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AST, ALT & ALP Lab. 5.

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Presentation on theme: "AST, ALT & ALP Lab. 5."— Presentation transcript:

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 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. 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. Mohammed Laqqan

5 Specimen Collection & Storage
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 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). Alanine + -Ketoglutarate Pyruvate + Glutamate Pyruvate + NADH + H Lactate + NAD LD ALT

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

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