Chowdhury 2000 Clin Chim Acta

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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.

» PMID: 10876012

Chowdhury SK, Drahota Z, Floryk D, Calda P, Houstek J (2000) Clin Chim Acta

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

O2k-Network Lab: CZ Prague Houstek J, CA Winnipeg Fernyhough P, CZ Hradec Kralove Cervinkova Z


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