Bioluminescent methods for determining metabolites in micro-samples of pig plasma.

Abstract

A highly sensitive and simplified method for the luminometric determination of plasma metabolites has been developed. Furthermore, the technique has been automated for the Dynatech ML2250 Microtiter Plate Luminometer and can be applied to the measurement of any plasma metabolite which may be coupled to a reaction involving the reduction of NAD+. Assays are described for lactose/galactose, beta-hydroxybutyrate and D-lactate, and have been validated with plasma samples. The assays require 1-2 microliters of plasma, and are capable of detecting concentrations below 5 mumol/l. Since luminometry is based on the kinetics of the luciferase/oxidoreductase enzyme system, components of complex biological samples may interfere with the rate of the reaction; necessitating the use of internal standards for individual samples. However, the need for internal standards to account for sample to sample variation in the luminescent response, has been eliminated with the present technique.