Abstract:
Techniques and protocols of assessment of mitochondrial properties are of physiological and physiopathological important significance. A precise knowledge of the advantages and limitations of the different protocols used to investigate the mitochondrial function, is therefore necessary. This report presents examples of how the skinned (or permeabilized) fibers technique could be applied for the polarographic determination of the actual quantitative and qualitative aspects of mitochondrial function in human muscle samples. We described and compared the main available respiration protocols in order to sort out which protocol seems more appropriate for the characterization of mitochondrial properties according to the questions under consideration: quantitative determination of oxidative capacities of a given muscle, characterization of the pattern of control of mitochondrial respiration, or assessment of a mitochondrial defect at the level of the respiratory chain complexes. We showed that while protocol A, using only two levels of the phosphate acceptor adenosine diphosphate (ADP) concentration and the adjunction of creatine, could be used for the determination of quantitative changes in very small amount of muscle samples, the ADP sensitivity of mitochondrial respiration was underestimated by this protocol in muscles with high oxidative capacities. The actual apparent Km for ADP and the role of functional activation of miCK in ATP production and energy transfer in oxidative muscles, are well-assessed by protocol B (in the absence of creatine) together with protocol C (in the presence of creatine) that use increasing concentrations of ADP ranging from 2.5-2000 microM. Protocol D is well-adapted to investigate the potential changes at different levels of the respiratory chain, by the use of specific substrates and inhibitors. As can be seen from the present data and the current review of previous reports in the literature, a standardization of the respiration protocols is needed for useful comparisons between studies.