Automated kinetic method for D-3-hydroxybutyrate in plasma or serum.

This assay for 3-hydroxybutyrate in plasma or serum, based on kinetic initial-rate techniques, has been optimized with respect to initial rate and linearity as follows: pH 8.5, hydroxybutyrate dehydrogenase 62.5 U/L, and NAD+ concentration 20 mmol/L. We have used the assay satisfactorily with both the Gilford 103 and Hitachi 705 discrete analyzers, obtaining results that compare well with those by a segmented-flow method. Within-assay precision (CV) varied from 7.8 to 0.6%, depending on both the analyzer used and on the concentration of 3-hydroxybutyrate. Analytical recovery was also dependent on 3-hydroxybutyrate concentration, varying from 99% at 2 mmol/L to 91% at 9.5 mmol/L. Lactate dehydrogenase/lactate interference in this direct assay is eliminated by incorporating oxalate in the assay reagents. Hydrazine, commonly used in equilibrium methods of analysis for 3-hydroxybutyrate, produced no significant advantage in this assay and was omitted. The mixed reagents for the assay are stable at 4 degrees C for at least a week. The advantages of analysis for this metabolite may now be realized, so that clinical and subclinical ketosis can be identified in humans and animals.