Flow cytometric detection of mitochondrial dysfunction in subpopulations of human mononuclear cells.
At 488 nm argon-ion laser excitation human mononuclear cells emit flavoprotein-related autofluorescence signals. Approximately 60% of these are caused by the mitochondrial flavoproteins alpha-lipoamide dehydrogenase and electron transfer flavoprotein, having differences in their fluorescence emission spectra. At the emission wavelength of 530 nm the redox changes of alpha-lipoamide dehydrogenase fluorescence in human mononuclear cells can be monitored by flow cytometry. This allows the estimation of the steady-state reduction level of this flavoprotein being in redox equilibrium with the mitochondrial NAD-system. We applied this method to elucidate the possible impairment of mitochondrial function in subpopulations of mononuclear cells of patients harboring deletions of the mitochondrial DNA in skeletal muscle. In the monocyte fraction of three patients and in the lymphocyte fraction of one patient we observed in the presence of the mitochondrial substrate octanoate elevated steady-state reduction levels of alpha-lipoamide dehydrogenase. This is an indication for the presence of respiratory chain-inhibited mitochondria in mononuclear cell subpopulations of the described patients. These data were confirmed by conventional determinations of maximal oxygen consumption rates of digitonin-permeabilized cells. Therefore, the flow cytometric determination of flavoprotein-caused autofluorescence changes is a useful and sensitive method for the detection of an impairment of mitochondrial respiratory chain in subpopulations of heterogeneous cell suspensions.
Kunz
D
D
Neurobiochemisches Labor der Klinik für Neurologie, Universität Magdeburg, Germany.
Luley
C
C
Winkler
K
K
Lins
H
H
Kunz
W S
WS
D002210:Caprylates; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis*; D005420:Flavoproteins / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology*; D017246:Ophthalmoplegia, Chronic Progressive External / Q000201:enzymology*; D010084:Oxidation-Reduction
D002210:Caprylates; D005420:Flavoproteins; D008058:Dihydrolipoamide Dehydrogenase; C031492:octanoic acid