人体肌肉组织中丙酮酸脱氢酶复合物的灵敏放射性同位素测定法。

A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue.

作者信息

Constantin-Teodosiu D, Cederblad G, Hultman E

机构信息

Department of Clinical Chemistry I, Huddinge University Hospital, Karolinska Institutet, Sweden.

出版信息

Anal Biochem. 1991 Nov 1;198(2):347-51. doi: 10.1016/0003-2697(91)90437-x.

DOI:10.1016/0003-2697(91)90437-x

PMID:1799221

Abstract

A radioactive assay for the determination of pyruvate dehydrogenase complex activity in muscle tissue has been developed. The assay measures the rate of acetyl-CoA formation from pyruvate in a reaction mixture containing NAD+ and CoASH. The acetyl-CoA is determined as [14C]citrate after condensation with [14C]-oxaloacetate by citrate synthase. The method is specific and sensitive to the picomole range of acetyl-CoA formed. In eleven normal subjects, the active form of pyruvate dehydrogenase (PDCa) in resting human skeletal muscle samples obtained using the needle biopsy technique was 0.44 +/- 0.16 (SD) mumol acetyl-CoA.min-1.g-1 wet wt. Total pyruvate dehydrogenase complex (PDCt) activity was determined after activation by pretreating the muscle homogenate with Ca2+, Mg2+, dichloroacetate, glucose, and hexokinase. The mean value for PDCt was 1.69 +/- 0.32 mumol acetyl-CoA.min-1.g-1 wet wt, n = 11. The precision of the method was determined by analyzing 4-5 samples of the same muscle piece. The coefficient of variation for PDCa was 8% and for PDCt 5%.

摘要

已开发出一种用于测定肌肉组织中丙酮酸脱氢酶复合物活性的放射性测定法。该测定法测量在含有NAD⁺和CoASH的反应混合物中由丙酮酸形成乙酰辅酶A的速率。通过柠檬酸合酶与[¹⁴C] -草酰乙酸缩合后，乙酰辅酶A被测定为[¹⁴C]柠檬酸。该方法对所形成的皮摩尔范围的乙酰辅酶A具有特异性和敏感性。在11名正常受试者中，使用针吸活检技术获得的静息人骨骼肌样本中，丙酮酸脱氢酶（PDCa）的活性形式为0.44±0.16（标准差）μmol乙酰辅酶A·min⁻¹·g⁻¹湿重。在用Ca²⁺、Mg²⁺、二氯乙酸、葡萄糖和己糖激酶预处理肌肉匀浆后，测定总丙酮酸脱氢酶复合物（PDCt）的活性。PDCt的平均值为1.69±0.32μmol乙酰辅酶A·min⁻¹·g⁻¹湿重，n = 11。通过分析同一块肌肉的4 - 5个样本确定该方法的精密度。PDCa的变异系数为8%，PDCt的变异系数为5%。