ABSTRACT
This study investigated the feasibility of using a phosphorescence oxygen analyser to measure cellular respiration [mitochondrial O[2] consumption] in foreskin samples and their fibroblast-rich cultures. Foreskin specimens from normal infants were collected immediately after circumcision and processed for measuring cellular respiration and for culture. Cellular mitochondrial O[2] consumption was determined as a function of time from the phosphorescence decay of the Pd [II] meso-tetra-[4-sulfonatophenyl]-tetrabenzoporphyrin. In sealed vials containing a foreskin specimen and glucose, O[2] concentration decreased linearly with time, confirming the zero-order kinetics of O[2] consumption by cytochrome oxidase. Cyanide inhibited O[2] consumption, confirming that the oxidation occurred mainly in the mitochondrial respiratory chain. The rate of foreskin respiration [mean +/- SD] was 0.074 +/- 0.02 microM O[2] min[-1] mg[-1] [n = 23]. The corresponding rate for fibroblast-rich cultures was 9.84 +/- 2.43 microM O[2] min[-1] per 10[7] cells [n = 15]. Fibroblast respiration was significantly lower in a male infant with dihydrolipoamide dehydrogenase gene mutations, but normalised with the addition of thiamine or carnitine. The foreskin and its fibroblast-rich culture are suitable for assessment of cellular respiration. However, the clinical utility of foreskin specimens to detect disorders of impaired cellular bioenergetics requires further investigation
ABSTRACT
We investigated the effect of caffeine on mitochondrial O[2] consumption in human promyelocytic leukemia [HL-60] cells. A phosphorescence analyzer that measures O[2] concentrations in cell suspensions as function of time was used for this purpose. O[2] concentrations were determined from the phosphorescence of Pd phosphor, calculated by fitting the phosphorescence decays to exponentials. In sealed vials, O[2] concentrations in the cell suspensions containing glucose declined linearly with time, showing zero-order kinetics for O[2] consumption. NaCN inhibited O[2] consumption, confirming the oxidation occurred in the mitochondrial respiratory chain. A rapid decline in the rate of respiration was observed when 50 [micro]M to 4.0 mM caffeine was added to HL-60 cells in cell growth media [containing 1.41 mM Ca[2+]] or phosphate-buffered-salts [containing 0.91 mM Ca[2+]]. This reversible inhibition was blocked by verapamil and was concentration-dependent, reaching a plateau [43 +/- 7% inhibition] at 50 [micro]M caffeine. The inhibition was not observed when cellular Can+ stores were depleted. T-cell lymphoma [Jurkat] cells and isolated mitochondria were less sensitive to caffeine. Thus, caffeine is a potent inhibitor of HL-60 respiration. This effect is possibly mediated by Ca[2+]-flooding into the cytosol and neighboring mitochondria