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CLINICAL BIO CHEMISTRY PRESENTATIONc

M. Zaharna Clin. Chem. Lab. 2009

Lab. 5

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

Aspartate aminotransferase (AST)

• AST involved in the transfer of an amino group between aspartate and -ketoacids.

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.

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.

Aspartate + -Ketoglutarate Oxaloacetate + Glutamate Oxaloacetate + NADH + H Malate + NAD

MD

AST

Alanine Aminotransferase (ALT)

• A transferase with enzymatic activity similar to AST• Converts alanine + α-ketoglutarate to pyruvate and glutamate

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 + NADLD

ALT

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.

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

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 availableMohammed Laqqan

M. Zaharna Clin. Chem. Lab. 2009