Journal of Neurochemistty Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry
Lipid Composition in Synaptic and Nonsynaptic Mitochondria from Rat Brains and Effect of Aging F. M. Ruggiero, F. Cafagna, V. Petruzzella, M. N. Gadaleta, and E. Quagliariello Department of Biochemistry and Molecular Biology and CNR Unit for the Study of Mitochondria and Bioenergetics, University of Bari, Bari, Italy
Abstract: The cholesterol, phospholipid, and fatty acid compositions in synaptic and nonsynaptic mitochondria from rat brains and the effect of aging were studied. Both cholesterol and phospholipid contents were found to be significantly different in synaptic compared to nonsynaptic mitochondria. In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%, respectively. Also, the phospholipid composition is significantly different in synaptic compared to nonsynaptic mito-
chondria. Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%)in nonsynaptic mitochondria from the brains of aged rats. Instead, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria. The 2 1% aging decrease in linoleic acid (18:2), observed only in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid. Key Words: Rat brain-Synaptosomes-Mitochondria-Lipids-Aging. Ruggiero F. M. et al. Lipid composition in synaptic and nonsynaptic mitochondria from rat brains and effect of aging. J. Neurochem. 59,487-491 (1992).
Brain is very rich in neutral lipids and, particularly, in cholesterol. The cholesterol found in the brain comes from in situ synthesis and from transfer of plasma cholesterol (S6rougne et al., 1976). Previous works have demonstrated that cholesterolincreases in the developing rat brain (Kishimoto et al., 1965; Bourre et al., 1990). A decrease in this compound during aging has been reported in gray and in white matter of the human brain (Soderberg et al., 1990). As a structural component of the membrane, cholesterol is involved in maintaining the integrity of the cells and in regulating the fluidity of the cellular membrane lipids. Relatively low cholesterol concentrations were found in mitochondria from heart and liver (Paradies and Ruggiero, 1990a,b). However, in erythrocyte membranes, which contain high levels of cholesterol (Ruggiero et al., 1984a, 1987), a new role of cholesterol was reported: determining the stability of the membrane contour so as to prevent endocytosis (Lange et al., 1980).The role of this major component of lipids in brain mitochondria is still unknown. Phospholipids are asymmetrically arranged in biological membranes and a cholesterol/phospholipid ratio peculiar to particular membranes can be detected (Van Deenen, 1965).To our knowledge, no data have
been reported on the phospholipid composition in both synaptic and nonsynaptic mitochondria. Aging is a ubiquitous biological phenomenon associated with histological, biochemical, and functional alterations. Recently, Gadaleta et al. (1990) have shown that the steady-state concentration of mitochondrial RNA undergoes an age-dependent decrease in rat brain and heart. Changes in membrane lipid content and functions appear to occur with aging (Noh1 and Kramer, 1980; Kim et al., 1988). Agelinked changes in the lipid composition in plasma, heart, and liver mitochondria have been reported recently (Paradiesand Ruggiero, 1990~; Ruggiero et al., 1990; Paradies and Ruggiero, 1991). The present study was designed to investigate, for the first time, the lipid composition in both synaptic and nonsynaptic mitochondria, prepared by a highly purified procedure, and the influence of aging on their lipid compositions.
Male Fisher rats 4 months (young), 12 months (mature), and 28 months (aged) old, fed ad libitum with a standard
Received October 22, 1991; accepted January 15, 1992. Address correspondence and reprint requests to Dr. F. M. Rug-
giero at Dipartimento di Biochimica e Biologia Molecolare, Via G . Amendola 165/A, 70126 Ban, Italy.
MATERIALS AND METHODS Isolation of rat brain mitochondria
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F. M. RUGGZERO ET AL.
488
TABLE 1. Cholesterol andphospholipid content in synaptic and nonsynaptic mitochondriafrom young, mature, and aged rats Synaptic mitochondria
Cholesterol Phospholipids Ratio cholesterol/phospholipids
Nonsynaptic mitochondria
Young
Mature
Aged
Young
Mature
Aged
179.7 f 10.3 448.6 f 19.7 0.40f 0.03
165.2 f 9.8 432.5 f 18.2 0.38f 0.04
130.9f 9.2" 392.3 f 15.4' 0.33 f 0.03'
121.4 f 10.5' 335.3 f 17.0' 0.36 ? 0.04'
114.0f 9.3 321.9f 16.9 0.35 f 0.03
88.0 f 8.3" 298.2 f 16.5' 0.29 f 0.02'
Each value represents the mean f SD obtained for five experiments with two rats from each group. Cholesterol is expressed as nmol/mg of protein, and phospholipids as nmol of lipid PJmg of protein. The p values were calculated by Student's f test. p < 0.00 1 vs. young rats. " p < 0.01 vs. young rats. 'p < 0.00 1 vs. synaptic mitochondria. taminated by synaptosomal and other membranous material as judged by the low recovery of acetylcholinesterase (0.5%of the homogenate activity). To obtain the synaptosomes, the first upper phase was carefully removed and layered on 6 ml of fresh biphase system, containingthe same components as the first one plus 2 g of medium B. The new phase system was mixed again and centrifuged, then the final upper phase was removed and carefully layered on top of 20 ml of medium A. Next the tubes were centrifuged at 8,000g for 8 min, with the synaptosomes being pelleted at the bottom. The purity of the synaptosomal fraction was checked both by electron microscopy and by determination of marker enzyme activities. Enzymes assayed were NADHJcytochrome c reductase (rotenone insensitive), as a marker for the external membrane and therefore for free mitochondrion contamination, and lactate dehydrogenase, as an indicator of synaptosomal membrane integrity (Lai and Clark, 1976; Booth et al., 1978).2',3'-Cyclic nucleotide-3'-phosphohydrolasewas analyzed according to the procedure of Olafson et al. (1 969). The extent of contamination of the synaptosomes by myelin was 0.5%. According to both structural and functional criteria, the purity of the synaptosomes was never below 80%. Synaptic mitochondria were prepared by suspending the synaptosomal pellet in 15 ml of 6 mMTris-HC1, pH 8.1, incubating the mixture on ice for 5 min, and homogenizing by hand. Sorbitol(1 M) was added up to a final concentration of 0.32 M, and the mixture centrifuged at 18,500 g for 10 min. The pellet, suspended in a small volume of medium
diet, were used throughout these studies. Free (nonsynaptic) and synaptic brain mitochondria were prepared by repeated partitioning in a two-phase system composed of dextranJ polyethylene glycol (Enriquez et al., 1990; Fernandez-Silva et al., 1991). The rats were beheaded, then the hemispheres were suspended in 15 ml of cold medium A (0.32 Msucrose, 10 mM Tris-HC1, pH 7.4, 1 mM potassium-EDTA) and homogenized in a Dounce-type glass homogenizer with 15 up-and-down strokes. The homogenate was centrifuged at 1,200 g for 6 min; then the supernatant was centrifuged at 12,000g for 10 min, producing a crude synaptosomal pellet. This pellet was resuspended in a small volume of medium B (0.32 M sorbitol, 5 mM potassium phosphate, pH 7.4, 0.1 mM potassium-EDTA). Two grams of a suspension of crude synaptosomes was added to 14 g of a biphase system prepared by mixing 5.12 g of 20% (wt/wt) dextran T-500, 2.56 g of 40% (wt/wt) polyethylene glycol 4000, 4.48 g of 1 M sorbitol, 0.36 g of 0.2 M potassium phosphate, pH 7.4, 0.2 g of 10 mM potassium-EDTA, and 1.24 g of distilled water. The phase system was mixed by 20 inversions of the tube and centrifuged at 1,200 g for 1 min, separating the mitochondria1 pellet in the two phases. The lower phase, containing free (nonsynaptic) mitochondria, was washed two times with 20 ml of medium B and centrifuged for 10 min at 8,000 g. Then the pellet was suspended in a small volume of medium C (10%glycerol, 35 mM Tris-HC1, pH 7.8, 20 mM NaCl, 5 mM MgCl,, 1 mg/ml bovine serum albumin). Electron microscopy showed that synaptosomes and myelin-like particles were virtually absent. The free mitochondria were minimallycon-
TABLE 2. Phospholipid composition in synaptic and nonsynaptic mitochondria from young, mature, and aged rats as determined by HPLC Distribution (mol%) Synaptic mitochondria
Nonsynaptic mitochondria
Phospholipid
Young
Mature
Aged
Young
Mature
Aged
DPG
8.7 f 0.8 45.9 f 1.8 6.5 f 0.5 11.0 f 1.0 27.9 f 1.2
8.9f 0.7 45.2f 1.5 6.9f 0.8 11.3 f 1.1 21.7f 1.3
8.5 f 0.9 45.0f 1.5 7.0 f 0.7 11.5 f 1.0 28.0 f 1.5
15.5 f 1.1" 36.6f 1.8" 3.8 f 0.4" 4.3 f 0.5" 39.8f 1.7"
14.0 f 1.0 37.0f 1.5 4.2 f 0.5 4.7 ? 0.6 40.1 f 1.8
1 1.4 f 0.9b 36.4 f 1.3 4.6 & 0.7 4.8 f 0.6 42.8 f 1.9
PE PI
PS PC
Each value represents the mean f SD obtained for five experiments with two rats from each group. DPG, cardiolipin; PE, phosphatidylethanolamine; PI, phosphatidylinositol;PS, phosphatidylserine;PC, phosphatidylcholine. " p < 0.001 vs. synaptic mitochondria. " p < 0.00 1 vs. young rats. J . Neurochem., Vol. 59, No. 2, 1992
LIPIDS IN RAT BRAIN MITOCHONDRIA DURING AGING B, was layered on top of a 14-gbiphase system. After mixing and centrifuging,the lower phase was diluted, washed twice with medium B, and finally, suspended in a small volume of incubation medium.
Extraction and separation of lipids Total lipids were extracted from synaptic and nonsynaptic mitochondria with chloroform/methanol by the Bligh and Dyer (1959)procedure. A portion of this extract was digested at 180°C with perchloric acid, then the phospholipid phosphorus content was determined by the Nakamura (1952)method. Phospholipids were separated by HPLC using a Beckman 344 gradient liquid chromatograph with an Ultrasil-Si column. The chromatographic system was programmed for gradient elution using the following mobile phases: solvent A, hexane/2-propanol (623,vol/vol); and solvent B, hexane/2-propanol/water (6:8:1.4,by vol). The percentage of solvent B in solvent A was increased in 15 min from 0 to 100%. The flow rate was 2 ml/min and detection was at 206 nm (Ruggiero et al., 1984b).Single phospholipid specieswere estimated quantitativelyby comparing the calcuIated peak areas with those of each phospholipid standard solution. Total cholesterol was analyzed with an Ultrasphere-ODS column as described by Ruggiero et al. (19846).First, synaptic and nonsynaptic mitochondria were saponified with alcoholicKOH and extracted with hexane. Then the extract was evaporated and the residue dissolved in 2-propanol, an aliquot of which was injected into the column. The mobile phase was 2-propanol/acetonitrile(5050,vol/vol) at a flow rate of 2 ml/min. Detection was at 200 nm. Fatty acids were extracted and analyzed by the method described by Ruggiero et al. (1984b). Briefly, synaptic and nonsynaptic mitochondria were saponified with KOH. Fatty acids were extracted with chloroform and esterified with m-methoxyphenacyl bromide. The column was an U1trasphere-ODS and the mobile phase was tetrahydrofuran/ by vol). acetonitrile/water (45:25:35,
489
ethanolamine (45.9%), phosphatidylcholine (27.9%), and phosphatidylserine (1 1.O%). In nonsynaptic mitochondria, the major phospholipids are phosphatidylcholine (39.8939, phosphatidylethanolamine (36.6%), and cardiolipin (15.5%). Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%) in nonsynaptic mitochondria from the brains of aged rats. A typical phospholipid separation in synaptic mitochondria, carried out by our HPLC method, is reported in Fig. 1. The order of elution in this system is neutral lipids (as a single peak), cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylNL
RESULTS The cholesterol and phospholipid contents in both types of brain mitochondria, reported in Table 1, are significantly different in synaptic compared to nonsynaptic mitochondria. The cholesterol content ranges from 179.7 nmol/mg of protein (synaptic mitochondria) to 12l .4 nmol/mg of protein (nonsynaptic mitochondria), whereas the phospholipid content ranges from 448.6 nmol of lipid Pi/mg of protein (synaptic mitochondria) to 335.3 nmol of lipid Pi/mg of protein (nonsynaptic mitochondria). In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%,respectively. The phospholipid composition in synaptic and nonsynaptic mitochondria from rat brains, shown in Table 2, is significantly different in synaptic compared to nonsynaptic mitochondria. In synapticmitochondria, the major phospholipids are phosphatidyl-
PC
I
0
4
0
12
16
20
24
Retention time (min)
FIG. 1. Chromatogram of phospholipidextract from synaptic mitochondria. NL,neutral lipids; DPG, cardiolipin; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine.The chromatographic system was programmed for gradient elution using the following mobile phases: solvent A, hexane/2-propanol (6:8, vol/vol); and solvent B, hexane/2-propanol/water (6:8:1.4, by vol). The percentageof solvent B in solvent A was increased in 15 min from 0 to 100%. Flow rate, 2 ml/min; detection, at 206 nm; chart speed, 30 crn/h; sensitivity, 0.5 AUFS.
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490
this study we found that the cholesterol content is very high in mitochondrial membranes from rat brains with respect to that found in mitochondria from heart and liver. Whereas the cholesterol content in synaptic mitochondria is high with respect to the corresponding value in nonsynaptic mitochondria, the cholesterol/phospholipidmolar ratio is almost the same in these two types of mitochondria. Previous works have reported that aging increases the cholesterol content in heart and liver mitochondria (Paradies and Ruuero, I990a, 1991). The cholesterol increase found in these membranes is probably caused by an altered cholesterol turnover and could be related to a cholesterol increase found in the plasma of aged rats (Ruggiero et al., 1990). In brain mitochondria we found a decrease in cholesterolcontent due to aging in both synaptic and nonsynaptic mitochondria. These results, which appear to conflict with previous results, may be explained by a different role of the cholesterol in these membranes. In fact, previous works have reported that cholesterol increases in the developing rat brain (Kishimoto et al., 1965; Bourre et al., 1990). A decrease in this compound during aging has been reported in gray and in white matter of the human brain (Siiderberg et al., 1990). SCrougneand Chevallier (1974) found that the hypothesis of a blood-brain barrier represented by the cerebral capillaries is not valid for cholesterol. Previous work has reported that an increase in the plasma cholesterol content of aged rats may lead to a preliminary condition of disease in the vascular system (Suzuki et al., 1985). We propose that an additional reason for the brain cholesterol decrease and the plasma cholesterol increase in aged rats (Ruggiero et al., 1990) can be attributed to a decrease in the exchange of cholesterol between the plasma and the brain. The age-dependent decrease in phospholipids in both types of brain mitochondria could be related to
choline. The gradient separation we used successfully separates the five phospholipid components in synaptic as well as in nonsynaptic mitochondria. On the other hand, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria (Table 3). The 2 1% aging decrease in linoleic acid ( I 8:2), observed exclusively in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid.
DISCUSSION The results reported here demonstrate that the lipid composition of synaptic mitochondria is very different from that of nonsynaptic mitochondria. These data are consistent with work clearly demonstrating that the activities of almost all the enzymes studied in synaptic mitochondria isolated from the three regions of the brain are significantly different from the corresponding values in nonsynaptic mitochondria isolated from the same regions (Leong et al., 1984). Cholesterol, phospholipids, and fatty acids are major factors influencing the physical properties and function of mitochondrial membranes. Cholesterol plays a key role because it appears to maintain the bilayer matrix in an intermediate fluid state by regulating the mobility of phospholipid fatty acyl chains (Demel and De Kruyff, 1976). An increase in cholesterol in relation to phospholipid has been shown to decrease fluidity in both biological and artificial membranes (Feo et al., 1975; Cooper et al., 1978). However, for erythrocyte membranes, which contain high levels of cholesterol, Lange et al. (1980) have reported a new role of cholesterol: determining the stability of membrane contour to prevent endocytosis. Brain mitochondria contain high levels of cholesterol (Demel and De b y & 1976); however, the role of this major component of lipids is still unknown. In
TABLE 3. Pattern of fatty acids in synaptic and nonsynaptic mitochondriafrom young, mature, and aged rats as determined by HPLC Distribution (mol%) Synaptic mitochondria
Nonsynaptic mitochondria
Fatty acid
Young
Mature
Aged
Young
Mature
Aged
16:O 18:O 18:l (n-9) 18:2 (n-6) 2 0 3 (n-6) 20:4 (n-6) 22:6 (n-3) UI 20:4f 18:2
28.1 f 1 . 1 17.0 f 0.8 18.1 f 1.0 14.2 f 0.8 0.6 & 0.2 21.5 f 1.5 0.5 ? 0.2 137.3 f 2.3 1.51 20.1
27.9 -t 1.2 16.8 f 1.0 18.4 f 1.1 14.0 ? 0.9 0.5 f 0.3 2 1 . 8 f 1.3 0.6 f 0.3 138.7 f 2.2 1.56 f 0.09
28.4 f 1.4 17.2 f 1.1 16.9 f 0.7 14.9 f 1.2 0.6 t 0.3 21.5 f 1.3 0.5 f 0.2 137.5 f 2.0 1.44 f 0.1
29.2 f 1.7 14.1 f 1.0 17.0 f 1.2 16.3 f 1.0 1.5 f 0.4 15.9 f 1.3" 6.0 f 0.5" 153.7 f 2.2" 0.97 0.09"
29.6 f 1.6 15.0 5 1.2 17.4 t 1.0 15.0 t 1 . 1 1.7 t 0.4 15.4 _t 1.0 5.9 t 0.6 149.5 t 2.3 1.03 -t 0.1
30.8 f 1.4 15.6 k 1.1 17.7 f 1.0 12.9 f l . l b 1.9 f 0.3 16.0k 1.5 5.1 f 0.6 143.8 f 2.0 1.24 f 0.09b
_+
Each value represents the mean f SD obtained for five experimentswith two rats from each group. The unsaturation index (UI) is defined as Zmol%of each fatty acid X number of double-bonds of the same fatty acid. ' p < 0.001 vs. synaptic mitochondria. p < 0.00 I vs. young rats.
'
J. Neuroehem., Vol. 59, No. 2. 1992
LIPIDS IN RAT BRAIN MITOCHONDRIA DURING AGING
the decrease observed in the total phospholipid content of the human brain by Soderberg et al. (1 990). Among phospholipids, only cardiolipin showed a significant decrease in nonsynaptic mitochondria due to aging. The function of cardiolipin in brain mitochondria is still unclear, but in liver this phospholipid, which is concentrated on the matrix side of the inner membrane (Krebs et al., 1979), has been shown to be necessary for optimal functioning of ATPase (Emster et al., 1977) and cytochrome oxidase (Vik et al., 1981). In conclusion, this study found that the lipid composition is different in synaptic and nonsynaptic mitochondria. The age-dependent decrease observed in the cholesterol content in both synaptic and nonsynaptic mitochondria could be related to an increase in plasma cholesterol (Suzuki et al., 1985; Ruggiero et al., 1990). Acknowledgment: This work was supported by the following funds: Minister0 Pubblica Istruzione (60%),Progetto Finalizzato Invecchiamento (CNR 92 1 106), and SigmaTau Company, Italy.
REFERENCES Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol 37, 91 1-917. Booth R. F. G. and Clark J. B. (1978) A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176,365-370. Bourre J. M., ClCment M., Gdrard D., Legrand R., and ChaudiGre J. ( 1990) Precursors for cholesterol synthesis (7-dehydrocholesterol, 7-dehydrodesmosterol,and desmosterol):cholesterol/7dehydrocholesterolratio as an index of development and aging in PNS but not in CNS. J. Neurochem. 54, 1196-1 199. Cooper R. A., Leslie M. F., Fischkoff S., Shinitzky M., and Sanford J. S. ( 1978)Factors influencingthe lipid composition and fluidity of red cell membranes in vitro: production of red cells possessing more than two cholesterolper phospholipids. Biochemistry 17, 327-33 1. Demel R. A. and De Kruyff B. (1 976) The function of sterols in membranes. Biochim. Biophys. Acta 457, 109-1 32. Enriquez J. A., SBnchez-PrietoJ., Muiiio Blanco M. T., Hernandez-Yago J., and Lbpez-PCrez M. J. (1990) Rat brain synaptosomes prepared by phase partition. J. Neurochem. 55, 18411849. Ernster L., Sandri G., Hundal T., Carlsson C., and Nerdenbrand K. (1977) Inhibitors as probes of mitochondrial adenosine triphosphatase, in Structure and Function of Energy- Transducing Membranes (Van Dam K. and Van Gelder, B. F., eds), pp. 209-222. Elsevier, Amsterdam. Feo F., Canuto R. A., Garcea R., and Gabriel L. (1975) Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver, fetal liver, cholesterol enriched liver and hepatomas AH-130,3924A and 5 123. Biochim. Biophys. Acta 413, 116-134. Fernandez-Silva P., Petruzzella V., Fracasso F., Gadaleta M. N., and Cantatore P. (199 1) Reduced synthesis of mtRNA in isolated mitochondria of senescent rat brain. Biochem. Biophys. Res. Commun. 176,645-653. Gadaleta M. N., Petruzzella V., Renis M., Fracasso F., and Cantatore P. ( 1990)Reduced transcription of mitochondrial DNA in the senescent rat. Tissue dependence and effect of acetyl-L-carnitine. Eur. J. Biochem. 187, 501-506. Kim J. H., Shrago E., and Elson C. E. (1988) Age-related changes in respiration coupled to phosphorylation. 11. Cardiac mitochondria. Mech. Ageing Dev. 46,219-290.
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Kishimoto Y., Davies W. E., and Radin N. S. ( 1 965) Developing rat brain: changes in cholesterol, galactolipids, and the individual fatty acids of gangliosides and glycerophosphatides. J. Lipid Res. 6, 532-536. Krebs J. J. R., Hanser H., and Carafoli E. (1979) Asymmetricdistribution of phospholipids in the inner membrane of beef heart mitochondria. J. Biol. Chem. 254,5308-5316. Lai J. C. K. and Clark J. B. (1976) Preparation and properties of mitochondria derived from synaptosomes. Biochem. J. 154, 423-432. Lange Y., Cutler H. B., and Steck T. L. (1980) The effect of cholesterol and other intercalated amphipaths on the contour and stability of the isolated red cell membrane. J. Biol. Chem. 255, 933 1-9337. Leong S. F., Lai J. C. K., Lim L., and Clark J. B. (1984) The activities of some energy-metabolising enzymes in nonsynaptic (free) and synaptic mitochondria derived from selected brain regions. J. Neurochem. 42, 1306- 1312. Lowry 0. H., Rosebrough N. J., Farr A. L., and Randall R. J. (1 95 1) Protein measurement with the Fohn phenol reagent. J. Biol. Chem. 193,265-275. Nakamura G. R. (1952) Microdetermination of phosphorus. Anal. Chem. 24, 1372. Noh1 H. and Warner R. ( I 980) Molecular basis of age-dependent changes in activity of adenine nucleotide translocase. Mech Ageing Dev. 14, 137-144. Olafson R. W., Drummond G. I., and Lee J. F. (1969) Studies on 2‘,3’-cyclicnucleotide-3’-phosphohydrolasefrom brain. Can J. Biochem. 47,961-966. Paradies G. and Ruggiero F. M. (1990~)Age-related changes in the activity of the pyruvate carrier and in the lipid composition in rat-heart mitochondria. Biochim. Biophys. Acta 1016, 207212. Paradies G. and Ruggiero F. M. (19906) Enhanced activity of the tricarboxylate camer and modification of lipids in hepatic mitochondria from hyperthyroid rats. Arch. Biochem. Biophys. 278,425-430. Paradies G. and Ruggiero F. M. (199 1) Effect of aging on the activity of the phosphate carrier and on the lipid composition in rat liver mitochondria. Arch. Biochem. Biophys. 284, 332-337. Ruggiero F. M., Landriscina C., Gnoni G. V., and Quagliariello E. ( 1984a)Alteration ofplasmaerythrocyte membrane lipid components in hyperthyroid rats. Horm. Metab. Res. 16, 37-40. Ruggiero F. M., Landriscina C., Gnoni G. V., and Quagliariello E. (1984b) Lipid composition of liver mitochondria and microsomes in hyperthyroid rats. Lipids 19, 171-178. Ruggiero F. M., Gnoni G. V., and Quagliariello E. (1987) Effect of hypothyroidism on the lipid composition of rat plasma and erythrocyte membranes. Lipids 22, 148- 151. Ruggiero F. M., Cafagna F., Gadaleta M. N., and Quagliariello E. ( 1990) Effect of aging and acetyl-L-carnitineon the lipid composition of rat plasma and erythrocytes. Biochem. Biophys. Res. Commun. 170,621-626. Skougne C. and Chevallier F. (1974) Microscopic radioautography of adult rat brain cholesterol. Problem of the blood-brain bamer. Exp. Neurol. 44, 1-9. SCrougne C., Lefevre C., and Chevallier F. (1976) Cholesterol transfer between brain and plasma in the rat: a model for the turnover of cerebral cholesterol. Exp. Neurol. 51, 229-240. Soderberg M., Edlund C., Kristensson K., and Dallner G. (1990) Lipid compositions of different regions of the human brain during aging. J. Neurochem. 54,415-423. Suzuki H., Kobayashi T., Hayakawa S., and Wada 0. (1985) Ageassociated changes in rat plasma lipids, platelet fatty acids and prostacyclin release. Biochim. Biophys. Acta 836, 394-396. Van Deenen L. L. M. (1965) Phospholipids and biomembranes, in Progress m Chemistry of Fats and Other Lipids, Vol. 8 (Holman R. T., ed), pp. 1-127. Pergamon Press, Oxford, England. Vik S. B., Georgevich G., and Capaldi R. A. (1981) Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 78, 14561460.
J. Neumhem.. Vol. 59, No. 2,1992
Lipid Composition in Synaptic and Nonsynaptic Mitochondria from Rat Brains and Effect of Aging F. M. Ruggiero, F. Cafagna, V. Petruzzella, M. N. Gadaleta, and E. Quagliariello Department of Biochemistry and Molecular Biology and CNR Unit for the Study of Mitochondria and Bioenergetics, University of Bari, Bari, Italy
Abstract: The cholesterol, phospholipid, and fatty acid compositions in synaptic and nonsynaptic mitochondria from rat brains and the effect of aging were studied. Both cholesterol and phospholipid contents were found to be significantly different in synaptic compared to nonsynaptic mitochondria. In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%, respectively. Also, the phospholipid composition is significantly different in synaptic compared to nonsynaptic mito-
chondria. Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%)in nonsynaptic mitochondria from the brains of aged rats. Instead, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria. The 2 1% aging decrease in linoleic acid (18:2), observed only in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid. Key Words: Rat brain-Synaptosomes-Mitochondria-Lipids-Aging. Ruggiero F. M. et al. Lipid composition in synaptic and nonsynaptic mitochondria from rat brains and effect of aging. J. Neurochem. 59,487-491 (1992).
Brain is very rich in neutral lipids and, particularly, in cholesterol. The cholesterol found in the brain comes from in situ synthesis and from transfer of plasma cholesterol (S6rougne et al., 1976). Previous works have demonstrated that cholesterolincreases in the developing rat brain (Kishimoto et al., 1965; Bourre et al., 1990). A decrease in this compound during aging has been reported in gray and in white matter of the human brain (Soderberg et al., 1990). As a structural component of the membrane, cholesterol is involved in maintaining the integrity of the cells and in regulating the fluidity of the cellular membrane lipids. Relatively low cholesterol concentrations were found in mitochondria from heart and liver (Paradies and Ruggiero, 1990a,b). However, in erythrocyte membranes, which contain high levels of cholesterol (Ruggiero et al., 1984a, 1987), a new role of cholesterol was reported: determining the stability of the membrane contour so as to prevent endocytosis (Lange et al., 1980).The role of this major component of lipids in brain mitochondria is still unknown. Phospholipids are asymmetrically arranged in biological membranes and a cholesterol/phospholipid ratio peculiar to particular membranes can be detected (Van Deenen, 1965).To our knowledge, no data have
been reported on the phospholipid composition in both synaptic and nonsynaptic mitochondria. Aging is a ubiquitous biological phenomenon associated with histological, biochemical, and functional alterations. Recently, Gadaleta et al. (1990) have shown that the steady-state concentration of mitochondrial RNA undergoes an age-dependent decrease in rat brain and heart. Changes in membrane lipid content and functions appear to occur with aging (Noh1 and Kramer, 1980; Kim et al., 1988). Agelinked changes in the lipid composition in plasma, heart, and liver mitochondria have been reported recently (Paradiesand Ruggiero, 1990~; Ruggiero et al., 1990; Paradies and Ruggiero, 1991). The present study was designed to investigate, for the first time, the lipid composition in both synaptic and nonsynaptic mitochondria, prepared by a highly purified procedure, and the influence of aging on their lipid compositions.
Male Fisher rats 4 months (young), 12 months (mature), and 28 months (aged) old, fed ad libitum with a standard
Received October 22, 1991; accepted January 15, 1992. Address correspondence and reprint requests to Dr. F. M. Rug-
giero at Dipartimento di Biochimica e Biologia Molecolare, Via G . Amendola 165/A, 70126 Ban, Italy.
MATERIALS AND METHODS Isolation of rat brain mitochondria
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TABLE 1. Cholesterol andphospholipid content in synaptic and nonsynaptic mitochondriafrom young, mature, and aged rats Synaptic mitochondria
Cholesterol Phospholipids Ratio cholesterol/phospholipids
Nonsynaptic mitochondria
Young
Mature
Aged
Young
Mature
Aged
179.7 f 10.3 448.6 f 19.7 0.40f 0.03
165.2 f 9.8 432.5 f 18.2 0.38f 0.04
130.9f 9.2" 392.3 f 15.4' 0.33 f 0.03'
121.4 f 10.5' 335.3 f 17.0' 0.36 ? 0.04'
114.0f 9.3 321.9f 16.9 0.35 f 0.03
88.0 f 8.3" 298.2 f 16.5' 0.29 f 0.02'
Each value represents the mean f SD obtained for five experiments with two rats from each group. Cholesterol is expressed as nmol/mg of protein, and phospholipids as nmol of lipid PJmg of protein. The p values were calculated by Student's f test. p < 0.00 1 vs. young rats. " p < 0.01 vs. young rats. 'p < 0.00 1 vs. synaptic mitochondria. taminated by synaptosomal and other membranous material as judged by the low recovery of acetylcholinesterase (0.5%of the homogenate activity). To obtain the synaptosomes, the first upper phase was carefully removed and layered on 6 ml of fresh biphase system, containingthe same components as the first one plus 2 g of medium B. The new phase system was mixed again and centrifuged, then the final upper phase was removed and carefully layered on top of 20 ml of medium A. Next the tubes were centrifuged at 8,000g for 8 min, with the synaptosomes being pelleted at the bottom. The purity of the synaptosomal fraction was checked both by electron microscopy and by determination of marker enzyme activities. Enzymes assayed were NADHJcytochrome c reductase (rotenone insensitive), as a marker for the external membrane and therefore for free mitochondrion contamination, and lactate dehydrogenase, as an indicator of synaptosomal membrane integrity (Lai and Clark, 1976; Booth et al., 1978).2',3'-Cyclic nucleotide-3'-phosphohydrolasewas analyzed according to the procedure of Olafson et al. (1 969). The extent of contamination of the synaptosomes by myelin was 0.5%. According to both structural and functional criteria, the purity of the synaptosomes was never below 80%. Synaptic mitochondria were prepared by suspending the synaptosomal pellet in 15 ml of 6 mMTris-HC1, pH 8.1, incubating the mixture on ice for 5 min, and homogenizing by hand. Sorbitol(1 M) was added up to a final concentration of 0.32 M, and the mixture centrifuged at 18,500 g for 10 min. The pellet, suspended in a small volume of medium
diet, were used throughout these studies. Free (nonsynaptic) and synaptic brain mitochondria were prepared by repeated partitioning in a two-phase system composed of dextranJ polyethylene glycol (Enriquez et al., 1990; Fernandez-Silva et al., 1991). The rats were beheaded, then the hemispheres were suspended in 15 ml of cold medium A (0.32 Msucrose, 10 mM Tris-HC1, pH 7.4, 1 mM potassium-EDTA) and homogenized in a Dounce-type glass homogenizer with 15 up-and-down strokes. The homogenate was centrifuged at 1,200 g for 6 min; then the supernatant was centrifuged at 12,000g for 10 min, producing a crude synaptosomal pellet. This pellet was resuspended in a small volume of medium B (0.32 M sorbitol, 5 mM potassium phosphate, pH 7.4, 0.1 mM potassium-EDTA). Two grams of a suspension of crude synaptosomes was added to 14 g of a biphase system prepared by mixing 5.12 g of 20% (wt/wt) dextran T-500, 2.56 g of 40% (wt/wt) polyethylene glycol 4000, 4.48 g of 1 M sorbitol, 0.36 g of 0.2 M potassium phosphate, pH 7.4, 0.2 g of 10 mM potassium-EDTA, and 1.24 g of distilled water. The phase system was mixed by 20 inversions of the tube and centrifuged at 1,200 g for 1 min, separating the mitochondria1 pellet in the two phases. The lower phase, containing free (nonsynaptic) mitochondria, was washed two times with 20 ml of medium B and centrifuged for 10 min at 8,000 g. Then the pellet was suspended in a small volume of medium C (10%glycerol, 35 mM Tris-HC1, pH 7.8, 20 mM NaCl, 5 mM MgCl,, 1 mg/ml bovine serum albumin). Electron microscopy showed that synaptosomes and myelin-like particles were virtually absent. The free mitochondria were minimallycon-
TABLE 2. Phospholipid composition in synaptic and nonsynaptic mitochondria from young, mature, and aged rats as determined by HPLC Distribution (mol%) Synaptic mitochondria
Nonsynaptic mitochondria
Phospholipid
Young
Mature
Aged
Young
Mature
Aged
DPG
8.7 f 0.8 45.9 f 1.8 6.5 f 0.5 11.0 f 1.0 27.9 f 1.2
8.9f 0.7 45.2f 1.5 6.9f 0.8 11.3 f 1.1 21.7f 1.3
8.5 f 0.9 45.0f 1.5 7.0 f 0.7 11.5 f 1.0 28.0 f 1.5
15.5 f 1.1" 36.6f 1.8" 3.8 f 0.4" 4.3 f 0.5" 39.8f 1.7"
14.0 f 1.0 37.0f 1.5 4.2 f 0.5 4.7 ? 0.6 40.1 f 1.8
1 1.4 f 0.9b 36.4 f 1.3 4.6 & 0.7 4.8 f 0.6 42.8 f 1.9
PE PI
PS PC
Each value represents the mean f SD obtained for five experiments with two rats from each group. DPG, cardiolipin; PE, phosphatidylethanolamine; PI, phosphatidylinositol;PS, phosphatidylserine;PC, phosphatidylcholine. " p < 0.001 vs. synaptic mitochondria. " p < 0.00 1 vs. young rats. J . Neurochem., Vol. 59, No. 2, 1992
LIPIDS IN RAT BRAIN MITOCHONDRIA DURING AGING B, was layered on top of a 14-gbiphase system. After mixing and centrifuging,the lower phase was diluted, washed twice with medium B, and finally, suspended in a small volume of incubation medium.
Extraction and separation of lipids Total lipids were extracted from synaptic and nonsynaptic mitochondria with chloroform/methanol by the Bligh and Dyer (1959)procedure. A portion of this extract was digested at 180°C with perchloric acid, then the phospholipid phosphorus content was determined by the Nakamura (1952)method. Phospholipids were separated by HPLC using a Beckman 344 gradient liquid chromatograph with an Ultrasil-Si column. The chromatographic system was programmed for gradient elution using the following mobile phases: solvent A, hexane/2-propanol (623,vol/vol); and solvent B, hexane/2-propanol/water (6:8:1.4,by vol). The percentage of solvent B in solvent A was increased in 15 min from 0 to 100%. The flow rate was 2 ml/min and detection was at 206 nm (Ruggiero et al., 1984b).Single phospholipid specieswere estimated quantitativelyby comparing the calcuIated peak areas with those of each phospholipid standard solution. Total cholesterol was analyzed with an Ultrasphere-ODS column as described by Ruggiero et al. (19846).First, synaptic and nonsynaptic mitochondria were saponified with alcoholicKOH and extracted with hexane. Then the extract was evaporated and the residue dissolved in 2-propanol, an aliquot of which was injected into the column. The mobile phase was 2-propanol/acetonitrile(5050,vol/vol) at a flow rate of 2 ml/min. Detection was at 200 nm. Fatty acids were extracted and analyzed by the method described by Ruggiero et al. (1984b). Briefly, synaptic and nonsynaptic mitochondria were saponified with KOH. Fatty acids were extracted with chloroform and esterified with m-methoxyphenacyl bromide. The column was an U1trasphere-ODS and the mobile phase was tetrahydrofuran/ by vol). acetonitrile/water (45:25:35,
489
ethanolamine (45.9%), phosphatidylcholine (27.9%), and phosphatidylserine (1 1.O%). In nonsynaptic mitochondria, the major phospholipids are phosphatidylcholine (39.8939, phosphatidylethanolamine (36.6%), and cardiolipin (15.5%). Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%) in nonsynaptic mitochondria from the brains of aged rats. A typical phospholipid separation in synaptic mitochondria, carried out by our HPLC method, is reported in Fig. 1. The order of elution in this system is neutral lipids (as a single peak), cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylNL
RESULTS The cholesterol and phospholipid contents in both types of brain mitochondria, reported in Table 1, are significantly different in synaptic compared to nonsynaptic mitochondria. The cholesterol content ranges from 179.7 nmol/mg of protein (synaptic mitochondria) to 12l .4 nmol/mg of protein (nonsynaptic mitochondria), whereas the phospholipid content ranges from 448.6 nmol of lipid Pi/mg of protein (synaptic mitochondria) to 335.3 nmol of lipid Pi/mg of protein (nonsynaptic mitochondria). In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%,respectively. The phospholipid composition in synaptic and nonsynaptic mitochondria from rat brains, shown in Table 2, is significantly different in synaptic compared to nonsynaptic mitochondria. In synapticmitochondria, the major phospholipids are phosphatidyl-
PC
I
0
4
0
12
16
20
24
Retention time (min)
FIG. 1. Chromatogram of phospholipidextract from synaptic mitochondria. NL,neutral lipids; DPG, cardiolipin; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine.The chromatographic system was programmed for gradient elution using the following mobile phases: solvent A, hexane/2-propanol (6:8, vol/vol); and solvent B, hexane/2-propanol/water (6:8:1.4, by vol). The percentageof solvent B in solvent A was increased in 15 min from 0 to 100%. Flow rate, 2 ml/min; detection, at 206 nm; chart speed, 30 crn/h; sensitivity, 0.5 AUFS.
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F. M. RUGGIERO ET AL.
490
this study we found that the cholesterol content is very high in mitochondrial membranes from rat brains with respect to that found in mitochondria from heart and liver. Whereas the cholesterol content in synaptic mitochondria is high with respect to the corresponding value in nonsynaptic mitochondria, the cholesterol/phospholipidmolar ratio is almost the same in these two types of mitochondria. Previous works have reported that aging increases the cholesterol content in heart and liver mitochondria (Paradies and Ruuero, I990a, 1991). The cholesterol increase found in these membranes is probably caused by an altered cholesterol turnover and could be related to a cholesterol increase found in the plasma of aged rats (Ruggiero et al., 1990). In brain mitochondria we found a decrease in cholesterolcontent due to aging in both synaptic and nonsynaptic mitochondria. These results, which appear to conflict with previous results, may be explained by a different role of the cholesterol in these membranes. In fact, previous works have reported that cholesterol increases in the developing rat brain (Kishimoto et al., 1965; Bourre et al., 1990). A decrease in this compound during aging has been reported in gray and in white matter of the human brain (Siiderberg et al., 1990). SCrougneand Chevallier (1974) found that the hypothesis of a blood-brain barrier represented by the cerebral capillaries is not valid for cholesterol. Previous work has reported that an increase in the plasma cholesterol content of aged rats may lead to a preliminary condition of disease in the vascular system (Suzuki et al., 1985). We propose that an additional reason for the brain cholesterol decrease and the plasma cholesterol increase in aged rats (Ruggiero et al., 1990) can be attributed to a decrease in the exchange of cholesterol between the plasma and the brain. The age-dependent decrease in phospholipids in both types of brain mitochondria could be related to
choline. The gradient separation we used successfully separates the five phospholipid components in synaptic as well as in nonsynaptic mitochondria. On the other hand, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria (Table 3). The 2 1% aging decrease in linoleic acid ( I 8:2), observed exclusively in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid.
DISCUSSION The results reported here demonstrate that the lipid composition of synaptic mitochondria is very different from that of nonsynaptic mitochondria. These data are consistent with work clearly demonstrating that the activities of almost all the enzymes studied in synaptic mitochondria isolated from the three regions of the brain are significantly different from the corresponding values in nonsynaptic mitochondria isolated from the same regions (Leong et al., 1984). Cholesterol, phospholipids, and fatty acids are major factors influencing the physical properties and function of mitochondrial membranes. Cholesterol plays a key role because it appears to maintain the bilayer matrix in an intermediate fluid state by regulating the mobility of phospholipid fatty acyl chains (Demel and De Kruyff, 1976). An increase in cholesterol in relation to phospholipid has been shown to decrease fluidity in both biological and artificial membranes (Feo et al., 1975; Cooper et al., 1978). However, for erythrocyte membranes, which contain high levels of cholesterol, Lange et al. (1980) have reported a new role of cholesterol: determining the stability of membrane contour to prevent endocytosis. Brain mitochondria contain high levels of cholesterol (Demel and De b y & 1976); however, the role of this major component of lipids is still unknown. In
TABLE 3. Pattern of fatty acids in synaptic and nonsynaptic mitochondriafrom young, mature, and aged rats as determined by HPLC Distribution (mol%) Synaptic mitochondria
Nonsynaptic mitochondria
Fatty acid
Young
Mature
Aged
Young
Mature
Aged
16:O 18:O 18:l (n-9) 18:2 (n-6) 2 0 3 (n-6) 20:4 (n-6) 22:6 (n-3) UI 20:4f 18:2
28.1 f 1 . 1 17.0 f 0.8 18.1 f 1.0 14.2 f 0.8 0.6 & 0.2 21.5 f 1.5 0.5 ? 0.2 137.3 f 2.3 1.51 20.1
27.9 -t 1.2 16.8 f 1.0 18.4 f 1.1 14.0 ? 0.9 0.5 f 0.3 2 1 . 8 f 1.3 0.6 f 0.3 138.7 f 2.2 1.56 f 0.09
28.4 f 1.4 17.2 f 1.1 16.9 f 0.7 14.9 f 1.2 0.6 t 0.3 21.5 f 1.3 0.5 f 0.2 137.5 f 2.0 1.44 f 0.1
29.2 f 1.7 14.1 f 1.0 17.0 f 1.2 16.3 f 1.0 1.5 f 0.4 15.9 f 1.3" 6.0 f 0.5" 153.7 f 2.2" 0.97 0.09"
29.6 f 1.6 15.0 5 1.2 17.4 t 1.0 15.0 t 1 . 1 1.7 t 0.4 15.4 _t 1.0 5.9 t 0.6 149.5 t 2.3 1.03 -t 0.1
30.8 f 1.4 15.6 k 1.1 17.7 f 1.0 12.9 f l . l b 1.9 f 0.3 16.0k 1.5 5.1 f 0.6 143.8 f 2.0 1.24 f 0.09b
_+
Each value represents the mean f SD obtained for five experimentswith two rats from each group. The unsaturation index (UI) is defined as Zmol%of each fatty acid X number of double-bonds of the same fatty acid. ' p < 0.001 vs. synaptic mitochondria. p < 0.00 I vs. young rats.
'
J. Neuroehem., Vol. 59, No. 2. 1992
LIPIDS IN RAT BRAIN MITOCHONDRIA DURING AGING
the decrease observed in the total phospholipid content of the human brain by Soderberg et al. (1 990). Among phospholipids, only cardiolipin showed a significant decrease in nonsynaptic mitochondria due to aging. The function of cardiolipin in brain mitochondria is still unclear, but in liver this phospholipid, which is concentrated on the matrix side of the inner membrane (Krebs et al., 1979), has been shown to be necessary for optimal functioning of ATPase (Emster et al., 1977) and cytochrome oxidase (Vik et al., 1981). In conclusion, this study found that the lipid composition is different in synaptic and nonsynaptic mitochondria. The age-dependent decrease observed in the cholesterol content in both synaptic and nonsynaptic mitochondria could be related to an increase in plasma cholesterol (Suzuki et al., 1985; Ruggiero et al., 1990). Acknowledgment: This work was supported by the following funds: Minister0 Pubblica Istruzione (60%),Progetto Finalizzato Invecchiamento (CNR 92 1 106), and SigmaTau Company, Italy.
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Kishimoto Y., Davies W. E., and Radin N. S. ( 1 965) Developing rat brain: changes in cholesterol, galactolipids, and the individual fatty acids of gangliosides and glycerophosphatides. J. Lipid Res. 6, 532-536. Krebs J. J. R., Hanser H., and Carafoli E. (1979) Asymmetricdistribution of phospholipids in the inner membrane of beef heart mitochondria. J. Biol. Chem. 254,5308-5316. Lai J. C. K. and Clark J. B. (1976) Preparation and properties of mitochondria derived from synaptosomes. Biochem. J. 154, 423-432. Lange Y., Cutler H. B., and Steck T. L. (1980) The effect of cholesterol and other intercalated amphipaths on the contour and stability of the isolated red cell membrane. J. Biol. Chem. 255, 933 1-9337. Leong S. F., Lai J. C. K., Lim L., and Clark J. B. (1984) The activities of some energy-metabolising enzymes in nonsynaptic (free) and synaptic mitochondria derived from selected brain regions. J. Neurochem. 42, 1306- 1312. Lowry 0. H., Rosebrough N. J., Farr A. L., and Randall R. J. (1 95 1) Protein measurement with the Fohn phenol reagent. J. Biol. Chem. 193,265-275. Nakamura G. R. (1952) Microdetermination of phosphorus. Anal. Chem. 24, 1372. Noh1 H. and Warner R. ( I 980) Molecular basis of age-dependent changes in activity of adenine nucleotide translocase. Mech Ageing Dev. 14, 137-144. Olafson R. W., Drummond G. I., and Lee J. F. (1969) Studies on 2‘,3’-cyclicnucleotide-3’-phosphohydrolasefrom brain. Can J. Biochem. 47,961-966. Paradies G. and Ruggiero F. M. (1990~)Age-related changes in the activity of the pyruvate carrier and in the lipid composition in rat-heart mitochondria. Biochim. Biophys. Acta 1016, 207212. Paradies G. and Ruggiero F. M. (19906) Enhanced activity of the tricarboxylate camer and modification of lipids in hepatic mitochondria from hyperthyroid rats. Arch. Biochem. Biophys. 278,425-430. Paradies G. and Ruggiero F. M. (199 1) Effect of aging on the activity of the phosphate carrier and on the lipid composition in rat liver mitochondria. Arch. Biochem. Biophys. 284, 332-337. Ruggiero F. M., Landriscina C., Gnoni G. V., and Quagliariello E. ( 1984a)Alteration ofplasmaerythrocyte membrane lipid components in hyperthyroid rats. Horm. Metab. Res. 16, 37-40. Ruggiero F. M., Landriscina C., Gnoni G. V., and Quagliariello E. (1984b) Lipid composition of liver mitochondria and microsomes in hyperthyroid rats. Lipids 19, 171-178. Ruggiero F. M., Gnoni G. V., and Quagliariello E. (1987) Effect of hypothyroidism on the lipid composition of rat plasma and erythrocyte membranes. Lipids 22, 148- 151. Ruggiero F. M., Cafagna F., Gadaleta M. N., and Quagliariello E. ( 1990) Effect of aging and acetyl-L-carnitineon the lipid composition of rat plasma and erythrocytes. Biochem. Biophys. Res. Commun. 170,621-626. Skougne C. and Chevallier F. (1974) Microscopic radioautography of adult rat brain cholesterol. Problem of the blood-brain bamer. Exp. Neurol. 44, 1-9. SCrougne C., Lefevre C., and Chevallier F. (1976) Cholesterol transfer between brain and plasma in the rat: a model for the turnover of cerebral cholesterol. Exp. Neurol. 51, 229-240. Soderberg M., Edlund C., Kristensson K., and Dallner G. (1990) Lipid compositions of different regions of the human brain during aging. J. Neurochem. 54,415-423. Suzuki H., Kobayashi T., Hayakawa S., and Wada 0. (1985) Ageassociated changes in rat plasma lipids, platelet fatty acids and prostacyclin release. Biochim. Biophys. Acta 836, 394-396. Van Deenen L. L. M. (1965) Phospholipids and biomembranes, in Progress m Chemistry of Fats and Other Lipids, Vol. 8 (Holman R. T., ed), pp. 1-127. Pergamon Press, Oxford, England. Vik S. B., Georgevich G., and Capaldi R. A. (1981) Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 78, 14561460.
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