Marine Plasmalogen and its Antioxidative Effect Intake of Dietary Fish

Chapter 7

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Marine Plasmalogen and its Antioxidative Effect Intake of Dietary Fish Oil into Membrane Lipid and Their Stability Against Oxidative Stress 1

2

Kozo Takama , Kazuaki Kikuchi , and Tetsuya Suzuki

1

Faculty of Fisheries, Department of Marine Bioresources Chemistry, Laboratory of Food Wholesomeness, Hokkaido University, Hakodate 041, Japan Yaizu Suisan Kagaku Company, Ogawa-Shinmachi, Yaizu, Shizuoka, Japan 1

2

Feeding marine lipids to rats increased n-3 PUFA level in myocardial phospholipids (MPL). Comparing PE subclasses (diacyl- and alkyl-PE), the PUFA content in PE-plasmalogen (PE -plasmalogen) was particularly high, suggesting the dietary PUFA was preferentially incorporated into PE-plasmalogen molecules. Levels of rat myocardial lipofuscin-like substances, and TBARS, in the marine lipid-fed group were lower than that of the lard-fed one. Those findings suggest antioxidative activity of PE-plasmalogen. The antioxidative effect of PE-subclasses was examined with Fe ascorbic acid system, by using multilamellar liposomes. Inserting PE-plasmalogen into the PC-liposomes prolonged the induction period. However, once peroxidation was induced, it rather accelerated the reaction. PE-plasmalogen in the liposome exhibited a protective effect on n-3 PUFA, only in the early stage of peroxidation. These results suggest that antioxidative effects of dietary marine lipids may be due to PE-plasmalogen. 2+

l-Alk-l-enyl-2-acylglycerophospholipid (general name; plasmalogen) is one of the lipid classes comprising membrane lipids. Generally, the C-2 position of the acyl moiety of glycerophospholipid is predominantly occupied by polyunsaturated fatty acids (PUFA) (7), which is assembled by an enzyme, acyltransferase (2,3). An interesting fact is that plasmalogen is rich in the tissues which demand high oxygen consumption; e.g., brain, heart, skeletal muscles, gills (4-7). In order to examine whether marine origin plasmalogen participates in the stabilization of membrane phospholipids or not, we made following studies; i.e., (a) comparison of intramolecular localization of PUFA in the rat myocardial membrane phospholipids before and after feeding diets of different marine lipid

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©1998 American Chemical Society Shibamoto et al.; Functional Foods for Disease Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

59 compositions., and (b) possible role of marine lipid origin plasmalogen as an antioxidant against oxidative damage of liposomes, as a biomembrane model. Dietary Lipid and Its Reflection on The Fatty Acid Compositions of Lipid Subclasses in Rat Heart Effect of Fish Oil Diet on Rat Heart Muscle Phospholipids. Two different types of diets were fed to male Wistar rats (5 weeks after weaning), for 4 weeks. The diets used in this study were: 1.) a soybean diet, consisting AIN-76 basal diet, and 5% of soybean oil (Asahi-Yushi KK. Asahikawa, Hokkaido), and 2.) a fish oil diet which was composed of AIN-76 diet and 4% of soybean oil and 1 % of sardine oil (Nihon Kagaku Shiryo, Hakodate, Hokkaido). After 4 weeks of feeding, the rats were fasted overnight, then euthanized under diethyl ether anesthesia by depleting whole blood. Next, the heart was taken out and washed thoroughly with PBS-saline to remove the blood. Hearts thus prepared were provided for lipid analysis. Heart lipid extracts were fractionated into phosphatidylcholine (PC), and phosphatidylethanolamine (PE) on a preparative TLC according to Horrocks et al. (8). These phospholipid fractions were further subfractionated into diacyl-type, alkenyltype and alkyl-type according to Waku et al. (9). They were provided for the analysis of fatty acid composition. As shown in Table I the proportion of n-3 series PUFA, represented by 20:5 and 22:6 in PE of any lipid subclasses, was higher than those of PC. The n-3 PUFA was remarkably high in alkenyl type PE (PE-plasmalogen) (Figure 1). It has been well established that the fatty acid composition of animal cell membranes is strongly influenced by dietary fat composition (10). Among them, the incorporation efficiency of PUFA into plasmalogen, especially into PE-plasmalogen was found extraordinary high. Comparison of lard diet, fish oil diet, and marine phospholipid diet on the fatty acid composition of heart muscle membrane lipid and TBARS levels. In another experiment, we fed rats three different types of diets; a) First, with a 1% of lard (Tsukishima Food Ind. Corp., Tokyo, Japan), b) secondly, fish oil (Nihon Kagakushiryo, Hakodate, Hokkaido, Japan), c) and thirdly, marine phospholipids were separately added to AIN-76 diet. The marine phospholipid fraction was prepared in our laboratory by extracting the crude lipid fraction with cold acetone from sardine gonads. Feeding was continued for 4 weeks, to male Wistar rats (6 weeks after weaning). Results showed that the fatty acid composition of myocardial lipids was closely dependent upon the fatty acid composition of dietary lipids (Table II). Lard diet fed group showed the highest TBARS level (77), whereas that of marine phospholipid diet fed group was the lowest (Table III). Comparison of Lipofuscin Levels of Rat Liver Muscle Lipofuscin is an amyloid complex formed by the reaction of lipid peroxide, amino acids, protein, and nucleic acids (72, 13). Lipofuscin has been regarded as an index of senescence ongoing because it accumulates in animal tissues accompanied with aging. A lipofuscin like substance is known to give bright yellow color under the fluorescence microscope. Heart muscle sections prepared by formalin fixation, and subsequent paraffin embedding

Shibamoto et al.; Functional Foods for Disease Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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Table I. Fatty Acid Composition (%) of PC- and PE- Subclasses in the Rat Heart. Diacyl Alkenyl Alkyl PE PC PE PC PE PC FO FO SO FO SO FO SO FO SO SO FO SO 25.4 28.9 17.5 18.3 1.9 1.1 19.6 21.1 10.1 11.5 9.9 10.1 16:0 2.2 4.8 2.3 3.0 0.5 0.6 1.3 1.9 0.3 0.4 0.1 0.1 16: 1 6.3 7.0 7.7 6.5 2.8 2.0 18:0 25.1 24.5 30.7 31.2 4.7 4.5 10.2 8.8 10.1 6.3 2.4 1.0 6.8 4.9 6.4 2.2 10.9 4.6 18 :2 20:4 27.5 21.5 17.4 8.4 49.1 35.1 43.9 20.3 25.2 19.3 13.5 10.9 0.7 6.1 1.1 3.6 0.5 5.5 1.2 3.5 0.1 1.8 0.2 3.1 20:5 4.1 0.9 1.1 tr 4.2 0.4 1.5 tr 1.1 0.1 22:4 0.8 0.1 1.1 3.1 12.4 11.7 6.6 9.4 3.3 4.7 2.5 2.1 1.7 3.5 22:5 22:6 4.7 13.2 23.8 37.8 12.9 27.5 29.5 53.4 7.3 10.8 17.8 24.0 43.2 42.5 29.0 30.5 44.8 45.6 40.8 42.7 17.9 17.0 7.4 6.0 I Sat I PUFA 46.2 46.2 53.3 53.2 76.1 76.6 89.2 91.4 48.3 45.7 60.9 61.0 39.6 26.4 26.9 11.5 58.6 40.9 52.5 23.0 38.8 28.1 29.9 19.3 I n-6 6.6 19.8 26.4 41.7 17.4 35.7 36.6 68.3 9.5 17.6 31.0 41.7 I n-3 PC: Phosphatidylcholine, PE: Phosphatidylethanolamine, SO: Soybean Oil, FO: Fish Oil . PC contents (u mol/ g heart) : SO fed group 12.5±0.7 [Diacyl 10.3±1.5, Alkenyl 0.5±0.3, Alkyl 0.2±0.2] ; FO fed group 13.0±0.9 [Diacyl 12.3±2.0, Alkenyl 0.8±0.1, Alkyl 0.3±0.3], PE contents (u mol/g heart) : SO fed group 11.2±0.9 [Diacyl 7.6±0.7, Alkenyl 3.1±0.5, Alkyl 0.2±0.1 ] ; FO fed group 11.7±1.2 [Diacyl 7.9±0.4, Alkenyl 3.1+0.1, Alkyl 0.2±0.1]. Fatty Acid

I

SO FO Diacyl

SO FO Alkenyl

SO FO

SO FO

Alkyl

Diacyl

SO FO Alkenyl

T SO FO Alkyl

Figure 1. Relative distributions of PUFA(n-6 or n-3) against total saturated fatty acid. • : n-6, • : n-3 ; SO: Soybean oil, FO: Fish oil


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Table II. Fatty Acid Composition (%) of Lipids in the Diet and Heart of the Rats after Feeding for Four Weeks. Fatty Acid Diet NPL PL Diet 14::0 0.4 1.9 0.1 1.6 13.1 25.5 17.6 15.3 16::0 0.2 0.4 8.0 1.5 16:: 1 3.1 32.6 3.5 4.9 18::0 18.4 29.5 3.0 22.1 18:: 1 18 :2 : 45.5 29.8 12.2 45.3 5.6 6.6 0.8 18::3 22.2 0.8 1.1 20::4 1.2 2.4 20::5 11.3 1.9 22::6 0.7 NPL: non-polar lipids, PL: polar lipids.

NPL 0.8 12.8 2.1 2.9 18.5 55.4 0.7 2.4 -

2.4

PL 0.2 15.9 0.1 31.4 2.3 16.8 0.1 18.2 0.4 13.5

Diet 1.1 22.9 0.4 3.0 24.8 38.6 4.8 0.3 0.4 3.7

NPL 0.7 16.3 2.3 5.4 22.2 42.7 0.8 0.3 0.4 3.7

PL 1.2 14.6 0.2 30.4 2.6 14.6 0.1 16.1 0.1 18.5

2+

Table III. Induction Period and Peroxidation Rate of PC-Liposome in Fe Ascorbate System. Tested lipid inserted into PC-liposomePC

PE

0.166 Induction Period 0.098 33.7 Oxidation Rate (nmol MDA/hr) 26.3 Oxidation level (nmol MDA/mg 51.2 60.0 lipid after lOhr) PC: Phosphatidycholine, PE: Phosphatidylethanolamine

PE-plasmalogen

0.226 40.9 71.2


62 were examined under the fluorescence microscope equipped with Video Enhanced Image Analyzer (ARGUS-100, Hamamatsu Photonics, KK., Hamamatsu, Japan). Integrated fluorescence intensities of heart muscle sections are presented in Table III. Undoubtedly, the lard diet fed section gave the highest fluorescence level, and the lowest level was observed for the marine phospholipid diet fed section. The results presented above are definite evidence to prove acyl moieties of dietary lipids. The n-3 series PUFA are especially incorporated into the acyl moieties of PE -plasmalogen. Furthermore, a very interesting result, was that lipid hydroperoxide level was kept low in the samples rich in n3 series PUFA contents, because n-3 series PUFA has been regarded highly susceptible to reactive oxygen attack. Susceptibility of Lipid Peroxidation and Participation of PE-plasmalogen in Liposomal System The results mentioned above suggest a possible relationship between PE-plasmalogen and suppression of membrane lipid peroxidation. Therefore, we carried out model experiments with an iron-ascorbic acid system, in diacyl-PE and alkenyl-PE embedded multilamellar liposomes, to examine the possibility whether our postulation would be worthy or not. PEplasmalogen embedded multilamellar liposomes were prepared from egg yolk phosphatidylcholine (Sigma Chemical Co., St. Louis, MO) and 10% (mol/mol) PEplasmalogen, by the method of Nagao and Mihara (14), with slight modification. In order to induce lipid peroxidation in the liposomes, egg yolk PC-OOH (15,16) was added at the concentration of 0.005% (mol/mol). Iron-ascorbic acid-induced lipid peroxidation was carried out in HEPES buffer (pH 7.4), at 37 °C At regular time intervals, certain volumes of reactant were collected for the measurement of TBARS, by the method of Uchiyama and Mihara (7 7). A relationship between the malondialdehyde (MDA) concentration, and incubation time, was plotted on a semi-logarithmic scale as shown in Figure 2. Since a linear relation was obtained in every experimental condition, the distance to the right of 0 along the xaxis, where lines intersects in the x-distance, should be regarded as the induction period. On the other hand, the gradient of each line is regarded to mean the rate of oxidation velocity. Obviously, the induction period was more effectively elongated in alkenyl-PE embedded liposomes than PE embedded system. However, once the lipid peroxidation was started, it was rather accelerated in the presence of plasmalogen. Major PUFA, consisting of PE (prepared from porcine liver, Doosan Serdary Res. Lab. Inc., Englewood Clifs, NJ) used in the present experiment, were linoleic acid (18:2) and arachidonic acid (20:4), occupying 9% and 16%, respectively. The ratio of PUFA to the total sum of saturated fatty acid was 0.9. While PE-plasmalogen (prepared from bovine brain, Doosan Serdary Res. Lab. Inc., Englewood Clifs, NJ) had linoleic acid (18:2) and cis-7,10,13,16-docosatetraenoic acid (22:4) as the major PUFA occupying 29% and 9%, respectively, and the ratio of PUFA to total saturated fatty acids was 1.9. Thus the composition of fatty acids varied by the materials.


63 801

Peroxidation period, log(hr) 2+

Figure 2. Peroxidation of PC-liposomes under Fe -ascorbate system at 37°C. • : Phosphatidylcholine, O: Phosphatidylethanolamine, #: PE-plasmalogen

Peroxidation period, log(hr)

Figure 3. PC-liposome peroxidation induced by AAPH at 37^. #: bovine brain PE-plasmalogen, O: starfish PE-plasmalogen


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Extension of initiation stage by PE-plasmalogen Although it is still unclear whether the compositional differences of fatty acids was the key to the antioxidative effect of PE-plasmalogen or not, an experiment using liposomes embedding PE-plasmalogen, which was prepared from starfish (Astropecten scoparius), showed us quite interesting results. As much as 74.7% of the PE fraction of the starfish was composed of alkenyl-type, or PE-plasmalogen. In this model experiment, peroxidation was started by the addition of an water soluble radical initiator, AAPH [2,2'azobis(2-amidinopropane)dihydrochloride]. In comparison with the previous experiment using bovine brain plasmalogen, the starfish-origin plasmalogen clearly showed a better effect on the extension of the induction period. At the same time, the rate and the extent of oxidation was also enhanced by the starfish-origin PE-plasmalogen than the bovine brain one. The starfish PE-plasmalogen contained arachidonic acid (34%) and eicosapentaenoic acid (20%), and the ratio of sum of PUFA to the total saturated fatty acid was 4.4. It is likely that higher content of PUFA in plasmalogen suppresses initiation of lipid peroxidation, or extends induction period. But once the peroxidation reaction moves into the propagation stage, PE-plasmalogen with higher PUFA content accelerates oxidation velocity. Antioxidative effects at the induction stage and a stimulation effect at the propagation stage of the PE-plasmalogen should be derivedfromthe vinyl-ether bond at C1 position of glycerol moiety. Kobayashi (18) commented in his review article the participation of vinyl-ether linkage during u.v.-induced oxidation reaction; i.e., continual U.V. irradiation leads to decomposition of plasmalogen in the liposomal system with time, and its decomposition is characteristic to the compound with a vinyl-ether linkage. That is, during peroxidation reaction in the liposomes plasmalogen molecules, or vinyl-ether bond, was served preferentially to be oxidized and decomposed by the singlet oxygen. In other reactions, the reaction mechanism with other reactive oxygen and free radicals should be the same. This is because induction periods were extended both in the AAPH-induced, and hydroperoxide-initiated systems. Furthermore, plasmalogen has been reported to participate in the protection of cell function of the Chinese hamster ovary (CHO) cells (19). Its protective function is explained by the high susceptibility of the molecule to reactive oxygen attack (20). In our experiment, a decrease of PUFA was recognized along with promoting propagation reaction in the liposome system (data not shown). In brief, plasmalogen serves itself as a sacrifice to protect other membrane lipid components. Literature Cited. (1) Horrocks, L.A. In Ether Lipids, Chemistry and Biology; Snyder, F. Ed.: Content,

composition, and metabolism of mammalian and avian lipid that contain ether groups; Academic Press: New York, NY, 1972, pp 233-237. (2) Svennerholm, L.; Stallberg-Stenhagen, S. J. Lipid Res. 1968, 9, 215-225. (3) Segler-Stahl, K.; Webster, J.C.; Brungraber, E.C. Gerontology. 1983, 29, 161-168.


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(4) Horrocks, L.A. In Ether Lipids, Chemistry and Biology; Snyder, F. Ed.: Content, composition, and metabolism of mammalian and avian lipid that contain ether groups; Academic Press: New York, NY, 1972, pp 179-224. (5) Davenport, J.B. Biochem. J. 1964, 90, 116-122. (6) Spanner, S. Nature, 1966, 270, 637. (7) Chapelle, S. Comp. Biochem. Physiol. 1986, 85B, 507-510. (8) Horrocks, L.A.; Sun, G.Y. In Research Methods in Neurochemistry, Rodnight, R.; Mariks, N . Ed.; Ehanolamine plasmalogens; Plenum Press: New York, NY, 1972; pp 223231. (9) Waku, K.; Ito, H.; Bito,T.; Nakazawa, Y. J. Biochem. 1974, 75, 1307-1312. (10) Scott, T.W.; Ashes, J.R.; Fleck, E.; Gulati, S.K. J. Lipid Res. 1993, 34, 827-835. (11) Fukunaga, K.; Suzuki, T.; Takama, K. J. Chromatogr. 1993, 621, 71-81. (12) Chio, K.S.; Reiss, U.; Fletcher, B.; Tappel, A.L. Science. 1969, 166, 1535-1536. (13) Kikugawa, K.; Ido, Y. Lipids. 1984, 19, 600-608. (14) Nagao, A.; Mihara, M . Biochem. Biophys. Res. Commun. 1978, 172, 385-389. (15) Terao, J.; Matsushita, S. Agric. Biol. Chem. 1981, 45, 587-593. (16) Terao, J.; Hirota, Y.; Kawakatsu, M.; Matsushita, S. Lipids. 1981, 16, 427-432. (17) Uchiyama, M.; Mihara, M . Anal. Biochem. 1978, 86, 271-278. (18) Kobayashi, T. In Function and Biosynthesis of Plasmalogens; Inoue, K., Nozawa, Y., Ed.; Phospholipid metabolism and disease in animal cells published as an extra number of Protein, Nucleic Acid and Enzyme; Kyoritsu Shuppan Kabushiki Kaisha: Tokyo, 1991, Vol. 2; pp 424-432. (in Japanese) (19) Zoeller, R.A.; Morand, O.H.; Raetz, C.R.H. J. Biol. Chem. 1988, 263, 11590-11596. (20) Morand, O.H.; Zoeller, R.A.; Raetz, C.R.H. J. Biol. Chem. 1988, 263, 11597-11606.


Marine Plasmalogen and its Antioxidative Effect Intake of Dietary Fish

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