Measurements of protein carbonyls, ortho- and metatyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats

Mitochondrial bioenergetic function is often reported to decline with age a nd the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of m itochondrial oxidative damage in aging. We hypothesized that, as radical pr oduction in mitochondria is greater than the rest of the cell, protein oxid ative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hy potheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta -tyrosine levels in protein hydrolysates were measured by a gas chromatogra phy/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxida tive damage in mitochondria. relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondr ial proteins on aging. Mitochondrial oxidative phosphorylation complex acti vities were also assessed revealing 39-42% decreases in F0F1 - ATP synthase activity in liver and heart on aging, but not in other oxidative phosphory lation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging. (C) 2001 Elsevier Science Inc.