Vol.
179,
No.
September
16,
BIOCHEMICAL
2, 1991
AND
BIOPHYSKAL
RESEARCH
COMMUNICATIONS Pages
1991
1077-1081
INHIBITORY EFFECT OF PHOSPHATIDYLSERINE ON IRON-DEPENDENT LIPID PEROXIDATION
Katsunori Yoshida 1, Junji Terao **, Tetsuya Suzuki 1 and Kozo Takama
1
1 Department of Food Science and Technology, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido, 041 Japan 2 National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, 305 Japan Received
August
1,
1991
SUMMARY. The effect of phospholipids on lipid peroxidation was investigated in liposomal suspension of egg yolk phosphatidylcholine. Both saturated and unsaturated phosphatidylserine effectively inhibited lipid peroxidation induced by ferrous-ascorbate system in the presence of phosphatidylcholine hydroperoxides. Studies on the iron trapping effect of phospholipids indicated that the effectiveness of inhibition depends on the charge of phosphatidylserine that binds to free ionic iron. Q 1991 Academic Pres5, Inc.
There are enough evidence that lipid peroxidation causes a great variety of diseases (1, 2). To better understand the mechanism of lipid peroxidation, many model systems have been employed. According to previous studies, both free and chelated iron induce lipid peroxidation (3-6). Vile and Winterbourn (7) have suggested that ionic iron binds to membrane lipids, which subsequently participates in peroxidation. Kunimoto et al. (8) have proposed that neutral or negatively charged membranes are more sensitive to iron catalyzed peroxidation than positively charged ones. Biological membranes have unique lipid compositions suggesting a specific role for many lipids (g-11). For example, acidic phospholipids have strong affinities toward divalent cations (12,13). Viani et a/. (14) reported that PA inhibits lipid
* To whom correspondence
should be addressed.
ABBREVIATIONS: EYPC, egg yolk phosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DPPE, dipalmitoyl phosphatidylethanolamine; DPPS, dipalmitoyl phosphatidylserlne; PLPC, pig liver phosphatidylcholine; PLPE, pig liver phosphatldylethanolamlne; BBPS, bovine brain phosphatidylserine; PA, phosphatidic add; MDA, malondialdehyde; AAPH, 2,2’azobis(2-amidinopropane)hydrochlotide.
1077
0006-291x/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
peroxidation of liposomes and rat brain synaptosomes. Despite the large number of investigations done, there has been very little information about the effect of phospholipids on membrane lipid peroxidation. In this study, we examined the effect of phospholipid polar head groups on liposomal lipid peroxidation. The results demonstrate that PS can inhibit peroxidation by trapping ionic iron responsible for the initiation process of lipid peroxidation.
MATERIALS
AND METHODS
Chemicals and Lipids: EYPC was purchased from Sigma Chemical Co., St. Louis, MO, and purified to remove hydroperoxides by chromatography on a glass column packed with Lichroprep RP-8 (Merck, Darmstadt, Germany) (15). EYPC hydroperoxides were prepared by photooxidation (16) and purified using Lichroprep RP-8 column. Purchased PLPC, PLPE and BBPS were further purified by reverse phase TLC. All phospholipids showed a single spot on thin layer chromatograms. Chelex 100 ion exchange resin purchased from Bio-Rad was used to remove trace contaminating metals from buffers and reagents. Preoaration of lioosomes: Multilamellar liposomes were prepared as described previously (17). Phospholipids in a chloroform solution were evaporated to a llpld film using nitrogen gas and finally vaccum pump. The dried lipid film was dispersed in 10 mM Tris-HCI buffer, pH 7.4, by vigorous shaking on a vortex mixer followed by ultrasonic irradiation. Final concentration in the liposomal solution was 4 mM EYPC, 0.4 mM additive phospholipids (PC, PE, PS) and 0.04 mM EYPC hydroperoxides which was not added in AAPH induced peroxidation system. Peroxidation of lioosomal liDids: Lipid peroxidation in liposomes was induced by the addition of an aqueous solution of 0.1 mM FeS04 and 1 .O mM ascorbic acid, or of 20 mM AAPH. Incubation was carried out at 37 “C with continuous shaking. Lipid peroxidation induced by ferrous-ascorbate was measured by MDA formation (18). EYPC hydroperoxides were determined by the method reported previously with a slight modification in which methanol/water (955, v/v) was used as an eluting solvent (19). Iron traooina effect of ohosDholioids : Liposomes of DPPC, DPPE and DPPS were prepared as described above. The same amount of FeSO4 used in peroxldation experiment was added to the liposome solution, and 0.9 ml of this solution was partitioned with 2 ml of chloroform/methanol (l:i, v/v). The Iron trapped by phospholipids passed over into the organic phase. The iron level in the organic phase was determined by flameless atomic absorption spectrometry (HITACHI 18030).
RESULTS
AND DISCUSSION
We prepared EYPC liposomes containing saturated phospholipids, then induced lipid peroxidation by adding ferrous-ascorbate (Fig. 1). An experiment using phospholipids of the same fatty acid constituents is useful for comparing the effect of their polar head groups. Liposomes used in this study contained 1 mol% EYPC hydroperoxides to unify the initial condition of peroxidation. Significant peroxidation was observed on EYPCIDPPC liposomes. Concerning EYPQDPPE liposomes, peroxidation progressed in the same way, however, the formation of MDA never exceeded that of EYPC/DPPC. In contrast, no significant peroxidation was 1078
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
02
RESEARCH
O
COMMUNICATIONS
2
6
TIME
(hrJ4
Fia. 1. Effect of saturated phospholipids on ferrous-ascorbate dependent peroxidation of EYPC liposomes. Incubations contained liposomes (4 mM c/PC, 0.4 mM additional phospholipids, 0.04 mM EYPC hydroperoxides) in 10 mM TrisHCI, pH 7.4. Lipid peroxidation was initiated in all incubations by the addition of 0.1 mM FeSO4 and 1.O mM ascorbic add. (mean * S.D. of triplicate determinations.) 0, DPPC; 0, DPPS 0, DPPE; Fin. 2. Effect of unsaturated phospholipids on ferrous-ascorbate dependent peroxidation of EYPC liposomes. The conditions are the same as described in Fig. ; (:I;; k S.D. of triplicate determinations.) ; .,PLPE; 0, BBPS
observed
on EYPC/DPPS
that DPPS inhibited lipid peroxidation induced by ferrous-ascorbate in comparison with DPPC and DPPE. To ensure that the inhibitory effect of PS in the liposomal system were not due to their acyl chain but polar head groups, the effect of unsaturated phospholipids was studied. EYPC liposomes containing unsaturated phosphdipids were subjected to lipid peroxidation by adding ferrous-ascorbate (Fig. 2). The ratio of unsaturated/saturated fatty acids of the phospholipids was 1.06 (PLPC), 1.16 (PLPE) and 1.20 @BPS). Inhibitory effect of unsaturated phospholipids on lipid peroxidation was similar to saturated ones. The highest peroxidation was observed in EYPC/PLPC liposomes throughout incubation, followed by EYPC/PLPE liposomes. BBPS effectively suppressed lipid peroxldation, too. From these results, it appears that peroxidation inhibitory effect of phospholipids is due to their polar head groups. In the next experiment, lipid peroxidation was induced by AAPH, a radical initiator well known to generate free radicals. The radical attacks lipid as a substrate and causes lipid hydroperoxides accumulation. EYPC hydroperoxides were estimated by conjugated diene increase using HPLC (19). As shown in Fig. 3, there was no remarkable difference in the effect of PC, PE and PS on AAPH induced lipid peroxidation. It therefore demonstrates that PS possesses little radical-scavenging ability in spite of its inhibitory effect on lipid peroxidation induced by ferrousascorbate system. How can the presence of PS inhibit iron dependent peroxidatlon of liposomes? Iron catalyzes
decomposition
liposomes.
It was found
of preformed 1079
lipld hydroperoxides
resulting
peroxy
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
PC
PE F
03
-O
1
3
4
TIMI? (hr)
Fia. 3. Effect of phospholipids on AAPH dependent peroxidation of EYPC liposomes. Incubations contained the same as Fig. 1. Lipid peroxidation was initiated by the addition of 20 mM AAPH. EYPC hydroperoxideswere omltted in the reaction system. (mean f S.D. of triplicate determinations.) 0, DPF’C; 0, DPPE; 0, DPPS Fia. 4. Iron trapping effect of phospholipids.0.1 mM FeS04 was added to DPPC, DPPE and DPPS liposomesand partitioned with chloroform/methanol(1:l , v/v). Iron level in organic phase was estimated by atomic absorption spectrometry. (mean k S.D. of triplicate determinations.)
and alkoxy radicals that lead to a chain reaction (20). Thus, we considered the possibility that the polar head groups of PS could bind to the ionic iron in that they reduce the amount of free ionic iron and suppress ferrous ion-induced decomposition of PC-hydroperoxides in the liposomes. We evaluated the amount of iron trapped by the phospholipids. As shown in Fig. 4, the rates of trapped Iron were 37 % (DPPC) and 45 % (DPPE). PS strongly bound to iron and the rate was nearly 100 %. These results agree with previous studies (12, 13).About 14 % of iron was distributed in the organic phase without phospholipids. PC and PE also bound to ionic iron but their binding effect is much weaker than that of PS. Thus PC and PE may not inhibit iron-dependent lipid peroxidation. PS has an acidic carboxyl group, PE and PS have a basic primary amine; their charge changes depending on pH values. PC possesses a quaternary amine which is positively charged at all pH values. The state of ionization of phospholipids at neutral pH is any of the following; the net charge is zero for PC, -1 for PS, PE may be neutral or slightly negatively charged (10). According to our results, the order of the inhibitory effect on lipid peroxldation was as follows; PS>>PE>PC. The more negatively charged the polar groups are, the greater they trap iron, which leads to inhibition of peroxidation. It is assumed that free ionic iron is bound to polar head groups of phospholipids by electrostatic force and that bound iron could not approach EYPC and EYPC hydroperoxides. Hence, iron may not be responsible for catalyzing hydroperoxides decomposition and lipid peroxidation chain reaction. Since AAPH has no charge in the solution, it is not affected by polar groups (Fig. 3), and its radical could freely attack substrate lipids. Viani et al. (14) have proposed that PA and free fatty acid complex bind to ferrous ion and inhibit iron’s capacity to promote lipid peroxidation. 1080
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
However, our results suggest that PS itself can bind to iron and suppress lipid peroxidation. It might be assumed that PS is responsible for the site-specific mechanism (21) of lipid peroxidation in biomembrane system because site-specific peroxidation is thought to be induced by transition metal ion on the membrane surface (22). Nevertheless, PS did not change during incubation with ferrousascorbate, which was confirmed by TLC and GLC analysis (not shown). Our results strongly suggests that PS exerts an inhibitory effect on biomembrane lipid peroxidation by acting as an iron trapper on the membrane surface.
REFERENCES 1. Halliwell, B. and Gutteridge, J. M. C. (1984) Biochem. J. 219 , l-14. 2. Slater, T. F. (1984) Biochem. J. 222 , l-15. 3. Svingen, B. A., Buege, J. A., O’Neal, F. 0. and Aust,S. D. (1979) J. Viol. Chem. 254 , 5892-5899. 4. Bucher, J. R., Tien, M. and Aust, S. D. (1983) Biochem. Biophys. Res. Commun. 111 , 777-784. 5. Wills, E. D. (1965) Biochim. Biophys. Acta 98, 238-251. 6. Kornbrust, D. J. and Mavis, R. D. (1980) Mol. Pharmacol. 17 , 400-407. 7. Vile, G. F. and Winterbourn, C. C. (1987) FEBS. 215 , 151-154. 8. Kunimoto, M., Inoue, K. and Nojima, S. (1981) Biochim. Biophys. Acta 848 , 169- 178. 9. Maeda, M., Nishijima, M., Takenaka, Y., Kuge, 0. and Akamatsu, Y. (1984) Biochim. Biophys. Acta 794 , 298-306. -
10. Boggs, J. M. (1980) Can. J. Biochem. 58, 755-770. 11. Green, D. E., Fry, M. and Blondin, G. A. (1980) Proc. Nat/. Acad. Sci, USA 77, 257-261. 12. Hauser, H. and Dawson, R. M. C. (1968) Biochem. J. 109 , 909-916. 13. Jacobson, K. and Papahadjopoulos, D. (1975) Biochemistry 14, 152-l 61. 14. Viani, P., Cervato, G., Fiorilli, A., Rigamonti, E. and Cestaro, B. (1990) Chem. Phys. Lipids 52 , 49-55.
15. Terao, J., Asano, I. and Matsushita, S. (1985) Lipids 20, 312-317. 16. Terao, J. and Matsushita, S. (1981) Agric. Ho/. Chem. 45 , 587-593. 17. Nagao, A. and Terao, J. (1990) Biochem. Biophys. Res, Commun. 172 , 385389.
18. Uchiyama, M. and Mihara, M. (1978) AnalBiochem. 88, 271-278. 19. Terao, J., Shibata, S.S., Yamada, K. and Matsushita, S. (1988) In Medical Biochemical and Chemical Aspects of Free Radicals (Hayaishi, O., Niki,E., Kondo, M. and Yoshikawa, T., eds.) Vol.8 , pp.781-788, Elsevier Science Publishers, B.V., Amsterdam. 20. Halliwell, B. and Gutteridge, J. M. C. (1988) In Medical Biochemical and Chemical Aspects of Free Radicals (Hayaishi, O., Niki, E., Kondo, M. and Yoshikawa, T., eds.), vol.1 , pp. 21-31, Elsevier Science Publishers, B.V., Amsterdam. 21. Czapski, G. and Goldstein, B. (1986) Free Radical Reseach Commun. 1 , 157161. 22. Fukuzawa, K., Kishikawa, K., Tadokoro, T., Tokumura, A., Tsukatani, H. and Gebicki, J. M. (1988) Arch. Biochem. Biophys. 280 , 152-160.
1081
179,
No.
September
16,
BIOCHEMICAL
2, 1991
AND
BIOPHYSKAL
RESEARCH
COMMUNICATIONS Pages
1991
1077-1081
INHIBITORY EFFECT OF PHOSPHATIDYLSERINE ON IRON-DEPENDENT LIPID PEROXIDATION
Katsunori Yoshida 1, Junji Terao **, Tetsuya Suzuki 1 and Kozo Takama
1
1 Department of Food Science and Technology, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido, 041 Japan 2 National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, 305 Japan Received
August
1,
1991
SUMMARY. The effect of phospholipids on lipid peroxidation was investigated in liposomal suspension of egg yolk phosphatidylcholine. Both saturated and unsaturated phosphatidylserine effectively inhibited lipid peroxidation induced by ferrous-ascorbate system in the presence of phosphatidylcholine hydroperoxides. Studies on the iron trapping effect of phospholipids indicated that the effectiveness of inhibition depends on the charge of phosphatidylserine that binds to free ionic iron. Q 1991 Academic Pres5, Inc.
There are enough evidence that lipid peroxidation causes a great variety of diseases (1, 2). To better understand the mechanism of lipid peroxidation, many model systems have been employed. According to previous studies, both free and chelated iron induce lipid peroxidation (3-6). Vile and Winterbourn (7) have suggested that ionic iron binds to membrane lipids, which subsequently participates in peroxidation. Kunimoto et al. (8) have proposed that neutral or negatively charged membranes are more sensitive to iron catalyzed peroxidation than positively charged ones. Biological membranes have unique lipid compositions suggesting a specific role for many lipids (g-11). For example, acidic phospholipids have strong affinities toward divalent cations (12,13). Viani et a/. (14) reported that PA inhibits lipid
* To whom correspondence
should be addressed.
ABBREVIATIONS: EYPC, egg yolk phosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DPPE, dipalmitoyl phosphatidylethanolamine; DPPS, dipalmitoyl phosphatidylserlne; PLPC, pig liver phosphatidylcholine; PLPE, pig liver phosphatldylethanolamlne; BBPS, bovine brain phosphatidylserine; PA, phosphatidic add; MDA, malondialdehyde; AAPH, 2,2’azobis(2-amidinopropane)hydrochlotide.
1077
0006-291x/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
peroxidation of liposomes and rat brain synaptosomes. Despite the large number of investigations done, there has been very little information about the effect of phospholipids on membrane lipid peroxidation. In this study, we examined the effect of phospholipid polar head groups on liposomal lipid peroxidation. The results demonstrate that PS can inhibit peroxidation by trapping ionic iron responsible for the initiation process of lipid peroxidation.
MATERIALS
AND METHODS
Chemicals and Lipids: EYPC was purchased from Sigma Chemical Co., St. Louis, MO, and purified to remove hydroperoxides by chromatography on a glass column packed with Lichroprep RP-8 (Merck, Darmstadt, Germany) (15). EYPC hydroperoxides were prepared by photooxidation (16) and purified using Lichroprep RP-8 column. Purchased PLPC, PLPE and BBPS were further purified by reverse phase TLC. All phospholipids showed a single spot on thin layer chromatograms. Chelex 100 ion exchange resin purchased from Bio-Rad was used to remove trace contaminating metals from buffers and reagents. Preoaration of lioosomes: Multilamellar liposomes were prepared as described previously (17). Phospholipids in a chloroform solution were evaporated to a llpld film using nitrogen gas and finally vaccum pump. The dried lipid film was dispersed in 10 mM Tris-HCI buffer, pH 7.4, by vigorous shaking on a vortex mixer followed by ultrasonic irradiation. Final concentration in the liposomal solution was 4 mM EYPC, 0.4 mM additive phospholipids (PC, PE, PS) and 0.04 mM EYPC hydroperoxides which was not added in AAPH induced peroxidation system. Peroxidation of lioosomal liDids: Lipid peroxidation in liposomes was induced by the addition of an aqueous solution of 0.1 mM FeS04 and 1 .O mM ascorbic acid, or of 20 mM AAPH. Incubation was carried out at 37 “C with continuous shaking. Lipid peroxidation induced by ferrous-ascorbate was measured by MDA formation (18). EYPC hydroperoxides were determined by the method reported previously with a slight modification in which methanol/water (955, v/v) was used as an eluting solvent (19). Iron traooina effect of ohosDholioids : Liposomes of DPPC, DPPE and DPPS were prepared as described above. The same amount of FeSO4 used in peroxldation experiment was added to the liposome solution, and 0.9 ml of this solution was partitioned with 2 ml of chloroform/methanol (l:i, v/v). The Iron trapped by phospholipids passed over into the organic phase. The iron level in the organic phase was determined by flameless atomic absorption spectrometry (HITACHI 18030).
RESULTS
AND DISCUSSION
We prepared EYPC liposomes containing saturated phospholipids, then induced lipid peroxidation by adding ferrous-ascorbate (Fig. 1). An experiment using phospholipids of the same fatty acid constituents is useful for comparing the effect of their polar head groups. Liposomes used in this study contained 1 mol% EYPC hydroperoxides to unify the initial condition of peroxidation. Significant peroxidation was observed on EYPCIDPPC liposomes. Concerning EYPQDPPE liposomes, peroxidation progressed in the same way, however, the formation of MDA never exceeded that of EYPC/DPPC. In contrast, no significant peroxidation was 1078
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
02
RESEARCH
O
COMMUNICATIONS
2
6
TIME
(hrJ4
Fia. 1. Effect of saturated phospholipids on ferrous-ascorbate dependent peroxidation of EYPC liposomes. Incubations contained liposomes (4 mM c/PC, 0.4 mM additional phospholipids, 0.04 mM EYPC hydroperoxides) in 10 mM TrisHCI, pH 7.4. Lipid peroxidation was initiated in all incubations by the addition of 0.1 mM FeSO4 and 1.O mM ascorbic add. (mean * S.D. of triplicate determinations.) 0, DPPC; 0, DPPS 0, DPPE; Fin. 2. Effect of unsaturated phospholipids on ferrous-ascorbate dependent peroxidation of EYPC liposomes. The conditions are the same as described in Fig. ; (:I;; k S.D. of triplicate determinations.) ; .,PLPE; 0, BBPS
observed
on EYPC/DPPS
that DPPS inhibited lipid peroxidation induced by ferrous-ascorbate in comparison with DPPC and DPPE. To ensure that the inhibitory effect of PS in the liposomal system were not due to their acyl chain but polar head groups, the effect of unsaturated phospholipids was studied. EYPC liposomes containing unsaturated phosphdipids were subjected to lipid peroxidation by adding ferrous-ascorbate (Fig. 2). The ratio of unsaturated/saturated fatty acids of the phospholipids was 1.06 (PLPC), 1.16 (PLPE) and 1.20 @BPS). Inhibitory effect of unsaturated phospholipids on lipid peroxidation was similar to saturated ones. The highest peroxidation was observed in EYPC/PLPC liposomes throughout incubation, followed by EYPC/PLPE liposomes. BBPS effectively suppressed lipid peroxldation, too. From these results, it appears that peroxidation inhibitory effect of phospholipids is due to their polar head groups. In the next experiment, lipid peroxidation was induced by AAPH, a radical initiator well known to generate free radicals. The radical attacks lipid as a substrate and causes lipid hydroperoxides accumulation. EYPC hydroperoxides were estimated by conjugated diene increase using HPLC (19). As shown in Fig. 3, there was no remarkable difference in the effect of PC, PE and PS on AAPH induced lipid peroxidation. It therefore demonstrates that PS possesses little radical-scavenging ability in spite of its inhibitory effect on lipid peroxidation induced by ferrousascorbate system. How can the presence of PS inhibit iron dependent peroxidatlon of liposomes? Iron catalyzes
decomposition
liposomes.
It was found
of preformed 1079
lipld hydroperoxides
resulting
peroxy
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
PC
PE F
03
-O
1
3
4
TIMI? (hr)
Fia. 3. Effect of phospholipids on AAPH dependent peroxidation of EYPC liposomes. Incubations contained the same as Fig. 1. Lipid peroxidation was initiated by the addition of 20 mM AAPH. EYPC hydroperoxideswere omltted in the reaction system. (mean f S.D. of triplicate determinations.) 0, DPF’C; 0, DPPE; 0, DPPS Fia. 4. Iron trapping effect of phospholipids.0.1 mM FeS04 was added to DPPC, DPPE and DPPS liposomesand partitioned with chloroform/methanol(1:l , v/v). Iron level in organic phase was estimated by atomic absorption spectrometry. (mean k S.D. of triplicate determinations.)
and alkoxy radicals that lead to a chain reaction (20). Thus, we considered the possibility that the polar head groups of PS could bind to the ionic iron in that they reduce the amount of free ionic iron and suppress ferrous ion-induced decomposition of PC-hydroperoxides in the liposomes. We evaluated the amount of iron trapped by the phospholipids. As shown in Fig. 4, the rates of trapped Iron were 37 % (DPPC) and 45 % (DPPE). PS strongly bound to iron and the rate was nearly 100 %. These results agree with previous studies (12, 13).About 14 % of iron was distributed in the organic phase without phospholipids. PC and PE also bound to ionic iron but their binding effect is much weaker than that of PS. Thus PC and PE may not inhibit iron-dependent lipid peroxidation. PS has an acidic carboxyl group, PE and PS have a basic primary amine; their charge changes depending on pH values. PC possesses a quaternary amine which is positively charged at all pH values. The state of ionization of phospholipids at neutral pH is any of the following; the net charge is zero for PC, -1 for PS, PE may be neutral or slightly negatively charged (10). According to our results, the order of the inhibitory effect on lipid peroxldation was as follows; PS>>PE>PC. The more negatively charged the polar groups are, the greater they trap iron, which leads to inhibition of peroxidation. It is assumed that free ionic iron is bound to polar head groups of phospholipids by electrostatic force and that bound iron could not approach EYPC and EYPC hydroperoxides. Hence, iron may not be responsible for catalyzing hydroperoxides decomposition and lipid peroxidation chain reaction. Since AAPH has no charge in the solution, it is not affected by polar groups (Fig. 3), and its radical could freely attack substrate lipids. Viani et al. (14) have proposed that PA and free fatty acid complex bind to ferrous ion and inhibit iron’s capacity to promote lipid peroxidation. 1080
Vol.
179,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
However, our results suggest that PS itself can bind to iron and suppress lipid peroxidation. It might be assumed that PS is responsible for the site-specific mechanism (21) of lipid peroxidation in biomembrane system because site-specific peroxidation is thought to be induced by transition metal ion on the membrane surface (22). Nevertheless, PS did not change during incubation with ferrousascorbate, which was confirmed by TLC and GLC analysis (not shown). Our results strongly suggests that PS exerts an inhibitory effect on biomembrane lipid peroxidation by acting as an iron trapper on the membrane surface.
REFERENCES 1. Halliwell, B. and Gutteridge, J. M. C. (1984) Biochem. J. 219 , l-14. 2. Slater, T. F. (1984) Biochem. J. 222 , l-15. 3. Svingen, B. A., Buege, J. A., O’Neal, F. 0. and Aust,S. D. (1979) J. Viol. Chem. 254 , 5892-5899. 4. Bucher, J. R., Tien, M. and Aust, S. D. (1983) Biochem. Biophys. Res. Commun. 111 , 777-784. 5. Wills, E. D. (1965) Biochim. Biophys. Acta 98, 238-251. 6. Kornbrust, D. J. and Mavis, R. D. (1980) Mol. Pharmacol. 17 , 400-407. 7. Vile, G. F. and Winterbourn, C. C. (1987) FEBS. 215 , 151-154. 8. Kunimoto, M., Inoue, K. and Nojima, S. (1981) Biochim. Biophys. Acta 848 , 169- 178. 9. Maeda, M., Nishijima, M., Takenaka, Y., Kuge, 0. and Akamatsu, Y. (1984) Biochim. Biophys. Acta 794 , 298-306. -
10. Boggs, J. M. (1980) Can. J. Biochem. 58, 755-770. 11. Green, D. E., Fry, M. and Blondin, G. A. (1980) Proc. Nat/. Acad. Sci, USA 77, 257-261. 12. Hauser, H. and Dawson, R. M. C. (1968) Biochem. J. 109 , 909-916. 13. Jacobson, K. and Papahadjopoulos, D. (1975) Biochemistry 14, 152-l 61. 14. Viani, P., Cervato, G., Fiorilli, A., Rigamonti, E. and Cestaro, B. (1990) Chem. Phys. Lipids 52 , 49-55.
15. Terao, J., Asano, I. and Matsushita, S. (1985) Lipids 20, 312-317. 16. Terao, J. and Matsushita, S. (1981) Agric. Ho/. Chem. 45 , 587-593. 17. Nagao, A. and Terao, J. (1990) Biochem. Biophys. Res, Commun. 172 , 385389.
18. Uchiyama, M. and Mihara, M. (1978) AnalBiochem. 88, 271-278. 19. Terao, J., Shibata, S.S., Yamada, K. and Matsushita, S. (1988) In Medical Biochemical and Chemical Aspects of Free Radicals (Hayaishi, O., Niki,E., Kondo, M. and Yoshikawa, T., eds.) Vol.8 , pp.781-788, Elsevier Science Publishers, B.V., Amsterdam. 20. Halliwell, B. and Gutteridge, J. M. C. (1988) In Medical Biochemical and Chemical Aspects of Free Radicals (Hayaishi, O., Niki, E., Kondo, M. and Yoshikawa, T., eds.), vol.1 , pp. 21-31, Elsevier Science Publishers, B.V., Amsterdam. 21. Czapski, G. and Goldstein, B. (1986) Free Radical Reseach Commun. 1 , 157161. 22. Fukuzawa, K., Kishikawa, K., Tadokoro, T., Tokumura, A., Tsukatani, H. and Gebicki, J. M. (1988) Arch. Biochem. Biophys. 280 , 152-160.
1081
