An enzymatic assay method for D(-)-3-hydroxybutyrate and acetoacetate involving acetoacetyl coenzyme A synthetase from Zoogloea ramigera.
An enzyme assay method for D(-)-3-hydroxybutyrate and acetoacetate involving acetoacetyl coenzyme A (CoA) synthetase was developed. To determine the concentration of D-3-hydroxybutyrate, it was oxidized with D-3-hydroxybutyrate dehydrogenase in the presence of nicotinamide adenine dinucleotide (NAD+) to acetoacetate, which was then converted to acetyl CoA via acetoacetyl CoA through the combined actions of acetoacetyl CoA synthetase and 3-ketothiolase in the presence of adenosine triphosphate (ATP) and CoA. To determine the concentration of acetoacetate, acetoacetyl CoA generated from acetoacetate with acetoacetyl CoA synthetase was reduced to 3-hydroxybutyryl CoA with 3-hydroxyacyl CoA dehydrogenase in the presence of NADH. The amount of D-3-hydroxybutyrate or acetoacetate was estimated from the increase or decrease in the absorbance at 340 nm, respectively. The present assay method seemed to be accurate and quick. Furthermore, as to the assaying of D-3-hydroxybutyrate, the omission of hydrazine, which is included for the standard method, may be preferable for routine assaying.
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
T
T
Saito
N
N
Takemura
M
M
Ito
K
K
Tomita
D020155:3-Hydroxybutyric Acid; D000090:Acetoacetates / Q000032:analysis*; D000818:Animals; D001419:Bacteria / Q000201:enzymology*; D003066:Coenzyme A Ligases; D006885:Hydroxybutyrates / Q000032:analysis*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D000090:Acetoacetates; D006885:Hydroxybutyrates; D003066:Coenzyme A Ligases; C023747:acetoacetyl-CoA synthetase; D020155:3-Hydroxybutyric Acid