Lab. 5

1. AST, ALT & ALP • Lab. 5

2. Aminotransferases • Aminotransferases or transaminases are a group of enzymes that catalyze the interconversion of amino acids and ketoacids (oxoacids) by transfer of amino group • The two aminotransferases of greatest clinical significance are: • Aspartate aminotransferase (AST), formerly termed glutamate oxaloacetate transaminase (GOT). • Alanine aminotransferase (ALT), formerly termed glutamate pyruvate transaminase (GPT). • Pyridoxal phosphate (P-5-P) is coenzyme • Mohammed Laqqan

3. Aspartate aminotransferase (AST) • AST involved in the transfer of an amino group between aspartate and -ketoacids. • Mohammed Laqqan

4. Clinical Significance • Aspartate aminotransferase (AST) is an enzyme found primarily in the heart, liver, and muscle. • Additional AST is released into the circulation after injury or death of cells. • This test is one of several that are performed when there has been damage to the heart muscle, as in myocardial infarction, and in assessing liver damage. • Infants levels approximately twice the adult level, these decline to adult levels by approximately 6 months of age. • Mohammed Laqqan

5. Specimen Collection & Storage • Specimen: • Serum, heparin plasma or EDTA plasma • Hemolysis should be avoided because it can dramatically increase serum AST concentrations • (RBCs contain 15 X the AST activity in serum) • Post AMI • Rises 6 – 8 hours • Peaks at 24 hours • Returns to normal by day 5 • AST levels are highest in acute hepatocellular disorders "viral hepatitis, cirrhosis. • Mohammed Laqqan

6. Assay for Enzyme activity • Measurement by Karmen method • A coupled reaction involving: • pyridoxal-5-phosphate (P-5-P) • and malate dehydrogenase (MDH) • at 37oC: • Decrease in absorbance at 340 nm is determined by continuous monitoring. AST Aspartate + -Ketoglutarate Oxaloacetate + Glutamate Oxaloacetate + NADH + H Malate + NAD MD • Mohammed Laqqan

7. Alanine Aminotransferase (ALT) • A transferase with enzymatic activity similar to AST • Converts alanine + α-ketoglutarate to pyruvate and glutamate • Mohammed Laqqan

8. Clinical Significance • It is found in the kidneys, heart, and skeletal muscle tissue but primarily in liver tissue. • The test is used mainly in the diagnosis of liver disease and to monitor the effects of hepatotoxic drugs. • Often higher than AST with liver damage and tend to remain elevated longer • Remains normal in AMI • Mohammed Laqqan

9. Specimen Collection • Specimen: • Serum, heparin plasma or EDTA plasma • Hemolysis should be avoided because it can increase serum ALT concentrations • (ALT in RBCs is roughly 5 X that of serum) • Mohammed Laqqan

10. ALT Alanine + -Ketoglutarate Pyruvate + Glutamate Pyruvate + NADH + H Lactate + NAD LD Assay for enzyme activity • The most common method in use today for measurement of ALT activity utilizes a coupled enzymatic procedure for monitoring disappearance of NADH. • In this approach lactate dehydrogenase (LDH) and its required cofactors are added and catalyze the conversion of pyruvate to lactate • This causes simultaneous oxidation of reduced nicotinamide adenine dinucleotide (NADH). • The disappearance of NADH is followed spectrophotometrically (at 340 nm).

11. Levels of AST & ALT • AST is assessed along ALT in monitoring liver damage. • These two values normally exist in an approximately 1:1 ratio. • As a rough guide: • AST>ALT in: • alcoholic hepatitis and cirrhosis, • metastatic cancer of the liver • non-biliary cirrhosis, • while ALT>AST in: • viral and drug hepatitis, • chronic hepatitis C • and hepatic obstruction. • Mohammed Laqqan

12. Alkaline Phosphatase (ALP) • Phosphatases transfer a phosphate moiety from one group to a second, forming an alcohol and a second phosphate compound. • The optimal reaction pH for ALP is between 9 and 10 and varies with the buffer and substrate. • ALP requires Mg2+ as an activator • Mohammed Laqqan

13. Isoenzymes • ALP exists as a number of isoenzymes • Major are those found in Liver, bone, placenta, and then intestinal fraction • Electrophoresis for isoenzyme analysis • Liver isoenzyme (fastest) • Bone isoenzyme • Placental isoenzyme • Intestinal isoenzyme (slowest) • Immunochemical methods now available • Mohammed Laqqan

14. Clinical Significance • Alkaline phosphatase (ALP) is an enzyme found in the liver, bone, placenta, intestine, and kidneys • Primarily in the cells lining the biliary tract and in the osteoblasts involved in the formation of new bone. • ALP is normally excreted from the liver in the bile. • Increased ALP levels are found most commonly during: • periods of bone growth (as in children), • in various types of liver disease, • and in biliary obstruction. • Serum ALP activity primarily reflects changes in bone and liver function, even though higher ALP activities can be found in other organs. • Mohammed Laqqan

15. Specimen Collection • Blood should be drawn after a fast of at least 8 hours. • Serum or heparinized plasma. • Slight hemolysis is tolerable, but gross hemolysis should be avoided. • These increases may be caused by: • the release of ALP from complexes with lipoproteins, • It is best to analyze ALP specimens the same day they are drawn. • ALP is inhibited by metal-complexing anticoagulants; EDTA, oxalate, and citrate inhibit the enzyme by complexing Mg2+ and should not be used. • Mohammed Laqqan

16. Assay for Enzyme activity • almost all assays for ALP employ p-nitrophenyl phosphate as the substrate. • Bowers and Combs method based on absorption of p-nitrophenol at 405 nm • At an alkaline pH, • p-nitrophenyl phosphate is colorless; • the product p-nitrophenol is intensely yellow