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Flashcards in Alanine Aminotransferase Deck (18)
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Method for the determination of Alanine Aminotransferase (ALT)

Kinetic method (Expert Panel of the International Federation of Clinical Chemistry)

1

Contain sodium azide

Enzyme reagent (R1) and starting reagent (R2)

2

Reference range

(Review)

3

Reaction principle

1 2-oxoglutarate + L-alanine -- (GPT) -- L-glutamate + pyruvate
2 Pyruvate + NADH + H -- (LDH) -- L-lactate + NAD

4

Contents

1 Reagent 1/Enzyme reagent
2 Reagent 2/Starting reagent

5

Reagent start stability

The reagents are stable even after opening up to the stated expiry date when stored from light at 2-8 Celsius

6

Procedure with sample start

For 12012: Pipette 2 mL from bottle 2 (starting reagent R2) into one bottle 1 (enzyme reagent, R1), mix thoroughly. The working reagent is stable for 4 weeks at 2-8 Celsius and 5 days at 15-25 Celsius.

7

Loss of activity

1 Within 3 days at 4 Celsius: 10%
2 At 20-25 Celsius: 17%

8

Specimen

1 Serum
2 Heparinized plasma
3 EDTA plasma

9

Optical path

1 cm

10

Wavelength

Hg 365 nm, 340 nm or Hg 334 nm

11

Temperature

1 25 Celsius
2 30 Celsius
3 37 Celsius

12

Measurement

Against air (decreasing absorbance)

13

Procedure with sample start

Semi-micro method
Mix, read the absorbance after 1 minute and at the same time start the stop watch. Read the absorbance again exactly after 1, 2, and 3 minutes.

14

Linearity

If the absorbance change per minute exceeds 0.160 at Hg 334 n/340 m or 0.080 at Hg 365 nm or if the activity is higher than 150 U/L at 25/30 Celsius or 280 U/L at 37 Celsius, dilute 0.1 mL of the sample with 0.9 mL physiological saline (0.9% and repeat the assay using this dilution). Multiply the result by 10.

15

Result of analyzing sera with very high activities

Initial absorbance may be very low as most of the NADH will have been consumed before taking the first reading

16

Factors for calculation

(Review)

17

Measurements for absorbance changes per minute within 0.06-0.08 (Hg 365 nm) or 0.12-0.16 (Hg 334 nm, 340 nm) (procedure 1+2)

Only from the first 2 minutes are used for calculation (1 minute incubation, 2 minutes measurement)