enzymes ast, alt & alp lab. 6. m. zaharna clin. chem. lab. 2009 aminotransferases...

EnzymesAST, ALT & ALP

Lab. 6

M. Zaharna Clin. Chem. Lab. 2009

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),


Aspartate Aminotransferase (AST)

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


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.


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 oC < 8%

• At 15-25 oC < 10%

• Stability at -20 oC: at lest 3 months


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


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.



• Specimen:• 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



• 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


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.


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.


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+


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.


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.



• 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.



• 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.


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

enzymes ast, alt & alp lab. 6. m. zaharna clin. chem. lab. 2009 aminotransferases...

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