Head-space gas-chromatographic determination of 3-hydroxybutyrate in plasma after enzymic reactions, and the relationship among the three ketone bodies

Abstract

In this sensitive, reproducible method for determination of D-3-hydroxybutyrate (3-OHB) in plasma, it is converted to acetone by use of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)/lactate dehydrogenase (EC 1.1.1.27) coupled with acetoacetate decarboxylase (EC 4.1.1.4). The resulting acetone is detected by head-space gas chromatography. The lowest concentration of 3-OHB detectable in plasma was 2 μmol/L. The calibration curve showed a linear relationship for 3-OHB concentration from 0 to 5 mmol/L (r = 0.999). Analytical recovery of 3-OHB (50 μmol/L) was 97.9 (SD 3.8)%. The method was developed for determination of the three ketone bodies in plasma. The ratio of acetone to acetoacetate was not significantly different (p > 0.2) between normals (n = 31) and diabetics (n = 86). In normal subjects, the ratio of 3-OHB to acetoacetate was 1.20 (SD 0.44). In diabetic patients, the ratio correlated with the logarithm of the total ketone body concentration (r = 0.828).