Aging Aging

238 Pallardo et al.age on mitochondria by flow cytometry methods which allow a direct measurementof the organellar function as shown by its membrane potential value. Thisvalue can be determined both on mitochondria isolated using standard techniquesand on those that remain in their normal environment inside the cells.Our own mitochondrial flow cytometry studies (6) on isolated rat hepatocyteshave shown, for the first time in intact cells, a correlation between age-relatedchanges in cell size and impaired mitochondrial function (Fig. 1). Specifically,we have observed that age is accompanied by a decrease in mitochondrial membranepotential (MMP), an increase in mitochondrial size, and a loss of organellarhomeostasis (resulting in raised levels of peroxide generation). The pathogeneticmechanisms responsible for these changes are not well understood, althoughas pointed out by Hagen et al. (8), loss of the membrane phospholipid cardiolipin(which is essential for respiratory chain work) may play a role in the agerelatedMMP decrease. Because MMP supports mitochondrial protein synthesis(9), its decrease with age may be linked to a general decline in the biochemicaland functional competence of mitochondria (2).It is well known that aging results in mitochondrial membrane changes thatincrease the vulnerability of these organelles to the stress caused by the isolationprocedure. Therefore, during isolation a considerable number of organelles(precisely those most altered by aging) may be lost, and therefore the dataobtained will not provide an accurate measurement of the functional state ofthe whole mitochondrial population. Nevertheless, because useful informationcan be obtained on isolated mitochondria, we will describe the methods used inour laboratory for flow cytometry study of isolated mitochondria and of mitochondriapresent in their normal environment in cells previously isolated fromtheir tissue. A detailed presentation of these methods may be of interest toworkers interested in the mechanisms of senescence of mitochondria of differentcell types, changes in the rate of aging caused by genetic or environmentalmodulation of mitochondrial development and function, and protection of themitochondrial membranes and DNA by dietary antioxidants and free radicalscavengers.The procedures presented here for the study of key parameters of mitochondrialmembrane function that change with age are as follows: (1) preparation ofthe respiration buffer for suspension of isolated mitochondria or cells and (2)determination of mitochondrial membrane mass, membrane potential, membranelipid composition, and peroxide production.Of course, a more complete understanding of the causes and effects ofmitochondrial aging will require determination of many other parameters,such as fine structural changes (see Chapter 17), NADH/NAD redox state,reduced and oxidized thiol contents, activity of the inner membrane respiratoryenzymes, state 3/state 4 respiratory control ratios (which indicate