N.S.

BIO-TEC
ALT/ALAT/GPT
Colorimetric determination of serum GPT

INTENDED USE SPECIMEN

NS Biotec ALT reagent is intended for the in vitro quantitative determination Serum is the only accepted specimen. Avoid hemolysis
of alanine aminotransferase (EC 2.6.1.2) activity in serum on manual
• Specimen Preparation & Stability
systems.
Separate serum from clot/cells within 8 hours at room temperature or
CLINICAL SIGNIFICANCE 0
48 hours at 2–8 C.
Alanine aminotransferase (glutamate pyruvate transaminase) belongs to the 0
ALT activity is stable at 2-8 C for 7 days. Freezing of the samples is not
group of transaminases, which catalyze the conversion of amino acids to
recommended.
the corresponding α-keto acids via the transfer of amino groups; they also
PROCEDURE
catalyze the reverse process. Although higher activities exist in the liver,
• Manual Procedure
minor activity can also be detected in the kidneys, heart, skeletal muscle,
Wavelength 546 nm (530 – 550)
pancreas, spleen, and lungs. Elevated levels of transaminases are Cuvette 1 cm light path
indicative of myocardial infarction, hepatopathies, muscular dystrophy, and Temperature
0
37 C
damage to internal organs. Increased levels of ALT however are generally a Zero adjustment against reagent blank
result of liver disease associated with some degree of hepatic necrosis such Specimen Serum
as cirrhosis, carcinoma, viral or toxic hepatitis, and obstructive jaundice. • Method (1) using table
Levels of ALT are only slightly elevated in patients following a myocardial
2
infarction . Blank Specimen
ASSAY PRINCIPLE
Colorimetric methods based on formation of the chromogenic R2 0.5 ml 0.5 ml
dinitrophenylhydrazone of pyruvate have been in wide use. However, the
Incubate at 370C for 5 minutes
accuracy of these methods is limited, and since dinitrophenylhydrazine
reacts with α-ketoglutarate as well as pyruvate, high reagent blanks are Specimen …… 100 µ l
1 Dist. H2O 100 µ l ……
obtained . The series of reactions involved in the assay system is as
follows: Mix, incubate for exactly 30 minutes at 370C.
1. The amino group is enzymatically transferred by ALT present in the
specimen from alanine to the carbon atom of α-oxoglutarate yielding R3 0.5 ml 0.5 ml
pyruvate and L-glutamate.
Mix, incubate for exactly 20 minutes at 20-25oC
2. Pyruvate formed is measured in its derivative form, 2.4-
dinitrophenylhydrazone. R4 (NaOH 0.4 N) 5.0 ml 5.0 ml
DL-Alanine + α-Oxoglutarate ALT/GPT Pyruvate + L-Glutamate
Mix, read the absorbance of specimen (Aspecimen) against reagent blank
The intensity of the color produced is directly proportional to the
after 5 minute.
enzyme activity. It is determined by measuring the increase in
The color development is stable for 60 minutes.
absorbance at 530 – 550 nm.
Obtain the activity of ALT activity in serum specimen from the
EXPECTED VALUES table (p.2)
Serum Up to 12 U/l • Method (2) using standard curve
Each laboratory should investigate the transferability of the expected • Standard curve.
values to its own patient population and if necessary determine its own 1. Label tubes for standard curve from 1 to 10.
reference range. For diagnostic purposes, the ALT results should 2. Pipette 200 µl of distilled water in all 10 standard tubes.
always be assessed in conjunction with the patient’s medical history, 3. Add R2 (buffer reagent) and R1 (pyruvate standard) to the
respective tubes as following:
clinical examination and other findings.
REAGENTS Pyruvate
Tube
Standard DI H2O (ml) Buffer R2 (ml)
#
R1 Pyruvate standard 2.0 mmol/l (ml)
R2 Phosphate buffer 1 0.00 0.2 1.00
pH 7.4 100 mmol/l
2 0.05 0.2 0.95
L-Alanine 100 mmol/l
α-oxoglutarate 4.0 mmol/l 3 0.1 0.2 0.9
R3 2,4-dinitrophenyl-
hydrazine 4.0 mmol/l 4 0.15 0.2 0.85
5 0.2 0.2 0.8
R4 NaOH 4.0 N
6 0.25 0.2 0.75
• Reagent Preparation & Stability
7 0.3 0.2 0.7
The pyruvate standard (R1), the buffer (R2) and the color reagent (R3)
are ready for use and stable up to the expiry date given on label when 8 0.35 0.2 0.65
0
stored at 2–8 C. The sodium hydroxide reagent (R4) should be 9 0.4 0.2 0.6
completed to 1 liter before use.
10 0.45 0.2 0.55
 Pipette 1000 µl of R3 (color reagent) in all tubes, and incubate at 20 QUALITY CONTROL
0
- 25 C for exactly 20 minutes.
It is recommended that controls (normal and abnormal) be included in:
4. Pipette 10 ml of 0.4 N NaOH in all tubes. Mix, read the absorbance
• Each set of assays, or
of all tubes against reagent blank after 5 minutes.
• At least once a shift, or
5. The color development is stable for one hour.
• When a new bottle of reagent is used, or
• Assay. • After preventive maintenance is performed or a clinical component
As same as Method (1) exactly but obtain the activity of ALT activity in
serum specimen from standard curve. is replaced.
CALCULATION Commercially available control material with established ALT/GPT

• For Method (1) values may be routinely used for quality control.
Obtain the activity of ALT in the serum specimen from the following Failure to obtain the proper range of values in the assay of control
table: material may indicate:
Absorbance U/l • Reagent deterioration,
0.025 4 • Instrument malfunction, or
0.050 8 • Procedure errors.
0.075 12 The following corrective actions are recommended in such situations:
0.100 17
• Repeat the same controls.
0.125 21
• If repeated control results are outside the limits, prepare fresh
0.150 25
control serum and repeat the test.
0.175 29
• If results on fresh control material still remain outside the limits,
0.200 34
then repeat the test with fresh reagent.
0.225 39
• If results are still out of control, contact NS Biotec Technical
0.250 43
0.275 48 Services.
0.300 52 INTERFERING SUBSTANCES
0.325 57 • Bilirubin:
0.350 62 No interference from free bilirubin up to a level of 15 mg/dl, and
0.375 67 from conjugated bilirubin up to level of 6.8 mg/dl.
0.400 72 • Drugs:
0.425 77 3
Youngs in 1990 has published a comprehensive list of drugs and
0.450 83 substances which may interfere with this assay.
0.475 88 • Haemoglobin:
0.500 94 No interference from haemoglobin up to a level of 500 mg/dl.
• For Method (2) • Haemolysis:
The absorbances of the increasing amounts of pyruvate standard Erythrocyte contamination may elevate results, since ALT/GPT
correspond to the following transaminase activities in U/l. activities in erythrocytes are three to five times higher than those in
normal sera.
Tube No.2 09 U/l
• Lipemia:
Tube No.3 18 U/l
Tube No.4 27 U/l No relevant interference.
Tube No.5 37 U/l • Pyruvate:
Tube No.6 46 U/l High levels of serum pyruvate may interfere with assay
Tube No.7 56 U/l performance.
Tube No.8 67 U/l
Tube No.9 77 U/l WARNING & PRECAUTIONS
Tube No.10 87 U/l • NS Biotec reagent is for in vitro diagnostic use. Normal precautions
The standard curve is obtained by plotting the measured absorbances exercised in handling laboratory reagents should be followed.
against the transaminase activities in U/l. • Incubation time and temperature are vital factor, instructions must
Ordinate: Absorbance be followed exactly.
Abscissa: Activity in U/l
• The reagent and sample volumes may be altered proportionally to
Obtain the activity of ALT in the serum specimen from the standard accommodate different spectrophotometer requirements.
curve.
• Don’t use the reagent if it is turbid.
LINEARITY BIBLIOGRAPHY
When run as recommended, the assay is linear up to absorbance 0.5 or 1. Reitman, S, and Frankel, S.(1957): Amer J Clin Path. 28: 56.
94 U/l.
2. Henry, JB, (1974): Clinical Diagnosis and Management by Laboratory
If result exceeds 94 U/l, specimen should be diluted 1+9 with 0.9%
NaCl solution and reassayed. Multiply the result by 10. Methods. W.B. Saunders and Co., Philadelphia, PA. p 361.

SENSTIVITY 3. Young, Ds (1990): Effects of Drugs on Clinical Laboratory Tests. Third

Edition. 1990: 3: 6-12.
The sensitivity is defined as the lower detection limit represents the
lowest measurable ALT/GPT concentration that can be distinguished
from zero.
When run as recommended the sensitivity of this assay is 4 U/l.

2 of 2