Latipää P M, Hassinen I E, Hiltunen J K
Department of Medical Biochemistry, University of Oulu, Finland.
Anal Biochem. 1988 May 15;171(1):67-72. doi: 10.1016/0003-2697(88)90125-x.
An enzymatic assay is presented for determining 3-hydroxyacyl-CoA and 2-trans-enoyl-CoA esters in tissue samples. The procedure includes extraction of acyl-CoA esters from frozen tissue samples with chloroform/methanol, stochiometric oxidation of the acyl esters to 3-keto-acyl-CoAs with 3-hydroxyacyl-CoA dehydrogenase in the presence or absence of enoyl-CoA hydratase, and an enzymatic cycling amplification of NADH produced for fluorometric determination. The procedure allows measurement of these intermediates of beta-oxidation at the picomole level. The method has been used successfully to measure the concentrations of 3-hydroxyacyl-CoA and 2-trans-enoyl-CoA esters in isolated rat hearts perfused with glucose or oleate or under anoxia.
本文介绍了一种用于测定组织样本中3-羟基酰基辅酶A和2-反式烯酰基辅酶A酯的酶促测定法。该方法包括用氯仿/甲醇从冷冻组织样本中提取酰基辅酶A酯，在存在或不存在烯酰辅酶A水合酶的情况下，用3-羟基酰基辅酶A脱氢酶将酰基酯化学计量氧化为3-酮酰基辅酶A，以及对产生的用于荧光测定的NADH进行酶循环扩增。该方法能够在皮摩尔水平上测量β-氧化的这些中间产物。该方法已成功用于测量灌注葡萄糖、油酸或处于缺氧状态下的离体大鼠心脏中3-羟基酰基辅酶A和2-反式烯酰基辅酶A酯的浓度。
