Chowdhury 2000 Clin Chim Acta
|Chowdhury SK, Drahota Z, Floryk D, Calda P, Houstek J (2000) Activities of mitochondrial oxidative phosphorylation enzymes in cultured amniocytes. Clin Chim Acta 298:157-73.|
Abstract: Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, 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 steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9–22 independent cultures of amniocytes.
• Keywords: Prenatal diagnosis, Amniocytes, Oxidative phosphorylation, Respiratory-chain enzymes, Mitochondrial membrane potential, TMRM cytofluorometry
Labels: MiParea: Respiration
Organism: Human Tissue;cell: Endothelial;epithelial;mesothelial cell, Fibroblast Preparation: Permeabilized cells
Coupling state: LEAK, ROUTINE, OXPHOS Pathway: N, S, CIV HRR: Oxygraph-2k