A sensitive radioisotopic assay of pyruvate dehydrogenase complex in human muscle tissue.
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%.
Constantin-Teodosiu
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Department of Clinical Chemistry I, Huddinge University Hospital, Karolinska Institutet, Sweden.
Cederblad
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Hultman
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D000105:Acetyl Coenzyme A / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002250:Carbon Radioisotopes; D003999:Dichloroacetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006593:Hexokinase / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000187:drug effects / Q000201:enzymology*; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry*; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D011768:Pyruvate Dehydrogenase Complex; D000105:Acetyl Coenzyme A; D003999:Dichloroacetic Acid; D006593:Hexokinase; D008274:Magnesium; D005947:Glucose; D002118:Calcium