1. Evaluation of Biochemical
Tests in Liver Disorders
By: Capt Arabinda Mohan Bhattarai
Guide: Col HS Batra

2. AIM
• To discuss the normal physiological function of liver.
• To evaluate abnormal liver function tests findings in liver
disorders.
• Approach to various liver disorders.

3. Introduction
• The liver has a central and critical biochemical role in
 metabolism
 digestion
 detoxification and
 elimination of substance from the body

4. DEGRADATION OF HEME TO BILIRUBIN
85% is derived from RBCs
In normal adults this
results in a daily load of
250-300 mg of bilirubin
Normal plasma conc is
less than 1 mg/dL
Hydrophobic – transported
by albumin to the liver for
further metabolism prior to
its excretion
“unconjugated” bilirubin

5. Bilirubin Metabolism

6. Excretory function
• Bilirubin:
 Orange-yellow pigment derived from heme, as a product of red blood
cell turnover.
 250-350 mg of bilirubin produced daily, 85% from RBCs.
Transported across the hepatocyte membrane, conjugated with
glucuronic acid and excreted into bile by an energy dependent process.

7. Serum Bilirubin – Estimation
• Principle: When diazotised sulfanilic acid reacts with bilirubin, it
forms ‘azobilirubin’, a purple coloured product measured
colorimetrically. This reaction is known as Van den Bergh reaction.
 Conjugated bilirubin
‘Direct positive’.
gives colour immediately (<1min)

Unconjugated bilirubin gives colour only after addition of
methanol ‘Indirect positive’(within 30 mins)

Both conjugated and unconjugated ‘Biphasic’(immediately
direct positive intensified by addition of alcohol indirect positive)

8. Fouchet’s Test
• Bilirubin in urine implies increased serum direct bilirubin and
excludes hemolysis as the cause
• Bile pigments adhere 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 and blue coloured cyanobilirubin forming pista green
colour.

9. Bile Salts
•
Source: cholesterol
• Primary bile acids: cholic and chenodeoxycholic acid.
• Metabolised by intestinal bacteria to secondary bile acids:
deoxycholic and lithocholic acid.
• Bile salts are Glycocholates and Taurocholates.
• Emulsification of Fatty acids

10. Detoxification function
•
Conversion of ammonia to Urea.
• Drug metabolism (Xenobiotics).
• Hippuric acid synthesis test

11. Synthetic Function
• Synthesis of Plasma proteins like Albumin, Transthyretin, Prothrombin
and Fibrinogen.
• Clotting factors except Von Willebrand factor and inhibitors of
coagulation, such as antithrombin.
• Post-translational carboxylation of (II, VII, IX and X) require vitamin
K, occurs within the hepatocyte

12. Synthetic Function
• Albumin:
 Synthesized exclusively by liver.
 Synthesis is inhibited by interleukin (IL)-6 in inflammatory
conditions.
 Decreased concentrations in cirrhosis, autoimmune hepatitis and
alcoholic hepatitis.
 Dye binding method (BCG) for estimation of albumin may give false
low values in patients with jaundice due to interference with bilirubin.

13. Synthetic Function
• Determination of total plasma proteins and A:G ratio.
• Severe or fulminant hepatic failure:
 concentration of short lived hepatic proteins (transthyretin and
prothrombin) fall quickly.
 minimal change in proteins with longer half lives.
• Decrease in fibrinogen levels <100 mg% seen in parenchymal liver
disease, acute hepatic necrosis.

14. Prothrombin Time
• It measures the activity of fibrinogen (I), Prothrombin (II) and factors
V, VII and X
• Prolonged PT indicates liver disease
• PT measures the time to clot after exposure of plasma to tissue factor.
• INR=[PT (patient)/PT (geometric mean of normal)]ISI

15. Prothrombin Time
• Prolonged due to lack of synthesis in hepatocellular disease or due to
lack of Vitamin-K absorption in obstruction
• Markedly prolonged PT indicates severe liver damage in hepatitis and
cirrhosis.
• Corrected within 24-48 hours by parenteral administration of vitamin
K (10mg/day for 3 days) in obstructive but not in hepatocellular
jaundice.

16. Carbohydrate metabolism
• Blood glucose levels maintained
 During short fasts by hepatic glycogenolysis.
 Prolonged fasts by hepatic gluconeogenesis
• Tests based on carbohydrate metabolism:
 Galactose tolerance test
• Hypoglycemia is a common complication in liver diseases like Reye’s
syndrome, fulminant hepatic failure and advanced cirrhosis

17. Lipid Metabolism
• Metabolism of cholesterol, synthesis, esterification, oxidation and excretion.
• Cholesterol-Cholesteryl ester ratio:
• Normal level- 150 mg to 250 mg, 60-70% as ester.
• Cholesterol endogenously synthesized in liver.
• Acute hepatic necrosis marked reduction in esters.
• Cirrhosis- decrease in HDL.
• Alcohol induced liver injury increase in HDL levels.

18. Serum Enzymes
• Plasma activities of several cytosolic, mitochondrial, and membrane
associated enzymes are measured.
• Ability of liver enzymes to assist in diagnosis depends on
 tissue specificity
 subcellular distribution
 relative activity of enzyme in liver and plasma
 patterns of release
 clearance from plasma.

19. AST( SGOT)
• Present in cytoplasm as well as mitochondria of hepatocytes.
• Mitochondrial isoenzyme represents a significant fraction of total AST within
hepatocytes, half lives of 87 hours.
• Require pyridoxal phosphate as cofactor.
• Plasma half lives of AST is 17 hours
• Upper reference range limits of 40 IU/L.
• Total cytoplasmic AST is present in highest activity in hepatocytes 7000 times higher
than plasma

20. ALT (SGPT)
• Plasma half life is 47 hours
• Liver specific activity in hepatocytes 3000 times higher than plasma
which is almost half of AST.
• Mitochondrial isoenzyme has very low half life making it insignificant
in diagnosis

21. Patterns of release
• In most forms of acute hepatocellular injury AST is higher than ALT
initially, due to higher activity of AST in hepatocytes
• Within 24-48 hours, if ongoing damage occurs ALT will become higher
than AST due to its longer half life.

22. Patterns of release
• In Alcoholic liver disease studies suggest that alcohol induces
mitochondrial damage.
• This causes release of mitochondrial AST which besides predominant
AST in hepatocytes has significant longer half life than
extramitochondrial AST and ALT causing AST:ALT ratio (De Ritis
Ratio of 3-4:1)

23. Activity
• AST/ALT ratio >2 with ALT <300 U/L suggestive of alcoholic
hepatitis.
• ALT more specific for liver disease.
• Greater increase in AST than ALT favor viral hepatitis, post hepatic
jaundice.

24. Alkaline Phosphatase
• Present in number of liver tissues including liver, bone, kidney and
intestine.
• Liver isoenzyme has half life of 3 days
• Normal range: 20-130 IU/L.
• Increased in cholestasis, obstructive jaundice.
• ALP and GGT are membrane bound glycoprotein enzymes found at
the canalicular membrane of hepatocytes

25. ϒ-Glutamyl Transferase
• Regulates the transfer of amino acids across cell membranes.
• If ALP and GGT are both elevated source is likely to be hepatic.
• Levels increased in about 60-70% chronic alcoholics.
• Normal Range: 5-40 IU/L

26. Mechanism of release
• Bile acids, solubilize the release of GGT and ALP from plasma
membrane.
• Ethanol, Phenytoin and Carbamazepine induce microsomal enzyme
synthesis lead to increase in GGT and ALP

27. 5’- Nucleotidase
• Increased in cholestatic disorders.
• No increase in activity in patients with bone disease
• Confirms the increase in ALP from hepatic source.

28. Lactate Dehydrogenase
• Cytosolic glycolytic enzyme catalyses the reversible oxidation of
lactate to pyruvate.
• Normal upper limit: 150 U/L
• Liver isoenzymes have half life of 4-6 hours and low activity (about
500) times than plasma.
• Space occupying lesions of liver, metastatic carcinoma lead to increase
in LDH> 500 IU/L and ALP> 250 IU/L.

29. Rate of clearance
• The half life of ALT is 47 hrs, cytoplasmic AST is 17 hrs.
• Liver isoenzyme of ALP is 3 days.
• GGT –10 days
• The removal of enzymes takes place by receptor- mediated endocytosis by
liver macrophages.
• Conjugated bilirubin binds covalently to albumin and is stays longer in the
blood.

30. Biliprotein/ Delta Bilirubin
• Formed by covalent attachment of Bilirubin monoglucuronide
with lysine residues of albumin or other proteins postsynthetically.
• Increased levels are markers of hepatic dysfunction.

31. Cirrhosis
• Earliest laboratory abnormalities are:
 fall in platelet count
 increase in PT
 decrease in albumin to globulin ratio <1
 increase in AST/ALT > 1
• End stage cirrhosis- massive tissue destruction, decrease in AST and
ALT.

32. Model for End Stage Liver Disease(MELD)
• MELD is calculated as
= 3.8[Ln Serum bilirubin (mg/dL)]+11.2 [Ln INR]+9.6 [Ln Serum Creatinine
(mg/dL)]+ 6.4
• Identify patients with advanced cirrhosis, candidates for liver
transplantation.
• Superior to Child-Pugh scoring in predicting short term survival
• Risk of death over 3 months is Low if score <10, intermediate if 10-20 and
high if >20

33. Viral Hepatitis
• Acute viral hepatitis is defined by the sudden onset of significant
aminotransferase elevation as a consequence of diffuse necroinflammatory
liver injury.
• This condition may resolve or progress to fulminant failure or chronic
hepatitis
• Chronic viral hepatitis is defined as the presence of persistent (at least 6
months) necroinflammatory injury that can lead to cirrhosis.
• Histopathologic classification of chronic viral hepatitis is based on etiology,
grade, and stage.

34. Features of viral hepatitis
Feature
HAV
HBV
HCV
HDV
HEV
Incubation (days) 15–45, mean 30
30–180, mean 60– 15–160, mean 50
90
30–180, mean 60– 14–60, mean 40
90
Onset
Acute
Insidious or acute Insidious
Insidious or acute Acute
Clinical
Severity
Mild
Occasionally
severe
Moderate
Occasionally
severe
Mild
Fulminant
0.1%
0.1–1%
0.1%
5–20%
1–2%
None
Occasional (1–
10%) (90% of
neonates)
Common (85%)
Common
None
Carrier
None
0.1–30%
1.5–3.2%
Variable
None
Cancer
None
+
+
±
None
Prognosis
Excellent
Worse with age,
debility
Moderate
Acute, good
Chronic, poor
Good
Progression to
chronicity

35. Algorithm for workup for Jaundice

36. Crigler-Najjar Syndrome
• Hereditary Glucuronyl Transferase Deficiency.
• Familial autosomal recessive disease (type I) and autosomal dominant
(type II)
• Indirect serum bilirubin is increased, appears first or second day of life
and persists for life.
• Type I complete enzyme deficiency, type II partial deficiency

37. Gilbert’s Syndrome
• Autosomal dominant
• Chronic, benign, intermittent, nonhemolytic and unconjugated
hyperbilirubinemia.
• Defective transport and conjugation of unconjugated bilirubin.
• Jaundice is accentuated by pregnancy, fever, exercise and various
drugs including alcohol.

38. Dubin-Johnson Syndrome
• Autosomal recessive disease.
• Conjugation of bilirubin-diglucuronide is normal
• Inability to transport bilirubin-glucuronide through hepatocytes into
canaliculi
• Symptoms: mild chronic recurrent jaundice and hepatomegaly
• Serum bilirubin (3-10 mg/dL rarely ≤ 30 md/dL), significant is direct.

39. Rotor’s Syndrome
• Autosomal recessive
• Asymptomatic, benign defective uptake and storage of conjugated
bilirubin, possibly in transfer of bilirubin from liver to bile.
• detected in adolescents or adults
• Jaundice accentuated by pregnancy, pills and alcohol
• Conjugated hyperbilirubinemia (<10 mg/dL)

40. Take Home Message
• Liver has central role in
metabolism, synthesis
and detoxification in
the body
• Interpretation of
liver function tests help in
early diagnosis of various
disorders.

41. References
• Tietze Textbook of clinical chemistry and molecular diagnostics-5th
Ed.
• Intepretation of Diagnostic Tests, Jacques Wallace-7th Ed.
• Textbook of Biochemistry with clinical correlations, Devlin-6th Ed
• Clinical Diagnosis and management by Lab methods- Henry 18th Ed

42. THANK YOU
Questions?