Semiautomatic Bioluminescence Determination of Glycerol Using a Computer Controlled Luminescence Analyser (Berthold LB 950 T)

Abstract

A semiautomatic determination of glycerol is described, in which luminescence produced by bacterial NADH-linked luciferase is measured by an automatic luminescence analyzer (Berthold LB 950 T). The glycerol determination is based on the enzymatic conversion of glycerol to 3-phosphoglycerate, made irreversible by the presence of arsenate. NADH, formed in the glycerol-3-phosphate and glyceraldehyde-3-phosphate dehydrogenase reactions, is subsequently determined by the bacterial luciferase system. Stable kinetics of light emission were obtained by reducing the catalytic concentration of NAD(P)H:FMN oxidoreductaase from 85 U/l to 8.5 U/l. This method was applied to serum samples and validated by comparison with an enzymatic fluorimetric method. The new method is .apprx. 10 times more sensitive than the fluorimetric one. It is simpler, more convenient, less time consuming and also less expensive than spectrophotometric, fluorimetric or radiochemical methods used for glycerol determination.