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 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 oC < 8% At 15-25 oC < 10% Stability at -20 oC: 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 37oC: Decrease in absorbance at 340 nm is determined by continuous monitoring. AST Aspartate + -Ketoglutarate Oxaloacetate + Glutamate Oxaloacetate + NADH + H Malate + NAD MD M. Zaharna Clin. Chem. Lab. 2009

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 Serum, heparin plasma or EDTA plasma Storage & Stability Loss of activity within 3 days At 2-8 oC < 10% At 15-25 oC < 17% Stability at -20 oC: 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 ALT Alanine + -Ketoglutarate Pyruvate + Glutamate Pyruvate + NADH + H Lactate + NAD LD 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 pH for ALP is between 9 and 10 ALP requires Mg2+ 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 Mg2+ 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 McCombs 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 M. Zaharna Clin. Chem. Lab. 2009