BY THE END OF THIS PRESENTATION YOU WILL BE ABLE TO ANSWER WHAT, WHY, WHICH ABOUT LIVER FUNCTION TESTS , WHAT IS JAUNDICE , METABOLISM OF HEME , FORMATION OF BILE PIGMENTS FROM HEME , TRASFER OF LILIRUBIN FROM BLOOD TO BILE , DETERMINATION OF SERUM BILIRUBIN, RETENTION JAUNDICE , REGURGITATION JAUNDICE ,DETERMINATION OF AMMONIA IN BLOOD ,ANTIPYRINE TEST, SERUM ENZYMES IN LIVER DISEASE, ASSESING EXTENT OF LIVER DAMAGE , DIAGNOSIS OF SUBCLINICAL JAUNDICE , BCG TEST , PLASMA PROTEINS , DETOXIFICATION FUNCTION OF LIVER

1. Liver Function Tests
Presented by: Moderator:
• Dr Premjeet Kaur
• Post Graduate Student
• Biochemistry Department
• SGRD Asr.
• Dr Shailja Gupta
• Associate Professor
• Biochemistry Department
• SGRD Asr.

2. Liver Function Tests
are useful in
• Detecting
• Diagnosing
• Evaluating severity
• Monitoring therapy
• Assessing the prognosis of liver diseases.

4. Functions of liver
Excretion Intermediary
metabolism
Synthesis Detoxification Storage
Bile pigment Carbohydrates Plasma proteins Ammonia Glycogen
Bile salts Amino acids Fibrinogen Antipyrine Vit A, K
Organic ions
like BSP
Conversion of
preprothrombin
to prothrombin

5. Tests based on excretory
function
1. Tests involving Bile Pigments (ie bilirubin & its
derivatives) in serum
2. Tests for Bile Pigments in urine
3. Tests using Bromosulphthalein

6. Globin
Mature RBC
Amino acid
pool
Heme
Iron
Biliverdin
Bilirubin
RE cells of spleenFerritin
Bilirubin +
albumin
Catabolism of heme produces bilirubin
Blood
Liver
After 120 days
85%
Ineffective
erythropoiesis
15%
Albumin
(Water soluble &
Unconjugated )Fe2+
(Water insoluble)
Metabolism of bilirubin
300mg/day

7. Blood Bilirubin +Albumin
( Unconjugated bilirubin )
Hepatocyte
Bilirubin
Bilirubin diglucuronide
( Conjugated bilirubin )
1. UPTAKE
2. CONJUGATION
•Crigler-Najjar syndrome
•Gilbert syndrome
Bile ductule
Bilirubin diglucuronide
3. SECRETION
Dubin-Johnson syndrome
Transfer of bilirubin from blood to bile
Glucuronyltransferase
INTESTINE

8. Bilirubin diglucuronide
Urobilinogen
beta
Glucuronidase
Bilirubin
Bacterial
Reduction
Stercobilinogen
(250-350 mg/day)
Reabsoption
Enterohepatic
Portal circulation
Urine
Urobilin
Stercobilin
Kidney
Faeces
Oxidation
Bile from
liver
Gut
20%
(4mg/day)

10. Determination of serum Bilirubin
1. Use of Diazo reaction :
bilirubin
diazotised sulphanilic acid
azobilirubin (2 molecules )
in acidic sol
red purple color
Direct Reaction results mainly in reaction of water
soluble conjugated bilirubin.To measure total bilirubin
a reagent termed as “accelarator “must be added.

11. contd
• Van den Bergh - used ethanol .
• Malloy and Evelyn -employed methanol .
• At present- caffeine or related substances like
dyphylline are used .
• Addition of alkaline tartrate increases
sensitivity by utilising enhanced absorption of
chromogen in alkaline solution .
• Most lab –use JENDRASSIK & GROF METHOD.
Bilirubin reacts with diazotized sulphanilic acid
the colour of which is measured at 530-560 nm
after 5min of incubation & is proportional to
bilirubin conc .

12. contd
Modern discrete analysers use 2,5 –
dichlorophenyldiazonium salt which is more
stable. A detergent is used as an accelerator .
• Among manual methods ,alkaine diazo methods
using sulphanilic acid are most popular.
• Interference by haemolysis :presence of
haemoglobin reduces the apparent bilirubin
concentrations. After splitting of bilirubin to
form one azobilirubin molecule and a dipyrole
the haemoglobin interfered with formation of
second molecule of azobilirubin .

13. •Right test tube showing haemolysis
Test should be repeated with clear sample .

14. •Contd.
Terms direct reacting and indirect reacting
bilirubin are related to but not completely
synonymous with conjugated and
unconjugated bilirubin .Pure unconjugated
bilirubin may give a colour in the direct reaction
upto 10 % of total bilirubin in some methods.
2. Spectrophotometric determination of
Native Bilirubin:
• Bilirubin in serum has an absorbance max
between 450 and 475 nm.
• The principal interfering substance is
oxyhaemoglobin
• Buffers like borate,phosphate etc are used.

15. contd
3.HPLC Methods for bilirubin :Overcomes the
problem of impurity and of different molecular
species present but stability of bilirubin on the
coloumn is doubtful .
• Bilirubin fractions in serum are separated by
gradient elution on reverse –phase coloumn
after removing globulins .
Fractions eluted were:
• unconjugated bilirubin (alpha )
• monoglucuronide(beta )
• diglucuronide(gama)
• another fraction(delta)

16. Contd
Delta fraction ->
• increase in all cases of conjugated
hyperbilirubinaemia and recovery phase of
hepatitis
• Low levels in normal subjects and in patients
with haemolytic disease .
NORMAL VALUES :
Bilirubin total = < 1 mg/dl
Direct (conjugated ) Bilirubin = o.o-o.25 mg/dl

17. Interpretation
Jaundice :a symptom; not disease
• Jaundice refers to yellow color of skin, nail
beds and sclerae
• Hyperbilirubinemia causes jaundice
• The increase may be due to excessive production
or decreased excretion of bilirubin.
• Icterus specifically is yellow discoloration of
eyes .
• Kernicterus is toxic bilirubin encephalopathy

18. contd

19. Jaundice is classified in several ways :
1. Nature of bile pigment
>unconjugated/conjugated
2. Anatomical site of disorder >pre hepatic ,
hepatic & post hepatic
3. Pathological cause>haemolysis, hepatocellular
or obstruction to bile flow
4. Nature of altered bilirubin metabolism>
• Retention of unconjugated bilirubin due to
excessive production
• Regurgitation of conjugated bilirubin due to
cholestasis

20. Retention jaundice : increased serum
unconjugated bilirubin without bilirubinuria
I. Prehepatic /haemolytic :
a) Congenital abnormalies of RBC-spherocytosis, abnormal Hb
like HbS & enzyme defects as G6PD def
b) Acquired haemolytic anaemias ,haemolytic diseases of newborn
& incompatible transfusion
c) Drugs-methyldopa
II. Defective metabolism in liver cell : It may be
a) Defective uptake : rifampicin
b) Defective conjugation :primaquine ,gilberts syndrome
,criglernajjar syndrome, physiological jaundice ,breast milk
jaundice

21. Regurgitation jaundice : Increase conjugate
bilirubin and bilirubinuria & stools may
become pale
I. Defective secretion :
a) Dubin Johnson syndrome ,
b) Rotor Syndrome &
c) familial non haemolytic jaundice
II. Intrahepatic cholestasis :
a) chronic active hepatitis c) biliary cirhosis
b) Lymphomas d) hepatoma
III. Extra hepatic cholestasis :
a) Gall stones

22. Mixed retention and regurgitation jaundice :
liver cell necrosis
• Disordered uptake, trasport & conjugation in
damaged cells ->retention
• Direct regurgitation of conjugated bilirubin from
damaged cells
CAUSES
• Viral Hepatitis A ,B ,nonA-nonB
• Drugs –CCL4 ,paracetamol

23. Interpretation of Hyperbilirubinemia
Acquired Congenital
1. UNCONJUGATED
HYPERBILIRUBINEMIA
(1)PHYSIOLOGICAL
a.jaundice of newborn.
b.breast milk jaundice
(2)HAEMOLYTIC jaundice
a.Newborn:haemolytic
disease of newborn
b.Adults
1.G6PD deficiency
2.Hereditary spherocytosis
3.Autoimmune haemolytic
anaemia
4.Toxins (CCLO4)
1. UNCONJUGATED :
• Gilberts syndrome
• Crigler -najjar syndrome
2. CONJUGATED;
• Dubin- Johnsons syndrome
• Rotor syndrome

24. 2.CONJUGATED HYPERBILIRUBINEMIA (Acquired)
INTRAHEPATIC EXTRAHEPATIC
1. Chronic hepatitis
2. Biliary cirrhosis
3. Lymphomas
4. Hepatoma
1. Gall stones
2. Carcinoma of head of
pancreas
3. Enlarged lymph glands

25. Familial hyperbilirubinneamias :genetically
determined disordered
Unconjugated Conjugated
• In 5% healthy population
without increased haemolysis
or hepatocellular disease
• Bilirubin range 17-50µmol/,
level persists ,no systematic
increase ,levels increase in
infection
1. Gilberts syndrome - most
common –defective
conjugation
2. Crigler-Najjar syndrome
• Type 1-absence of conjugation
• Type 2-severe deficiency of
conjugation
Jaundice mild
1. Dubin-Johnson
syndrome –
• cojugation normal ,
• defective transport into bile
capillaries
• accumulation of brown
pigment –lipofuscin
2. Rotor syndrome –same
but without other features

26. Neonatal jaundice
A. If unconjugated bilirubin present –
• Physiological jaundice -retention jaundice due to
temporary insufficient glucuroyl transferase
activity esp in premature , max around 5 days
• Rhesus isoimmunisation – haemolytic
• Breast feeding jaundice-due to pregnanediol
B. If conjugated bilirubin present-
• Septicaemia –most common
• Biliary atresia

27. Treatment of Physiological Jaundice
Phototherapy Eyes and genital parts covered

28. Tests for Bile Pigments in Urine
1. BILIRUBIN :
Fouchets test
Ictotest
Reagent strips
2. UROBILINOGEN
Ehrlichs test
Reagent strips

29. Tests using Bromosulphathalein
• Very sensitive test ,abnormal before occurance of jaundice .
• Tests the transport ,conjugation and excretory abilities of the
liver .
• BSP excretion test : I/V injection of a single bolus of a
solution of BSP (50gm%) and measurement of serum
concentration 45min later .
• Determination of BSP: BSP is purple in alkaline solution and
colourless in acid .The difference in absorbance at 580nm in
acid and alkaline solution is a measure of BSP.
• Interpretation :In normal retention after 45min is less than
5%.
• Higher levels found in old age ,cholestasis or impairment of
liver function.

30. Tests based on Intermediary
metabolic function
1. Galactose elimination tests
2. Serum amino acids in liver
disease

31. Galactose Elimination Test
• Method Galactose in blood is measured using galactose
oxidase. Alternatively production of NADH by aciton of
galactose dehydrogenase on galactose can be measured
using commercial reagents (Boehringer) and read at
340nm.
• 350mg galactose/kg body wt given i/v after overnight fast.
A morning samlpe is collected for control . Blood samples
are collected after 5min at ½ hourly interval for 2hrs .
• Interpretation:
a)In normal the curve falls steeply within one hour
b)In obstructive jaundice similar results are obtained .
c)In parenchymal diseases with liver cell damage the fall in
bld galactose takes place more slowly.

32. Serum amino acids in liver disease
• An abnormal plasma amino acid profile is found
in hepatic coma .
• Ratio of aromatic amino acids (phenylalanine,
tyrosine and methionine )to branched amino
acids (leucine, isoleucine and valine) has shown
some prognostic value in chronic active hepatitis
• Methods :a)Automated colorimetric assay for
methionine in assesing progress of acute liver
failure .b)Analysis of plasma amino acids by ion-
exchange chromatography or more recently
HPLC.

33. Tests based on the synthetic
function
•Plasma proteins synthesis

34. contd
1. Electrophoretic separation of plasma proteins
normal subjects liver diseases
• albumin……........ 55.2%........decreased in cirrhosis
• globulin…………….44.8%
α1-globulin..….1.23%
α2-globulin..….5.3%
β-globulin…..…13.4%
γ-globulin….….11.0%........increased in hepatitis
• fibrinogen………...6.5% ……..decreased in necrosis

35. 2) Flocculation tests
a. Thymol turbidity test:Degree of turbidity is measured
against standards when serum is mixed with buffered
solution of thymol.
normal:0-4 units
infective hepatitis:5-20 units
obstructive jaundice:only 8%give +ve result
thymol floccution test :after turbidity keep tubes in dark
& read floccution if any .Its graded as –ve,+ve,++ ,+++
b) Zinc sulphate turbidity test :is done for γ-globulin
Serum is diluted with ZnSOч ,amount of turbidity
develops is proportional to γ-globulin .
NV=2-8 units ,increased in infective hepatitis ,+ve in
other cases of increased γ -globulin

36. 3)BCG dye method:Albumin is the major plasma
protein regulating distribution of extracellular fluid
• Principle/Method :Albumin binds
Bromocresol green at ph 4.2 .The blue green
colour formed is proportional to conc of albumin
which is read at 625nm.
• NV=3.8-5.0 mg/dl
• Interpretation:
Increased :dehydration
Decreased :excessive protein loss ,decreased
synthesis due to dietry def. ,increased catabolism
in fever etc

37. Levels of plasma proteins & clinical significance in liver
Plasma proteins &
Normal values
Decreased Increased
1. Albumin =
3.8-5.0 gm/dl
Acute phase of Reaction
to trauma or infection
2. albumin/globuin =
2:1
Advanced parenchymal
liver disease
3. Fibrinogen=
200-400mg%
Severe liver disease Infection and acute
phase reaction
4. Prothrombin
P Time=10-16sec
1.Dependent on Vit K
availability which is
decreased in Ostructive
conditions
2.Hepatocellular damage
5. Alpha –Fetoprotein
<1μg/100ml
Primary hepatoma
6.Ceruloplasmin =
25-45mg/dl
Wilsons disease

38. Tests based on detoxification
function
Blood ammonia
Aminopyrine

40. Determination of Ammonia in blood and plasma
• Blood ammonia levels help in monitoring deterioration or
improvement in patient with acute hepatic failure .
• Enzymatic kit method: employing glutamate
dehydrogenase
GLDH
Alpha –Oxoglutarate+NH4+ +NAD(P)H ----L-Glutamate+NAD(P)H+H2O
• Disappearance of NAD(P)H is measured
spectrophotometrically.
• Normal levels :40-75µg/100ml blood
• Interpretation :Increased in parenchymal liver disease like
cirrhosis and hepatic coma

42. Antipyrine breath test
• Antipyrine is metabolised in liver by N-
demethylation to give CO2 .
• Method :Overnight fast, 2 mg labelled
aminopyrine is given orally .Dried breath is
bubbled through a solution which changes color
in presence of CO2.
• Interpretation CO2 excretion is reduced in
parenchymal liver disease like
cirrhosis,hepatitis and neoplasia

43. Serum enzymes in liver disease
Aminotransferases
Alkaline phosphatase
Gammaglutamyl transpeptidase
Others;5-nucleotidase,cholinesterase

44. Alanine aminotransferases
ALT
L-Alanine +α-Ketoglutarate ----------Pyruvate +L-Glutamate
LDH
Pyruvate +NADH + H+ -------------L-Lactate + NAD+
Aspartate aminotransferases
AST
L-aspartate + α-Ketoglutarate ---------Oxaloacetate + L-glutamate
MDH
Oxaloacetate +NADH+H-----------L-malate + NAD+
NAD+ formed is read at 340 nm after 1 min
incubation

45. Normal value: ALT/SGPT=upto 46U/L
:AST/SGOT=upto 49U/L
• Both ALT & AST increase in parenchymal liver
cell damage .
• ALT>AST In viral & toxic hepatitis
• AST>ALT In alchoholic hepatitis & cirrhosis

46. Alkaline phosphatase
• Isozyme of liver origin arises from lining of bile canaliculi &
sinusoidal surfaces of hepatocytes
• PNPP Method measures the yellow color devoloped by p-
nitrophenol at 405nm
ALP
• p-Nitrophenyl phosphate +H2O------------p-nitrophenol +Phosphate
(yellow)
• Normal levels :60-170U/L(25c) (adults)
151-471U/L(25c)(children)
• Interpretation -Levels increase in post hepatic obstruction .
physiological increase is seen in pregnancy

47. Gammaglutamyl transpeptidase
γGT
γGlutamyl p-Nitroanilide +Glycylglycine -----------
LγGlutamyl-Glycylglycine + p-nitroaniline.
P-nitroaniline absorbs light at 405 nm
Normal levels :men:9-52IU/dl (37C)
women:5-32IU/dl (37C)
Interpretation :Increased in post hepatic obstruction
In differentiating bone & liver disorder

48. Others
1. 5 –nucleotidase:
5-nucleotidase
Adenosine 5-P------------------ Adenine + i-P
NV = 2-17 IU/L
Interpretation Raised in obstructive biliary tract disease
ADV:over ALP is that it is not affected in bone diseases
2.Cholinesterase :Hydrolyse esters of choline to give choline & acid
2 Types 1.True cholinestrase found nerve tissue
2.psuedo cholinestrase found in various tissues
NV = 2.17-5.17IU/dl
Interpretation :reduced in liver cell damage .

49. contd
3. Serum lactate dehydrogenase (LDH)
LDH
Pyruvate + NADH + H+ --------- Lactate +NAD+
Rate of oxidation of NADH+ to NAD+ is measured as decrease in
absorbance which is proportional to LDH activity
NV=70-240 IU/L
Dis ADV: LDH is less-specific as it is widely distributed cardiac &
skeletal muscle ,liver ,kidney & RBC
Isozymes of LDH are separated on basis of electrophoretic mobility .
Interpretation :In liver diseases increased activity is found in
infectious hepatitis

50. Contd
4. Serum Iso-citrate Dehydrogenase (ICD): found in liver only .
NV= 0.9-4.0 IU/L
Levels increase in infectious hepatitis & normal in obstructive
jaundice .
5.Serum Ornithine Carbamoyl transferase (OCT)
Ornithine + Carbamoyl-P--------citruline + PO4
NV= 8 -20 mIU
Interpretation:increased in viral hepatitis
Serum OCT appears to be specific and sensitive measure for
hepatocellular injury as enzyme is exclusively found in liver
6.Serum Leucine Amino Peptidase (LAP):splits N-terminal residue
from certain L-peptides & amides having free NH2 gp.
NV= 15-56 mIU

51. Contd
Interpretation :increased in viral hepatitis,marked rise in
Hepatoma
7.Serum hydoxy Butyrate Dehydrogenase (SHBD)
NV = 56-125 IU/l
Inerpretation :increased in viral hepatitis
8. Serum Aldolase and Phosphohexose Isomerase : Both
are markedly increased in acute hepatitis.
9. Serum Amylase :low levels in Liver diseases like
infectious hepatitis
10.Serum Sorbitol Dehydrganase (SDH):NV=<0.2mIU
Sorbitol + NAD+ <-----------------> fructose +NADH
Interpretation :increased in Acute viral hepatitis

52. Use of liver tests in liver diseases
1. Differential diagnosis of jaundice: Biochemical
tests such as serum bilirubin & enzymes helps
distinguish
Between haemolytic jaundice ,acute hepatitis or post
hepatic obstruction
Between extrahepatic cholestasis for which surgery is
usual course of action and intrahepatic cholestasis
for which medical treatment is appropriate
2. Assesing extent of liver damage:
Fall in serum albumin & rise in Igs gives an indication
of extent of liver damage .

53. contd
3. Diagnosis of sub-clinical liver disease :
Liver has considerable functional reserve and
regenerating capacity in chronic liver disease.
Biochemical tests can indicate the default even
before the occurance of clinically apparent
disease .
BSP test is considered most sensitive test
x – x – x

54. THANQ?