Diagnostic Biochemistry

Practicals
Lab. Practical 1 Blood Glucose Estimation

Lab. Practical 2 Oral Glucose Tolerance
Lab. Practical 3 Glycated Hemoglobin Estimation
Lab. Practical 4 easurment o! Triglycerides
Lab. Practical " easurement o! Total #$olesterol % H&L and L&L
Lab. Practical ' (enal )unction Tests* B+, and #reatinine estimation
Lab. Practical - #reatinine #learance Estimation
Lab. Practical . Li/er )unction Tests 1* Bilirubin 0 Total and &irect
Lab. Practical 1 Li/er )unction Tests 2* En2ymes0 3LT% 34T and GGT
Lab. Practical 15 3lbumin and Total Protein Estimation
Lab. Practical 11 Bone 6ro!ile Testes
Lab. Practical 12 #ardiac 6ro!ile testes
Lab. Practical 13 1" Tutorials
Introduction To Applied Biochemistry
General Comments about testing
T$ere are so many di!!erent met$ods used to analy2e di!!erent c$emical com6ounds t$at to state one
met$od o/er anot$er is un!air. 3not$er issue is t$at your body c$emistry c$anges t$roug$out t$e day in
res6onse to e7ternal conditions suc$ as e7ercise and internal conditions suc$ as 8idney !unction. T$is
ma8es com6arisons among /arious tests di!!icult to do. One met$od to lessen t$ese /ariables is to try to
$a/e your tests done by t$e same laboratory so t$at com6arisons o! test /alues are 6ossible. 9t is also
bene!icial t$en to $a/e your tests dra:n under t$e same conditions ;!asting<non !asting% early
morning<late a!ternoon% etc.= so t$at you can eliminate t$ese inter!erences :$en you loo8 at your
results.
Practices of Clinical Biochemistry Part II:
Estimation of Blood Glucose
Introduction:
T$e im6ortance o! testing t$e blood glucose le/el comes !rom t$e !act t$at t$e brain cells are
/ery de6endent on t$e e7tracellular glucose concentration !or t$eir energy su66ly0 $y6oglycemia is li8ely
to im6air cerebral !unctions as :ell as do t$e $y6erglycemia es6ecially o! ra6id onset% :$ic$ can cause
cerebral dys!unction by a!!ecting e7tracellular osmolarity.
Obecti!es:
>To 8no: t$e di!!erent met$ods !or estimation o! blood glucose
>To 8no: t$e 6recautions needed to get accurate results and better inter6retation o!
glycemic status in relation to disease condition.
"ethods:
any met$ods :ere de/elo6ed to estimate t$e glucose le/el in body !luids among :$ic$ t$e
commonly used no:adays% t$e en2ymatic met$ods. T$ese met$ods can be summari2ed and
categori2ed into
3= (eduction met$ods : T$ese met$ods de6end on t$e reducti/e 6ro6erty o! glucose;aldose=
1>Ferriccyanide( Hoffmans) method: using !erricyanide :$ic$ is reduced by t$e
glucose .
)e
???
)e
??
;color c$ange !rom yello: to colorless solution t$at :ill
diminis$ t$e absorbance measured 6$otometerically =

2#Copper sulfate methods :
Benedict* T$e reagent contains ,a>citrate @,a carbonate :it$ #u4O
4
. 9t gi/es
color acc. To conc. o! glucose ;green>>>>>yello:>>>>>bro:n>>>>>red=.
)e$ling * using AOH @,a<A tartrate :it$ #u4O
4
)olin> Bu * 3l8aline #u 4O
4
?P$os6$omolybdic acid molybdenum blue
by reducing #u
2
O #uO
2
3>Smogi-Nelson method : using 3rsenomolybdate
,.B. T$e reduction met$ods need al8aline medium @$eat
T$ese met$ods are Cualitati/e @ semi>Cuantitati/e.
B= 3romatic amines met$od :
O>toludine ?glucose ;ald$yde=
$eat @acidity
glucosamine ;colored =
#= En2ymatic met$ods*
1-Hexokinase methods(The reference method=.
Bit$ 6re>de6roteini2ation o! sam6le or :it$out.
Glucose ?3TP ?HA3&P?G'P
G'P ?,3& ?G'P& ' P>gluconolactone ?$A%&'&
;measured at 345=
2- Glucose oxidase methods*
>TrinderDs ;En2.>&ye #olorimetric = met$od*
:$ic$ is colorimetric eit$er by s6ectro6$otometer or re!ractrometer
;re!ractrometeric met$ods eit$er in a !ilm !orm E8oda8 Ectac$emF or a stri6 !orm
E&ry c$emistryF =.
>Ainetic met$od*
by measuring t$e increase in absorbance t$roug$ increase in ,3&H?H
>Polarigra6$ic met$od*
using O2 electrode to detect O2 utili2ation.
,.B. GO&<PO& met$od can not used !or detection o! urine glucose because t$e urine
contains inter!ering substances !or 6ero7idase ;PO&= .
- To use t$is met$od treatment o! t$e urine sam6le eit$er by 4omogi ,elson !iltrate or 9on
E7c$ange (esin is ta8en be!ore running . 3lso% using GO&<PO& met$od in urine :it$
modi!ication li8e % Polarigra6$ic determination :it$ 6ost>reaction elimination o! H
2
O
2
by*
et$anol @ catalase or 9odide @ molybdate.
3- Glucose Dehydrogenase Method:
Glucose ?,3&
G&H
Gluconolactone ?,3&H?H ;measured at 345=
Glucose O(idase for blood glucose estimation )E(periment *+,
P-I$CIP.E O/ T&E "ET&O%
Glucose o7idase ;GO&= catalyses t$e o7idation o! glucose to gluconic acid. T$e !ormed
$ydrogen 6ero7ide ;H2O2=% is detected by a c$romogenic o7ygen acce6tor% 6$enol>
amino6$ena2one in t$e 6resence o! 6ero7idase ;PO&=*
Principle: ;TrinderDs met$od =
>&>glucose
utarotase
>&>glucose
>&>glucose ?H
2
O?O
2
Glucose o7idase
&>gluconic acid?H
2
O
2
H
2
O
2
? 4>amino6$ena2one?6$enol
Pero7idase
Guinonemine ?4 H
2
O
T$e intensity o! t$e color !ormed is 6ro6ortional to t$e glucose concentration in t$e sam6le.
C.I$ICA. 0IG$I/ICA$CE
Glucose is a maHor source o! energy !or most cells o! t$e body0 insulin !acilitates glucose entry into t$e
cells. &iabetes is a disease mani!ested by $y6erglycemia0 6atients :it$ diabetes demonstrate an
inability to 6roduce insulin. #linical diagnosis s$ould not be made on a single test result0 it s$ould
integrate clinical and ot$er laboratory data.
P(EP3(3T9O,
Bor8ing reagent ;B(=*
&issol/e t$e contents o! one /ial ( 2 En2ymes in one bottle o! ( 1 Bu!!er.
#a6 and mi7 gently to dissol/e contents.
T$e reagent is stable 1 mont$ a!ter reconstitution in t$e re!rigerator ;2>.I#= or - days at room
tem6erature ;1">2"I#=.
0igns of reagent deterioration:
1> Presence o! 6articles and turbidity.
2> Blan8 absorbance ;3= at "5" nm 5.15.
(eCuirements:
J4am6les*
>Blood sam6les
B$ole blood
4erum
Plasma ;:it$ #a.o7alates<,a)=% :$ic$ is t$e 6re!erred sam6le
>)res$ urine by double /oid collection tec$niCueKK.L
>#4) collected in sterile clean container and to be done immediately or centri!uged to get cell
!ree !luid.
9nstrumentation*
>P$otometer adHusted on :a/elengt$ "45 nm
>#u/ette ;lig$t 6at$= 1 cm
>Bater bat$ at 3- I#
>3utomatic 6i6ettes% dis6osable test tubes % rac8s and dis6osable ti6s !or t$e
dis6ensers.
P-OCE%1-E
1
1. 3ssay conditions*
Ba/elengt$* . . . . . . . . . . . . . .. . "5" nm ;415>""5=
#u/ette* . . . . . . . . . . . . . . . . . . . . .. 1 cm lig$t 6at$
Tem6erature. . . . . . . . . . . . . . . . . . . 3-I# < 1">2"I#
12. 3dHust t$e instrument to 2ero :it$ distilled :ater.
23. Pi6ette into a cu/ette*
Blan8
B( ;mL= 1.0
4tandard ;L= --
4am6le ;L= --
14. i7 and incubate !or 15 min at 3-I# or 1">25 min at room tem6erature ;1">2"I#=.
1". (ead t$e absorbance ;3= o! t$e sam6les and standard% against t$e Blan8.
T$e colour is stable !or at least 35 minutes.
CA.C1.ATIO$0
;3= 4am6le 7 155 ;4tandard conc.= M mg<dL glucose in t$e sam6le
;3= 4tandard
Con!ersion factor: mg<dL 7 5.5"""M mmol<L.
JLinearity o! t$e test M 455 mg<dl ;4am6les gi/e $ig$er le/el must be retested :it$ dilution
by suitable bu!!er or dist. H
2
O=
-esult* 3bs. O! t$e 4tandard N 5.3
3s t$e concentration o! glucose standard M 155 mg<dl
T$e Glucose concentration in t$e sam6le M 333 O 3bs. O! t$e 4am6le
$ormal -ange:
Blood glucoseK )astingM -5 > 115 mg<dl @ 2 $rs. Post6randial M 115 > 145 mg<dl
+rine glucose .. P detectable limit ;,il=
#4) glucose N '5 > 15 mg<dl
,.B. To e76ress t$e result in mmol<L di/ide by 1. ; B o! Glucose M1.5=
Interpretation:
I #&ypoglycemia * T$e 6atient considered critically $y6oglycemic i!*
B$ole Blood glucose le/el P 45mg<dl
4erum<Plasma glucose le/el P 4"mg<dl
3> Bell )ed 4tate Hy6oglycemia*
1> E7cessi/e 9nsulin (elease*
a. (eacti/e Hy6oglycemia
b. 3limentary Hy6erinsulinism
c. Leucine Hy6ersensiti/ity
2> 9n$erited En2yme &e!ect*
a. Galactose >1> P$os6$ate
b. )ructose >1> P$os6$ate
3> )ed 4tatus )unctional Hy6oglycemia*
B> )asting Hy6oglycemia*
1>Organic Hy6oglycemia*
a>Pancreatic B>#ell disease<#3
b>,on>Pancreatic Tumors
c>3nterior Pituitary Hy6o>!unction
d>3drenocortical Hy6o>!unction
e>9ngestion o! 38ee )ruit
2> )unctional )asting Hy6oglycemia
a> On s6eci!ic $e6atic en2yme de!iciency*

1> Genetic &e!iciency or &elayed aturation o! En2ymes in Pre>
mature Babies
2> Glycogen 4torage &isease
b> 9nduced by E7ogenous 3gents*
1>3lco$ol 9nta8e
2>E7cessi/e 9nsulin 3dministration
3>E7cessi/e 4ul!onylurea 3dministration
II # &yperglycemia :
- &iabetes ellitus
- Hemoc$romatosis
- Hy6o8alemia
- 4tress
- P$eoc$romocytoma
- 3nest$esia
- Pregnancy
- Hy6ert$yroidism
- #us$ing disease
- Hy6er6ituitarism ;gigantism=
%iscussion:
JPhysiological 2 Biochemical Bac3ground*
Glucose metabolism% 9nsulin action and ot$er $ormonal e!!ects on glucose in
t$e $uman body.
4Pathological 2 %isease Correlation: &iabetes ellitus% #us$ing
disease %Hy6ert$yroidism K..etc
5uestions:
1> B$at is t$e basis o! reduction met$ods !or glucose estimation L
2> Gi/e s$ort notes on TrinderDs met$od !or glucose estimation.
3> B$en does a 6erson considered $y6oglycemicL
4> B$at are t$e ty6es o! $y6oglycemia L
"> Gi/e an account on t$e 6rinci6le o! glucose o7idase met$od !or glucose estimation.
O-A. G.1CO0E TO.E-A$CE TE0T
Introduction:
On standard oral glucose dose% t$e res6onse o! t$e body regarding t$e absor6tion and
metabolism o! glucose said to be tolerant on meeting t$e normal ele/ation and return. B$ereas
abnormal and im6ro6er glucose metabolism is termed glucose intolerance. T$is used to diagnose
diseases :$ere t$e glucose metabolism is im6aired as in &iabetes mellitus. Oral glucose tolerance test
;OGTT= $as been :idely used as t$e golden standard !or diagnosing diabetes mellitus in clinically
doubt!ul cases. Lately% t$oug$t% t$e use o! OGTT in 6rimary care $as been Cuestioned !or se/eral
reasons. 9t $as lo: re6roducibility and is /ery e76ensi/e. Ho:e/er% !or t$e detection o! diabetes in
6regnant :omen% it is still recommended.
Obecti!es:
9t is to 6ractice t$e OGTT and 8no:ing t$e uses and inter6retation regarding t$e diagnostic
bene!its o! t$is laboratory test.
Indications:
1> Borderline !asting blood sugar !or Q2 times ;N 115 12"mg<dl=
2> &iagnosis o! Gestational &iabetes ;G&= at 24 2. :ee8s o! gestation es6ecially !or t$ose
$a/e a !amily $istory o! diabetes.
3> 3!ter deli/ery !or t$ose :as su!!ering !rom G&.
OGTT )E(periment * 6,:
4Patient preparation )Per7uisites, 0
3cti/ity>>&onRt smo8e or e7ercise strenuously !or . $ours be!ore t$e test or during t$e
test.
&iet>>Eat a $ig$>carbo$ydrate diet ;Q 1"5 g<day= !or 3 days% t$en !ast !or 15 to 12 $ours
be!ore t$e test. &onRt drin8 co!!ee or alco$ol !or . $ours be!ore t$e test.
&rugs ;medicines=>9n!orm t$e 6erson 6er!orming t$e test to omit any medications listed%
as under ta8ing t$ese drugs t$e test results may di!!er ;contrace6ti/es to be sto66ed one
cycle be!ore t$e 6er!ormance o! OGTT=.
T$e test must be 6er!ormed at daytime ;morning=.
4 General description of test
Test usually ta8es 3 $ours but can last as long as ' $ours ;e7tended OGTT=.
&rin8 :ater !reCuently during t$e test ;t$e only allo:ed !luid to drin8=.
T$e !irst blood sam6le and t$e !irst urine sam6le are collected bet:een - 3.. and 1
3..% a!ter you $a/e !asted !or 12 $ours.
O6erator gi/es a test load o! glucose% usually -" 155 gram de7trose < 355 ml :ater%
lemon !la/ored . &rin8 t$e entire solution in " minutes.
Blood and urine sam6les are collected at 35 min.% '5 min.% 15 min.%125 min. and 3 $ours
and sometimes immediately a!ter drin8ing oral glucose solution.
%ose of Oral Glucose*
&e7trose* 1 1.-" g<8g. body :t. ;!or adults5 and not e7ceeds 155 g.
9t is to be dissol/ed in 2"5 355 ml lemon !la/ored :ater.
)ortical * 113 ml com6leted to 355 ml :ater
Luco2ade* 3"5 ml. ;ready to use=
J0amples:
Blood sam6les 0 !asting;basal= sam6le% 35min. a!ter oral glucose load% '5min% 15min%
125min. ;in e7tended OGTT anot$er 2 sam6les :ill be ta8en at 2S$our and 3 $ours=.
+rine sam6les 0 !irst !asting urine and t$e $ourly collected urine sam6les.
Calculation: t$ere are di!!erent met$ods to calculate and inter6ret t$e glucose le/els ;mg<dl=in OGTT*
Glucose sam6le Bil8erson #riteria )aHan>conn criteria (e/ised 4ummation
)asting Q 135 1 6oint >
J9! T o! results
;) ? '5min. ? 15 min. ? 125
min.= Q '55 mg<dl M
&iabetic
J9! T o! results P '55 M non
diabetic
35 min. > >
'5 min. Q115 S 6oint Q115 ?1
15 min. > Q 1'" ?1
125 min. Q145 S 6oint Q 145 ?1
2 S $our Q135 1 6oint >
#alculation o! (esults
2 3 6oint &iabetic
S > 1 S 6oint 4us6ect
Uero ,on diabetic
3 &iabetic
1 2 4us6ect
Uero ,on diabetic

-esults and %iagnosis: Glucose tolerance tests may lead to one of the follo8ing diagnoses:
$ormal -esponse
3 6erson is said to $a/e a normal res6onse :$en t$e 2>$our glucose le/el
is less t$an or eCual to 115 mg<dl% or !ollo:ing t$is normal le/els.
Time Pregnancy Other Adults Child
)asting P155 P115 P135
35% '5 @ 15 minutes P255 P255 P255
125 minutes P14" P145 P145
Impaired /asting Glucose
B$en a 6erson $as a !asting glucose eCual to or greater t$an 115 and less
t$an 12' mg<dl% t$ey are said to $a/e im6aired !asting glucose. T$is is
considered a ris8 !actor !or !uture diabetes% and :ill li8ely trigger anot$er
Impaired Glucose Tolerance
3 6erson is said to $a/e im6aired glucose tolerance :$en t$e 2>$our
glucose results !rom t$e oral glucose tolerance test are greater t$an or
eCual to 145 but less t$an 255 mg<dl. T$is is also considered a ris8 !actor
!or !uture diabetes. T$ere $as recently been discussion about lo:ering t$e
u66er /alue to 1.5 mg<dl to diagnose more mild diabetes to allo: earlier
inter/ention and $o6e!ully 6re/ention o! diabetic com6lications.
%iabetes
3 6erson $as diabetes :$en oral glucose tolerance tests s$o: t$at t$e
blood glucose le/el at 2 $ours is eCual to or more t$an 255 mg<dl. T$is
must be con!irmed by a second test ;any o! t$e t$ree= on anot$er day.
T$ere $as recently been discussion about lo:ering t$e u66er /alue to 1.5
mg<dl to diagnose more 6eo6le :it$ mild diabetes to allo: earlier
inter/ention and $o6e!ully 6re/ention o! diabetic com6lications.
Gestational %iabetes
3 :oman $as gestational diabetes :$en s$e is pregnant and $as any t:o
o! t$e !ollo:ing* a !asting 6lasma glucose o! more t$an 15" mg<dl% a 1>$our
glucose le/el o! more t$an 115 mg<dl% a 2>$our glucose le/el o! more t$an
1'" mg<dl% or a 3>$our glucose le/el o! more t$an 14" mg<dl.

%iscussion:
%rugs may affect OGTT results
3m6$etamines.
3rginine.
Ben2odia2e6ines.
Beta>adrenergic bloc8ers.
#$lort$alidone.
#lo!ibrate.
#orticosteroids.
&e7trot$yro7ine.
&ia2o7ide.
E6ine6$rine.
)urosemide.
Glucose 9.V.
9nsulin.
Lit$ium.
3O in$ibitors.
,icotinic acid ;large doses=.
Oral contrace6ti/es ;estrogen>6rogestogen combination=.
Oral $y6oglycemics.
P$enol6$t$alein.
P$enot$ia2ines.
P$enytoin.
T$ia2ide diuretics.
Triamterene.
Other factors that may affect test results
Et$anol.
#a!!eine.
(ecent in!ection.
)e/er.
Pregnancy.
3cute illness.
Peo6le o/er age "5 tend to:ard decreasing carbo$ydrate tolerance% :$ic$ may cause
con!licting results.
#us$ingRs disease% $emoc$romo>cytosis% 6$eoc$romocytoma% inHury to central ner/ous system%
tumor o! 6ancreas islet cells% malabsor6tion% 3ddisonRs disease% $y6ot$yroidism% $y6o6ituitarism.
(educed carbo$ydrate inta8e !or se/eral days be!ore t$e test.
)ailure to !ollo: dietary and e7ercise restrictions.
51E0TIO$0:
a. B$at are t$e indications o! OGTT L
b. B$at are t$e 6rereCuisites o! OGTT L
c. &ra: a gra6$ o! a normal glucose tolerance.
Glycated &emoglobins
Introduction:
Glyco$emoglobin ;GHb% glycated $emoglobin% glycosylated $emoglobin= is a generic term !or
$emoglobin bound irre/ersibly ;8etoamine !orm= to glucose. O!ten% t$e term is used to mean total
glycated $emoglobin% and sometimes to mean $emoglobin 31c.
Total glycated $emoglobin ;Total GHb= re!ers to all t$e glycated $emoglobins% including glycated
$emoglobin /ariants. Total glycated $emoglobin is usually determined by a!!inity c$romatogra6$y or
immunassays.
Hemoglobin 31c ;Hb31c= is t$e maHor sub!raction o! t$e glycated normal $emoglobin ;Hb31=.
&etermination o! Hb31c is usually ac$ie/ed by ion>e7c$ange HPL# or gel electro6$oresis.
Obecti!es:
9t is to 8no: t$e im6ortance o! glycated $emoglobin as a long term monitoring test :$ic$ may be used
to $el6 controlling t$e treatment o! diabetes mellitus.
Types of Glycated hemoglobins*
Hb31a
1
M Hb ? )ructose>1%'>bis6$os6$ate ;)BP=
Hb31a
2
M Hb ? Glucose>'>6$os6$ate
Hb31b M Hb ? Pyru/ic acid
Hb31c M Hb ? Glucose ;,>terminal o! W>c$ain=
Hb31d M Hb ? Glucose ; internal a.a. o! X<W> c$ain=
1sing G&b :
onitoring blood glucose is a 8ey com6onent o! success!ul diabetes management. Bit$ t$e a/ailability
o! sel!>monitoring and Hb3
1#
testing% laboratory testing !or !asting glucose and 2>$our 6ost>-"g glucose
load s$ould no longer be used routinely to assess glucose control. Laboratory measurement o! glucose%
$o:e/er% may be use!ul to /eri!y t$e accuracy o! $ome glucose monitoring eCui6ment or :$en t$ere $as
been a loss o! diabetic control.
Hb3
1#
measurement 6ro/ides a Cuantitati/e and reliable measure o! glycemic status and control o/er an
e7tended 6eriod o! time% t$ereby com6lementing day>to>day monitoring. Hb3
1#
le/els are a better ;and
less e76ensi/e= measure o! long>term glucose control t$an re6eated !asting and 6.c. glucose le/els.
O/er t$e li!e o! a red blood cell ;:$ic$ a/erages 125 days=% a !raction o! $emoglobin :ill become
co/alently bound to glucose and ot$er sugar molecules. T$is reaction occurs non>en2ymatically and at a
rate :$ic$ is 6ro6ortional to t$e concentration o! glucose in t$e blood. Hb3
1#
is t$e largest single
com6onent o! t$ese glycated $emoglobins.
,.B. Blood Glucose le/el re!lects t$e 6re/ious !e: $ours glycemic state% glycated 3lbumin re!lects 15
14 days glycemic state% :$ile Hb31c re!lects t$e longest ;2>3 mont$s= glycemic state.
"ethods:
T$ere are currently !our main tec$niCues !or determining glycated $emoglobins*
1. #ation>e7c$ange c$romatogra6$y > se6arates $emoglobins using HPL# based on net c$arge
as a result o! glycation0
2. Gel electro6$oresis0
3. 3!!inity c$romatogra6$y > se6arates total glycated $emoglobins by binding to solid>6$ase
di$ydro7yborate0
4. 9mmunoassay > based on binding to s6eci!ic antibodies.
E(periment * 9: Estimation of &bA+c by using affinity chromatography column
Principle:
9n a c$romatogra6$y column% t$e $emoglobins in a $emolysed sam6le is bound by di!!erent a!!inity to
di$ydro7yborate. Elution o! Hb31c is carried out by 6$os6$ate bu!!er% :$ile t$e ot$er $emoglobins
se6arate ;elute= a!ter by sodium c$loride solution.
Procedure:
Calculation: Y Hb31c M
A+ : +;;
<
A+ ' ) =>?@ : A6,

-eference ranges:
%egree of glucose control Total G&b &b A+c
,ormal ;non>diabetic= P -Y P 'Y
,ear normoglycemic - to .Y ' to -Y
&iabetes #ontrol and #om6liance Trial ;&##T= t$era6eutic goal Less t$an -Y
9n good control . to 1Y - to .Y
3ctions suggested 1 to 11Y . to 1Y
,ot in control Q 11 Y Q 1Y
T$e determination o! a glycated $emoglobin le/el may assist in t$e initial diagnosis o! diabetes% or it
may be used to indicate t$e degree o! long>term diabetic control in diabetic 6atients. T$e signi!icance o!
a lo: glycated $emoglobin le/el $as not been establis$ed.
3nnual Hb31c P 1.1 times t$e u66er limit o! normal ;...Y=% suggesting less li8ely occurring
com6lications.
3nnual Hb31c Q 1.- times t$e u66er limit o! normal ;13."Y=% suggesting more li8ely occurring
com6lications.
Correlation 8ith "ean Blood Glucose .e!els
3 single !asting blood glucose measurement only gi/es an indication o! t$e 6atientRs immediate 6ast
;last 1 to 2 $ours= condition% and may not re6resent t$e true status o! blood glucose regulation. 9n
contrast% t$e le/el o! glycated $emoglobin is directly related to t$e a/erage glucose concentration o/er
t$e li!e>s6an o! t$e $emoglobin in t$e circulation.
Various !ormulae $a/e been 6ro6osed to demonstrate t$e correlation bet:een t$e mean blood glucose
;BG= and Hemoglobin 31c ;Hb31c=.
BG mg<dl M ;33.3 O Hb31c= > .'
Or% BG mg<dl M 15 ; Hb31c ?4 =
%iscussion:
#auses o! ele/ated Hb31c*
- +ncontrolled &..
- Z Hb)
- Z Triglycerides
- Lead to7icity
- [ iron anemia
- 46lenectomy
- #() \ Hemodialysis
#auses o! decreased Hb31c*
- #auses o! [ (B#s li!e s6an ; $emolytic or $emorr$agic=
- Hemodilution ;e.g. 6regnancy=
5uestions:
- Gi/e t$e obHecti/es o! glycated $emoglobin estimation.
- Brite do:n di!!erent met$ods !or t$e determination o! glycated $emoglobin.
- B$at is t$e 6rinci6le !or t$e determination o! glycated $emoglobin by c$romatogra6$y L
.ipid Profile
Introduction:
4ome bene!icial as6ects o! li6ids include t$e !ollo:ing* energy course% !unction and structural
com6onents o! cell membranes% and 6recursor com6ound to many im6ortant substances suc$
as /itamin & and steroid ;se7= $ormones.
Bit$ e/idence o! a lin8 bet:een ele/ated li6ids and at$erosclerosis ;also 8no:n as
arteriosclerosis or at$erot$rombosis=% t$ere is increase interest !rom bot$ t$e medical and lay
community in t$e battery o! tests commonly ordered as a li6id 6ro!ile. Pre6aration !or $a/ing
blood collected !or li6id testing s$ould include a 12>14 $our o/ernig$t !ast.
Obecti!es:
> T$e contribution o! $y6erc$olesterolemia to coronary $eart disease ;#H&= ris8% including t$e
im6ortance o! ele/ations in total c$olesterol% L&L c$olesterol% H&L c$olesterol% ratio o! total to
H&L c$olesterol.
> T$e classi!ication o! dysli6idemias% including :$o to screen% and $o: o!ten
> T$e a/ailable diagnostic studies and t$eir use% 6articularly determinations o! H&L% L&L and
total c$olesterol% as :ell as t$e need to test !or ot$er cardio/ascular ris8 !actors .
E(periment * =:Glycerol#Phosphate O(idase method for Triglycerides
4am6le triglycerides incubated :it$ li6o6roteinli6ase ;LPL=% liberate glycerol and !ree !atty acids.
Glycerol is con/erted to glycerol>3>6$os6$ate ;G3P= and adenosine>">di6$os6$ate ;3&P= by glycerol
8inase and 3TP. Glycerol>3>6$os6$ate ;G3P= is t$en con/erted by glycerol 6$os6$ate de$ydrogenase
;GPO= to di$ydro7yacetone 6$os6$ate ;&3P= and $ydrogen 6ero7ide ;H2O2=. 9n t$e last reaction%
$ydrogen 6ero7ide ;H2O2= reacts :it$ 4>amino6$ena2one ;4>3P= and 6>c$loro6$enol in 6resence o!
6ero7idase ;PO&= to gi/e a red colored dye*
Principle:
Triglycerides ? H2O
li6ase

Glycerol ? ))3
Glycerol ? 3TP
glycerol 8inase
Glycerol>3>6$os6$ate ? 3&P
Glycerol>3>6$os6$ate ? O2
Glycerol>P$os6$ate O7idase
&H3P ? H2O2
H2O2 ? 4> 3minoanti6yrine ? #$loro6$enol
6ero7idase
Guinoneimine
T$e intensity o! t$e color !ormed is 6ro6ortional to t$e triglycerides concentration in t$e sam6le.

Triglycerides are !ats t$at 6ro/ide energy !or t$e cell. Li8e c$olesterol% t$ey are deli/ered to t$e bodyDs
cells by li6o6roteins in t$e blood. 3 diet :it$ a lot o! saturated !ats or carbo$ydrates :ill raise t$e
triglyceride le/els. T$e increases in serum triglycerides are relati/ely non>s6eci!ic. )or e7am6le li/er
dys!unction resulting !rom $e6atitis% e7tra $e6atic biliary obstruction or cirr$osis% diabetes mellitus is
associated :it$ t$e increase #linical diagnosis s$ould not be made on a single test result0 it s$ould
integrate clinical and ot$er laboratory data.
P-EPA-ATIO$
Bor8ing reagent ;B(=* &issol/e ;= t$e contents o! one /ial ( 2 En2ymes into one bottle o! ( 1 Bu!!er.
Bor8ing reagent ;B(=* &issol/e ;= t$e contents o! one /ial ( 2 En2ymes in 15 mL o! ( 1 Bu!!er.
B( stability* ' :ee8s at 2>.I# or 1 :ee8 at room tem6erature ;1">2"I#=.
0A"P.E0
4erum or $e6arini2ed or E&T3 6lasma1.
4tability o! t$e sam6le* " days at 2>.I# .
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . .. . . . . "5" nm ;415>""5=
#u/ette* . . . . . . . . . . . . . . . . . . . . . . . . 1 cm lig$t 6at$
Tem6erature . . . . . . . . . . . . . . . . . . . . 3-I# < 1">2"I#
Blan8 4tandard
B( ;mL= 1.0 1.0
4tandard ;L= -- 10
4am6le ;L= -- --
4. i7 and incubate !or " min. at 3-I# or 15 min. at room tem6erature.
". (ead t$e absorbance ;3= o! t$e sam6les and 4tandard% against t$e Blan8. T$e colour is stable !or at
least 35 minutes.
CA.C1.ATIO$0
A Sample x 200 (Standard conc.) = mg/dL triglycerides in the sample
A Standard
Conversion factor: mg/dL x 0.0113= mmol/L.
-eference ranges:
,ormal )asting blood triglycerides M '5 1'5 mg<dl
9t is considered normal as long as it is belo: 255 mg<dl
%iscussion:
>Ty6es o! $y6erli6idaemias
51E0TIO$0:
a. Gi/e t$e di!!erent met$ods !or t$e determination o! triglycerides.
b. Brite a s$ort note on $y6ertriglyceridemia.
c. Gi/e t$e u66er cut o!! /alue o! triglyceride !or a diagnosis o!
$y6ertriglyceridemia.
%ETE-"I$ATIO$ O/ C&O.E0TE-O.:
I$T-O%1CTIO$:
#$olesterol is a :a7y substances used in e/ery cell membrane you $a/e and as a base !or
se/eral $ormones. T$e recommended daily allo:ance !or dietary c$olesterol inta8e is 355 milligrams.
ost cells $a/e some ca6acity to synt$esi2e c$olesterol. T$e largest 6ercentage o! synt$esi2ed
c$olesterol is made in t$e li/er. #$olesterol lo:ering medications 6rescribed by 6$ysicians in$ibit t$e
synt$esis o! c$olesterol by t$e li/er% t$ereby reducing t$e le/el in t$e blood stream.
OBAECTIBE0:
T$e estimation o! c$olesterol along :it$ ot$er 6arameters o! li6id 6ro!ile is necessary !or t$e
classi!ication and diagnosis o! li6emias
T$e c$olesterol 6resent in t$e sam6le originates a coloured com6le7% according to t$e !ollo:ing
reaction* T$e intensity o! t$e color !ormed is 6ro6ortional to t$e c$olesterol concentration in t$e sam6le
Principle )E(periment *@,:
#$olesterol esters ? H
2
O
#$olesterol esterase
#$olesterol ? )3
#$olesterol ? O
2

#$olesterol O7idase
#$olesterol>3>one ? H
2
O
2
H
2
O
2
? 4>33P ? P$enol
Pero7idase
Guinonimine
#$olesterol is a !at>li8e substance t$at is !ound in all body cells. T$e li/er ma8es all o! t$e c$olesterol
t$e body needs to !orm cell membranes and to ma8e certain $ormones. T$e determination o! serum
c$olesterol is one o! t$e im6ortant tools in t$e diagnosis an classi!ication o! li6emia. Hig$ blood
c$olesterol is one o! t$e maHor ris8 !actors !or $eart disease"%'. #linical diagnosis s$ould not be made
on a single test result0 it s$ould integrate clinical and ot$er laboratory data.
P-EPA-ATIO$
Bor8ing reagent ;B(=* &issol/e ;= t$e contents o! one /ial ( 2 En2ymes in one bottle o! ( 1 Bu!!er.
;B(= is stable* 4 mont$s at 2>.I# or 45 days at 1">2"I#. 3/oid direct sunlig$t.
0A"P.E0
4erum or 6lasma1%2* 4tability o! t$e sam6le !or - days at 2>.I# or !ree2ing at 25I# :ill 8ee6 sam6les
stable !or a !e: mont$s.
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . .. . . "5" nm ;"55>""5=
#u/ette* . . . . . . . . . . . . . . . . . . . . .. 1 cm lig$t 6at$
Tem6erature . . . . . . . . . . . . . . . .. . . . .3-I# <1">2"I#
Blan8 4tandard 4am6le
B( ;mL= 1.0 1.0 1.0
4tandard ;L= -- 10 --
4am6le ;L= -- -- 10
14. i7 and incubate !or " min. at 3-I# or 15 min. at room tem6erature.
2". (ead t$e absorbance ;3= o! t$e sam6les and 4tandard% against t$e Blan8. T$e colour is stable
!or at least '5 minutes.
CA.C1.ATIO$0
3 ;4am6le= 7 255 ;4tandard conc.= M mg<dL c$olesterol in t$e sam6le
3 ;4tandard=
Con!ersion factor: mg<dL 7 5.52".M mmol<L.
J,ormal range* &esirable blood c$olesterol le/el P 255 mg<dl
4us6ect to /ascular and #H& 255 245 mg<dl
Hig$ ris8 grou6 !or #H& Q 245 mg<dl
Risk evaluation:
Less than 200 mg/dL Normal
200-239 mg/dL Borderline
240 mg/dL and above igh
.%. Cholesterol

&irect determination o! serum L&Lc ;lo:>density li6o6rotein c$olesterol= le/els :it$out t$e need !or
any 6re>treatment or centri!ugation ste6s. T$e assay ta8es 6lace in t:o ste6s.
1I Elimination o! li6o6rotein no>L&L

2
O
#$olesterol ? )3
#$olesterol ? O
2

#$olesterol O7idase
2
O
2
H
2
O
2

catalase
2 H
2
O ? O
2
2I easurement o! L&Lc
2
O
#$olesterol ? )3
#$olesterol ? O
2

#$olesterol O7idase
2
O
2
H
2
O
2
? 4>33P ? P$enol
Pero7idase
Guinonimine ? 4 H
2
O
2
T$e intensity o! t$e color !ormed is 6ro6ortional to t$e L&Lc concentration in t$e sam6le.
T$e L&Lc 6article is li6o6roteins t$at trans6ort c$olesterol to t$e cells. O!ten called ]bad c$olesterol^
because $ig$ le/els are ris8 !actor !or coronary $eart disease and are associated :it$ obesity% diabetes
and ne6$rosis 1%"%'. #linical diagnosis s$ould not be made on a single test result0 it s$ould integrate
clinical and ot$er laboratory data.
P-EPA-ATIO$
> - + and - 6* 3re ready to use. >
&%.c<.%.c CA.: &issol/e t$e contents :it$ 1 mL o! distilled :ater.
#a6 /ial and mi7 gently to dissol/e contents.
0A"P.E0
4erum * 3!ter sam6ling% t$e test s$ould be 6er!ormed :it$out delay. (e6eated !ree2ing and t$a:ing
s$ould be a/oided. 4tability o! t$e sam6le* - days at 2>.I# .
P-OCE%1-E
. 3ssay conditions*
Ba/elengt$* . . . . . . . . . . . . . .. . . '55 ;"15>-55= nm
#u/ette* . . . . . . . . . . . . . . . . . . . . .. . .1 cm. lig$t 6at$
Tem6erature . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-I#
(1;L! 355 355 355
4tandard ;L= >>>>>>> 4 >>>>>>
4am6le ;L! >>>>>>> >>>>>>>> 4
4. i7 and incubate !or " min. at 3-I#.
". 3dd*
(2 ;L! 155 155 155
'. i7 and incubate !or " min. at 3-I#. -. (ead t$e absorbance ;3=% against t$e Blan8.
CA.C1.ATIO$0
;3= 4am6le 7 4tandard.conc. M mg<dL o! L&Lc in t$e sam6le
;3= 4tandard
Con!ersion factor: mg<dL 7 5.52".'
. -E/E-E$CE BA.1E0
Le/els o! t$e ris8
&esirable P 155 mg<dL
edium 135>1'5 mg<dL
Hig$ Q 1'5 mg<dL
&%. cholesterol

T$e /ery lo: density ;VL&L= and lo: density ;L&L= li6o6roteins !rom serum or 6lasma are 6reci6itated
by 6$os6$otungstate in t$e 6resence o! magnesium ions. 3!ter remo/ed by centri!ugation t$e clear
su6ernatant containing $ig$ density li6o6roteins ;H&L= is used !or t$e determination o! H&L c$olesterol

H&L 6articles carry c$olesterol !rom t$e cells bac8 to t$e li/er. H&L is 8no:n as ]good c$olesterol^
because $ig$ le/els are t$oug$t to lo:er t$e ris8 o! $eart disease. 3 lo: H&L c$olesterol le/els% is
considered a greater $eart disease ris8. #linical diagnosis s$ould not be made on a single test result0 it
s$ould integrate clinical and ot$er laboratory data.
Procedure :

PT3 ? B. sam6le
(T incubation !or 15 min
#entri!ugation !or 15 min. at 4555 r6m
4u6ernatant c$olesterol ? #$ol.O7idase reagent > H&L> #$ol. #onc.
0A"P.E0
4erum or 6lasma1* )ree o! $emolysis. (emo/ed !rom t$e blood clot as soon as 6ossible.
4tability * H&L #$olesterol is stable !or - days at 2>.I# .
P-OCE%1-E
Precipitation
11. Pi6ette into a centri!uge tube*
12. i7 :ell0 allo: to stand !or 15 min at room tem6erature.
23. #entri!uge at 4555 r.6.m. !or 25 min or 2 min at 12555 r.6.m..
34. #ollect t$e su6ernatant and test H&Lc.
( ;L= 155
4am6le ;mL= 1.5
Test
)ollo:ing t$e #$olesterol reagent instructions.
CA.C1.ATIO$0
> Bit$ )actor*
3"5" nm 4am6le 7 325 M mg<&l H&Lc in t$e sam6le.
3"4' nm 4am6le 7 4-" M mg<&l H&Lc in t$e sam6le
Calculation of .%.#cholesterol
3ccording to t$e )riede:ald )ormula*

L&L c$olesterol M Total c$olesterol > Triglycerides >H&L c$olesterol

5uestions:
a. Brite a s$ort note on Hy6erli6idemia.
b. B$ic$ one o! t$e t:o H&L<L&L is more dangerous to $ealt$ and
gi/e reason.
c. B$ic$ diet can cause increase in H&L>#$ol L

Crite the e7uation for &%.#cholesterol calculated from TGD Total Chol>2 .%.>
.aboratory -enal /unction Tests
I# 1rea Estimation 2 Blood 1rea $itrogen )B1$,
Introduction:
Aidney 6roblems are /ery common in clinical medicine. Essentially all seriously sic8 6atients :ill
need t$eir 8idney !unction e/aluated during t$e course o! t$eir illnesses.
3!ter $istory and 6$ysical e7am are com6lete% t$e initial ste6s in c$ec8ing 6atientsR 8idneys are
6er!orming t$e !ollo:ing tests* ;1= urinalysis ;2= serum creatinine ;3= serum urea )Eblood
urea nitrogenED EB1$E,. ,e7t% you may c$ec8 ;4= ability to concentrate urine.
Bot$ creatinine and B+, are included on t$e common c$emical 6ro!iles. _ou can c$ec8 t$e
ability to concentrate urine using a $ygrometer% re!ractrometer% or di6stic8.
Obecti!es:
"ethods:
+# Chemical )direct, method:
+rea ? &iacetyl mono7ime;&3= &iacetyl>+rea
&iacetyl>+rea ? )e
3?
?acidic 6H _ello: &ia2ine ;measured at "45=
6#EnFymatic )indirect, method:
+rea ? H
2
O
+rease
#O
2
? $&
9
_ello: Orange
;at "45 nm=
E(periment * G )"odified BerthlotHs -eaction,:
NH
3
" indi#ator
d$e %dr$
#hemistr$!
&mmonia
dete#ting
ele#trode 'ond(#tivit$
di))eren#e bet*een
non-ioni+ed (rea and
ioni+ed ammonia
,ineti#
-(lti-
en+$mati#
method
Berthlots
% g.2
/,.!
+rea in t$e sam6le is $ydroli2ed en2ymatically into ammonia ;,H4?= and carbon dio7ide ;#O2=.
3mmonia ions !ormed reacts :it$ salicylate and $y6oc$lorite ;,a#lO=% in 6resence o! t$e catalyst
nitro6russide% to !orm a green indo6$enol*
T$e intensity o! t$e color !ormed is 6ro6ortional to t$e urea concentration in t$e sam6le
+rea is t$e !inal result o! t$e metabolism o! 6roteins0 it is !ormed in t$e li/er !rom its destruction.
Ele/ated urea can a66ear in blood ;uremia= in* diets :it$ e7cess o! 6roteins% renal diseases% $eart
!ailure% gastrointestinal $emorr$age% de$ydration or renal obstruction1%'%-. #linical diagnosis s$ould not
be made on a single test result0 it s$ould integrate clinical and ot$er laboratory data.
Principle*
+rea ? H
2
O
+rease
#O
2
? ,H
3
,H
3
? ,a>salicylate ? ,a>$y6oc$lorite ?,a>nito6russide 9ndo6$enol
P-EPA-ATIO$
> Bor8ing reagent ;B(=* &issol/e one tablet ( 3 En2ymes in one bottle o! ( 1 Bu!!er. #a6 and mi7
gently to dissol/e contents.
4tability* 4 :ee8s in t$e re!rigerator ;2>.I#= or - days at room tem6erature ;1">2"I#=.
> ( 2 ,a#lO is ready to use
*
0A"P.E0
1> 4erum or $e6arini2ed 6lasma* &o not use ammonium salts or !luoride as anticoagulants.
2> +rine* &ilute sam6le 1<"5 in distilled :ater. i7. ulti6ly results by "5 ;dilution !actor=. Preser/e
urine sam6les at 6H P 4.
+rea is stable at 2>.I# !or " days0
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . .. . . . .. . . . . . ".5 nm
#u/ette* . . . . . . . . . . . . . . . . . . . . .. 1 cm lig$t 6at$
Tem6erature. . . . . . . . . . . . . . . . . .. . . 3-I# < 1">2"I#
Blan8
B( ;mL= 1.5
4tandard ;L= >>
4am6le ;L= >>
14. i7 and incubate " min at 3-I# or 15 min at room tem6erature ;1">2"I#=.
2". Pi6ette*
Blan8
( 2 ;mL=
1'. i7 and incubate " min at 3-I# or 15 min at room tem6erature ;1">2"I#=.
2-. (ead t$e absorbance ;3= o! t$e sam6les and calibrator% against t$e Blan8. T$e colour is
stable !or at least 35 minutes at 1">2"I#.
CA.C1.ATIO$0
;3= 4am6le 7 "5 ;4tandard conc.= M mg<dL urea in t$e sam6le
;3= 4tandard
15 mg<L urea B+, di/ided by 5.4'' M 21 mg<L urea M 5.3' mmol<L urea.
Con!ersion factor: mg<dL 7 5.1''" M mmol<L.
-E/E-E$CE BA.1E01
4erum * 1"> 4" mg<dL ;2.41>-.41 mmol<L=
+rine * 25 > 3" gr<24 $.
Blood +rea ,itrogen ;B+,= . 2" mg<dl
Interpretation:
9nter6retation o! t$e B+, is usually straig$t!or:ard% t$oug$ t$ere are a !e: t$ings to remember.
*Increased B1$ is% by de!inition% a2otemia. 9t is due eit$er to increased 6rotein catabolism or im6aired
8idney !unction.
*Increased protein cataolism results !rom*
a really $ea/y 6rotein meal ;Aebda% El>Be8% etc.=
se/ere stress ;myocardial in!arction% $ig$ !e/er% etc.=
u66er G9 bleeding ;blood being digested and absorbed=
*Impaired !idney function may be `6rerenal`% `renal`% or `6ostrenal`.
Prerenal aFotemia results !rom underperfusion o! t$e 8idney* de$ydration%
$emorr$age% s$oc8% congesti/e $eart !ailure
-enal aFotemia $as se/eral !amiliar causes* acute tubular necrosis% c$ronic
interstitial ne6$ritis% glomerulone6$ritis% etc.
Postrenal aFotemia results !rom ostruction of urinary flo"* 6rostate
trouble% stones% surgical mis$a6s% tumors
,.B. 9n acute renal !ailure% B+, increases around 25 mg<dL eac$ day ;Jestimates
/ary0 range o! increase is 15>"5 mg<dL daily=.
*%ecreased B1$
#ac! of protein ;celiac disease% some 6atients :it$ ne6$rotic syndrome=
Se$ere li$er disease ;end>stage cirr$osis% yello: atro6$y% really bad $e6atitis%
$alot$ane or acetamino6$en to7icity% en2yme de!ects=
%$erhydration ;iatrogenic% 6syc$ogenic :ater>drin8ing=
%iscussion*
>P$ysiological @ Bioc$emical Bac8ground*
>Pat$ological @ &isease #orrelation*
5uestions:
- Brite t$e di!!erent met$ods !or estimation o! blood ureaL
- #alculate B+, on estimation o! blood urea.
> ention causes o! $y6era2otemia.
II# Plasma Creatinine Estimation
Introduction:
#reatinine is t$e end 6roduct o! muscle metabolism. 9t is e7creted t$roug$ t$e 8idneys and c$anges in
creatinine are an early indicator o! 8idney disease as :ell as being seen in se/ere muscle damage or
:asting diseases or :it$ many medications suc$ as antibiotics. t$is test can be 6er!ormed on
s6ecimens dra:n !rom 6atients in eit$er t$e !asting or non !asting state.
"ethods:
1> %irect Chemical methods*
a= &affe method * 4ee t$e 6rinci6le and 6rocedure ;E76eriment =
) 'N( method (used in dry chemistry:
#reatinine ?&initroben2oic acid ?al8aline 6H 6ur6lis$ rose
6# Indirect EnFymatic methods:
a= &eaminase met$od ;One en2yme ste6 met$od=*
#reatinine
&eaminase
met$yl $ydantoin ? ,H3
;detected by di!!erent tec$niCues=
b= 3mido$ydrolase met$od ; multi>en2ymatic met$od=*
> #reatinine
#reatinine. 3mido$ydrolase
#reatine
> #reatine
#reatine 8inase
#reatine >6
> #reatine>6 ?3TP #reatine ?3&P
> 3&P ? P>enol 6yro/ate ;PEP= 3TP ?Pyru/ate
> Pyru/ate ? ,3&H?H
?

L&H
Lactate ? $A%
;:it$ diminis$ed absorbance at 345 nm=
T$e assay is based on t$e reaction o! creatinine :it$ sodium 6icrate as described by aa!!b. #reatinine
reacts :it$ al8aline 6icrate !orming a red com6le7. T$e time inter/al c$osen !or measurements a/oids
inter!erences !rom ot$er serum constituents. T$e intensity o! t$e color !ormed is 6ro6ortional to t$e
creatinine concentration in t$e sam6le.
Principle )AaffeH "ethod,:
#reatinine ? Picric acid ? al8aline 6H 2%4%' trinitro6$enol
;aano/s8iDs com6le7=
measured at "25 nm
#reatinine is t$e result o! t$e degradation o! t$e creatine% com6onent o! muscles% it can be trans!ormed
into 3TP% t$at is a source o! $ig$ energy !or t$e cells. T$e creatinine 6roduction de6ends on t$e
modi!ication o! t$e muscular mass% and it /aries little and t$e le/els usually are /ery stable. 9s e7creted
by t$e 8idneys. Bit$ 6rogressi/e renal insu!!iciency t$ere is retention in blood o! urea% creatinine and
uric acid. Ele/ate creatinine le/el may be indicati/e o! renal insu!!iciency1%4%". #linical diagnosis s$ould
not be made on a single test result0 it s$ould integrate clinical and ot$er laboratory data.
P-EPA-ATIO$
i7 eCual /olumes o! ( 1 Picric (eagent and ( 2 3l8aline reagent.
T$e :or8ing reagent is stable !or 15 days at 1">2"I#.
2> Blan8 absorbance ;3= at 412 nm 1..5.
0A"P.E0
1> 4erum or $e6arini2ed 6lasma.
#reatinine stability* 24 $ours at 2>.I#.
1> +rine* &ilute sam6le 1<"5 :it$ distilled :ater. i7. ulti6ly results by "5 ;dilution !actor=0
#reatinine stability* - days at 2>.I#.
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . . . . . 412 nm ;415>"15=
#u/ette* . . . . . . . . . . . . . . . . . . . . . . . . 1 cm. lig$t 6at$
Tem6erature. . . . . . . . . . . . . . . . . . . . . 3-I# < 1">2"I#
Blan8 4tandard
B( ;mL= 1.0
4tandard ;L= --
4am6le ;L= --
1
4. i7 and start sto6:atc$.
2". (ead t$e absorbance ;31= a!ter 35 seconds and a!ter 15 seconds ;32= o! t$e sam6le addition.
3'. #alculate* 3M 32 31.
CA.C1.ATIO$0
0 3 4am6le 0 3 Blan8 7 2 ;4tandard conc.= M mg<dL o! creatinine in t$e sam6le
03 4tandard 0&Blan1

Con!ersion factor: mg<dL 7 ...4 M mol<L. l
-E/E-E$CE BA.1E0
4erum or 6lasma
ale 023 - 124 mg<dL M ;'1.. 123.-=c mol<L
)emale 024 - 121 mg<dL M ;"3.5 1-.2 = cmol<L
+rine* 1">2" mg<Ag<24 $
ale 10 - 20 mg<Ag<24 $ M .. 1--c mol<Ag<24 $
)emale 5 6 15 mg<Ag<24 $ M -1 1-- cmol<Ag<24 $
9nter6retation* #auses o! renal !ailure
&iscussion*
P$ysiological @ Bioc$emical Bac8ground*
Ot$er (enal )unction Tests*
Tutorial
Creatinine clearance * is :idely used to a66ro7imate glomerular !iltration. _ou need a timed urine
sam6le and a blood sam6le.
T$e clearance o! a substance is t$e /olume o! 6lasma `cleared` o! t$at substance 6er unit time.
Clearance I )conc> in urine, ( )urine !olume,
)conc> in plasma, : time of urine collection )min>,>
9n deciding $o: to `time` your collection% remember t$at you donRt really need to collect urine !or
a !ull 24 $ours. One grou6 got more reliable results by a controlled collection o/er 4 $ours%
monitoring body 6osition ;8e6t t$em lying do:n= and $ydration :it$ body sur!ace area
measurement.
#reatinine clearance is not a 6er!ect measure o! G)(% because some is not !iltered and some is
secreted into t$e 6ro7imal tubule. T$ese !ractions tend to cancel eac$ ot$er out in $ealt$% but
:$en G)( dro6s belo: 35 mL<min% tubular secretion a66roac$es or e/en e7ceeds t$e amount
!iltered at t$e glomerulus.
J3lso% lots and lots o! red meat ;6rotein and creatinine>ric$= can lead to o/erestimates ;maybe
35Y= in G)( in renal !ailure 6atients.
(e!erence range !or creatinine clearance is 15>125 mL<min !or young adults0 /alues tend to !all
by around 5." mL<year o/er age 25% :orse !or $y6ertensi/es .
J)ormulas to adHust `normal` !or body sur!ace area $a/e been de/ised% etc. )or 8ids% a
$eig$t<creatinine ratio o! 2.1 or less is normal. G)( !or adults can be estimated by /arious
!ormulas0 try 1.12 7 #reatinine #learence > 25.'.
3not$er !ormula to correct t$e clearance according to body sur!ace area is d#orrected
#r.#l. M #r.#l. O ;1.-< body sur!ace area=e
JB$et$er or not `corrections` are a66lied% creatinine clearance is a 6retty good estimate o!
glomerular !iltration rate e7ce6t at /ery lo: /alues% :$en tubular secretion o! creatinine
become 6ro6ortionately greater.
%ETE-"I$ATIO$ O/ 1-IC ACI%
I$T-O%1CTIO$:
+ric acid is a6urine com6ound t$at circulates in 6lasma as sodium urate and is e7creted by
8idney. 9t is deri/ed !rom t$e brea8 do:n o! nucleic acids t$at are ingested or come !rom t$e destruction
o! tissue cells0 it is also synt$esi2ed in t$e body !rom sim6le com6ounds as s$o:n in !igure.
OBAECTIBE0:
To 8no: t$e uric acid le/el in t$e body
To diagnose a case o! Hy6eruricemia ;Gouts,
"ET&O%0:
Chemical "ethod )Phosphotungestic acid "ethodJ,
EnFymatic "ethod
P-I$CIP.E d EnFymatic Colorimetric )1ricase "ethod,K:
+ric acid is o7idi2ed by uricase to allantoine and $ydrogen 6ero7ide ;2H2O2=% :$ic$ under t$e in!luence
o! PO&% 4amino6$ena2one ;4>3P= and 2>4 &ic$loro6$enol sul!onate ;&#P4= !orms a red Cuinoneimine
com6ound*
+ric acid ? 2H2O ? O2 +ricase 3llantoine ? #O2 ? 2H2O2
2H2O2 ? 4>3P ? &#P4 PO& Guinoneimine? 4H2O
T$e intensity o! t$e red color !ormed is 6ro6ortional to t$e uric acid concentration in t$e sam6le.
+ric acid and its salts are end 6roducts o! t$e 6urine metabolism. Bit$ 6rogressi/e renal insu!!iciency%
t$ere is retention in blood o! urea% creatinine and uric acid. Ele/ate uric acid le/el may be indicati/e o!
renal insu!!iciency and is commonly associated :it$ gout #linical diagnosis s$ould not be made on a
single test result0 it s$ould integrate clinical and ot$er laboratory data.
P-EPA-ATIO$
Bor8ing reagent ;B(=* &issol/e t$e contents o! one /ial ( 2 En2ymes in one bottle ( 1 Bu!!er. #a6
and mi7 gently to dissol/e contents. ;B(= is stable a!ter reconstitution 1 mont$ at 2>.I# or 15 days at
room tem6erature.
0A"P.E0 >
4erum or 6lasma* 4tability 3>" days at 2>.I# or ' mont$s at 25I#.
> +rine ;24 $=1* 4tability 4 days at 1">2"I#% 6H Q.. &ilute sam6le 1<"5 in distilled :ater. i7. ulti6ly
results by "5 ;dilution !actor=0
9! urine is cloudy0 :arm t$e s6ecimen to '5I# !or 15 min to dissol/e 6reci6itated urates and uric acid. &o
not re!rigerate.
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . . . . . . . ."25 nm ;415>""5=
#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . 1 cm lig$t 6at$
Tem6erature . . . . . . . . . . . . . . . . . . . . . . 3-I# < 1">2"I#

B( ;ml= 1.5 1.5 1.5
4tandard ;L= >>>>>>> 2" >>>>>>>>
4am6le ;L= >>>>>>> >>>>>>>>> 2"
4. i7 and incubate !or " min at 3-I# or 15 min at 1">2"I#.
". (ead t$e absorbance ;3= o! t$e sam6les and 4tandard% against t$e Blan8.
CA.C1.ATIO$0
4erum or 6lasma
;3= 4am6le7 ' ;4tandard conc.=M mg<dL uric acid in t$e sam6le
;3= 4tandard
+rine 24 $
3= 4am6le7 7 ' 7 /ol. ;dL= urine 24 $ Mmg<24 $ uric acid
4tandard
Con!ersion factor: mg<dL 7 "1."M mol<L.
-E/E-E$CE BA.1E0:
4erum or 6lasma*
Bomen 2." > '.. mg<dL M 141 45" c mol<L
en 3.' > -.- mg<dL M214 4". c mol<L
+rine* 2"5 > -"5 mg<24 $ M 1.41 > 4." mmol<24 $
C.I$ICA. 0IG$I/ICA$CE:
&yperuricemia )Gout,
%I0C100IO$:
#auses o! ele/ated uric acidemia
51E0TIO$0:
1.Gi/e t$e 6rinci6le !or t$e determination o! serum uric acid by uricase
met$od.
2. Brite a s$ort note on +ric acid metabolism.
3. E76lain t$e $y6eruricemia.
PRERENAL VS. RENAL AZOTEMA !
3 /ery common clinical 6roblem is to distinguis$ 6rerenal a2otemia ;due to s$oc8% de$ydration%
#H) >> also `$e6atorenal syndrome`= !rom renal a2otemia ;acute tubular necrosis% `renal
s$utdo:n`.=
Eit$er could be t$e cause :$en a 6atient $as been $y6otensi/e and no: is a2otemic and
oliguric. T$e management is di!!erent.
One older tec$niCue is to calculate t$e B1$<creatinine ratio. T$is is normally bet:een 15 and
25.
Values o/er 25% suggest 6rerenal a2otemia rat$er t$an acute tubular necrosis.
Hig$ /alues are also seen 6ostrenal a2otemia and u66er G9 bleeding.
9n !act% a $ig$ B+,<creatinine ratio is a common !inding% es6ecially in t$e elderly% and a mar8er
!or ill>$ealt$ .
3not$er a66roac$ is to measure sodium on a urine specimen>
9n 6rerenal a2otemia% urine sodium is lo: ;t$e 8idney res6onds to lo: blood !lo: by `trying to
retain all t$e sodium it can.`=
9n acute tubular necrosis% urine sodium is $ig$er ;t$e renal tubules are unable to concentrate or
dilute t$e glomerular !iltrate e!!ecti/ely.=
+rinary sodium under 25 mEC<L suggests 6rerenal a2otemia ;or $e6atorenal syndrome% etc.=0
urinary sodium o/er 45 mEC<L suggests acute tubular necrosis.
J3 !urt$er re!inement% currently 6o6ular% is to measure t$e fractional e(cretion of filtered
sodiumD a66ro7imated by*
Values less t$an 1Y indicate 6rerenal a2otemia0 /alues o/er 2Y indicate acute tubular
necrosis.
4e/eral ot$er !actors can com6licate t$e 6icture in suc$ 6atients.
&iuretics :ill increase t$e e7cretion o! !iltered sodium% :$ile secondary $y6eraldosteronism ;as
in cirr$osis= :ill decrease sodium e7cretion.
9n acute tubular necrosis due to myoglobinuria% sodium e7cretion is lo: ;t$e tubules are
6lugged% not damaged.=
JTi6* 9! you obtain urine by sCuee2ing a dia6er or t$e absor6ti/e balls you 6laced into t$e dia6er% your
estimate o! urine creatinine :ill be lo: because t$ese t$ings absorb creatinine .
1rine Protein<creatinine ratio
+rine 6rotein<creatinine ratio ;+P<+#r= is used to calculate urine 6rotein loss into t$e urine
:it$out a need !or 24 $our urine collection. #om6ared to con/entional 24 $our> urinary 6rotein
/alue% +P<+#r is less time consuming and less accurate.
Generally :it$ 6roteinuria% +P<+#r is greater t$an 1.5.
#)SS F*+I#I*, ,)N*# F-NC.I%N .)S.S
+> Blood p&
#$anges in acid>base balance is obser/ed !reCuently in renal !ailure es6ecially :$en ad/anced.
6> .ipids
Hy6erli6idaemia can occur :it$ renal disease% suc$ as ne6$rotic syndrome. 9ncreased $e6atic
li6o6rotein synt$esis and $y6oalbuminaemia is 6ro6osed in t$e 6at$ogenesis.
9> Plasma protein
Generally t$e concentration o! 6lasma 6rotein is ele/ated due to de$ydration but can be
reduced in 6rimary glomerular diseases suc$ as glomerulone6$ritis and renal amyloidosis.
=> Amylase and lipase
Ele/ated 6lasma li6ase and amylase le/els can be obser/ed in dogs :it$ renal disease%
because t$ese t:o en2ymes are eliminated by t$e 8idneys.
@> Total -ed blood cell
9n c$ronic renal disease% non>regenerati/e anemia is commonly obser/ed. 9t is mainly due to a
reduced eryt$ro6oietin le/el secondary to t$e loss o! renal 6arenc$yma.Ot$er causes o! anemia
in renal disease include $aemorr$age% s$orter t$e li!e s6an o! eryt$rocytes and bone marro:
de6ression.

G> $#acetyl#beta#%#glucosaminidase )EglucosaminidaseED $AG= is a lysosomal en2yme ;B
145%555= !ound in serum and urine. +rinary ,3G is a 6ro6osed mar8er !or tubular disease%
es6ecially subtle industrial 6oisoning% acute 6yelone6$ritis% early acute tubular necrosis% and
early trans6lant reHection. ;T$ese !unctions are no: largely ta8en o/er by beta>2 microglobulin=.
?> Adenosine %eaminase Binding Protein is an en2yme !rom t$e brus$ borders o! t$e
6ro7imal tubule. Li8e ,3G% its 6resence in urine indicates tubular disease.
L> Al3aline phosphatase in urine comes !rom t$e 6ro7imal tubular brus$ border .
M> Beta#6 microglobulin )beta#6#m, is t$e s$ort c$ain o! t$e HL3 class 9 6roteins. 9n $ealt$% it is
!reely !iltered by t$e glomerulus% and !ully reabsorbed by t$e 6ro7imal tubule.
4erum beta>2>m $as been suggested as a measure o! glomerular !iltration rate% similar
to creatinine. Ob/iously t$is isnRt a good idea !or 6atients :it$ tissue necrosis%
lym6$omas% etc.
+rine beta>2>m $as !ound :ides6read acce6tance as an researc$ tool. 9t a66ears i!
le/els in t$e serum and glomerular !iltrate e7ceed :$at t$e 6ro7imal tubule can
reabsorb ;more t$an 4." mg<L= or i! t$ere is renal tubular disease. 9t is /ery sensiti/e as
an indicator o! t$e latter.
+;> Tubular functions* 1rinary amino acids and ma(imum concentrating ability are
sensiti/e screens !or tubular damage. .ithium clearance is a researc$erRs :ay o! estimating
deli/ery to t$e distal tubule.
++> Isotope scans e7ist to com6are t$e !unction o! t$e 8idneys. T$ese may 6ro/e a /aluable
su66lement to t$e intra/enous 6yelogram. ore recently% t$e +6> color %oppler sonogram%
:$ic$ is c$ea6 and 6ortable% $as 6ro/ed e/en more use!ul t$an t$ese scans in trans6lant
6atients. ost recent o! all% t$ereRs a Tc11 scanner t$at monitors glomerular !iltration minute by
minute% suitable !or t$e intensi/e care .
+9> Positron emission tomography is t$e latest :ay o! measuring renal blood !lo:.
0PECI/IC G-ABITN O/ 1-I$E
B$ile not a `blood test`% c$ec8ing urine s6eci!ic gra/ity 6ro/ides /ery im6ortant in!ormation
about tubular !unction and $ydration.
Peo6le in our culture drin8 relati/ely little !luid. T$us `normal` 6eo6le $a/e !airly concentrated
urine ;4G greater t$an 1.515=. O! course% t$e same is true o! 6atients in 6rerenal a2otemia ;$ig$
urinary s6eci!ic gra/ity% lo: or 2ero urinary sodium=.
Patients :it$ tubular disease ;`renal a2otemia`% i.e.% acute tubular necrosis% really bad bilateral
6yelone6$ritis or interstitia ne6$ritis% or on diuretics% or :it$ end>stage 8idney= :ill $a/e
isost$enuria.
Patients getting lots o! !luid by 9V% or :it$ diabetes insi6idus% or ent$usiastic :ater>drin8ers
;ast$matics% cra2ies= :ill $a/e lo: urine s6eci!ic gra/ity.
0erum and 1rine Osmolality
T$e term osmolality re!ers to t$e osmotic concentration o! a !luid. T$e osmolality o! serum% urine% or any
ot$er body !luid de6ends on t$e number o! acti/e ions or molecules in a solution. 9n laboratory re6orts%
osmolality is e76ressed as `so many` milliosmoles 6er 8ilogram o! :ater ;mOsm<8g :ater=. Bit$ a
standard measurement o! osmoles and o! milliosmoles !or clinical studies% t$e 6recise concentration o!
acti/e solutes in t$e serum and urine can be calculated. Tests o! bot$ serum and urine osmolality can
yield im6ortant in!ormation about a 6atientRs ability to maintain a normal !luid balance status.
4odium% blood urea nitrogen% and blood glucose le/els are maHor !actors in determining serum
osmolality. 9n se/ere de$ydration serum osmolality :ill be increased% as t$ere is less :ater in 6ro6ortion
to solutes in t$e serum or blood. +rine osmolality% li8e s6eci!ic gra/ity% is a measurement o! t$e
concentration o! urine. +rine osmolality re!lects t$e total number o! osmotically acti/e 6articles in t$e
urine% :it$out regard to t$e si2e or :eig$t o! t$e 6articles. 4ubstances suc$ as glucose% 6roteins% or
dyes increase t$e urinary s6eci!ic gra/ity. T$ere!ore% urine osmolality is a more accurate measurement
o! urine concentration t$an s6eci!ic gra/ity% and urine osmolality can be com6ared :it$ t$e serum
osmolality to obtain an accurate 6icture o! a 6atientRs !luid balance.
-eference !alues for osmolality*
4erum osmolality* 2.2 > 21" mOsm<8g :ater0 a serum osmolality o! 2." mOsm correlates :it$
a urine s6eci!ic gra/ity o! 1.515
+rine osmolality* e7treme range o! "5 > 1455 mOsm<8g :ater% but a/erage is about "55 > .55
mOsm. 3!ter an o/ernig$t !ast% t$e urine osmolality s$ould be at least 3 times t$e serum
osmolality
Increased serum and urine osmolality )hyperosmolality, le/els are seen in*
(enal disease
#ongesti/e $eart !ailure
3ddisonRs disease
&e$ydration
&iabetes insi6idus
Hy6ercalcemia
&iabetes mellitus<$y6erglycemia
Hy6ernatremia
3lco$ol ingestion
annitol t$era6y
32otemia
%ecreased serum and urine osmolality )hypoosmolality, le/els are seen in*
4odium loss due to diuretic use and a lo: salt diet
Hy6onatremia
3drenocortical insu!!iciency
493&H
E7cessi/e :ater re6lacement<o/er$ydration<:ater into7ication
Panic /alues !or serum osmolality are /alues o! less t$an 245 mOsm or greater t$an 321 mOsm. 3
serum o! osmolality o! 3.4 mOsm 6roduces stu6or. 9! t$e serum osmolality rises o/er 455 mOsm% t$e
6atient may $a/e grand mal sei2ures. Values greater t$an 425 mOsm are !atal.
B$en t$e serum osmolality is normal or increased% t$e 8idneys are conser/ing :ater. 3s t$e serum
osmolality rises% t$e urine osmolality s$ould also rise. T$e $ig$er t$e number o! millosmoles in t$e urine%
t$e more concentrated t$e urine0 t$is is t$e e76ected 6$ysiological res6onse to de$ydration
T$is table s$o:s t$e relations$i6 bet:een serum and urine osmolality and t$e clinical signi!icance o!
laboratory /alues.
0erum Osmolality 1rine Osmolality Clinical 0ignificance
,ormal /alues*
2.2>21"mOsm
,ormal /alues*
"55>.55mOsm

,ormal or increased 9ncreased )luid /olume de!icit
&ecreased &ecreased )luid /olume e7cess
,ormal &ecreased 9ncreased !luid inta8e or diuretics
9ncreased or normal &ecreased ;:it$ no increase in
!luid inta8e=
Aidneys unable to concentrate
urine or lac8 o! 3&H ;diabetes
insi6idus=
&ecreased 9ncreased 493&H
1rine Concentration tests*
3n increase in 6lasma osmolarity stimulates 3&H secretion by t$e 6osterior 6ituitary gland. 3&H
stimulates renal :ater resor6tion and increases urine 4G. T$ese tests are designed to identi!y
concentrating de!ects in t$e 8idney. T$ey are indicated in animals t$at s$o: 6olydi6sia<6olyuria ;P&<P+=
:it$out a2otaemia and de$ydration and are contraindicated in de$ydrated animals% 6regnant animals or
a2otaemic animals :it$ diluted urine.

.i!er /unction Tests )./T,
Albumin estimation
IIIIIIIIIII
Introduction:
3lbumin is made in t$e li/er and is res6onsible !or maintaining 6ro6er !luid balances. Too little albumin
may result in !luids `lea8ing` out o! t$e blood /essels into surrounding s6aces suc$ as t$e abdomen.
&ecreased amounts o! albumin can occur :$en t$e li/er is not ma8ing enoug$ or i! albumin is being lost
t$roug$ t$e 8idneys. 9ncreases in albumin do not occur naturally but can be seen in 6atients :$o $ad
recei/ed albumin sus6ensions.
"ethods
1>Preci6itation met$od
2>Electro6$oresis
3>Globulin Try6to6$an content met$od
4>9mmunoc$emical met$ods.
">&ye binding met$ods
I, Precipitation method
J+se serum only
J,ot a66lied !or automation
J+sed no: !or se6aration @ manu!acturing albumin .
JPreci6itation is done by salting out of globulins @t$en albumin in t$e su6ernatant is
measured using a 6rotein estimation met$od.

II, Electrophoresis
MMMMMMMMMMM
J4e6aration o! 3lbumin !rom t$e maHor classes o! 6rotein in an electrical !ield @ t$e staining Yis
obtained .
Calculation of Albumin I O Albumin : Total Protein
J&i!!icult met$od !or 3utomation.
J9t is a Cuantitati/e met$od but tends to o$er estimate *lumin because albumin is t$e best binder o!
staining dyes @t$e band density o! alb. scanned by densitometer.
III, Globulin Tryptophan content method
IIIIIIIIII
J9n t$is met$od Try6to6$an content o! t$e globulin is !ist estimated as !ollo:ing0
Glyco7ylic acid ?try6to6$an ;Globulin=Pur6le c$romogen ;measured at "45=
#alculation o! 3lbumin M T.Protein > Globulin
IB, Immune chemical methods
IIIIIIIIIIIIII
A=>Electro #immune#diffusion )EI%=* #onsidered t$e Reference method %uantitati!e @Manual"
Jigration o! 6rotein !ractions in an electrical !ield t$roug$ a medium contains s6eci!ic antibodies to
albumin. T$e $eig$t o! t$e Rocket #reci#itin line is correlated to albumin conc.
J+sed !or serum only.
B=>-adial immune diffusion )-I%=*By measuring t$e diameter o! 6reci6itin ring bet:een albumin @its
antibodies incor6orated in agarose gel.
J+sed !or serum @#4). Ta8es long time.
C=>Turbidimetry* T$e reaction bet:een albumin and its s6eci!ic antibodies !orm com6le7es %t$at :ill
decrease t$e lig$t transmission t$roug$ t$e reaction 6$ase more t$an !ree albumin ;antigen=.
%=>$ephelometry:>
E,>(adio immune assay ;(93=.
/,>En2yme immune assay ;EL943=

3lbumin in t$e 6resence o! bromcresol green at a slig$tly acid 6H% 6roduces a colour c$ange o! t$e
indicator !rom yello:>green to green>blue. T$e intensity o! t$e color !ormed is 6ro6ortional to t$e
albumin concentration in t$e sam6le
.
One o! t$e most im6ortant serum 6roteins 6roduced in t$e li/er is albumin. T$is molecule $as an
e7traordinarily :ide rage o! !unctions% including nutrition% maintenance o! oncotic 6ressure and trans6ort
o! #a??% bilirubin% !ree !atty acid% drugs and steroids. Variation in albumin le/els indicate li/er diseases%
malnutrition% s8in lesions suc$ as dermatitis and burns or de$ydration. #linical diagnosis s$ould not be
made on a single test result0 it s$ould integrate clinical and ot$er laboratory data.
P-EPA-ATIO$
(eagent and calibrator are ready to use
2> Blan8 absorbance ;3= at '35 nm 5.45.
0A"P.E0
4erum or 6lasma% !ree o! $emolysis* 4tability 1 mont$ at 2>.I# or 1 :ee8 at 1">2"I#.
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . .. . '35 nm ;'55>'"5=
#u/ette* . . . . . . . . . . . . . . . . . . . . . . 1 cm lig$t 6at$
Tem6erature . . . . . . . . . . . . . . . . . . . .. . . . 1">2"I#
Blan8
( ;mL= 1.0
4tandard ;L= --
4am6le ;L= --
14. i7 and incubate !or 15 min at room tem6erature ;1">2"I#=.
2". (ead t$e absorbance ;3= o! t$e sam6les and 4tandard% against t$e Blan8.
T$e colour is stable 1 $our at room tem6erature.

CA.C1.ATIO$0
;3= 4am6le 7 " ;4tandard conc.= M g<dL albumin in t$e sam6le
;3= 4tandard
Con!ersion factor: g<dL 7 144.1 M mol<L

-E/E-E$CE BA.1E0
9>@ to @>; g<d.>
Total protein estimation
Introduction:
3 Total Protein can be done on eit$er a !asting or non !asting s6ecimen. 9t is usually done as a general
screening assay since it is com6osed o! t:o maHor !ractions ;albumin and globulin=. Ele/ations or
decreases in a total 6rotein must be in/estigated to !ind out :$ic$ o! t$e t:o com6onents is causing t$e
6roblem. 4ince many o! t$e ne7t le/el tests may be re6orted as 6ercentages or ratios% it is necessary to
$a/e t$e total 6rotein rerun at t$e time t$ese tests are 6er!ormed. O/erall% a general re!erence range is
".5 > ..5 gram<dL.. 4ince t$is is a stable assay% t$e range o! /ariation is Cuite small. 3cce6table /ariation
is 1.5
9! bot$ t$e albumin and globulin are ele/ated% one 6ossibility is de$ydration or a slo: do:n o! blood
!lo:. 9! bot$ are decreased% t$e most common cul6rit is li/er !unction. 4ince bot$ albumin and globulin
can be assayed indi/idually% t$ey are sometimes re6orted as an `3G ratio`. ;4ee albumin and globulin
!or s6eci!ics.=
Patients :it$ Baldenstrfms macroglobulinemia may $a/e total 6roteins abo/e ..". T$ey s$ould
consider $a/ing tests 6er!ormed on urine s6ecimens as t$is :ill lessen t$e clotting 6roblem !ound in t$e
s6ecimen but still 6ro/ide adeCuate ans:ers to t$e 6$ysician.
1>+ltra/iolet absor6tion met$od
2>46eci!ic gra/ity met$ods !or T.P. a=P$illi6s
or b=Lo:ry @Hunter
3>(e!ractrometry.
4>AHelda$l nitrogen detection met$od a=Titration or b= 8inetic
">#u4O4 ;#u>Pr com6le7= et$ods a=Lo:ry or b= Biuret
Normal (,eference),anges:
> 3mmonia ;Plasma on He6arin=M 1">"1 ug<dl
> T.P Premature babies M begin !rom 3.' g<dl
,e:borns M 4.' >".- g<dl
-mont$s >1yr. M ".1 >-.3 g<dl
1>2yrs. M ".- >-." g<dl
3dults M '.5 > ..5 g<dl
> E7ercise @3mbulatory 5." g<dl to T.P ;by e7tra/asation o! 6roteins=
+# -ltra$iolet *sorption:

2-5 215 nm
255 >22" nm
J+sed !or 4olutions rat$er t$an serum.
J+sing Guart2 #u/ette ;:it$ no scratc$es=
On using serum % &ilute 1*1555 :it$ ,a#l 5.1" mol<L.
T$is met$od de6ends on Try6to6$an @Tyrosine content o! t$e 6rotein.
J9nter!erence by !ree tyrosine%try6to6$an% bil.@+.3.
6-Specific gra$ity method for .otal proteins:
a)/hillips et al0:
Jdro6s o! serum are allo:ed to !all into `+ni/ersal`containers !illed :it$ #u4O4 soln. eac$ o! 8no:n
s6.gr.
;4toc8 soln.M1"1<L 46.grM1.1=%t$en serial dilutions are made to get solns. o! s6.gr bet:een 1.51"
1.53"
3t certain s6eci!ic gra/ity a dro6 :ill not mo/e neit$er u6 or do:n;M46.gr.o! interest=
Total 6rotein;g<dl= M 3'";46.gr.o! 9nt. g 1.55- =
J#an estimate T.P bet:een 3.3 g15.3 g<dl
b,Column method (#o"ry1Hunter):
J+sing only single gradient column o! mi7ed organic liCuids :it$ #u4O4 Hac8et ;maintaining constant
tem6.=.
J9t reCuires only one dro6%:$ic$ :ill be $anged at certain gradient .
JLi8e in P$illi6s met$od T.P can be calculated.
9#,efractometry method:
JT$is met$od is based on t$e re!raction o! incident lig$t by total dissol/ed solids.
J3 large dro6 o! serum or urine is allo:ed to s6read bet:een slide @t$in !ilm and re!racted rays ma8e
s$ar6 line di/iding t$e dar8 @ lig$t !ields.
J#an estimate T.P bet:een 3." g11 g<dl
2-34eldahl +ethod:
JT$e re!erence met$od
Jusing 6rotein !ree !iltrate.
J&e6end on estimation o! 6rotein nitrogen.
Protein
H24O4?#atalyst
?,a>olybdate
$&='

3l8aline PH
$&9
H#l;standard 4ol.= $A%P& '2o7oglutarate
; eit$er=
;Titration=

,eutral PH $A%P ' glutamate
;,isseleri2ation= ;onitor abs.c$ange at 345 nm=
#oncentration o! total 6roteinM detected nitrogen O155<1'
M detected nitrogen O '.2"
J!actor '.2" is t$e result o! 155<1' as eac$ 155 g 6rt.1' g ,itrogen.
J #orrected 6r.conc. M ;6r.,2>,P,= O '.2"
5-*l!aline C-S%2 soln0+ethods:
0ample
,aOH ? #u4O4>
#o66er '>6e6tide bond 6rotein com6le7
)olin;)enol=? A@,aTartarate;Mcolor stabili2er=
#io>#alteau;PT3?P$>olbdic a.=

olybdinum blue ? Violet color o! #u>Pr.#om6le7
Tungesten blue;at '"5> -"5nm= ;at "4'nm=
#%6,7Hs "ethod (I-,).Hs "ethod
0ensiti!ity: +;; times P BiuretHs good for Pr> 6#+6 g
0pecificity: .ess specific specific

$o> of reagents: 6 -eagents One reagent

%rug Interference %ependence on Tryptophan2Tyrosine
)salicylatesDsulfa2tetracyclines, e>g Alb>I;>6 O Tryptophan by 8t>
Glob>I6O Tryptophan by 8t>
Proteins gi/e an intensi/e /iolet>blue com6le7 :it$ co66er salts in an al8aline medium. 9odide is
included as an antio7idant. T$e intensity o! t$e color !ormed is 6ro6ortional to t$e total 6rotein
concentration in t$e sam6le

T$e 6roteins are macromolecular organic com6ounds% :idely distributed in t$e organism. T$ey act li8e
structural and trans6ort elements. T$e 6roteins o! t$e serum are di/ide in t:o !ractions% albumin and
globulins T$e determination o! total 6roteins is use!ul in t$e detection o!* > Hig$ 6rotein le/els caused by
$emoconcentration li8e in t$e de$ydrations or increase in t$e concentration o! s6eci!ic 6roteins. > Lo:
6rotein le/el caused by $emodilution by an im6ared synt$esis or loss ;as by $emorr$age= or e7cessi/e
6rotein catabolism. #linical diagnosis s$ould not be made on a single test result0 it s$ould integrate
clinical and ot$er laboratory data
P-EPA-ATIO$
T$e reagents are ready to use
2> Blan8 absorbance ;3= at "45 nm 5.22.
0A"P.E0
4erum or $e6arini2ed 6lasma* 4tability o! t$e sam6le* 1 mont$ at re!rigerator ;2>.I#=.
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . . . . . . . . . . . "45 ;"35>""5= nm
#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 1 cm. lig$t 6at$
Tem6erature . . . . . . . . . . . . . . . . . . . . . . . . . .3-I# < 1">2"I#
( ;mL= 1.5 1.5 1.5
4tandard ;L= >>>>>> 2" >>>>>>>
4am6le;L= >>>>>> >>>>>>>>> 2"
14. i7 and incubate " min at 3-I# or 15 min at room tem6erature.
2". (ead t$e absorbance ;3= o! t$e sam6les and 4tandard% against t$e Blan8.
CA.C1.ATIO$0
;3= 4am6le7 - ;4tandard conc.=M g<dL o! total 6rotein in t$e sam6le
;3= 4tandard
-E/E-E$CE BA.1E0
Ad!lts" #.# $ %.3 g/dL
&e'(orn" .2 $ ).1 g/dL
Bilirubin estimation
Introduction:
Bilir(bin is the end "rod(#t o) red #ell l$sis and re#$#ling o) hemoglobin *hi#h is "er)ormed
in the liver. 7he test 8(anti)ies t*o di))erent )orms o) bilir(bin2 one is the )inal "rod(#t *hile
the other is an intermediate )orm.
7he b(ild (" o) bilir(bin in the blood stream is #alled 9a(ndi#e and is a general sign o) liver
disease. -an$ medi#ations2 gall bladder disease as *ell as vir(ses s(#h as in)e#tio(s
monon(#leosis and he"atitis *ill have 9a(ndi#e. -an$ in)ants are born *ith less than )(ll$
mat(re livers. &s a #onse8(en#e2 )or the )irst several da$s2 the$ ma$ sho* :neonatal 9a(ndi#e:
*hi#h is a b(ild (" o) bilir(bin in the blood stream. 7his sho(ld go a*a$ as the liver mat(res.
Bilir(bin determinations are (sed to st(d$ liver )(n#tion and red #ell metabolism
"LN"AL S#N$"AN"E
Bilirubin is a brea8do:n 6roduct o! $emoglobin. 9t is trans6orted !rom t$e s6leen to t$e li/er and
e7creted into bile. Hy6erbilirubinemia results !rom t$e increase o! bilirubin concentrations in 6lasma.
#auses o! $y6erbilirubinemia* Total bilirubin ;T=* 9ncrease $emolysis% genetic errors% neonatal Haundice%
ine!!ecti/e eryt$r6oiesis% and drugs. &irect bilirubin ;&=* He6atic c$olestasis% genetic errors%
$e6atocellular damage1%"%'. #linical diagnosis s$ould not be made on a single test result0 it s$ould
integrate clinical and other laborator$ data.
"ethods for Bilirubin estimation
IIIIIIIIIIIIIII
+# %irect 0pectrophotometry:
4-estricted to ne8born)Q 6L days, up to 9 monthsD because their serum contains no
Carotenes>
Jeasuring absorbency o! Bilirubin in serum at 2 :a/e lengt$s 4"5 @ "45 nm
The difference in the absorbance represents bilirubin absorbance )A=@; # A@=; ,
4That is because &emoglobin reads the same at both C>. 8hile bilirubin reads at =@; nm>
6# %irect 03in Bilirubinometer:
J(estricted also to ne:borns u6 to 3 mont$s.
J,eeds calibration using*>et$yl Orange solution.
Or >)ilter ultilayered color glass.
34"5 > 3 "45 M 3bsorbance o! bilirubin
9# 0pectral shift change method:
J46ectral s$i!t t$roug$ adding $ydro6$obic cationic 6olymer.
J+sed by AO&3A E#T3#3 only.
=#&P.C)&igh Purity .i7uid Chromatography,:
+sing ,ormal 4ilica #$romatogra6$y .
T$e (e!erence met$od.
@#Colometric methods )E(periment * L,:
JT$e most commonly used met$ods.
&e6end on reaction o! bilirubin@ &ia2oti2ed 4ul!anilic acid ;&43=

"E..ON 2EBE.N$ "> AE$%-A00I.#G-O/ ">
4Accelerator: "ethanol or 1rea Caffeine 2$a>BenFoate
4P& : $eutral Al3aline)P&I+6,
4C>. : GG; @G; nm
4Color : -ed purple red
Coloumetric -eaction:
4Bil.Glucuronides ' %0A AFobilirubin 'H2O?#o2?Hcl
)'irect (iliruin)
4Bil.Glucuronides '%0A '*ccelerator AFobilirubin
'H2O?#o2?Hcl).otal (iliruin)
G#Bilirubin O(idase "ethod :
J46eci!ic !or Bilirubin only.
JBil. ?Bil. O7idase Bili/erdin ;measured at 45" nm=
PRN"PLE O$ THE METHO%
Bilir(bin is #onverted to #olored a+obilir(bin b$ dia+oti+ed s(l)anili# a#id and meas(red
"hotometri#all$. ;) the t*o )ra#tions "resents in ser(m2 bilir(bin-gl(#(romide and )ree
bilir(bin loosel$ bo(nd to alb(min2 onl$ the )ormer rea#ts dire#tl$ in a8(eo(s sol(tion
%bilir(bin dire#t!2 *hile )ree bilir(bin re8(ires sol(bili+ation *ith dimeth$ls(l)o<ide %=->;!
to rea#t %bilir(bin indire#t!. .n the determination o) indire#t bilir(bin the dire#t is also
determined2 the res(lts #orres"ond to total bilir(bin. 7he intensit$ o) the #olor )ormed is
"ro"ortional to the bilirr(bin #on#entration in the sam"le
P-EPA-ATIO$
3ll t$e reagents are ready to use
2> #olor de/elo6ment in ( 2.
0pecimen Precautions:
IIIIIIIIIIIIIIIIIII
+# 0erum or Plasma
6# A!oid &emolysis
9# A!oid light e(posure
=# 0torage for 9 days in dar3 2 refrigerator )for months if freeFed at ?;R C,
@# 1rine sample either -andom or 6= hrs> not stored for P6= hrs>
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . K . . . . """ nm ;"35>".5=
#u/ette* . . . . . . . . . . . . . . . ... . . K K . . .1 cm lig$t 6at$
Tem6erature . . . . . . . . . . . . . . . . . . . . . . . . . . . 1">2"I#
Blan8
.
Total B Blan8 &irect
( 1 ;&= ;mL= -- -- 1.? 1.?
( 2 ;T= ;mL= 1.? 1.? --
( 3 ;L = -- ?0 -- ?0
4am6le 100 100 100 100
1
4. i7 and incubate e7actly !or @ minutes at 1">2"I#.
2". (ead t$e absorbance ;3=.
CA.C1.ATIO$0
Cith /actor:
;;3= 4am6le > ;3= 4am6le Blan8= 7 /actor4 M mg<dL bilirubin in t$e sam6le
: Theoretical factor: Bilirubin ;T= M 11%1 0 Bilirubin ;&= M 14
Con!ersion factor: mg<dL 7 1-.1 M mol<L.
REFERENCE VALUES
*ilir!(in +otal
,p to 1.10 mg/dLM1%.%1 mol/L
*ilir!(in -irect
,p to 0.2 mg/dL=..2/ mol/L
5uestions:
- Brite causes o! Haundice
- B$at are t$e commonest met$ods o! estimating serum bilirubin in neonatesL
- ention t$e di!!erent causes o! ele/ated direct and indirect bilirubins.
Catalytic )EnFymatic, acti!ities of .i!er )E./T,
+#Gamma Glutamyl Transferase )GGT,
Introduction:
T$is en2yme used to metaboli2e materials in t$e 8idney% li/er% gall bladder% and 6ancreas. 9t is an
e7ce6tionally sensiti/e indicator o! stress in t$ese sites. 3s a conseCuence% /ariations in results may be
Cuite common. 3lco$ol consum6tion ;e/en a little= and many medications are t$e c$ie! causes o! t$ese
s:ings. T$is test is used to !ollo: 8idney% li/er or 6ancreatic !unction
P-I$CIP.E O/ T&E "ET&O% ;Ainetic test ;42as2=
Gamma>glutamyl trans!erase ;h>GT= catalyses t$e trans!er o! h>glutamyl grou6 !rom h>glutamyl>6>
nitroanilide to acce6tor glycylglycine% according to t$e !ollo:ing reaction*
h>>L>Glutamyl>3>carbo7y>4>nitroanilide ? Glycylglycine h>GT
h>L>Glutamyl>glycylglycine ? 2>,itro>">aminoben2oic acid
T$e rate o! 2>nitro>">aminoben2oic acid !ormation% measured 6$otometrically% is 6ro6ortional to t$e
catalytic concentration o! h>GT 6resent in t$e sam6le
Gamma>glutamyl trans!erase ;h>GT= is a cellular en2yme :it$ :ide tissue distribution in t$e body%
6rimarily in t$e 8idney% 6ancreas% li/er and 6rostate.
easurements o! gamma>glutamyl trans!erase ;h>GT= acti/ity are used in t$e diagnosis and treatment
o! $e6atobiliary diseases suc$ biliary obstruction% cirr$osis or li/er tumours
#linical diagnosis s$ould not be made on a single test result0 it s$ould integrate clinical and ot$er
laboratory data.
P-EPA-ATIO$
&issol/e one tablet o! ( 2 4ubstrate in one /ial o! ( 1 Bu!!er.
4tability* 21 days at 2>.I# or " days at room tem6erature ;1">2"I#=.
2> Blan8 absorbance ;3= at 45" nm i 1.25.
0A"P.E0
4erum. hjGT is stable !or at least 3 days at 2>.I#% . $ours at 1">2"I# and 1 mont$ at 25I#.
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45" nm
#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . . . 1 cm lig$t 6at$
#onstant tem6erature . . . . . . . . . . . . . . .2"I# <35I# < 3-I#
2. 3dHust t$e instrument to 2ero :it$ distilled :ater or air.
14. i7% :ait !or 1 minute.
2". (ead initial absorbance ;3= o! t$e sam6le% start t$e sto6:atc$ and read absorbances at 1
minute inter/als t$erea!ter !or 3 minutes.
3'. #alculate t$e di!!erence bet:een absorbances and t$e a/erage absorbance di!!erences 6er
minute ;k3<min=.
CA.C1.ATIO$0
ean 3M ;l3=<min M ;31?32?33= < 3
En2yme acti/ity ;+<L= M l 3 O )actor
1nits: One international unit ;9+= is t$e amount o! en2yme t$at trans!orms 1 cmol o! substrate 6er
minute% in standard conditions. T$e concentration is e76ressed in units 6er litre o! sam6le ;+<L=.
-E/E-E$CE BA.1E0
2"I# 35I#
Bomen 4>1. +<L ">2" +<L
en '>2. +<L .>3. +<L
k3<min 7 1115 M +<L o! h>GT
6# Alanine Transaminase )A.T,
3lanine aminotran!erase ;3LT= o Glutamate 6yru/ate transaminase ;GPT= catalyses t$e re/ersible
trans!er o! an amino grou6 !rom alanine to X>8etoglutarate !orming glutamate and 6iru/ate.
T$e 6iru/ate 6roduced is reduced to lactate by lactate de$ydrogenase ;L&H= and ,3&H*
X>8etoglutarate ? L>3lanine
3LT ;GPT=
L>Glutamate ? Pyru/ate
Pyru/ate ? ,3&H?H?
Lactate de$ydrogenase ;L&H=
L>Lactate ? ,3&
T$e rate o! decrease in concentration o! ,3&H% measured 6$otometrically% is 6ro6ortional to t$e
catalytic concentration o! 3LT 6resent in t$e sam6le
CLINICAL SIGNIFICANCE
T$e 3LT is a cellular en2yme% !ound in $ig$est concentration in li/er and 8idney. Hig$ le/els are
obser/ed in $e6atic disease li8e $e6atitis% diseases o! muscles and traumatisms% its better a66lication is
in t$e diagnosis o! t$e diseases o! t$e li/er.
B$en t$ey are used in conHunction :it$ 34T aid in t$e diagnosis o! in!arcts in t$e myocardium% since t$e
/alue o! t$e 3LT stays :it$in t$e normal limits in t$e 6resence o! ele/ated le/els o! 34T
laboratory data.
P-EPA-ATIO$
&issol/e one tablet o! (2 4ubstrate in one /ial o! (1.
4tability* 21 days at 2>.I# or -2 $ours at room tem6erature ;1">2"I#=.
2> Blan8 absorbance ;3= at 345 nm P 1.55.
0A"P.E0
4erum or 6lasma* 4tability - days at 2>.I#..
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 nm
#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 1 cm lig$t 6at$
#onstant tem6erature . . . . . . . . .. . . . . .2"I# < 35I# < 3-I#
14. i7% incubate !or 1 minute.
2". (ead initial absorbance ;3= o! t$e sam6le% start t$e sto6:atc$ and read absorbances at 1>
minute ;k3<min=.
CA.C1.ATIO$0
l3 ;mean di!!erence o! readings= M ;31 ?32?33= < 3
&L7 en+$me a#tivit$ %@/L! A 0& B Ca#tor %C1!
-E/E-E$CE BA.1E0
2"I# 35I# 3-I#
en u6 to 22 +<L 21 +<L 45 +<L
Bomen u6 to 1. +<L 22 +<L 32 +<L
,ormal ne:borns $a/e been re6orted to s$o: a re!erence range o! u6 to double t$e adult% attributed to
t$e neonateDs $e6atocytes. T$ese /alues decline to adult le/els by a66ro7imately 3 mont$s o! age.
k3<min 7 1-"5 M +<L o! 3LT

9# Aspartate Transaminase )A0T,
Introduction:
3s6artate Transaminase ;34T= is also 8no:n by its older name% 4GOT% t$is en2yme is needed in t$e
utili2ation o! energy sources. 9t is !ound in $ig$ concentrations in muscle ;cardiac and ot$ers=% li/er% and
ot$er organs. T$is test usually is ordered to !ollo: cardiac and muscle disease .
T$is test can be 6er!ormed on s6ecimens !rom 6atients :$o are eit$er in a !asting or non !asting. 3dult
re!erence ranges /ary :idely :it$ di!!erent instruments.
3s6artate aminotrans!erase ;34T= !ormerly called glutamate o7aloacetate ;GOT= catalyses t$e
re/ersible trans!er o! an amino grou6 !rom as6artate to X>8etoglutarate !orming glutamate and
o7alacetate. T$e o7alacetate 6roduced is reduced to malate by malate de$ydrogenase ;&H= and
,3&H*
X>8etoglutarate ? L>3s6artate
34T ;GOT=
L>Glutamate ? O7aloacetate
O7aloacetate ? ,3&H?H?
alate de$ydrogenase ;&H=
alate? ,3&
T$e rate o! decrease in concentration o! ,3&H% measured 6$otometrically% is 6ro6ortional to t$e
catalytic concentration o! 34T 6resent in t$e sam6le.
T$e 34T is a cellular en2yme% is !ound in $ig$est concentration in $eart muscle% t$e cells o! t$e li/er% t$e
cells o! t$e s8eletal muscle and in smaller amounts in ot$er :ea/es.
3lt$oug$ an ele/ated le/el o! 34T in t$e serum is not s6eci!ic o! t$e $e6atic disease% is used mainly to
diagnostic and to /eri!y t$e course o! t$is disease :it$ ot$er en2ymes li8e 3LT and 3LP.
3lso it is used to control t$e 6atients a!ter myocardial in!arction% in s8eletal muscle disease and ot$er
laboratory data.
P-EPA-ATIO$
&issol/e one tablet o! (.2 4ubstrate :it$ one /ial o! (1 Bu!!er.
4tability* 21 days at 2>.I# or -2 $ours at room tem6erature ;1">2"I#=.
2> Blan8 absorbance ;3= at 345 nm P 1.55.
0A"P.E0
4erum or 6lasma* 4tability - days at 2>.I#..
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 nm
#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . . . .1 cm. lig$t 6at$
#onstant tem6erature . . . . . . . . . . . . . . .2"I# <35I# < 3-I#
minute ;k3<min=.
CA.C1.ATIO$0
l3 ;mean di!!erence o! readings=<min. M ;31 ?32?33= < 3
34T en2yme acti/ity ;+<L= M l3 O )actor ;)1=
-E/E-E$CE BA.1E0
2"I# 35I# 3-I#
en u6 to 11 +<L 2' +<L 3. +<L
Bomen u6 to 1' +<L 22 +<L 31 +<L

k3<min 7 1-"5 M +<L o! 34T
Bone Profile Testes
Calcium %etermination
Introduction:
#alcium is reCuired !or cell !unction o/erall and !or bone metabolism. Too little calcium gets you eit$er a
loss o! tissue !unction or so!t bones ;osteo6orosis= :$ile too muc$ gi/es you tetni ; cardiac arrest and<or
loc8 Ha: is !rom o/er clenc$ing o! muscles= or o/er brittle bones. #$anges in calcium are used to assess
bone !unction. Hig$er blood le/els usually mean lo:er bone le/els. +sually 6er!ormed in conHunction
:it$ P$os6$orous determinations.
OBAECTIBE0:
#9oni2ed calcium constitutes 4. to "2 Y o! t$e total calcium% t$e un>ioni2ed di!!usible !orm
constitutes " Y a66ro7. and about 43 4- Y o! t$e total 6lasma calcium is 6rotein bound% 6rimarily to
albumin% but also to some e7tant to t$e X>% W> and h>globulins.
>To 8no: t$e status body calcium ;Tetany=
"ET&O%0:
i. #$elation :it$ o>#resol6$t$alein #om6le7one;#olorimetric=
ii. 3tomic absor6tion 46ectro6$otometry ;334=
iii. )lame 6$otometer
i/. 94E
T$e measurement o! calcium in t$e sam6le is based on !ormation o! color com6le7 bet:een calcium
and o>cresol6$talein in al8aline medium*
#a?? ? o>#resol6$talein OH #olored com6le7
O>#resol6$t$alein #om6le7 one gi/es /iolet color in al8aline medium.
T$e intensity o! t$e colour !ormed is 6ro6ortional to t$e calcium concentration in t$e sam6le
#alcium is t$e most abundant and one o! t$e most im6ortant minerals in t$e $uman body.
366ro7imately 11Y o! body calcium is !ound in bones. 3 decrease in albumin le/el causes a decrease
in serum calcium. Lo: le/els o! calcium are !ound in $y6o6arat$yroidism% 6seudo$y6o6arat$yroidism%
/itamin & de!iciency% malnutrition and intestinal malabsortion. 3mong causes o! $y6ercalcemia are
cancers% large inta8e o! /itamin &% en$aced renal retention% osteo6orosis% sarcosidosis% t$yroto7icosis%
$y6er6arat$yroidism. #linical diagnosis s$ould not be made on a single test result0 it s$ould integrate
clinical and ot$er laboratory data.
P-EPA-ATIO$
3ll t$e reagents are ready to use. To 6re6are monoreagent% mi7 according to t$is 6ro6ortion* "5 /ol. o!
(1 and 1 /ol. o! (2.
0A"P.E0 >
4erum or 6lasma* 4e6arated !rom cells as ra6idly as 6ossible. Blood anticoagulants :it$ o7alate or
E&T3 are not acce6table since t$ese c$emicals :ill strongly c$elate calcium.
> +rine* #ollect 24 $our urine s6ecimen in calcium !ree containers. T$e collecting bottles s$ould contain
15 ml o! diluted ,itric acid ;"5Y /</=. (ecord t$e /olume.
&ilute a sam6le 1<2 in distilled :ater. i7. ulti6ly results by 2 ;dilution !actor=. 4tability o! t$e sam6les*
#alcium is stable 15 days at 2>.I#.
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . .. . . "-5 nm ;""5>"15=
#u/ette* . . . . . . . . . . . . . . . . . . . . .. 1 cm. lig$t 6at$
Tem6erature . . . . . . . . . . . . . . . . . . . 3-I# < 1">2"I#
(1 ;mL= 2.5 2.5 2.5
(2 ;2 dro6= 1 1 1
4tandard ;L= >>>>> 25 >>>>>
4am6le ;L= >>>>> >>>>>> 25
4. i7 and incubate !or " min at 3-I# < 1">2"I#. ". (ead t$e absorbance ;3= o! t$e sam6les and
calibrator% against t$e Blan8.
T$e color is stable !or at least 45 minutes.
CA.C1.ATIO$0
4erum and 6lasma ;3= 4am6le 7 15 ;4tandard conc.= M mg<dL calcium
;3= 4tandard
Con!ersion factor: mg<dL 7 5.2"M mmol<L.
-E/E-E$CE BA.1E0
4erum or 6lasma*
3dults ..">15." mg <dL M 2.1>2.' mmol<L
#$ildren 15 >12 mg<dL M 2." > 3 mmol<L
,e:borns . >13 mg<dL M 2 > 3.2" mmol<L

-E01.T0:
C.I$ICA. 0IG$I/ICA$CE:
4 &NPOCA.CE"IA
1. Vitamin & de!iciency
2. Hy6o6arat$yroidism
3. 3l8alosis ;3l8alemia=
4 &NPE-CA.CE"IA
1. Hy6er6arat$yroidism
2. alignancy o! bone
3. T$yroto7icosis
4. Vitamin & into7ication
". 9dio6at$ic
%I0C100IO$:
TETA$N
P-ECA1TIO$0:
+> 3/oid /enous stasis ;9ncrease 6rotein @ calcium=
6> &o not use contaminated glass :are ;9ncrease calcium=
9> Li6emic 4am6les ;Pre6are blan8 5.5" ml sam6le ? 2." &.C,
51E0TIO$0:
1. B$at is H_PO#3L#E93L Brite a s$ort note on Tetany.
2. Gi/e t$e 6rinci6le !or t$e determination o! serum calcium by colorimetric met$od.
3. Enumerate di!!erent met$ods !or t$e determination o! serum calcium.
5uantitati!e determination of phosphorus
9norganic 6$os6$orus reacts :it$ molybdic acid !orming a 6$os6$omolybdic com6le7. 9ts subseCuent
reduction in al8aline medium originates a blue molybdenum colour.
T$e intensity o! t$e color !ormed is 6ro6ortional to t$e inorganic 6$os6$orus concentration in t$e sam6le
P$os6$orus is an essential mineral !or tissue bone !ormation and is reCuired by e/ery cell in t$e body
!or normal !unction. 366ro7imately ."Y o! t$e body 6$os6$orus is !ound in bone and in teet$.
Lo: le/els o! 6$os6$orus% can be caused by $y6er/itaminosis 5% 6rimary $y6er6arat$yroidism% renal
tubular disorders% antacids or malabsortion.
Hig$ le/els o! 6$os6$orus can be caused by diet% bone metastases% li/er disease% alco$ol ingestion%
diarr$ea and /omiting
laboratory data.
P-EPA-ATIO$
i7 eCual /olumes o! ( 1 ;olybdic= and ( 2 ;#ataly2er=
4tability* 24 $ at 2>.#% 6rotected !rom lig$t.
0A"P.E0
> 4erum*
)ree o! $emolysis. 4erum s$ould be remo/ed !rom t$e clot as Cuic8ly as 6ossible to a/oid ele/ation o!
serum 6$os6$orus !rom $ydrolysis or lea8age o! 6$os6$ate 6resent in eryt$rocytes.
4tabilitr - days at 2>.#.
> +rinem ;24 $=*
#ollect t$e s6ecimen into a bottle containing 15 mL o! 15Y !$! $ydroc$loric acid ;H#9= to a/oid
6$os6$ate 6reci6itations. 3dHust to 6H 2. &ilute t$e sam6le %$%& :it$ distilled :ater. i7. ulti6ly t$e
result by 15 ;dilution !actor=. 4tability* 15 days at 2>Bo#.
P-OCE%1-E
1> 3ssay conditions*
Ba/elengt$* .................-15 nm ;'25>-"5=
#u/ette* ............................1 cm. lig$t 6at$
Tem6erature ....................3-n# % 1">2"n#
2> 3dHust t$e instrument to 2ero :it$ distilled :ater.
3> Pi6ette into a cu/ette*
4. i7 and incubate !or 15 min at 3-n# or 35 min at room tem6erature ;1">35n#=.
"> (ead t$e absorbance ;3= o! t$e sam6les and calibrator% against t$e Blan8.
T$e colour is stable !or at least 2 $ours.
CA.C1.ATIO$0
4erum
;3= 4am6le 7 " ;#alibrator cone.= M mg<dL o! 6$os6$orus in t$e sam6le
;3= #alibrator
#on/ersion !actor* mg<dL 7 5.323 M mmol$'"
-E/E-E$CE BA.1E0
4erum*
#$ildren * 4.5 > -.5 mg<dL M ;1.3 > 2.2 mmol<L=
3dults * 2." > ".5 mg<dL M ;O.. > 1.. mmo<lL=

B( ;mL= 1.? 1.? 1.?
4tandard ;cL= - ?0 --
4am6le ;cL= -- -- ?0
5uantitati!e determination of al3aline phosphatase )A.P,

3l8aline 6$os6$atase ;3LP= catalyses t$e $ydrolysis o! 6>nitro6$enyl 6$os6$ate at 6H 15.4% liberating 6>
nitro6$enol and 6$os6$ate% according to t$e !ollo:ing reaction*
6>,itro6$enyl6$os6$ate ? H
2
5 6>,itro6$enol ? P$os6$ate
T$e rate o! 6>,itro6$enol !ormation% measured 6$otometrically% is 6ro6ortional to t$e catalytic
concentration o! al8aline 6$os6$atase 6resent in t$e sam6le
3l8aline 6$os6$atase is an en2yme 6resent in almost all :ea/es o! t$e organism% being 6articularly
$ig$ in bone% li/er% 6lacenta% intestine and 8idney.
Bot$ increases and decreases o! 6lasma 3LP are o! im6ortance
clinically. #auses o! increased 6lasma 3LP* PagetRs disease o!
bone% obstructi/e li/er disease% $e6atitis% $e6atoto7icity caused by
drugs or osteomalacia. #auses o! decreased 6lasma 3LP*
#retinism and /itamin # de!iciency1%"%'. #linical diagnosis s$ould
not be made on a single test result0 it s$ould integrate clinical and
ot$er laboratory data.
P-EPA-ATIO$
8or3ing reagent )C-,:
&issol/e one tablet o! ( 2 4ubstrate in one /ial o! ( 1 Bu!!er.
0A"P.E0
4erum or $e6arin2ed. 6lasma o+se un$emoly2ed .serum% se6arated !rom t$e clot as soon as 6ossible.
4tability* 3 days at 2>.n#.
P-OCE%1-E
1>3ssay conditions*
Ba/elengt$* ................. .. . ...................45" nm
#u/ette* . . . . . . . . . . . . . . . . . . . . .. . .. . 1 cm lig$t 6at$
#onstant tem6erature ...................2"n# 3&() 3*()
2>3dHust t$e instrument to 2ero :it$ distilled :ater or air.
3>Pi6ette into a cu/ette*
B( ;mL= 1.2
;cL=4am6le 25
">(ead initial absorbance ;3= o! t$e sam6le% start t$e sto6:atc$ and read absorbances at 1 minute
inter/als t$erea!ter !or 3 minutes.
'>#alculate t$e di!!erence bet:een absorbances and t$e a/erage absorbance di!!erences 6er minute
;l3<min=.
CA.C1.ATIO$0
;l3<min= 7 3355 M +<L de 3LP
+nits* One international unit ;9+= is t$e amount o! en2yme t$at trans!orms 1 cmol o! substrate 6er
-E/E-E$CE BA.1E0
+

2"n#
3-n#
#$ildren ;1>14 years= P 455 +<L P 4.5 +<L P '4" +<L
3dults '5 > 1-5 +<L -3 > 25- +<L 1. > 2-1 +<L
)actors a!!ecting 3LP acti/ities in a normal 6o6ulation include e7ercise% 6eriods o! re6aid gro:t$ in
c$ildren and 6regnancy.

Cardiac profile Testes
5uantitati!e determination of creatin 3inase )CS,
#reatine 8inase is a cellular en2yme :it$ :ide tissue distribution in t$e body. 9ts 6$ysiological role is
associated :it$ adenosine tri6$os6$ate ;3TP= generation !or contractile or trans6ort systems.
Ele/ated #A /alues are obser/ed in diseases o! s8eletal muscle and a!ter myocardial in!arction
laboratory data.
#reatine 8inase ;#A= catalyses t$e re/ersible trans!er o! a 6$os6$ate grou6 !rom 6$os6$ocreatine to
3&P. T$is reaction is cou6led to t$ose catalysed by $e7o8inase ;HA= and glucose>'>6$os6$ate
de$ydrogenase ;G'P>&H=*
P$os6$ocreatine ? 3&P #A #reatine ? 3TP
3TP ? Glucose HA 3&P ? Glucose>'>6$os6$ate
G'P ? ,3&P
?
G'P>&H '>P$os6$ogluconate ? ,3&PH ? H
?
T$e rate o! ,3&PH !ormation% measured 6$otometrically% is 6ro6ortional to t$e catalytic concentration o!
#A 6resent in t$e sam6le
P-EPA-ATIO$
&issol/e 1 tablet o! ( 2 4ubstrate :it$ one /ial o! ( 1.
4tability* " days at 2>.I# or 24 $ours at room tem6erature ;1">2"I#=.
0TO-AGE A$% 0TABI.ITN
3ll t$e com6onents o! t$e 8it are stable until t$e e76iration date on t$e label :$en stored tig$tly closed
at 2>.I#% 6rotected !rom lig$t and contaminations 6re/ented during t$eir use.
&o not use t$e tablets i! a66ears bro8en.
&o not use reagents o/er t$e e76iration date.
2> Blan8 absorbance ;3= at 345 nm i 1.'5.
0A"P.E0
4erum or 6lasma* 4tability - days at 2>.I#% 6rotected !rom lig$t.
T$e creatin 8inase acti/ity decreases 15Y a!ter 1 day at 2>"I# or a!ter 1 $our at 1">2"I#.
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 nm
#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . . . .1 cm lig$t 6at$
2" > 35I#
B( ;mL=
4am6le ;cL=
14. i7% incubate !or 2 minutes.
minute ;k3<min=.
CA.C1.ATIO$0
k3 < min 7 412- M +<L #A
k3 < min 7 .51" M +<L #A
-E/E-E$CE BA.1E0

2"I# 35I# 3-I#
en% u6 to .5 +<L 135 +<L 11" +<L
Bomen% u6 to -5 +<L 115 +<L 1-5 +<L
5uantitati!e determination of creatine 3inase "B )CS#"B,
3n antibody to t$e anti #A> in$ibits com6letely #A> and subunit ;= o! t$e #A>B. T$e acti/ity o!
t$e non>in$ibited #A>B subunit is t$en assayed by t$e !ollo:ing series o! reactions*
P$os6$ocreatine ? 3&P #A #reatine ? 3TP
3TP ? Glucose HA 3&P ? Glucose>'>6$os6$ate
G'P ? ,3&P
?
G'P>&H '>P$os6$ogluconate ? ,3&PH ? H
?
T$e rate o! ,3&PH !ormation% measured 6$otometrically% is 6ro6ortional to t$e catalytic concentration o!
#A>B 6resent in t$e sam6le
#A>B is an en2yme !ormed by t$e association o! t:o subunits !rom muscle ;= and ner/e cells ;B=.
#A>B is usually 6resent in serum at lo: concentration0 it is increases a!ter an acute in!arct o!
myocardium and later descends at normal le/els. 3lso is increased% rarely% in s8eletal muscle damage.
laboratory data.
P-EPA-ATIO$
> Bor8ing reagent ;B(=
&issol/e one tablet o! ( 2 in one /ial o! ( 1.
4tability* . days at 2>.I# or 24 $ours at 1">2"I#.
2> Blan8 absorbance ;3= at 345 nmi 1.'5.
0A"P.E0
4erum or 6lasma* 4tability - days at 2>.I#% 6rotected !rom lig$t.
#A>B acti/ity decreases a 15Y a!ter 24 $ours at 4I# or 1 $our at 2"I#.
P-OCE%1-E
Ba/elengt$* . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 nm
#u/ette* . . . . . . . . . . . . . . . . . . . . .. . . . . 1 cm lig$t 6at$
B( ;mL= 1.
0
4am6le ;cL= 40
4. i7. 9ncubate !or 15 minute.
". (ead initial absorbance ;3= o! t$e sam6le% start t$e sto6:atc$ and read again a!ter " minutes ;32=.
'. #alculate t$e di!!erence bet:een absorbances * l3M 32 31.
CA.C1.ATIO$0
03 7 .2" M +<L de #A>B l3 7 1'"1 M +<L de #A>B
1nits: One international unit ;9+= is t$e amount o! en2yme t$at trans!orms 1 mol o! substrate 6er
Percentage of CS#"B acti!ity in sample*

#A>B 3cti/ity
Y #A>B 3cti/ity M 7 155
#A total 3cti/ity
-E/E-E$CE BA.1E0
Heart in!arct 6robability is $ig$ at t$e !ollo:ing conditions*
2"I# 35I# 3-I#
#A>B Q 15 +<L Q 1" +<L Q 24 +<L
#A>B acti/ity is bet:een ' and 2"Y o! total #A acti/ity.

Appendi( )+,
Collecti$e 3no"ledge of +ost Common #a0.ests
(lood .ests
Glucose: Glucose is the primary blood sugar test and indicates blood sugar le!el at the time
blood 8as dra8n> &igh !alues are seen in diabetics> In addition to pancreatic functionsD Glucose
may be altered by diet and medication> $ormal fasting !alue is ?;#++;>
/ructosamine: Indicates blood sugar le!els o!er the past one to three 8ee3s>
&GB A+C )Glycohemoglobin,: Indicates blood sugar acti!ity for the past three months>
B1$: B1$ stands for Blood 1rea $itrogen and is a 8aste product 8hich should be remo!ed
from the blood by the 3idneys> This test measures 3idney function> $ormal range is G#6;>
Creatinine: Creatinine is a 8aste product 8hich should be remo!ed from the blood by the
3idneys> This test measures 3idney function> $ormal range is ;>@#+>6>
A0AT<A.T: "aterial found in the li!er cells and muscle )heart, cells> %amage to these cells 8ill
increase !alues> $ormal range is +;#G;>
.%&: .%& is a material found in blood cells and li!er cells> Brea3do8n of the blood cells as in
heart disease or li!er damage may increase !alues> $ormal range is M+#+L;>
Al3aline Phosphorus: A material found in the blood related to li!er and bone> $ormal range for
adult males is 6;#+6@T normal range for adult females is =6#+6=>
0GOTD 0GPT: T8o measures of li!er functionT occasionally affected by muscle inury>
GGTP: The earliest li!er function to become abnormal>
Total Bilirubin: The le!el of pigment in the blood> Ele!ations can be associated 8ith li!er disease
or brea3do8n or red blood cells> 0light increases are sometimes seen 8ithout significance>
0ome people normally ha!e isolated ele!ations of bilirubin called GilbertUs disease> $ormal
range is +>;#+>6>
Total Protein: This is a combination of albumin and globulinD 8hich are proteins> Abnormal
!alues occur in li!er disease and poor nutrition> $ormal range is G>?#L>;>
Globulin: Globulin helps to combat infection on a normal le!el> It is the total protein !alue minus
albumin !alue> $ormal range is 6>9#=>;>
A<G -atio: Albumin !alue di!ided by the globulin !alue> $ormal range is ;>L#6>=>
Calcium: The most abundant mineral found in the human body> Abnormalities are found in loss
of boneD 3idney disease and lac3 of Bitamin %> $ormal range is L>@#+;>@>
Phosphorous: -elated to bone acti!ity and usually follo8s e(act opposite of calcium> $ormal
range is 6>@#=>G>
1ric Acid: A material 8hichD if in e(cessD can deposit stones in the 3idney or in the oints and
cause gout> $ormal range for males is =>;#?>;T normal range for females is 6>;#G>;>
Cholesterol: A blood fat related in part to eating animal fats such as eggsD cheeseD creamD li!erD
por3D beefD etc> Increased !alues may indicate a tendency to ha!e hardening of the arteries>
Balues of +L; or less are associated 8ith least ris3 of heart diseaseT in addition to diet and
e(ercise>
.ipoproteins: Proteins combined 8ith lipids that ser!e as carriers of cholesterol> .%. )EBadE
Cholesterol,T &%. )EGoodE Cholesterol,> The higher the !alueD the less li3ely that cholesterol
deposits are in the blood stream and the less li3ely the chance of coronary heart disease>
Cholesterol<&%. ratio measures the coronary ris3 factors>
Triglycerides: A blood fat related to calories and starch )s8eets, in the diet> &igh le!els can
impair circulation and lead to hardening of the arteries> Alcohol also 8ill increase the !alue> /ast
o!ernight test for accurate test results> $ormal range for males is =;#+G;T normal range for
feales is 9@#+9@>
"agnesium: An element absorbed in the intestine> Abnormal le!els are found in pancreatitisD
alcoholism and AddisonUs disease> $ormal range is +>L#6>=>
0ocium: A body saltD also termed electrolyte> Sidney disease and some diseases of the adrenal
gland and dehydration can cause abnormal results> $ormal range is +9@#+=@>
Potassium: A body salt or electrolyte found mostly inside of cells> ECater pillsE may lo8er
potassium and increase 3idney damage> $ormal range is 9>G#@>;>
Chloride: A body salt<electrolyteD it usually follo8s the same pattern as sodium> $ormal range is
+;+#+++>
Co6: Buffer system 8hich assists in the transport of carbon dio(ide from the tissue to the lungs>
$ormal range is 6+#9+>
&IB antibody: Presence of antibody is associated 8ith ha!ing been infected by the !irus 3no8n
to cause AI%0 )Ac7uired Immune %eficiency 0yndrome,>
P0A: Abnormal le!els in the serum are associated 8ith clinical abnormalities of the prostateD
including prostate cancer> Because P0A is found in normalD malignant and benign prostatic
tissueD clinical discrimination is based upon its serum le!el>
Complete (lood Count
CBC )Chite Blood Cells,: Chite blood count is the number of 8hite blood cells> It helps combat
infection> $ormal range is =>L#+;>L>
-BC )-ed Blood Cells,: -ed blood count is the number of red blood cells> It relates to anemia
and o(ygen transport> $ormal range for males is =>?#G>+T normal range for females is =>6#@>=>
&GB<&CT: &emoglobin is an iron#bearing protein 8hich is the red coloring matter found in blood>
$ormal range for males is +=#+LT normal range for females is +6#+G>
"C&<"CB<"C&C: "athematical relationship bet8een red blood count siFeD red blood count
number and hemoglobin concentration>
Platelets: Platelets deal 8ith hemostasis and blood coagulation> $ormal range is +9;#=;;>
-rine .ests
CBC )Chite Blood Cells,: Indicates possible infection of urinary tractD bladder or 3idney>
-BC )-ed Blood Cells,: Possible 3idney stoneD 3idney infection or tumor>
Casts: Possible 3idney infection or disease>
Glucose: 0ugar in the urineD possibility of glucose intolerance or lo8 renal threshold>
Protein: Possible 3idney infection or disease>
Appendi( )6,
Common Blood Profiles
(e!erence /alues !or t$e more commonly em6loyed laboratory tests are gi/en in t$e !ollo:ing table. T$e
re!erence /alues are in t$e units currently o!ten used and in t$e 9nternational 4ystem ;49= o! +nits.
Test Current units /actor 0I units
%iabetic 0creen
Glucose% !asting
'">115 mg<dl
5.5""
3."->'.5" mmol<L
Glucose % random
-1>1.5mg<dl 5.5"" 3.1>15.5 mmol<L
Glycosylated $emoglobin
; Hba1c =
"." > .."Y
&eart disease ris3 factors
)fasting lipids ,
Total #$olesterol
P255 mg<dl 5.52"1 P".2 mmol<L
H&L c$olesterol
Q3" mg<dl 5.52"1 Q5.1 mmol<L
L&L c$olesterol
P1"5 mg<dl 5.52"1 P3.1 mmol<L
Triglyceride
P25" mg<dl 5.5113 P2.3 mmol<L
Total c$olesterol<H&L ratio
P".. 1 P"..
.i!er function tests
Total Bilirubin
5.2">1." mg<dl 1-.1 4.3>2".' mol<L
&irect Bilirubin
5>5." mg<dl 1-.1 5 > . mol<L
9ndirect Bilirubin
5>5.1 mg<dl 1-.1 5>14 mol<L
Total Protein
'.">.." gm<dl 15 '">." gm<L
3lbumin
3.">4.. gm<dl 5.1"4 5."4 > 5.-4mmol<L
Globulin
2.5>3.1 g<dl 15 25>31 g<L
3lbumin<Globulin ratio
1>2." 1 1>2."
g >Glutamy trans6e6tidase
;GGT= >ale
11>"5 9+<L 1.'- O 15
>.
1.>.4 O 15
>.
Aatal<L
g >Glutamy trans6e6tidase
;GGT= >)emale
->3" 9+<L 12>". O 15
>.
Aatal<L
3l8aline P$os6$atase
4">12" 9+<L;u6 to
1555 9+<L in young
c$ildren=
1.'- O 15
>.
5.-">2.1 O 15
>.
Aatal<L
3lanine aminotrans!erase
;4GPT < 3LT=
">3" 9+<L 1.'- O 15
>.
..4 >". O 15
>.
Aatal<L
3s6artate aminotrans!erase
;4GOT< 34T=
">45 9+<L 1.'- O 15
>.
..4 >'- O 15
>.
Aatal<L
-enal<Sidney /unction Tests
+rea ;B+,=
.>2" mg<dl 5.3"- 2.1>..1 mmol<L
#reatinine
5.'>1.- mg<dl ...4 "3>1"5 mol<L
+ric acid
3.">..5 mg<dl 5.5"1 5.21>5.4- mmol<L
Potassium
3.3>4.1 mmol<L 1 3.3>4.1 mmol<L
4odium
13">14" mmol<L 1 13">14" mmol<L
Total #alcium
..1>15.3 mg<dl 5.2" 2.23>2."- mmol<L
)ree #alcium
4.">".5 mg<dl 5.2" 1.12>1.2" mmol<L
P$os6$ate
2.">4." mg<dl 5.323 5..>1." mmol<L
Other Common 0erum Chemistries<EnFymatic Acti!ities
3mmonia ;6lasma=
11>3" mol<L 1 11>3" mol<L
Blood gases ;arterial% :$ole
blood= > 6H
-.3">-.4" 1 -.3">-.4"
blood= > PO2
.5>15" mmHg 5.133 15.'>14.5 8Pa
blood= > P#O2
3" > 4" mmHg 5.133 4.->'.5 8Pa
blood= >#arbon dio7ide
content
22>31 mmol<L 1 22>31 mmol<L
p>#arotene
"5>355 g<dl 5.51.' 5.1>".' mol<L
#eru6lasmin
5.23>5.". gm<L '.- 1.">3.1 mol<L
#$loride
1">15" mEC<L 1 1">15" mmol<L
#o66er > ale
-5>145 g<dl 5.1"- 11.5>22 mol<L
#o66er > )emale
.">1"" g<dl 5.1"- 13.3>24.3 mol<L
#om6lement ;total% $emolytic=
11.>22'#H"5+<ml
#3
.1>1-" mg<dl 5.51 5..>1.-"gm<L
#4
12>34 mg<dl 5.51 5.12>5.34 gm<L
#reatinine #learence '5>125 ml<min
)erritin > #$ildren 13>14" ng<ml 2.2" 21>32' 6mol<L
)erritin >ale% adult 2">245 ng<ml "'>"45 6mol<L
)erritin > )emale% adult 12>135 ng<ml 2->212 6mol<L
)ibrinogen 1"5>3'5 mg<dl 5.51 1.">3.' gm<L
)olate > Plasma 1.->12.' ng<ml 2.2- 3.1>21 nmol<L
)olate > (ed cell 1"3>'52 ng<ml 34->13'- nmol<L
Ha6toglobin 155>355 mg<dl 5.51 1.15>3.55 gm<L
9mmunoglobulin > 9g3 31>3". mg<dl 5.51 5.31>3.". gm<L
9mmunoglobulin > 9g 33>221 mg<dl 5.51 5.33>2.21 gm<L
9mmunoglobulin > 9gG '-1>1"3- mg<dl 5.51 '.-1>1".3- gm<L
9ron > ale .5>1'5 g<dl 5.1-1 14.3>2..' mol<L
9ron > )emale '5>13" g<dl 15.->24.2 mol<L
9ron > Binding ca6acity 2"5>3"5 g<dl 44.->'2.' mol<L
9ron > Trans!errin saturation 1'>"-Y 1 1'>"-Y
Lactate ;6lasma= 5.3>1.3 mmol<L 1 5.3>1.3 mmol<L
agnesium
1.">2.1 mEC<L 5." 5.->1.1 mmol<L
Osmolality
2-5>215 mOsm<8g 1 2-5>215 mOsm<8g
Protein electro6$oresis >
3l6$a> 1> globulin
5.1>5." gm<dl 15 1>" gm<L
Protein electro6$oresis > 3l6$a
>2> globulin
5.3>1.2 gm<dl 15 3>12 gm<L
Protein electro6$oresis > Beta
globulin
5.->1.- gm<dl 15 ->1- gm<L
Protein electro6$oresis >
Gamma globulin
5.->1.- gm<dl 15 ->1- gm<L
Vitamin 3
35>1" g<dl 5.53" 1.5">3.32 mol<L
Vitamin B12
255>.55 6g<ml 5.-31 14.>"11 6mol<L
Other common serum enFymatic acti!ities
3ldolase
1.">..1 9+<L 1.'- O 15
>.
2.">13." O 15
>.
Aatal<L
3mylase
2">11" 9+<L 1.'- O 15
>.
42>112 O 15
>.
Aatal<L
#reatine 8inase > ale
+6 to 1." 9+<L 1.'- O 15
>.
+6 to 351O15
>.
Aatal<L
#reatine 8inase > )emale
+6 to 1"5 9+<L 1.'- O 15
>.
+6 to 2"1O15
>.
Aatal<L
Lactic de$ydrogenase
15>2"5 9+<L 1.'- O 15
>.
1"5>41- O 15
>.
Aatal<L
Li6ase
4>24 9+<dl 15 45>245 9+<L
"R > ,ucleotidase
2>1' 9+<L 1.'- O 15
>.
3>2- O 15
>.
Aatal<L
P$os6$atase% acid
+6 to 5.- 9+<L 1.'- O 15
>.
+6 to 1.2O15
>.
Aatal<L
Common 0erum &ormone Balues
3#TH% !asting ;. 3=
25>155 6g<ml 5.22 4.4>22 6mol<L
3ldosterone
15>1'5 ng<L 2.-- 2.>443 mmol<L
#ortisol ;6lasma% morning=
.>2" g<dl 5.52- 5.22>5."- mol<L
)4H > ale
+6 to 25 9+<L 1 +6 to 25 9+<L
)4H )emale > )ollicular
+6 to 25 9+<L 1
+6 to 25 9+<L
)4H )emale > Luteal
+6 to 1" 9+<L 1 +6 to 1" 9+<L
)4H )emale > idcycle
1">35 9+<L 1 1">35 9+<L
)4H )emale >
Postmeno6ausal
Q45 9+<L 1 Q45 9+<L
Gastrin% !asting
+6 to 135 6g<ml 1 +6 to 135 ng<L
Gro:t$ $ormone% !asting
P" ng<ml 1 P" g<L
1->Hydro7y6rogesterone >
Pre6ubertal
3>15 ng<dl
ale% adult
2->111 ng<dl
)emale > )ollicular
1">-5 ng<dl
)emale > Luteal
3">215 ng<dl
9nsulin% !asting ;-2 $r=
P15 m+<L 1 P15 m+<L
LH > ale +6 to 2" 9+<L 1 +6 to 2" 9+<L
LH > )emale >)ollicular +6 to 45 9+<L 1 +6 to 45 9+<L
LH > )emale > Luteal +6 to 2" 9+<L 1 +6 to 2" 9+<L
LH > )emale > idcycle "5>1"5 9+<L 1 "5>1"5 9+<L
LH > )emale >
Postmeno6ausal
Q35 9+<L 1 Q35 9+<L
Parat$yroid $ormone 2>15 +<ml 1 2>15 arb units
Progesterone > ale +6 to 155 ng<dl
Progesterone > )emale
>)ollicular
+6 to 1"5 ng<dl
Progesterone > )emale >
Luteal
2"5>2.55 ng<dl
Progesterone > )emale >1
st

trimester
1355>"555 ng<dl
Prolactin > ale 2>12> ng<ml 1 2>12 ug<L
Prolactin > )emale 2>25 ng<ml 1 2>25 ug<L
(enin acti/ity ;6lasma= 5.1>3.3 ng<ml<$r 5.2-. 5.2>5.1 ng<L.s
Testosterone% total > ale 2.5>1555 ng<dl 5.534' 15>3" nmol<L
Testosterone% total > )emale 25 >125 ng<dl 1>4 nmol<L
Testosterone% !ree > ale "2>2.5 6g<ml 5.5534' 5.1.>1.5 nmol<L
Testosterone% !ree > )emale 1.1>'.3 6g<ml 4>22 O 15
>
q nmol<L
T$yro7ine% total ; T4 = ".5>11.5 ug<dl 12.1 '4>142 nmol<L
T$yro7ine% !ree 1.5>2.3 ng<dl 12.1 13>35 6mol<L
T3 resin u6ta8e 3">4"Y 5.51 5.3">5.4" arb units
Triiodot$yronine ;T3= 155>21' ng<dl 5.51"4 1."4>3.23 nmol<L
T4 inde7 1.-">4.1" 1 1.-">4.1" arb units
T4H +6 to 15 +<ml 1 +6 to 15 m+<L
Vitamin &% 1%2" di$ydro7y 25>-' 6g<ml
Vitamin &% 2" $ydro7y 15>"" ng<m
Common 1rinary Chemistries
>aminole/ulinic acid 1.3>-.5 mg<day -.' 1.1>"3 mol<day
3mylase 5.54>5.35 9+<min 1 5.54>5.35 9+<min
#alcium P 2"5mg<day 5.52" P '.2" nmol<day
#atec$olamines P 13" g<day 1 +6 to 13" g<day
&o6amine 15>445 g<day
E6ine6$rine P 13 g<day "." +6 to -1 nmol<day
,ore6ine6$rine 11>.' g<day ".1 '">"5- nmol<day
#o66er 1">"5 g<day 5.51"- 5.2>5.-. mol<day
#ortisol% !ree 25>15 g<day 2.-' ""> 24. nmol<day
#reatinine > ale 1.5>2.5 gm<day 5.55.. 5.551>5.51. mmol<day
#reatinine > )emale 5.'>1." gm<day

5.55">5.513 mmol<day
"Hydro7yindoleacetic acid 1..>' mg<day ".3 1.">31.. mol<day
Hydro7y6roline% total 2">-- mg<day -.'3 111>".. mol<day
etane6$rine 5.3>1.5 mg<day
O7alate +6 to 45 mg<day -.13 P 31- mol<day
Por6$yrin>#o6ro6or6$yrin 1">12" g<day 1."3 23>111 nmol<day
Por6$yrin>+ro6or6$yrin P 35 g<day 1.2 +6 to 3' nmol<day
Protein P 1"5 mg<day 5.551 +6 to 5.1"5 gm<day
Vanillylmandelic acid ;V3= 5.">- mg<day ".5" 2.">3".3 mol<day
Common &ematologic 0tudies
#oagulation studies > Bleeding
time
2.">1." min '5 1"5>"-5 sec
#oagulation studies >Partial
t$rombo6lastin time
2">41 sec 1 2">41 sec
#oagulation studies >
Prot$rombin time
15..>13.5 sec 1 15..>13.5 sec
#oagulation studies >
T$rombin time
11.3>1.." sec 1 11.3>1.." sec
Hematocrit > ale 45.->"5.3Y 5.51 5.4>5."53 arb units
Hematocrit > )emale 3'.1>44.3Y 5.3'>5.44 arb units
Hemoglobin > ale 13..>1-.2 gm<dl 5.'2 .."'>15.- mmol<L
Hemoglobin > )emale 12.1>1".1 gm<dl -."5>1.3' mmol<L
Eryt$rocyte < (B# count >
ale
4.">".- O15
'
< l 15
'
4.">".- O 15rs<L
Eryt$rocyte < (B# count >
)emale
3.1> ".5O15
'
< l

3.1>".5 O 15rs<L
Leu8ocyte count 3..>1..O 15q< l 15
'
3..>1.. O 15
1
<L
Leu8ocyte 6ro!ile >
Lym6$ocytes
1.2>3.3O 15q< l
Leu8ocyte 6ro!ile >
ononuclear cells
5.1>5.-O15q< l
Leu8ocyte 6ro!ile >
Granulocytes
1..>'.'O 15q< l
Platelet count 115 >45" O15q< l 15
'
115>45" O 15
1
<L
Eryt$rocyte indices > ean
cor6uscular $emoglobin
2'.->33.- 6g<cell 5.5'2 1.''>2.51 !mol<cell
cor6uscular $emoglobin
concentration
32.->3"." gm<dl 5.'2 25.3>22.5 mmol<L
cor6uscular /olume
.5.5>1-.' cu .5.5>1-.' !l
Eryt$rocyte indices > (ed cell
distribution :idt$
11..>14.'Y

Eryt$rocyte 4edimentation
rate
+6 to 35 mm<$r 1 +6 to 35 mm<$r
(eticulocyte count
5.2>2.5Y
Practical 0heet *) ,
Practical E(periment: ...........................................................
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Obecti!es*
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Principle*
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-esults*
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CO""E$T0 2 51E0TIO$0
KKKKKKKK.

Diagnostic Biochemistry

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Diagnostic Biochemistry

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Practicals

Lab. Practical 1 Blood Glucose Estimation

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