Oxoglutarate + L-Alanine L-Glutamate + Pyruvate

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ALT STABILITY AND PREPARATION OF REAGENTS

R1. Buffer
Alanine Aminotransferase Contents ready for use. Stable up to the expiry date
Manual specified when stored at +2 to +8C.

INTENDED USE R2. 2,4-dinitrophenyl-hydrazine


For the quantitative in vitro determination of Alanine Contents ready for use. Stable up to the expiry date
Aminotransferase (ALT) in serum. This product is suitable for specified when stored at +2 to +8C.
Manual use.
R3. Sodium Hydroxide
Cat. No. Make 1 vial of sodium hydroxide 3 up to 1000 ml with
AL 146 R1. Buffer 1 x 100 ml redistilled water in a volumetric flask. Stable up to
200 tests R2. 2,4-Dinitrophenylhydrazine 1 x 100 ml expiry date when stored at +2 to +8C.
R3. Sodium Hydroxide 1 x 100 ml
CAL. Pyruvate Standard 1 x 10 ml CAL. Pyruvate Standard
Contents ready for use. Stable up to expiry date when
GTIN: 05055273200188 stored at +2 to +8C. To construct a calibration curve
for GPT, use the Pyruvate Standard undiluted.
Colorimetric method (Reitman and Frankel) for determination
of serum alanine aminotransferase. MATERIALS PROVIDED
Buffer
PRINCIPLE 2,4-Dinitrophenylhydrazine
ALT Sodium Hydroxide
-oxoglutarate + L-alanine L-glutamate + pyruvate Pyruvate Standard
Alanine Aminotransferase is measured by monitoring the MATERIALS REQUIRED BUT NOT PROVIDED
concentration of pyruvate hydrazone formed with Randox Human Assayed Multi-Sera Control Level 2 (Cat. No.
2,4-dinitrophenyl-hydrazine. HN1530).
SAMPLE MATERIAL PROCEDURE NOTE
Serum. Transaminase activities in some sera are stimulated by high
concentrations of aldehydes, ketones, or oxo acids.
REAGENT COMPOSITION Measurement against a sample blank (Procedure 2) instead of
a reagent blank (Procedure 1) avoids the risk of finding such
Contents Initial Concentration of Solutions artifacts.
R1. Buffer PROCEDURE
Phosphate buffer 100 mmol/l, pH7.4
L-alanine 200 mmol/l
Wavelength: Hg 546 nm (530 - 550 nm)
-oxoglutarate 2.0 mmol/l
Cuvette: 1cm light path
R2. 2,4-dinitrophenylhydrazine 2.0 mmol/l
Incubation Temperature: 37C
R3. Sodium Hydroxide 4.0 mol/l
CAL. Pyruvate Standard See lot specific insert
1. Measurement against Reagent Blank

Pipette into test tubes:


SAFETY PRECAUTIONS AND WARNINGS
For in vitro diagnostic use only. Do not pipette by mouth.
Exercise the normal precautions required for handling Reagent Blank Sample
laboratory reagents.
Sample --- 0.1 ml
Solution R1 contains Sodium Azide. Avoid ingestion or Buffer (R1) 0.5 ml 0.5 ml
contact with skin or mucous membranes. In case of skin Distilled Water 0.1 ml ---
contact, flush affected area with copious amounts of water. In Mix, incubate for exactly 30 min. at 37C
case of contact with eyes or if ingested, seek immediate
medical attention. 2,4-DNP (R2) 0.5 ml 0.5 ml

Sodium Azide reacts with lead and copper plumbing, to form Mix, allow to stand for exactly 20 min. at 20 to 25C
potentially explosive azides. When disposing of such reagents
flush with large volumes of water to prevent azide build up. Sodium Hydroxide (R3) 5.0 ml 5.0 ml
Exposed metal surfaces should be cleaned with 10% sodium
hydroxide. Mix, read the absorbance of sample (Asample) against the
reagent blank after 5 minutes.
Health and Safety data sheets are available on request.

The reagents must be used only for the purpose


intended by suitably qualified laboratory personnel,
under appropriate laboratory conditions.

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MANUAL AL146

2. Measurement against Sample Blank CALIBRATION USING STANDARD


Pyruvate Standard is used to construct a calibration curve
Pipette into test tubes: when readings cannot be taken at 546 nm. Increasing amounts
of pyruvate are allowed to react with 2,4-
Sample Blank Sample dinitrophenylhydrazine. The concentration of hydrazone
Sample --- 0.1 ml formed is approximately proportional to the amount of
Buffer (R1) 0.5 ml 0.5 ml pyruvate reacted.
QUALITY CONTROL
Mix, incubate for exactly 30 min. at 37C Randox Human Assayed Multi –Sera Control Level 2 is
recommended for daily quality control. The value obtained
2,4-DNP (R2) 0.5 ml 0.5 ml should fall within a specified range. If the value falls outside the
Sample 0.1 ml --- range and repetition excludes error, the following steps
should be taken:
1. Check instrument settings and light source.
Mix, allow to stand for exactly 20 min. at 20 to 25C 2. Check cleanliness of all equipment in use.
3. Check water Contaminants i.e. bacterial growth may
Sodium Hydroxide (R3) 5.0 ml 5.0 ml contribute to inaccurate results.
4. Check reaction temperature.
Mix, read the absorbance of the sample (Asample) against the 5. Check expiry date of kit and contents.
6. Contact Randox Laboratories Technical
sample blank after 5 minutes. Services, Northern Ireland +44 (0) 28 94451070.
PROCEDURE CALCULATION
Obtain the activity of ALT in the serum from the table:
Wavelength 490-560 nm
Absorbance U/l Absorbance U/l
Cuvette 1 cm light path
Temperature 20 - 25C 0.025 4 0.275 48
0.050 8 0.300 52
Pipette into test tubes: 0.075 12 0.325 57
0.100 17 0.350 62
0.125 21 0.375 67
Tube Pyruvate Redistilled Buffer 0.150 25 0.400 72
No Standard Water Solution 0.175 29 0.425 77
(ml) (ml) (ml) 0.200 34 0.450 83
0.225 39 0.475 88
1 0.00 0.2 1.00 0.250 43 0.500 94
2 0.05 0.2 0.95
3 0.10 0.2 0.90 INTERFERENCE
4 0.15 0.2 0.85 Haemolysis interferes with the assay.
5 0.20 0.2 0.80
6 0.25 0.2 0.75 Physiological changes in serum or plasma analyte
7 0.30 0.2 0.70 concentrations can be caused by a number of substances.
Comprehensive discussion of possible interfering substances,
8 0.35 0.2 0.65 their serum or plasma concentrations, and their possible
9 0.40 0.2 0.60 physiological involvements is beyond the scope of this
10 0.45 0.2 0.55 document. The listed reference contains specific details on
known potential interfering substances (2). The user must
remain vigilant to the possible effect on results of unknown
interferences from medications or endogenous substances. All
Mix and pipette into each tube 1.0 ml of Reagent Solution R2. patient results must be evaluated in light of the total clinical
Mix and incubate for 20 min at 20 to 25C. Add 10 ml of status of the patient.
Sodium Hydroxide soln to each tube. Mix and read
absorbance against blank (tube no 1) after 5 mins. NORMAL VALUES
Serum up to 12 U/l
The absorbances of the increasing amounts of pyruvate (0.05 - It is recommended that each laboratory establish its own
0.45 ml Pyruvate Standard) correspond to the following reference range to reflect the age, sex, diet and geographical
transaminase activities in U/l. location of the population.

Tube No. GPT U/l LINEARITY


If the absorbance exceeds 0.5 dilute 0.1 ml of sample with
0.9 ml of 0.9% NaCl solution and reassay. Multiply the result
2 9 by 10.
3 18
4 27 REFERENCE
5 37 1. Reitman, S., and Frankel, S., Amer. J. Clin. Path., 1957; 28:
6 46 56.
2. Young DS. Effects of Drugs on Clinical Laboratory Tests.
7 56 5th ed. Washington, DC: AACC Press; 2000.
8 67
9 77 The presence of a vertical bar in the margin indicates a technical
10 87 update from the previous revision.

The calibration curve is obtained by plotting the measured Randox Teoranta, Meenmore,
absorbances against the transaminase activities in U/l. Dungloe, Donegal,
F94 TV06, Ireland
ordinate = absorbance
abscissca = activity in U/l. Revised 26 Apr 16 bi
Rev. 002

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