Rapid acetoacetate analysis on the LKB 8600 Reaction Rate Analyser.

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Abstract
Automation of acetoacetate analysis has previously been by continuous flow methods using colorimetric techniques. This method uses trichloroacetic acid as protein precipitant (100 g/l), a 50 .mu.l sample volume, a reaction rate mode of measurement, and a 1 min measuring time. D(-)-.beta.-Hydroxybutyrate dehydrogenase [EC 1.1.1.30] from Rhodopseudomonas sphaeroides catalyzes the reaction: acetoacetate + NADH + H+ .dblarw. D(-)-.beta.-hydroxybutyrate + NAD+. Acetoacetate is quantitated by the initial rate of decrease in absorbance of NADH at 340 nm. The advantages which this assay system has over previous methods are that it is more specific, the analysis time is very short, the method is semiautomated using a commonly available instrument, it is a discrete system with a minimum of reagent wastage, and the analysis is performed directly on the deproteinized supernatant without the requirement of an initial step in which protein precipitant is removed. [Measurement of acetoacetate in human diabetic ketoacidosis and lactic acidosis is discussed.].