Lipids
Dietary Essential Fatty Acids Change the Fatty Acid Profile of Rat Neural Mitochondria Over Time1 Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Canada M5S 1A8 evident in the phospholipid cardiolipin (CL), also called diphosphatidyl glycerol, because it contains by far the highest concentrations of 18:2(i2-6) in the brain. Cardiolipin is found almost exclusively in the mitochondria (4). This present report demonstrates that the fatty acid profile of neural mitochondria could be altered by dietary fat in a relatively short time. In the work presented in this paper, the time course of this relationship was explored more fully.
ABSTRACT This experiment examined the time course over which the amount of dietary essential fatty acids (EPA) affects brain mitochondria! fatty acids. Weanling rats were fed 20% (wt/wt) fat diets that contained either 4 or 15% (wt/wt of diet) EPA for 1, 2, 3 or 6 wk or a 10% EPA diet for 3 or 6 wk. The EPA ratio (18: 2(n-6)/18:3(n-3)] of all diets was -30. Fatty acid analysis of brain mitochondria! phosphatidylethanolamine, phosphatidylcholine and cardiolipin revealed that the largest dietary effect was on 18:2(n-6), which was 30% higher in rats fed the 15 vs. 4% EPA diets after 1 wk. This difference increased to twofold by 3 wk and was still twofold after 6 wk. These results demonstrate several facts: 1) the response of 18:2(n-6) in cardiolipin to dietary EPA is very fast and large, relative to changes in other quantitatively major fatty acids observed in weanling rats; 2) the 18:2(n-6) level in neural cardiolipin stabilizes after 3 wk of feeding at a level dependent upon the amount of dietary EPA; and 3) at least one neural fatty acid, 18:2(n-6), is very sensitive to amounts of dietary EPA that are well above the animal's EPA re quirement. J. Nutr. 121: 1548-1553, 1991.
MATERIALS AND METHODS
INDEXING KEY WORDS:
•essential fatty adds •neural fatty acids •rats •cardiolipin •llnolelc add
Recently, we found that the level of linoleic acid [18:2(a-6)] in the brain of weanling rats could be al tered twofold by dietary fatty acid composition after 4 wk of feeding (Dyer, J. R. and Greenwood, C. E., unpublished data). The specific dietary fat character istic responsible for this effect was later identified as being the total amount of the two essential fatty acids (EFA)4 18:2(B-6) and oc-linolenic acid [18:3(u-3)] (1). The diets used in these experiments were not defi cient in EFA and, therefore, the neural 18:2(u-6) was responding to changes in the dietary EFA that were well above what is presently considered to be the animal's requirement (2) and within the range typi cally consumed by humans (3). The change in the neural 18:2(n-6) level was most
Materials. All nonfat dietary ingredients were pur chased from Teklad Diets (Madison, WI) except cornstarch (St. Lawrence Starch, Mississauga, ON, Can ada). The beef tallow was generously donated by Canada Packers Co. (Toronto, ON). The dietary oils were all cold-pressed and were bought from several local outlets. All chemicals were purchased from Sigma Chemical (St. Louis, MO), except fatty acid methyl ester standards (NuChek Prep, Elysian, MN) and sucrose (Analar, BDH Chemicals Canada, To ronto, ON). Chloroform, methanol and hexane were double-distilled and obtained from the University of Toronto's Solvent Distillation Service. Iso-octane (HPLC grade) was purchased from Fisher Chemicals (Fair Lawn, NJ). Thin layer chromatography plates and gas chromatography capillary column were
'Supported by grants from the Medical Research Council of Canada and the International Life Sciences Institute-Nutrition Foundation. JRD was the recipient of a Medical Research Council Studentship. 2Present address: Neurological Research Unit, Livingston Hall, Room 110, Montreal General Hospital, 1625 Pine Ave., Montreal, PQ, Canada H3G 1A4. ^o whom reprint requests should be addressed. 'Abbreviations used: CL, cardiolipin; EFA, essential fatty acids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PUPA, polyunsaturated fatty acids; UI, unsaturatÃ-on index.
0022-3166/91 $3.00 ©1991 American Institute of Nutrition. Received 14 January 1991. Accepted 15 April 1991. 1548
Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
JOHN R. DYER2 Ano CAROL E. GREENWOOD3
DIETARY EFA AND NEURAL FATTY ACID CHANGES
OVER TIME
1549
TABLE 1 Dietary fatty add composition.1 Diet
SFA2
MUTA
PUTA
P/S
18:2(n-6)
diet7.74.92.74.29.915.00.541.996.69g/1004.19.614.5g g
18:3|n-3)
EFA ratio3
diet0.120.320.5233.730.228.2
'All diets contained 20% (wt/wt) fat; 18:2(n-6) and 18:3(n-3) were the only (n-6) and (n-3) fatty acids present in significant amounts. Fatty acid composition was determined by gas chromatography. Abbreviations used: EFA, essential fatty acids; MUFA, monounsaturated fatty acids; PUF A, polyunsaturated rated fatty acids/saturated fatty acids, wt/wt; SFA, saturated fatty acids. 3EFA ratio = 18:2(n-6)/18:3(n-3), wt/wt.
obtained through Chromatographie Specialties (Brockville, ON). Experimental design. The purpose of this ex periment was to determine the time course over which the amount of dietary EFA alters the fatty acid profile of mitochondria in the brain. Rats were divided into eleven groups of six rats based on body weight. One group was killed at the beginning of the experiment to establish the initial neural fatty acid profile. Eight groups were fed diets containing 4 or 15% (wt/wt) EFA for 1, 2, 3 or 6 wk. The remaining two groups were fed a 10% EFA diet for 3 or 6 wk. At the end of the experimental periods, animals were decapitated (900-1000 h) and their brains quickly re moved, wrapped in foil, and covered with ice until membrane fractionation was conducted. Diets. Semipurified diets (as described in réf.5) were made, each containing (g/100 g): protein as casein (23), comstarch (40), mineral mixture (5.1, Bemhart-Tomarelli, réf.6), fiber (5, alpha-cellulose), vitamin mixture (2.5, TD67231, réf.7), and supple mental L-methionine (0.25). All diets contained 20% (wt/wt) fat. The diets contained different proportions of beef tallow, safflower oil and linseed oil in order to vary the amount of EFA, but keep the EFA ratio [18: 2(n-6)/18:3(n-3)] at -30 (Table 1). AU diets were ade quate in EFA. The fatty acid composition of the fat mixtures were determined by gas chromatography as described below. Animals. Male weanling Wistar rats (Charles River Breeding Labs, St. Constant, PQ, Canada), initially •weighing 50-70 g, were individually housed in a room with a 12-h lightrdark cycle (lights on 700-1900 h) and allowed free access to both water and food. Body weights and food intakes were monitored throughout the experimental period. Membrane fractionation and lipid analysis. Mem brane fractionation on sucrose density gradients was conducted immediately following decapitation, using the method of Whittaker and Barker (8). Mitochondria were collected and kept at -70°Cuntil lipids were extracted. Lipid was extracted from each membrane fraction by the method of Folch et al. (9). Cardiolipin,
fatty acids; P/S - polyunsatu-
phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were separated by TLC using a modified version of the method of Kennedy et al. (10). Fatty acid methyl esters were prepared from each phospholipid by the method of Morrison and Smith (11) and analyzed by gas chromatography. The 18:l(n-9) and 18:l(i2-7) did not reliably separate and are reported as the total 18:1 isomers. Details of these methods were given previously (5). Statistical analysis. Statistical analysis was done using SAS 6.03 (SAS Institute, Cary, NC) for the microcomputer. The effects of die amount of dietary EFA and the duration of feeding (time) on individual fatty acids and die unsaturation index (UI),5 were tested using multivariate and univariate ANOVA for each phospholipid. Groups fed different levels of EFA were compared using Student's t test.
RESULTS The energy intake, body weight gain and brain weight were similar for animals fed each diet, except for body weight gain at 2 wk (Table 2). The multivariate ANOVA tested the effects of diet and time on all of die fatty acids togedier and re vealed that dietary EFA, time and dieir interaction had significant effects on die mitochondrial fatty acid profile in each phospholipid (P < 0.001), except for die interaction in PE (P > 0.05). Time was also significant in the univariate analyses of individual fatty acids, dieir ratios and die UI in all cases, except 22: 6(n-3) in PC (P = 0.066). Dietary EFA had a very pronounced influence on certain fatty acids, but not on otiiers. The greatest impact of dietary EFA on the mitochondrial fatty acid composition was evident in 18:2(n-6) (Fig. 1). In each
5Unsaturation index (UI) - Ejnumber of double bonds in each fatty acid) x (mol/100 mol of each fatty acid).
Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
4% EFA10% EFA15% EFA7.75.02.2g/100
DYER AND GREENWOOD
1550
TABLE 2 Energy intake, body weight gain and brain weight of weanling rats fed different levels of essential fatty acids (EFA) for 6 wk' Diet (% EFA)
Time (wk)
Energy intake
Body weight gain
Brain weight Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
1.50 ±0.02
41541541015410151122333666144314433389343156325519520512,41412,48912,606±±±±±±±±±±331392672051131
'Values are means ±SEM(n - 6). Energy intake and body weight gain are cumulative Significant
difference between
values for the time periods specified.
diets (P < 0.05 by t test).
phospholipid, only 1 wk of feeding was sufficient to result in significantly greater levels of 18:2(n-6) in the high compared with the low EFA groups {P < 0.05, t test). By wk 3, a twofold difference was present that seems maximal because the difference was of the same magnitude at 6 wk. The divergence in the 18: 2(n-6) levels resulted from the combination of a decrease from baseline (0 wk) in animals fed the low EFA diet and an increase in animals fed the high EFA diet. The percentage of 18:2(n-6) in the mitochondria from animals in the intermediate EFA group fell roughly midway between the percentages of 18: 2(n-6) in the other two groups at both 3 and 6 wk. Therefore, stable 18:2(n-6) levels were reached after a 3-wk feeding period, regardless of the amount of EFA in the diet. The mitochondria! saturated fatty acids were unaf fected by the amount of EFA in the diet, with the exception of inconsistent changes in 16:0 from CL. Approximately 10, 6 and 42% of the fatty acids in CL, PE and PC, respectively, were present as 16:0; 5, 25 and 10% of the fatty acids in CL, PE and PC, respec tively, were present as 18:0 (data not shown). Mod erate differences in the monounsaturated fatty acids occurred in response to the dietary EFA, particularly in CL where they are the most abundant. The levels of 16:l(B-7) (data not shown) and the total 18:1 isomers in the low EFA group were 5-15% higher than in the high EFA group (Fig. 1). By wk 1, the difference in 18:1 levels in CL was maximal and it remained constant through wk 6. Animals fed the intermediate EFA diet had values of 18:1 isomers in CL that were between those of the other two dietary groups at wk 3 and 6. In contrast to the other fatty acids, 22:5(n-6) was altered in PE and PC (but not in CL) by dietary EFA (Fig. 2). With time, the level of 22:5(n-6) increased in
all phospholipids, but the low EFA diet elevated 22: 5(fl-6)levels in PE and PC more than did the high EFA diet. The effect was greater in PE than in PC (50 vs. 25%), but was too erratic in CL to be significant. Not all of the PUFA in the membrane reacted to the amount of EFA in the diet. Diet had no effect on either 20:4(c-6) (Fig. 1) or 22:4(iz-6)(Fig. 2). Only in CL was 22:6(n-3) influenced by dietary EFA, with levels in animals fed the low EFA diet being -15% greater than those in animals fed the high EFA diet by wk 2 (Fig. 2). The dietary EFA had no impact on the membrane UI; however, the overall (n-6)/(n-3) ratio was signifi cantly greater in animals fed the high EFA diet. Changes in the membrane (n-6)/(n-3) ratio paralleled those observed in membrane 18:2(n-6) and were greater in CL due to the magnitude of changes in 18: 2(jQ-6)in this phospholipid (data not shown).
DISCUSSION Dietary fatty acid composition can alter the neural fatty acid profile in rats (for reviews see réf.12, 13). Numerous reports (14-16) have demonstrated that the 20- and 22-carbon PUFA in the brain are affected by a deficiency of EFA. In addition, many studies (17-19) have shown that the proportion of (12-6)and (fl-3)22-carbon PUFA in the brain respond to the ratio of EFA in the diet, hi both cases, the effect of dietary fat on the brain of rats is greater when imposed during the fetal and neonatal stages than after weaning be cause in rats the majority of the neural 20- and 22-carbon PUFA have been deposited in the brain by weaning (20). The speed and magnitude of the change in neural 18:2(n-6) reported here, and its sensitivity to
DIETARY
EFA AND
CARDIOLIPIN
40 38
18:1
NEUR'
T. FATTY
ACID
CHANGES
OVER TIME
PHOSPHATIDYLETHANOLAMINE
1551
PHOSPHATIDYLCHOLINE
Total
36 32 30 22
18:2fn6i
< 18
m 14 u. 0s 10
22 20
20:4fn61
12 20
18
19
16
18
14
11 10
17 1
o
23456
1
Weeks on Diet FIGURE 1 The effect of the amount of dietary essential fatty acids (EFA) over time on the 18- and 20-carbon fatty acids in neural mitochondria from weanling rats. Animals were fed 20% (wt/wt) fat diets containing either 4, 10 or 15% (wt/wt diet) EFA for the specified time periods. Fatty acid profiles of individual mitochondrial phospholipids were measured by gas-liquid chromatography. Fatty acid values are expressed as the percentage (wt/wt) of total fatty acids,- values are means ±SEM (n - 6/ diet x time). Statistical analysis by ANOVA (P < 0.05): A - significant effect of EFA amount; I - significant diet by time interaction; time was significant in all cases.
amounts of dietary EFA within a range considered adequate, are novel. That is, these data demonstrate that major changes in neural fatty acid profile, in response to changes in dietary fatty acid composition, occur even beyond periods of brain growth and rapid fatty acid accretion, with alterations observed in as little as 1 wk of feeding. Furthermore, these data show that an EFA deficiency need not be imposed for neural fatty acid profile to be influenced. Rather, the dietary fatty acids fed in this experiment are represen tative of fats typically consumed by humans (3). The response of 18:2(n-6) in CL to dietary EFA was very fast and large relative to changes in other quanti tatively major fatty acids observed in weanling rats. After only 1 wk, the level of 18:2(n-6) in the 15% EFA group was one-third higher than in the 4% EFA group, and there was a twofold difference by wk 3. In com parison, it took 12 wk for the dietary EFA ratio (1.8 vs. 165) to cause a 20% difference in the levels of 22: 6(n-3) from synaptosomal PE (19). Only one other
study (21) has examined the effect of dietary fat com position on the fatty acid profile of neural CL over such a short time period, and it also found a twofold difference in 18:2(n-6) levels after only 10 d of feeding young rats either com or sardine oil diets. A feeding period of longer than 3 wk did not cause any greater divergence in the neural 18:2(n-6) levels of animals fed the low, moderate or high EFA diets. Therefore, 3 wk was sufficient time for the mitochon drial fatty acid changes to reach their maximum. Furthermore, there was no indication that any com pensatory mechanisms were activated in the body (or brain) to reverse the effect of dietary EFA, because the levels of membrane 18:2(n-6) in each dietary group were constant relative to each other at 3 and 6 wk. Generally, dietary EFA altered the fatty acid com position of CL from neural mitochondria more than that of PE or PC because the largest absolute and relative effect of dietary EFA was on 18:2(n-6) and it is concentrated in CL. Consequently, the level of 18:
Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
34
DYER AND
1552 CARDIOLIPIN 0.8
GREENWOOD
22:4(n6i
1.8
1.4
0.7
1.0
3.0
0.5
2.6
0.6
32
10
22:5(n6>
13 O < 0.8
0.4
10
22:6fn31
30 28 26 1
o
23456
1
Weeks on Diet FIGURE 2 The effect of the amount of dietary essential fatty acids (EFA) over time on the 22-carbon fatty acids in neural mitochondria from weanling rats. Animals were fed 20% (wt/wt) fat diets containing either 4, 10 or 15% (wt/wt diet) EFA for the specified time periods. Fatty acid profiles of individual mitochondrial phospholipids were measured by gas-liquid chromatography. Fatty acid values are expressed as the percentage (wt/wt) of total fatty acids; values are means ±SEM (n - 6/ diet x time). Statistical analysis by ANOVA (P < 0.05): A - significant effect of EFA amount; I - significant diet by time interaction,- time was significant in all cases.
2(n-6) is very probably affected directly by the dietary EFA, whereas the changes in the proportions of the other fatty acids in CL occur secondarily. In this case, there does not seem to be a reciprocal replacement of fatty acids of a similar type, as there is with the 22-carbon PUPA. The large rise in the percentage of 18:2(u-6) is counterbalanced by smaller decreases in the percentages of several other fatty acids of varying chain length and unsaturatìon,such as 16:l(n-7), the 18:1 isomers and 22:6(n-3). Not all of the fatty acids in CL were changed, however, so the replacement is not completely random and possibly is based on the positions of the fatty acids in CL. The speed with which dietary EFA altered the neural 18:2(n-6) levels is surprising, as is the obser vation that the effective range of dietary EFA was well above the rat's requirement for EFA (2). A defi ciency of EFA alters the neural fatty acid profile, but diets adequate in EFA have not been studied in this respect. Effects on the brain that result from varying
dietary EFA over a range of levels that provide the animal with adequate amounts of EFA are more relevant to humans than changes resulting from EFA deficiency. People rarely consume inadequate amounts of EFA, unless they have a defect in lipid absorption or are receiving one of the older parenteral nutrition formulations (22). The functional significance of altering the level of 18:2(n-6) in neural CL is unknown, although there is circumstantial evidence to support the hypothesis that the function of the brain is affected by the di etary fatty acid manipulations described in this paper. First, CL is notable for having by far the highest concentrations of 18:2(n-6) of any phospholipid, and for its almost exclusive occurrence in mitochondria (4). Second, several mitochondrial enzymes involved in oxidative respiration (23-27) and the transport of fatty acids across the inner mitochondrial membrane (28-31) are specifically associated with CL and/or require CL for optimal activity. Finally, modifications
Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
0.6
« 1.2
PHOSPHATIDYLCHOLINE
PHOSPHATIDYLETHANOLAMINE
DIETARY EPA AND NEURAL FATTY ACID CHANGES
LITERATURE CITED 1. Dyer, J. R. &.Greenwood, C. E. (1991) The level of linoleic acid in neural cardiolipin is linearly correlated to the amount of essential fatty acids in the diet of the weanling rat. J. Nutr. Biochem. (in press) 2. Rivers, J.P.W. &. Frankel, T. L. (1981) Essential fatty acid deficiency. Br. Med. Bull. 37: 59-64. 3. FAO (1982) Monthly Bull. Stat. 5: 11-20. 4. Sastry, P. S. (1985) Lipids of nervous tissue: composition and metabolism. Prog. Lipid Res. 24: 69-176. 5. McGee, C. D. &. Greenwood, C. E. (1989) Effects of dietary fatty acid composition on macronutricnt selection and synaptosomal fatty acid composition in rats. J. Nutr. 119: 1561-1568. 6. Bemhart, F. W. &. Tomarelli, R. M. (1969) A salt mixture supplying the National Research Council estimates of the mineral requirement of the rat. J. Nutr. 89: 495-500. 7. Musten, B., Peace, D. &. Anderson, G. H. (1974) Food intake regulation in the weanling rat. J. Nutr. 104: 563-572. 8. Whittaker, V. P. &. Barker, L. A. (1972) The subcellular fractionation of brain tissue with special reference to the prep aration of synaptosomes and their component organdÃ-es. In: Methods of Neurochemistry (Fried, R., ed.), vol. 2, pp. 1-52. Marcel Dekker, New York, NY. 9. Folch, J., Lees, M. &. Stanley, G.H.S. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509. 10. Kennerly, D. A., Sullivan, T. J. &. Parker, C. W. (1979) Acti vation of phospholipid metabolism during mediator release from stimulated rat mast cells. J. Immunol. 122: 152-159. 11. Morrison, W. R. &.Smith, L. M. (1964) Preparation of fatty acid methyl esters and dimethylacetals from lipides with boron trifluoride-methanol. J. Lipid Res. 5: 600-608. 12. Johnston, P. V. (1979) Nutrition and neural lipids. In: Advances in Nutritional Research (Draper, H. H., ed.), vol. 2, pp. 149-180. Plenum Press, New York, NY. 13. Menon, N. K. &. Dhopeshwarkar, G. A. (1982) Essential fatty acid deficiency and brain development. Prog. Lipid Res. 21: 309-326. 14. Mohrhauer, H. &.Holman, R. T. (1963) Alterations of the fatty acid composition of brain lipids by varying levels of dietary essential fatty acids. J. Neurochem. 10: 523-530. 15. Sinclair, A. J. &. Crawford, M. A. (1973) The effect of a low-fat maternal diet on neonatal rats. Brit. J. Nutr. 29: 127-137. 16. Ailing, C., Bruce, A, Karlsson, I. &. Svennerholm, L. (1974) The effect of different dietary levels of essential fatty acids on lipids of rat cerebrum during maturation. J. Neurochem. 23:
1553
1263-1270. 17. Lamptey, M. S. &.Walker, B. L. (1976) A possible essential role for dietary linolenic acid in the development of the young rat. J. Nutr. 106: 86-93. 18. Bourre, J. M., Pascal, G., Durand, G., Masson, M., Dumont, O. & Picio 11i, M. (1984) Alterations in the fatty acid composition of rat brain cells (neurons, astrocytes, and oligodendrocytes) and of subcellular fractions (myelin and synaptosomes) in duced by a diet devoid of n-3 fatty acids. J. Neurochem. 43: 342-348. 19. Dyer, J. R. &. Greenwood, C. E. (1991) Neural 22-carbon fatty acids respond rapidly and specifically to a range of dietary linoleic to a-linolenic fatty acid ratios. J. Neurochem. 56: 1921-1931. 20. Sinclair, A J. &. Crawford, M. A (1972) The accumulation of arachidonate and docosahexaenoate in the developing rat brain. J. Neurochem. 19: 1753-1758. 21. Yamaoka, S., Urade, R. &. Kito, M. (1988) Mitochondrial function in rats is affected by modification of membrane phospholipids with dietary sardine oil. J. Nutr. 118: 290-296. 22. Yamanaka, W. K., Clemans, G. W. & Hutchinson, M. L. (1981) Essential fatty acid deficiency in humans. Prog. Lipid Res. 19: 187-215. 23. Aswathi, Y. C., Chuang, T. F., Keenan, T. W. Ä.Crane, F. L. (1971) Tightly bound cardiolipin in cytochromc oxidase. Biochim. Biophys. Acta 226: 42-52. 24. Vik, S. B., Georgevich, G. & Capaldi, R. A (1981) Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase. Proc. Nati. Acad. Sci. USA 78: 1456-1460. 25. Robinson, N. C., Strey, F. & Talbert, L. (1980) Investigation of the essential boundary layer phospholipids of cytochrome c oxidase using Triton X-100 delipidation. Biochemistry 19: 3656-3661. 26. Fry, M. &. Green, D. E. (1980) Cardiolipin requirement by cytochrome oxidase and the catalytic role of phospholipid. Biochem. Biophys. Res. Comm. 93: 1238-1246. 27. Santiago, E., Lopez-Moratalla, N. &. Segovia, J. L. (1973) Corre lation between losses of mitochondrial ATPase activity and cardiolipin degradation. Biochem. Biophys. Res. Comm. 53: 439-445. 28. Bieber, L. L. &. Farrell, S. (1983) Camitine acyltransferases. In: The Enzymes (Boyer P.D., ed.), vol. 16, pp. 627-644. Academic Press, New York, NY. 29. Fiol, C. J. &. Bieber, L. L. (1984) Sigmoid kinetics of purified beef heart mitochondrial camitine palmitoyltransferase. f. Biol. Chem. 259: 13084-13088. 30. Pande, S. V., Murthy, M.S.R. & Noel, H. (1986) Differential effects of phosphatidylcholine and cardiolipin on camitine palmitoyltranferasc activity. Biochim. Biophys. Acta 877: 223-230. 31. Noel, H. &. Pande, S. V. (1986) An essential requirement of cardiolipin for mitochondrial camitine acylcarnitine translocase activity. Eur. J. Biochem. 155: 99-102. 32. Abuirmeileh, N. M. & Elson, C. E. (1980) The influence of linoleic acid intake on membrane-bound respiratory activities. Lipids 15: 918-924. 33. Abuirmeileh, N. M. &. Elson, C. E. (1980) The influence of linoleic acid intake on electron transport system components. Lipids 15: 925-931. 34. Innis, S. M. & Clandinin, M. T. (1981) Dynamic modulation of mitochondrial membrane physical properties and ATPase ac tivity by diet lipid. Biochem. J. 198: 167-175. 35. McMurchie, E. J., Abeywardena, M. Y., Chamock, J. S. & Gibson, R. A. (1983) Differential modulation of rat heart mito chondrial membrane-associated enzymes by dietary lipid. Bi ochim. Biophys. Acta 760: 13-24.
Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
to dietary fat that include changes in the amount of EFA have been shown in peripheral tissues to affect oxidative respiration and the activity of some of the enzymes that bind CL (21, 32-35). The lack of research concerning neural CL means that our experiments linking dietary EFA to 18: 2(n-6) in the brain have opened up a new line of investigation in the area of dietary fat and the brain. The findings reported here are exciting because the magnitude of the change that occurs in membrane 18: 2(n-6) is very large, and 18:2(n-6) responds to dietary fat much more quickly than other fatty acids, in cluding the 22-carbon PUFA. This undermines the idea that the mature brain is relatively indifferent to changes in dietary fat or to diet in general, and forces us to modify our thinking about how the developed brain reacts to dietary fat.
OVER TIME
Dietary Essential Fatty Acids Change the Fatty Acid Profile of Rat Neural Mitochondria Over Time1 Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Canada M5S 1A8 evident in the phospholipid cardiolipin (CL), also called diphosphatidyl glycerol, because it contains by far the highest concentrations of 18:2(i2-6) in the brain. Cardiolipin is found almost exclusively in the mitochondria (4). This present report demonstrates that the fatty acid profile of neural mitochondria could be altered by dietary fat in a relatively short time. In the work presented in this paper, the time course of this relationship was explored more fully.
ABSTRACT This experiment examined the time course over which the amount of dietary essential fatty acids (EPA) affects brain mitochondria! fatty acids. Weanling rats were fed 20% (wt/wt) fat diets that contained either 4 or 15% (wt/wt of diet) EPA for 1, 2, 3 or 6 wk or a 10% EPA diet for 3 or 6 wk. The EPA ratio (18: 2(n-6)/18:3(n-3)] of all diets was -30. Fatty acid analysis of brain mitochondria! phosphatidylethanolamine, phosphatidylcholine and cardiolipin revealed that the largest dietary effect was on 18:2(n-6), which was 30% higher in rats fed the 15 vs. 4% EPA diets after 1 wk. This difference increased to twofold by 3 wk and was still twofold after 6 wk. These results demonstrate several facts: 1) the response of 18:2(n-6) in cardiolipin to dietary EPA is very fast and large, relative to changes in other quantitatively major fatty acids observed in weanling rats; 2) the 18:2(n-6) level in neural cardiolipin stabilizes after 3 wk of feeding at a level dependent upon the amount of dietary EPA; and 3) at least one neural fatty acid, 18:2(n-6), is very sensitive to amounts of dietary EPA that are well above the animal's EPA re quirement. J. Nutr. 121: 1548-1553, 1991.
MATERIALS AND METHODS
INDEXING KEY WORDS:
•essential fatty adds •neural fatty acids •rats •cardiolipin •llnolelc add
Recently, we found that the level of linoleic acid [18:2(a-6)] in the brain of weanling rats could be al tered twofold by dietary fatty acid composition after 4 wk of feeding (Dyer, J. R. and Greenwood, C. E., unpublished data). The specific dietary fat character istic responsible for this effect was later identified as being the total amount of the two essential fatty acids (EFA)4 18:2(B-6) and oc-linolenic acid [18:3(u-3)] (1). The diets used in these experiments were not defi cient in EFA and, therefore, the neural 18:2(u-6) was responding to changes in the dietary EFA that were well above what is presently considered to be the animal's requirement (2) and within the range typi cally consumed by humans (3). The change in the neural 18:2(n-6) level was most
Materials. All nonfat dietary ingredients were pur chased from Teklad Diets (Madison, WI) except cornstarch (St. Lawrence Starch, Mississauga, ON, Can ada). The beef tallow was generously donated by Canada Packers Co. (Toronto, ON). The dietary oils were all cold-pressed and were bought from several local outlets. All chemicals were purchased from Sigma Chemical (St. Louis, MO), except fatty acid methyl ester standards (NuChek Prep, Elysian, MN) and sucrose (Analar, BDH Chemicals Canada, To ronto, ON). Chloroform, methanol and hexane were double-distilled and obtained from the University of Toronto's Solvent Distillation Service. Iso-octane (HPLC grade) was purchased from Fisher Chemicals (Fair Lawn, NJ). Thin layer chromatography plates and gas chromatography capillary column were
'Supported by grants from the Medical Research Council of Canada and the International Life Sciences Institute-Nutrition Foundation. JRD was the recipient of a Medical Research Council Studentship. 2Present address: Neurological Research Unit, Livingston Hall, Room 110, Montreal General Hospital, 1625 Pine Ave., Montreal, PQ, Canada H3G 1A4. ^o whom reprint requests should be addressed. 'Abbreviations used: CL, cardiolipin; EFA, essential fatty acids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PUPA, polyunsaturated fatty acids; UI, unsaturatÃ-on index.
0022-3166/91 $3.00 ©1991 American Institute of Nutrition. Received 14 January 1991. Accepted 15 April 1991. 1548
Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
JOHN R. DYER2 Ano CAROL E. GREENWOOD3
DIETARY EFA AND NEURAL FATTY ACID CHANGES
OVER TIME
1549
TABLE 1 Dietary fatty add composition.1 Diet
SFA2
MUTA
PUTA
P/S
18:2(n-6)
diet7.74.92.74.29.915.00.541.996.69g/1004.19.614.5g g
18:3|n-3)
EFA ratio3
diet0.120.320.5233.730.228.2
'All diets contained 20% (wt/wt) fat; 18:2(n-6) and 18:3(n-3) were the only (n-6) and (n-3) fatty acids present in significant amounts. Fatty acid composition was determined by gas chromatography. Abbreviations used: EFA, essential fatty acids; MUFA, monounsaturated fatty acids; PUF A, polyunsaturated rated fatty acids/saturated fatty acids, wt/wt; SFA, saturated fatty acids. 3EFA ratio = 18:2(n-6)/18:3(n-3), wt/wt.
obtained through Chromatographie Specialties (Brockville, ON). Experimental design. The purpose of this ex periment was to determine the time course over which the amount of dietary EFA alters the fatty acid profile of mitochondria in the brain. Rats were divided into eleven groups of six rats based on body weight. One group was killed at the beginning of the experiment to establish the initial neural fatty acid profile. Eight groups were fed diets containing 4 or 15% (wt/wt) EFA for 1, 2, 3 or 6 wk. The remaining two groups were fed a 10% EFA diet for 3 or 6 wk. At the end of the experimental periods, animals were decapitated (900-1000 h) and their brains quickly re moved, wrapped in foil, and covered with ice until membrane fractionation was conducted. Diets. Semipurified diets (as described in réf.5) were made, each containing (g/100 g): protein as casein (23), comstarch (40), mineral mixture (5.1, Bemhart-Tomarelli, réf.6), fiber (5, alpha-cellulose), vitamin mixture (2.5, TD67231, réf.7), and supple mental L-methionine (0.25). All diets contained 20% (wt/wt) fat. The diets contained different proportions of beef tallow, safflower oil and linseed oil in order to vary the amount of EFA, but keep the EFA ratio [18: 2(n-6)/18:3(n-3)] at -30 (Table 1). AU diets were ade quate in EFA. The fatty acid composition of the fat mixtures were determined by gas chromatography as described below. Animals. Male weanling Wistar rats (Charles River Breeding Labs, St. Constant, PQ, Canada), initially •weighing 50-70 g, were individually housed in a room with a 12-h lightrdark cycle (lights on 700-1900 h) and allowed free access to both water and food. Body weights and food intakes were monitored throughout the experimental period. Membrane fractionation and lipid analysis. Mem brane fractionation on sucrose density gradients was conducted immediately following decapitation, using the method of Whittaker and Barker (8). Mitochondria were collected and kept at -70°Cuntil lipids were extracted. Lipid was extracted from each membrane fraction by the method of Folch et al. (9). Cardiolipin,
fatty acids; P/S - polyunsatu-
phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were separated by TLC using a modified version of the method of Kennedy et al. (10). Fatty acid methyl esters were prepared from each phospholipid by the method of Morrison and Smith (11) and analyzed by gas chromatography. The 18:l(n-9) and 18:l(i2-7) did not reliably separate and are reported as the total 18:1 isomers. Details of these methods were given previously (5). Statistical analysis. Statistical analysis was done using SAS 6.03 (SAS Institute, Cary, NC) for the microcomputer. The effects of die amount of dietary EFA and the duration of feeding (time) on individual fatty acids and die unsaturation index (UI),5 were tested using multivariate and univariate ANOVA for each phospholipid. Groups fed different levels of EFA were compared using Student's t test.
RESULTS The energy intake, body weight gain and brain weight were similar for animals fed each diet, except for body weight gain at 2 wk (Table 2). The multivariate ANOVA tested the effects of diet and time on all of die fatty acids togedier and re vealed that dietary EFA, time and dieir interaction had significant effects on die mitochondrial fatty acid profile in each phospholipid (P < 0.001), except for die interaction in PE (P > 0.05). Time was also significant in the univariate analyses of individual fatty acids, dieir ratios and die UI in all cases, except 22: 6(n-3) in PC (P = 0.066). Dietary EFA had a very pronounced influence on certain fatty acids, but not on otiiers. The greatest impact of dietary EFA on the mitochondrial fatty acid composition was evident in 18:2(n-6) (Fig. 1). In each
5Unsaturation index (UI) - Ejnumber of double bonds in each fatty acid) x (mol/100 mol of each fatty acid).
Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
4% EFA10% EFA15% EFA7.75.02.2g/100
DYER AND GREENWOOD
1550
TABLE 2 Energy intake, body weight gain and brain weight of weanling rats fed different levels of essential fatty acids (EFA) for 6 wk' Diet (% EFA)
Time (wk)
Energy intake
Body weight gain
Brain weight Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
1.50 ±0.02
41541541015410151122333666144314433389343156325519520512,41412,48912,606±±±±±±±±±±331392672051131
'Values are means ±SEM(n - 6). Energy intake and body weight gain are cumulative Significant
difference between
values for the time periods specified.
diets (P < 0.05 by t test).
phospholipid, only 1 wk of feeding was sufficient to result in significantly greater levels of 18:2(n-6) in the high compared with the low EFA groups {P < 0.05, t test). By wk 3, a twofold difference was present that seems maximal because the difference was of the same magnitude at 6 wk. The divergence in the 18: 2(n-6) levels resulted from the combination of a decrease from baseline (0 wk) in animals fed the low EFA diet and an increase in animals fed the high EFA diet. The percentage of 18:2(n-6) in the mitochondria from animals in the intermediate EFA group fell roughly midway between the percentages of 18: 2(n-6) in the other two groups at both 3 and 6 wk. Therefore, stable 18:2(n-6) levels were reached after a 3-wk feeding period, regardless of the amount of EFA in the diet. The mitochondria! saturated fatty acids were unaf fected by the amount of EFA in the diet, with the exception of inconsistent changes in 16:0 from CL. Approximately 10, 6 and 42% of the fatty acids in CL, PE and PC, respectively, were present as 16:0; 5, 25 and 10% of the fatty acids in CL, PE and PC, respec tively, were present as 18:0 (data not shown). Mod erate differences in the monounsaturated fatty acids occurred in response to the dietary EFA, particularly in CL where they are the most abundant. The levels of 16:l(B-7) (data not shown) and the total 18:1 isomers in the low EFA group were 5-15% higher than in the high EFA group (Fig. 1). By wk 1, the difference in 18:1 levels in CL was maximal and it remained constant through wk 6. Animals fed the intermediate EFA diet had values of 18:1 isomers in CL that were between those of the other two dietary groups at wk 3 and 6. In contrast to the other fatty acids, 22:5(n-6) was altered in PE and PC (but not in CL) by dietary EFA (Fig. 2). With time, the level of 22:5(n-6) increased in
all phospholipids, but the low EFA diet elevated 22: 5(fl-6)levels in PE and PC more than did the high EFA diet. The effect was greater in PE than in PC (50 vs. 25%), but was too erratic in CL to be significant. Not all of the PUFA in the membrane reacted to the amount of EFA in the diet. Diet had no effect on either 20:4(c-6) (Fig. 1) or 22:4(iz-6)(Fig. 2). Only in CL was 22:6(n-3) influenced by dietary EFA, with levels in animals fed the low EFA diet being -15% greater than those in animals fed the high EFA diet by wk 2 (Fig. 2). The dietary EFA had no impact on the membrane UI; however, the overall (n-6)/(n-3) ratio was signifi cantly greater in animals fed the high EFA diet. Changes in the membrane (n-6)/(n-3) ratio paralleled those observed in membrane 18:2(n-6) and were greater in CL due to the magnitude of changes in 18: 2(jQ-6)in this phospholipid (data not shown).
DISCUSSION Dietary fatty acid composition can alter the neural fatty acid profile in rats (for reviews see réf.12, 13). Numerous reports (14-16) have demonstrated that the 20- and 22-carbon PUFA in the brain are affected by a deficiency of EFA. In addition, many studies (17-19) have shown that the proportion of (12-6)and (fl-3)22-carbon PUFA in the brain respond to the ratio of EFA in the diet, hi both cases, the effect of dietary fat on the brain of rats is greater when imposed during the fetal and neonatal stages than after weaning be cause in rats the majority of the neural 20- and 22-carbon PUFA have been deposited in the brain by weaning (20). The speed and magnitude of the change in neural 18:2(n-6) reported here, and its sensitivity to
DIETARY
EFA AND
CARDIOLIPIN
40 38
18:1
NEUR'
T. FATTY
ACID
CHANGES
OVER TIME
PHOSPHATIDYLETHANOLAMINE
1551
PHOSPHATIDYLCHOLINE
Total
36 32 30 22
18:2fn6i
< 18
m 14 u. 0s 10
22 20
20:4fn61
12 20
18
19
16
18
14
11 10
17 1
o
23456
1
Weeks on Diet FIGURE 1 The effect of the amount of dietary essential fatty acids (EFA) over time on the 18- and 20-carbon fatty acids in neural mitochondria from weanling rats. Animals were fed 20% (wt/wt) fat diets containing either 4, 10 or 15% (wt/wt diet) EFA for the specified time periods. Fatty acid profiles of individual mitochondrial phospholipids were measured by gas-liquid chromatography. Fatty acid values are expressed as the percentage (wt/wt) of total fatty acids,- values are means ±SEM (n - 6/ diet x time). Statistical analysis by ANOVA (P < 0.05): A - significant effect of EFA amount; I - significant diet by time interaction; time was significant in all cases.
amounts of dietary EFA within a range considered adequate, are novel. That is, these data demonstrate that major changes in neural fatty acid profile, in response to changes in dietary fatty acid composition, occur even beyond periods of brain growth and rapid fatty acid accretion, with alterations observed in as little as 1 wk of feeding. Furthermore, these data show that an EFA deficiency need not be imposed for neural fatty acid profile to be influenced. Rather, the dietary fatty acids fed in this experiment are represen tative of fats typically consumed by humans (3). The response of 18:2(n-6) in CL to dietary EFA was very fast and large relative to changes in other quanti tatively major fatty acids observed in weanling rats. After only 1 wk, the level of 18:2(n-6) in the 15% EFA group was one-third higher than in the 4% EFA group, and there was a twofold difference by wk 3. In com parison, it took 12 wk for the dietary EFA ratio (1.8 vs. 165) to cause a 20% difference in the levels of 22: 6(n-3) from synaptosomal PE (19). Only one other
study (21) has examined the effect of dietary fat com position on the fatty acid profile of neural CL over such a short time period, and it also found a twofold difference in 18:2(n-6) levels after only 10 d of feeding young rats either com or sardine oil diets. A feeding period of longer than 3 wk did not cause any greater divergence in the neural 18:2(n-6) levels of animals fed the low, moderate or high EFA diets. Therefore, 3 wk was sufficient time for the mitochon drial fatty acid changes to reach their maximum. Furthermore, there was no indication that any com pensatory mechanisms were activated in the body (or brain) to reverse the effect of dietary EFA, because the levels of membrane 18:2(n-6) in each dietary group were constant relative to each other at 3 and 6 wk. Generally, dietary EFA altered the fatty acid com position of CL from neural mitochondria more than that of PE or PC because the largest absolute and relative effect of dietary EFA was on 18:2(n-6) and it is concentrated in CL. Consequently, the level of 18:
Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
34
DYER AND
1552 CARDIOLIPIN 0.8
GREENWOOD
22:4(n6i
1.8
1.4
0.7
1.0
3.0
0.5
2.6
0.6
32
10
22:5(n6>
13 O < 0.8
0.4
10
22:6fn31
30 28 26 1
o
23456
1
Weeks on Diet FIGURE 2 The effect of the amount of dietary essential fatty acids (EFA) over time on the 22-carbon fatty acids in neural mitochondria from weanling rats. Animals were fed 20% (wt/wt) fat diets containing either 4, 10 or 15% (wt/wt diet) EFA for the specified time periods. Fatty acid profiles of individual mitochondrial phospholipids were measured by gas-liquid chromatography. Fatty acid values are expressed as the percentage (wt/wt) of total fatty acids; values are means ±SEM (n - 6/ diet x time). Statistical analysis by ANOVA (P < 0.05): A - significant effect of EFA amount; I - significant diet by time interaction,- time was significant in all cases.
2(n-6) is very probably affected directly by the dietary EFA, whereas the changes in the proportions of the other fatty acids in CL occur secondarily. In this case, there does not seem to be a reciprocal replacement of fatty acids of a similar type, as there is with the 22-carbon PUPA. The large rise in the percentage of 18:2(u-6) is counterbalanced by smaller decreases in the percentages of several other fatty acids of varying chain length and unsaturatìon,such as 16:l(n-7), the 18:1 isomers and 22:6(n-3). Not all of the fatty acids in CL were changed, however, so the replacement is not completely random and possibly is based on the positions of the fatty acids in CL. The speed with which dietary EFA altered the neural 18:2(n-6) levels is surprising, as is the obser vation that the effective range of dietary EFA was well above the rat's requirement for EFA (2). A defi ciency of EFA alters the neural fatty acid profile, but diets adequate in EFA have not been studied in this respect. Effects on the brain that result from varying
dietary EFA over a range of levels that provide the animal with adequate amounts of EFA are more relevant to humans than changes resulting from EFA deficiency. People rarely consume inadequate amounts of EFA, unless they have a defect in lipid absorption or are receiving one of the older parenteral nutrition formulations (22). The functional significance of altering the level of 18:2(n-6) in neural CL is unknown, although there is circumstantial evidence to support the hypothesis that the function of the brain is affected by the di etary fatty acid manipulations described in this paper. First, CL is notable for having by far the highest concentrations of 18:2(n-6) of any phospholipid, and for its almost exclusive occurrence in mitochondria (4). Second, several mitochondrial enzymes involved in oxidative respiration (23-27) and the transport of fatty acids across the inner mitochondrial membrane (28-31) are specifically associated with CL and/or require CL for optimal activity. Finally, modifications
Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
0.6
« 1.2
PHOSPHATIDYLCHOLINE
PHOSPHATIDYLETHANOLAMINE
DIETARY EPA AND NEURAL FATTY ACID CHANGES
LITERATURE CITED 1. Dyer, J. R. &.Greenwood, C. E. (1991) The level of linoleic acid in neural cardiolipin is linearly correlated to the amount of essential fatty acids in the diet of the weanling rat. J. Nutr. Biochem. (in press) 2. Rivers, J.P.W. &. Frankel, T. L. (1981) Essential fatty acid deficiency. Br. Med. Bull. 37: 59-64. 3. FAO (1982) Monthly Bull. Stat. 5: 11-20. 4. Sastry, P. S. (1985) Lipids of nervous tissue: composition and metabolism. Prog. Lipid Res. 24: 69-176. 5. McGee, C. D. &. Greenwood, C. E. (1989) Effects of dietary fatty acid composition on macronutricnt selection and synaptosomal fatty acid composition in rats. J. Nutr. 119: 1561-1568. 6. Bemhart, F. W. &. Tomarelli, R. M. (1969) A salt mixture supplying the National Research Council estimates of the mineral requirement of the rat. J. Nutr. 89: 495-500. 7. Musten, B., Peace, D. &. Anderson, G. H. (1974) Food intake regulation in the weanling rat. J. Nutr. 104: 563-572. 8. Whittaker, V. P. &. Barker, L. A. (1972) The subcellular fractionation of brain tissue with special reference to the prep aration of synaptosomes and their component organdÃ-es. In: Methods of Neurochemistry (Fried, R., ed.), vol. 2, pp. 1-52. Marcel Dekker, New York, NY. 9. Folch, J., Lees, M. &. Stanley, G.H.S. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509. 10. Kennerly, D. A., Sullivan, T. J. &. Parker, C. W. (1979) Acti vation of phospholipid metabolism during mediator release from stimulated rat mast cells. J. Immunol. 122: 152-159. 11. Morrison, W. R. &.Smith, L. M. (1964) Preparation of fatty acid methyl esters and dimethylacetals from lipides with boron trifluoride-methanol. J. Lipid Res. 5: 600-608. 12. Johnston, P. V. (1979) Nutrition and neural lipids. In: Advances in Nutritional Research (Draper, H. H., ed.), vol. 2, pp. 149-180. Plenum Press, New York, NY. 13. Menon, N. K. &. Dhopeshwarkar, G. A. (1982) Essential fatty acid deficiency and brain development. Prog. Lipid Res. 21: 309-326. 14. Mohrhauer, H. &.Holman, R. T. (1963) Alterations of the fatty acid composition of brain lipids by varying levels of dietary essential fatty acids. J. Neurochem. 10: 523-530. 15. Sinclair, A. J. &. Crawford, M. A. (1973) The effect of a low-fat maternal diet on neonatal rats. Brit. J. Nutr. 29: 127-137. 16. Ailing, C., Bruce, A, Karlsson, I. &. Svennerholm, L. (1974) The effect of different dietary levels of essential fatty acids on lipids of rat cerebrum during maturation. J. Neurochem. 23:
1553
1263-1270. 17. Lamptey, M. S. &.Walker, B. L. (1976) A possible essential role for dietary linolenic acid in the development of the young rat. J. Nutr. 106: 86-93. 18. Bourre, J. M., Pascal, G., Durand, G., Masson, M., Dumont, O. & Picio 11i, M. (1984) Alterations in the fatty acid composition of rat brain cells (neurons, astrocytes, and oligodendrocytes) and of subcellular fractions (myelin and synaptosomes) in duced by a diet devoid of n-3 fatty acids. J. Neurochem. 43: 342-348. 19. Dyer, J. R. &. Greenwood, C. E. (1991) Neural 22-carbon fatty acids respond rapidly and specifically to a range of dietary linoleic to a-linolenic fatty acid ratios. J. Neurochem. 56: 1921-1931. 20. Sinclair, A J. &. Crawford, M. A (1972) The accumulation of arachidonate and docosahexaenoate in the developing rat brain. J. Neurochem. 19: 1753-1758. 21. Yamaoka, S., Urade, R. &. Kito, M. (1988) Mitochondrial function in rats is affected by modification of membrane phospholipids with dietary sardine oil. J. Nutr. 118: 290-296. 22. Yamanaka, W. K., Clemans, G. W. & Hutchinson, M. L. (1981) Essential fatty acid deficiency in humans. Prog. Lipid Res. 19: 187-215. 23. Aswathi, Y. C., Chuang, T. F., Keenan, T. W. Ä.Crane, F. L. (1971) Tightly bound cardiolipin in cytochromc oxidase. Biochim. Biophys. Acta 226: 42-52. 24. Vik, S. B., Georgevich, G. & Capaldi, R. A (1981) Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase. Proc. Nati. Acad. Sci. USA 78: 1456-1460. 25. Robinson, N. C., Strey, F. & Talbert, L. (1980) Investigation of the essential boundary layer phospholipids of cytochrome c oxidase using Triton X-100 delipidation. Biochemistry 19: 3656-3661. 26. Fry, M. &. Green, D. E. (1980) Cardiolipin requirement by cytochrome oxidase and the catalytic role of phospholipid. Biochem. Biophys. Res. Comm. 93: 1238-1246. 27. Santiago, E., Lopez-Moratalla, N. &. Segovia, J. L. (1973) Corre lation between losses of mitochondrial ATPase activity and cardiolipin degradation. Biochem. Biophys. Res. Comm. 53: 439-445. 28. Bieber, L. L. &. Farrell, S. (1983) Camitine acyltransferases. In: The Enzymes (Boyer P.D., ed.), vol. 16, pp. 627-644. Academic Press, New York, NY. 29. Fiol, C. J. &. Bieber, L. L. (1984) Sigmoid kinetics of purified beef heart mitochondrial camitine palmitoyltransferase. f. Biol. Chem. 259: 13084-13088. 30. Pande, S. V., Murthy, M.S.R. & Noel, H. (1986) Differential effects of phosphatidylcholine and cardiolipin on camitine palmitoyltranferasc activity. Biochim. Biophys. Acta 877: 223-230. 31. Noel, H. &. Pande, S. V. (1986) An essential requirement of cardiolipin for mitochondrial camitine acylcarnitine translocase activity. Eur. J. Biochem. 155: 99-102. 32. Abuirmeileh, N. M. & Elson, C. E. (1980) The influence of linoleic acid intake on membrane-bound respiratory activities. Lipids 15: 918-924. 33. Abuirmeileh, N. M. &. Elson, C. E. (1980) The influence of linoleic acid intake on electron transport system components. Lipids 15: 925-931. 34. Innis, S. M. & Clandinin, M. T. (1981) Dynamic modulation of mitochondrial membrane physical properties and ATPase ac tivity by diet lipid. Biochem. J. 198: 167-175. 35. McMurchie, E. J., Abeywardena, M. Y., Chamock, J. S. & Gibson, R. A. (1983) Differential modulation of rat heart mito chondrial membrane-associated enzymes by dietary lipid. Bi ochim. Biophys. Acta 760: 13-24.
Downloaded from https://academic.oup.com/jn/article-abstract/121/10/1548/4743989 by University of Rhode Island user on 02 December 2018
to dietary fat that include changes in the amount of EFA have been shown in peripheral tissues to affect oxidative respiration and the activity of some of the enzymes that bind CL (21, 32-35). The lack of research concerning neural CL means that our experiments linking dietary EFA to 18: 2(n-6) in the brain have opened up a new line of investigation in the area of dietary fat and the brain. The findings reported here are exciting because the magnitude of the change that occurs in membrane 18: 2(n-6) is very large, and 18:2(n-6) responds to dietary fat much more quickly than other fatty acids, in cluding the 22-carbon PUFA. This undermines the idea that the mature brain is relatively indifferent to changes in dietary fat or to diet in general, and forces us to modify our thinking about how the developed brain reacts to dietary fat.
OVER TIME
