83

ARTICLE

I

I

Synthesis of a Phosphatidyl Derivative of Vitamin E and Its Antioxidant Activity in Phospholipid Bilayers Takuro Koga a,*, Akihiko Nagao b, Junji Terao b, Kohei Sawada c and Kazuo Mukai c aNoda Institute for Scientific Research, Noda-shi, Chiba 278, bNational Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba-shi, Ibaraki 305 and CDepartment of Chemistry, Faculty of Science, Ehime University, Matsuyama-shi, Ehime 790, Japan

A novel phospholipid containing a chromanol structure at its polar h e a d group w a s s y n t h e s i z e d f r o m e g g y o l k p h o s p h a t i d y l c h o l i n e and 2,5,7,8-tetramethyl-6-hydroxy2 - ( h y d r o x y e t h y l ) c h r o m a n by t r a n s p h o s p h a t i d y l a t i o n cata l y z e d by p h o s p h o l i p a s e D f r o m Streptomyces lydicus. T h e s t r u c t u r e of the p r o d u c t s y n t h e s i z e d w a s s h o w n by s p e c t r a l a n a l y s i s to b e 1,2-diacyl-sn-glycero-3-phospho-2'hydroxyethyl-2',5',7',8'-tetramethyl-6'-hydroxychroman. The p h o s p h a t i d y l c h r o m a n o l (PCh) s h o w e d a n t i o x i d a n t activity against radical chain oxidation of methyl l i n o l e a t e in s o l u t i o n in a m a n n e r s i m i l a r to that ofd-~-toc o p h e r o l (a-Toc) and 2,2,5,7,8-pentamethyl-6-chromanoL H o w e v e r , P C h w a s less effective as a c h a i n - b r e a k i n g ant i o x i d a n t t h a n w a s a-Toc w h e n u n i l a m e l l a r e g g y o l k p h o s p h a t i d y l c h o l i n e l i p o s o m e s w e r e e x p o s e d to e i t h e r a w a t e m s o l u b l e or a lipid-soluble radical initiator. It is l i k e l y that t h e p h o s p h o l i p i d n a t u r e of P C h affects t h e loc a t i o n and the m o b i l i t y o f t h e c h r o m a n o l m o i e t y in the m e m b r a n e b i l a y e r r e s u l t i n g in a d e c r e a s e in a n t i o x i d a n t activity. On t h e o t h e r hand, the a n t i o x i d ~ n t a c t i v i t y of P C h w a s little different f r o m that of a-Toc in , n i l a m e l l a r l i p o s o m e s w h e n e x p o s e d to a lipid-soluble radical initiator in t h e p r e s e n c e of a s c o r b i c acid. It a p p e a r s that P C h in p h o s p h o l i p i d b i l a y e r s can b e r e g e n e r a t e d by a s c o r b i c acid in a q u e o u s p h a s e as c a n be a-Toc. The n e w p h o s p h o lipid, p h o s p h a t i d y l c h r o m a n o l , s h o u l d p r o v e useful as a c h a i n - b r e a k i n g a n t i o x i d a n t in p h o s p h o l i p i d m e m b r a n e s . Lipids 29, 83-89 (1994).

Lipid peroxidation in biomembranes contributes significantly to oxidative damage in biological systems (1). It is also known t h a t biomembranes possess an antioxidant defense against lipid peroxidation (2), in which d-u-tocopherol (a-Toc) is thought to act as a potent chain-breaking reagent (3). The mechanism of radical scavenging by a-Toc in homogenous solution is well understood (4,5). It is also known t h a t a-Toc prevents phospholipid peroxidation in biomembranes (6-8). Although a n u m b e r of studies have been u n d e r t a k e n using liposomes as a m e m b r a n e model (6-10), the mechanism of antioxidant action of r in biomembranes is still not entirely *To whom correspondence should be addressed at Noda Institute for Scientific Research, 399 Noda, Noda-Shi, Chiba 278, Japan. Abbreviations: AAPH, 2,2'-azobis(2-amidinopropane) hydrochloride; AMVN, 2,2'-azobis(2,4-dimethylvaleronitrile); AsA, ascorbic acid; DTPA, diethylenetriaminepentaaeetic acid; HPLC, high-performance liquid chromatography; LUV, large unilamellar vesicles; MeL-OOH, methyl linoleate hydroperoxides; NMR, nuclear magnetic resonance; PA, phosphatidic acid; PC, phosphatidylcholine; PCh, phosphatidylchromanol; PC-OOH, phosphatidylcholine hydroperoxides; PLD, phospholipase D; PMC, 2,2,5,7,8-pentamethyl6-chromanol; SIMS, sputtered ion mass spectrometry; TLC, thinlayer chromatography; a-Toc, d-a-tocopherol; Toc-Et, 2,5,7,8tetramethyl-6-hydroxy-2-(hydroxyethyl)chroman. Copyright 9 1994 by AOCS Press

oh X=C oh

co-To(:

oh.

HO~cH

CH~

3

x=c

HOOCH, H~C

PMC

CH3

2--CH2~OH CH=

Toc-Et

oh

0

oh --oh - - o - - P --o--oh

L-o-..

PCh

I ~O ~RI oh (R1,R2: acylgroup)

FIG. 1. Molecular structures of d-a-tocopherol, (a-Toc), 2,2,5,7,8-pentamethyl-6-chromanol (PMC), 2,5,7,8-tetramethyl-6-hydroxy-2-(hydroxyethyl)chroman (Toc-Et) and phosphatidylchromanol (PCh).

clear. In particular, the location in the phospholipid bilayer of the chromanol ring, which is the active moiety of a-Toc for radical-scavenging t h a t interferes with the reaction with chain-propagating lipid peroxyl radicals, is still a subject of discussion (10-13). The transphosphatidylation activity of phospholipase D (PLD) is commonly used to modify the polar head group of phospholipids (14,15). We previously described the synthesis of 6-phosphatidyl-L-ascorbic acid catalyzed by PLD (16). This phosphatidyl derivative of ascorbic acid was shown to prevent liposomal lipid oxidation more effectively t h a n ascorbic acid (17). This, in turn, prompted us to synthesize a phospholipid containing a chromanol ring, as such an a-Toc analogue would seem helpful in investigating the antioxidant action of a-Toc in membranes. In the present work, we report on the synthesis of a new phospholipid containing a chromanol ring (Fig. 1), as well as on the antioxidant activity of the phosphatidylchromanol (PCh) synthesized in unilamellar liposomes and in hexane/isopropanol solution.

MATERIALS AND METHODS

Chemicals. Phosphatidylcholine (PC) from egg yolk and dimyristoyl PC were purchased from Sigma Chemical Co. (St. Louis, MO). d-a-Toc and 2,2,5,7,8-pentamethylLIPIDS, Vol. 29, no. 2 (1994)

84 T. KOGAET AL. 6-chromanol (PMC) (Fig. 1) were obtained from Eisai Co. (Tokyo, Japan). 2,5,7,8-Tetramethyl-6-hydroxy-2(hydroxyethyl)chroman (Toc-Et) was kindly supplied by Kuraray Co. Ltd. (Kurashiki, Japan) (Fig. 1). PLD from Streptomyces lydicus was kindly supplied by Honen Co. (Yokohama, Japan). 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) were obtained from Wako Pure Chemical Co. (Osaka, Japan). All other chemicals used were of reagent grade. Preparation of PCh. PCh was synthesized by reacting 2.5 mL of 25 mM egg yolk PC and 25 mM Toc-Et in diethyl ether and 25 mL of 10 U of PLD and 10 mM CaC12 in 10 mM acetate buffer (pH 5.1) at 37~ with continuous shaking. After 2 h of incubation, 3 mL of 6 N HC1 was added, and the product was extracted three times with 30 mL of chloroform/methanol (2:1, vol/vol). The combined chloroform layers were washed with 20 mL of water, and evaporated in a rotary evaporator. The residue was dissolved in chloroform/methanol (95:5, vol/vol) at 10 mg/mL and placed onto a silica gel column (Lichroprep Si 60 40--63 lam; Merck, Darmstadt, Germany) that had previously been equilibrated with the same solvent. The product was eluted at a flow rate of 1.8 mL/min, 1.0-mL fractions were collected, and fractions were monitored by thin-layer chromatography (TLC) (Silica gel 60; Merck) using chloroform/methanol (8:2, vol/vol) as developing solvent. Phospholipids were visualized by spraying with Dittmer's reagent (18). Reducing activity in TLC fractions was detected by exposure to ferric ion reagent (19). Fractions containing PCh were redissolved in methanol, and then stored at -20~ until use.

Assay of PCh and phosphatidic acid (PA) in the reaction mixture. PCh and PA were assayed by high-performance liquid chromatography (HPLC) on an aminopropane-bonded silica column (Shim-Pack FLC-NH2, 4.6 x 50 mm, 3 ~m; Shimadzu Co., Kyoto, Japan) using n-hexane/isopropanol/10% phosphoric acid (70:30:0.5, by vol) at a flow rate of 2.0 mL/min. PCh and PA eluted at 1.5 and 2.2 min, and were monitored at 295 and 206 nm, respectively. Spectral analyses. Infrared spectra were measured on KBr pellets using a JASCO FT/IR-7300 infrared spectrophotometer (Japan Spectroscopic Co., Ltd., Tokyo, Japan). Mass spectra were measured in the sputtered ion mass spectrometry (SIMS) mode using a Hitachi M80B instrument (Hitachi Co., Tokyo, Japan). IH and ~3C nuclear magnetic resonance (NMR) spectra were measured with a JEOL GSX-270 FT NMR spectrometer (Japan Electronic Optics Laboratory Co., Tokyo, Japan) at 270 and 67.9 MHz, respectively. Samples were dissolved in CDC13/CD3OD (2:1, vol/vol), and tetramethylsilane was used as internal standard. Peroxyl radical-scavenging activity in solution. Peroxyl radical-scavenging activities of a-Toc, PMC and PCh in homogeneous solution were determined by measuring the inhibition of free radical oxidation of methyl linoleate that was initiated by a lipid-soluble azo compound (17,20). The reaction mixture contained 110 ~mol of methyl linoleate and 0.1 pmol of antioxidant in 1.0 LIPIDS, Vol. 29, no. 2 (1994)

mL of n-hexane/isopropanol (7:3, vol/vol). Oxidation was initiated by adding 110 mM AMVN (100 ~L) in the same solvent. The mixture was incubated at 37~ with continuous shaking in the dark and aliquots were withdrawn at regular intervals to measure methyl linoleate hydroperoxides (MeL-OOH) (21). Preparation of unilamellar liposomes. Liposomes [large unilamellar vesicles (LUV)] were prepared by an extrusion method (22). First, PC was purified to remove contaminant peroxides by reverse-phase column chromatography as described previously (23). A stock solution of purified PC in chloroform, with or without antioxidants, was placed into a test tube and was evaporated under a stream of nitrogen, then in vacuo. The thin lipid film on the glass wall was dispersed with a vortex mixer for 1 min in 0.7 mL Tris-HC1 buffer (0.01 M, pH 7.4) containing 0.5 mM diethylenetriaminepentaacetic acid (DTPA) followed by ultrasonic irradiation with a Heat Systems Sonifier (Model W-380; Farmingdale, NY) for 30 s. LUV were obtained by extruding the suspension through polycarbonate filters mounted in an extrusion apparatus (LiposoFast, Avestin, Inc., Ottawa, Canada), and the LUV were diluted with the same volume of Tris-buffer.

Lipid peroxidation of LUV initiated by radical initiators. In the experiments with a water-soluble radical initiator, oxidation was started by the addition of AAPH to the LUV suspension. Oxidation was carried out in the dark at 37~ under air with continuous shaking. In the experiments with lipid-soluble radical initiator, AMVN was mixed with PC before the preparation of the liposomes, and the reaction mixture was kept at 50~ during the oxidation. Aliquots (10 1JL) were withdrawn at specific time intervals and phosphatidylcholine hydroperoxides (PC-OOH) were quantified by HPLC as described previously (24). The octane-bonded silica column (TSK-gel OCTYL-80TS column, 6 • 150 mm, 5 ]am; TOSOH, Tokyo, Japan) was eluted with methanol/water (93.5:6.5, vol/vol). Standard PC-OOH was prepared as described (24).

Determination of antioxidants during liposomal peroxidation, a-Toc, PMC and PCh were measured by HPLC on an octodecane-bonded silica column (YMCpack ODS, 6 x 150 mm, 5 pm; Yamamura Chemical Laboratories, Kyoto, Japan) that was eluted with acetonitrile/ethanol (60:40, vol/vol) at a flow rate of 2.5 mL/min. The eluent was monitored fluorometrically at an excitation wavelength of 298 nm and an emission wavelength of 325 nm using a JASCO Spectrofluorometer 821-FP (Tokyo, Japan). (x-Toc, PMC and PCh were eluted at 4.5, 1.5 and 1.7 min, respectively. Ascorbic acid (AsA) was assayed by HPLC using a cation exchange column (TSK gel SCX, 7.8 x 300 mm, 5 }lm, TOSOH) with 2 mM phosphoric acid as eluent at a flow rate of 1.2 mL/min. The eluent was monitored at 254 nm; AsA was eluted at 6.6 min. RESULTS

Synthesis of PCh. The time course of the enzymatic reaction for the synthesis of PCh catalyzed by PLD is

85 ANTIOXIDANT A C T M T Y OF PHOSPHATIDYLCHROMANOL TABLE 1

4

O E :L

3

V

2 n

0 m 0 eel.

1 9

A

;i

"-

"-

|

I

U

i

I

I

4

8

24

48

72

aB

0

-"

J

"nine (h) FIG. 2. Time course for the s y n t h e s i s o f phosphatidylchrom a n o l catalyzed by p h o s p h o l i p a s e D. The a m o u n t s of PCh and p h o s p h a t i d i c acid (PA) w e r e c a l c u l a t e d from high-performance l i q u i d chromatography p e a k areas u s i n g calibration curves; (~), PCh; (A), PA.

shown in Figure 2. The reaction reached a plateau after 1.5 h of incubation, and PC was converted to the new product in high yield (approximately 94%). Little PA accumulated throughout the reaction period, indicating that little hydrolysis occurred. The new product gave a blue spot on TLC with Dittraer reagent and a red spot with ferric ion reagent, indicating that the new compound was a phospholipid with reducing activity. Spectral analyses were carried out to identify the structure of the phospholipid synthesized from 1,2dimyristoyl-sn-glycero-3-phosphocholine. The SIMS spectrum showed an [M + H +] ion at m / z 825. The infrared spectrum indicated the presence of a phosphate group and of hydroxyl at the chromanol ring (KBr, cm -1 : 1745, C=O; 1457,-OH; 1246, P=O; 1027, P-O-C). To elucidate the structure in detail, laC NMR chemical shifts were assigned to the carbons of the dimyristoylglycerophosphate and the chroman ring moieties (Table 1). The 13C chemical shifts of the dimyristoylglycerol moiety of the product were essentially the same as those of 1,2-dimyristoyl-sn-glycero-3-phosphocholine(16). The 13C chemical shifts of the chroman ring moiety were essentially the same as those of Toc-Et except for the C-I' and C-2' shifts. The C-2' signal was shifted downfield (3.24 ppm) relative to that of Toc-Et, while the C-I' signal was shifted slightly upfield (1.43 ppm). Such 13C chemical shifts for carbons proximal to phosphate were also observed in the spectra of 6-phosphatidyl-L-ascorbic acid (16) and other phosphoesters (14). Based on these data and on 1H NMR spectra (data not shown), the compound was identified as 1,2-dimyristoyl-sn-glycero3-phospho-2'-hydroxyethyl-2',5',7', 8'-tetramethyl-6'-hydroxy-chroman.

Peroxyl radical-scavenging activity of PCh, a-Toc and PMC in solution. Peroxyl radical-scavenging activities of PCh and of related compounds were evaluated by measuring the inhibition of methyl linoleate peroxidation initiated by AMVN in hexane/isopropanol solution. A

13C NMR S p e c t r a of PCh~ Dimyristoyl PC and Toc-Et a Chemical shifts (5) Carbons PCh Dimyrist0yl PC b Toc-Et Dimyristoyl glycerol moiety 63.01 CH20 (sn-1) 62.85 70.74 CHO (sn-2) 70.78 63.93 CH2OP (sn-3) 63.86 174.30 CO ester 174.27 173.93 173.90 34.55-22.96 (CH2)n 34.48-22.99 14.21 CH 3 terminal 14.22 Choline moiety CH2OP CH2N (CH3)3N

59.34 66.79 54.39

Toc-Et moiety 75.49 C-2 73.63 31.85 C-3 32.40 20.51 C-4 20.92 118.72 C-5 117.25 144.72 C-6 145.40 121.20 C-7 120.93 122.35 C-8 122.51 23.04 C-2a 23.92 117.15 C-4a 123.45 11.27 C-5a 11.60 12.05 C-7a 11.91 12.05 C-8a 12.56 145.09 C-8b 145.40 41.96 C-I' 40.53 59.25 C-2' 62.49 aChemical shifts are given downfield from tetramethylsilane. bData for dimyristoyl PC are those reported by Nagao et al. (16). Abbreviations: NMR, nuclear magnetic resonance; PCh, phosphatidylchromanol; Dimyristoyl PC, 1, 2-dimyristoyl-sn-glycero-3phosphocholine; Toc-Et, 2, 5, 7, 8-tetramethyl-6-hydroxy-2-(hydroxyethyl) chroman.

4-

3A

X

E

-to o o ui

2-

1.

a

O, 0

SO

100

150

200

250

T i m e (min)

FIG. 3. Inhibition o f the 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN)-initiated oxidation of m e t h y l linoleate in solution by a-Toc, PMC and PCh. Th e reaction system consisted of m e t h y l linoleate (100 mM), antioxidant (90.9 pLM) a n d AMVN (10 mM) in n-hexane/isopropanol (7:3, vol/vol); (O), no addition; (C)), a-Toc; ([~), PMC; (A), PCh. See F i g u r e 1 for other abbreviations; MeL-OOH, m e t h y l linoleate hydroperoxides.

LIPIDS, Vol. 29, no. 2 (1994)

86

T. KOGA ET AL. 0,8-

0,8

A 0.6,

0.6

i

g 0,4

o

g

0.2

O.2

0.0,

~

i

i

i

i

1~

1~

2~

0.0

i

250

0

100

Time (min)

100 I

200

300

T i m e (min)

B

B

lOO, A

i-

m "I

60.

a

w

|

m

4o

S re

20. 0

~_,~ 0

S0

100 150 T i m e (min)

..~.

~

200

."q...,

200

250

FIG. 4. A c c u m u l a t i o n o f p h o s p h a t i d y l c h o l i n e h y d r o p e r o x i d e s (PC-OOH) (A) and loss o f ~-Toc, PMC a n d P C h (B) duri n g p h o s p h o l i p i d p e r o x i d a t i o n in l a r g e u n i l a m e l l a r v e s i c l e s e x p o s e d to 2,2'-azobis(2oamidinopropane)hydrochloride (AAPH). The r e a c t i o n s y s t e m c o n s i s t e d o f PC (5 raM), ~-Toc, PMC or P C h (10 ~LM), d i e t h y l e n e t r i a m i n e p e n t a a c e t i c a c i d

(0.5 raM) and AAPH (10 raM) in 1.1 mL of Tris-HC1 buffer;, (@), n o addition; (9 ~-Toc; (D), PMC; (A), PCh. See Figure 1

~ 0

lOO

2o0

[] 3o0

T i m e (rain)

FIG. 5. A c c u m u l a t i o n o f PC-OOH (A) a n d loss o f ~-Toc, PMC a n d PCh (B) during p h o s p h o l i p i d p e r o x i d a t i o n in l a r g e - n i l a m e l l a r v e s i c l e s e x p o s e d to AMVN. The r e a c t i o n s y s t e m c o n s i s t e d o f PC (5 mM), a-Toc, PMC or P C h (5 pfl~I), DTPA (0.5 raM), and M V N (1 mM) in 1.1 mL of Tris-HCl buffer; (O), n o addition; (O), a-Toc; ([B), PMC; (A), PCh. See Figures 1, 3 a n d 4 for a b b r e v i a t i o n s .

for o t h e r a b b r e v i a t i o n s .

typical example of the inhibition seen is shown in Figure 3. F r o m the d a t a we obtained from three independent experiments, we calculated kinetic p a r a m e t e r s by the method of Ingold and colleagues (4,25). PCh, (x-Toc a n d PMC showed almost the s a m e induction periods (tinh; 7190 _+ 610 s, 7090 _+420 S and 7290 +_ 490 s for aToc, PMC and PCh, respectively). The r a t e s of oxidation (Rinh) d u r i n g the induction period were (9.97 _+ 3.16)x 10 -9 M/s, (4.79 +_ 1.56)• 10 -9 M/s, (5.69 _+ 1.02)x 10 -9 M/s for PCh, a-Toc and PMC, respectively. This indicates t h a t the n u m b e r of radicals t r a p p e d by PCh (n) was the s a m e as t h a t t r a p p e d by a-Toc and PMC as n is a function of tinh. The kin h /kp values, the ratio of the rate constant for the inhibition reaction vs. t h a t of the propagation reaction, were calculated from tin h and Rin h as described previously (26). For PCh, kinh/k p was 1480 _+ 400, which was slightly lower t h a n for a-Toc (3040 _+ 770) and PMC (2520 _+ 280).

Free radical-scavenging activity of PCh, a-Toc and PMC in unilamellar liposomes. We used LUV and estim a t e d the antioxidant activities of a-Toc and its ana-

LIPIDS, Vol. 29, no. 2 (1994)

logues against phospholipid peroxidation initiated by free radical generators. Figure 4 shows a typical example of the inhibition of the peroxidation initiated by AAPH by a-Toc and its analogues. The water-soluble radical initiator produces peroxyl radicals in the aqueous phase and thereby attacks phospholipids from the aqueous phase at the m e m b r a n e surface (27). PC-OOH, the p r i m a r y products of peroxidation, accumulated in the initial stage of the reaction without antioxidants. (xToc suppressed the accumulation of PC-OOH with a pronounced induction period (65 min). Although P C h and PMC also r e t a r d e d lipid peroxidation, their induction periods were shorter t h a n t h a t of u-Toc (36 min for PCh, and 39 min for PMC). In addition, both PCh and PMC were consumed faster t h a n a-Toc. When we used a lipid-soluble azo compound to initiate peroxyl radical-driven lipid peroxidation within the liposomal m e m b r a n e s , a-Toc showed a longer induction period (126 min) t h a n P C h (94 min) and PMC (97 min), and ct-Toc was consumed less rapidly t h a n were the latter two compounds (Fig. 5). Thus, PCh was less effective

87 ANTIOXIDANTACTIVITYOF PHOSPHATIDYLCHROMANOL acid was consumed in the initial stage, and the levels of a-Toc or its analogues always decreased after ascorbic acid was consumed. Interestingly, there were only small differences in the induction periods and in the consumption of ascorbic acid between the three compounds. Thus, in the presence of AsA PCh exerted the same antioxidant activity as did a-Toc.

1.21.0

9

o.] ~)

0.6-

o. nO 0.4-

DISCUSSION 0.20.0 !

9

.

21111

0

9

400

600

!

800

Time (min) 100

B t~

"o ee o 0

100,

2o0

DA

o

A

40o Time (min)

!

!

600

8O0

C

o2O~ o

D

~g o

b Q A ~

400 Z~ []

re 0 0

I

|

200

400

~

!

I

600

800

(mini FIG. 6. A c c u m u l a t i o n o f PC-OOH (A), loss of ascorbic acid (AsA) (B), and loss of a-Toc, PMC or PCh (C) d u r i n g phosp h o l i p i d peroxidation in large unflamellar vesicles exposed to AMVN. The reaction system consisted of PC (5 mM), AsA (100 pflVl),cc-Toc,PMC or PCh (5 pdVD,d i e t h y l e n e t r i a m i n e pentaacetic acid (0.5 mM), and AMVN (1 raM) in 1.1 m L of Tris-HCl buffer; (e), no addition; (D), a-Toc; (A), PMC; (O), PCh. See Figures 1, 3 and 4 for abbreviations.

than a-Toc as a chain-breaking antioxidant when either a water-soluble or a lipid-soluble initiator was used to induce the radical chain reaction.

Effect of ascorbic acid on the inhibition by PCh, a-Toc and PMC in liposomal phospholipid peroxidation. Figure 6 shows the inhibition of PC-OOH accumulation by a-Toc or its analogues in the presence of ascorbic acid. It is apparent that the presence of ascorbic acid increased the induction period significantly as indicated by comparison of the data in Figure 6 and Figure 5. Ascorbic

We previously synthesized phosphatidyl AsA (16) and showed that this phospholipid derivative could be incorporated into phospholipid bilayers, and that it exerted antioxidant activity against oxidative attack from the aqueous phase more efficiently than did ascorbic acid (17). Because vitamin E is a major lipophilic chainbreaking antioxidant in cellular membranes, we now synthesized phosphatidyl vitamin E by the transphosphatidylation catalyzed by PLD. The reaction proceeded with a high yield, and there was little hydrolysis (Fig. 2) as was reported for the hydrolysis of phosphatidylinositol catalyzed by this enzyme (28). Resistance against PLD-catalyzed hydrolysis appears to be due to steric hindrance at the polar head group moiety. a-Toc consists of two structural domains. One is the chromanol structure responsible for radical scavenging activity and the other is the phytyl side chain which is required for anchoring a-Toc within phospholipid bilayers (29). The product we synthesized by the PLD reaction contains a phospholipid moiety and chromanol moiety, and thus can be considered an a-Toc analogue in which the phytyl side chain is replaced by a phosphatidyl group. The product thus is lipophilic, much like a-Toc. However, introduction of the phosphatidyl group could affect the radical scavenging activity of the chromanol moiety by changing its location in the phospholipid bilayer. It has already been shown that the phytyl side chain does not affect the free radical scavenging activity of aToc in solution (30). Our data show that PMC and a-Toc gave similar kinetic parameters, i.e., kinh/k p and n values, for free radical oxidation in solution, and this is consistent with the view that the phytyl side chain is not responsible for the inherent antioxidant activity of chromanol. The fact that PCh gave the same n value furthermore suggests that PCh scavenges free radicals by the same mechanism as does a-Toc. The slightly smaller kink /k_ value for PCh may be due to the lower mobility of PC~ in solution or to steric hindrance of scavenging peroxyl radicals as the phospholipids exist in the aggregated form in apolar solvents (31). Niki and his co-workers (7,10) have shown that the antioxidant effect of a-Toc in phospholipid membranes is lower than in homogeneous solution, and that PMC is more effective than a-Toc in scavenging free radicals in membranes. The higher activity of PMC in membranes could be explained by differences between PMC and aToc in location and mobility. Previous studies suggest that the chromanol moiety of a-Toc is located in the hydrophobic region near the membrane surface and that the mobility of chromanol is restricted by the hydrophoLIPIDS, Vol. 29, no. 2 (1994)

88 T. KOGAETAL. bic side chain, while PMC can move m o r e freely within the m e m b r a n e (10-13). Our results show t h a t PCh is less active as a chain-breaking antioxidant in phospholipid m e m b r a n e s , regardless of the site of the chain initiation reaction. When a radical g e n e r a t o r is used for initiating lipid peroxidation in m e m b r a n e s , a-Toc can scavenge chain-initiating peroxyl radicals as well as chain-propagating lipid peroxyl radicals. We p r e s u m e t h a t the chromanol moiety of P C h is located on the m e m b r a n e surface and t h a t the phospholipid moiety is inserted into the phospholipid bilayer, thereby restricting the mobility of P C h within the hydrophobic bilayer region. This location of P C h in m e m b r a n e s would lower the reactivity with chain-propagating lipid peroxyl radicals. The lateral diffusion r a t e of phospholipids in bilayers was reported to be about 270 times lower t h a n t h a t of a-Toe (32,33). Therefore, the lateral diffusion of the chromanol group of P C h would also be likely to be restricted within phospholipid bilayers resulting in the app a r e n t lower activity with scavenging lipid peroxyl radicals. We found t h a t PMC is also less effective t h a n (~-Toc in preventing free radical initiated lipid peroxidation in the m e m b r a n e model (Figs. 4 and 5) in spite of its ability to move freely within the bilayer. In addition, PMC and P C h were consumed m o r e rapidly t h a n a-Toc, a-Toc donates a hydrogen a t o m to a peroxyl radical to form an a-tocopheroxyl radical (8). Subsequent reaction of the da-tocopheroxyl radical with a second peroxyl radical gives both a stable product and an unstable product t h a t m a y produce an alkoxyl radical (34-36). The lower effectiveness of PMC and P C h could be explained by the assumption t h a t the reaction t h a t produces the unstable products occurs quite easily in the case of PMC and PCh. Among the three compounds, a-Toc acts most efficiently as chain-breaking antioxidant in phospholipid bilayers. This supports the idea t h a t the phytyl side chain of aToc a r r a n g e s the chromanol moiety at a site suitable for scavenging chain-carrying lipid peroxyl radicals in biological m e m b r a n e s (7,10). On the other hand, w h e n the liposomal peroxidation was initiated in the presence of ascorbic acid, the antioxidant activity of P C h and PMC was the s a m e as t h a t of a-Toc (Fig. 6). Although ascorbic acid cannot inhibit lipid peroxidation within liposomes directly, it is known t h a t it acts in the aqueous p h a s e as a synergist by regenerating ~-Toc (37,38). Bowry et al. (39) pointed out t h a t the regeneration of a-Toc by ascorbic acid i s required to prevent the prooxidant effect of (~-Toc. We also found t h a t ascorbic acid prolonged the induction period of a-Toc and its analogues in liposomal suspensions. It should also be e m p h a s i z e d t h a t there is no difference in the antioxidant activity between a-Toc and PCh, although P C h is less effective in the absence of ascorbic acid. It a p p e a r s t h a t the regeneration by ascorbic acid is quite i m p o r t a n t in d e t e r m i n i n g antioxidant activity in phospholipid bilayers w h e n ascorbic acid is present in the aqueous phase. In conclusion, we synthesized a new v i t a m i n E phospholipid with antioxidant activity, which works as a chain-breaking antioxidant in phospholipid bilayers. Its LIPIDS, Vol. 29, no. 2 (1994)

activity is comparable to t h a t of v i t a m i n E in the presence of ascorbic acid, b u t its activity is lower in the absence of ascorbic acid. The new v i t a m i n E phospholipid should be active in biological systems, provided the chromanol moiety is r e g e n e r a t e d by ascorbic acid.

ACKNOWLEDGMENTS The authors thank Sinbo M. and Shiono M. for kindly supplying PLD and Toc-Et, respectively. We are very grateful to Ishida N., Yamashita C., Matsudo T. and Uchida R. for their help in spectral analyses and for valuable discussions.

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