AST/ALT || C1LP8924BAAONTVD

 Can be separated by Electrophoresis or Paper

INTRODUCTION Chromatography
 Isoenzyme analysis is NOT routinely performed in
 Aminotransferases constitute a group of enzymes
the clinical laboratory (most done in research)
that catalyze the interconversion of amino acid to 2
oxo-acids by transfer of amino group Clinical Significance of AST and ALT Determination
 Specificity of the individual enzymes derives from
the particular amino acid that serves as the other AST ALT
donor of an amino group Myocardial Infarction Many dss. Related to
 Reaction catalyzed by the enzyme is the reversible liver damage
transfer of an –NH2 from an amino acid to keto acid Hepatocellular Disease Viral Hepatitis
Skeletal Muscle Disorder
 In the body, the processes serve to provide sources
Pulmonary Embolism
of nitrogen for the urea cycle
and Muscular Dystrophy
Reaction  ALT is more elevated than AST in various
inflammatory conditions of the liver
 ALT  Evaluation of hepatic disorders
 Screen in blood banking for donors
L-alanine + a-ketoglutarate <---->  Assay of ALT will often be requested along with that
Pyruvate + L-glutamate of AST to help determine the source of elevated AST
level and to detect liver involvement concurrent
*Pyruvate formed is metabolized further through with myocardial injury
the citric acid cycle to provide biochemical energy or
is involved in the synthesis of fatty acids ASPARTATE AMINOTRANSFERASE

Description
 AST
L–aspartate + a-ketoglutarate <---->  Is an enzyme belonging to the class of transferases
Oxaloacetate + L-glutamate  Commonly referred as a transaminase and is
involved in the transfer of an amino group between
*Oxaloacetate is also used in the citric acid cycle aspartate and alpha-ketoacids (a-ketoglutarate) to
form oxaloacetate and glutamate
In each of the reaction, glutamate is formed which is  Characterized by the movement of an amine or a
then deaminated to: phosphate group from one molecule to another
 Serum Glutamic-Oxaloacetic Transaminase (Old
1. Produce ammonia (for the urea cycle) term)
2. Generate a-ketoglutarate  Pyridoxal Phosphate serves as coenzyme
- A form of Vit. B6 derived from pyridoxamine which is
Two Aminotransferases
essential for aminotransferases activity
 Aspartate Aminotransferase (SGOT)
OLDER TERMINOLOGY Serum Glutamic
 Alanine Aminotransferase (SGPT)
Oxaoacetic
Transaminase (SGOT)
- In the parallel measurement of ALAT and ASAT is EC NAME L-Aspartate: 2
applied to distinguish Liver from Head or Skeletal oxoglutarate
Damages Aminotransferase
- Increased AST than ALT that is out of proportion EC NUMBER 2.6.1.1
 Caused by a differential reduction in OPTIMUM PH 7.4 (7.3-7.8)
L–aspartate + a-ketoglutarate <----> Oxaloacetate + L-glutamate
hepatic ALT due to deficiency of the
cofactor P-5-P
COOH COOH
- The ALAT/ASAT ratio is used in the differential COOH C=O AST COOOH HC=NH2
diagnosis of liver diseases HC=NH2 + CH2 <----> C=O + CH2
 <1 = Mild liver damage CH2 CH2 CH2 CH2
 >1 = Severe/Chronic liver damage COOH COOH COOH COOH
AA I KA II KA I AA II
Isoenzymes of Aminotransferases  The transamination reaction is important in
intermediary metabolism because of its function in
 2 Fractions the synthesis and degradation of amino acids
 The Ketoacids formed by the reaction
- Cell Cytoplasm (Soluble) (Pyruvate and Oxaloacetate) are ultimately
 Predominant form occurring in the oxidized by the Tricarboxylic Acid Cycle
serum (AKA Citric Acid Cycle/Krebs Cycle) to
- Mitochondria provide a source of energy
 Markedly elevated in disorders
producing cellular necrosis (Liver
Cirrhosis)
- Both fractions are detected in sera in patients with
coronary and hepatobiliary diseases
Tissue Sources Methods

 AST is widely distributed in human tissue  Manometric

Highest Concentration Liver and Cardiac Tissue
(Heart)
Significant Amounts Skeletal Muscle and
Kidney
Smaller Amounts Spleen, Pancreas, Lungs
 Erythrocytes has 10-15X AST than in serum, thus
HEMOLYSIS SHOULD BE AVOIDED
Diagnostic Significance
 The clinical use of AST is limited mainly to the evaluation
of hepatocellular disorders and skeletal muscle
involvement
 Increased AST levels can occur in connection with damages
of heart or skeletal muscle as well as of liver parenchyma
 In AMI, AST levels begin to rise within 6-8 hours,
peak at 24 hours and generally return to normal
within 5 days
 However, because of the wide tissue
distribution, AST levels are not useful in the
diagnosis of MI
 AST elevations are frequently seen in pulmonary embolism
(blockage in the artery of the lungs; blood clot, fat globule
or a bubble of air)
 Following congestive heart failure, AST levels also may be
increased, probably reflecting liver involvement as a result
of inadequate blood supply to the organ
 AST levels are highest in Acute Hepatocellular Disorders
 In Viral Hepatitis, levels may reach 100 times ULN (Upper
Limit of Normal)
 In cirrhosis, only moderate levels (Approx 4 times ULN) are
detected
 Skeletal Muscle disorders such as muscular dystrophies,
and inflammatory conditions also cause increase in AST
levels (4-8x ULN)
 AST exists as two isoenzyme fractions located in the cell
cytoplasm and mitochondria
 The intracellular concentration of AST may be
7000 times higher than the extracellular
concentration
- Convenient and accurate method for following reactions in
which one of the component is a gas
 Oxidase (O2 uptake)
 Decarboxylase (CO2 uptake)
- Oxaloacetate is converted into CO2 using aniline citrate
- CO2 formed is measured manometrically
 Chromatographic
- Paper Chromatography used to isolate glutamate
 Spectrophotometric
- Authored by Karmen
- Based on the reduction of oxaloacetate to malate by
NADH and MDH
 Fluorometric
- Is the result of electronic transition
- Converts absorbing molecules into an excited state
- NAD formed is measured fluorometrically
 Colorimetric
- Method of Reitman and Frankel
- Oxaloacetate reacts with a color reagent to form a colored
product which is measured photometrically
 General Principles Employed in the Measurement of Total
Activity
- Assays for both AST and ALT utilize the same type of
reactions for detection of products
- 3 Major Approaches which involve the measure of the
formation of ketoacid produced in the reaction
A. Reaction with 2,4-Dinitrophenylhydrazone
B. Coupling with Diazonium Salts
C. Coupled Enzyme Assay

Assay for Enzyme Activity

 Assay methods for AST are generally based on the

principle by Karmen Method (Author Karmen 1955) which
incorporates a coupled enzymatic reaction using Malate
Dehydrogenase as the indicator reaction and monitors the
change in absorbance at 340nm continuously as NADH is
oxidized to NAD+
 Optimal pH = 7.3-7.8

1. Aspartate + Oxaloacetate +

a-ketoglutarate <---AST---> Glutamate

2. Oxaloacetate + NADH <--MDH---> Malate + NAD+

 Oxaloaceate will inhibit AST with time

Sources of Error

 HEMOLYSIS (False High; 10-15X in RBCs)

 TURBID or ICTERIC samples (Difficulties in Blanking)
- Will have light scattering
 STABILITY IN SERUM (3-4 days at refrigerated
temperatures)

Reference Range

 5-30 U/L at 370C

 Reaction with 2,4-Dinitrophenylhydrazone Coupling with Diazonium Salts Coupled Enzyme Assay
ASSAY Colorimetric Assay UV Spectrophotometric Assay
(Continuous Kinetic Monitoring)
AUTHOR Reitman and Frankel Karmen (1955)
Mod. By Henry, Chiamori, Gulob,
Bertman (1960)
DESCRIPTION Involves a reaction between the color The coupling of keto acid product Preferred Method for both ALT/AST
reagent and the ketoacid formed with diazonium salt forming a
diazonium derivative

6-Benzamido-4-Methoxy-m-toluidine
diazonium chloride

 Reacts specifically with

oxaloacetic acid and can be
utilized only by the AST
substrate
PRINCIPLE Oxaloacetate formed in the reaction is Oxaloacetate is determined by a coupled
converted by 2,4-dinitrophenylhydrazone enzymatic reaction using malate
into a corresponding dinitrophenyl- dehydrogenase as indicator enzyme and
hydrazone derivative which gives a strong monitors the change in absorbance at
blue color measured at 505 nm 340nm as NADH is oxidized to NAD

Intensity of Color is directly proportional

to concentration
REACTION 1. Aspartate + a-ketoglutarate -AST-> 1. Asparate + a-ketoglutarate –AST->
Oxaloacetate + Glutamate Oxloacetate + Glutamate
(2) Oxaloacetate + 2,4-DNPH -----> 2. Oxaloacetate + NADH –MDH--> Malate
Oxaloacetatedinitrophenylhydrazone +NAD

 2,4-DNPH = Indicator Enzyme = will convert the

activator/chromogen/dye product of the first reaction/primary
reaction (Oxaloacetate) into a measured
product(NAD+) in a linear manner (every
molecule of primary product is
instantaneously converted regardless of
the substrate concentration)

NADH = Hydrogen Donor and coenzyme

MAJOR Lack specificity Some determination of blank values High absorbance reading due to NADH
PROBLEMS - Any keto compound present is necessary so that the final activity
contributes to the color reaction may be corrected for there are other
(false high) materials that will react with the
- The substate ketoacid (a- color reagent
ketoglutarate) reacts with the
color reagent to some extend
UNIT OF Rate of decrease in absorbance at 340nm
ACTIVITY resulting from oxidation of NADH/unit to
NAD+
REFERENCE 5-35U/L (with pyridoxal-5-phosphate)
RANGE
Role of Pyridoxal Phosphate OLDER TERMINOLOGY Serum Glutamic
Pyruvic Transaminase
 Steps in Transamination Reaction (SGPT)
EC NAME L-Alanine: 2 oxoglutarate
Amino Acid I + Pridoxal <--AST--> Keto Acid I Aminotransferase
Keto Acid + Pyridozel <--AST--> Amino Acid II EC NUMBER 2.6.1.2
OPTIMUM PH 7.4 (7.3-7.8)
- This modification of assay has made the L–alanine + a-ketoglutarate <----> Pyruvate + L-glutamate
measurement of AST and ALT more reliable
COOH COOH
ALANINE AMINOTRANSFERASE COOH C=O AST COOOH HC=NH2
HC=NH2 + CH2 <----> C=O + CH2
Description
CH3 C00H CH3 CH2
COOH
 Is a transferase with enzymatic activity similar to AST
AA I KA II KA I AA II
- Pyridoxal-5-Phosphate acts as a coenzyme
 Specifically, it catalyzes the transfer of an amino group
from alanine to alpha-ketoglutarate with the formation of
glutamate and pyruvate
 Serum Glutamic-Pyruvic Transaminase (Old term)
Tissue Sources  Cardiac tissue contains small amount of ALT activity, but
the serum level usually remains normal in AMI unless
 ALT is distributed in many tissues with highest subsequent liver damaged has occurred
Highest Concentration Liver - ALT level have historically been compared with levels of
Lower Levels Heart Skeletal Muscles AST to help determine the source of an elevated AST level
Significant Levels Kidney and to detect liver involvement concurrent with
- It is considered the more liver-specific enzyme of the myocardial injury
transferases and a valuable indicator of obstructive liver
disease Assay for Enzyme Activity
- Erythrocytes contain 5-8x as much as in serum therefore
HEMOLYSIS MUST BE AVOIDED  The typical assay procedure for ALT consists of Coupled
Enzymatic Reaction using LD as the indicator enzyme,
Diagnostic Significance which catalyzes the reduction of pyruvate to lactate with
simultaneous oxidation of NADH
 Clinical elevations of ALT assays are confined mainly to  The change in absorbance at 340nm measured
evaluation of hepatic disorders continuously is directly proportional to ALT activity
 Higher levels are found in hepatocellular disorders rather  The optimal pH is 7.3-7.8
than extra-hepatic or intrahepatic obstructive disorders  The reaction proceeds:
 In acute inflammatory conditions of the liver, ALT
elevations are frequently higher than those of AST and Alanine + a-ketoglutarate <--ALT--> pyruvate + glutamate
tend to remain elevated longer as a result of the longer
half-life or those of ALT in serum (16 hours , and 24 hours Pyruvate + NADH + H+ <--LD--> Lactate + NAD+
respectively)
Sources of Error
 STABILITY = 3-4 Days at 40C Reference Range
 HEMOLYSIS = False elevation
 TURBID OR ICTERIC (pertaining to Jaundice) Samples =  ALT = 6-37 U/L at 370C
difficulty in blanking

Measurement of Total Activity (Same with AST)

Reaction with 2,4-Dinitrophenylhydrazone Coupling with Diazonium Salts Coupled Enzyme Assay
ASSAY Colorimetric Assay Uv Spectrophotometric Assay
AUTHOR Reitman and Frankel Karmen
DESCRIPTION Involves a reaction between the color The coupling of keto acid product Preferred Method for both ALT/AST
reagent and the ketoacid formed with diazonium salt forming a
diazonium derivative
PRINCIPLE Pyruvate formed in the reaction is converted Pyruvate is determined by a coupled
by 2,4-dinitrophenylhydrazone into a enzymatic reaction using Lactate
corresponding dinitrophenyl-hydrazone dehydrogenase as indicator enzyme
derivative which gives a strong blue color which catalyzes the reduction of
measured at 505 nm pyruvate to lactate with simultaneous
oxidation of NADH. The change in Abs at
340nm is directly proportional to ALT
acitivty
REACTION 1. Alanine + a-ketoglutarate -AST-> 1. Alanine + a-ketoglutarate –AST->
Pyruvate + Glutamate Pyruvate + Glutamate
(2) Pyruvate + 2,4-DNPH -----> 2. Pyruvate + NADH –LDH--> Lactate
Pyruvate dinitrophenylhydrazone +NAD
MAJOR Lack specificity Some determination of blank values High absorbance reading due to NADH
PROBLEMS - Any keto compound present is necessary so that the final activity
contributes to the color reaction may be corrected for there are other
(false high) materials that will react with the
- The substate ketoacid reacts with color reagent
the color reagent to some extend
REFERENCE Less than 55 U/L (with P-5-P)
RANGE
Optimized UV-Test Acoording to the IFCC (International Federation of Clinical Chemistry and Laboratory Medicine)

AST ALT
PRINCIPLE L-aspartate +2-oxoglutarate <--AST--> L-glutamate + L-alanine +2-oxoglutarate <--AST--> L-glutamate + pyruvate
oxaloacetate Pyruvate + NADH + H+ <--LDH--> Lactate + NAD+
Oxaloacetate + NADH + H+ <--MDH--> Malate + NAD+ - The addition of Pyridoxal-5-Phosphate (P-5-P)
- The addition of Pyridoxal-5-Phosphate (P-5-P) stabilizes the activity of transaminases and avoids
stabilizes the activity of transaminases and avoids falsely low values in samples containing insufficient
falsely low values in samples containing insufficient endogenous P-5-P
endogenous P-5-P  Myocardial Infarction
 Myocardial Infarction  Liver Disease
 Liver Disease  Intensive Care Patients
 Intensive Care Patients

REAGENTS R1 TRIS Buffer (pH 7.65) R1 TRIS Buffer (pH 7.15)

- Tris hydroxymethyl
aminomethane
L-aspartate = substrate
MDH = indicator enzyme
LDH = decrease interference of
endogenous pyruvate
R2 Oxoglutarate = amine acceptor
NADH = coenzyme/H+ donor
P-5-P Fluid Stable Good’s Buffer (pH 9.6)
Pyridoxal-5-Phosphate
- Conezyme
- Stimulate APO-AST
REAGENT PREP Without Pyridoxal-5-Phosphate
- Mix 4 parts of R1 + 1 Part R2 = Monoreagent
Stability = 4 weeks at 2-30C; 5 Days at 15-250C
- The monoreagent must be protected from light
- NaCl solution (9g/L)
L-alanine = substrate
LDH = indicator enzyme
R2 Oxoglutarate
NADH
P-5-P Fluid Stable Good’s Buffer (pH 9.6)
Pyridoxal-5-Phosphate
Without Pyridoxal-5-Phosphate
- Mix 4 parts of R1 + 1 Part R2 = Monoreagent
Stability = 4 weeks at 2-30C; 5 Days at 15-250C
- The monoreagent must be protected from light
- NaCl solution (9g/L)

STORAGE The reagents are stable up to the end of the indicated month The reagents are stable up to the end of the indicated month
INSTRUCTION of expiry, if stored at 2-80C, protected from light, and of expiry, if stored at 2-80C, protected from light, and
AND STABILITY contamination is avoided contamination is avoided

SPECIMEN  Serum  Serum

 Heparin plasma  Heparin plasma
 EDTA plasma  EDTA plasma
STABILITY  4 days at 20-250C  3 days at 20-250C
 7 days at 4-80C  7 days at 4-80C
 3 months at -200C  7 days at -200C
DISCARD CONTAMINATED SPECIMENS DISCARD CONTAMINATED SPECIMENS
FREEZE ONLY ONCE to prevent denaturation FREEZE ONLY ONCE to prevent denaturation
ASSAY  Wavelength: 340nm, 365nm,334  Wavelength: 340nm, 365nm,334
 Optical Path: 1cm  Optical Path: 1cm
 Temperature: 370C  Temperature: 370C
 Measurement: Against AIR BLANK (Decreasing Abs)  Measurement: Against AIR BLANK (Decreasing Abs)

SAMPLE START  Sample: 100uL  Sample: 100uL

 Monoreagent: 1000uL  Monoreagent: 1000uL
 Mix, read absorbance after 1 min and start  Mix, read absorbance after 1 min and start
stopwatch stopwatch
 Read absorbance again1,2,3 minutes (4 min total)  Read absorbance again1,2,3 minutes (4 min total)

CALCULATION AST (U/L) = A/min x Factor AST (U/L) = A/min x Factor

FACTORS  340nm = 1745  340nm = 1745
 334nm = 1780  334nm = 1780
 365nm = 3235  365nm = 3235
CONVERSION ukat/L = U/L x 0.0167 ukat/L = U/L x 0.0167
FACTORS
REFERENCE  Without P-5-P Activation  Without P-5-P activation
RANGE - Women: <31U/L ; 0.52ukat/L - Women: <31U/L ; 0.52ukat/L
- Men: <35U/L ; 0.58ukat/L - Men: <41U/L ; 0.58ukat/L
PERFORMANCE  With P-5-P activation  With P-5-P activation
CHAR. - 700 U/L (on automated systems) - 600 U/L (on automated systems)
 Manual Procedure  Manual Procedure
- A/min of 0.16 at 340nm - A/min of 0.16 at 340nm
- A/min of 0.08 at 365nm - A/min of 0.08 at 365nm
 If values are exceeded, the samples should be  If values are exceeded, the samples should be
diluted 1+9 with NaCl (9g/L) solution and results are diluted 1+9 with NaCl (9g/L) solution and results
multiplied by 10 are multiplied by 10

 Limit of Detection/Sensitivity: 2U/L AST  Limit of Detection/Sensitivity: 4U/L ALT

INTERFERENCES  ASCORBIC ACID > 30mg/dL  ABSCORBIC ACID > 30mg/dL

 HEMOGLOBIN >100mg/dL = high interference  HEMOGLOBIN >200mg/dL = high interference
 BILIRUBIN >40mg/dL  BILIRUBIN >40mg/dL
 LIPEMIA (Triglycerides) >2000mg/dL  LIPEMIA (Triglycerides) >2000mg/dL
SGOT/SGPT TRANSAMINASE METHOD

METHOD Reitman and Frankel Method

SAMPLE Serum
CSF
REAGENTS Phosphate Buffer pH 7.4
SGOT Substrate
- a-ketoglutarate
- L-aspartate
SGPT Substrate
- a-ketoglutarate
- L-alanine
COLOR REAGENT
- 2,4-dynitrophenylhydrazine
Sodium Hydroxide 0.4N
PROCEDURE 1. Set the blank tube at 100% transmittance at 505mu
2. Read the % transmittance of the test
3.The results are calculated using the calibration curve
4. If a value over 200 units is obtained, the test should be repeated using 0.2mL of a 1:5 dilution of the
sample. The result must be multiplied by 5