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[27] Radioisotopic assay of acetylcarnitine and acetyl-CoA

1986; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(86)23029-3

1557-7988

Shri V. Pande,

Cancer, Hypoxia, and Metabolism

This chapter examines the radioisotopic assay of acetylcarnitine and acetyl-CoA. Acetyl-CoA is stoichiometrically converted to [14C]citrate using citrate synthase and excess of freshly generated [U-14C]ox-aloacetate of high specific radioactivity. The leftover [14C]oxaloacetate is then converted back to [14C]aspartate by adding glutamate and aspartate aminotransferase. The [14C]aspartate is then removed from the reaction mixture by the addition of a cation exchanger and the radioactivity of the supernatant due to the [14C]citrate formed is measured. Because of the high affinity of the citrate synthase for acetyl-CoA and oxaloacetate and of favorable equilibrium of the reaction toward citrate formation, near quantitative conversion of picomoles of acetyl-CoA to citrate is obtained in a short time even in the presence of only a modest excess of [14C]oxaloacetate. On inclusion of CoASH and carnitine acetyltransferase the same procedure enables the estimation of acetylcarnitine. To minimize complications from the spontaneous decarboxylation of oxaloacetate in solutions, the [14C]oxaloacetate is generated last and the oxaloacetate-enzyme premixes are used immediately after their preparation. Because of the large dilution of tissue extracts needed for the present assays owing to their high sensitivity, it is unlikely that interfering substances of tissue origin would invalidate the assay. With samples containing nanomole quantities, both acetyl-CoA and acetylcarnitine can be assayed by following changes in NADH concentration spectrophotometrically or fluorimetrically by coupling of the citrate synthase reaction to that of malate dehydrogenase.