Activities of mitochondrial oxidative phosphorylation enzymes in cultured amniocytes

Amniocytes represent a population of foetal cells that can be used for pren atal diagnosis in families with suspected mitochondrial oxidative phosphory lation (OXPHOS) defects. In this paper, we present a complex protocol for e valuation of the function of mitochondrial OXPHOS enzymes in cultured amnio cytes using three independent and complementary methods: (a) spectrophotome try as a tool for determination of the capacities of mitochondrial respirat ory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glyceroph osphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzy me functions in situ using digitonin-permeabilised amniocytes (rotenone-sen sitive oxidation of pyruvate + malate, antimycin A-sensitive oxidation of s uccinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under stea dy-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9-22 independent cultures of amniocytes. (C) 2000 Elsevier Science B.V. All rights reserved.