Download Enzymes

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
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Aspartate aminotransferase (AST)
• AST involved in the transfer of an amino group between
aspartate and -ketoacids.
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Clinical Significance
• Aspartate aminotransferase (AST) is an enzyme found
primarily in the heart, liver, and muscle.
• Additional AST is released into the circulation after
injury or death of cells.
• 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.
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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.
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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
Oxaloacetate + Glutamate
MD
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Malate + NAD
Alanine Aminotransferase (ALT)
• A transferase with enzymatic activity similar to AST
• Converts alanine + α-ketoglutarate to pyruvate and glutamate
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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
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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)
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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 + NADH + H
ALT
LD
Pyruvate + Glutamate
Lactate + NAD
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.
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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
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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
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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.
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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.
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Assay for Enzyme activity
• almost all assays for ALP employ p-nitrophenyl phosphate as
the substrate.
• Bowers and Combs method based on absorption of pnitrophenol at 405 nm
• At an alkaline pH,
• p-nitrophenyl phosphate is colorless;
• the product p-nitrophenol is intensely yellow