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

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

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

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

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

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.

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 600 nm.

Jendrassik-Grof Advantages – Not affected by pH changes – Maintains optical sensitivity at low bilirubin concentrations – Insensitive to high protein concentrations Jendrassik-Grof Animation – E BFF8F344F92903AF

Reference Ranges for Bilirubin

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

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

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

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

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

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

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

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

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

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.

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

Hepatitis B Virus

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)

References Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. antigen.cfm _sero.htm _sero.htm Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson.