Enzymes AST ALT ALP Lab 6 Aminotransferases Aminotransferases

Enzymes AST, ALT & ALP Lab. 6

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), • and alanine aminotransferase (ALT), formerly termed glutamate pyruvate transaminase (GPT), M. Zaharna Clin. Chem. Lab. 2009

Aspartate Aminotransferase (AST) • AST involved in the transfer of an amino group between aspartate and ketoacids. M. Zaharna Clin. Chem. Lab. 2009

Diagnostic Significance • AST is an enzyme found primarily in the heart, liver, and muscle. • It is released into the circulation after injury or death of cells. • Thus, 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. M. Zaharna Clin. Chem. Lab. 2009

Specimen Collection & Storage • Specimen: • Serum, heparin plasma or EDTA plasma • Hemolysis should be avoided because it can dramatically increase serum AST concentrations • (RBCs contain high AST activity) • Storage & Stability • Loss of activity • At 2 -8 o. C • At 15 -25 o. C < 8% < 10% • Stability at -20 o. C: at lest 3 months M. Zaharna Clin. Chem. Lab. 2009

Assay for Enzyme activity • Measurement by Karmen method • A coupled reaction involving: • pyridoxal-5 -phosphate (P-5 -P) • and malate dehydrogenase (MDH) • at 37 o. C: • Decrease in absorbance at 340 nm is determined by continuous monitoring. Aspartate + -Ketoglutarate Oxaloacetate + NADH + H AST MD M. Zaharna Clin. Chem. Lab. 2009 Oxaloacetate + Glutamate Malate + NAD

Alanine Aminotransferase (ALT) • A transferase with enzymatic activity similar to AST • Converts alanine + α-ketoglutarate to pyruvate and glutamate M. Zaharna Clin. Chem. Lab. 2009

Diagnostic 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. M. Zaharna Clin. Chem. Lab. 2009

Specimen Collection & Storage • Specimen: • Serum, heparin plasma or EDTA plasma • Storage & Stability • Loss of activity within 3 days • At 2 -8 o. C • At 15 -25 o. C < 10% < 17% • Stability at -20 o. C: at lest 3 months • A marked decrease in ALT activity is seen following freeze/thaw cycles M. Zaharna Clin. Chem. Lab. 2009

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). M. Zaharna Clin. Chem. Lab. 2009

Assay for Enzyme activity Alanine + -Ketoglutarate Pyruvate + NADH + H ALT LD Pyruvate + Glutamate Lactate + NAD • The disappearance of NADH is followed spectrophotometrically (at 340 nm). M. Zaharna Clin. Chem. Lab. 2009

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 • and non-biliary cirrhosis, • while ALT>AST in: • viral and drug hepatitis, • chronic hepatitis C • and hepatic obstruction. M. Zaharna Clin. Chem. Lab. 2009

Levels of AST & ALT • The degree of increase in these enzyme levels provides information as to the possible source of the problem. • A twofold increase is suggestive of an obstructive problem, often requiring surgical intervention. • A 10 -fold increase of ALT and AST indicates a probable medical problem such as hepatitis. M. Zaharna Clin. Chem. Lab. 2009

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 p. H for ALP is between 9 and 10 • ALP requires Mg 2+ M. Zaharna Clin. Chem. Lab. 2009

Diagnostic 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. M. Zaharna Clin. Chem. Lab. 2009

Diagnostic Significance • Serum ALP activity primarily reflects changes in bone and liver function, even though higher ALP activities can be found in other organs. • Individuals with blood types B and O exhibit increases in serum activities of intestinal ALP approximately 2 h after eating a fatty meal. • ALP is often interpreted as “abnormal, ” particularly in children, because of the use of inappropriate reference intervals by a laboratory. M. Zaharna Clin. Chem. Lab. 2009

Specimen Collection & Storage • 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. • Certain sample storage conditions tend to increase serum ALP. • There is a significant increase in activity after warming of previously refrigerated or frozen sera. • The ALP activity in fresh serum increases by up to 2% in 6 h at 25° C. • Increases of up to 30% of ALP activity occur after frozen serum is thawed, and in lyophilized specimens after reconstitution. M. Zaharna Clin. Chem. Lab. 2009

Specimen Collection & Storage • 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 Mg 2+ and should not be used. M. Zaharna Clin. Chem. Lab. 2009

Assay for Enzyme activity • almost all assays for ALP employ p-nitrophenyl phosphate as the substrate. • Bowers and Mc. Combs method based on absorption of p-nitrophenol at 405 nm • At an alkaline p. H, • p-nitrophenyl phosphate is colorless; • the product p-nitrophenol is intensely yellow M. Zaharna Clin. Chem. Lab. 2009