Kinetic measurement of the combined concentrations of acetoacetate and β-hydroxybutyrate in serum

Abstract

This is an automated method for the kinetic measurement of the combined concentrations of acetoacetate and β-hdyroxybutyrate in a single channel of the 'Multistat III' centrifugal analyzer. Acetoacetate is first reduced with high concentrations of NADH by catalysis with 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30). This reaction mixture is diluted with excess NAD+. The endogenous β-hydroxybutyrate and that resulting from acetoacetate are then measured kinetically. Comparing the combined concentration of acetoacetate and β-hydroxybutyrate (x) with the sum of acetoacetate and β-hydroxybutyrate measured as described by Hansen and Freier (Clin Chem 1978;24:475) (x) yielded the relationship: y = 0.99x - 0.57 (r = 0.93, n = 25). The run-to-run CVs for low (5 mmol/L) and high (15 mmol/L) acetoacetate controls were 12% and 6%, respectively. The method is useful for determining the concentration of ketone bodies in 2-μL samples of serum of patients with diabetic ketoacidosis. The sensitivity can be increased to determine ketone body concentration in nonketotic individuals by increasing sample volume to 10 μL.