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Evaluation of Biochemical
Tests in Liver Disorders
By: Capt Arabinda Mohan Bhattarai
Guide: Col HS Batra
AIM
• To discuss the normal physiological function of liver.

• To evaluate abnormal liver function tests findings in live...
Introduction
• The liver has a central and critical biochemical role in
 metabolism
 digestion

 detoxification and
 e...
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 Metabolism
Excretory function
• Bilirubin:
 Orange-yellow pigment derived from heme, as a product of red blood
cell turnover.

 250...
Serum Bilirubin – Estimation
• Principle: When diazotised sulfanilic acid reacts with bilirubin, it
forms ‘azobilirubin’, ...
Fouchet’s Test
• Bilirubin in urine implies increased serum direct bilirubin and
excludes hemolysis as the cause
• Bile pi...
Bile Salts
•

Source: cholesterol

• Primary bile acids: cholic and chenodeoxycholic acid.
• Metabolised by intestinal bac...
Detoxification function
•

Conversion of ammonia to Urea.

• Drug metabolism (Xenobiotics).

• Hippuric acid synthesis tes...
Synthetic Function
• Synthesis of Plasma proteins like Albumin, Transthyretin, Prothrombin
and Fibrinogen.
• Clotting fact...
Synthetic Function
• Albumin:

 Synthesized exclusively by liver.
 Synthesis is inhibited by interleukin (IL)-6 in infla...
Synthetic Function
• Determination of total plasma proteins and A:G ratio.
• Severe or fulminant hepatic failure:
 concen...
Prothrombin Time
• It measures the activity of fibrinogen (I), Prothrombin (II) and factors
V, VII and X
• Prolonged PT in...
Prothrombin Time
• Prolonged due to lack of synthesis in hepatocellular disease or due to
lack of Vitamin-K absorption in ...
Carbohydrate metabolism
• Blood glucose levels maintained
 During short fasts by hepatic glycogenolysis.
 Prolonged fast...
Lipid Metabolism
• Metabolism of cholesterol, synthesis, esterification, oxidation and excretion.
• Cholesterol-Cholestery...
Serum Enzymes
• Plasma activities of several cytosolic, mitochondrial, and membrane
associated enzymes are measured.
• Abi...
AST( SGOT)
• Present in cytoplasm as well as mitochondria of hepatocytes.
• Mitochondrial isoenzyme represents a significa...
ALT (SGPT)
• Plasma half life is 47 hours

• Liver specific activity in hepatocytes 3000 times higher than plasma
which is...
Patterns of release

• In most forms of acute hepatocellular injury AST is higher than ALT
initially, due to higher activi...
Patterns of release
• In Alcoholic liver disease studies suggest that alcohol induces
mitochondrial damage.
• This causes ...
Activity
• AST/ALT ratio >2 with ALT <300 U/L suggestive of alcoholic
hepatitis.
• ALT more specific for liver disease.
• ...
Alkaline Phosphatase
• Present in number of liver tissues including liver, bone, kidney and
intestine.
• Liver isoenzyme h...
ϒ-Glutamyl Transferase
• Regulates the transfer of amino acids across cell membranes.
• If ALP and GGT are both elevated s...
Mechanism of release
• Bile acids, solubilize the release of GGT and ALP from plasma
membrane.
• Ethanol, Phenytoin and Ca...
5’- Nucleotidase
• Increased in cholestatic disorders.
• No increase in activity in patients with bone disease
• Confirms ...
Lactate Dehydrogenase
• Cytosolic glycolytic enzyme catalyses the reversible oxidation of
lactate to pyruvate.
• Normal up...
Rate of clearance
• The half life of ALT is 47 hrs, cytoplasmic AST is 17 hrs.
• Liver isoenzyme of ALP is 3 days.
• GGT –...
Biliprotein/ Delta Bilirubin
• Formed by covalent attachment of Bilirubin monoglucuronide
with lysine residues of albumin ...
Cirrhosis
• Earliest laboratory abnormalities are:
 fall in platelet count
 increase in PT
 decrease in albumin to glob...
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 Se...
Viral Hepatitis
• Acute viral hepatitis is defined by the sudden onset of significant
aminotransferase elevation as a cons...
Features of viral hepatitis
Feature

HAV

HBV

HCV

HDV

HEV

Incubation (days) 15–45, mean 30

30–180, mean 60– 15–160, m...
Algorithm for workup for Jaundice
Crigler-Najjar Syndrome
• Hereditary Glucuronyl Transferase Deficiency.

• Familial autosomal recessive disease (type I) a...
Gilbert’s Syndrome
• Autosomal dominant
• Chronic, benign, intermittent, nonhemolytic and unconjugated
hyperbilirubinemia....
Dubin-Johnson Syndrome
• Autosomal recessive disease.
• Conjugation of bilirubin-diglucuronide is normal
• Inability to tr...
Rotor’s Syndrome
• Autosomal recessive

• Asymptomatic, benign defective uptake and storage of conjugated
bilirubin, possi...
Take Home Message
• Liver has central role in
metabolism, synthesis
and detoxification in
the body
• Interpretation of
liv...
References
• Tietze Textbook of clinical chemistry and molecular diagnostics-5th
Ed.
• Intepretation of Diagnostic Tests, ...
THANK YOU
Questions?
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Approach to evaluation of liver disorders

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  • 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
  • Transcript of "Approach to evaluation of liver disorders"

    1. 1. Evaluation of Biochemical Tests in Liver Disorders By: Capt Arabinda Mohan Bhattarai Guide: Col HS Batra
    2. 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. 3. Introduction • The liver has a central and critical biochemical role in  metabolism  digestion  detoxification and  elimination of substance from the body
    4. 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. 5. Bilirubin Metabolism
    6. 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. 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. 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. 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. 10. Detoxification function • Conversion of ammonia to Urea. • Drug metabolism (Xenobiotics). • Hippuric acid synthesis test
    11. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 35. Algorithm for workup for Jaundice
    36. 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. 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. 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. 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. 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. 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
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