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Liver function test

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Liver function test

  1. 1. Dr. Bahoran Singh Moderator- Dr. Swati LIVER FUNCTION TESTS
  2. 2. Functions of the Liver • Metabolic • Storage- Glycogen, vitamins (all Fat soluble and few water soluble), iron • Excretory/Secretory – bile excretion • Protective (eg. kuffer cells) • Coagulation – production of clotting factors • Detoxification of drugs via cytochromes.
  3. 3. Metabolic functions • Carbohydrate metabolism – Gluconeogenesis – Glycogenolysis and glycogenesis • Hormone metabolism • Lipid Metabolism – Synthesis of fatty acids, cholesterol, lipoproteins – Ketogenesis • Drug Metabolism • Protein Metabolism – Synthesis of plasma proteins – Urea synthesis
  4. 4. LFTs are classified as: • Excretory function tests: Bile pigments,salts,acids,bilirubin and BSP • Metabolic functions tests : Carbohydrates, Protiens, Fats • Synthetic capabilities : Protiens(albumin), coagulation factors • Detoxification : Ammonia, drugs • Tests of liver injury : Enzyme assays,autoimmune markers,markers of hepatitis virus infections
  5. 5. Uses of LFTs • Diagnosis of type of jaundice- etiology • Assess severity & follow trend of liver disease • Detect latent liver disease • Screening of infective hepatitis • Screen drug hepatotoxicity
  6. 6. Indication and limitation of LFT • Indication- • Screen for liver diseases • Identifying the nature of liver diseases( hepatocellular, cholestatic, or infiltrative.) • Assess severity and prognosis of liver disease • Follow up the course of liver disease. • Limitations – • Do not necessarily assess liver function. • Lack sensitivity • Lack specificity
  7. 7. Test that assess excretory function • Jaundice – yellowish discoloration of skin, sclera, and mucous membrane. Evident when bilirubin >2 mg/dl. • Classification of jaundice- 1. According to type of bilirubin increased- – Unconjugated hyperbilirubinemia ( unconj. Bilirubin > 85% of total) – Conjugate hyperbilirubinemia ( conj. Bilirubin > 20%)
  8. 8. • According to site of disease • Prehepatic- • Hemolytic • Ineffective erythropoesis • Resorption of large hematoma • Hepatic • Predominantly unconjugated
  9. 9. Bilirubin • Bilirubin is the major metabolites of heme • Sources:-hemoglobin, myoglobin & cytochromes. • 250-350mg of bilirubin is produced daily • Normal levels upto 1mg% • Strenuous exercise –significant increase in bilirubin values .
  10. 10. Bilirubin Metabolism
  11. 11. Lab tests for bilirubin • Bilirubin is measured using diazotised sulfanilic acid -form a reddish purple coloured complex- spectrophotometrically • total bilirubin=unconjugated +conjugated +delta bilirubin • direct bilirubin assays measure: – C bilirubin +delta bilirubin +small% of UC bilirubin
  12. 12. Bilirubin - interpretation • Normal bilirubin level- upto 1mg% • Direct bilirubin upto 0.3mg% • direct bilirubin <20% of total-hemolytic jaundice =20-40%of total- hepatocellular jaundice >50%of total- post hepatic jaundice
  13. 13. Bile Salt Assay • Analysis done in fasting state • Assay done using chromatographic metods, HPLC • Sulphur Test- – Principle: BS ↓ surface tension of urine – Method: urine(10ml)+sulphur powder sprinkled →particles sink to bottom- BS present → particles float- BS absent
  14. 14. Clinical condition Normal Bile Salts absent Bile Pigments absent Urobilinog en trace Prehepatic Absent Absent Very high + ++ to++++ Hepatic Present trace to+++ + Present Increased + + Post hepatic Present ++ to ++++ Present Absent
  15. 15. Determination of bile pigments • Harrison spot test : • urine sediment + Fouchet's reagent→ No change in colour – BP absent change in colour to green – BP present Positive result graded as trace - ++++ as per intensity of colour of sediment
  16. 16. Urobilinogen determination • Freshly collected normal fasting urine sample- +ve reaction for urobilinogen • On air exposure oxidized to urobilin (pinkish brown) • Test – – Urine + Ehrlich's reagent→ pale pink urobilinogen normal – cherry red- urobilinogen ↑↑↑ – graded as per colour intensity
  17. 17. Metabolic functions:- protein and ammonia metabolism • Ammonia derived from amino acid and nucleic acid metabolism. • Metabolised only in the liver: • Urea cycle or Krebs Henseleit cycle • Ammonia Urea . • Liver damage >80%- ↑ NH3 & arginine conc. Hepatic encephalopathy • Degree of hepatic encephalopathy is proportional to NH3 conc. in arterial blood .
  18. 18. Assays for Ammonia • Enzyme assay •Arterial blood is prefered for assay •Specimen should be kept in ice water until separation of cells from plasma α ketoglutarate + NH3 glutamate Glutamate dehydrogenase NADPH NADP indicator colour •Dry slide method Alkaline pH buffers convert ammonium ions to ammonia gas – bromophenol blue - used indicator
  19. 19. Cholesterol and other lipids • Lipoprotein sythesis • Esterification of cholesterol • Liver injury: ↓ HDL, ↓ LCAT, ↓ lipoprotein lipase • ↑ TGs -↑ unesterified cholesterol, ↑phospholipids • Cirrhotics with poor nutrition – ↓ cholesterol • Alcohol induced liver injury: ↑ HDL ↑ apoA-1 protein • Cirrhosis : ↓ apoA-1 protein • apoA-1 protein – PGA index used to differentiate ALD & cirrhosis ( PT, GGT, and Apo- A1 protein)
  20. 20. Drug metabolism • Xenobiotics are metabolised – microsomes of liver CYP 450 • Detoxification – 2 phases phase 1-oxidation/hydroxlation phase 2-conjugation with polar compound • Severe liver injury - ↓ability to metabolise drugs- measure extent of liver damage in known liver disease
  21. 21. Assay : Breath tests • Radiolabelled drug (usually C13 labelled) administered – measure exhaled CO2 in pt breath • Breath tests – based on rate limiting step in metabolism- 2 groups • 1st – independent of hepatic blood flow (only microsomal enzymatic activity) – eg: aminopyrine, caffeine, diazepam • 2nd – dependent on rate of hepatic blood flow – eg: methacetin, phenacetin, erythromycin
  22. 22. Synthetic functions:- protein synthesis • Liver – site for synthesis for most plasma proteins( 100% albumin) exceptions- immunoglobulins & vWF • Extensive liver destruction-↓serum total proteins and albumin • Cirrhosis - + ↓ delivery of amino acids • Common causes of ↓ S.proteins : renal diseases, liver disease, malnutrition, protein losing enteropathy, chronic inflammatory diseases • ↓S.proteins levels –depend on t½ of protein eg: albumin- 20 days , transthyretin – 1-2 days factor VII- 4-6hrs , transferrin – 6 days
  23. 23. Protein Assays • Biuret method : • peptide backbone C=O +copper • Dye binding method : protein + Coomassie blue dye • Albumin + bromocresol green/ purple COLOURED COMPLEX Spectrophotometric quantitation Normal total protein levels : 6-7.8 g/dl Albumin levels: 3.5- 5 g/dl
  24. 24. Protein Assay • Thymol turbidity test : determination of serum proteins -Principle: reaction &ɣ β glogulin with phenolic group of thymol -Serum + buffered soln Thymol → Turbidity - measured
  25. 25. Protein electrophoresis • Cirrhosis : – ↓↓albumin, – ↓alpha-1,alpha-2 & beta band – ↑ polyclonal immunoglobulins: IgG & IgA (beta-gamma bridging pattern) • Autoimmune hepatitis: -↓ albumin -↑↑ polyclonal IgG • Primary biliary cirrhosis: -↑ polyclonal IgM
  26. 26. Other proteins alpha-1-antitrypsin • Most abundant alpha-1 globulin • Most imp protease inhibitor in plasma • Inhibits trypsin & other serine proteases • Coded by Pi gene on Chromosome14 • Mutation- ↓protein glycation → ↑conc. In hepatocytes – periportal- discrete cytoplasmic bodies- Neonatal hepatitis- cirrhosis in 3% ↓conc. In plasma - emphysema
  27. 27. Ceruloplasmin • Copper containing protein in serum • Enzyme present in highest circulating conc. • Ferroxidase – converts Fe++ →Fe+++ -allow binding to transferrin • ↓ levels in Wilson´s disease-mutation in Chr13
  28. 28. Coagulation factors • Liver – clotting factor synthesis – inhibitors of coagulation synthesis – fibrin degradation product catabolism • Most common coagulopathy in liver disease - Disseminated Intravascular Coagulopathy • DIC: ↑consumption of clotting factors &platelets ↑ PT,↑ PTT, ↓ platelets,↑d-Dimer levels • Liver failure (some cases): ↓clotting factors- ↓decreased synthesis ↓platelets – sequestration in spleen fibrin split products- 80% • pt s without fibrinolysis, ↑ PT,↑ PTT, d-Dimer levels-NOT elevated
  29. 29. Prothrombin time (PT) • Most frequently used – liver associated coagulation abnormalities- best index of severity • Efficacy of extrinsic clotting system- factor VII • Factor VII- synthesized in liver- evaluate liver function • PT part of MELD score- Model for End stage Liver Disease -evaluating priority- Liver Transplantation - predicts 3 month mortality for cirrhotic pts -computed no.- based on values of bilirubin, creatinine and PT (INR)
  30. 30. Problems with using PT • Non-specific – elevated in most coagulation disorders • In cholestasis – with normal hepatocyte fn ↑ PT - ↓ bile salts - ↓ absorption of vit K - ↓ factors II, VII, IX, X cholestasis- precursor forms of clotting factors increased
  31. 31. Tests of Liver Injury Plasma Enzyme Levels • Aspartate Aminotransferase (AST) • Alanine Aminotransferase (ALT) • Lactate Dehydrogenase (LDH) • Alkaline Phosphatase (ALP) • Gamma Glutamyl Transferase (GGT) • 5‘- Nucleotidase
  32. 32. Cellular location of enzymess
  33. 33. Algorithm for Diagnosis of Liver Diseases
  34. 34. Aminotransferases (Transaminases) • Aspartate Aminotransferase(AST) =Serum Glutamate Oxaloacetate Transaminase (SGOT) • Alanine Aminotransferase(ALT) = Serum Glutamate Pyruvate Transaminase(SGPT)
  35. 35. AST- liver, heart skeletal muscle, kidneys, brain, RBCs • In liver 20% activity is cytosolic and 80% mitochondrial • Clearance performed by sinusoidal cells, • AST cytosolic t½ -17hrs,7000 X plasma conc. • AST mitochondrial t½ - 87hrs • Used for monitoring therapy with hepatotoxic drugs if levels >3 X normal stop therapy ALT – more specific to liver, very low concentrations in kidney and skeletal muscles. • In liver totally cytosolic. • t½ - 47hrs , 3000 X plasma conc. • Used in non alcoholic, asymptomatic pts.
  36. 36. •Acute hepatocellular injury - < 24hrs – AST > ALT > 24hrs - AST < ALT since ALT has longer half life •Alcoholic hepatitis (alcohol induced hepatocyte injury)- AST >ALT - mitochondrial damage induced – ASTm released – has a longer t½ & is the predominant AST in hepatocyte - ↑↑ AST:ALT → 3-4 : 1 – DeRitis Ratio - ↑ ASTm is s/o advanced alcoholic liver disease •Chronic liver injury (mainly cirrhosis) – ALT > AST -but as fibrosis progresses – ALT↓ & ↑ AST:ALT •End stage liver cirrhosis – AST and ALT levels NOT elevated - massive tissue destruction •Acute fulminant hepatic failure - ↑↑AST and ↑↑ ALT , AST:ALT > 1 Aminotransferases (cont..)
  37. 37. Assay for AST & ALT • Vit B6 important requirement for AST and ALT assays • Normal serum levels upto 40 IU/dl for both ↑ aspartate in reaction → glutamate α ketoglutararte Glutamate dehydrogenase NAD NADH(Colour indicator) Oxaloacetate malate malate dehydrogenase NAD NADH (Colour indicator) Measured spectrophotometrically
  38. 38. Mild Chronic Elevation in Serum Aminotransferases • Step one – Medications and supplements – Alcohol use – Viral hepatitis B and C – Hemochromatosis – Fatty liver (hepatic steatosis and steatohepatitis )
  39. 39. Mild Chronic Elevation in Serum Aminotransferases • Step two – Muscle disorders – Thyroid disease – Celiac disease (less) – Adrenal insufficiency (less) – Anorexia nervosa (less)
  40. 40. Mild Chronic Elevation in Serum Aminotransferases • Step three – Autoimmune hepatitis – Wilson's disease – Alpha-1-antitrypsin deficiency
  41. 41. Mild Chronic Elevation in Serum Aminotransferases • Step four – A liver biopsy is often considered in patients in whom all of the above testing has been unyielding. – However, in some settings, the best course may be observation.
  42. 42. Lactate dehydrogenase (LDH) • Cytosolic glycolytic enzyme • Lactate Pyruvate • 5 major isomers : LD1 – LD5 LD1 & LD2 – cardiac muscle,kidney,RBCs LD4 & LD5 – liver , skeletal muscle • t½ - 4-6hrs , 500X plasma conc. • Normal levels upto 150 IU/dl • ↑↑ hepatitis- transient-normal –clinical presentation • ↑↑total LDH > 500 IU/dl ↑↑ALP > 250 IU/dl in absense of abnormality in AST or ALT Indicate SOL – s/o metastatic Ca, HCC, hemangioma oxidation
  43. 43. Enzymes – canalicular injury Markers of Cholestasis Alkaline Phosphatase (ALP) – • liver and bone (placenta, kidneys, intestines or WCC) • Hepatic ALP present on surface of bile duct epithelia- canalicular surface and accumulating bile salts increase its release from cell surface – in biliary dysfunction (not cell injury). • Takes time for induction of enzyme levels so may not be first enzyme to rise and half-life is 3 days • ALP isoenzymes, 5-NT or gamma GT may be necessary to evaluate the origin of ALP • Normal = 30-120 IU/L • Causes of ↑ in ALP: biliary tract obstruction –eg: Stones hepatitis ascending cholangitis
  44. 44. • Obstructive jaundice : ALP > 2X UNL ≈ rate of ↑ in bilirubin • Partial obstruction : ↑ ALP ≈ ↑ C.bilirubin :Dissociated jaundice • Passive congestion & hepatic injury: mod. ↑ALP • high mol.wt ALP : malignant disease involing liver • Intestinal ALP: -↑ in disorders of intestinal tract &cirrhosis - discriminate intrahepatic &extrahepatic jaundice-absent in extrahepatic jaundice -lacks sensitivity • Cholestasis relieved – ALP levels ↓ more slowly than bilirubin Alkaline phosphatase (cont..)
  45. 45. Clinical significance of AST: ALT ratio
  46. 46. Gamma Glutamyl transferase (GGT) • Regulates transport of AA across cell membrane • hepatocytes and biliary epithelial cells, pancreas, renal tubules and intestine • Confirm hepatic source for a raised ALP • Half life -10 days • Very sensitive but Non-specific • Raised in ANY liver disease hepatocellular or cholestatic • Highest values – 10 x –chronic cholestasis: PBC, SC • Chronic alcohol abuse – 60-70% show ↑ GGT -correlation between amount of alcohol intake & GGT activity -remains elevated 1 month after alcohol abstinence -t½ ↑ to 28 days • Cholestasis of pregnancy : ↑ ALP, but GGT remains normal
  47. 47. • GGT is ↑ in:-alcoholics without liver diseas – obese pts – high conc.of drugs: acetaminophen, phenytoin, carbamazepine(GGT ↑ to restore glutathione used to metabolise drugs) • Isolated increase does not require any further evaluation, suggest watch and repeated quarterly,or if other LFT’s become abnormal then investigate • GGT Assay: substrate - ɣ glutamyl-p nitroanilide → p nitroaniline liberated (chromogenic) – measured spectrophotometrically
  48. 48. Other canalicular enzymes • 5‘ Nucleotidase • Leucine aminopeptidase ↑ in cholestatic disorders
  49. 49. Autoimmune markers • Primary Biliary cirrhosis : AMA • Primary Sclerosing Cholangitis:- p ANCA - ANA - ASMA • Autoimmune hepatitis: - ANA - ASMA - anti-LKM1
  50. 50. Markers of Hepatitis Viral Infections Hepatitis A : • Anti-HAV - Measures IgG and IgM Indicates exposure and immunity • Anti-HAV, IgM -Indicates acute infection
  51. 51. Hepatitis B
  52. 52. Hepatitis C • Anti-HCV - Indication infection with HCV. Does not indicate immunity • HCV RNA - Active Virus. Used to detect HCV when anti-HCV is negative
  53. 53. Hepatitis E: • Anti-HEV IgM – recent or current infection • Anti-HEV IgG – current or past infection • HEV RNA-PCR – definite indicator of acute infection
  54. 54. Diagnosing & following cirrhosis, fibrosis & necroinflammation of Liver • Definitive diagnosis of fibrosis, necrosis,& inflammation of the liver is Liver Biopsy- invasive • Indices -Levels of serum analytes measuring liver function- used to follow these disease process Non-invasively • Pro-collagen type III pro-peptide (PIIIP) – follow active cirrhosis
  55. 55. • PGA Index:- –prothrombin time –Gamma Glutamyl transferase –Apolipoprotein A1 Higher PGA scores correlate with degree of hepatic fibrosis & severity of cirrhosis -judged by clinical grading & liver biopsy -good correlation with levels of PIIIP
  56. 56. • Fibrotest & Actitest : measurement of 6 analytes –Apolipoprotein A1 –Gamma Glutamyl transferase –Haptoglobin –Total bilirubin –Alpha-2 macroglobulin –ALT Also includes pt s age and gender
  57. 57. • Correlations with liver biopsy results –then performed using an artificial intelligence algorithm – Fibrotest scores – computed on a scale of 0 – 1.0 – histopathological staging system – METAVIR – Actitest scores- computed on scale 0-1.0 correlated with necroinflammatory activity aslo using METAVIR grading system • More effective and of value in detecting fibrosis False +ve results with – Treatment of hepatitis C with ribavirin – Gilberts disease – Extrahepatic cholestasis – Acute inflammation
  58. 58. Other Indices • FIBROspect II-tissue inhibitors metalloproteinases , alpha-2 macroglobulin, hyaluronic acid • Forms index: age, platelet count, GGT, cholesterol,-correlated –cirrhosis • AST:ALT,INR,APRI

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