PROCEDURE

GPT GPT/ALT = 5-30 WU/ml (2.4 -14.5 U/L)

Substrate GPT (R.1) 0.5 ml
Preincubate for 5 min at 37°C.
Serum 100 µL REFERENCES
Mix. Return to bath for 1. Reitman S., Frankel S.,Am. Clin. Pathol., 28,56 (1957)
30 min 2. Tietz, NW.,Fund of Clinical Chem., 446 (1970)
GPT DNPH R.2 0.5 ml 3. Schmidt, E., Enzymology Biol.Clin., 3,1 (1963)
Colorimetric test. Reitman-Frankel Mix. Allow to stand for 20 min at room temperature.
NaOH 0.4 N 5.0 ml
For in vitro diagnostic use only MIX. Let stand for 15 min at room temperature. ATLAS Medical
Read at 505 nm against a water blank. William James House,
Store at 2-8ºC The colour is stable at least 60 minutes. Cowley Road, Cambridge, CB4 0WX, UK
Tel: ++44 (0) 1223 858 910
INTENDED USE Fax: ++44 (0) 1223 858 524
For the determination of GPT concentration in human serum. CALCULATIONS
From absorbencies, read units of GPT from corresponding curves. PPI767A01
PRINCIPLE Rev A(28.10.2015)
The glutamic transaminase enzymes, serum glutamic oxalacetic CALIBRATION (mL) Catalogue Number Store at
(GOT) and serum glutamic pyruvic (GPT), catalyze the transfer of
For In-Vitro Diagnostic
the amino group of glutamic acid to oxalacetic acid and pyruvic 1 2 3 4 5 6 Caution
use
acid in reversible reactions. The transaminase activity is Water 0.2 0.2 0.2 0.2 0.2 0.2 Number of tests in the Read product insert
proportional to the amount of oxaloacetate pyruvate formed GOT R.1 1.0 0.9 0.8 0.7 0.6 0.5 pack before use
over a definite period of time and is measured by a reaction with Pyruvic stand. -- 0.1 0.2 0.3 0.4 0.5
2.4-dinitrophenylhydrazine (DNPH) in alkaline solution. Lot (batch) number Manufacturer
DNPH R.2 1.0 1.0 1.0 1.0 1.0 1.0
Mix. Allow to stand for 20 min at room temperature. Fragile,
REAGENTS NaOH 0.4 N 10 10 10 10 10 10
Expiry date
handle with care
Materials Provided Manufacturer fax Do not use if
MIX. Allow to stand for at least 15 min. number package is damaged
Content Concentration Read against water blank at 505 nm.
Reagent -1 Substrate GPT (ALT) Manufacturer telephon
Plot a calibrator curve of the absorbances found vs. the
e number
DL-Alanine 200 mmol/L corresponding units, on a graph paper, according to the following
Ketoglutarate 2 mmol/L concentrations:

Reagent -2 Colour reagent GPT WU/mL 0 25 50 83 126 --

2.4-dinitrophenylhydrazine 1 mmol/L U/L 0 12 24 40 62 --

Standard Pyruvic standard. 1.2 mmol/L LINEARITY

When GPT exceeds 126 WU/Ml (61 u/l) repeat test using a 1:10
Materials Required But Not Provided dilution of serum with saline sol.(9g/L). Multiply the result by 10.
UNITS
Content Concentration
1 WU/mL = 1U/L x 2.07
Soduim Hydroxide (NaOH) 4N 1 U/L = 1WU/mL x 0.483

NORMAL VALUES
SAMPLES
Serum, free of hemolysis.
 PREPARATION PROCEDURE
 Working reagent (WR): Mix: 4 ml of (R1) + 1ml (R2) 1. Peincubate working reagent, samples and controls to
 Stability: 4 weeks at 2-8°C. Protect from light reaction temperature
GPT (ALT) 2. Set the photometer to 0 absorbance with distilled water
NADH. Kinetic UV. IFCC Liquid. STORAGE AND STABILITY 3. Pipette into a cuvette:
Quantitative determination of alanine aminotransferase GPT (ALT)  All the components of the kit are stable until the Reaction
For in -vitro diagnostic use only. expiration date on the label when stored tightly closed 37°C 30°C
temperature
at 2-8 ° C, protected from light and contaminations WR (mL) 1.0ml 1.0ml
Store at 2-8ºC. prevented during their use.
Sample (μL) 50 μL 100 μL
 Do not use reagents over the expiration date.
4. Mix gently by inversion. Insert cuvette into the cell holder
PRINCIPLE OF THE METHOD  Signs of reagent deterioration:
and start stopwatch.
Alanine aminotransferase (ALT/GPT) catalyzes the transfer of  Presence of particles and turbidity.
5. Incubate for 1 minute and record initial absorbance
the amino group from alanine to oxoglutarate with the  Blank absorbance (A) at 340 nm < 1.000 in 1cm cuvette.
reading.
formation of glutmate and pyruvate. The latter is reduced to
6. Repeat the absorbance readings exactly after 1,2 and 3
lactate by lactate dehydrogenase (LDH) in the presence of SAMPLES
minutes.
reduced nicotinamide adenine dinucleotide (NADH). The Serum .EDTA or heparinized plasma free or hemolysis. ALT is
7. Calculate the difference between absorbances.
reaction is monitored kinetically at 340 nm by the rate of stable in serum or plasma 24 hours at room temperature
8. Calculate the mean of the results to obtain the average
decrease in absorbance resulting from the oxidation of and for 1 week at 2-8°C.
change in absorbance per minutes (ΔA/min).
NADH to NAD+, proportional to the activity of ALT present in
the sample. INTERFERENCES
CALCULATIONS
L-Alanine + 2-oxoglutarate ALT /GPT L-Glutamate + Pyruvate  Lipemia (intralipid >15 g/L) does not interfere.
U/L= ΔA/min x 3333 (37°C)
 Bilirubin (>30 mg/dL) does not interfere.
U/L= ΔA/min x 1746 (30°C)
Pyruvate + NADH + H+ MDH Lactate + NAD+  Hemoglobin (>10g/dL) does not interfere.
Samples with ΔA/min exceeding 0.160 at 340 nm should be
 Other drugs and substances may interfere.
diluted 1:10 with saline and assayed again . Multiply the
This test has been formulated according the standardized
results by 10.
method described by IFCC. EQUIPMENTS NEEDED BUT NOT PROVIDED
If the results are to be expressed as SI units apply :
 Photometer or spectrophotometer with a
U/L x 0.01667=μkat/L.
REAGENTS thermostatted cell compartment set at 30/37°C ,
TRIS buffer pH 7.3 150 mmol/L capable of reading at 340 nm .
R1 REFERENCE VALUES
Lactate dehydrogenase (MDH) >1350U/L  Stopwatch, strip-chart record or printer.
ALT Substrate Up to 40U/L(0.67
L-Alanine 750 mmol/L  Cuvettes with 1-cm pathlength 37°C
μkat/L.)
R2  Pipettes to measure reagent and samples. Adults
NADH 1.3 mmol/L Up to 25U/L(0.42
ALT 30°C
2-oxoglutarate 75 mmol/L μkat/L.)
coenzyme
Levels approximately twice the adult level are seen in ANALYTICAL PERFORMANCE 2. International Federation of clinical Chemistry (IFCC). Clin
neonates and infants; these decline to adult level -Detection Limit: 7.95 U/L. Chem Lab 1998;36:185.
approximately 6 months of age. -Linearity: Up to 500 U/L. 3. Young DS. Effect of drugs on clinical laboratory tests. 5th
It is recommended that each laboratory establishes its own ed . AACC Press,2000.
reference range. -Precision: 4. Tietz. Textbook of Clinical Chemistry, 2 edition. Burtis CA,
(U/L) Within-run Between-run Ashwood ER.W.B.Saunders Co. 1994.
QUALITY CONTROL Mean 32.4 140.8 32.4 140.8
Control sera are recommended to monitor the performance SD 0.79 1.41 0.97 2.77
of assay procedures: ATLAS H Normal and Pathologic. If CV (%) 2.43 1.00 3.49 1.97
control values are found outside the defined range, check ATLAS MEDICAL
N 10 10 10 10
the instrument, reagents and technique for problems. Each -Sensitivity: 0.280 mA/min/U/L GPT. Unit 4, William James House
laboratory should establish its own Quality Control scheme -Correlation: this assay (y) was compared with similar Cowley Rd, Cambridge, CB4 0WX
and corrective actions if controls do not meet the acceptable commercial method (x). Tel: ++44 (0) 1223 858 910
tolerances. The result were : Fax: ++44 (0) 1223 858 524
N=50 r=0.99 y=1.041 x + 1.447 PPI446A01
CLINICAL SIGNFINANCE Rev D (10.11.2015)
The group of enzymes called transaminase exist in tissues of The analytical performances have been generated using on
Product Reference For in-vitro
many organs. Necrotic activity in these organs causes a automatic instrument. Results may very depending on the
No. diagnostic use.
release of measured. Since heart tissue is rich in AST instrument.
increased serum levels appear in patients after myocardial Caution. Store at 2 - 8°C.
infraction, as well as in patients with muscle disease. Notes:
Read product
Muscular dystrophy and dermatomyositis . 1. The method may be used with different instruments. Any Manufacturer.
insert before use.
The liver is especially rich in ALT, being this enzyme application to an instrument should be validated to
Lot (batch) Manufacturer
measurement used primarily as a test for infectious and toxic demonstrate method. It is recommended to validate
number. telephone number.
hepatitis, although high levels of both ALT and AST may also periodically the instrument. Contact to the distributor for
Manufacturer fax
be found in cases of liver cell damage and acute pancreatitis, any question on the application method. Expiry date.
number.
suggesting that the obstruction of billary tree by the 2. Clinical diagnosis should not be made on findings of a
adematous pancreas and the presence of associate hepatic single test result. But should integrate both clinical and
disease may contribute to elevated AST levels these patients. laboratory data.
Slight or moderate elevations of AST and ALT activities may
be observed after intake of alcohol and after administration REFERENCES
of various drugs, such as salicylates, opiates, and ampicilin. 1. Winn-Deen E.S ,David H, Singer G, and Chavez R. Clin
Chem 1988;34:2005.
 EQUIPMENTS NEEDED BUT NOT PROVIDED conditions. The concentration is expressed in units per liter of
 Spectrophotometer or colorimeter sample (U/L).
measuring at 340 nm.
Temperature conversion factors
 Thermostatic bath at 25°C, 30°C, 37° To correct results to other temperatures multiply by:
C (± 0.1°C).
ATLAS GPT (ALT) TEST Assay Conversion factor to
 Matched cuvettes 1.0 cm light path.
(Kinetic)  General laboratory equipment. temperature 25°C 30°C 37°C
For in -vitro diagnostic use only.
25°C 1.00 1.32 1.82
PREPARATION
 Working reagent (WR): 30°C 0.76 1.00 1.39
Store at 2-8C
 Dissolve one tablet of R2 Substrate in 2mL of 37°C 0.55 0.72 1.00
INTENDED USE R1.cap and mix gently to dissolve contents.
 Stability: 21 days at 2-8°C or 72 hours at room QUALITY CONTROL
For the determination of GPT (ALT) concentration in human
temperature (15-25°C). Control sera are recommended to monitor the performance
serum or plasma.
of assay procedures.
INTRODUCTION STORAGE AND STABILITY If control values are found outside the defined range, check
The ALT is a cellular enzyme, found in highest concentration in  All the components of the kit are stable until the the instrument, reagents and technique for problems.
liver and kidney. High levels are observed in hepatic disease like expiration date on the label when stored tightly Each laboratory should establish its own Quality Control
hepatitis, diseases of muscles and traumatisms, its better closed at 2-8°C, protected from light and scheme and corrective actions if controls do not meet the
application is in the diagnosis of the diseases of the liver. contaminations prevented during their use. acceptable tolerances.
When they are used in conjunction with AST aid in the diagnosis  Do not use the tablets if appears broken.
REFERENCE VALUES4'5
of infarcts in the myocardium, since the value of the ALT stays  Do not use reagents over the expiration date.
25°C 30°C 37°C
within the normal limits in the presence of elevated levels of  Signs of reagent deterioration:
AST. Men up to 22 U/L 29 U/L 40 U/L
 Presence of particles and turbidity.
Clinical diagnosis should not be made on a single test result;  Blank absorbance (A) at 340 nm < 1.00. Women up to 18 U/L 22 U/L 32 U/L
it should integrate clinical and other laboratory data.
Normal newborns have been reported to show a reference
SAMPLES
range of up to double the adult, attributed to the neonate's
PRINCIPLE OF THE METHOD Serum or plasma': Stability 7 days at 2-8°C..
hepatocytes. These values decline to adult levels by
Alanine aminotranferase (ALT) o Glutamate pyruvate approximately 3 months of age.
PROCEDURE
transaminase (GPT) catalyses the reversible transfer of an These values are for orientation purpose; each laboratory
1. Assay conditions:
amino group from alanine to α-ketoglutarate forming should establish its own reference range.
Wavelength: …………………………….340 nm
glutamate and piruvate.
Cuvette :………………………………..1 cm light path
The piruvate produced is reduced to lactate by lactate PERFORMANCE CHARACTERISTICS
Constant temperature ………………25°C / 30°C / 37°C
dehydrogenase (LDH) and NADH: Measuring range:
2. Adjust the instrument to zero with distilled water
Alanine + α-Ketoglutarate ALT Glutamate + Piruvate From detection limit of 1,20 U/L to linearity limit of 262 U/L. If
or air.
the results obtained were greater than linearity limit, dilute
3. Pipette into a cuvette:
Piruvate + NADH + H LDH Lactate + NAD' the sample 1/10 with NaCl 9 g/L and multiply the result by
WR (mL) 1.0
10.
Sample (μL) 100
Precision:
The rate of decrease in concentration of NADH, measured
photometrically, is proportional to the catalytic concentration 4. Mix, incubate for 1 minute. Intra-assay (n=20)
of ALT present in the sample'. 5. Read initial absorbance (A) of the sample, start the Mean U/L 34 .9 11 8.4
stopwatch and read absorbances at 1-minute
SD 0.64 1.17
REAGENTS intervals thereafter for 3 minutes.
6. Calculate the difference between absorbances and CV (%) 1.84 0.99
R1 TRIS pH 7.8 100 mmol/L
Buffer L-Alanine 500 mmol/L the average absorbance differences per minute 1

(ΔA/min). Inter-assay (n=20)

NADH 0.18 mmol/L 34.1 118.3
R2 CALCULATIONS
Lactate dehydrogenase (LDH) 1200 U/L
Substrate ΔA/min x 1750 = U/L of ALT 1.03 1.53
α-Ketoglutarate 15 mmol/L
3.04 1.29
Units: One international unit (IU) is the amount of enzyme that
transforms 1 μmol of substrate per minute, in standard
Sensitivity:
Atlas Medical
1 U/L = 0.000557 ΔA / min.
William James House,
Accuracy:
Cowley Road, Cambridge, CB4 0WX, UK
Results obtained using ATLAS reagents (y) did not show
Tel: ++44 (0) 1223 858 910
systematic differences when compared with other
Fax: ++44 (0) 1223 858 524
commercial reagents (x). The results obtained using 50
PPI194A01
samples were the following:
Rev B (10.11.2015)
Correlation coefficient (r): 0.99.
Regression equation: y= 0.98x + 0.38.
The results of the performance characteristics depend on the Catalogue Number Store at
analyzer used. For In-Vitro Diagnostic
Caution
use
INTERFERENCES Number of tests in the Read product insert
Anticoagulants currently in use like heparin, EDTA, oxalate pack before use
and fluoride do not affect the results. Hemolysis interferes
with the assay'. Lot (batch) number Manufacturer
A list of drugs and other interfering substances with ALT Fragile, handle with
determination has been reported by Young et Al. Expiry date
care
Manufacturer fax Do not use if
REFERENCES number package is damaged
1. Murray R. Alanine aminotransferase. Kaplan A
Manufacturer
et al. Clin Chem The C.V. Mosby Co. St Louis. telephone number
Toronto. Princeton 1984; 1088-1090.
2. Young DS. Effects of drugs on Clinical Lab. Tests,
4th ed AACC Press, 1995.
3. Young DS. Effects of disease on Clinical Lab.
Tests, 4th ed AACC 2001.
4. Burtis A et al. Tietz Textbook of Clinical
Chemistry, 3rd ed AACC 1999.
5. Tietz N W et al. Clinical Guide to Laboratory
Tests, 3rd ed AACC 1995.