1LIVER FUNCTION TESTS

A clinician noticed pallor & icterus on a 12 year old boy who came to OPD. General physical examination revealed the boy was poorly built and nourished. On abdominal examination, boy had spleenomegaly with ulcers around ankle.

What is your probable diagnosis? Suggest the biochemical investigations to

confirm your diagnosis.

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Classification – Liver Function Test Based on Liver Function & Clinical Utility

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Group I Markers of liver dysfunction

Serum bilirubin – Total and ConjugatedUrine – Bile salts, Bile pigments, Urobilinogen

Group II Synthetic function Total protein, Albumin and A/G ratioProthrombin timeSerum Cholesterol

Group III Markers of hepatocellular injury

Alanine transaminase (ALT)Aspartate transaminase (AST)

Group IV Markers of obstructive liver disease

Alkaline phosphataseGamma glutamyl transferase

Group V Metabolic capacity Blood ammoniaGalactose tolerance test

Group VI Excretory & Detoxifying capacity

Bromsulfthalein excretory testHippuric acid test

Tests based on Excretory capacity of liver

Bromsulpthalein (BSP) test Dye used to assess excretory function of the liver Non-toxic compound exclusively excreted through

bile Dose: 5mg/Kg body weight Administration: Intravenously Procedure: Serum concentration is measured at 45

min and at 2 hours Result: > 5% of dye retained abnormality in

excretory function of the liver4

Tests based on Detoxifying capacity of liver

Hippuric acid test Hippuric acid synthesis assess detoxification function Benzoic acid + Glycine = Hippuric acid Procedure:

- 6g sodium benzoate dissolved in 250ml water is given orally 2hrs after light breakfast and after emptying bladder- Urine is collected for next 4 hours- Amount of hippuric acid excreted is estimated- Normal: > 4.5 g of hippuric acid (60% of sodium benzoate)- Abnormal: < 3g indicates hepatic dysfunction

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Serum Bilirubin Excretory product Formation: Catabolism of heme Types:

- Unconjugated: water insoluble- Conjugated: water soluble

Estimation: ‘van den Bergh’ reaction Reagents:

- Diazotised sulfanilic acid: Sulfanilic acid in hydrochloric acid + sodium nitrite- Methanol

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Serum Bilirubin – Estimation Principle: When diazotised sulfanilic acid

reacts with bilirubin, it forms ‘azobilirubin’, a purple coloured product

Conjugated bilirubin gives colour immediately ‘Direct positive’

Unconjugated bilirubin gives colour only after addition of methanol ‘Indirect positive’

Both conjugated and unconjugated ‘Biphasic’

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Serum Bilirubin – Applications

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Type of Bilirubin

van den Bergh Reaction

Class of Jaundice

Causes

Unconjugated Indirect positive Pre-hepatic or Hemolytic

Neonatal jaundiceSickle cell anaemiaThalassemiaGilbert’s syndromeCrigler-Najjar syndromeMismatch blood transfusion

Both Unconjugated and Conjugated

Biphasic Hepatic Viral hepatitisAlcoholic hepatitisDrug induced hepatitis

Conjugated Direct positive Post-hepatic or Obstructive

Gall stonesBiliary atresiaTumours of biliary tractCarcinoma head of pancreas

Urine – Urobilinogen Ehrlich’s test Principle: Urobilinogen reacts with p-

dimethylamino-benzaldehyde in chloroform to form a pink coloured aldehyde complex.

Reagents:- Ehrlich’s reagent- Saturate sodium acetate- Chloroform

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Urine – Urobilinogen Procedure: 5 ml Urine + 5 ml Ehrlich’s reagent Mix and

allow to stand for 10 min + 5 ml saturated sodium acetate and mix + 5 ml chloroform shake vigourously and allow layers to separate

Appearance of pink colour in the chloroform layer indicates presence of urobilinogen

Colour is easily detected when viewed from top of the test tube

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Urine – Bile salts & Bile pigments Bile salts: Hay’s test Principle: Bile salts have the property of

lowering surface tension. Hence when sulphur poweder is sprinkled to urine containing bile salts, it sinks to the bottom.

Bile pigments: Fouchet’s test Principle: Bile pigments adhare to the

precipitate of barium sulphate. On addition of Fouchet’s reagent, ferric chloride in the presence of trichloroacetic acid oxidises yellow colour bilirubin to green colour biliverdin.

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A 45 year female patient was admitted to casualty with the complaints of severe pain in the right hypocondriac region, decreased appetite and vomiting. She also complained of pruritis. General physical examination revealed that the female was obese. Per abdomen examination showed tenderness in right hypochondriac region.

What is your probable diagnosis? Suggest the biochemical investigations to

confirm your diagnosis.12

Urinary findings in Jaundice

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Type of Jaundice

Urobilinogen Bile salts Bile pigments

Pre-hepatic or Hemolytic

+++ NIL NIL

Hepatic Normal / + ++

Post-hepatic of Obstructive

NIL +++ ++++

Tests based on synthetic function Seum total proteins: estimated by ‘Biuret method’ Serum alboumin: estimated by BCG-dye binding

method Total proteins – Albumin = Globulin Calculation of Albumin/Globulin ratio Prothrombin Time

- ½ life of prothrombin is 6 hours- indicates present functioning status of liver

Serum cholesterol: estimation by ‘cholesterol oxidase’ method

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Application of tests based on synthetic functions of liver

- Serum albumin levels- Serum total protein levels- Reversal of A/G ratio- Serum total cholesterol levels- Prolonged prothrombin time

Chronic diseases of liver and in cirrhosis of liver

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Enzymes indicating Hepatocellular damage and Obstructive liver disease

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Name of Enzyme Diseases associated with increase

Alanine transaminase (ALT) Acute viral hepatitisAlcoholic liver diseaseAutoimmune hepatitisChronic viral hepatitisNon-alcoholic steatohepatitis

Aspartate transaminase (AST)

Alkaline Phosphatase Cholestatic jaundiceHepatic carcinomaGall stonesBiliary atresiaTumours of Biliary tractCarcinoma head of the pancreas

Gamma Glutamyl transferase Chronic alcoholism

5’ Nucleotidase Biliary obstruction

Tests based on Metabolic capacity of liver

Blood Ammonia levels Index of urea synthesis by the liver Sample: Arterial blood Accumulation of ammonia levels in CNS

coma Use: Hepatic encephalopathy

Galactose tolerance test

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Liver Function Test - Summary

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Estimation of serum bilirubin van den Bergh reaction

Conjugated bilirubin , alkaline phosphatase with bile salts and bile pigments

Obstructive jaundice

Estimation of urobilinogen Ehrlich’s test

Urobilinogen Hemolytic jaundice

Markers of Hepatocellular damage ALT & AST

Marker of alcoholic liver disease Gamma glutamyl transferase

Tests to detect synthetic functions of liver Serum albuminProthrombin time

Test to assess excretory function of liver Bromsulphthalein Test

Test to assess detoxification function of liver Hippuric acid test

Blood ammonia levels Hepatic encephalopathy

Examination Tips• Classify liver function tests. Describe any three

tests with its clinical importance.

• van den Bergh reaction• Test based on synthetic functions of the liver• Enzyme markers for liver disease

• Ehrlich’s test• Bromsulpthalein test• Hippuric acid test

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