In Macrophages mainly in spleen, methemoglobin from red cells split to give free globin chains and heme. The porphyrin of heme is oxidized by the microsomal hemeoxygenase, producing the straight chain compound biliverdin and releasing iron. Biliverdin is reduced to bilirubin by NADPH dependent enzyme, biliverdinreductase. Bilirubin bound mainly to albumin transported to portal system to the liver where it enters the hepatocyte.
Organic componds of both endogenous and exogenous origin are extracted from the sinusoidal blood, biotransformed and excreted into the bile or urine.Bilirubin is transported from sites of production spleen, loosely bound to albumin, unconjugated form
provide surface active detergent molecule
Normal fibrinogen level 200-400 mg%. Patterns depend on the type, severity and duration of liver injury. In cirrhosis besides hepatocyte destruction, portal hypertension is the cause of diminished protein production by decreased delivery of amino acids to liver
ISI international sensitivity index
Galactose is a monosaccharide almost exclusively metabolized by the liver. Subject is given iv galactose about 300mg/day and blood is drawn at 10 minutes interval for the next 2 hours and galactose is estimated. Half life of galactose is 10-15 mins. markedly elevated in hepatocellular damage
Obstructive jaundice no change. Hepatocellular jaundice decreases. Acute hepatic necrosis marked reduction in esters.HDL levels increase by the expression of apolipoproteinapo A-I protein.
and ALT 47, ALT activity in hepatocytes is 300 times than plasma.
ALT is typically higher than AST because of slower clearance
Because of higher activity of AST in hepatocytes.
It catalyses the transfer of a glutamyl group from glutathione to a free amino acid
Model for End Stage Liver Disease
Model for end stage liver disease
Approach to evaluation of liver disorders
Evaluation of Biochemical
Tests in Liver Disorders
By: Capt Arabinda Mohan Bhattarai
Guide: Col HS Batra
• To discuss the normal physiological function of liver.
• To evaluate abnormal liver function tests findings in liver
• Approach to various liver disorders.
• The liver has a central and critical biochemical role in
elimination of substance from the body
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
Orange-yellow pigment derived from heme, as a product of red blood
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.
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.
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)
• 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
• 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
Conversion of ammonia to Urea.
• Drug metabolism (Xenobiotics).
• Hippuric acid synthesis test
• Synthesis of Plasma proteins like Albumin, Transthyretin, Prothrombin
• 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
Synthesized exclusively by liver.
Synthesis is inhibited by interleukin (IL)-6 in inflammatory
Decreased concentrations in cirrhosis, autoimmune hepatitis and
Dye binding method (BCG) for estimation of albumin may give false
low values in patients with jaundice due to interference with bilirubin.
• 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.
• 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
• 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
• Corrected within 24-48 hours by parenteral administration of vitamin
K (10mg/day for 3 days) in obstructive but not in hepatocellular
• 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
• 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.
• Plasma activities of several cytosolic, mitochondrial, and membrane
associated enzymes are measured.
• Ability of liver enzymes to assist in diagnosis depends on
relative activity of enzyme in liver and plasma
patterns of release
clearance from plasma.
• 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
• 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
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.
Patterns of release
• In Alcoholic liver disease studies suggest that alcohol induces
• 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)
• AST/ALT ratio >2 with ALT <300 U/L suggestive of alcoholic
• ALT more specific for liver disease.
• Greater increase in AST than ALT favor viral hepatitis, post hepatic
• Present in number of liver tissues including liver, bone, kidney and
• 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
• 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
Mechanism of release
• Bile acids, solubilize the release of GGT and ALP from plasma
• Ethanol, Phenytoin and Carbamazepine induce microsomal enzyme
synthesis lead to increase in GGT and ALP
• Increased in cholestatic disorders.
• No increase in activity in patients with bone disease
• Confirms the increase in ALP from hepatic source.
• 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.
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
• Conjugated bilirubin binds covalently to albumin and is stays longer in the
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.
• 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
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
• Identify patients with advanced cirrhosis, candidates for liver
• 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
• Acute viral hepatitis is defined by the sudden onset of significant
aminotransferase elevation as a consequence of diffuse necroinflammatory
• This condition may resolve or progress to fulminant failure or chronic
• 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.
Features of viral hepatitis
Incubation (days) 15–45, mean 30
30–180, mean 60– 15–160, mean 50
30–180, mean 60– 14–60, mean 40
Insidious or acute Insidious
Insidious or acute Acute
10%) (90% of
Worse with age,
• Hereditary Glucuronyl Transferase Deficiency.
• Familial autosomal recessive disease (type I) and autosomal dominant
• 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
• Autosomal dominant
• Chronic, benign, intermittent, nonhemolytic and unconjugated
• Defective transport and conjugation of unconjugated bilirubin.
• Jaundice is accentuated by pregnancy, fever, exercise and various
drugs including alcohol.
• Autosomal recessive disease.
• Conjugation of bilirubin-diglucuronide is normal
• Inability to transport bilirubin-glucuronide through hepatocytes into
• Symptoms: mild chronic recurrent jaundice and hepatomegaly
• Serum bilirubin (3-10 mg/dL rarely ≤ 30 md/dL), significant is direct.
• 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)
Take Home Message
• Liver has central role in
and detoxification in
• Interpretation of
liver function tests help in
early diagnosis of various
• Tietze Textbook of clinical chemistry and molecular diagnostics-5th
• 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