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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 oC
• At 15-25 oC
< 8%
< 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.
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 oC
• At 15-25 oC
< 10%
< 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
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 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