Free Radical. and Aging ed. by I. Emerit &. B. Chance ©1992 BirkhOuser Verlag Basel/Switzerland
Vitamin E requirement in relation to dietary fish oil and oxidative stress in elderly Mohsen Meydani Antioxidant Research Laboratory, USDA-Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA Summary. A growing body of evidence shows that oxygen radicals and other products of free radical reactions are involved in aging and age-related degenerative diseases. Recent studies have suggested that fish oils (FO) have a potentially beneficial effect on age-associated diseases. Consumption of FO may increase requirement for vitamin E, especially under conditions where oxidative stress is increased. Vitamin E requirement increases wtih increased intake of dietary polyunsaturated fatty acids (PUFA). This relationship may be exaggerated in elderly subjects. Our studies, as well as those of others, have shown that plasma lipid peroxides are significantly higher in older subjects compared to young subjects. Thus, in conditions where the percentage of highly unsaturated fatty acid increases in the membrane, older subjects may be more susceptible to oxidative damage. In a series of human studies, we found that older women, receiving FO supplements for 3 months exhibited a greater increase in plasma PUFA compared to young subjects. By substituting membrane fatty acids with the potentially unstable (n-3) fatty acids of FO, older subjects were found to be at greater risk of oxidative stress than young subjects. In addition, when exposed to eccentric exercise-induced oxidative stress, older men, receiving vitamin E supplements for 48 days, exhibited significantly lower levels of lipid peroxides in urine compared to placebo control. These data indicate that older subjects are more susceptible to oxidative stress and may benefit from the antioxidant protection provided by vitamin E.
Introduction
Animal and human studies suggest that the formation of oxygen free radicals and lipid peroxides occur as continuous biological processes in which a number of defensive enzymatic and non-enzymatic systems are involved. Free radicals, i.e., atoms and molecules with an unpaired electron can damage molecules having important roles in cellular homeostasis, resulting in a total loss of cellular funtion crucial to the survival of organism. According to the free radical theory of aging, the aging process and degenerative changes associated with this process may occur due to accumulative effect of the random free radical reactions that are continuously occurring at the cellular level (Harman, 1980; Samarajski et aI., 1968). A growing body of evidence indicates that oxygen-derived species such as 0;, H 0·, H2 O2 , ••• , and other products of free radical and lipid peroxidation reactions play an important role in the pathogenesis of
412 several age-associated disorders (Halliwell, 1987; Meydani et ai., 1990). During the reduction of oxygen which occurs in the respiratory chain in the inner mitochondrial membrane, superoxide radicals are formed. In normal, balanced homeostatic conditions, cells are continuously exposed to free radicals. These radicals have the capacity to trigger chain reactions and therefore, produce membrane lipid peroxidation (Harman, 1984). However, in oxidative stress conditions, higher levels of oxygen radicals are produced, exceeding homeostatic antioxidant protection of cells, and thereby resulting in peroxidation of biological membranes (Davies et ai., 1982; Dillard et ai., 1978). Lipid peroxidation of the biological membranes can lead to loss of integrity of membrane and dysfunction of receptors and membrane-bound enzymes. Lipid peroxidation events also release reactive free radicals. and toxic aldehydes which can then completely inactivate enzymes and other cell components (Bowles et ai., 1991). There are multiple cellular enzymatic and non-enzymatic antioxidant defense systems in cells which function to protect the membranes and other cell organelles from the deleterious effects of free radical reactions. Vitamin E is the major fat-soluble, chain breaking antioxidant in biological membranes. It protects membrane polyunsaturated fatty acids (PUFA) from lipid peroxidation (Chow, 1991). Early studies have demonstrated that the requirement for this antioxidant increases with increased consumption of PUFA (Witting, 1974). The ability of vitamin E to protect membranes from oxidative damage is dependent on the magnitude and duration of oxidative stress. Fatty acid composition of a diet influences the fatty acid composition of membrane phospholipids (Witting, 1974). An increased requirement for vitamin E has been suggested in cases of high intake of PUF A (Draper, 1980). In addition, a high intake of PUFA may interfere with vitamin E absorption from the intestine (Gallo-Torres, 1980; Leka et ai., 1989). A relative increase in mono- and poly-unsaturated fatty acids has been recommended by the U.S. National Cholesterol Education Panel as an approach for reducing the risk of cardiovascular disease among Americans. Experimental and epidemiological data suggest that the level and the type of dietary fat playa significant role in the incidence and pathogenesis of age-associated degenerative diseases such as atherosclerosis and coronary artery disease. Several population based studies have recommended increasing physical fitness and physical exercise to attain a variety of beneficial health effects, for both young and elderly people. In a series of human studies which involved fish oil supplementation and used eccentric exercise to induce oxidative stress, the potential of dietary vitamin E supplementation in young and older subjects was investigated.
413 Fish oil and oxidative stress
Fish and fish oil concentrates containing long chain (n-3) fatty acids have been indicated to protect humans against coronary artery disease (Herold and Kinsella, 1986; Glomset, 1985). Recently, accumulated evidence from animal experiments and human trials have widely promoted (n-3) fatty acids of fish oils in the prevention and treatment of various pathological conditions, including rheumatoid arthritis, psoriasis, cancer and other inflammatory diseases (J:Unsella et aI., 1990). With renewed interest in fish oil as a preventative measure and treatment of various conditions, its potentially harmful effects have been overlooked. Without adequate antioxidant protection, the substitution of membrane fatty acids with highly oxidizable (n-3) fatty acids of fish oil, i.e. eicosapentaenoic acid (EPA) with 5 double bonds and docosahexaenoic acid (DHA) with 6 double bonds, may potentiate peroxidation of cellular membranes (Odeley and Watson, 1991). Fish oil capsules currently available on the market contain variable levels of EPA and DHA. In order to prevent the oxidation of EPA and DHA in fish oil products and extend capsules shelf-life, manufacturers add vitamin E in a variety of forms. The level of ex-tocopherol contained in fish oil products was measured in our laboratory (Chee et aI., 1990) and ranged from 180 f.lg to 2240 f.lg per g of fish oil; certain products available on the market also contain y-tocopherol which has 1/10 the biological activity of ex-tocopherol. In animal studies, Meydani et al. (1987) reported that in both young and old mice supplemented with different levels of vitamin E, fish oil fed animals had significantly lower ex-tocopherol concentrations in plasma and tissues compared to mice fed corn oil or coconut oil. This reduced status of vitamin E in fish oil fed animals might be due to decreased absorption of vitamin E and/or its increased demand and utilization by other tissues. We have found that percent lymphatic appearance of intact 14-C-ex-tocopherol orally administered in fish oil to rats was significantly (p < 0.05) lower that when administered in either corn oil or olive oil (fish oil: 19.9 ± 16.3%, corn oil: 41.7 ± 22.4%, and olive oil: 49.4 ± 24.8%) (Leka et aI., 1989). The absorption of ex-tocopherol by the intestine was reduced and a significant portion of the administered dose decomposed before lymphatic absorption. Therefore, consumption of meals comprised of fish or fish oil prodcuts, may necessitate increased intake of vitamin E, at least in part to compensate for the partial destruction and reduction in absorption of ex-tocopherol from the gut. Oxidative damage to tissues has been suggested as the bases for the pathology of several human diseases (Ralliwell, 1987). Long-term fish oil supplementation without adequate antioxidant protection, may result in in vivo peroxidation of (n-3) fatty acid and thereby contribute to
414 Table I. Change of plasma fatty acids ratio, ex-tocopherol and lipid peroxides following fish oil supplementation
Young (22-35 y) Older (51-71 y)
PUFA/SFA
ex-Tocopherol/ (EPA+DHA)
+10.1%** +18.6%***
-72%** -79%***
+29.4%* +63.1%*
a% change after 2 months of fish oil supplementation. Significant change from pre-supplementation level, *p < 0.05, **p < 0.001, ***p < 0.0001.
the onset and/or progression of some of age-associated diseases. We investigated the potential change of plasma lipid peroxides of 15 young (22-35 years) and 10 healthy older (51-71 years) women taking 6 capsules of fish oil with their meals daily for a three-month period (Meydani et aI., 1991). The fish oil capsules used in the study provided 1.68 g of EPA, 0.72 g of DHA, 0.6 g of other fatty acids and 6 IU of vitamin E per day. After three months of supplementation, older women exhibited a significantly higher increase in plasma EPA and DHA and showed a dramatic increase in PUFA to saturated fatty acids (SFA) ratio compared to young women. Although plasma vitamin E levels did not change, fish oil supplementation significantly decreased the plasma vitamin E/(EPA + DHA) and significantly increased the plasma level of lipid peroxides (Table 1). Increase in plasma lipid peroxides due to fish oil supplementation was significantly higher in the older women compared to young women. Even though the long-term supplementation with fish oil in this study was found to be beneficial in reducing plasma total triglyceride, without providing adequate antioxidant protection by vitamin E, an alteration in fatty acid composition of plasma, and most likely of other tissues to more highly unsaturated fatty acids with a higher potential for peroxidation, could interfere with the homeostatic balance between antioxidants and prooxidants. Exercise and oxidative stress
Animal and human studies have shown that unaccustomed exercise as well as strenuous and exhaustive exercise can induce oxidative damage and cause muscle injury. Exercise influences oxidative metabolism and produces reactive oxygen species which appear to play a key role in changing the membrane fatty acid composition, permeability and leakage of enzymes, all of which eventually cause the injury to muscle membrane. There is evidence indicating that the generation of free radicals and lipid peroxides increases with the increased respiration during exercise (Oohil et aI., 1986; Ji et aI., 1990; Sumida et aI., 1989).
415 This increase may last for quite some time following an unaccustomed exercise. In vitamin E deficient animals, exercise increases susceptibility to free radical damage and results in premature exhaustion, greater fragility of lysosomal membrane, and marked depression of muscle mitochondrial respiratory control (Davies et aI., 1982; Gohil et ai. 1986; Quintanilha et aI., 1982). Bowles et ai. (1991) reported that a single bout of exercise at 70% of V0 2max in untrained rats resulted in 30% decline in vitamin E content of the quadricep muscle. Davis et ai. (1982) have shown that exhaustive exercise in animals causes lipid peroxidation and mitochondrial damage. The limited human studies conducted to date indicate that vitamin E supplementation reduces oxidative stress and lipid peroxidation events following exercise. This implies that vitamin E requirements may increase with exercise (Pincemail et aI., 1988; Simon-Schnass and Pabst, 1988). In order to investigate the protective role of vitamin E supplementation against exercise-induced oxidative stress, nine young (22-29 years) and 12 older (55-74 years) healthy men were studied following supplementation with either 800 IU of vitamin E or placebo for 48 days. Volunteers were then subjected to a 45-min bout of eccentric exercise, running downhill on a treadmill at 75% of maximum of their heart rate (Meydani et aI., 1991). After 48 days, the concentration of plasma O(-tocopherol increased (56% in young, 70% in older subjects) and y-tocopherol decreased (99% in young, 60% in older subjects). Using an eccentric exercise model in which the muscle is contracted while lengthening, showed a decrease of 0(- and y-tocopherol levels in the muscle biopsies obtained from the vastus lateralis muscle of young subjects (Table 2). The change observed in the vitamin E status of muscle following exercise was accompanied with 148% increase in total muscle lipid conjugated dienes placebo groups compared to a 38% increase in the subjects who received vitamin E supplements (Table 2). Therefore, significant decreases in both 0(- and y-tocopherol and concomitant increase of lipid conjugated dienes in muscle was demonstrated immediately following exercise. This indicated that acute exercise increases active oxygen radicals species and lipid peroxidation.
Table 2. Change in vitamin E and lipid peroxidation index in muscle biopsy obtained from young subjects following eccentric exercise
IX-Tocopherol y-Tocopherol Conjugated dienes
Placebo
Vitamin E-supplemented
-15%* -20% +145%
-23%* -29% +38%
*significant (p < 0.05) decrease from pre-exercise, both groups combined.
416 Furthermore, these results imply that during this process, vitamin E is utilized in the muscle to counteract deleterious action of free radicals and prevent oxidative injury to membrane lipids. Similar results have been reported in exercised animals (Gohil et at, 1986, 1987; Allessio et at, 1988; Allessio et at, 1988). The protective effect of vitamin E was further substantiated by measuring 24 h urinary TBA-adducts as an index of whole body response to oxidative stress (Draper et at, 1984; Ekstrom et at, 1986; Wu et at, 1990). The protective effect of vitamin E extended up to 12 days post exercise (Meydani et at, 1991) at which time muscle protein injury and remodeling was evident by excretion of 3-methylhistidin (Cannon et at, 1991). Vitamin E supplemented subjects excreted less TBA-adducts as compared to placebo groups (p < 0.05). Percent increase in urinary TBA-adducts at 12 days post-exercise was 32.7 ± 13.3 and 16.9 ± 22.4 in young and older vitamin E supplemented subjects, respectively; whereas percent increase of urinary TBA-adducts in placebo groups was 58.7 ± 24.5 and 76.8 ± 34.9 in young and older subjects, respectively. Conclusion
Epidemiological studies reveal an inverse association between antioxidant nutrient intake and incidence of various age-associated disorders (Gey et at, 1991; Esterbauser et at, 1991). Increased levels of dietary antioxidants significantly reduce the tissue level of lipid peroxides in animal and humans (Pubelle et at, 1982; Meydani et at, 1985; Meydani et at" 1988; Meydani et at, 1986; Chavance et at, 1984; Wartanowicz et at, 1984; Lemoyne et at, 1987). The level of plasma lipid peroxides increases with age (Pubelle et at, 1982) which may be due, in part, 'to diminished status of dietary antioxidants. Low intake and/or low plasma antioxidant levels have been observed in older adults (Campbell et at, 1989). Factors such as inadequate antioxidant protection, longterm exposure to pro-oxidant toxicants, pollutants, and drugs, high intake of specific nutrients such as iron, copper, long-term intake of fish oil concentrates and performing unaccustomed and acute exercise, can increase the potential for cellular and tissue lipid peroxidation and damage. These environmental conditions can contribute significantly to the acceleration of the aging process and age-associated degenerative disorders. Therefore, higher than recommended levels of dietary antioxidants such as vitamin E might be beneficial in older adults. Allessio, H. M" and Goldfarb, A. H. (1988) Lipid peroxidation and scavenger enzymes during exercise: adaptive response to training. J. Appl. Physiol. 64: 1333-1336. Allessio, H. M., Goldfarb, A. H., and Cutler, R. G. (1988) MDA content increases in fastand slow-twitch skeletal muscle with intensity of exercise in rat. Cell Physiol. 24: C874C877.
417 Bowles, D. K., Torgan, C. E., Ebner, S., Kehrer, J. P., Ivy, J. L., and Starnes, J. W. (1991) Effects of acute, submaximal exercise on skeletal muscle vitamin E. Free Rad. Res. Comms. 14: 139-143. Campbell, D., Bunkder, V. W., Yhomas, A. J., and Clayton, B. E. (1989) Selenium and vitamin E status of healthy and institutionalized elderly subjects: analysis of plasma, erythrocytes and platelets. Br. J. Nutr. 62: 221-227. Cannon, J. G., Meydani, S. N., Fielding, R. A., Fiatarone, M. A., Meydani, M., Farhangmehr, M., Orencole, S. F., Blumberg, J. B., and Evans, W. (1991) The acute phase response in exercise. II. Associations between vitamin E, cytokines and muscle proteolysis. Am J. Physio!. 29: RI235-RI240. Chee, K. M., Gong, J. X., Good Ress, D. M., Meydani, M., Ausman, L., Johnson, J., Signel, E. N., and Schaefer, E. J. (1990) Fatty acid content of marine oil capsules. Lipids 25: 523-528. Chevance, M., Brubacher, G., Herbeth, B., Vernbes, G., Mikstacki, T., Dete, F., Fournier, C., and Janot, C. (1984) Immunological and nutritional status among the elderly, in: Lymphoid Cell Functions in Aging. DeWick, AI, ed. Eurage, Paris, pp. 231-237. Chow, C. K. (1991) Vitamin E and oxidative stress. Free Radic. Bio!. Med. ll: 215-22. Davies, K. J. A., Quintanilha, A. T." Brooks, G. A., and Packer, L. (1982) Free radicals and tissue damage produced by exercise. Biochem. Biophys. Res. Commun. 107: 1198-1205. Dillard, C. H., Litov, R. E., Savin, W. M., Dumelin, E. E., and Tappel, A. L. (1978) Effect of exercise, vitamin E and ozone on pulmonary function and lipid peroxidation. J. App!. Physio!.: Respirat. Environ. Exer. Physio!. 45: 927-932. Draper, H. H. (1980) Nutrient interrelationships, in: Vitamin E, A Comprehensive Treatise. Machlin, L. J., ed. Marcel Dekker, New York, pp. 272-288. Draper, H. H., Polensek, L., Hadley, M., and McGirr, L. G. (1984) Urinary malonaldehyde as an indicator of lipid peroxidation in the rat and in the tissues. Lipids 19: 836-843. Ekstrom, T., Stahl, A., Sigvardsson, K., and Hogberg, J. (1986) Lipid peroxidation in vivo monitored as ethane exhalation and malondialdehyde excretion in urine after oral administration of chloroform. Acta Pharmaco!. Toxico!. 58: 289-296. Esterbauser, H., Dieber-Robtheneder, M., Strieg, G., and Waeg, G. (1991) Role of vitamin E in preventing the oxidation of low-density lipoprotein. Am. J. Clin. Nutr. 53: 314S-2IS. Gallo-Torres, H. E. (1980) Absorption, in: Vitamin E, A Comprehensive Treatise. Machlin, L. J., ed. Marcel Dekker, New York, pp. 170-267. Glomset, J. A. (1985) Fish, fatty acids, and human health. N. Eng!. J. Med. 312: 1253-1255. Gey, K. F., Puska, P., Jordan, P., and Moser, U. K. (1991) Inverse correlation between plasma vitamin E and mortality from ischemic heart disease in cross-cultural epidemiology. Am. J. Clin. Nutr. 53: 326S-34S. Gohil, K., Rothfuss, L., Lang, J., and Packer, L. (1987) Effect of exercise training on tissue vitamin E and ubiquinone content. J. App!. Physio!. 63: 1638-1641. Gohil, K., Packer, L., deLumen, B., Brooks, G. A., and Terblanche, G. E. (1986) Vitamin E deficiency and vitamin C supplements: exercise and mitochondrial oxidation. J. App!. Physio!. 60: 1986-1991. Halliwell, B. (1987) Oxidants and human disease: some new concepts. FASEB J. I: 358-364. Harman, D. (1980) Free radical theory of aging: beneficial effect of antioxidant on the life span of male NZB mice; role of free radical reactions in the deterioration of the immune system with aging and in the pathogenesis of systemic lupus erythrematous. Age 3: 64-73. Harman, D. (1984) Free radicals and the origination, evolution and present status of free radical theory of aging, in: Free Radicals in Molecular Biology, Aging and Disease. Armstrong, D., Cutler, R. G., Sohal, R. S., and Slater, T. F., eds. Ravenswood Press, New York, pp. 1-12. Herold, P. M., and Kinsella, J. E. (1986) Fish oil consumption and decreased risk of cardiovascular disease: a comparison of findings from animal and human feeding trials. Am. J. Clin. Nutr. 43: 566-598. Ji, L. L., Dillon, D., and Wu, E. (1990) Alteration of antioxidant enzymes with aging in rat skeletal muscle and. liver. Am. J. Physio!. 258: R918-R923. Kinsella, J. E., Lokesh, B., Stone, R. A. (1990) Dietary n-3 polyunsaturated fatty acids and amelioration of cardiovascular disease: possible mechanisms. Am. J. Clin. Nutr. 52: 1-28. Leka, L. S., Remaly, K. M., Bizinkauskas, P. A., and Meydani, M. (1989) Effects of fish oil on intestinal absorption of vitamin E in the rat. FASEB J. 3: A951.
418 Lemoyne, M., Van Gossen, A., Kurian, R., Ostro, M., Axler, J., and Jeejeebhoy, K. N. (1987) Breath pentane analysis as an index of lipid peroxidation: a functional test of vitamin E status. Am. J. Qin. Nutr. 46: 267-272. Meydani, S. N., Shapiro, A. C., Meydani, M., Macauley, J. B., and Blumberg, J. B. (1987) Effect of age and dietary fat (fish, corn and coconut oils) on tocopherol status of C57BLJ6Nia mice. Lipids 22: 345-350. Meydani, S. N., Barklund, M. P., Liu, S., Meydani, M., Miller, R. A., Cannon, J. G., Morrow, F. D., Rocklin, R., and Blumberg, J. B. (1990) Vitamin E supplementation enhances cell-mediated immunity in healthy elderly subjects. Am. J. Clin. Nutr. 52: 557-563. Meydani, M., Verdon, C. P., and Blumberg, J. B. (1985) Effect of vitamin E, selenium and age on lipid peroxidation events in rate cerebrum. Nutr. Res. 5: 1227-1236. Meydani, M., Macauley, J. B., and Blumberg, J. B. (1988) Effect of dietary vitamin E and selenium on susceptibility of brain regions to lipid peroxidation. Lipids 23: 405-409. Meydani, M., Natiello, F., Goldin, B., Free, N., Woods, M., Schaefer, E., Blumberg, J. B., and Gorbach, S. L. (1991) Effect of long-term fish oil supplementation on vitamin E status and lipid peroxidation in women. J. Nutr. 121: 484-491. Meydani, M., Evans, W., Handelman, G., Biddle, L., Fielding, R. A., Meydani, S. N., Orencole, S. F., Fiatarolle, M. A., Blumberg, J. B., and Cannon, J. G. (1991) Vitamin E protection of exercise-induced oxidative damage in young and elderly subjects. FASEB J. 5: A919. Meydani, S. N., Cathcart, E. S., Hopkins, R. E., Meydani, M., Hayes, K. c., and Blumberg, J. B. (1986) Antioxidants in experimental amyloidosis of young and old mice, in: Fourth International Symposium of Amyloidosis. Glenner, G. G., Asserman, E. P., Benditt, E., Calkins, E., Cohen, As., and Zucker-Franklin, D., eds. Plenum Press, New York, pp. 683-692. Odeleye, O. E., Watson, R. R. (1991) Health implication of the n-3 fatty acids. Am. J. Qin. Nutr. 53: 177-178. Pincemail, J., Deby, C., Camus, G., Pirnay, F., Bouchez, R., Massaux, L., and Goutier, R. (1988) Tocopherol mobilization during intensive exercise. Eur. J. Appl. Physiol. 57: 189-191. Pubelle, P., Chantreuil, J., Bensadous, J., Blotman, F., Simon, L., and Crastes de Paule, A. (1982) Plasma lipoperoxides and aging. Biomed. 36: 164-166. Quintanilha, A. T., Packer, L., Davies, J. M. S., Racanelli, T., Davies, K. J. A. (1982) Membrane effects of vitamin E deficiency: bioenergetics and surface charge density studies of skeletal muscle and liver mitochondria. Ann. NY Acad. Sci. 399: 32-47. Samarajski, T., Ordy, J. M., and Rudy-Reimer, P. (1968) Lipofuscin pigment accumulation in the nervous system of aging. Anat. Rec. 160: 555-574. Simon-Schnass, I., and Pabst, H. (1988) Influence of vitamin E on physical performahce. Internat. J. Vito Nutr. Res. 58: 49-54. Sumida, S., Tanaka, K., Kitao, H., and Nakadomo, F. (1989) Exercise-induced lipid peroxidation and leakage of enzymes before and after vitamin E supplementation. Int. J. Biochem. 21: 835-838. Wartanowicz, M., Panczenko-Kresowska, B., Ziemlanski, S., Kowalska, M., and Okolska, G. (1984) The effect of alpha-tocopherol and ascorbic acid on the serum lipid peroxide level in elderly people. Ann. Nutr. Metab. 28: 186-191. Witting, L. A. (1974) Vitamin E-polyunsaturated lipid relationship in diet and tissues. Am. J. Qin. Nutr. 27: 952-959. Wu, W-H., Meydani, M., Meydani, S. N., Burklund, P. M., Blumberg, J. B., Munro, H. N. (1990) Effect of dietary iron overload on lipid peroxidation, prostaglandin synthesis and lymphocyte proliferation in young and old rats. J. Nutr. 120: 280-289.
Vitamin E requirement in relation to dietary fish oil and oxidative stress in elderly Mohsen Meydani Antioxidant Research Laboratory, USDA-Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA Summary. A growing body of evidence shows that oxygen radicals and other products of free radical reactions are involved in aging and age-related degenerative diseases. Recent studies have suggested that fish oils (FO) have a potentially beneficial effect on age-associated diseases. Consumption of FO may increase requirement for vitamin E, especially under conditions where oxidative stress is increased. Vitamin E requirement increases wtih increased intake of dietary polyunsaturated fatty acids (PUFA). This relationship may be exaggerated in elderly subjects. Our studies, as well as those of others, have shown that plasma lipid peroxides are significantly higher in older subjects compared to young subjects. Thus, in conditions where the percentage of highly unsaturated fatty acid increases in the membrane, older subjects may be more susceptible to oxidative damage. In a series of human studies, we found that older women, receiving FO supplements for 3 months exhibited a greater increase in plasma PUFA compared to young subjects. By substituting membrane fatty acids with the potentially unstable (n-3) fatty acids of FO, older subjects were found to be at greater risk of oxidative stress than young subjects. In addition, when exposed to eccentric exercise-induced oxidative stress, older men, receiving vitamin E supplements for 48 days, exhibited significantly lower levels of lipid peroxides in urine compared to placebo control. These data indicate that older subjects are more susceptible to oxidative stress and may benefit from the antioxidant protection provided by vitamin E.
Introduction
Animal and human studies suggest that the formation of oxygen free radicals and lipid peroxides occur as continuous biological processes in which a number of defensive enzymatic and non-enzymatic systems are involved. Free radicals, i.e., atoms and molecules with an unpaired electron can damage molecules having important roles in cellular homeostasis, resulting in a total loss of cellular funtion crucial to the survival of organism. According to the free radical theory of aging, the aging process and degenerative changes associated with this process may occur due to accumulative effect of the random free radical reactions that are continuously occurring at the cellular level (Harman, 1980; Samarajski et aI., 1968). A growing body of evidence indicates that oxygen-derived species such as 0;, H 0·, H2 O2 , ••• , and other products of free radical and lipid peroxidation reactions play an important role in the pathogenesis of
412 several age-associated disorders (Halliwell, 1987; Meydani et ai., 1990). During the reduction of oxygen which occurs in the respiratory chain in the inner mitochondrial membrane, superoxide radicals are formed. In normal, balanced homeostatic conditions, cells are continuously exposed to free radicals. These radicals have the capacity to trigger chain reactions and therefore, produce membrane lipid peroxidation (Harman, 1984). However, in oxidative stress conditions, higher levels of oxygen radicals are produced, exceeding homeostatic antioxidant protection of cells, and thereby resulting in peroxidation of biological membranes (Davies et ai., 1982; Dillard et ai., 1978). Lipid peroxidation of the biological membranes can lead to loss of integrity of membrane and dysfunction of receptors and membrane-bound enzymes. Lipid peroxidation events also release reactive free radicals. and toxic aldehydes which can then completely inactivate enzymes and other cell components (Bowles et ai., 1991). There are multiple cellular enzymatic and non-enzymatic antioxidant defense systems in cells which function to protect the membranes and other cell organelles from the deleterious effects of free radical reactions. Vitamin E is the major fat-soluble, chain breaking antioxidant in biological membranes. It protects membrane polyunsaturated fatty acids (PUFA) from lipid peroxidation (Chow, 1991). Early studies have demonstrated that the requirement for this antioxidant increases with increased consumption of PUFA (Witting, 1974). The ability of vitamin E to protect membranes from oxidative damage is dependent on the magnitude and duration of oxidative stress. Fatty acid composition of a diet influences the fatty acid composition of membrane phospholipids (Witting, 1974). An increased requirement for vitamin E has been suggested in cases of high intake of PUF A (Draper, 1980). In addition, a high intake of PUFA may interfere with vitamin E absorption from the intestine (Gallo-Torres, 1980; Leka et ai., 1989). A relative increase in mono- and poly-unsaturated fatty acids has been recommended by the U.S. National Cholesterol Education Panel as an approach for reducing the risk of cardiovascular disease among Americans. Experimental and epidemiological data suggest that the level and the type of dietary fat playa significant role in the incidence and pathogenesis of age-associated degenerative diseases such as atherosclerosis and coronary artery disease. Several population based studies have recommended increasing physical fitness and physical exercise to attain a variety of beneficial health effects, for both young and elderly people. In a series of human studies which involved fish oil supplementation and used eccentric exercise to induce oxidative stress, the potential of dietary vitamin E supplementation in young and older subjects was investigated.
413 Fish oil and oxidative stress
Fish and fish oil concentrates containing long chain (n-3) fatty acids have been indicated to protect humans against coronary artery disease (Herold and Kinsella, 1986; Glomset, 1985). Recently, accumulated evidence from animal experiments and human trials have widely promoted (n-3) fatty acids of fish oils in the prevention and treatment of various pathological conditions, including rheumatoid arthritis, psoriasis, cancer and other inflammatory diseases (J:Unsella et aI., 1990). With renewed interest in fish oil as a preventative measure and treatment of various conditions, its potentially harmful effects have been overlooked. Without adequate antioxidant protection, the substitution of membrane fatty acids with highly oxidizable (n-3) fatty acids of fish oil, i.e. eicosapentaenoic acid (EPA) with 5 double bonds and docosahexaenoic acid (DHA) with 6 double bonds, may potentiate peroxidation of cellular membranes (Odeley and Watson, 1991). Fish oil capsules currently available on the market contain variable levels of EPA and DHA. In order to prevent the oxidation of EPA and DHA in fish oil products and extend capsules shelf-life, manufacturers add vitamin E in a variety of forms. The level of ex-tocopherol contained in fish oil products was measured in our laboratory (Chee et aI., 1990) and ranged from 180 f.lg to 2240 f.lg per g of fish oil; certain products available on the market also contain y-tocopherol which has 1/10 the biological activity of ex-tocopherol. In animal studies, Meydani et al. (1987) reported that in both young and old mice supplemented with different levels of vitamin E, fish oil fed animals had significantly lower ex-tocopherol concentrations in plasma and tissues compared to mice fed corn oil or coconut oil. This reduced status of vitamin E in fish oil fed animals might be due to decreased absorption of vitamin E and/or its increased demand and utilization by other tissues. We have found that percent lymphatic appearance of intact 14-C-ex-tocopherol orally administered in fish oil to rats was significantly (p < 0.05) lower that when administered in either corn oil or olive oil (fish oil: 19.9 ± 16.3%, corn oil: 41.7 ± 22.4%, and olive oil: 49.4 ± 24.8%) (Leka et aI., 1989). The absorption of ex-tocopherol by the intestine was reduced and a significant portion of the administered dose decomposed before lymphatic absorption. Therefore, consumption of meals comprised of fish or fish oil prodcuts, may necessitate increased intake of vitamin E, at least in part to compensate for the partial destruction and reduction in absorption of ex-tocopherol from the gut. Oxidative damage to tissues has been suggested as the bases for the pathology of several human diseases (Ralliwell, 1987). Long-term fish oil supplementation without adequate antioxidant protection, may result in in vivo peroxidation of (n-3) fatty acid and thereby contribute to
414 Table I. Change of plasma fatty acids ratio, ex-tocopherol and lipid peroxides following fish oil supplementation
Young (22-35 y) Older (51-71 y)
PUFA/SFA
ex-Tocopherol/ (EPA+DHA)
+10.1%** +18.6%***
-72%** -79%***
+29.4%* +63.1%*
a% change after 2 months of fish oil supplementation. Significant change from pre-supplementation level, *p < 0.05, **p < 0.001, ***p < 0.0001.
the onset and/or progression of some of age-associated diseases. We investigated the potential change of plasma lipid peroxides of 15 young (22-35 years) and 10 healthy older (51-71 years) women taking 6 capsules of fish oil with their meals daily for a three-month period (Meydani et aI., 1991). The fish oil capsules used in the study provided 1.68 g of EPA, 0.72 g of DHA, 0.6 g of other fatty acids and 6 IU of vitamin E per day. After three months of supplementation, older women exhibited a significantly higher increase in plasma EPA and DHA and showed a dramatic increase in PUFA to saturated fatty acids (SFA) ratio compared to young women. Although plasma vitamin E levels did not change, fish oil supplementation significantly decreased the plasma vitamin E/(EPA + DHA) and significantly increased the plasma level of lipid peroxides (Table 1). Increase in plasma lipid peroxides due to fish oil supplementation was significantly higher in the older women compared to young women. Even though the long-term supplementation with fish oil in this study was found to be beneficial in reducing plasma total triglyceride, without providing adequate antioxidant protection by vitamin E, an alteration in fatty acid composition of plasma, and most likely of other tissues to more highly unsaturated fatty acids with a higher potential for peroxidation, could interfere with the homeostatic balance between antioxidants and prooxidants. Exercise and oxidative stress
Animal and human studies have shown that unaccustomed exercise as well as strenuous and exhaustive exercise can induce oxidative damage and cause muscle injury. Exercise influences oxidative metabolism and produces reactive oxygen species which appear to play a key role in changing the membrane fatty acid composition, permeability and leakage of enzymes, all of which eventually cause the injury to muscle membrane. There is evidence indicating that the generation of free radicals and lipid peroxides increases with the increased respiration during exercise (Oohil et aI., 1986; Ji et aI., 1990; Sumida et aI., 1989).
415 This increase may last for quite some time following an unaccustomed exercise. In vitamin E deficient animals, exercise increases susceptibility to free radical damage and results in premature exhaustion, greater fragility of lysosomal membrane, and marked depression of muscle mitochondrial respiratory control (Davies et aI., 1982; Gohil et ai. 1986; Quintanilha et aI., 1982). Bowles et ai. (1991) reported that a single bout of exercise at 70% of V0 2max in untrained rats resulted in 30% decline in vitamin E content of the quadricep muscle. Davis et ai. (1982) have shown that exhaustive exercise in animals causes lipid peroxidation and mitochondrial damage. The limited human studies conducted to date indicate that vitamin E supplementation reduces oxidative stress and lipid peroxidation events following exercise. This implies that vitamin E requirements may increase with exercise (Pincemail et aI., 1988; Simon-Schnass and Pabst, 1988). In order to investigate the protective role of vitamin E supplementation against exercise-induced oxidative stress, nine young (22-29 years) and 12 older (55-74 years) healthy men were studied following supplementation with either 800 IU of vitamin E or placebo for 48 days. Volunteers were then subjected to a 45-min bout of eccentric exercise, running downhill on a treadmill at 75% of maximum of their heart rate (Meydani et aI., 1991). After 48 days, the concentration of plasma O(-tocopherol increased (56% in young, 70% in older subjects) and y-tocopherol decreased (99% in young, 60% in older subjects). Using an eccentric exercise model in which the muscle is contracted while lengthening, showed a decrease of 0(- and y-tocopherol levels in the muscle biopsies obtained from the vastus lateralis muscle of young subjects (Table 2). The change observed in the vitamin E status of muscle following exercise was accompanied with 148% increase in total muscle lipid conjugated dienes placebo groups compared to a 38% increase in the subjects who received vitamin E supplements (Table 2). Therefore, significant decreases in both 0(- and y-tocopherol and concomitant increase of lipid conjugated dienes in muscle was demonstrated immediately following exercise. This indicated that acute exercise increases active oxygen radicals species and lipid peroxidation.
Table 2. Change in vitamin E and lipid peroxidation index in muscle biopsy obtained from young subjects following eccentric exercise
IX-Tocopherol y-Tocopherol Conjugated dienes
Placebo
Vitamin E-supplemented
-15%* -20% +145%
-23%* -29% +38%
*significant (p < 0.05) decrease from pre-exercise, both groups combined.
416 Furthermore, these results imply that during this process, vitamin E is utilized in the muscle to counteract deleterious action of free radicals and prevent oxidative injury to membrane lipids. Similar results have been reported in exercised animals (Gohil et at, 1986, 1987; Allessio et at, 1988; Allessio et at, 1988). The protective effect of vitamin E was further substantiated by measuring 24 h urinary TBA-adducts as an index of whole body response to oxidative stress (Draper et at, 1984; Ekstrom et at, 1986; Wu et at, 1990). The protective effect of vitamin E extended up to 12 days post exercise (Meydani et at, 1991) at which time muscle protein injury and remodeling was evident by excretion of 3-methylhistidin (Cannon et at, 1991). Vitamin E supplemented subjects excreted less TBA-adducts as compared to placebo groups (p < 0.05). Percent increase in urinary TBA-adducts at 12 days post-exercise was 32.7 ± 13.3 and 16.9 ± 22.4 in young and older vitamin E supplemented subjects, respectively; whereas percent increase of urinary TBA-adducts in placebo groups was 58.7 ± 24.5 and 76.8 ± 34.9 in young and older subjects, respectively. Conclusion
Epidemiological studies reveal an inverse association between antioxidant nutrient intake and incidence of various age-associated disorders (Gey et at, 1991; Esterbauser et at, 1991). Increased levels of dietary antioxidants significantly reduce the tissue level of lipid peroxides in animal and humans (Pubelle et at, 1982; Meydani et at, 1985; Meydani et at" 1988; Meydani et at, 1986; Chavance et at, 1984; Wartanowicz et at, 1984; Lemoyne et at, 1987). The level of plasma lipid peroxides increases with age (Pubelle et at, 1982) which may be due, in part, 'to diminished status of dietary antioxidants. Low intake and/or low plasma antioxidant levels have been observed in older adults (Campbell et at, 1989). Factors such as inadequate antioxidant protection, longterm exposure to pro-oxidant toxicants, pollutants, and drugs, high intake of specific nutrients such as iron, copper, long-term intake of fish oil concentrates and performing unaccustomed and acute exercise, can increase the potential for cellular and tissue lipid peroxidation and damage. These environmental conditions can contribute significantly to the acceleration of the aging process and age-associated degenerative disorders. Therefore, higher than recommended levels of dietary antioxidants such as vitamin E might be beneficial in older adults. Allessio, H. M" and Goldfarb, A. H. (1988) Lipid peroxidation and scavenger enzymes during exercise: adaptive response to training. J. Appl. Physiol. 64: 1333-1336. Allessio, H. M., Goldfarb, A. H., and Cutler, R. G. (1988) MDA content increases in fastand slow-twitch skeletal muscle with intensity of exercise in rat. Cell Physiol. 24: C874C877.
417 Bowles, D. K., Torgan, C. E., Ebner, S., Kehrer, J. P., Ivy, J. L., and Starnes, J. W. (1991) Effects of acute, submaximal exercise on skeletal muscle vitamin E. Free Rad. Res. Comms. 14: 139-143. Campbell, D., Bunkder, V. W., Yhomas, A. J., and Clayton, B. E. (1989) Selenium and vitamin E status of healthy and institutionalized elderly subjects: analysis of plasma, erythrocytes and platelets. Br. J. Nutr. 62: 221-227. Cannon, J. G., Meydani, S. N., Fielding, R. A., Fiatarone, M. A., Meydani, M., Farhangmehr, M., Orencole, S. F., Blumberg, J. B., and Evans, W. (1991) The acute phase response in exercise. II. Associations between vitamin E, cytokines and muscle proteolysis. Am J. Physio!. 29: RI235-RI240. Chee, K. M., Gong, J. X., Good Ress, D. M., Meydani, M., Ausman, L., Johnson, J., Signel, E. N., and Schaefer, E. J. (1990) Fatty acid content of marine oil capsules. Lipids 25: 523-528. Chevance, M., Brubacher, G., Herbeth, B., Vernbes, G., Mikstacki, T., Dete, F., Fournier, C., and Janot, C. (1984) Immunological and nutritional status among the elderly, in: Lymphoid Cell Functions in Aging. DeWick, AI, ed. Eurage, Paris, pp. 231-237. Chow, C. K. (1991) Vitamin E and oxidative stress. Free Radic. Bio!. Med. ll: 215-22. Davies, K. J. A., Quintanilha, A. T." Brooks, G. A., and Packer, L. (1982) Free radicals and tissue damage produced by exercise. Biochem. Biophys. Res. Commun. 107: 1198-1205. Dillard, C. H., Litov, R. E., Savin, W. M., Dumelin, E. E., and Tappel, A. L. (1978) Effect of exercise, vitamin E and ozone on pulmonary function and lipid peroxidation. J. App!. Physio!.: Respirat. Environ. Exer. Physio!. 45: 927-932. Draper, H. H. (1980) Nutrient interrelationships, in: Vitamin E, A Comprehensive Treatise. Machlin, L. J., ed. Marcel Dekker, New York, pp. 272-288. Draper, H. H., Polensek, L., Hadley, M., and McGirr, L. G. (1984) Urinary malonaldehyde as an indicator of lipid peroxidation in the rat and in the tissues. Lipids 19: 836-843. Ekstrom, T., Stahl, A., Sigvardsson, K., and Hogberg, J. (1986) Lipid peroxidation in vivo monitored as ethane exhalation and malondialdehyde excretion in urine after oral administration of chloroform. Acta Pharmaco!. Toxico!. 58: 289-296. Esterbauser, H., Dieber-Robtheneder, M., Strieg, G., and Waeg, G. (1991) Role of vitamin E in preventing the oxidation of low-density lipoprotein. Am. J. Clin. Nutr. 53: 314S-2IS. Gallo-Torres, H. E. (1980) Absorption, in: Vitamin E, A Comprehensive Treatise. Machlin, L. J., ed. Marcel Dekker, New York, pp. 170-267. Glomset, J. A. (1985) Fish, fatty acids, and human health. N. Eng!. J. Med. 312: 1253-1255. Gey, K. F., Puska, P., Jordan, P., and Moser, U. K. (1991) Inverse correlation between plasma vitamin E and mortality from ischemic heart disease in cross-cultural epidemiology. Am. J. Clin. Nutr. 53: 326S-34S. Gohil, K., Rothfuss, L., Lang, J., and Packer, L. (1987) Effect of exercise training on tissue vitamin E and ubiquinone content. J. App!. Physio!. 63: 1638-1641. Gohil, K., Packer, L., deLumen, B., Brooks, G. A., and Terblanche, G. E. (1986) Vitamin E deficiency and vitamin C supplements: exercise and mitochondrial oxidation. J. App!. Physio!. 60: 1986-1991. Halliwell, B. (1987) Oxidants and human disease: some new concepts. FASEB J. I: 358-364. Harman, D. (1980) Free radical theory of aging: beneficial effect of antioxidant on the life span of male NZB mice; role of free radical reactions in the deterioration of the immune system with aging and in the pathogenesis of systemic lupus erythrematous. Age 3: 64-73. Harman, D. (1984) Free radicals and the origination, evolution and present status of free radical theory of aging, in: Free Radicals in Molecular Biology, Aging and Disease. Armstrong, D., Cutler, R. G., Sohal, R. S., and Slater, T. F., eds. Ravenswood Press, New York, pp. 1-12. Herold, P. M., and Kinsella, J. E. (1986) Fish oil consumption and decreased risk of cardiovascular disease: a comparison of findings from animal and human feeding trials. Am. J. Clin. Nutr. 43: 566-598. Ji, L. L., Dillon, D., and Wu, E. (1990) Alteration of antioxidant enzymes with aging in rat skeletal muscle and. liver. Am. J. Physio!. 258: R918-R923. Kinsella, J. E., Lokesh, B., Stone, R. A. (1990) Dietary n-3 polyunsaturated fatty acids and amelioration of cardiovascular disease: possible mechanisms. Am. J. Clin. Nutr. 52: 1-28. Leka, L. S., Remaly, K. M., Bizinkauskas, P. A., and Meydani, M. (1989) Effects of fish oil on intestinal absorption of vitamin E in the rat. FASEB J. 3: A951.
418 Lemoyne, M., Van Gossen, A., Kurian, R., Ostro, M., Axler, J., and Jeejeebhoy, K. N. (1987) Breath pentane analysis as an index of lipid peroxidation: a functional test of vitamin E status. Am. J. Qin. Nutr. 46: 267-272. Meydani, S. N., Shapiro, A. C., Meydani, M., Macauley, J. B., and Blumberg, J. B. (1987) Effect of age and dietary fat (fish, corn and coconut oils) on tocopherol status of C57BLJ6Nia mice. Lipids 22: 345-350. Meydani, S. N., Barklund, M. P., Liu, S., Meydani, M., Miller, R. A., Cannon, J. G., Morrow, F. D., Rocklin, R., and Blumberg, J. B. (1990) Vitamin E supplementation enhances cell-mediated immunity in healthy elderly subjects. Am. J. Clin. Nutr. 52: 557-563. Meydani, M., Verdon, C. P., and Blumberg, J. B. (1985) Effect of vitamin E, selenium and age on lipid peroxidation events in rate cerebrum. Nutr. Res. 5: 1227-1236. Meydani, M., Macauley, J. B., and Blumberg, J. B. (1988) Effect of dietary vitamin E and selenium on susceptibility of brain regions to lipid peroxidation. Lipids 23: 405-409. Meydani, M., Natiello, F., Goldin, B., Free, N., Woods, M., Schaefer, E., Blumberg, J. B., and Gorbach, S. L. (1991) Effect of long-term fish oil supplementation on vitamin E status and lipid peroxidation in women. J. Nutr. 121: 484-491. Meydani, M., Evans, W., Handelman, G., Biddle, L., Fielding, R. A., Meydani, S. N., Orencole, S. F., Fiatarolle, M. A., Blumberg, J. B., and Cannon, J. G. (1991) Vitamin E protection of exercise-induced oxidative damage in young and elderly subjects. FASEB J. 5: A919. Meydani, S. N., Cathcart, E. S., Hopkins, R. E., Meydani, M., Hayes, K. c., and Blumberg, J. B. (1986) Antioxidants in experimental amyloidosis of young and old mice, in: Fourth International Symposium of Amyloidosis. Glenner, G. G., Asserman, E. P., Benditt, E., Calkins, E., Cohen, As., and Zucker-Franklin, D., eds. Plenum Press, New York, pp. 683-692. Odeleye, O. E., Watson, R. R. (1991) Health implication of the n-3 fatty acids. Am. J. Qin. Nutr. 53: 177-178. Pincemail, J., Deby, C., Camus, G., Pirnay, F., Bouchez, R., Massaux, L., and Goutier, R. (1988) Tocopherol mobilization during intensive exercise. Eur. J. Appl. Physiol. 57: 189-191. Pubelle, P., Chantreuil, J., Bensadous, J., Blotman, F., Simon, L., and Crastes de Paule, A. (1982) Plasma lipoperoxides and aging. Biomed. 36: 164-166. Quintanilha, A. T., Packer, L., Davies, J. M. S., Racanelli, T., Davies, K. J. A. (1982) Membrane effects of vitamin E deficiency: bioenergetics and surface charge density studies of skeletal muscle and liver mitochondria. Ann. NY Acad. Sci. 399: 32-47. Samarajski, T., Ordy, J. M., and Rudy-Reimer, P. (1968) Lipofuscin pigment accumulation in the nervous system of aging. Anat. Rec. 160: 555-574. Simon-Schnass, I., and Pabst, H. (1988) Influence of vitamin E on physical performahce. Internat. J. Vito Nutr. Res. 58: 49-54. Sumida, S., Tanaka, K., Kitao, H., and Nakadomo, F. (1989) Exercise-induced lipid peroxidation and leakage of enzymes before and after vitamin E supplementation. Int. J. Biochem. 21: 835-838. Wartanowicz, M., Panczenko-Kresowska, B., Ziemlanski, S., Kowalska, M., and Okolska, G. (1984) The effect of alpha-tocopherol and ascorbic acid on the serum lipid peroxide level in elderly people. Ann. Nutr. Metab. 28: 186-191. Witting, L. A. (1974) Vitamin E-polyunsaturated lipid relationship in diet and tissues. Am. J. Qin. Nutr. 27: 952-959. Wu, W-H., Meydani, M., Meydani, S. N., Burklund, P. M., Blumberg, J. B., Munro, H. N. (1990) Effect of dietary iron overload on lipid peroxidation, prostaglandin synthesis and lymphocyte proliferation in young and old rats. J. Nutr. 120: 280-289.
