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Age related changes in the lipids of the microsomal and the mitochondrial membranes of rat liver and kidney
Mechanisms of Ageing and Development, 6 (1977) 197-205 ©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
197
A G E R E L A T E D C H A N G E S IN T H E LIPIDS O F T H E M I C R O S O M A L A N D T H E M I T O C H O N D R I A L M E M B R A N E S O F R A T L I V E R AND K I D N E Y
LYNN S. GRINNA Department of Biology, University of California, Los Angeles, California 90024 (U.S.A.) (Received August 15, 1976; in revisedform November8, 1976)
SUMMARY The lipid contents of the microsomal and mitochondrial membrane fractions of liver and kidney were determined in 6 and 24 month old rats. A significant age related increase in the molar ratio of cholesterol/phospholipid was observed in all membrane fractions. A significant age related reduction of phospholipid was noted in the microsomal fractions of liver and kidney. The relative amount of phosphatidylethanolamine was found to decrease in all membrane fractions during aging. Membrane glyceride content, however, remained relatively constant with age. Significant increase in oleic acid was seen in the neutral lipid of both liver and kidney and in the polar lipid of kidney. Significant increase in docosahexaenoic acid and significant decrease in linoleic acid were seen in the polar lipid of the liver membrane fractions. Possible alterations in membrane physiochemical properties and in membrane function due to these age related lipid changes are discussed.
INTRODUCTION The physical and functional characteristics of biological membranes are influenced by the lipid components of the membranes. Membrane lipids, to a large extent, determine the properties of membrane fluidity, hydrophobicity and permeability [ 1--4]. Additionally, lipids are thought to contribute to the chemical and functional asymmetry of membranes [5, 6]. Membrane lipid has been shown to be required for the activity of many membrane bound enzymes and has been found to control, influence or modify enzyme thermal and kinetic behavior [7-9]. Changes in membrane lipids have been noted in several pathological conditions and also during aging [10-25]. Although several of these studies have been extensive in their analyses, none have provided detailed data on membranes where extensive functional change exists. Correlation between chemical and functional change, therefore, requires that such detailed data be collected on the lipids of both young and old animal membrane fractions which are known to be functionally different.
198 Extensive age related changes occur in the activities of microsomal and mitochondrial enzymes of rat liver and kidney [ 11, 18, 26-28]. Results obtained in Arrhenius studies of membrane bound enzymes indicate that the phase behavior of membranes changes with age [28]. Kinetic analysis and thermal inactivation studies have indicated that the interactions of enzyme protein with membrane lipid may be altered with age and that the incorporation of certain proteins into the membrane may be reduced during aging [26, 27]. Collectively the available information gives support to the suggestion that the age related functional changes in membranes may be related to lipid changes and to resultant alterations in the interactions of membrane lipid and protein components. In the present study the detailed lipid composition of the liver and kidney microsomal and mitochondrial membranes was determined. These tissues and membrane fractions were selected because they are not only functionally and chemically different from one another but are also functionally altered during aging. Also, it was of interest to compare the lipid changes of these fractions with those reported for other tissues and species during aging.
METHODSAND MATERIALS Male Sprague-Dawley rats maintained on Purina chow and water ad libitum were used in all experiments. The animals were killed by stunning, exsanguination and decapitation. For each experiment the tissues of 2-3 animals were pooled prior to homogenization. Five separate experiments were performed on each age group. The livers and kidneys were homogenized by hand, using a Potter-Elvehjem type homogenizer, and were diluted 1:10 with 0.25 M sucrose. Whole cells, nuclei and debris were removed at 1000 × g for 10 min. Mitochondrial fractions were obtained by centrifugation of the 1000 × g supernatant for 15 min at 12,000 × g. The crude mitochondrial fractions were resuspended to the original volume with sucrose and centrifugations at 1000 × g and 12,000 × g were repeated. The fluffy material overlaying the mitochondrial pellet was discarded along with the supematant. The microsomal fractions were obtained by centrifugation of the first 12,000 × g supernatant for 90 min at 105,000 × g. The microsomal fractions were resuspended to original volume in sucrose and centrifugations at 12,000 × g and 105,000 × g were repeated. Fraction purity was determined as previously described [11 ]. All pellets were stored at - 2 0 °C. Lipid extractions were performed according to procedures described by Folch et al. [29] and by Bligh and Dyer [30]. The lipid extracts were separated into neutral and polar fractions using columns of 1 g silicic acid (Clarkson Chemical Co., Inc.) as described by Colbeau et al. [31]. All samples were concentrated under a stream of nitrogen and analysed immediately. Phospholipid was determined by the method of Raheja et al. [32] using phosphatidylcholine (Sigma Chemical Co.) as standard. Cholesterol was analyzed by the method of Zlatkis and Zak [33]. Glyceride was determined by the enzymatic method of Garland and Randall [34] following alkaline hydrolysis of the samples as
199 described by Chernick [35]. Tripalmitin (Sigma Chemical Co.) was used as standard for glyceride determination. Methyl esters of fatty acids were prepared using 14% boron trifluoride-methanol (Applied Science Laboratories, Inc.). The prepared methyl esters were analyzed on a Perkin-Elmer 990 gas chromatograph equipped with 15% EGSS-X column, flame ionization detector and Infotronics digital integrator. The column was temperature programmed from 170-200 °C at 4 °C/rain following 4 min at 170 °C. Fatty acid standards (Applied Science Laboratories, Inc.) were used to identify the major fatty acids. Polar lipids were separated by two-dimensional thin-layer chromatography using precoated silica gel G plates (Analtech, Inc.). Chloroform/methanol/water~28% ammonia (130:70:8:0.5) was used as first dimension solvent and chloroform/acetone/methanol/ acetic acid/water (100:20:10:10:2) was used as second dimension solvent. Spots were detected with iodine vapor and/or 2,7-dichlorofluorescein (Applied Science Laboratories, Inc.) and were eluted and analyzed as described by Raheja et al. [32].
RESULTS The content o f phospholipid in the microsomal membranes of liver and kidney was found to be higher than the phospholipid content of the mitochondrial membranes of these tissues (Table I). During aging, the phospholipid contents of the microsomal membranes of both liver and kidney decreased approximately 18-20%. The phospholipid contents of the mitochondrial membranes of both liver and kidney were found to be constant during aging. TABLE I PHOSPHOLIPID, CHOLESTEROL AND GLYCERIDE CONTENTS OF LIVER AND KIDNEY MICROSOMES AND MITOCHONDRIA FROM 6 AND 24 MONTH OLD RATS
Membrane and Tissue
Age /months)
Phospholipid-P Cholesterol protein protein (nmol/mg) (nmol/mg)
Cholesterol Glyceride phospholipid-P protein (M} /nmol/mg)
6 24 6 24
443 366 461 403
0.212 -+0.009 0.301 ± 0.017t 0.516 ± 0.010 0.590 ± 0.031t
32.2 29.9 20.6 17.1
6 24 6 24
157 ± 20 152 ± 15 212 ± 16 232 ± 20
0.083 0.117 0.157 0.243
10.1 ± 1.8 8.1 ± 1.3 10.3 ± 1.8 13.0 ± 0.5
Microsomes
Liver Kidney
± 28* ± 17T ± 10 ± 19t
93.7 110.2 238.4 237.6
± 3.3 t 5.9 ± 5.3 ± 8.7
± 3.1 ± 2.0 -+ 1.3 -+0.7
Mitochondria Liver Kidney
*Values are the average o f 5 determinations + SD.
t p < 0.05 24 months vs. 6 months.
13.0 ± 0.2 17.8 ± 0.4t 33.3 ± 3.0 56.4 ± 4.9t
± 0.005 -+0.009t ± 0.007 ± 0.003t
200 The cholesterol content o f microsomal membranes is 6 - 7 times higher than the cholesterol content o f mitochondrial membranes (Table I). Also, the membranes of kidney contain 2-3 times more cholesterol than membranes o f liver. During aging the cholesterol content o f the microsomal membranes remains constant. In contrast, the cholesterol contents of the mitocfiondrial membranes o f liver and kidney increase 35 and 70% respectively during aging. The molar ratio of cholesterol/phospholipid is of considerable importance to the physiochemical properties o f membranes. Expressed as the molar ratio o f cholesterol/ phospholipid, the relative amount o f cholesterol increased significantly in all membrane fractions during aging (Table I). In the roicrosomal fractions the increase in the ratio was due to decrease in membrane phospholipid, whereas in the mitochondrial fractions the increase in the ratio was due to the increase in the content o f membrane cholesterol. The glyceride content of the membranes, which includes mono-, di-, and triglycerides, did not change with age (Table I). The glyceride content of the microsomal membranes was higher than that of the mitochondrial membranes. The glyceride content o f the mitochondrial membranes o f liver and kidney were similar; however, the glyceride content of the microsomal membranes of liver was approximately 50% higher than that o f kidney. The content o f individual phospholipid species of the membranes is reported in Table II. In agreement with others, phosphatidylcholine was the most abundant phospholipid of microsomal membranes and also o f liver mitochondrial membranes [31, 36, 37]. In kidney mitochondrial membranes the contents of phosphatidylcholine and TABLE II PHOSPHOLIPID CLASS COMPOSITION OF LIVER AND KIDNEY MICROSOMES AND MITOCHONDRIA FROM 6 AND 24 MONTH OLD RATS
Membrane and Tissue
Age (months)
PC (%)t
PE (%)
C [%)
6 24 6 24
62.5 65.7 44.2 44.9
± 5.4** ± 4.3 ± 2.3 ± 3.2
26.9 22.6 31.6 30.6
± 3.4 ± 2.3 ± 3.1 ± 3.8
6 24 6 24
45.7 48.5 37.7 43.0
± 2.5 ± 2.0 +- 2.6 ± 3.0
33.6 30.2 39.9 37.7
± 1.7 ± 1.4 ± 1.7 ± 1.8
Other* (%)
Microsomes Liver Kidney
Mitochondria Liver Kidney
10.6 11.7 24.2 24.5
16.9 13.9 16.8 13.5
PC = Phosphatidylcholine; PE = phosphatidylethanolamine; C = cardiolipin. Phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatides. Percentage of total phospholipid. **Average of 3-4 determinations ± SD.
~
± 3.6 ± 2.0 ± 3.0 ± 1.2
3.8 7.4 5.6 5.8
201 phosphatidylethanolamine were similar. Cardiolipin was a major component of mitochondrial membranes but was absent in microsomal membranes. Phosphatidylserine, phosphatidylinositol, sphingomyelin and lysophosphatides were collectively measured and were found to contribute only a small percentage to the total lipid of the liver microsomes but a larger percentage in the kidney microsomes. These lipids contribute in only a minor way to the total phospholipid of the mitochondrial membranes. The relative amount of phosphatidylethanolamine appeared to decrease slightly with age. The decrease, which was approximately 10% in liver and 5% in kidney, was not statistically significant, but was observed in all analyses performed. In mitochondrial fractions of liver and kidney, cardiolipin decreased approximately 20% between 6 and 24 months of age. These changes in the relative amounts of the individual phospholipids, although small, occurred both in the presence of (microsomes) and in the absence of (mitochondria) age related changes in the amount of phospholipid in the membranes. Previous studies have indicated that the fatty acid compositions of certain membranes change with age [12, 16-18]. The fatty acid compositions of the isolated neutral and polar lipid fractions of the membranes were therefore determined. As seen in Table III, there were significant changes in the fatty acids of the polar lipid of the membranes of liver and kidney. In the liver there was significant decrease in linoleic acid (18:2) and increase in docosahexaenoic acid (22:6) with age. By contrast, in the polar lipid fraction of kidney membranes there was significant increase in oleic acid (18:1) with age. Minor changes in other fatty acids were also .noted. Neutral lipid was found to have a fatty acid composition which was different from that seen in the polar lipid fractions of the same tissue and age related changes in fatty TABLE III FATTY ACIDS OF POLAR LIPID FROM LIVER AND KIDNEY MICROSOMES AND MITOCHONDRIAFROM 6 AND 24 MONTHOLD RATS Tissue Age and Membrane (months)
16:0 (%) ~
18:0 (%)
18:1 (%)
18:2 (%)
20:4 (%)
22:6 (%)
6 24 6 24
27.6t 24.7 22.0 24.5
24.7 23.9 17.1 18.6
10.3 10.1 10.3 10.4
15.5 ± 1.2"* 10.3 ± 0.5* 21.2 ± 2.0 17.0 ± 0.7 ¢
20.6 20.3 20.0 17.4
2.0±1.0"* 8.4±1.1' 5.1±1.1 8.9±0.4*
6 24 6 24
26.9 27.0 23.1 23.5
20.3 17.4 19.4 17.4
8.0±1.4"* 12.1±1.7' 8.7±0.3 11.0±1.0'
10.3 9.4 16.6 14A
28.1 24.6 28.1 26.8
1.8 1.6 1.5 1.7
Liver Microsomes Mitochondria Kidney Microsomes Mitochondria
*Area percentage. tAverage of 3-5 determinations, SD given only where P < 0.05. **Average of 3-5 determinations ± SD. *P < 0.05 24 months vs. 6 months.
202 TABLE IV FATTY ACIDS OF NEUTRAL LIPID FROM LIVER AND KIDNEY MICROSOMES AND MITOCHONDRIA FROM 6 AND 24 MONTH OLD RATS Tissue and Membrane
Age (months)
16:0 + 16:1 (%) *
18:0 (%)
18:1 (%)
6 24 6 24
35.5t 34.4 40.9 40.9
6.3 4.7 6.1 5.7
28.5 34.9 25.0 29.7
6 24 6 24
32.4 28.0 31.1 26.8
12.8 11.8 16.2 13.5
18:2 (%)
20:4 (%)
23.7 22.2 20.3 17.2
3.1 2.6 2.3 2.1
Liver Microsomes Mitochondria
+- 1.7"* +- 1.5 :~ +- 0.7 +- 0.9*
Kidney Microsomes Mitochondria
22.3 -+ 2.1 28.3 -+0.3* 18.8 -+0.9 23.8 +- 2.0*
20.9 21.8 20.4 17.6
7.4 8.4 11.2 -+ 2.1 18.2 +-2.4*
*Area percentage. tAverage of 3-5 determination, SD given only where P < 0.05. **Average of 3-5 determinations -+SD. *P < 0.05 24 months vs. 6 months. acids were also noted in the neutral fractions (Table IV). In the neutral lipid of both liver and kidney membranes the content of oleic acid (18:1) increased significantly with age. In the kidney mitochondrial fractions there was, additionally, an increase in the content of arachadonic acid (20:4) with age. This fatty acid was found to occur at much higher levels in kidney neutral and polar lipid than in similar fractions from liver.
DISCUSSION Age related lipid changes were noted in the microsomal and mitochondrial fractions of rat liver and kidney. These lipid changes are of general interest as they support the premise that membrane lipid change may be a general characteristic associated with aging [11, 12, 2 6 - 2 8 ] . The changes are of specific interest as they may relate to previously reported age related alteration in enzyme function in the microsomal and mitochondrial fractions of liver and kidney [ 11,18, 2 6 - 2 8 ] . The lipid values of the microsomal and mitochondrial fractions of the 6 month old animals are in agreement with published values [31,36, 37]. The values of the 24 month old animals indicate a significant increase in the molar ratio of cholesterol/phospholipid in all membrane fractions, significant changes in the fatty acid composition of all membrane fractions, and a significant decrease in the phospholipid of the microsomal membrane fractions. Although not reported here, the lipid contents of membrane fractions obtained from animals between 6 and 24 months of age were consistant with the results presented.
203 Of the lipid changes noted during aging, increase in the molar ratio of cholesterol• phospholipid is considered the most important since the increase was observed in all membrane fractions examined and since it is known that the ratio has effects on membrane physiochemical properties. When the cholesterol content is increased the thermal motion of the hydrocarbon portion of membranes is reduced [4, 38, 39]. This results in a reduction of the fluidity of membranes and reduction in component intermixing and mobility [4, 38, 39]. Increase in cholesterol also has been found to decrease the permeability of model membranes and to decrease the expansion of phospholipid monolayers thereby hindering the insertion of new compounds into the monolayers [38, 40]. Increase in membrane cholesterol also alters and depresses the activities of certain membrane bound enzymes [38, 39]. Cholesterol has been reported to increase with age in rat liver and skeletal muscle [13, 25], in mouse brain [16] and in human red blood cells [19, 20]. Increase in membrane cholesterol, and the physiochemical alterations such an increase would produce, have been suggested as the initiation factors in the development of atherosclerosis [38, 41]. Age related reduction in the phospholipid content of the microsomal membranes of the liver and kidney is considered important as it may result in alteration in enzyme activities, reduction of the bulk lipid and change in permeability properties of the membranes [7, 42]. In vitro the reduction of membrane phospholipid produces dramatic changes (usually reduction) in membrane bound enzyme activities [7, 42]. Decrease in phospholipid content with age has been reported in rat liver and pancreas [21] and in mouse liver microsomes [ 12]. Of less obvious consequence are the changes seen in the fatty acid profiles and the individual phospholipid species of the membranes during aging. In the presentcstudy the fatty acid composition was found to change slightly towards a more unsaturated system during aging. The importance of the specific fatty acid changes is unknown, although it is known that unsaturated fatty acids contribute to membrane fluidity [4, 39]. The changes observed might represent a compensatory response to the (proposed) decrease in membrane fluidity. It is of interest that in all cases the changes seen in the fatty acid composition were in those fatty acids which predominantly (90%) occupy position 2 of the phospholipids [43]. The changes in the fatty acids of the polar lipid appeared to be organ specific whereas the changes in the neutral lipid were specific for oleic acid. The phospholipid composition of the membranes changed slightly with age such that phosphatidylethanolamine was reduced in all membrane fractions. The change occurred independently of the extent of change in the total amount of phospholipid in the membranes. The role that the phosphoryl group may play in the maintenance of biological membrane fluidity and asymmetry are currently matters of controversy and speculation [4, 6, 39,44]. If the distribution of phospholipids within cellular membranes is proven to be asymmetric then the age related changes observed in the phospholipid class composition of the membranes would have considerable functional significance. Similar decrease in membrane phosphatidylethanolamine has been reported for rat liver plasma membrane [23], rat skeletal muscle sarcoplasmic reticulum [22] and human aorta
204 [24]. However, phosphatidylethanolamine has been reported to increase with age in mouse liver microsomes [12] and in human red blood cells [19]. The results presented suggest that during aging, changes in microsomal and mitochondrial lipid produce membranes which are less fluid and asymmetrically altered. It is also suggested that this change is a general one during aging and is causally related to the functional decline which is known to occur in these membranes during aging.
ACKNOWLEDGEMENTS The author wishes to thank Dr. Albert A. Barber for his continued interest and support throughout this project. Supported by USPHS grant AG 00477-01 from the National Institute of Child Health and Human Development, Aging Branch.
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205 19 M. Gold and H. Altschuler, Red blood ceils and plasma phospholipids in aging humans, J. GerontoL, 27 (1972)444-450. 20 S. Malhotra and D. Kritchevsky, Cholesterol exchange between the red blood cells and plasma of young and old rats, Mech. Ageing Develop., 4 (1975) 137-145. 21 K. G. Prasannan, Influence of age on the total lipid, phospholipid and cholesterol contents of pancreas and liver in albino rats, Experientia, 29 (1972) 946-947. 22 H. A. Bertrand, B. P. Yu and E. J. Masoro, The effect of rat age on the composition and functional activities of skeletal muscle sarcoplasmic reticulum membrane preparations, Mech. Ageing Develop., 4 (1975) 7-17. 23 M. S. Rubin, N. I. Swislocki and M. Sonenberg, Changes in rat liver plasma membrane phospholipids during aging, Proc. Soc. Exp. BioL Med., 142 (1973) 1008-1010. 24 G. Rouser and R. D. Solomon, Changes in phospholipid composition of human aorta with age, Lipids, 4 (1969) 232-234. 25 J. A. Story, S. A. Tepper and D. Kritchevsky, Lipid metabolism in aging rats, Adv. Exp. BioL Med., 61 (1975) 281. 26 L. S. Grinna and A. A. Barber, Kinetic analysis of the age related differences in glucose-6 phosphatase, Exp. GerontoL, 10 (1975) 319-323. 27 L. S. Grinna and A. A. Barber, Age related thermolability of liver and kidney glucose-6.phosphatase, Exp. Gerontol., 10 (1975) 239-240. 28 L. S. Grinna~ Age related alteration in membrane lipid and protein interactions: Arrhenius studies of microsomal glucose-6-phosphatase, Gerontology, in press. 29 J. Folch, M. Lees and G. H. Sloane-Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497-509. 30 E. G. Bligh and W. J. Dyer, A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol., 37 (1959) 911-917. 31 A. Colbeau, J. Nachbaur and P. M. Vignais, Enzymatic characterization and lipid composition of rat liver subcellular membranes, Biochim. Biophys. Acta, 249 (1971) 462--492. 32 R. K. Raheja, C. Kaur, A. Singh and I. S. Bhatia, New colorimetric method for the quantitative estimation of phospholipids without acid digestion, J. Lipid Res., 14 (1973) 695-697. 33 A. Zlatkis and B. Zak, Study of a new cholesterol reagent, Anal Biochem., 29 (1969) 143-138. 34 P. B. Garland and P. J. Randle, A rapid enzymatic assay for glycerol, Nature, 196 (1962) 987-988. 35 S. S. Chernick, Determination of glycerol in acyl glycerols, in J. M. Lowensteio.(ed.), Methods in Enzymology, Vol. 14, Academic Press, New York, 1969, pp. 627-630. 36 L. D. Bergelson, E. V. Dyatlovitskaya, I. B. Sorokina and N. P. Gorkova, Phospholipid composition of mitochondria and microsomes from regenerating rat liver and hepatomas of different growth rate, Biochim. Biophys. Acta, 360 (1974) 361-365. 37 D. S. White, The phospholipid composition of mammalian tissues, in G. B. Ansell, J. N. Hawthorne and R. M. Dawson (eds), Form and Function o f Phospholipids. Bioehim. Biophys. Acta Library, Vol. 3, Elsevier, Amsterdam, 1973, pp. 441-482. 38 D. Papahadjopoulos, M. Cowden and H. Kimelberg, Role of cholesterol in membranes: effects on phospholipid-protein interactions, membrane permeability and enzymatic activity, Biochim. Biophys. Acta, 330 (1973) 8-26. 39 H. K. Kimelberg and D. Papahadjopoulos, Effects of phospholipid acyl chain fluidity, phase transitions and cholesterol on (Na÷K÷)-stimulated adenosine triphosphatase, J. Biol. Chem., 249 (1974) 1071-1080. 40 K. Inoue and T. Kitagawa, Effect of lipid composition on sensitivity of lipid membranes to Triton X-100, Biochim. Biophys. Acta, 426 (1976) 1-16. 41 D. Papahadjopoulos, Cholesterol and cell membrane function: a hypothesis concerning the etiology of atherosclerosis, J. Theor. BioL, 43 (1974) 329-337. 42 J. B. Finean, Phospholipids in biological membranes and the study of phospholipid-protein interactions, in G. B. Ansell, J. N. Hawthorne and R. M. C. Dawson (eds), Form and Function Of Phospholipids: Biochim. Biophys. Acta Library, Vol. 3, Elsevier, Amsterdam, 1973, pp. 171-204. 43 T. C. Lee and F. Snyder, Phospholipid metabolism in rat liver endoplasmic reticulum: structural analysis, turnover studies and enzymic activities, Biochim. Biophys. Acta, 291 (1973) 71-82. 44 D. M. Michaelson, A. F. Horwitz and M. P. Klein, Head group modulation of membrane fluidity in sonieated phospholipid dispersions, Biochemistry, 13 (1974) 2605-2612.
197
A G E R E L A T E D C H A N G E S IN T H E LIPIDS O F T H E M I C R O S O M A L A N D T H E M I T O C H O N D R I A L M E M B R A N E S O F R A T L I V E R AND K I D N E Y
LYNN S. GRINNA Department of Biology, University of California, Los Angeles, California 90024 (U.S.A.) (Received August 15, 1976; in revisedform November8, 1976)
SUMMARY The lipid contents of the microsomal and mitochondrial membrane fractions of liver and kidney were determined in 6 and 24 month old rats. A significant age related increase in the molar ratio of cholesterol/phospholipid was observed in all membrane fractions. A significant age related reduction of phospholipid was noted in the microsomal fractions of liver and kidney. The relative amount of phosphatidylethanolamine was found to decrease in all membrane fractions during aging. Membrane glyceride content, however, remained relatively constant with age. Significant increase in oleic acid was seen in the neutral lipid of both liver and kidney and in the polar lipid of kidney. Significant increase in docosahexaenoic acid and significant decrease in linoleic acid were seen in the polar lipid of the liver membrane fractions. Possible alterations in membrane physiochemical properties and in membrane function due to these age related lipid changes are discussed.
INTRODUCTION The physical and functional characteristics of biological membranes are influenced by the lipid components of the membranes. Membrane lipids, to a large extent, determine the properties of membrane fluidity, hydrophobicity and permeability [ 1--4]. Additionally, lipids are thought to contribute to the chemical and functional asymmetry of membranes [5, 6]. Membrane lipid has been shown to be required for the activity of many membrane bound enzymes and has been found to control, influence or modify enzyme thermal and kinetic behavior [7-9]. Changes in membrane lipids have been noted in several pathological conditions and also during aging [10-25]. Although several of these studies have been extensive in their analyses, none have provided detailed data on membranes where extensive functional change exists. Correlation between chemical and functional change, therefore, requires that such detailed data be collected on the lipids of both young and old animal membrane fractions which are known to be functionally different.
198 Extensive age related changes occur in the activities of microsomal and mitochondrial enzymes of rat liver and kidney [ 11, 18, 26-28]. Results obtained in Arrhenius studies of membrane bound enzymes indicate that the phase behavior of membranes changes with age [28]. Kinetic analysis and thermal inactivation studies have indicated that the interactions of enzyme protein with membrane lipid may be altered with age and that the incorporation of certain proteins into the membrane may be reduced during aging [26, 27]. Collectively the available information gives support to the suggestion that the age related functional changes in membranes may be related to lipid changes and to resultant alterations in the interactions of membrane lipid and protein components. In the present study the detailed lipid composition of the liver and kidney microsomal and mitochondrial membranes was determined. These tissues and membrane fractions were selected because they are not only functionally and chemically different from one another but are also functionally altered during aging. Also, it was of interest to compare the lipid changes of these fractions with those reported for other tissues and species during aging.
METHODSAND MATERIALS Male Sprague-Dawley rats maintained on Purina chow and water ad libitum were used in all experiments. The animals were killed by stunning, exsanguination and decapitation. For each experiment the tissues of 2-3 animals were pooled prior to homogenization. Five separate experiments were performed on each age group. The livers and kidneys were homogenized by hand, using a Potter-Elvehjem type homogenizer, and were diluted 1:10 with 0.25 M sucrose. Whole cells, nuclei and debris were removed at 1000 × g for 10 min. Mitochondrial fractions were obtained by centrifugation of the 1000 × g supernatant for 15 min at 12,000 × g. The crude mitochondrial fractions were resuspended to the original volume with sucrose and centrifugations at 1000 × g and 12,000 × g were repeated. The fluffy material overlaying the mitochondrial pellet was discarded along with the supematant. The microsomal fractions were obtained by centrifugation of the first 12,000 × g supernatant for 90 min at 105,000 × g. The microsomal fractions were resuspended to original volume in sucrose and centrifugations at 12,000 × g and 105,000 × g were repeated. Fraction purity was determined as previously described [11 ]. All pellets were stored at - 2 0 °C. Lipid extractions were performed according to procedures described by Folch et al. [29] and by Bligh and Dyer [30]. The lipid extracts were separated into neutral and polar fractions using columns of 1 g silicic acid (Clarkson Chemical Co., Inc.) as described by Colbeau et al. [31]. All samples were concentrated under a stream of nitrogen and analysed immediately. Phospholipid was determined by the method of Raheja et al. [32] using phosphatidylcholine (Sigma Chemical Co.) as standard. Cholesterol was analyzed by the method of Zlatkis and Zak [33]. Glyceride was determined by the enzymatic method of Garland and Randall [34] following alkaline hydrolysis of the samples as
199 described by Chernick [35]. Tripalmitin (Sigma Chemical Co.) was used as standard for glyceride determination. Methyl esters of fatty acids were prepared using 14% boron trifluoride-methanol (Applied Science Laboratories, Inc.). The prepared methyl esters were analyzed on a Perkin-Elmer 990 gas chromatograph equipped with 15% EGSS-X column, flame ionization detector and Infotronics digital integrator. The column was temperature programmed from 170-200 °C at 4 °C/rain following 4 min at 170 °C. Fatty acid standards (Applied Science Laboratories, Inc.) were used to identify the major fatty acids. Polar lipids were separated by two-dimensional thin-layer chromatography using precoated silica gel G plates (Analtech, Inc.). Chloroform/methanol/water~28% ammonia (130:70:8:0.5) was used as first dimension solvent and chloroform/acetone/methanol/ acetic acid/water (100:20:10:10:2) was used as second dimension solvent. Spots were detected with iodine vapor and/or 2,7-dichlorofluorescein (Applied Science Laboratories, Inc.) and were eluted and analyzed as described by Raheja et al. [32].
RESULTS The content o f phospholipid in the microsomal membranes of liver and kidney was found to be higher than the phospholipid content of the mitochondrial membranes of these tissues (Table I). During aging, the phospholipid contents of the microsomal membranes of both liver and kidney decreased approximately 18-20%. The phospholipid contents of the mitochondrial membranes of both liver and kidney were found to be constant during aging. TABLE I PHOSPHOLIPID, CHOLESTEROL AND GLYCERIDE CONTENTS OF LIVER AND KIDNEY MICROSOMES AND MITOCHONDRIA FROM 6 AND 24 MONTH OLD RATS
Membrane and Tissue
Age /months)
Phospholipid-P Cholesterol protein protein (nmol/mg) (nmol/mg)
Cholesterol Glyceride phospholipid-P protein (M} /nmol/mg)
6 24 6 24
443 366 461 403
0.212 -+0.009 0.301 ± 0.017t 0.516 ± 0.010 0.590 ± 0.031t
32.2 29.9 20.6 17.1
6 24 6 24
157 ± 20 152 ± 15 212 ± 16 232 ± 20
0.083 0.117 0.157 0.243
10.1 ± 1.8 8.1 ± 1.3 10.3 ± 1.8 13.0 ± 0.5
Microsomes
Liver Kidney
± 28* ± 17T ± 10 ± 19t
93.7 110.2 238.4 237.6
± 3.3 t 5.9 ± 5.3 ± 8.7
± 3.1 ± 2.0 -+ 1.3 -+0.7
Mitochondria Liver Kidney
*Values are the average o f 5 determinations + SD.
t p < 0.05 24 months vs. 6 months.
13.0 ± 0.2 17.8 ± 0.4t 33.3 ± 3.0 56.4 ± 4.9t
± 0.005 -+0.009t ± 0.007 ± 0.003t
200 The cholesterol content o f microsomal membranes is 6 - 7 times higher than the cholesterol content o f mitochondrial membranes (Table I). Also, the membranes of kidney contain 2-3 times more cholesterol than membranes o f liver. During aging the cholesterol content o f the microsomal membranes remains constant. In contrast, the cholesterol contents of the mitocfiondrial membranes o f liver and kidney increase 35 and 70% respectively during aging. The molar ratio of cholesterol/phospholipid is of considerable importance to the physiochemical properties o f membranes. Expressed as the molar ratio o f cholesterol/ phospholipid, the relative amount o f cholesterol increased significantly in all membrane fractions during aging (Table I). In the roicrosomal fractions the increase in the ratio was due to decrease in membrane phospholipid, whereas in the mitochondrial fractions the increase in the ratio was due to the increase in the content o f membrane cholesterol. The glyceride content of the membranes, which includes mono-, di-, and triglycerides, did not change with age (Table I). The glyceride content of the microsomal membranes was higher than that of the mitochondrial membranes. The glyceride content o f the mitochondrial membranes o f liver and kidney were similar; however, the glyceride content of the microsomal membranes of liver was approximately 50% higher than that o f kidney. The content o f individual phospholipid species of the membranes is reported in Table II. In agreement with others, phosphatidylcholine was the most abundant phospholipid of microsomal membranes and also o f liver mitochondrial membranes [31, 36, 37]. In kidney mitochondrial membranes the contents of phosphatidylcholine and TABLE II PHOSPHOLIPID CLASS COMPOSITION OF LIVER AND KIDNEY MICROSOMES AND MITOCHONDRIA FROM 6 AND 24 MONTH OLD RATS
Membrane and Tissue
Age (months)
PC (%)t
PE (%)
C [%)
6 24 6 24
62.5 65.7 44.2 44.9
± 5.4** ± 4.3 ± 2.3 ± 3.2
26.9 22.6 31.6 30.6
± 3.4 ± 2.3 ± 3.1 ± 3.8
6 24 6 24
45.7 48.5 37.7 43.0
± 2.5 ± 2.0 +- 2.6 ± 3.0
33.6 30.2 39.9 37.7
± 1.7 ± 1.4 ± 1.7 ± 1.8
Other* (%)
Microsomes Liver Kidney
Mitochondria Liver Kidney
10.6 11.7 24.2 24.5
16.9 13.9 16.8 13.5
PC = Phosphatidylcholine; PE = phosphatidylethanolamine; C = cardiolipin. Phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatides. Percentage of total phospholipid. **Average of 3-4 determinations ± SD.
~
± 3.6 ± 2.0 ± 3.0 ± 1.2
3.8 7.4 5.6 5.8
201 phosphatidylethanolamine were similar. Cardiolipin was a major component of mitochondrial membranes but was absent in microsomal membranes. Phosphatidylserine, phosphatidylinositol, sphingomyelin and lysophosphatides were collectively measured and were found to contribute only a small percentage to the total lipid of the liver microsomes but a larger percentage in the kidney microsomes. These lipids contribute in only a minor way to the total phospholipid of the mitochondrial membranes. The relative amount of phosphatidylethanolamine appeared to decrease slightly with age. The decrease, which was approximately 10% in liver and 5% in kidney, was not statistically significant, but was observed in all analyses performed. In mitochondrial fractions of liver and kidney, cardiolipin decreased approximately 20% between 6 and 24 months of age. These changes in the relative amounts of the individual phospholipids, although small, occurred both in the presence of (microsomes) and in the absence of (mitochondria) age related changes in the amount of phospholipid in the membranes. Previous studies have indicated that the fatty acid compositions of certain membranes change with age [12, 16-18]. The fatty acid compositions of the isolated neutral and polar lipid fractions of the membranes were therefore determined. As seen in Table III, there were significant changes in the fatty acids of the polar lipid of the membranes of liver and kidney. In the liver there was significant decrease in linoleic acid (18:2) and increase in docosahexaenoic acid (22:6) with age. By contrast, in the polar lipid fraction of kidney membranes there was significant increase in oleic acid (18:1) with age. Minor changes in other fatty acids were also .noted. Neutral lipid was found to have a fatty acid composition which was different from that seen in the polar lipid fractions of the same tissue and age related changes in fatty TABLE III FATTY ACIDS OF POLAR LIPID FROM LIVER AND KIDNEY MICROSOMES AND MITOCHONDRIAFROM 6 AND 24 MONTHOLD RATS Tissue Age and Membrane (months)
16:0 (%) ~
18:0 (%)
18:1 (%)
18:2 (%)
20:4 (%)
22:6 (%)
6 24 6 24
27.6t 24.7 22.0 24.5
24.7 23.9 17.1 18.6
10.3 10.1 10.3 10.4
15.5 ± 1.2"* 10.3 ± 0.5* 21.2 ± 2.0 17.0 ± 0.7 ¢
20.6 20.3 20.0 17.4
2.0±1.0"* 8.4±1.1' 5.1±1.1 8.9±0.4*
6 24 6 24
26.9 27.0 23.1 23.5
20.3 17.4 19.4 17.4
8.0±1.4"* 12.1±1.7' 8.7±0.3 11.0±1.0'
10.3 9.4 16.6 14A
28.1 24.6 28.1 26.8
1.8 1.6 1.5 1.7
Liver Microsomes Mitochondria Kidney Microsomes Mitochondria
*Area percentage. tAverage of 3-5 determinations, SD given only where P < 0.05. **Average of 3-5 determinations ± SD. *P < 0.05 24 months vs. 6 months.
202 TABLE IV FATTY ACIDS OF NEUTRAL LIPID FROM LIVER AND KIDNEY MICROSOMES AND MITOCHONDRIA FROM 6 AND 24 MONTH OLD RATS Tissue and Membrane
Age (months)
16:0 + 16:1 (%) *
18:0 (%)
18:1 (%)
6 24 6 24
35.5t 34.4 40.9 40.9
6.3 4.7 6.1 5.7
28.5 34.9 25.0 29.7
6 24 6 24
32.4 28.0 31.1 26.8
12.8 11.8 16.2 13.5
18:2 (%)
20:4 (%)
23.7 22.2 20.3 17.2
3.1 2.6 2.3 2.1
Liver Microsomes Mitochondria
+- 1.7"* +- 1.5 :~ +- 0.7 +- 0.9*
Kidney Microsomes Mitochondria
22.3 -+ 2.1 28.3 -+0.3* 18.8 -+0.9 23.8 +- 2.0*
20.9 21.8 20.4 17.6
7.4 8.4 11.2 -+ 2.1 18.2 +-2.4*
*Area percentage. tAverage of 3-5 determination, SD given only where P < 0.05. **Average of 3-5 determinations -+SD. *P < 0.05 24 months vs. 6 months. acids were also noted in the neutral fractions (Table IV). In the neutral lipid of both liver and kidney membranes the content of oleic acid (18:1) increased significantly with age. In the kidney mitochondrial fractions there was, additionally, an increase in the content of arachadonic acid (20:4) with age. This fatty acid was found to occur at much higher levels in kidney neutral and polar lipid than in similar fractions from liver.
DISCUSSION Age related lipid changes were noted in the microsomal and mitochondrial fractions of rat liver and kidney. These lipid changes are of general interest as they support the premise that membrane lipid change may be a general characteristic associated with aging [11, 12, 2 6 - 2 8 ] . The changes are of specific interest as they may relate to previously reported age related alteration in enzyme function in the microsomal and mitochondrial fractions of liver and kidney [ 11,18, 2 6 - 2 8 ] . The lipid values of the microsomal and mitochondrial fractions of the 6 month old animals are in agreement with published values [31,36, 37]. The values of the 24 month old animals indicate a significant increase in the molar ratio of cholesterol/phospholipid in all membrane fractions, significant changes in the fatty acid composition of all membrane fractions, and a significant decrease in the phospholipid of the microsomal membrane fractions. Although not reported here, the lipid contents of membrane fractions obtained from animals between 6 and 24 months of age were consistant with the results presented.
203 Of the lipid changes noted during aging, increase in the molar ratio of cholesterol• phospholipid is considered the most important since the increase was observed in all membrane fractions examined and since it is known that the ratio has effects on membrane physiochemical properties. When the cholesterol content is increased the thermal motion of the hydrocarbon portion of membranes is reduced [4, 38, 39]. This results in a reduction of the fluidity of membranes and reduction in component intermixing and mobility [4, 38, 39]. Increase in cholesterol also has been found to decrease the permeability of model membranes and to decrease the expansion of phospholipid monolayers thereby hindering the insertion of new compounds into the monolayers [38, 40]. Increase in membrane cholesterol also alters and depresses the activities of certain membrane bound enzymes [38, 39]. Cholesterol has been reported to increase with age in rat liver and skeletal muscle [13, 25], in mouse brain [16] and in human red blood cells [19, 20]. Increase in membrane cholesterol, and the physiochemical alterations such an increase would produce, have been suggested as the initiation factors in the development of atherosclerosis [38, 41]. Age related reduction in the phospholipid content of the microsomal membranes of the liver and kidney is considered important as it may result in alteration in enzyme activities, reduction of the bulk lipid and change in permeability properties of the membranes [7, 42]. In vitro the reduction of membrane phospholipid produces dramatic changes (usually reduction) in membrane bound enzyme activities [7, 42]. Decrease in phospholipid content with age has been reported in rat liver and pancreas [21] and in mouse liver microsomes [ 12]. Of less obvious consequence are the changes seen in the fatty acid profiles and the individual phospholipid species of the membranes during aging. In the presentcstudy the fatty acid composition was found to change slightly towards a more unsaturated system during aging. The importance of the specific fatty acid changes is unknown, although it is known that unsaturated fatty acids contribute to membrane fluidity [4, 39]. The changes observed might represent a compensatory response to the (proposed) decrease in membrane fluidity. It is of interest that in all cases the changes seen in the fatty acid composition were in those fatty acids which predominantly (90%) occupy position 2 of the phospholipids [43]. The changes in the fatty acids of the polar lipid appeared to be organ specific whereas the changes in the neutral lipid were specific for oleic acid. The phospholipid composition of the membranes changed slightly with age such that phosphatidylethanolamine was reduced in all membrane fractions. The change occurred independently of the extent of change in the total amount of phospholipid in the membranes. The role that the phosphoryl group may play in the maintenance of biological membrane fluidity and asymmetry are currently matters of controversy and speculation [4, 6, 39,44]. If the distribution of phospholipids within cellular membranes is proven to be asymmetric then the age related changes observed in the phospholipid class composition of the membranes would have considerable functional significance. Similar decrease in membrane phosphatidylethanolamine has been reported for rat liver plasma membrane [23], rat skeletal muscle sarcoplasmic reticulum [22] and human aorta
204 [24]. However, phosphatidylethanolamine has been reported to increase with age in mouse liver microsomes [12] and in human red blood cells [19]. The results presented suggest that during aging, changes in microsomal and mitochondrial lipid produce membranes which are less fluid and asymmetrically altered. It is also suggested that this change is a general one during aging and is causally related to the functional decline which is known to occur in these membranes during aging.
ACKNOWLEDGEMENTS The author wishes to thank Dr. Albert A. Barber for his continued interest and support throughout this project. Supported by USPHS grant AG 00477-01 from the National Institute of Child Health and Human Development, Aging Branch.
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