beta-Oxidation of [9,10(n)-H-3]palmitate by human leukocytes: A simple in situ assay to assess mitochondrial toxicity in the presence of toxins

We utilized the beta-oxidation of [9,10(n)-H-3]palmitate in human leukocyte s as a. simple assay to assess mitochondrial toxicity. Advantages of the us e of tritiated fatty acids in lieu of C-14-labeled fatty acids include less interassay variation, higher specific activities obtained at a lower cost, and the relatively small number of cells required. In our assay, only 50 m u g cellular protein (approximate to 5 x 10(4) leukocytes) are required per beta-oxidation reaction as opposed to 1 x 10(6) Leukocytes required in the beta-oxidation reaction with C-14-labeled fatty acids. The higher specific activities obtained substantially reduced assay volumes, and 96 well micro titer plates could be used to conduct the assays. Rotenone (a mitochondrial complex I inhibitor) and antimycin (a mitochondrial complex III inhibitor) acutely inhibited the beta-oxidation of [9,10(n)-H-3]palmitate, thereby va lidating our assay. This assay offers an in situ or even a pseudo in vivo r endering of mitochondrial inhibition while potential effects of the cell me mbrane or cytosol or both on the toxicology of the compounds are simultaneo usly taken into account. We exploited these properties of our assay to inve stigate the in. situ mitochondrial inhibitory properties of the Parkinsonia n-inducing drug MPTP and its neurotoxic pyridinium metabolite, MPP+.