The effect of aging and increasing ascorbate concentrations on respiratory chain activity in cultured human fibroblasts.

The specific activities of Complexes I-III, II-III, and IV of the respiratory chain, and citrate synthase, were determined in mitochondrial sonicates of six control passage 5 fibroblast cultures, cultivated in growth medium containing fetal calf serum as the only source of ascorbate. The enzymes were also assayed in serially subcultured fibroblasts which were characterized as aged at passage 20 and beyond. Results indicated a significant loss of all enzyme activities in aged cells at passage 20, 25, and 30. Further studies involved maintenance of serially subcultured cells in serum free media to which increasing ascorbate concentrations (100, 200, and 300 micromol 1(-1)) were added. Results indicated that ascorbate at 100 micromol 1(-1) was not sufficient to restore any of the enzyme activities in aged cells. An ascorbate concentration of 200 micromol 1(-1) however, could totally restore Complex IV and citrate synthase activities, but had no effect on complexes I-III and II-III activities which required 300 micromol 1(-1) ascorbate to be partially or totally restored respectively. In conclusion, this study demonstrates an age related drop in mitochondrial respiratory chain activity in cultured human fibroblasts. Enzyme activities could be completely or partially restored in the presence of double or triple normal human plasma ascorbate concentrations. 2010 John Wiley & Sons, Ltd.