- Email: [email protected]
Lipid metabolism in aging
Mechanisms of Ageing and Development
Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
LIPID METABOLISM IN AGING*
DAVID KRITCHEVSKY The Wistar Institute of Anatomy and Biology, Philadelphia, Pa. 19104 (U.S.A.)
(Received August 4th, 1972)
With relatively few exceptions (considering the breadth of the literature), studies of lipid metabolism in aging have consisted of compendia of the lipid content of various tissues. These data are important to the understanding of the chemical state of the organism as it grows older. However, studies of lipid metabolism per se encompass a wide area. There are several important classes of lipids and in the case of each class we must consider the influence of aging on synthesis, excretion, degradation and metabolic interconversions in vivo. There is a growing body of literature in this area but it is still sparse. Among the classes of lipids, particular compounds command more attention than others because of their central positions in the etiology of some diseases. Cholesterol, for instance, has been the subject of many studies because of its association with atherosclerosis, a disease considered to be concomitant with aging in some quarters. In this paper I will attempt to avoid discussions of age-related diseases and will concentrate on what is known of the lipid content of tissues and of lipid metabolism in normal animals as they age. The most recently reported study of cholesterol levels in man (Table I) details data obtained from more than 3000 visitors to the 1968 San Francisco Health Fair 1. It is evident from these data that in both sexes cholesterol levels rise in the third decade of life. In men the cholesterol levels appear to reach a plateau at 50-59 years of age, whereas in women there is a sharp rise in cholesterol levels after age 50. The sharp increase in cholesterol levels with age has also been reported in a study made in India 2, but here the rise occurs in the 30-39-year group. A similar pattern has been found in men in the Abkhazian Republic of Russia 3. This study of 300 men included 26 each in the 90-99-year and lO0-year-plus age groups. In the Russian study, blood phospholipid levels rose from 167 rag/100 ml in the 20-29-year group to 201 mg/ml in the 30-39-year group. The lipids of aging rats (1-18 months) have been studied by Carlson and co* This work was supported, in part, by U.S. Public Health Service Research Grant HE-03299, and Research Cancer Award HE-0734 from the National Heart and Lung Institute. Mech. Age. Dev., 1 (1972) 275-284
275
workers 4 with the results shown in Table II. In serum there is an increase in plasma triglycerides, cholesterol and phospholipids between the fourth and ninth months. These changes are not reflected in either heart or liver lipids, suggesting an inability to clear circulating lipid rather than deposition. These studies were carried out on Sprague-Dawley rats. Studies with inbred rats 5 show different basal (30-day) serum cholesterol levels and also indicate different patterns with age. Thus at 90 days the cholesterol levels fall in the BN and Lewis strains and are unchanged in the DA strain
TABLE I SERUM CHOLESTEROL
After Werner
et
L E V E L S IN M A N
al. 1. N.S., not significant.
Age (years)
Mean
cholesterol level (mg/ lO0 ml) Significance
Male
Female
0-12 13-19 20~29 30-39 40~49 50-59 60 69 70-79
194 197 227 242 246 254 259 258
197 198 224 230 215 272 285 275
(t) N.S. N.S. N.S. 0.05 <0.001 < 0.001 < 0.001 < 0.05
T A B L E 1I B L O O D A N D T I S S U E L I P I D S IN A G I N G
RATS
After Carlson et al.4.
Age (months) 1
4
9
18
0.45 0.51 96 130
0.55 0.83 94 142
0.44 2.50 218 271
0.57 2.55 307 353
Liver: Triglycerides (pmoles/g) Cholesterol (mg/g) Phospholipid (mg/g)
8.4 3.4 30.9
6.6 4.3 27.2
7.3 4.9 24.3
8.1 6.7 21.7
Heart: Triglycerides (pmoles/g) Cholesterol (mg/g) Phospholipids (mg/g)
2.6 2.1 22.6
1.4 2.0 26.4
2.0 2.2 24.0
1.5 2.1 24.6
Plasma:
Free fatty acids (mmole/I) Triglycerides (mmoles/l) Cholesterol (mg/lO0 ml) Phospholipids (mg/lO0 ml)
276
Mech. Age. Dev., 1 (1972) 275 284
rat. Wistar rats show a slight rise in cholesterol levels at 3 months of age. The data suggest yet another parameter to be considered in animal experiments, namely, the strain. Zucker and Zucker6, 7 have developed data to show that the accretion of lipid in rats is more a function of body weight than of age. In the study of Das and Bhattacharya 2 the weight of the men sampled fell in the sixth decade of life as did their cholesterol levels. In the gray rabbit there is a distinct rise in brain lipid with age; also a rise in lung lipid, and relatively few changes in liver, muscle and plasma lipids s. The observations of increased brain lipids have been confirmed in the N e w Zealand rabbit 9. The greatest changes take place between the first and 14th days of life. During this time there is a 5-fold increase in cholesterol and a 12-fold increase in cerebroside. The type of sterol present in the brain also changes with age. Brain of newborn rats contains an appreciable level of 24-dehydrocholesterol (desmosterol) which disappears by the time myelination has been completed 10. The rise in blood cholesterol which is observed with age can be due to increased synthesis, decreased catabolism and excretion, or increased absorption. The effect of age on cholesterol synthesis was studied over a quarter of a century ago. In the earliest T A B L E III INFLUENCE OF AGE ON INCORPORATION CHOLESTEROL
OF
[1-2H]ACETATE INTO LIVER
After Bloch et al. n.
Rat weight (g)
No.
230 150-170 120-130
4 3 2
Atom % excess ZH 0.09 i 0.01 0.32 4- 0.07 1.02 (0.73 - 1.30) :[: 0.29
T A B L E IV EFFECT OF AGE ON BILIARY OUTPUT
OF CHOLESTEROL
IN RATS
After Rosenman and Shibata 12. Age (weeks)
No.
Weight (g )
Plasma Bile cholesterol (mg/lO0 ml) Vol.
(mO
6 8 9 20 28 44
9 6 12 12 10 10
116 181 184 310 373 406
Mech. Age. Dev., 1 (1972) 275-284
65 43 48 64 47 47
7.1 11.1 12.0 14.5 15.7 15.3
Cholesterol mg/lO0 ml
mg total
mg/g body wt
23.6 26.6 24.1 16.8 12.8 12.5
1.7 2.9 3.0 2.4 2.0 1.9
0.014 0.016 0.016 0.007 0.005 0.004
277
days of work o n cholesterol biosynthesis, Bloch e t al. H showed a significant decrease in liver sterol synthesis with age (Table III). I n a study involving a p a r a m e t e r of biosynthesis other t h a n isotope i n c o r p o r a t i o n , viz. biliary o u t p u t of cholesterol (Table IV), R o s e n m a n a n d Shibata 1-~ showed almost a 50 % decrease in cholesterol o u t p u t between 6 a n d 44 weeks of life. Calculation of mg cholesterol excreted per g body weight shows a relatively high level at 6-9 weeks of life (0.014 mg) and a drop to a b o u t 0.006 mg at 20-44 weeks of life. T r o u t e t al. 13 showed that the skin, t e n d o n a n d aorta of y o u n g rats (under 4 weeks) incorporate more [14C]acetate into free and ester sterol than do tissues of rats
TABLE V STEROL SYNTHESIS FROM [1-14C]ACETATE IN RAT TISSUES (COMPARISON OF OLD (1.25-3 YEAR) TO YOUNG RATS) After Trout et al. 13. Tissue
Ratio o f activities (old/young) Old/O.~4-week rats r
Skin free sterol Skin ester sterol Liver free sterol Liver ester sterol Tendon free sterol Aorta free sterol
OId/6-week rats
~
28 10 23 27 1 7
60 70 34 35 7 77
TABLE VI STEROL METABOLISM IN RATS After Yamamoto and Yamamura15. Conditions
14C as percent o f that in 2-month-old rats 5-month-old
8-month-old
57 45
37 27
60 58 55
30 32 32
Fecal 14C after intraperitoneal [14C]cholesterol: Total 61 Bile acids 54 Non-saponifiable 63
41 36 41
278
Mech. Age. Dev., 1 (1972) 275-284
Biosynthesis from acetate: In vivo In vitro
Biliary 14C after intravenous [14C]cholesterol: Total Bile acids Non-saponifiable
TABLE VII ENDOCRINE INFLUENCE ON CHOLESTEROL TURNOVER IN RATS After Hruza TM. Treatment
Hypophysectomy Thyroidectomy Thyroxine Insulin
Ratio of cpm in serum (Control vs. treated) Young (6 weeks)
Old (13 months)
+55* +48* --22* --29*
+40* --13 + 15 + 15
* Significant difference.
TABLE VIII LIPID METABOLISM IN EPIDIDYMAL FAT OF RATS After Benjamin et al. 19. Glucose
Young (90-40 g)
OM (350-400 g)
Rate of acetate incorporation (Mmoles/3h/g)
--
20
1.9
Palmitic acid -+ triglycerides (/~moles/2 h/g)
-+
1.8 15.1
0.8 5.2
Palmitic acid ~ CO2 (/~moles oxidised/2 h/g)
-+
Lipolysis -(/~equivs free fatty acid/2 h/g) +
0.20 0.06
0.04 0.04
3.5 2.6
3.5 1.3
6-10 weeks of age (Table V). Their findings with y o u n g rat skin confirm those of Srere et al. 14. Y a m a m o t o a n d Y a m a m u r a 15 studied synthesis a n d excretion of cholesterol in rats aged 2, 5 a n d 8 months. A t 5 m o n t h s there is a b o u t a 50-60 ~ drop i n all three parameters, while at 8 m o n t h s the r e d u c t i o n is in the range of 60-70 ~o (Table VI). There is one report, however, which claims that there is n o difference in cholesterol synthesis in y o u n g or old rats 16. HruzaZ7, is has studied cholesterol t u r n o v e r in y o u n g a n d old rats. He f o u n d a decreased t u r n o v e r in older rats. Cholesterol t u r n o v e r in y o u n g rats is reduced by h y p o p h y s e c t o m y a n d t h y r o i d e c t o m y a n d increased by thyroxine or insulin treatment. I n older rats h y p o p h y s e c t o m y significantly increases cholesterol t u r n o v e r b u t the Mech. Age. Dev., 1 (1972) 275-284
279
other treatments have relatively little effect (Table VII). These findings indicate the interrelationships between endocrine factors and lipid metabolism. Benjamin et al. 19, studying adipose tissue metabolism in the rat, found reduced lipid synthesis and oxidation (Table VIII) with aging. Analysis of the triglyceride fatty acids showed a marked reduction in C10, CI~ and C14 fatty acids with aging, and an increase in Cls:I and Cls:z. In animals being fed laboratory chow for most of their lives, these changes may merely reflect the composition of the dietary fat. In a study of human adipose tissue, Insull and Bartsch 20 found increases in palmitic and oleic acids with age and attributed these changes primarily to the dietary pattern. Lipolysis of triglycerides is the major metabolic source of free fatty acids. Since aging seems to affect other aspects of lipid metabolism, its influence upon lipolytic systems is of interest. Zemplenyi and Grafnetter zl found that rat aorta slices were able to hydrolyze the lipids present in lipemic human serum. The extent of lipolysis was assayed by measurement of the free fatty acids released. In two series of experiments (Table IX) they demonstrated significantly greater lipolytic activity in aortas of 7month-old rats than in aortas of rats aged 2, 12 or 24 months. The lowest lipolytic activity was found in the 2-month-old rats. The lipolytic activity of the myocardium for 2-, 7- and 12-month-old rats (10 per group) was 11.27, 12.80 and 13.42, respectively. The difference between the 2- and 7-month-old groups was highly significant (P < 0.001). Dury 22 also found a greater degree of lipolysis (using a fat emulsion as substrate) in old (15-18-month-old) compared to young (3-4-month-old) rat aortas. Heparin, administered 15 min before the aorta was taken, significantly enhanced lipolysis in the young aortas but had no effect on the old ones. In man, Nikkila and Niemi 23 found that the activity of heparin-induced lipoprotein lipase was significantly higher in the sera of subjects aged 20-39 years when compared to subjects aged 63-92 years (P < 0.01). The time required for clearing of a standard fat meal is three times higher in old patients z4. The foregoing data suggest a progressive inability to metabolize lipid in the
TABLE IX L1POLYTIC ACTIVITY OF RAT AORTA After Zemplenyi and Grafnetter21. Rat age
No.
Lipolysis (mequivs fatty acid/g tissue ~- S.E.)
Experiment 1 : 3 months 7 months 12 months
10 7 7
4.38 i 0.19 7.43 ~ 1.14 5.90 i 0.42
Experiment 2: 2 months 7 months 24 months
7 8 8
4.84 i 0.74 8.77 ~ 0.49 6.01 ~ 0.52
280
Mech. Age. Dev., 1 (1972) 275-284
aging organism. This is seen in b o t h synthesis and degradation o f fat. In the face o f dietary fat, the decreased synthesis cannot compensate for decreased catabolism. The development and availability of h u m a n diploid cell strains 25 provides a unique system for the study of fundamental biochemical p h e n o m e n a at the cellular level. The observation 26 that these cells display a limited capacity for doubling (which m a y be interpreted as senescence at the cellular level) makes them an interesting system in which to study biochemical aspects o f aging. We have studied~7, 2s the lipid composition of early and late passage WI-38 cells. There is an increase in total lipid o f late passage cells, but the neutral lipid: phospholipid ratio is unchanged. The only significant differences between specific lipid components are in the phospholipid fraction. The older cells show significant decreases in phosphatidylethanolamine (P < 0.025) and phosphatidylinositol (P < 0.025) and an increase in lecithin (P < 0.005) (Table X). These changes m a y reflect differences in m e m b r a n e characteristics, but this possibility has not yet been investigated. The fatty acid spectra of the neutral lipids and phospholipids o f the cells (Table XI) are similar. The WI-38 cells synthesize cholesterol f r o m [14C]acetate when maintained in
TABLE X PERCENTAGE* LIP1DS OF EARLY AND LATE PASSAGE WI-38 CELLS Reprinted with permission from Kritchevsky and Howard 2s.
Total lipid (mg/100 mg dry wt): Total neutral lipid:
Early passage
Late passage
(19 i 1.5)
(25 ~2.6)
30 4- 2.2
32 i 2.5
% total neutral lipM
Cholesteryl ester Triglyceride Free fatty acid Diglyceride Cholesterol Monoglyceride Total phospholipid:
9.3 4- 2.6 11 4- 1.9 29 + 1.9 11 4- 2.6 32 + 1.7 7.1 t 1.3
11 ± 1.4 14 :k 1.7 32 ± 4.1 7.7 ± 0.9 30 4- 2.2 10 4- 4.4
69 4- 2.2
68 ± 2.5
% totalphospholipM
Phosphatidylethanolamine Phosphatidylinositol Phosphatidylserine Lecithin Sphingomyelin Lysolecithin
12 -4- 1.7 15 zk 2.7 8.0 zk 0.9 53 -4- 3.7 9.6 -4- 1.9 2.5 ~- 0.5
6.9 4- 1.7 8.4 ± 1.7 8.9 4- 3.4 70 ± 3.8 9.6 ± 0.9 2.5 ± 0.4
* Mean -4- S.E. of 8 determinations. Mech. Age. Dev., 1 (1972) 275-284
281
serum-containing medium 29, but the synthetic rate is very slow. The conclusion that this might be due to the presence of exogenous cholesterol has been proven experimentally by Rothblat et al. 3°. These authors showed that WI-38 cells can synthesize significant amounts of cholesterol when grown in the absence of exogenous lipid (delipidized serum protein)31, 32. The addition of cholesterol to this medium can inhibit endogenous cholesterogenesis by more than 95 ~. Chang 33 has studied lipid metabolism in human amnion cells. The uptake of [14C]cholesterol or [14C]palmitate by these cells increases with age. Thus, in two experiments the uptake of [14C]cholesterol by 42- and 70-day-old cells was 381 and 4 5 4 ~ of that observed in 14-day-old cells. The uptake of [1-14C]palmitate by aging cells was increased, and its oxidation to 14CO2 was decreased, resulting in a net accumulation of cellular lipid. The 14C-labeled lipid:14CO2 ratio for 14-, 42- and T A B L E XI F A T T Y A C I D S O F E A R L Y A N D L A T E P A S S A G E WI-38 C E L L S Reprinted with permission f r o m Kritchevsky a n d H o w a r d 2s.
Fatty acid
Myristic Myristoleic Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic Arachidonic
Carbon No.
Neutral lipids (%)
Phospholipids (%)
Early passage (3) *
Late passage (3) *
Early passage (5) *
Late passage (6) *
14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 20:4
2.1 ± trace 22 ± trace 17 ± 33 -2 7 -2 trace 20 -2
0.41 -t- 0.08 trace 20 -2 0.91 0.40 -2 0.08 19 -2 2.1 31 -2 1.3 10 -2 0.91 trace 19 -2 1.5
3.2 -2 trace 23 -2 trace 17 -2 32 -2 6.1 -2 trace 19 -2
1.8 -2 0.60 t race 23 -2 2.0 0.31 -2 0.04 20 -2 1.5 30 -2 2.5 7.5 -2_ 0.36 1.9 -2 0.20 17 -2 1.9
0.57 2.2 2.2 0.4 1.3 3.2
0.16 1.8 1.9 1.4 0.87 2.7
* N u m b e r o f determinations.
T A B L E XII E F F E C T O F H Y D R O C O R T I S O N E O N [ 2 6 - 1 4 C ] C H O L E S T E R O L A N D [1-14C]OLEIC A C I D M E T A B O L I S M BY P H A C U L T U R E S After Y u a n a n d C h a n g a L
Age (days)
31 59 87 115
282
No. expts
4 4 2 1
Cells )< 104 per culture
Oleic acid (Lipid/C02)
--
+
--
32 17 8 6
146 155 102 69
15.6 10.2 21.1 20.7
Cholesterol uptake (cpm) ÷
4.3 3.4 6.8 2.8
--
+
303 428 1625 3014
267 229 424 1180
Mech. Age. Dev., 1 (1972) 275-284
70-day-old cells was 0.3, 6.7 a n d 8.3, respectively. O x i d a t i o n o f [6-14C]gIucose was n o t r e d u c e d in the aging cells, indicating an i m p a i r m e n t o f the fatty acid o x i d a t i o n system, b u t n o t o f the tricarboxylic acid cycle. Recently Y u a n a n d C h a n g 34 have o b s e r v e d that the a d d i t i o n o f h y d r o c o r t i s o n e to a m n i o n cells abolishes the a g e - d e p e n d e n t increase in 14C-labeled lipid :14CO2 ratio in cells fed [l-14C]oleic acid a n d r e t a r d s the increase in cholesterol u p t a k e seen with aging (Table XII). H y d r o c o r t i s o n e a n d cortisone have also been shown to extend the life-span o f WI-38 h u m a n diploid cells 35. The m e c h a n i s m by which these changes t a k e place has n o t been elucidated. In s u m m a r y , aging animals exhibit a progressive inability to handle lipid. W h i l e they synthesize less lipid, they also m e t a b o l i z e less, the result being an ager e l a t e d increase o f lipid in tissues a n d blood. H u m a n d i p l o i d cells, which seem to age in vitro, also exhibit some o f the same m e t a b o l i c characteristics as intact organisms with r e g a r d to lipid m e t a b o l i s m . These cells offer a unique o p p o r t u n i t y to study the mechanisms underlying the changes in lipid m e t a b o l i s m o b s e r v e d with aging. W e have a c c u m u l a t e d e n o u g h d a t a relating to the lipid c o n t e n t o f aging tissues a n d these d a t a are generally consistent regardless o f species. W e need n o w to u n d e r s t a n d why these changes t a k e place a n d h u m a n diploid cells offer a u n i q u e substrate for such investigations.
REFERENCES 1 M. Werner, R. E. Tolls, J. V. Hultin and J. Mellecker, Influence of sex and age on the normal range of eleven serum constituents, Z. Klin. Chem. Klin. Biochem., 8 (1970) 105-115. 2 B. C. Das and S. K. Bhattacharya, Variation in lipoprotein level with changes in age, weight and cholesterol ester, Gerontologia, 5 (1961) 25-34. 3 N. N. Kipshidze, Quantitative changes in lipid, protein and carbohydrate metabolism in relation to age, Thule International Symposium on Cancer and Ageing, 1967, pp. 49-57. 4 L. A. Carlson, S. O. FrSberg and E. R. Nye, Effect of age on blood and tissue lipid levels in the male rat, Gerontologia, 14 (1968) 65-79. 5 D. Kritchevsky and S. A. Tepper, Serum cholesterol levels of inbred rats, Am. J. Physiol., 207 (1964) 631-633. 6 T. F. Zucker and L. M. Zucker, Fat accretion and growth in the rat, J. Nutr., 80 (1963) 6-19. 7 T. F. Zucker and L. M. Zucker, Phosphatides and cholesterol in the rat body: Effects of growth, diet and age, J. Nutr., 80 (1963) 20-24. 8 J. P. Hrachovec and M. Rockstein, Age changes in lipid metabolism and their medical implications, Gerontologia, 3 (1959) 305-326. 9 K. B. Dalal and E. R. Einstein, Biochemical maturation of the central nervous system. I. Lipid changes, Brain Res., 16 (1969) 441-451. 10 D. Kritchevsky and W. L. Holmes, Occurrence of desmosterol in developing rat brain, Biochem. Biophys. Res. Commun., 7 (1962) 128-131. 11 K, Bloch, E. Borek and D. Rittenberg, Synthesis of cholesterol in surviving liver, J. Biol. Chem., 162 (1946) 441-449. 12 R. H. Rosenman and E. Shibata, Effect of age upon hepatic synthesis of cholesterol in the rat, Proc. Soc. Exp. Biol. Med., 81 (1952) 296-298. 13 E. C. Trout, Jr., K. T. Kao, C. A. Hizer and T. H. McGavack, In vitro synthesis of free andester cholesterol in various tissues of young and old rats, J. Gerontol., 17 (1962) 363-368. 14 P. A. Srere, I. L. Chaikoff, S. S. Treitman and L. S. Burstein, The extrahepatic synthesis of cholesterol, J. Biol. Chem., 182 (1950) 629-634. 15 M. Yamamoto and Y. Yamamura, Changes of cholesterol metabolism in the aging rat, Atherosclerosis, 13 (1971) 365-374. Mech. Age. Dev., 1 (1972) 275-284
283
16 K. A. Tretiakova and D. E. Grodsensky, The rate of cholesterol and fatty acid synthesis in the adrenals, testicles and liver of young and old rats both normal and irradiated, Biokhemia, 25 (1960) 399-403. 17 Z. Hruza and M. Wachtlova, Decrease of cholesterol turnover in old rats, Exp. Gerontol., 4 (1969) 245-250. 18 Z. Hruza, Effect of endocrine factors on cholesterol turnover in young and old rats, Exp. Gerontol., 6 (1971) 199-204. 19 W. Benjamin, A. Gellhorn, M. Wagner and H. Kundel, Effects of aging on lipid composition and metabolism in the adipose tissues of the rat, Am. J. Physiol., 201 (1961) 540-546. 20 W. Insull, Jr. and G. E. Bartsch, Fatty acid composition of human adipose tissue related to age, sex and race, Am. J. Clin. Nutr., 20 (1967) 13-23. 21 T. Zemplenyi and D. Grafnetter, The lipolytic activity of the aorta: Its relation to aging and to atherosclerosis, Gerontologia, 3 (1959) 55-64. 22 A. Dury, Lipolytic activity of aorta of young and old rats and influence of heparin in vivo, J. Gerontol., 16 (1961) 114 117. 23 E. A. Nikkila and T. Niemi, Effect o f a g e o n the lipemia clearing activity of serum after administration of heparin to human subjects, J. Gerontol., 12 (1957) 44-47. 24 G. H. Becker, J. Meyer and H. Necheles, Fat absorption in young and old age, Gastroenterology, 14 (1950) 80-90. 25 L. Hayflick and P. S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res., 25 (1961) 585-621. 26 L. Hayflick, The limited in vitro lifetime of human diploid cell strains, Exp. Cell Res., 37 (1965) 614-636. 27 D. Kritchevsky and B. V. Howard, The lipids of human diploid cell strain W1-38, Ann. Med. Exp. Fenn., 44 (1966) 343-347. 28 D. Kritchevsky and B. V. Howard, Lipid metabolism in human diploid ceils, in E. Hole6kov~t and V. J. Cristofalo (Eds)., Aging in Cell and Tissue Culture, Plenum Press, New York, 1970, pp. 57-82. 29 B. V. Howard and D. Kritchevsky, The lipids of normal diploid (WI-38) and SV40 transformed human cells, Int. J. Cancer, 4 (1969) 393-402. 30 G. H. Rothblat, R. Boyd and C. Deal, Cholesterol biosynthesis in WI-38 and WI-38VA13A tissue culture cells, Exp. Cell Res., 67 (1971) 436-440. 31 G. H. Rothblat, M. K. Buchko and D. Kritchevsky, Cholesterol uptake by L5178Y tissue culture cells: Studies with delipidized serum, Biochim. Biophys. Acta, 164 (1968) 327-338. 32 G. H. Rothblat, The effect of serum components on sterol biosynthesis in L cells, J. Cell. Physiol., 74 (1969) 163-170. 33 R. S. Chang, Metabolic alterations with senescence of human cells, Arch. Int. Med., 110 (1962) 563-568. 34 G. C. Yuan and R. S. Chang, Effect of bydrocortisone on age-dependent changes in lipid metabolism of primary human amnion cells in vitro, Proc. Soc. Exp. Biol. Med., 130 (1969) 934-936. 35 V. J. Cristofalo, Metabolic aspects of ageing in human diploid cells, in E. Hole6kov~i and V. J. Cristofalo (Eds.), Aging in Cell and Tissue Culture, Plenum Press, New York, 1970, pp. 83-119.
284
Mech. Age. Dev., 1 (1972) 275-284
Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
LIPID METABOLISM IN AGING*
DAVID KRITCHEVSKY The Wistar Institute of Anatomy and Biology, Philadelphia, Pa. 19104 (U.S.A.)
(Received August 4th, 1972)
With relatively few exceptions (considering the breadth of the literature), studies of lipid metabolism in aging have consisted of compendia of the lipid content of various tissues. These data are important to the understanding of the chemical state of the organism as it grows older. However, studies of lipid metabolism per se encompass a wide area. There are several important classes of lipids and in the case of each class we must consider the influence of aging on synthesis, excretion, degradation and metabolic interconversions in vivo. There is a growing body of literature in this area but it is still sparse. Among the classes of lipids, particular compounds command more attention than others because of their central positions in the etiology of some diseases. Cholesterol, for instance, has been the subject of many studies because of its association with atherosclerosis, a disease considered to be concomitant with aging in some quarters. In this paper I will attempt to avoid discussions of age-related diseases and will concentrate on what is known of the lipid content of tissues and of lipid metabolism in normal animals as they age. The most recently reported study of cholesterol levels in man (Table I) details data obtained from more than 3000 visitors to the 1968 San Francisco Health Fair 1. It is evident from these data that in both sexes cholesterol levels rise in the third decade of life. In men the cholesterol levels appear to reach a plateau at 50-59 years of age, whereas in women there is a sharp rise in cholesterol levels after age 50. The sharp increase in cholesterol levels with age has also been reported in a study made in India 2, but here the rise occurs in the 30-39-year group. A similar pattern has been found in men in the Abkhazian Republic of Russia 3. This study of 300 men included 26 each in the 90-99-year and lO0-year-plus age groups. In the Russian study, blood phospholipid levels rose from 167 rag/100 ml in the 20-29-year group to 201 mg/ml in the 30-39-year group. The lipids of aging rats (1-18 months) have been studied by Carlson and co* This work was supported, in part, by U.S. Public Health Service Research Grant HE-03299, and Research Cancer Award HE-0734 from the National Heart and Lung Institute. Mech. Age. Dev., 1 (1972) 275-284
275
workers 4 with the results shown in Table II. In serum there is an increase in plasma triglycerides, cholesterol and phospholipids between the fourth and ninth months. These changes are not reflected in either heart or liver lipids, suggesting an inability to clear circulating lipid rather than deposition. These studies were carried out on Sprague-Dawley rats. Studies with inbred rats 5 show different basal (30-day) serum cholesterol levels and also indicate different patterns with age. Thus at 90 days the cholesterol levels fall in the BN and Lewis strains and are unchanged in the DA strain
TABLE I SERUM CHOLESTEROL
After Werner
et
L E V E L S IN M A N
al. 1. N.S., not significant.
Age (years)
Mean
cholesterol level (mg/ lO0 ml) Significance
Male
Female
0-12 13-19 20~29 30-39 40~49 50-59 60 69 70-79
194 197 227 242 246 254 259 258
197 198 224 230 215 272 285 275
(t) N.S. N.S. N.S. 0.05 <0.001 < 0.001 < 0.001 < 0.05
T A B L E 1I B L O O D A N D T I S S U E L I P I D S IN A G I N G
RATS
After Carlson et al.4.
Age (months) 1
4
9
18
0.45 0.51 96 130
0.55 0.83 94 142
0.44 2.50 218 271
0.57 2.55 307 353
Liver: Triglycerides (pmoles/g) Cholesterol (mg/g) Phospholipid (mg/g)
8.4 3.4 30.9
6.6 4.3 27.2
7.3 4.9 24.3
8.1 6.7 21.7
Heart: Triglycerides (pmoles/g) Cholesterol (mg/g) Phospholipids (mg/g)
2.6 2.1 22.6
1.4 2.0 26.4
2.0 2.2 24.0
1.5 2.1 24.6
Plasma:
Free fatty acids (mmole/I) Triglycerides (mmoles/l) Cholesterol (mg/lO0 ml) Phospholipids (mg/lO0 ml)
276
Mech. Age. Dev., 1 (1972) 275 284
rat. Wistar rats show a slight rise in cholesterol levels at 3 months of age. The data suggest yet another parameter to be considered in animal experiments, namely, the strain. Zucker and Zucker6, 7 have developed data to show that the accretion of lipid in rats is more a function of body weight than of age. In the study of Das and Bhattacharya 2 the weight of the men sampled fell in the sixth decade of life as did their cholesterol levels. In the gray rabbit there is a distinct rise in brain lipid with age; also a rise in lung lipid, and relatively few changes in liver, muscle and plasma lipids s. The observations of increased brain lipids have been confirmed in the N e w Zealand rabbit 9. The greatest changes take place between the first and 14th days of life. During this time there is a 5-fold increase in cholesterol and a 12-fold increase in cerebroside. The type of sterol present in the brain also changes with age. Brain of newborn rats contains an appreciable level of 24-dehydrocholesterol (desmosterol) which disappears by the time myelination has been completed 10. The rise in blood cholesterol which is observed with age can be due to increased synthesis, decreased catabolism and excretion, or increased absorption. The effect of age on cholesterol synthesis was studied over a quarter of a century ago. In the earliest T A B L E III INFLUENCE OF AGE ON INCORPORATION CHOLESTEROL
OF
[1-2H]ACETATE INTO LIVER
After Bloch et al. n.
Rat weight (g)
No.
230 150-170 120-130
4 3 2
Atom % excess ZH 0.09 i 0.01 0.32 4- 0.07 1.02 (0.73 - 1.30) :[: 0.29
T A B L E IV EFFECT OF AGE ON BILIARY OUTPUT
OF CHOLESTEROL
IN RATS
After Rosenman and Shibata 12. Age (weeks)
No.
Weight (g )
Plasma Bile cholesterol (mg/lO0 ml) Vol.
(mO
6 8 9 20 28 44
9 6 12 12 10 10
116 181 184 310 373 406
Mech. Age. Dev., 1 (1972) 275-284
65 43 48 64 47 47
7.1 11.1 12.0 14.5 15.7 15.3
Cholesterol mg/lO0 ml
mg total
mg/g body wt
23.6 26.6 24.1 16.8 12.8 12.5
1.7 2.9 3.0 2.4 2.0 1.9
0.014 0.016 0.016 0.007 0.005 0.004
277
days of work o n cholesterol biosynthesis, Bloch e t al. H showed a significant decrease in liver sterol synthesis with age (Table III). I n a study involving a p a r a m e t e r of biosynthesis other t h a n isotope i n c o r p o r a t i o n , viz. biliary o u t p u t of cholesterol (Table IV), R o s e n m a n a n d Shibata 1-~ showed almost a 50 % decrease in cholesterol o u t p u t between 6 a n d 44 weeks of life. Calculation of mg cholesterol excreted per g body weight shows a relatively high level at 6-9 weeks of life (0.014 mg) and a drop to a b o u t 0.006 mg at 20-44 weeks of life. T r o u t e t al. 13 showed that the skin, t e n d o n a n d aorta of y o u n g rats (under 4 weeks) incorporate more [14C]acetate into free and ester sterol than do tissues of rats
TABLE V STEROL SYNTHESIS FROM [1-14C]ACETATE IN RAT TISSUES (COMPARISON OF OLD (1.25-3 YEAR) TO YOUNG RATS) After Trout et al. 13. Tissue
Ratio o f activities (old/young) Old/O.~4-week rats r
Skin free sterol Skin ester sterol Liver free sterol Liver ester sterol Tendon free sterol Aorta free sterol
OId/6-week rats
~
28 10 23 27 1 7
60 70 34 35 7 77
TABLE VI STEROL METABOLISM IN RATS After Yamamoto and Yamamura15. Conditions
14C as percent o f that in 2-month-old rats 5-month-old
8-month-old
57 45
37 27
60 58 55
30 32 32
Fecal 14C after intraperitoneal [14C]cholesterol: Total 61 Bile acids 54 Non-saponifiable 63
41 36 41
278
Mech. Age. Dev., 1 (1972) 275-284
Biosynthesis from acetate: In vivo In vitro
Biliary 14C after intravenous [14C]cholesterol: Total Bile acids Non-saponifiable
TABLE VII ENDOCRINE INFLUENCE ON CHOLESTEROL TURNOVER IN RATS After Hruza TM. Treatment
Hypophysectomy Thyroidectomy Thyroxine Insulin
Ratio of cpm in serum (Control vs. treated) Young (6 weeks)
Old (13 months)
+55* +48* --22* --29*
+40* --13 + 15 + 15
* Significant difference.
TABLE VIII LIPID METABOLISM IN EPIDIDYMAL FAT OF RATS After Benjamin et al. 19. Glucose
Young (90-40 g)
OM (350-400 g)
Rate of acetate incorporation (Mmoles/3h/g)
--
20
1.9
Palmitic acid -+ triglycerides (/~moles/2 h/g)
-+
1.8 15.1
0.8 5.2
Palmitic acid ~ CO2 (/~moles oxidised/2 h/g)
-+
Lipolysis -(/~equivs free fatty acid/2 h/g) +
0.20 0.06
0.04 0.04
3.5 2.6
3.5 1.3
6-10 weeks of age (Table V). Their findings with y o u n g rat skin confirm those of Srere et al. 14. Y a m a m o t o a n d Y a m a m u r a 15 studied synthesis a n d excretion of cholesterol in rats aged 2, 5 a n d 8 months. A t 5 m o n t h s there is a b o u t a 50-60 ~ drop i n all three parameters, while at 8 m o n t h s the r e d u c t i o n is in the range of 60-70 ~o (Table VI). There is one report, however, which claims that there is n o difference in cholesterol synthesis in y o u n g or old rats 16. HruzaZ7, is has studied cholesterol t u r n o v e r in y o u n g a n d old rats. He f o u n d a decreased t u r n o v e r in older rats. Cholesterol t u r n o v e r in y o u n g rats is reduced by h y p o p h y s e c t o m y a n d t h y r o i d e c t o m y a n d increased by thyroxine or insulin treatment. I n older rats h y p o p h y s e c t o m y significantly increases cholesterol t u r n o v e r b u t the Mech. Age. Dev., 1 (1972) 275-284
279
other treatments have relatively little effect (Table VII). These findings indicate the interrelationships between endocrine factors and lipid metabolism. Benjamin et al. 19, studying adipose tissue metabolism in the rat, found reduced lipid synthesis and oxidation (Table VIII) with aging. Analysis of the triglyceride fatty acids showed a marked reduction in C10, CI~ and C14 fatty acids with aging, and an increase in Cls:I and Cls:z. In animals being fed laboratory chow for most of their lives, these changes may merely reflect the composition of the dietary fat. In a study of human adipose tissue, Insull and Bartsch 20 found increases in palmitic and oleic acids with age and attributed these changes primarily to the dietary pattern. Lipolysis of triglycerides is the major metabolic source of free fatty acids. Since aging seems to affect other aspects of lipid metabolism, its influence upon lipolytic systems is of interest. Zemplenyi and Grafnetter zl found that rat aorta slices were able to hydrolyze the lipids present in lipemic human serum. The extent of lipolysis was assayed by measurement of the free fatty acids released. In two series of experiments (Table IX) they demonstrated significantly greater lipolytic activity in aortas of 7month-old rats than in aortas of rats aged 2, 12 or 24 months. The lowest lipolytic activity was found in the 2-month-old rats. The lipolytic activity of the myocardium for 2-, 7- and 12-month-old rats (10 per group) was 11.27, 12.80 and 13.42, respectively. The difference between the 2- and 7-month-old groups was highly significant (P < 0.001). Dury 22 also found a greater degree of lipolysis (using a fat emulsion as substrate) in old (15-18-month-old) compared to young (3-4-month-old) rat aortas. Heparin, administered 15 min before the aorta was taken, significantly enhanced lipolysis in the young aortas but had no effect on the old ones. In man, Nikkila and Niemi 23 found that the activity of heparin-induced lipoprotein lipase was significantly higher in the sera of subjects aged 20-39 years when compared to subjects aged 63-92 years (P < 0.01). The time required for clearing of a standard fat meal is three times higher in old patients z4. The foregoing data suggest a progressive inability to metabolize lipid in the
TABLE IX L1POLYTIC ACTIVITY OF RAT AORTA After Zemplenyi and Grafnetter21. Rat age
No.
Lipolysis (mequivs fatty acid/g tissue ~- S.E.)
Experiment 1 : 3 months 7 months 12 months
10 7 7
4.38 i 0.19 7.43 ~ 1.14 5.90 i 0.42
Experiment 2: 2 months 7 months 24 months
7 8 8
4.84 i 0.74 8.77 ~ 0.49 6.01 ~ 0.52
280
Mech. Age. Dev., 1 (1972) 275-284
aging organism. This is seen in b o t h synthesis and degradation o f fat. In the face o f dietary fat, the decreased synthesis cannot compensate for decreased catabolism. The development and availability of h u m a n diploid cell strains 25 provides a unique system for the study of fundamental biochemical p h e n o m e n a at the cellular level. The observation 26 that these cells display a limited capacity for doubling (which m a y be interpreted as senescence at the cellular level) makes them an interesting system in which to study biochemical aspects o f aging. We have studied~7, 2s the lipid composition of early and late passage WI-38 cells. There is an increase in total lipid o f late passage cells, but the neutral lipid: phospholipid ratio is unchanged. The only significant differences between specific lipid components are in the phospholipid fraction. The older cells show significant decreases in phosphatidylethanolamine (P < 0.025) and phosphatidylinositol (P < 0.025) and an increase in lecithin (P < 0.005) (Table X). These changes m a y reflect differences in m e m b r a n e characteristics, but this possibility has not yet been investigated. The fatty acid spectra of the neutral lipids and phospholipids o f the cells (Table XI) are similar. The WI-38 cells synthesize cholesterol f r o m [14C]acetate when maintained in
TABLE X PERCENTAGE* LIP1DS OF EARLY AND LATE PASSAGE WI-38 CELLS Reprinted with permission from Kritchevsky and Howard 2s.
Total lipid (mg/100 mg dry wt): Total neutral lipid:
Early passage
Late passage
(19 i 1.5)
(25 ~2.6)
30 4- 2.2
32 i 2.5
% total neutral lipM
Cholesteryl ester Triglyceride Free fatty acid Diglyceride Cholesterol Monoglyceride Total phospholipid:
9.3 4- 2.6 11 4- 1.9 29 + 1.9 11 4- 2.6 32 + 1.7 7.1 t 1.3
11 ± 1.4 14 :k 1.7 32 ± 4.1 7.7 ± 0.9 30 4- 2.2 10 4- 4.4
69 4- 2.2
68 ± 2.5
% totalphospholipM
Phosphatidylethanolamine Phosphatidylinositol Phosphatidylserine Lecithin Sphingomyelin Lysolecithin
12 -4- 1.7 15 zk 2.7 8.0 zk 0.9 53 -4- 3.7 9.6 -4- 1.9 2.5 ~- 0.5
6.9 4- 1.7 8.4 ± 1.7 8.9 4- 3.4 70 ± 3.8 9.6 ± 0.9 2.5 ± 0.4
* Mean -4- S.E. of 8 determinations. Mech. Age. Dev., 1 (1972) 275-284
281
serum-containing medium 29, but the synthetic rate is very slow. The conclusion that this might be due to the presence of exogenous cholesterol has been proven experimentally by Rothblat et al. 3°. These authors showed that WI-38 cells can synthesize significant amounts of cholesterol when grown in the absence of exogenous lipid (delipidized serum protein)31, 32. The addition of cholesterol to this medium can inhibit endogenous cholesterogenesis by more than 95 ~. Chang 33 has studied lipid metabolism in human amnion cells. The uptake of [14C]cholesterol or [14C]palmitate by these cells increases with age. Thus, in two experiments the uptake of [14C]cholesterol by 42- and 70-day-old cells was 381 and 4 5 4 ~ of that observed in 14-day-old cells. The uptake of [1-14C]palmitate by aging cells was increased, and its oxidation to 14CO2 was decreased, resulting in a net accumulation of cellular lipid. The 14C-labeled lipid:14CO2 ratio for 14-, 42- and T A B L E XI F A T T Y A C I D S O F E A R L Y A N D L A T E P A S S A G E WI-38 C E L L S Reprinted with permission f r o m Kritchevsky a n d H o w a r d 2s.
Fatty acid
Myristic Myristoleic Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic Arachidonic
Carbon No.
Neutral lipids (%)
Phospholipids (%)
Early passage (3) *
Late passage (3) *
Early passage (5) *
Late passage (6) *
14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 20:4
2.1 ± trace 22 ± trace 17 ± 33 -2 7 -2 trace 20 -2
0.41 -t- 0.08 trace 20 -2 0.91 0.40 -2 0.08 19 -2 2.1 31 -2 1.3 10 -2 0.91 trace 19 -2 1.5
3.2 -2 trace 23 -2 trace 17 -2 32 -2 6.1 -2 trace 19 -2
1.8 -2 0.60 t race 23 -2 2.0 0.31 -2 0.04 20 -2 1.5 30 -2 2.5 7.5 -2_ 0.36 1.9 -2 0.20 17 -2 1.9
0.57 2.2 2.2 0.4 1.3 3.2
0.16 1.8 1.9 1.4 0.87 2.7
* N u m b e r o f determinations.
T A B L E XII E F F E C T O F H Y D R O C O R T I S O N E O N [ 2 6 - 1 4 C ] C H O L E S T E R O L A N D [1-14C]OLEIC A C I D M E T A B O L I S M BY P H A C U L T U R E S After Y u a n a n d C h a n g a L
Age (days)
31 59 87 115
282
No. expts
4 4 2 1
Cells )< 104 per culture
Oleic acid (Lipid/C02)
--
+
--
32 17 8 6
146 155 102 69
15.6 10.2 21.1 20.7
Cholesterol uptake (cpm) ÷
4.3 3.4 6.8 2.8
--
+
303 428 1625 3014
267 229 424 1180
Mech. Age. Dev., 1 (1972) 275-284
70-day-old cells was 0.3, 6.7 a n d 8.3, respectively. O x i d a t i o n o f [6-14C]gIucose was n o t r e d u c e d in the aging cells, indicating an i m p a i r m e n t o f the fatty acid o x i d a t i o n system, b u t n o t o f the tricarboxylic acid cycle. Recently Y u a n a n d C h a n g 34 have o b s e r v e d that the a d d i t i o n o f h y d r o c o r t i s o n e to a m n i o n cells abolishes the a g e - d e p e n d e n t increase in 14C-labeled lipid :14CO2 ratio in cells fed [l-14C]oleic acid a n d r e t a r d s the increase in cholesterol u p t a k e seen with aging (Table XII). H y d r o c o r t i s o n e a n d cortisone have also been shown to extend the life-span o f WI-38 h u m a n diploid cells 35. The m e c h a n i s m by which these changes t a k e place has n o t been elucidated. In s u m m a r y , aging animals exhibit a progressive inability to handle lipid. W h i l e they synthesize less lipid, they also m e t a b o l i z e less, the result being an ager e l a t e d increase o f lipid in tissues a n d blood. H u m a n d i p l o i d cells, which seem to age in vitro, also exhibit some o f the same m e t a b o l i c characteristics as intact organisms with r e g a r d to lipid m e t a b o l i s m . These cells offer a unique o p p o r t u n i t y to study the mechanisms underlying the changes in lipid m e t a b o l i s m o b s e r v e d with aging. W e have a c c u m u l a t e d e n o u g h d a t a relating to the lipid c o n t e n t o f aging tissues a n d these d a t a are generally consistent regardless o f species. W e need n o w to u n d e r s t a n d why these changes t a k e place a n d h u m a n diploid cells offer a u n i q u e substrate for such investigations.
REFERENCES 1 M. Werner, R. E. Tolls, J. V. Hultin and J. Mellecker, Influence of sex and age on the normal range of eleven serum constituents, Z. Klin. Chem. Klin. Biochem., 8 (1970) 105-115. 2 B. C. Das and S. K. Bhattacharya, Variation in lipoprotein level with changes in age, weight and cholesterol ester, Gerontologia, 5 (1961) 25-34. 3 N. N. Kipshidze, Quantitative changes in lipid, protein and carbohydrate metabolism in relation to age, Thule International Symposium on Cancer and Ageing, 1967, pp. 49-57. 4 L. A. Carlson, S. O. FrSberg and E. R. Nye, Effect of age on blood and tissue lipid levels in the male rat, Gerontologia, 14 (1968) 65-79. 5 D. Kritchevsky and S. A. Tepper, Serum cholesterol levels of inbred rats, Am. J. Physiol., 207 (1964) 631-633. 6 T. F. Zucker and L. M. Zucker, Fat accretion and growth in the rat, J. Nutr., 80 (1963) 6-19. 7 T. F. Zucker and L. M. Zucker, Phosphatides and cholesterol in the rat body: Effects of growth, diet and age, J. Nutr., 80 (1963) 20-24. 8 J. P. Hrachovec and M. Rockstein, Age changes in lipid metabolism and their medical implications, Gerontologia, 3 (1959) 305-326. 9 K. B. Dalal and E. R. Einstein, Biochemical maturation of the central nervous system. I. Lipid changes, Brain Res., 16 (1969) 441-451. 10 D. Kritchevsky and W. L. Holmes, Occurrence of desmosterol in developing rat brain, Biochem. Biophys. Res. Commun., 7 (1962) 128-131. 11 K, Bloch, E. Borek and D. Rittenberg, Synthesis of cholesterol in surviving liver, J. Biol. Chem., 162 (1946) 441-449. 12 R. H. Rosenman and E. Shibata, Effect of age upon hepatic synthesis of cholesterol in the rat, Proc. Soc. Exp. Biol. Med., 81 (1952) 296-298. 13 E. C. Trout, Jr., K. T. Kao, C. A. Hizer and T. H. McGavack, In vitro synthesis of free andester cholesterol in various tissues of young and old rats, J. Gerontol., 17 (1962) 363-368. 14 P. A. Srere, I. L. Chaikoff, S. S. Treitman and L. S. Burstein, The extrahepatic synthesis of cholesterol, J. Biol. Chem., 182 (1950) 629-634. 15 M. Yamamoto and Y. Yamamura, Changes of cholesterol metabolism in the aging rat, Atherosclerosis, 13 (1971) 365-374. Mech. Age. Dev., 1 (1972) 275-284
283
16 K. A. Tretiakova and D. E. Grodsensky, The rate of cholesterol and fatty acid synthesis in the adrenals, testicles and liver of young and old rats both normal and irradiated, Biokhemia, 25 (1960) 399-403. 17 Z. Hruza and M. Wachtlova, Decrease of cholesterol turnover in old rats, Exp. Gerontol., 4 (1969) 245-250. 18 Z. Hruza, Effect of endocrine factors on cholesterol turnover in young and old rats, Exp. Gerontol., 6 (1971) 199-204. 19 W. Benjamin, A. Gellhorn, M. Wagner and H. Kundel, Effects of aging on lipid composition and metabolism in the adipose tissues of the rat, Am. J. Physiol., 201 (1961) 540-546. 20 W. Insull, Jr. and G. E. Bartsch, Fatty acid composition of human adipose tissue related to age, sex and race, Am. J. Clin. Nutr., 20 (1967) 13-23. 21 T. Zemplenyi and D. Grafnetter, The lipolytic activity of the aorta: Its relation to aging and to atherosclerosis, Gerontologia, 3 (1959) 55-64. 22 A. Dury, Lipolytic activity of aorta of young and old rats and influence of heparin in vivo, J. Gerontol., 16 (1961) 114 117. 23 E. A. Nikkila and T. Niemi, Effect o f a g e o n the lipemia clearing activity of serum after administration of heparin to human subjects, J. Gerontol., 12 (1957) 44-47. 24 G. H. Becker, J. Meyer and H. Necheles, Fat absorption in young and old age, Gastroenterology, 14 (1950) 80-90. 25 L. Hayflick and P. S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res., 25 (1961) 585-621. 26 L. Hayflick, The limited in vitro lifetime of human diploid cell strains, Exp. Cell Res., 37 (1965) 614-636. 27 D. Kritchevsky and B. V. Howard, The lipids of human diploid cell strain W1-38, Ann. Med. Exp. Fenn., 44 (1966) 343-347. 28 D. Kritchevsky and B. V. Howard, Lipid metabolism in human diploid ceils, in E. Hole6kov~t and V. J. Cristofalo (Eds)., Aging in Cell and Tissue Culture, Plenum Press, New York, 1970, pp. 57-82. 29 B. V. Howard and D. Kritchevsky, The lipids of normal diploid (WI-38) and SV40 transformed human cells, Int. J. Cancer, 4 (1969) 393-402. 30 G. H. Rothblat, R. Boyd and C. Deal, Cholesterol biosynthesis in WI-38 and WI-38VA13A tissue culture cells, Exp. Cell Res., 67 (1971) 436-440. 31 G. H. Rothblat, M. K. Buchko and D. Kritchevsky, Cholesterol uptake by L5178Y tissue culture cells: Studies with delipidized serum, Biochim. Biophys. Acta, 164 (1968) 327-338. 32 G. H. Rothblat, The effect of serum components on sterol biosynthesis in L cells, J. Cell. Physiol., 74 (1969) 163-170. 33 R. S. Chang, Metabolic alterations with senescence of human cells, Arch. Int. Med., 110 (1962) 563-568. 34 G. C. Yuan and R. S. Chang, Effect of bydrocortisone on age-dependent changes in lipid metabolism of primary human amnion cells in vitro, Proc. Soc. Exp. Biol. Med., 130 (1969) 934-936. 35 V. J. Cristofalo, Metabolic aspects of ageing in human diploid cells, in E. Hole6kov~i and V. J. Cristofalo (Eds.), Aging in Cell and Tissue Culture, Plenum Press, New York, 1970, pp. 83-119.
284
Mech. Age. Dev., 1 (1972) 275-284