ALT an enzyme found in the liver that helps convert proteins into energy for the liver cells. When the liver is damaged, ALT is released into the bloodstream and levels increase.
Kinetic UV method
Kinetic UV method based on IFCC recommendations
SGPT (ALT) catalyzes the transfer of amino group betwee L-Alanine and a-ketoglutarate to form pyruvate and glutamate. The pyruvate formed reacts with NADH in the presence of Lactate Dehydrogenase to form NAD. The rate of oxidation of NADH to NAD is measured as a decrease in absorbance which is proportional to the SGPT (ALT) activity in the sample.
- R 1 : ALT Enzyme Reagent
- Buffer Tris, pH = 7.5 —– 125 mmol/L
- L-alanine ———— 600 mmol/L
- LDH ————– ≥ 1.7 KU/L
- R 2 : ALT Substrate Reagent
- NADH —– 0.18 mmol/L
- α -ketoglutarate —- 15 mmol/L
- Detergent, preservative.
Test Requirements :
- Test Tubes
- Bio-Chemistry analyzer
- ALT Test Kit
Reagent Preparation and Stability :
4 Part of R1 (0.8 ml)
1 Part of R2 (0.2 ml)
and Mix well
Avoid direct exposure to light. Stability of working reagent: 3 days at 2 – 8 °C.
Specimen collection and handling:
Analyzer Parameter Required :
|Reaction Type||Kinetic (decreasing)|
|Wavelength||340 nm (334nm – 365nm)|
|Delay Time||60 sec|
|Interval Time||60 sec|
|No. Of reading||2|
|Zero Setting||Deionised Water|
|Light Path||1 cm|
Pipette into clean dry test tube labeled as (T) and Calibrator Label (c) and QC Label (QC) or as your required:
|Specimin||(T)||(C) / (QC)|
|Working Reagent||1.0 ml||1.0 ml|
|Pt. Sample||0.1 ml||–|
|Calibrator or QC||–||0.1 ml|
|Mix well and read the initial absorbance after 1 min and repeat the absorbance reading after every 1, & 2 mins. Calculate the mean absorbance change per minute (Δ OD/min.).|
CALCULATION : ALT activity (U/L) = D A/min. x 1746
NOTE: Samples having a very high activity show a very low initial absorbance as most of the NADH is consumed prior to the start of the measurement. If this is suspected then dilute the sample and repeat the assay
The procedure is linear upto 300 U/L.
if the activity exceeds this limit dilute the sample with normal saline (NaCl 0.9 %) and multiply result by dilution factor.
QUALITY CONTROL :
For accuracy it is necessary to run known controls with every assay.
Normal Value :
Serum : < 40 U/L
Note: Each Laboratory or Each Company Kits should establish it’s own normal range.