Liver function test and jaundice

For MBBS, BDS, Medicine, post-graduate students

1. JAUNDICE & LFT Prof (Dr.) Viyatprajna Acharya MBBS, MD PhD

2. Diagnosis?? A nurse working in the infectious diseases ward complains of upper abdominal pain, recurrent vomiting and fever. On examination she was icteric and had an enlarged liver. Biochemical findings were as follows: S. Bilirubin – total 12.5 mg%; Direct 6.5 mg%. ALP – 200 U/L, AST – 140 U/L, ALT- 290 U/L. Bile pigments ++ and bile salts +. HbS –ve. What is your probable diagnosis-?

3. One 8- year old child was brought to the hospital with jaundice and severe bone pain. This was 3 rd such attack in the last 5 years and his brother had also died of a similar attack 3 years before. The boy was severely anaemic and the treating physician ordered for an LFT and a haemoglobin electrophoresis.

4. A lady delivered a baby at home safely. After three days, she noticed icterus over the skin and sclera of the child and so, she took the child to hospital. The child was otherwise normal. The laboratory investigations done in the hospital showed, Serum total Bilirubin- 6 mg/dl Unconjugated Bilirubin - 5.2 mg/dl What can be the possible diagnosis?

5. An obese woman presented with acute abdomen and jaundice. She gave a history of indigestion and vomiting after intake of food for past 2 years. She was treated symptomatically off and on with permanent relief. On examination, she’d rashes all over her body and her biochemical profile was TB- 12mg%, DB – 8 mg%, ALP- 500 IU / L, ALT- 35 IU/L, AST- IU / L, urinary bile salts +++. On ERCP, CBD stones were found.

6. Jaundice / icterus - Cardinal manifestation of several diseases due to affection of liver and biliary tract / a hemolytic disorder Clinically- Yellow discoloration of sclera, skin, mucous membranes due to deposition of bile pigment (Hyperbilirubinemia) Biochemically – Serum bilirubin > 2mg/dl

7. Fate of hemoglobin Humans- 250-400 mg bilirubin produced everyday (80% from heme and 20% from non-heme proteins) Senescent RBC rupture (80%) , premature erythroid cell damage in BM (10%) , Myoglobin and Cytochrome damage (10%) – pool of heme 1g Hb = 35 mg bilirubin

8. Hemoglobin Heme Globin Microsomal heme oxygenase system CO 3O 2 + 5 NADPH Biliverdin Bilirubin Biliverdin reductase NADPH + H + NADP + This process takes place in RE system (Liver and spleen) and bilirubin produced is water insoluble.

9. Biliverdin

10. Bilirubin transport & conjugation ALB= Albumin, BT- Bilirubin transporter, GST = glutathione-S- transferase, BMG= bilirubin monoglucuronide, BDG = bilirubin diglucuronide

11. Bilirubin binds to albumin- travel to hepatocytes Albumin is left, BT carries UCB into the hepatocytes Ligandin / Protein Y / glutathione-S- transferase – binds to UCB and prevents its efflux Conjugation with Glucuronic acid with the help of bilirubin Uridine diphosphate glucuronyl transferase - BMG= bilirubin monoglucuronide, BDG = bilirubin diglucuronide

12. Bilirubin in intestine Conjugated bilirubin poured into 2 nd part of duodenum through bile Unchanged through proximal intestine Distal ileum & Colon- deconjugated by bacteria reduced to urobilinogen Urobilinogen further reduced Setrcobilinogen & Mesobilinogen

13. 80% of these excreted either unchanged or converted to urobilins , stercobilins Feces, urine (impart colour) 20% of these – enterohepatic circulation A small fraction (< 3 mg/ dl) – in urine If bacterial flora reduced, bilirubin is not converted to urobilinogen instead re-oxidized to Biliverdin → green stool

14. Alternative routes of bilirubin elimination In the absence of bilirubin glucuronidation a fraction of bilirubin is excreted as hydroxylated products – may be by Microsomal P450 system or mitochondrial bilirubin oxidase system in liver and other tissues

15. Normal level of Serum bilirubin- 0.2-1.0 mg /dl 80% UC bilirubin – 0.2-0.8 mg/dl 20% C bilirubin – 0- 0.2 mg/dl Latent jaundice – 1-2 mg/dl Jaundice - > 2 mg / dl

16. Biochemical test Van den Bergh reaction UCB – indirect CB – direct Hepatic jaundice – biphasic reaction

17. What causes  bilirubin? Overproduction by reticuloendothelial system Failure of hepatocyte uptake Failure to conjugate or excrete Obstruction of biliary excretion into intestine

18. Classification of jaundice Inherited hyperbilirubinemia Acquired hyperbilirubinemia

19. Inherited hyperbilirubinemia Crigler - Najjar’s syndrome – i ) Type I – complete absence of glucuronyl transferase ii) Type II – partial absence of glucuronyl transferase Gilbert’s syndrome- impaired hepatic intake & ↓conjugation Dubin - Johnson’s syndrome- defective excretion of conjugated bilirubin; black pigmentation Rotor syndrome- defective excretion of conjugated bilirubin; no pigmentation

20. Acquired hyperbilirubinemia Pre-hepatic / Haemolytic jaundice Hepatocellular / Hepatic jaundice Obstructive jaundice Physiological jaundice of the newborn

21. Pre-hepatic / hemolytic jaundice A. Hemolytic disease of the newborn Rh incompatibility Erythroblastosis fetalis B. Hemolytic disease due to other causes Congenital spherocytosis G-6-PD deficiency Incompatible blood transfusion Hereditary spherocytosis

22. Features of Pre-hepatic / hemolytic jaundice Mild jaundice Maximum unconjugated bilirubin Urobilinogen +++ in urine and stool Urine colour is normal- absence of bilirubin ↑ AST & ALT; ALP normal

23. Hepatic / hepatocellular jaundice Viral hepatitis Toxic chemicals – alcohol, chloroform, CCl4 Drugs Cirrhosis

24. Features of hepatic jaundice UC &C bilirubin both are present ↓ urobilinogen in urine and feces Bilirubin in urine + Biphasic reaction in Van den Bergh reaction ↑ALT, AST; ALP moderately high Cirrhosis will give a picture of both hepatocellular as well as post-hepatic jaundice

25. Post-hepatic / obstructive jaundice Cholestasis - stagnation of bile Intrahepatic cholestasis- Chronic active hepatitis Biliary cirrhosis Lymphomas Primary hepatoma Obstructive stage of viral hepatitis Extrahepatic cholestasis- stones, stricture in CBD, CA head of pancreas, enlarged LN at porta hepatis

26. Features of obstructive disease Regurgitation of bile → biliary canaliculi damage → infiltrate to lymph → blood circulation ↑ Conjugated bilirubin in blood UBG is decreased ; in complete obstruction it’s absent Clay coloured stool Bile salts in urine

27. Pre-hepatic / hemolytic jaundice Hepatic / Hepatocellular Post-hepatic / Obstructive Aetiology Excessive hemolysis Parenchymal disease Obstruction to biliary passage Degree of jaundice Low Mod. to severe Mod. to severe Feces Dark Pale Clay Van den Bergh reaction Indirect Biphasic Direct Pigment in circulation UC Bilirubin C & UC C Bilirubin in urine nil + ++ Urobilinogen in urine ++ + or ± ↓ or absent Fecal Setrcobilinogen ↑↑ ↓ ↓ or absent Steatorrhoea ─ + ++

28. Pre-hepatic / hemolytic jaundice Hepatic / Hepatocellular Post-hepatic / Obstructive Bile salt in urine ─ + ++ Prothrombin time ± ↑ ↑, normal after vit K inj. ALT / AST ++ +++ to ++++ ↑ to ++; never exceeds 300 U /L ALP ± + +++

29. Physiological jaundice of the newborn After 2 nd day of life jaundice appears Mild jaundice Due to accelerated RBC hemolysis Immature hepatic system → conjugation of bilirubin fails Unconjugated hyperbilirubinemia Hardly crosses > 15 mg/ dl Disappears by 2 nd wk of life

30. kernicterus Kern= Nucleus of brain Icterus = jaundice Bilirubin > 20 mg / dl At lower level of bilirubin also kernicterus can occur due to presence of sulfonamides, coumarin or radio-contrast dye – they prevent albumin-bilirubin binding by competitive or allosteric displacement.

31. Hyperbilirubinemia- induced toxic encephalopathy – mental retardation Phototherapy – E- isomerization of bilirubin- makes bilirubin more water soluble Phenobarbital – induces bilirubin metabolizing system

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33. Liver function test

34. SYNTHETIC FUNCTIONS PLASMA PROTEINS COAGULATION FACTORS MANY GLOBULINS CHOLESTEROL TAG LIPOPROTEIN METABOLIC FUNCTIONS CARBOHYDRATE KETOGENESIS TCA CYCLE PROTEIN CATABOLISM NUCLEOTIDE DETOXIFICATION AND EXCRETION AMMONIA TO UREA BILIRUBIN CHOLESTEROL DRUG HOMEOSTASIS STORAGE AID IN DIGESTION- BS

35. Clinical manifestations of liver diseases Jaundice Portal hypertension Ascites

36. INDICATIONS OF LFT Jaundice Suspected liver metastasis Alcoholic liver disease Any undiagnosed chronic disease Annual check up for diabetes patients Coagulation disorders Therapy with Statins to check hepatotoxicity

37. Classification of lft Tests of hepatic excretory function Plasma proteins (tests for synthetic function of liver) Liver enzyme panel Special tests

38. i.Tests of hepatic excretory function Serum bilirubin- Total, UC, C Urine – bile pigments, bile slats, urobilinogen

39. ii. Plasma proteins (tests for synthetic function of liver) Albumin - ↓ in chr. Liver diseases, A:G ratio inversed Globulins – ↑ gammaglobulins in chr liver diseases ↑ IgG in autoimmune hepatitis; ↑IgM in primary biliary cirrhosis Prothrombin time – prolonged in liver diseases

40. AFP – tumour marker for hepatitis & cirrhosis Ceruloplasmin - ↑ level in active hepatitis, biliary cirrhosis, hemochromatosis, obstructive biliary disease Transthyretin / prealbumin- indicator of early disease Alpha-1-antitrypsin – liver cirrhosis Haptoglobin - assess the recent changes in liver

41. iii. Liver enzyme panel Hepatocellular damage – ALT & AST (5-40 U/L) very high levels – viral & toxic hepatitis ALT is more specific for liver than AST; but in alcoholic hepatitis – AST elevated Moderate elevation (100-300 U /L)- alcoholic hepatitis, AI hepatitis, Wilson’s disease, Non-alcoholic chr hepatitis Minor elevation (100 U/ L)- Chr viral hepatitis, fatty liver & in nonalcoholic steatohepatitis

42. Obstructive liver diseases - ↑ALP & GGT Cholestasis or Hepatic carcinoma Parenchymal disease – mild elevation Very high elevation (10-12 times)- obstructive jaundice Drastic elevation (10-25 times)- Bone diseases ALP normal value – 80-125 U /l

43. Liver specific enzymes- GGT (?) & 5’ Nucleotidase GGT- sensitive to alcohol abuse ↑ levels in Chr alcoholism, pancreatic disease, MI, renal failure, COPD, DM 5’ Nucleotidase – More specific for obstructive liver disease GST (Glutathione- S- Transferase)- very specific

44. iv. Tests based on metabolic function of liver Galactose tolerance test BSP excretion test Blood ammonia estimation - Gives impression of liver’s capacity to generate urea from ammonia ↑NH3- cirrhosis, portocaval anastomosis Obsolete

45. Immunological tests ↑IgG – Chr. Hepatitis, alcoholic & AI hepatitis ↑ IgM – Primary biliary cirrhosis, viral hepatitis ↑ IgA – Alcoholic cirrhosis & Primary biliary cirrhosis

46. Pre-hepatic / hemolytic jaundice Hepatic / Hepatocellular Post-hepatic / Obstructive Aetiology Excessive hemolysis Parenchymal disease Obstruction to biliary passage Degree of jaundice Low Mod. to severe Mod. to severe Feces Dark Pale Clay Van den Bergh reaction Indirect Biphasic Direct Pigment in circulation UC Bilirubin C & UC C Bilirubin in urine nil + ++ Urobilinogen in urine ++ + or ± ↓ or absent Fecal Setrcobilinogen ↑↑ ↓ ↓ or absent Steatorrhoea ─ + ++

47. Pre-hepatic / hemolytic jaundice Hepatic / Hepatocellular Post-hepatic / Obstructive Bile salt in urine ─ + ++ Prothrombin time ± ↑ ↑, normal after vit K inj. ALT / AST ++ +++ to ++++ ↑ to ++; never exceeds 300 U /L ALP ± + +++

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49. LFT profile- principles and procedures Bilirubin- Total and Direct AST ALT ALP GGT TP Albumin Immune markers

50. alt Alanine aminotransferase (ALT) catalyzes the transamination of L-alanine to α -ketoglutarate ( α -KG), forming L-glutamate and pyruvate. The pyruvate formed is reduced to lactate by lactate dehydrogenase (LDH) with simultaneous oxidation of reduced nicotinamide -adenine dinucleotide (NADH). The change in absorbance is directly proportional to the ALT activity and is measured using a bichromatic (340, 700 nm) rate technique. ALT L-alanine+ a-KG → L-glutamate + pyruvate P5P, Tris, pH 7.4   LDH Pyruvate + NADH +H + → Lactate + NAD +

51. ast Aspartate aminotransferase (AST) catalyzes the transamination from L-aspartate to a-ketoglutarate, forming L-glutamate and oxaloacetate . The oxaloacetate formed is reduced to malate by malate dehydrogenase (MDH) with simultaneous oxidation of reduced nicotinamide adenine dinucleotide (NADH). The change in absorbance with time due to the conversion of NADH to NAD is directly proportional to the AST activity and is measured using a bichromatic (340, 700 nm) rate technique. AST L-aspartate + a-ketoglutarate ————> L-glutamate + Oxalacetate pH 7.8   MDH Oxaloacetate + NADH ————> Malate + NAD    

52. Alkaline phosphatase Alkaline phosphatase catalyzes the transphosphorylation of p- nitrophenylphosphate (p-NPP) to p- nitrophenol (p-NP) in the presence of the transphosphorylating buffer, 2-amino-2-methyl-1-propanol (AMP). The reaction is enhanced through the use of magnesium and zinc ions. The change in absorbance at 405 nm due to the formation of p-NP is directly proportional to the ALP activity, since other reactants are present in non-rate limiting quantities and is measured using a bichromatic (405, 510 nm) rate technique. ALP, Mg/Zn p-NPP + AMP → p-NP + AMP + PO 4 pH 10.35

53. Total protein Modified method of Biuret reaction This method incorporates tartrate as a complexing agent to prevent precipitation of Cu(OH) 2 . Serum blanking increases method sensitivity and minimizes spectral interference from lipemia . Cupric ion (Cu++) reacts with the peptide linkages (-C-NH-CH-C-NH-) of protein in a basic solution. || | || R O O   The blue copper (II) protein complex thus formed is proportional to the total protein concentration in the sample and is measured using a bichromatic (540, 700 nm) endpoint technique. OH – Cu ++ + Protein ————> complex (absorbs at 540 nm)

54. albumin Method: adaptation of BCP (Bromocresol purple ) dye binding method In the presence of a solubilizing agent, BCP binds to albumin at pH 4.9. The amount of albumin-BCP complex is directly proportional to the albumin concentration. The complex absorbs at 600 nm and is measured using a polychromatic (600, 540, 700 nm) endpoint technique.   pH 4.9 Albumin + BCP dye → Albumin-BCP complex (non-absorbing at 600 nm) (absorbs at 600 nm)

55. direct bilirubin Modified Doumas reference method Diazotized sulfanilic acid is formed by combining sodium nitrite and sulfanilic acid at low pH. The sample is diluted in 0.05M HCl. A blank reading is taken to eliminate interference from non-bilirubin pigments. Upon addition of the diazotized sulfanilic acid, the conjugated bilirubin is converted to diazo -bilirubin, a red chromophore which absorbs at 540 nm and is measured using a bichromatic (540, 700 nm) endpoint technique. Conjugated bilirubin + Diazotized sulfanilic acid → Red chromophore (absorbs at 540 nm)

56. Total bilirubin Diazotized sulfanilic acid is formed by combining sodium nitrite and sulfanilic acid at low pH. Bilirubin in the sample, including the delta form is solubilized by dilution in a mixture of caffeine/benzoate/acetate/EDTA. Upon addition of the diazotized sulfanilic acid, the solubilized bilirubin is converted to diazo -bilirubin, a red chromophore which absorbs at 540nm and is measured using a bichromatic (540, 700 nm) endpoint technique. Solubilized bilirubin + Diazotized sulfanilic acid ———> Red chromophore (absorbs at 540 nm)

57. Gamma-glutamyl transferease The method uses the substrate L-gamma-glutamyl-3-carboxy-4-nitranilide with glycylglycine . Gamma-glutamyl transferase catalyzes the transfer of the glutamyl moiety from Gamma-glutamyl-3-carboxy-4-nitranilide (GCNA) to glycylglycine thereby releasing 5-amino-2-nitrobenzoate which absorbs at 405 nm. This change is proportional to the Gamma-glutamyl transferase activity and is measured using a bichromatic (405, 600 nm) rate technique. GGT GCNA + glycylglycine → L-g-glutamyl- glycylglycine + 5-amino-2-nitrobenzoate

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