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explained in terms of an age-linked increase in the proportion of polyunsaturated fatty acids and a decrease in the proportion of monounsaturated fatty acids in the mitochondrial lipids. Death within the colony is observed only in animals in excess of 24 months of age. About four-fifths of the animals survive to at least 26 months of age. Thus the observed trends are not due to survival factors in individual animals since the trends are established before death in the population is observed. This work was supported by a grant from the Medical Research Council. Barber, A. A. & Bernheim, F. (1967) Aduan. Gerontol. Res. 2,355-403 Chappell,J. B. & Hansford, R. G. (1969) in Sub-cellularComponents:Preparationsand Fractionation (Birnie, G . D. & Fox, S. M., eds.), 1st edn., pp. 43-56, Butterworths, London Crawford, N. (1958) Clin. Chim. Acta 3,357-367 Fortney, S. R. & Lynn, W. S. (1964) Arch. Biochem. Biophys. 104,241-247 Horton, A. A. &Packer, L. (1970) J. Gerontol. 25,199-204 Hunter, F. E., Scott, A., Hoffsten, P. E., Guerra, F., Weinstein, J., Schneider, A., Schutz, B., Fink, J., Ford, L. &Smith, E. (1964) J. Biol. Chem. 239,604-613 Lapetina, E. G. & Michell, R. H. (1972) Biochem. J. 126,1141-1 147 Ottolenghi, A. (1959) Arch. Biochern. Biophys. 79,355-363 Patton, S. & Kurtz, G. W. (1951) J. Dairy Sci. 34,669-674 Sinnhuber, R. 0. & Yu, T. C. (1958) FoodTechnol. 12,9-I2 Stocks, J. & Dormandy, T. L. (1971) Brit. J. Haematol. 20,95-111 Wilbur, K. M., Bernheim, F. & Shapiro, 0. W. (1949) Arch. Biochern. 24,305-313 Yasuda, M. (1931) J. Biol. Chem. 94,401-409

The Effect of Age on Mitochondria1 Ultrastructure and Enzyme Cytochemistry PATRICIA D. WILSON and L. M. FRANKS Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, U.K. There is considerable evidence to suggest that there are mitochondrial changes in aging and in tumours (Tauchi et al., 1964; Chen et al., 1972; Menzies &Gold, 1972; Tribe & Ashurst, 1972; Siliprandi et al., 1973; Bernhard, 1969; Frolkis & Bogatskaya, 1968; Glew et al., 1973; Martin et al., 1974; Wilson, 1972, 1973), but there are few detailed studies. In this paper we describe age-associated changes in mitochondrial ultrastructure in the mouse liver in our colony of aging mice, and compare the cytochemical localization of cytochrome c oxidase (an inner-membrane enzyme) and malate dehydrogenase (a matrix enzyme). We also point out some problems in the interpretation of data obtained from mitochondria isolated from the livers of young and old mice. In the parenchymal cells of the livers of young mice, the mitochondria were small and the majority were elongated and rod-like, with long, regularly arranged cristae and dense homogeneous matrix. This is the ‘classical’ mitochondrial structure. In old mice the liver-cell mitochondria were enlarged and rounded with a light ‘foamy’ vacuolated matrix. Quantitative studies showed a significant increase in the mean size and an increased proportion of larger mitochondria in intact 30-month-old perfused livers. Endothelial and Kupffer cell mitochondria were smaller than those of the parenchymal cells and had a characteristic cristal pattern. The mitochondria in these cells showed similar age-associated changes. The age-associated changes did not affect all cells or all mitochondria in a single cell to the same extent. In some cells, apparently severely affected and apparently normal mitochondria could be found side by side. The isolated mitochondrial fractions were prepared by a modification of the method of Schneider (Falcone & Hadler, 1968). They were relatively pure, consisting of mitochondria with some glycogen contamination. There was little if any difference in the structure 1975

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of nlitochondria isolated from young or old mice. They were rounded and showed the ‘condensed‘ configuration with dense matrix and swollen cristae. There was no significant increase in the size of mitochondria isolated from the old livers compared with those isolated from the young livers. The size distribution of mitochondria was different in the 6-month- and 30-month-old perfused liver (Fig. la). The 30-month-old tissue contained a lower proportion of small mitochondria and a higher proportion of larger mitochondria than the livers of 6-month-old animals. Fig. 1(b)shows that the size distributionsfor the mitochondria isolated from 6-monthold and 30-month-old livers were different. Although the mitochondria isolated from 30-month-old livers also contained a lower proportion of small mitochondria and a higher proportion of large mitochondria than those isolated from 6-month-old livers, there were no very large mitochondria present in either fraction. The cytochemical localization of cytochrome c oxidase and malate dehydrogenase was not altered significantly with age. A factor to be considered when using biochemical techhiques to examine changes in mitochondria with age is the morphological demonstration that the changes may not affect all mitochondria to the same extent. Grossly altered and normal mitochondria have been seen side by side in the same cell. Cytochemistry may be considered to overcome some of the disadvantagesof homogenization and fractionation procedures. An important finding of these morphological studies is the evidence of a selectiveloss of the large, abnormal mitochondria during this procedure of cell fractionation and mitochondriaI isolation. This must be taken into consideration when considering the use of isolated mitochondria for functional studies. In this case, despite considerable differences in structure, and perhaps function of the mitochondria in the intact tissue, after the isolation p r d u r e only the small more normal mitochondria remain. Thus, in the case of the old m o w liver, the sample of isolated mitochondria obtained is not representative of the tissue as a whole. At present we have no information as to whether the abnormal mitochondria are destroyed during the isolation procedure, or whether they could be recovered in another fraction. In conclusion, we suggest that morphology has an important role to play alongside conventional biochemical techniques in the elucidation of possible functional changes of mitachondria with age. 35

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A transparent sheet of equally spaced dots was superimposed over electron micrographs of the same magnification and the percentage of mitochondria was plotted against the ‘size’ as estimated by the number of dots touching any mitochondrion. There were eight mice in each age group. (a) Perfused mouse liver; (6) isolated mitochondria. Bars represent standard deviations. , 6 months; o, 30 months. Vd. 3

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Bernhard, W. (1969) in Handbook of Molecular Cytology (Lima de Faria, A., ed.), pp. 1-99, North-Holland, Amsterdam Chen, J. C . , Warshaw, J. B. &Sanadi,D. R. (1972)J. CeNPhysiol. 80,141-148 Falcone, A. B. & Hadler, H. I. (1968) Arch. Biochem. Biophys. 124,91-I09 Frolkis, W. V. & Bogatskaya, L. N. (1968) Exp. Gerontol. 3,199-210 Glew, R. H., Zatzkin, J. B. & Kayman, S. C. (1973) Cancer Res. 33,2135-2142 Martin, A. P., Cornbleet, P. J., Lucas, F. V., Morris, H. P. & Vorbeck, M. L. (1974) Cancer Res. 34,850-858

Menzies, R. A. &Gold, P. H. (1972) J. Neurochem. 19,1671-1683 Siliprandi, D., Siliprandi, M., Scutari, G. & Zoccarato, F. (1973) Biochem. Biophys. Res. Commun. 55,563-568

Tauchi, H., Sato, T., Hashno, M., Kobayarhi, H., Ada Chi, F., Aoki, J. & Masuko, T. (1964) in Age with a Future (Hansen, P. F., ed.), pp. 203-235, Munksgaard, Copenhagen Tribe,M. A. &Ashurst,D.E. (1972)J. CeNSci. 10,443-469 Wilson, P. D. (1972) Gerontologia 18,36-45 Wilson, P. D. (1973) Gerontologiu19,79-125

Age-Related Changes in Gluconeogenesis and the Metabolism of Alanine in the Perfused Liver of Neonatal Rats KEITH SNELL* and DERYCK G . WALKER Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K. The milk diet of the suckling neonatal rat is low in carbohydrate. Blood glucose concentrations throughout the suckling period are, however, maintained at normo- or slightly hyper-glycaemic values as compared with adult values. This places the burden for maintaining glycaemia on gluconeogenic processes in the neonate. The capacity to perform gluconeogenesis develops rapidly immediately after parturition (Snell & Walker, 1973a,b)and an increased gluconeogenic flux, as measured in vivo, is maintained throughout the suckling period (Walker & Snell, 1973).This is accompanied by elevated activities of a number of hepatic enzymes known to be involved in this process (see Snell & Walker, 1973~).Previous measurements of gluconeogenesis from potential substrates in the neonatal rat involved the use of liver slices (Vernon et al., 1968)and were of limited value for various reasons. Recently the technique of liver perfusion has been adapted to studying net glucose synthesis in the neonatal rat and the increased conversion of a number of substrates into glucose has been demonstrated (Snell, 1974). The present study extends this work. All animals were starved overnight (16-20h) before perfusion of the livers in situ as described by Snell(l974). Measurements of apparent endogenous rates of glucose synthesis in the absence of added substrates revealed a somewhat higher value in the 10-dayold neonatal rat as compared with the adult. When rates of gluconeogenesis in the presence of added substrate (10mM) were corrected for the endogenous rates the 10-day-old rat showed a significantly greater (P

The effect of age on mitochondrial ultrastructure and enzyme cytochemistry.

126 BIOCHEMICAL SOCIETY TRANSACTIONS explained in terms of an age-linked increase in the proportion of polyunsaturated fatty acids and a decrease in...
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