Transferase Liver Enzymes

AST, ALT and GGT

 Outline of Lecture: Transferases
• Introduction
• Source
• Clinical Significance
• Methods of Analysis and Calculations
• Specimen
• Interpretation of Results
• Sources of Error
• Summary
 Introduction to Transferases
• Catalyze interconversion of functional groups.
– Transaminase- Aminoacids to -oxoacids
- GGT Catalyzes the transfer of the y-glutamyl
group from peptides and compounds that contain
it to an acceptor.
• Names:
- AST; formerly Glutamate Oxaloacetate
transaminase, SGOT
-ALT; formerly Glutamate Pyruvate Transaminase,
SGPT
 Alanine Transaminase (ALT) Biochemical
Theory & Metabolic Pathways
Transaminase - transfers amino groups
• Other descriptive names:
– serum glutamic pyruvate transaminase
(SGPT)
– alanine aminotransferase ( ALAT)
• ALT catalyzes the reaction:
L-Alanine + -Oxoglutarate  Pyruvate +
L-Glutamate
 Sources
• Transaminases are widely distributed throughout the
body.
• AST is found primarily in the heart->liver->skeletal->
muscle ->kidney.
• ALT is found primarily in the liver -> kidney
• ALT is exclusively cytoplasmic; but small mitochondrial
ALT are found i.e. isoenzymes (ALTc &ASTm)
• both mitochondria and cytoplasmic forms of AST are
found in cells.
• About 5% to 10% of the AST activity in serum from
healthy individuals is of mitochondrial origin.
 Clinical Significance of ALT
• Hepatocellular necrosis releases mitochondrial ALT
– Associated with liver inflammation (hepatitis)
• Drugs overdose or toxicity
• Infections from Viruses or bacteria
• Alcohol, cirrhosis
• Monitor for liver toxicity in anti-retroviral
therapy (ART).
• Rise before jaundice
 Clinical Significance of ALT and AST
Damage to tissue can release different types of
enzymes based on their location.
• Mild inflammation of the liver
• Reversibly increases the permeability of the cell
membrane releases cytoplasmic enzymes: AST
• Necrosis releases mitochondrial ALT & AST
• Enzyme activity levels in body fluids can reflect
leakage from cells due to cellular injury, changes
in enzyme production rate, metabolic or genetic
states or proliferation of tumors
 Analytical Methods of ALT
Continuous Monitoring Method
• Coupled reaction at 37 0 C
• l-Alanine + a-oxoglutarate ALT, P-5’-P l-
glutamate + pyruvate
• Pyruvate + NADH + H+ LD lactate +
NAD+
• Decrease in Abs. 340 nm (multi-point
analysis).
Calculating Absorbance Per Minute
• In ALT the absorbance decreases over time
• The result is -A X F
Min
• Where F340 = -1746
• So final activity is a positive number U/L
 Continuous Monitoring Analysis Results
Example
• The following results for ALT were determined:
Time (min) Absorbance

0 1.350

1 1.300

2 1.250

3 1.201

• Are the results progressing in the expected direction?

Answer: yes, they are decreasing

 Continuous Monitoring Results
of ALT analysis
What is the  Abs for each minute?

Answer: -0.050/min; -0.050/min; -0.049 /min

Are the results consistent?

Answer: Yes, absorbance decreases

consistently
Average = -0.050 /min
 Fixed End-Point Assay for ALT
• Photometric Method
• ALT + 2,4 dintrophenylhydrazine 
dintrophenylhydrazone (chromogenic
product)
• Product measured photometrically
 Specimen for ALT
• Non-hemolyzed serum or plasma.
• Heparinized plasma
• < 2 day old samples
• Fasting specimen is preferred
• Half-life of ALT (27+10 hours)
 Reference Values of ALT

Serum or plasma reference ranges vary with

method
• Reference ranges reflect the normal amount
in serum or plasma:
• Adult male <45 U/L
• Adult female <34 U/L
 Sources of Error in ALT
• Nonlinear results from side reactions can be repeated
with diluted sample
• Hemolyzed samples cause false positive
• Loss of activity if specimen is stored at room
temperature
• Unstable analytical temperature (deviation from 37 0C)
• Unstable photometer Substrate exhaustion due to high
levels of enzyme activity
 Problem-Solving Enzyme Analysis

What should be considered to solve the

problem of unstable  Abs/ min?

Answer: Look at results and see if

Abs/min is consistently decreasing.
That may indicate substrate exhaustion
and need to dilute.
If Abs/min fluctuates randomly it may
indicate unstable temperature.
 Aspartate Transaminase (AST)
Biochemical Theory & Metabolic
Pathway
Serum glutamic oxaloacetic transaminase (SGOT) or
aspartate aminotransferase (ASAT/AAT)
• Transfers amino groups to form oxaloacetate
• Found in serum from various tissues
• Associated with hepatocytes
 Clinical Significance of AST
• Hepatocellular inflammation releases cytoplasmic
AST
• Hepatocellular necrosis releases mitochondrial
AST.
– Associated with liver inflammation (hepatitis)
• Drugs overdose or toxicity
• Infections from Viruses or bacteria
• Alcohol
• Other organ diseases
– Myocardial infarction
 Analytical Method of AST
• Coupled reaction at 37 0 C
• Amino acid + substrate -(AST,
coenzyme)amino acid + product
• Substrate (product of 1st) + coenzyme -(2nd
enzyme) product + reduced coenzyme
• Decrease in Abs. 340 nm (continuous
monitoring).
 Analytical Method of AST
• Continuous Monitoring Method
• l-Aspartate + a-oxoglutarate -(AST, P-5’-P)l-
glutamate + oxaloacetate
• Oxaloacetate + NADH + H+ (MDH) >l-malate +
NAD+
• Decrease in Abs. 340 nm (multi-point assay)
Calculating Absorbance Per Minute
• In AST the absorbance decreases over time
• The result is -A X F
Min
• Where F340 = -value
• So final activity is a positive number U/L
 Fixed End-Point Assay for AST
• Photometric Method
• AST is coupled with 2,4 dintrophenylhydrazine
 dintrophenylhydrazone (chromogenic
product)
• Product measured photometrically
 Specimen for AST
• Nonhemolyzed serum or plasma.
• Heparinized plasma
• < 2 day old samples
• Nonfasting may falsely increase
• AST activity in serum is stable for up to 48
hours at 4°C.
 Reference Ranges of AST
Serum or plasma reference ranges vary with
method
• Reference range:
• Adult male <35 U/L
• Adult female <31 U/L
 Interpretation of Transaminases
HIV treatment, especially the reverse transcriptase
inhibitors are associated with metabolic
complications:
• Pancreatitis, hypertriglyceridemia, and lactic
acidosis
• Hepatomegaly, and hepatic inflammation
Patients taking these medications will have liver
enzyme levels monitored every few months to
monitor for these complications
 Here is a question for you:
List the initial substrate and the final product(s)
for AST.

Answer: initial substrate = -oxoglutarate

and final products= lactate + NAD+
 Analytical Errors for AST Activity:
Sources of errors in testing for AST:
• Unstable temperature (deviation from 37 0 C
• Unstable photometer
• Substrate exhaustion due to high
levels of enzyme activity
 Sources of Error in AST
• Presence of ammonia will consume the NADH
• Nonlinear results from side reactions can be
repeated with diluted sample
• Hemolyzed specimens cause false increase
• Nonfasting specimens may falsely increase
• Loss of activity if stored at room
temperature for > a day
 Gamma-Glutamyl Transferase (GGT)
Biochemical Theory & Metabolic Pathway
• Involved in the transfer of glutamyl group
• Glutathione metabolism
• Cysteine product preserves intracellular
homeostasis of oxidative stress
 Source
• Found in biliary ducts of liver, kidney tubules,
and prostate
 Clinical Significance of GGT
• Associated with disease in the liver such as
hepatobiliary obstruction or inflammation
• Elevated because of alcoholism
 Specimen for GGT
• Nonhemolyzed serum
• EDTA plasma
 Analytical Methods for GGT
I. Serum Start Method:
• Peptide GGPNA substrate – GGT 
p-nitroaniline
– Increased in Abs at 405 nm
 Analytical Methods for GGT

I. Continuous Monitoring method/Substrate Start

Method
• Coupled reaction at 37 0C
• Gamma-glutamyl-p-nitroanalide + (HCl)
glycylglycine –(GGT, pH 8.2)--> Gamma-
glutamylgylcylglycine + p-nitroaniline.
• Measure increase Abs. 405 nm as determined
by continuous or 2- point monitoring with a
manual or automated spectrophotometer
Calculating Absorbance Per Minute
• In GGT the absorbance inreases over time
• The result is A X F
Min
• Where F405 = value
• So final activity is a positive number U/L
 Interpretation of GGT
• Serum levels increase in hepatobiliary
obstruction or inflammation or in alcoholism
• Serum or plasma reference ranges vary with
method.
Reference range
• Adult Male<55 U/L
• Adult female <38 U/L
 Quality Control
• A normal & abnormal quality control sample
should be analyzed along with patient samples,
using Westgard or other quality control rules for
acceptance or rejection of the analytical run.
– Assayed known samples
– Commercially manufactured (Humastar)

• Validate patient results

• Detects analytical errors.
Sources of Errors for GGT Activity:

• Loss of activity if stored at room temperature

for longer than a day
• Heparinized plasma produces turbid samples
• Other anticoagulants( citrate, oxalate,
fluoride) depress activity of enzyme
 Pre-analytical Errors for GGT
• Hemolysis
• Samples > 2 days old not stored in refrigerator
or freezer.
 Analytical Errors for GGT

• Substrate exhaustion from extremely elevated

enzyme activity
• Unstable temperature during analysis
• Unstable photometer