Rapid acetoacetate analysis on the LKB 8600 Reaction Rate Analyser.

Automation of acetoacetate analysis has previously been by continuous flow methods using colorimetric techniques (Klein and Oklander, 1966; Dickie and Gibson, 1976). The most specific methods appear to be enzymatic using the fl-hydroxybutyrate dehydrogenase (fi HBDH, E.C.1.1.1.30)/NADH system (Williamson et al., 1962). These end-point methods, however, do not readily permit the analysis of large numbers of samples, and the incubation time of an enzymatic end-point assay can take as long as 50 minutes (Gibbard and Watkins, 1968). A kinetic technique would reduce this time considerably. The LKB 8600 Reaction Rate Analyser has many advantages including precise temperature control, ease of use, reliability (Smith et al., 1970), and accuracy The following method uses trichloroacetic acid as protein precipitant (100 g/l), a 50 ,IL sample volume, a reaction rate mode of measurement, and a one-minute measuring time. D(-)-+l-Hydroxybutyrate dehydrogenase catalyses the reaction: acetoacetate + NADH + H+ = D(-)-P-hydroxybutyrate + NAD+. Acetoacetate is quan- titated by the initial rate of decrease in absorbance of NADH at 340 nm.