Cytochrome oxidase (COX) activity, selectively evidenced by preferential diaminobenzidine cytochemistry, has been measured by computer-assisted morphometric methods in the cerebellar cortex of adult and old rats. We calculated the ratio (R) between the area of the precipitate due to the cytochemical reaction and the overall area of each mitochondrion. The value of R is reported to provide information on the fraction (%) of the inner mitochondrial membrane actively involved in adenosine triphosphate (ATP) provision. Thus, since COX is the terminal enzyme of the mitochondrial respiratory chain, R can be considered a reliable index of the mitochondrial metabolic competence (MMC), i.e. the capacity of each organelle to provide ATP. The data from each animal were ordered by increasing values of mitochondrial area, then they were divided into quartiles. While in adult rats we documented an inverse correlation between mitochondrial size and R values (r = -0.905), in old animals we found increasing values of R as the mitochondrial area increases (r =0.561). Paired-quartile comparisons of the R values from adult and old animals documented a marked age-related impairment of MMC in small (I quartile: -31.6%) and medium-sized (II quartile: -26.4; III quartile: -16.4) mitochondria, while large organelles showed the lowest age-related decrease (IV quartile: -3.0%). Mitochondrial decrease in number and increase in size are consistently reported as a general trend in old organisms. Mitochondrial enlargement is supposed to constitute a compensating reaction to the numeric loss since it extends the inner membrane area potentially involved in the respiratory chain. This compensation has been reported to be due to an increased complement of oversized mitochondria in discrete cellular or tissue compartments, however it is still debated whether these enlarged organelles are capable of providing adequate amounts of ATP according to their enlarged size. By matching quantitative preferential cytochemistry of COX and classical morphometric procedures, the present findings support that in adulthood as well as in aging the complement of oversized mitochondria appears to represent a weak compensating reaction since in both these conditions their R value is about 0.28 vs. 0.39 and 0.26 of the small-sized organelles in adult and old animals, respectively. Conceivably, our results support that a marked dysfunction of small and medium-sized mitochondria contributes to the significant decay of energy metabolism well documented in physiological aging.
mitochondrial metabolic competence
cytochrome oxidase activity