockett KA,
Alpers MP, Clark IA. Association between serum
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
levels of reactive nitrogen intermediates and coma in
children with cerebral malaria in Papua New
Guinea. Trans R Soc Trop Med Hyg 1996; 90: 270 –3.
381. Collinosdoby P, Nickols GA, Osdoby P. Bone cell
function, regulation, and communication: a role for
nitric oxide. J Cell Biochem 1995; 57: 399 – 408.
382. Ralston SH. The Michael Mason Prize Essay 1997.
Nitric oxide and bone: what a gas. Br J Rheumatol
1997; 36: 831– 8.
383. Sobrevia L, Mann GE. Dysfunction of the endothelial
nitric oxide signalling pathway in diabetes and hy-
perglycaemia. Exp Physiol 1997; 82: 423–52.
384. McDaniel ML, Kwon G, Hill JR, Marshall CA, Cor-
bett JA. Cytokines and nitric oxide in islet inflam-
mation and diabetes. Proc Soc Exp Biol Med 1996;
211: 24 –32.
385. Murphy C, Newsholme P. The enemy within. The
role played by immunostimulated macrophages in
the pathogenesis of insulin dependent diabetes mel-
litus. Biochem Soc Trans 1997; 25: 368S.
386. Suarez-Pinzon WL, Szabo C, Rabinovitch A. Devel-
opment of autoimmune diabetes in NOD mice is
associated with the formation of peroxynitrite in
pancreatic islet beta-cells. Diabetes 1997; 46: 907–
11.
387. Cosentino F, Hishikawa K, Katusic ZS, Luscher TF.
High glucose increases nitric oxide synthase expres-
sion and superoxide anion generation in human
aortic endothelial cells. Circulation 1997; 96: 25– 8.
388. Moldovan S, Livingston E, Zhang RS, Kleinman R,
Guth P, Brunicardi FC. Glucose-induced islet hyper-
emia is mediated by nitric oxide. Am J Surg 1996;
171: 16 –20.
389. Lekakis J, Papamichael C, Anastasiou H, et al.
Endothelial dysfunction of conduit arteries in insu-
lin-dependent diabetes mellitus without microalbu-
minuria. Cardiovasc Res 1997; 34: 164 – 8.
390. Soulis T, Cooper ME, Sastra S, et al. Relative contri-
butions of advanced glycation and nitric oxide syn-
thase inhibition to aminoguanidine mediated reno-
protection in diabetic rats. Diabetologia 1997; 40:
1141–51.
391. Wessels J, Peake P, Pussell BA, Macdonald GJ.
Nitric oxide synthase inhibition in a spontaneously
hypertensive rat model of diabetic nephropathy. Clin
Exp Pharmacol Physiol 1997; 24: 451–3.
392. Vlassara H. Recent progress in advanced glycation
end products and diabetic complications. Diabetes
1997; 46: S19 –25.
393. Hill MJ, Elliott P, Joossens JV, et al. Twenty-four
hour urinary nitrate excretion in 48 populations
from 30 countries: an ECP-INTERSALT collabora-
tive study. Int J Epidemiol 1996; 25: 505–12.
394. Parslow RC, McKinney PA, Law GR, Staines A,
Williams R, Bodansky HJ. Incidence of childhood
diabetes mellitus in Yorkshire, Northern England, is
associated with nitrate in drinking water: an ecolog-
ical analysis. Diabetologia 1997; 40: 550 – 6.
395. Virtanen SM, Jaakkola L, Rasanen L, et al. Nitrate
and nitrite intake and the risk for Type I Diabetes in
Finnish children. Childhood Diabetes in Finland
Study Group. Diabet Med 1994; 11: 656 – 62.
396. Macilwain C. US report raises fears over nitrate
levels in water. Nature 1995; 377: 4.
397. Morales Suarez-Varela M, Llopis Gonzalez A, Tejer-
izo Perez ML, Ferrandiz Ferragud J. Concentration
of nitrates in drinking water and its relationship
219
 ELLIS
with bladder cancer. J Environ Pathol Toxicol Oncol
1993; 12: 229 –36.
398. Ward MH, Mark SD, Cantor KP, Wesenberger DD,
Correa-Villasenor A, Zahm SH. Drinking water ni-
trate and the risk of non-Hodgkin’s lymphoma. Epi-
demiology 1996; 7: 465–71.
399. Weisenberger DD. Potential health consequences of
ground-water contamination by nitrates in Ne-
braska. Neb Med J 1993; 78: 7–12.
400. Morales-Suarez-Varela MM, Llopis-Gonzales A,
Tejerizo-Perez ML. Impact of nitrates in drinking
water on cancer mortality in Valencia, Spain. Eur J
Epidemiol 1995; 11: 15–21.
401. Anonymous. Spontaneous abortions possibly related
to ingestion of nitrate-contaminated well water—
LaGrange County, Indiana, 1991–1994. MMWR
1996; 45: 569 –72.
402. Tsezou A, Kitsiou-Tzeli S, Galla A, et al. High nitrate
content in drinking water: cytogenetic effects in
exposed children. Ach Environ Health 1996; 51:
458 – 61.
403. van Maanen JM, van Dijk A, Mulder K, et al.
Consumption of drinking water with high nitrate
levels causes hypertrophy of the thyroid. Toxicol Lett
1994; 72: 365–74.
404. Moller H. Occurence of carcinogens in the external
environment: epidemiological investigations. Phar-
macol Toxicol 1993; 72 (Suppl 1): 39 – 45.
405. McGeehin MA, Reif JS, Betcher JC, Mangione EJ.
Case-control study of bladder cancer and water dis-
infection methods in Colorado. Am J Epidemiol
1993; 138: 492–501.
406. Steindorf K, Schlehofer B, Becher H, Hornig G,
Wahrendorf J. Nitrate in drinking water. A case-
control study on primary brain tumours with an
embedded drinking water survey in Germany. Int J
Epidemiol 1994; 23: 451–7.
407. Preston-Martin S, Pogoda JM, Mueller BA, Holly
EA, Lijinsky W, Davis RL. Maternal consumption of
cured meats and vitamins in relation to pediatric
brain tumors. Cancer Epidemiol Biomarkers Prev
1996; 5: 599 – 605.
408. van Loon AJM, Botterweck AAM, Goldbohm RA,
Brants HAM, van den Brandt PA. Nitrate intake and
gastric cancer risk: results from the Netherlands
cohort study. Cancer Lett 1997; 114: 259 – 61.
409. van Maanen JM, Welle IJ, Hageman G, Dallinga JW,
220
ET AL.
Mertens PL, Kleinjans JC. Nitrate contamination of
drinking water: relationship with HPRT variant fre-
quency in lymphocyte DNA and urinary excretion of
N-nitrosamines. Environ Health Perspect 1996; 104:
522– 8.
410. Aruoma OI, Whiteman M, England TG, Halliwell B.
Antioxidant action of ergothioneine: assessment of
its ability to scavenge peroxynitrite. Biochem Bio-
phys Res Commun 1997; 231: 389 –91.
411. Hoglen NC, Waller SC, Sipes IG, Liebler DC. Reac-
tions of peroxynitrite with gamma-tocopherol. Chem
Res Toxicol 1997; 10: 401–7.
412. Fiala ES, Sodum RS, Bhattacharya M, Li H. (-)-
Epigallocatechin gallate, a polyphenolic tea antioxi-
dant, inhibits peroxynitrite-mediated formation of
8-oxodeoxyguanosine and 3-nitrotyrosine. Experien-
tia 1996; 52: 922– 6.
413. Christen S, Woodall AA, Shigenaga MK, Southwell-
Keely PT, Duncan MW, Ames BN. Gamma-toco-
pherol traps mutagenic electrophiles such as NO(X)
and complements alpha-tocopherol: physiological im-
plications. Proc Natl Acad Sci USA 1997; 94: 3217–
22.
414. Hegesh E, Shiloah J. Blood nitrates and infantile
methemoglobinemia. Clin Chim Acta 1982; 125:
107–15.
415. Bruning-Fann CS, Kaneene JB. The effects of ni-
trate, nitrite and N-nitroso compounds on human
health: a review. Vet Hum Toxicol 1993; 35: 521–38.
416. Lutynski R, Steczek-Wojdyla M, Wojdyla Z, Kroch S.
The concentrations of nitrates and nitrites in food
products and environment and the occurrence of
acute toxic methemoglobinemias. Przegl Lek 1996;
53: 351–5.
417. Fan AM, Steinberg VE. Health implications of ni-
trate and nitrite in drinking water: an update on
methemoglobinemia occurrence and reproductive
and developmental toxicity. Regul Toxicol Pharma-
col 1996; 23: 35– 43.
418. Lonnqvist PA, Jonsson B, Winberg P, Frostell CG.
Inhaled nitric oxide in infants with developing or
established chronic lung disease. Acta Paediat 1995;
84: 1188 –92.
419. Kennedy N, Smith CP, McWhinney P. Faulty sau-
sage production causing methaemoglobinaemia.
Arch Dis Child 1997; 76: 367– 8.
CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998