2.2.

Transferase Liver
Enzymes

AST, ALT and GGT

Learning Objectives

Upon completion of the lectures and the exercises, the

student will be able to:
 Describe the biochemical theory & metabolic pathways,
and biochemical properties of transferases:
 Aspartate transaminase, ( AST)
 Alanine transaminase ( ALT)
 Gamma-glutamyl transferase ( GGT)
 Discuss the normal & abnormal states affecting levels of
AST, ALT and GGT
Learning Objectives

Upon completion of the lectures and the exercises, the

student will be able to:
 Describe the principles of ALT and AST analyses in
terms of key reagents and their role.
 Suggest possible steps to prevent preanalytical,
analytical and postanalytical errors in transaminase
analysis.
 Correlate the assessment of serum enzymes, with
pathology of the liver.
Outline of Lecture: Transferases

 Introduction
 Source
 Clinical Significance
 Methods of Analysis and Calculations
 Specimen
 Interpretation of Results
 Quality Control
 Sources of Errors
 Reporting and Documentation
 Summary
Introduction to Transferases

 Catalyze interconversion of functional groups.

 Transaminase- Aminoacids to -oxoacids
 - insert for GGT

o Names:
- AST; formerly Glutamate Oxaloacetate
transaminase, GOT
-ALT; formerly Glutamate Pyruvate Transaminase,
GPT
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
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
Clinical Significance of ALT

 Hepatocellular necrosis releases mitochondrial

ALT
 Associated with liver inflammation (hepatitis)
 Drugs overdose or toxicity
 Infections from Viruses or bacteria
 Alcohol
Analytical Methodology 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 is coupled with 2,4 dintrophenylhydrazine
 dintrophenylhydrazone (chromogenic
product)
 Product measured photometrically
Specimen for ALT

 Nonhemolyzed serum or plasma.

 Heparinized plasma
 < 2 day old samples
 Fasting specimen is preferred
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
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.
 Assayedknown samples
 Commercially manufactured (Humastar)

 Validate patient results

 Detects analytical errors.
Quality Control Analysis
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 0 C
 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
 Myocardia infarction
Analytical Methodology 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 Methodology 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
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+
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
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.
 Assayedknown samples
 Commercially manufactured (Humastar)

 Validate patient results

 Detects analytical errors.
Analytical Errors for
ASTActivity:

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
Clinical Significance of GGT

 Found in biliary ducts of liver, kidney tubules,

and prostate
 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 Methodology: GGT by

II. 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 or plasma reference ranges vary with

method.
Reference range
 Adult Male <55 U/L
 Adult female <38 U/L

Serum levels increase in hepatobiliary obstruction

or inflammation or in alcoholism
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.
 Assayedknown 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
Automated analysis of Transferase
Enzymes
Reporting of Transferase
Enzyme Analysis Results
 Before Reporting:
 Check that quality control samples are accepted.
 Check that results correlate well with other results

 Avoid these mistakes in reporting:

 Wrong name
 Incorrect units or reference range
 Reference range not matched to gender
 Transcribing wrong number
 Report too late
Documentation of
Transferase Enzyme Analysis
 Record patient results in result logbook
 Record QC results in QC logbook
 Retain records for recommended time
Summary
This lecture provided information about:
 Principles of alanine and aspartate transaminase
analyses in terms of key reagents and their role.
 Sources of interferance and methods to validate
results
 Interpretion of serum liver enzymes with liver
pathologies
References

 Burtis, Carl A., and Ashwood, Edward R.. Tietz:

Fundamentals of Clinical Chemistry. Philadelphia, 2001
 Arneson, W and J Brickell: Clinical Chemistry: A
Laboratory Perspective 1st ed. 2007 FA Davis