The patient, a 45-year-old female, presented with right upper quadrant pain, decreased appetite, vomiting, and pruritis. On examination, she was obese and the right hypochondriac region was tender. The probable diagnosis is obstructive jaundice. Biochemical investigations to confirm include elevated conjugated bilirubin and alkaline phosphatase on liver function tests, and bile salts and bile pigments in the urine.

1. LIVER FUNCTION TESTS 1

2.  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.
2

3. Classification – Liver Function Test
 Based on Liver Function & Clinical Utility
Group I Markers of liver Serum bilirubin – Total and
dysfunction Conjugated
Urine – Bile salts, Bile pigments,
Urobilinogen
Group II Synthetic function Total protein, Albumin and A/G ratio
Prothrombin time
Serum Cholesterol
Group III Markers of hepatocellular Alanine transaminase (ALT)
injury Aspartate transaminase (AST)
Group IV Markers of obstructive Alkaline phosphatase
liver disease Gamma glutamyl transferase
Group V Metabolic capacity Blood ammonia
Galactose tolerance test
Group VI Excretory & Detoxifying Bromsulfthalein excretory test
capacity Hippuric acid test 3

4. 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 liver
4

5. 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
5

6. 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
6

7. 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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8. Serum Bilirubin – Applications
Type of van den Class of Causes
Bilirubin Bergh Jaundice
Reaction
Unconjugated Indirect positive Pre-hepatic or Neonatal jaundice
Hemolytic Sickle cell anaemia
Thalassemia
Gilbert’s syndrome
Crigler-Najjar syndrome
Mismatch blood transfusion
Both Biphasic Hepatic Viral hepatitis
Unconjugated Alcoholic hepatitis
and Conjugated Drug induced hepatitis
Conjugated Direct positive Post-hepatic or Gall stones
Obstructive Biliary atresia
Tumours of biliary tract
Carcinoma head of pancreas
8

9. 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
9

10. 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
10

11. 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.
11

12.  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

13. Urinary findings in Jaundice
Type of Urobilinogen Bile salts Bile pigments
Jaundice
Pre-hepatic or +++ NIL NIL
Hemolytic
Hepatic Normal / ↓ + ++
Post-hepatic of NIL +++ ++++
Obstructive
13

14. 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
14

15. 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
15

16. Enzymes indicating Hepatocellular damage
and Obstructive liver disease
Name of Enzyme Diseases associated with increase
Alanine transaminase (ALT) Acute viral hepatitis
Aspartate transaminase (AST) Alcoholic liver disease
Autoimmune hepatitis
Chronic viral hepatitis
Non-alcoholic steatohepatitis
Alkaline Phosphatase Cholestatic jaundice
Hepatic carcinoma
Gall stones
Biliary atresia
Tumours of Biliary tract
Carcinoma head of the pancreas
Gamma Glutamyl transferase Chronic alcoholism
5’ Nucleotidase Biliary obstruction 16

17. 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
17

18. Liver Function Test - Summary
Estimation of serum bilirubin van den Bergh reaction
↑Conjugated bilirubin , ↑ alkaline Obstructive jaundice
phosphatase
with bile salts and bile pigments
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 albumin
Prothrombin 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
18

19. 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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