1. MLAB 2401: Clinical Chemistry Keri Brophy-Martinez Assessment of Liver Function

2. Liver Panel Albumin Bilirubin, total Bilirubin, direct AST/SGOT ALT/SGPT Alkaline Phosphatase

3. History of Bilirubin Analysis Ehrlich(1883) – Described the reaction of bilirubin with diazotized sulfanilic acid= DIAZO REACTION Malloy and Evelyn (1937) – Diazo reaction with 50% methanol as an accelerator Jendrassik and Grof ( 1938) – Diazo reaction with caffeine-benzoate-acetate as accelerator – Increased sensitivity

4. Measured vs. Calculated Measured Analytes – Total Bilirubin – Conjugated bilirubin (DIRECT) Calculated Analytes – Unconjugated bilirubin (INDIRECT)

5. Fractions & Their Characteristics Conjugated/Direct – Polar – Water-soluble – Found in plasma, unbound or free – Reacts with diazotized sulfanilic acid without an accelerator Unconjugated/Indirect – Nonpolar – Water-insoluble – Found in plasma, bound to albumin – Reacts with diazotized sulfanilic acid with an accelerator Delta – Conjugated bilirubin bound to albumin – Observed in hepatic obstructions

6. Specimen Collection and Storage Serum or plasma preferred Temperature sensitive Fasting sample preferred – Lipemia increases bilirubin concentrations No hemolysis – Hemolysis decreases the reaction of bilirubin with the diazo reagent Light sensitive – Bilirubin levels decrease by 30-50% per hour.

7. Methods of Bilirubin Analysis Jendrassik-Grof – Measures Total and Conjugated bilirubin – Principle Bilirubin pigments in serum react with a diazo reagent which results in the production of azobilirubin( a purple product). Measured at 540 nm. Caffeine -benzoate accerlerates the coupling of bilirubin with the diazo reagent. Ascorbic acid stops the reaction. Alkaline tartrate converts the purple azobilirubin to a blue azobilirubin. This product is measured spectrophotometrically @ 600 nm.

8. Jendrassik-Grof Advantages – Not affected by pH changes – Maintains optical sensitivity at low bilirubin concentrations – Insensitive to high protein concentrations Jendrassik-Grof Animation – http://webcls.utmb.edu/lo/publicdl.asp?616404F 6782E84851260BFF8F344F92903AF

9. Reference Ranges for Bilirubin

10. Urine Bilirubin Presence indicates conjugated hyperbilirubinemia Detected using urine dipsticks – Have a diazo reagent imbedded in the strip – Follows the Ehrlich principle – (Chemstrip/Multistix) Fresh urine should be used – Avoid light and oxidation

11. Urobilinogen End product of bilirubin metabolism Majority excreted in feces, some reabsorbed and returned to the liver Increased – Hemolytic disease – Defective liver-cell function Decreased – Biliary obstruction – Carcinoma

12. Determination of Urobilinogen Ehrlich’s reaction – Ehrlich’s reagent=p-dimethyl aminobenzaldehyde – Urobilinogen + Ehrlich’s reagent = Red color – Performed on fresh urine Reference Range – 0.1-1.0 Ehrlich units in two hours

13. Enzymes Liver damage results in the release of enzymes into the circulation Differentiate between functional or mechanical causes of disease Significant enzymes – AST – ALT – ALP – GGT – 5’ nucleotidase – LDH

14. Enzymes Aminotransferases – ALT and AST rise rapidly in most diseases of the liver and stay elevated for up to 2-6 weeks – Highest levels seen with hepatitis, hepatic ischemia and drug/toxin-induced necrosis Phosphatases – ALP differentiates hepatobiliary disease from bone disease – 5’-Nucleotidase is elevated in hepatobiliary disease

15. Enzymes GGT elevated in biliary obstruction and in chronic alcoholism LDH/LD serves as a nonspecific marker of cellular injury

16. Enzymes: Points to Remember Elevated Liver enzymes are as easy as ABC – Alcoholism – Biliary Obstruction – Cirrhosis

17. Misc. Liver Function Tests Prothrombin time – Elevated in liver disease Ammonia – Elevated in liver disease Glucose/Galactose Tolerance – Assess the liver’s ability to metabolize carbohydrates

18. Disease States ConditionASTALTALPGGTAlbumin Alcoholic hepatits IINIIIIN Acute Hepatitis IIIIIIIN Biliary Obstruction NI III CirrhosisNI D Reye’s Syndrome IIN I = Increased N= Normal

19. Hepatitis A Markers Performed by serological antibodies – IgM indicates acute infection and can persist for 3-6 months – IgG appears shortly after IgM, and confers lifelong immunity.

20. Hepatitis B Markers HBsAG: Hepatitis B Surface Antigen Detected prior to onset of symptoms HBcAG: Hepatitis B Core Antigen Found in an acute infection HBeAg: Hepatitis B Envelope Antigen Found in acute and chronic infections

21. Hepatitis B Virus

22. Hepatitis C Testing Two methods currently used – Anti-HCV detection by EIA (Screen) A positive test indicates exposure to HCV, it can not determine a current infection versus a past infection – Quantitative nucleic acid PCR for HCV RNA (Confirmatory)

23. References Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. http://www.abbottdiagnostics.co.uk/About_Us/UK/hepatitis_ antigen.cfm http://depts.washington.edu/labweb/Divisions/Viro/Hepatitis _sero.htm http://depts.washington.edu/labweb/Divisions/Viro/Hepatitis _sero.htm http://tmp.kiwix.org:4201/A/Hepatitis_B.html Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson.