Fluxes of nicotinamide adenine dinucleotides through mitochondrial membranes in human cultured cells

We report on the loss of mitochondrial nicotinamide adenine dinucleotides in human cultured cells along with cell culture and acidification of the culture medium. This was established both by the direct measure ment of the decrease in the mitochondrial NAD content and by the alteration of the oxidative properties of the mitochondria. In situ, this loss could be reversed in less than 2 h by changing the culture medium or by readjusting the pH of the medium at physiological pH values. By studying the oxidative properties of intact, but NAD-depleted, mitochondria in digitonin-permeabilized cells, we found that a rapid influx of NAD could replenish the mitochondrial NAD pool. This allowed the restoration of an active NAD(+)-dependent substrate oxidation. Depletion of mitochondrial NAD in cells grown under quiescent conditions was further confirmed by fluorimetric measurement of mitochondrial NAD, as was the influx of NAD(+) into the mitochondrial matrix. These data constitute the first evidence of rapid fluxes of NAD through mitochondrial membranes in animal cells. They also point to the possible confusion between a loss of mitochondrial NAD and a defect of respiratory chain complex I in the context of screening procedures for respiratory chain disorder in human.