A fast enzymatic analysis for plasma ammonia
i) Ammonia is converted to L-glutamate by glutamate dehydrogenase (GLDH) in the presence of α-ketoglutarate and excess NADH in a brief pre-reaction:
(ii) Under the conditions of the assay, the rate of oxidation of excess NADH by peroxidase (POD) is inversely proportional to ammonia concentration:
A sample blank allows for interference by endogenous NADH consuming reactions, etc.
|Sample volume||>||200 µl (2 x 100 µl)|
|Analysis time||>||20 seconds (from injection)|
|Linearity||>||0 - 800 µmol/L (0 - 13.5 µg/ml)|
|Precision||>||C.V. of 2% @ 500 µmol/L (ca. 8.5 µg/ml)|
|Accuracy||>||Method comparison vs Spectrophotometric UV (Sigma):
y (Analox) = 0.97x + 0.28 µg/ml, r = 0.968, n = 33
|Detection limit||>||18 µmol/L (ca. 0.3 µg/ml)|
|Reagent Stability||>||Shelf-life unopened: 9 months stored at 0-5°C.
Shelf-life reconstituted: NADH/α-ketoglutarate, 7 - 10 days stored at 0-5°C; POD reagent 5-6 weeks at 0-5°C.
A sample blank allows for interference by endogenous NADH consuming reactions.