Determination of acetoacetate in plasma by a combination of enzymatic decarboxylation and head-space gas chromatography.

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Abstract
Acetoacetate [a ketone body] concentration in plasma was determined by headspace gas chromatography after decarboxylation by the use of acetoacetate decarboxyase, which was recently isolated from Bacillus polymyxa A-57 strain. Partial purification of acetoacetate decarboxylase solution was carried out by DEAE-cellulose column chromatography of the cell-free extract, which was prepared by ultrasonication. The properties of the enzyme were particularly suitable for acetoacetate assay (optimum ph, 5.8; Vmax, 230 .mu.mol/min per mg; Km, 5.9 .times. 10-4 M). Plasma was deproteinized by Somogyi''s method. Acetone in the mildly basic supernatant was assayed by head-space gas chromatography. Acetoacetate was calculated by subtracting blank acetone from total acetone after enzymatic decarboxylation of acetoacetate. The minimal detectable concentration was 1 .mu.M. This method gave better reproducibility (CV [coefficient of variation] = 2.0-8.0%) and recovery (96.0-101.8%) than chemical decarboxylation with perchloric acid. Normal subjects (n = 31) showed plasma acetone levels of 7.2 .+-. 3.4 .mu.M and acetoacetate levels of 22.5 .+-. 9.7 .mu.M. Diabetic patients (n = 44), treated by diet control alone wihtout drug theapy, gave plasma acetone levels of 8.1 .+-. 3.5 .mu.M and acetoacetate levels of 25.0 .+-. 8.0 .mu.M. There was no significant difference between the 2 groups.