Dec 15, 2017
ALT/GPT Detection Assay
- 1Universidade de São Paulo
External link: https://labtest.com.br/reagentes/altgpt-liquiform/
Protocol Citation: Zumira Carneiro 2017. ALT/GPT Detection Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.k9ncz5e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 14, 2017
Last Modified: March 15, 2018
Protocol Integer ID: 9230
Keywords: ALT; Quantification of hepatic injury
Abstract
Intended use:
Kinetic system for Alanine Aminotransferase (ALT/ GPT) activity determination.
Teste Principle:
ALT catalyzes the transfer of amino groups from Alanine to Ketoglutarate, yielding pyruvate and Glutamate. The pyruvate is reduced to lactate by for action of the lactate dehydrogenase (LDH) which oxidizes NADH to NAD+.
The reduction of the absorbance at 340 nm, as consequence of NADH oxidation, must be photometricaly monitored, and is proportional to the ALT activity in the sample.
Aminotransferase determination involves the following reactions:
L-alanine + Ketoglutarate ---(ALT) → Pyruvate + L-Glutamate
Pyruvate + NADH ---(LDH)→ NAD + L-lactate
Attachments
Guidelines
Reagents are for “in vitro” diagnostic use. Use the reagents according to the working procedures for clinical laboratories.
Storage and stability - Unopened reagents, when stored at indicated temperature, are stable up to expiration date shown on the label.
Materials
MATERIALS
Reagent 1- TRIS buffer (132.5 mmol/L), L-Alanine (687.5 mmol/L); LDH (≥2300 U/L) and sodium azide (0.095%).
Reagent 2 - TRIS buffer (20 mmol/L), NADH (1320 µmol/L); Ketoglutarate (82.5 mmol/L) and sodium azide (0.095%).
Reagent 3 - TRIS buffer (20 mmol/L), pyroxal phosphate (11.1 mmol/L); sodium azide (0.095%).
Safety warnings
The reagents contain sodium azide as preservative. Avoid ingestion. In case of eyes contact, immediately flush eyes with plenty of water and get prevent azide accumulation.
Before start
Spectrophotometer.
- Micropipettes and pipettes for measuring the stated volumes
- Water bath at temperature indicated under PROCEDURE.
- Stopwatch.
Step 1
Step 1
- In order to achieve traceable results to IFCC1 Procedure, is needed the use of the two-reagent method, to occur the enzyme total activation by the pyridoxal phosphate.
- Preparation the reagent: Add 0.300 mL of the Reagent 3 to a bottle of Reagent 1(24 mL) and mix. Stability: 21 days at 2 – 8 0C and 24 hours 15 –25 0C when no chemical or microbial contamination occurs. Optionally, a lower volume of the mixture (Reagent 1 + Reagent 3) may be prepared by using one part of the Reagent 3 to 80 parts of
Reference: 'Reference procedure for the Measurement of catalytic Concentration of Alanine Aminotransferase. Cin Chem Lab Med 2002, 40 (70): 718-24.'
Step 2
Step 2
- In a test tube labeled “test” or “Calibrator”, Add 0.160 mL of the mixture Reagent 1 + Reagent 3.
Step 3
Step 3
- Add 0.020 mL of the sample or enzymes calibrator, homogenize and incubate a water- bath at 37 ± 0.2 0C. Wait five minutes. After this incubation it is possible wait until 30 minutes to start the kinetic determination with the addition of the Reagent 2.
Step 4
Step 4
Perform a water Blank measurement at 340 nm.
Step 5
Step 5
- 0.040 mL of the Reagent 2, homogenize and transfer immediately to a cuvette at 37 ± 0.2 0C. Wait one minute.
Step 6
Step 6
- Measure the initial absorbance (A1). and start simultaneously the timer. Measure the absorbance again after 2 minutes (A2).
CALCULATIONS
CALCULATIONS
It is a usual procedure calculated the enzymatic activity results using a theoretical factor achieved in reaction optimum conditions, described below:
Wavelength: 340 nm
Cuvette at 37 ± 0.2 0C, 10 mm light path.
Pass band ≤2 nm
Sray light ≤ 0.1%
If one of the correlated parameters is modified, it is recommended to apply an enzymes calibrator indicated by the reagent manufacturer. Labtest Diagnostica recommends Calibra series to perform the ALT/GPT system calibration.
DA/minute(test or calibrator) = (A1-A2)/2
Factor: (Calibrator activity)/(DA/minute (test) x factor
ALT activity (U/L) = DA/minute (test) x Factor
If all the correlated parameters are fulfilled, the theoretical factor (1746) can be applied.