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Acetoacetate concentration in plasma was determined by head-space gas chromatography after decarboxylation by the use of acetoacetate decarboxylase, 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 found to be particularly suitable for acetoacetate assay (optimum pH, 5.8 ; Vmax, 230μmol/min/mg ; Km, 5.9×10-4M). 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 1μM. This method gave better reproducibility (CV=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±3.4μM and acetoacetate levels of 22.5±9.7μM. Diabetic patients (n=44), treated by diet control alone without drug therapy, gave plasma acetone levels of 8.1±3.5μM and acetoacetate levels of 25.0±8.0μM. There was no significant difference between the two groups.

Gliclazide, 1-(3-azabicyclo[3.3.0]oct-3-yl)-3-(p-tolylsulfonyl)urea, an oral hypoglycemic drug having the sulfonylurea structure, was completely degraded in a medium containing more than 0.2M chloride or bromide ion at 37°C after 24h. Other sulfonylureas, tolazamide and glycopyramide, were also degraded in the presence of chloride ion, but tolbutamide, acetohexamide, chlorpropamide and glibenclamide were not. Fluoride, carbonate, acetate, sulfate and phosphate ions and both sodium and potassium cations induced slight degradation of gliclazide. Two degradation products were isolated, crystallized, and identified as a hydrazine compound (3-azabicyclo[3.3.0]oct-3-yl-amine monohydrochloride) and p-toluenesulfonamide based on mp, ultraviolet, infrated, proton nuclear magnetic resonance, mass spectra and high performance liquid chromatography data. The anion-induced gliclazide degradation was dose-dependently depressed by human serum albumin or other proteins. It was completely depressed by albumin at over 0.3 mg/ml. Other serum proteins, α, β and γ-globulins, depressed the anion (Cl-)-induced gliclazide degradation similarly to albumin.