Saito T, Takemura N, Ito M, Tomita K
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Chem Pharm Bull (Tokyo). 1990 Jun;38(6):1627-9. doi: 10.1248/cpb.38.1627.
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.
开发了一种涉及乙酰乙酰辅酶A（CoA）合成酶的D（-）-3-羟基丁酸酯和乙酰乙酸酯的酶测定方法。为了测定D-3-羟基丁酸酯的浓度，在烟酰胺腺嘌呤二核苷酸（NAD +）存在下，用D-3-羟基丁酸脱氢酶将其氧化为乙酰乙酸酯，然后在三磷酸腺苷（ATP）和CoA存在下，通过乙酰乙酰辅酶A合成酶和3-酮硫解酶的联合作用，乙酰乙酸酯通过乙酰乙酰辅酶A转化为乙酰辅酶A。为了测定乙酰乙酸酯的浓度，在NADH存在下，用3-羟基酰基辅酶A脱氢酶将由乙酰乙酰辅酶A合成酶从乙酰乙酸酯生成的乙酰乙酰辅酶A还原为3-羟基丁酰辅酶A。分别根据340nm处吸光度的增加或减少来估算D-3-羟基丁酸酯或乙酰乙酸酯的量。本测定方法似乎准确且快速。此外，对于D-3-羟基丁酸酯的测定，省略标准方法中包含的肼可能更适合常规测定。
