Enzymic determination of d(−)-β-hydroxybutyric acid and acetoacetic acid in blood

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Abstract
D([long dash])-[beta]-Hydroxybutyric dehydrogenase of Rhodopseudomonas spheroides has been partially purified. The enzyme is water-soluble and stable. The purified material is free from reduced diphos-phopyridine nucleotide oxidase but still contains some malic and polyol (sorbitol, mannitol) dehydrogenase. Unlike the similar enzymes from Rhodospirillum rubrum and Bacillus megaterium, the activity of the purified enzyme is not increased by the addition of magnesium or manganese. The use of the enzyme for the spectrophotometric determination of D([long dash])-[beta]-hydroxybutyrate in 0.02-0.20 umole quantities is described. The reduction of diphosphopyridine nucleotide is measured at pH 8.5 in the presence of hydrazine. The enzyme can also be used for the estimation of acetoacetate by measuring the disappearance of reduced diphosphopyridine nucleotide at pH 7.0. The ratio of D([long dash])-u-hydroxybutyrate to acetoacetate in fasting human subjects was found to be 2.73[plus or minus]0.73 (S.D.) irrespective of total concentration of ketone bodies over the range 0.046-0.876 umole/ml.