Copper-induced lipid peroxidation in liposomes, micelles, and LDL: Which is the role of vitamin E?

Free RadicalBiology& Medicine,Vol. 18, No. I, pp. 67-74, 1995 Copyright© 1994 ElsevierScienceLtd Printed in the USA. All rights re~rved 0891-5849/95 $9.50 + .00

Pergamon

0891-5849(94)00103-0

Original Contribution COPPER-INDUCED LIPID PEROXIDATION IN LIPOSOMES, MICELLES, AND LDL: WHICH IS THE ROLE OF VITAMIN E? MATILDE MA1OR1NO,* ADRIANA ZAMBURLINI,* ANTONELLA ROVERI,* and FULV10 URSINIt *Department of Biological Chemistry, University of Padova, Padova, Italy; and tDepartment of Chemical Sciences and Technology, University of Udine, Udine, Italy (Received l0 August 1993; Revised 21 December 1993; Re-revised 6 May 1994; Accepted 9 May 1994)

A b s t r a c t Liposomes, containing phospholipid hydroperoxides, are peroxidised in the presence of Cu ÷÷. Peroxidation starts after a period of resistance to oxidation, which is abolished by the shift of lipid organisation from bilayer to micellar dispersion. Independently from ongoing peroxidation, vitamin E in liposomes also reacts with Cu ++, and it is consumed. The evidence that phospholipid hydroperoxides induce an acceleration of vitamin E consumption rate and that the consumption of vitamin E and phospholipid hydroperoxides are stoichiometric indicates that, in liposomes, the rate-limiting reaction is the interaction between radicals generated by copper from vitamin E and phospholipid hydroperoxides. In micelles, on the other hand, vitamin E is directly oxidised by copper at a much faster rate; thus, the concerted consumption of phospholipid hydroperoxides does not take place. Moreover, in micelles challenged with Cu ++, vitamin E plays a pro-oxidant effect (M. Maiorino et al. FEBS Lens., 330(2): 174-176; 1993). In LDL, incubation with Cu ÷+ promotes vitamin E consumption at a fast rate, as in micelles, but not the concerted disappearance of lipid hydroperoxides, as in liposomes. However, the direct vitamin E oxidation by Cu ++, observed in micelles and liposomes, does not lead to a pro-oxidant effect in LDL. The kinetics of peroxidation, indeed, is identical in native and vitamin E-depleted LDL. These results argue against an involvement of vitamin E, both as antioxidant or pro-oxidant in LDL challenged with Cu ++, and suggest that other factors, besides antioxidant content, must be relevant in determining LDL oxidative resistance. Keywords--Vitamin E, c~-tocopherol, Copper, Lipid peroxidation, Phospholipid hydroperoxides, Low density lipoproteins, Atherosclerosis, Free radicals

INTRODUCTION

Cu ÷+, the lipid peroxidation rate increases after a period o f relative resistance to oxidation, during which the formation o f oxidation products is m i n i m a l and antioxidants are consumed. 6 T h e relevance o f vitamin E as a m a j o r antioxidant in L D L is b a s e d on the observation that it is c o n s u m e d first, with a fast rate. On the other hand, its i m p o r t a n c e on length o f the induction p e r i o d has been recently questioned. 7-9 M o r e o v e r , we have recently shown that, in detergent dispersion, a - t o c o p h e r o l is able to reduce Cu ÷+, thus playing a pro-oxidant effect) ° T h e a i m o f this article is a d e e p e r characterisation o f the role p l a y e d b y vitamin E as antioxidant or prooxidant in Cu++-induced lipid peroxidation. A l t h o u g h b e i n g eventually addressed to L D L , this study has been organised to take a d v a n t a g e from informations obtained in m o d e l s y s t e m s - - m i c e l l a r dispersions and unilamellar liposomes. R e p o r t e d results indicate that, in the p r e s e n c e o f Cu +÷, vitamin E is p r o - o x i d a n t in micelles, and a m i n o r antioxidant in liposomes, whereas neither o f these effects is relevant in LDL.

Peroxidation o f low density lipoproteins ( L D L ) and their resistance to o x i d a t i v e degradation have been anal y s e d with special e m p h a s i s in the last few years, since p e r o x i d a t i o n has been suggested to p l a y a relevant role in the modification o f these lipoproteins, eventually leading to an increased uptake b y cells present in the arterial wall. T h e uptake o f o x i d a t i v e l y m o d i f i e d L D L through the s c a v e n g e r receptor, indeed, and their cytotoxic effect, have been suggested to p l a y a k e y role in atherogenesis, t-4 T o define factors accounting for o x i d a t i v e resistance o f L D L , 5 different o x i d a t i v e challenges have been used. A m o n g these, peroxidation b y Cu ÷+ has b e c o m e very p o p u l a r b e c a u s e the m o d e l is simple and reproducible. W h e n L D L are incubated in the presence o f

Address correspondence to: Matilde Maiorino, Department of Biological Chemistry, via Trieste, 75, 35121--Padova, Italy.

67

68

M. MAIORIr~O et al.

MATERIALS AND METHODS

Preparation of Palrnitoyl Linoleoyl Phosphatidylcholine Hydroperoxides Palmitoyl Linoleoyl Phosphatidylcholine Hydroperoxides (PLPCOOH) was prepared by enzymatic hydroperoxidation of PLPC (Sigma Chemical Co.) as previously reported ~l and purified by HPLC.

Preparation of micelles An appropriate amount of PLPC and PLPCOOH was dried by solvent evaporation under argon and resuspended in 10 mM NazHPO4, 0.16 M NaC1, pH 7.4, 0.1% sodium deoxycholate. When present, vitamin E was added to phospholipids before solvent evaporation. PLPC, PLPCOOH, and vitamin E concentrations in micelles were approximately 1 mg/ml, 20 nmoles/ ml, and 10 nmoles/ml, respectively.

Purification of phospholipids and vitamin E Preparation of LDL 1. PLPC was loaded on a 4.6 × 250 mm Ultrasphere ODS column. Elution was carried out with 100% methanol at a flow rate of 1 ml/min, with simultaneous detection at 210 and 233 nm (Beckman 168 diode array detector module). PLPC, absorbing at 210 nm, was eluted after 12.5 min. 2. PLPCOOH was purified under identical conditions and was eluted as main peak absorbing at 233 nm after 5.6 min. 3. DPPC (Sigma Chemical Co.) was used as supphed, without further purification. PLPC and PLPCOOH, collected from several chromatographic runs, were concentrated by solvent evaporation under argon and quantiffed by phosphorous contentLzand enzymatic titration of hydroperoxides, j3 4. Vitamin E (D-L ot-tocopherol, Merck) was purified by HPLC under the same chromatographic conditions, with detection at 292 nm. Retention time was 11 min. The peak, collected from several runs, was concentrated under argon and quantified spectrophotometrically by using the extinction coefficient E292 = 3144 M-~cm -~.

Preparation of liposomes Liposomes containing PLPC and PLPCOOH were prepared mixing appropriate amounts of methanolic solutions of phospholipids, leading to a PLPC and PLPCOOH final concentration in liposomes of approximately I mg/ml and 20 nmoles/ml, respectively. The methanolic solution was dried and resuspended in 10 mM NazHPO4, 0.16 M NaCI, pH 7.4, frozen and thawed three times. Liposomes were finally prepared by extrusion at room temperature, under argon pressure.14 When present, vitamin E was added to phospholipid solution to obtain a concentration in liposomes of 10 nmoles/ml. DPPC liposomes were prepared as noted, but extrusion temperature was raised to 40°C.

LDL (d. 1019-1063), prepared from a pool of fresh blood from healthy donors as described ~5were exhaustively dialysed against 10 mM Na2HPO4, 0.16 M NaCI, pH 7.4, at 4°C and were used within 24 h after dialysis. Aged LDL were produced by leaving dialysed particles to sit at 4°C up to 50-60 days. This treatment increases phospholipid hydroperoxides without significantly affecting vitamin E content. ~6 Cu ++-dependent peroxidation Liposomes (1 mg PL/ml), micelles (1 mg PL/ml), and LDL (0.1 mg protein/ml) prepared as noted earlier were incubated with 50 #M CuSO4 at room temperature in 10 mM NazHPO4, 0.16 M NaCI, pH 7.4.

Analysis of substrates and oxidation products in liposomes and micelles Analysis of phospholipid (PLPC and PLPCOOH) and vitamin E was carried out on 50 #1 aliquots, withdrawn from the incubation mixture and directly injected to HPLC column, Ultrasphere ODS (4.6 × 250 mm), mobile phase 100% methanol, flow rate 1 ml/ min. The serial alignment of UV (Beckman 168 diode array detector module, recording simultaneously at 210 and 233 nm) and fluorescence (Shimadzu RF. 530 detector module, recording at ex. 286 - em. 330) detectors, allowed the simultaneous monitoring of PLPCOOH, ot-tocopherol and PLPC, whose retention times were 5.6, 10, and 12.5 min, respectively. The formation of vitamin E oxidation products (8ce hydroxyl-tocopherone and tocopheryl-quinone) in liposomes or micelles was searched after extraction. To the sample containing 2 mg of phospholipids, 25 mM SDS, 5% (w/v) ascorbic acid, 2 mM butylated hydroxytoluene, 1.5 sample volumes of ethanol were added, and lipophylic components extracted twice with 2 ml of hexane and hexane phases pooled. Dried ex-

Lipid peroxidationby Cu÷+ and vitamin E tract was resuspended in a small volume of chloroform-methanol (1:1) and injected in the noted chromatographic system, recording simultaneously at 240 and 270 nm.

Analysis of phospholipid hydroperoxides and vitamin E in LDL Lipid extract, 17 from an amount of LDL containing 0.2 mg of protein, was dried under argon, resuspended in 150/~1 n-hexane-isopropanol (4:6) and applied to 1 ml L.C. Diol Supelclean cartridge (Supelco). This was previously conditioned, in series, with methanolwater (9:1); toluol-ethyl acetate (1:1); n-hexane. Cartridge was eluted with two column volumes of n-hexane, toluol-ethyl acetate (1:1), and eventually methan o l - w a t e r (9:1). The last fraction was collected and, after reduction of the volume by solvent evaporation, analysed by HPLC on an Ultrasphere ODS column (4.6 × 250 mm) for its phospholipid and phospholipid hydroperoxide content. Js The chromatographic run was carried out with a linear gradient of methanol, acetonitrile, water from 79.5:0.5:20 to 90.5:8.8:0.7. Both solutions contained 20 mM choline. Gradient was generated over a 35-min period, after 5 min under isocratic conditions, the flow rate was 1 rnl/min. The different molecular species of phospholipid hydroperoxides and phospholipids were separated by this chromatographic system. Retention times were from 36 to 42 and from 43 to 58 min, respectively. Quantitation of lipid hydroperoxide was made on the basis of a standard of PLPCOOH. The recovery of pure PLPCOOH processed through the complete procedure ranged from 70% to 80%. This incomplete recovery was apparently due to sample loss and not rearrangement, as indicated by the identical ratio of PLPCOOH measured as hydroperoxide versus organic phosphorus obtained at the beginning and at the end of the procedure. Vitamin E content in LDL was measured by HPLC (fluorimetric detection) after extraction. RESULTS AND DISCUSSION

Liposomes and lipid micelles Peroxidation of PLPC liposomes--containing PLPCOOH (16 nmoles/mg P L ) - - w i t h C u r r results in two phases (Fig. 1). During the first phase, the rate of lipid hydroperoxide formation is rather slow, becoming progressively faster in the second phase. In the presence of deoxycholate, a fast linear rate of lipid hydroperoxide formation is apparent from the beginning of the reaction. The observation that lipid peroxidation is faster in

69

60 5O a_

~

40

......

y L i ¸!i

i ¸

30

t~ 20 10

0

50

100 150 200 Time, minutes

250

300

Fig. 1. Effectof DOC on Cu÷+-inducedphospholipidhydroperoxide formation in liposomes. PLPC liposomes containing PLPCOOH, were incubated with Cu+÷, in the absence (-.-) or in the presence (-II-) of 0.1% deoxycholate.PLPCOOH formation was measured by HPLC on aliquots withdrawn at different times. The minimal formation of PLPCOOH, taking place in the absence of Cu+*, has been subtracted. Results represent a mean of two separate experiments (Variation < 3%).

the presence of detergents, has been previously reported for other peroxidation systems 19 and has been accounted for by the shift from bilayer to micellar structure, associated with a higher accessibility to the oxidant. This view seems to fit this peroxidation model as well, supporting the concept that the kinetics of Curt-induced lipid peroxidation is affected by physical state of the substrate undergoing peroxidation. The ability of C u r r to peroxidise liposomes and micelles depends on the following reactions: PLPCOOH + Cu ÷r ~ PLPCOO" + Cu ÷

(1)

PLPCOOH + Cu r ~ PLPCO" + Cu r÷.

(2)

Moreover, it is worth noting that, in the experimental conditions of Figure 1, a kind of lag phase is apparent even though no antioxidants have been added to liposties. Incubation of liposomes containing vitamin E with Cu ÷r results in a vitamin E consumption that is slow when liposomes contain saturated lipids and it is accelerated by phospholipid hydroperoxides (Fig. 2). The vitamin E consumption observed in liposomes, containing nonoxidable lipids, is thus independent from the initiation of lipid peroxidation, and it is apparently simply due to its oxidation by Cu r÷, a reaction that has been recently described to take place in detergent dispersions of vitamin E, L° the rate of vitamin E consumption being faster in dispersions. This indicates that, as phospholipids, vitamin E as well is more acces-

70

M. MAIORINOe t

The acceleration o f vitamin E c o n s u m p t i o n by P L P C O O H (Fig. 2) and the 1:1 stoichiometry o f the c o n s u m p t i o n o f the two c o m p o u n d s (Fig. 3A) is reasonably accounted for b y the reactions a m o n g the products o f reactions 1, 2, and 3, the rate o f which is apparently stimulated b y r e m o v a l o f the products.

.~ 4 ,

"" .....

al.

o!

> 3 "~

PLOO" + a - T O C - O " - " A D D U C T

(4)

PLO" + c~ - TOC-O" "-')ADDUCT.

(5)

= 1

OF 0

50

100 150 Time, minutes

200

250

Fig. 2. Effects of lipid composition and phospholipid hydroperoxide content on the kinetics of vitamin E consumption induced by Cu ÷+ in liposomes. Vitamin E was incorporated in DPPC (-O-), PLPC (-A-), DPPC/PLPCOOH (-*-), PLPC/PLPCOOH (-II-) liposomes. During incubation with Cu ÷+, vitamin E consumption was measured by HPLC on aliqnots withdrawn at different times. Percentage of residual vitamin E at each time point was calculated, and the logarithm of this value was plotted against time to obtain a straight line. Vitamin E initial concentration in liposomes was 10 nmoles/mg of PL. PLPCOOH, when present, was 20 nmoles/mg of PL. Results represent a mean of two separate experiments (Variation < 1%).

22

10

20

8

0 r~

E 6 18

16

= 0 u0 14 sible to Cu r÷ in m i c e l l e s than in bilayers. The e v i d e n c e that the rate o f vitamin E c o n s u m p t i o n is relatively faster in l i p o s o m e s containing unsaturated lipids (Fig. 2) further suggests the i n v o l v e m e n t o f a p h y s i c a l constraint o f the e n v i r o n m e n t affecting the interaction o f vitamin E with Cu r+. The c o n s u m p t i o n o f vitamin E b y Cu r÷ is accounted for b y the reaction: a - T O C - O H + Cu +÷ ---, ot - TOC-O" + Cu ÷. (3) A similar reaction has been d e s c r i b e d for F e 3÷ in micelles containing vitamin E. 2°'2j In these e x p e r i m e n t a l conditions, however, a m u c h higher, s u p r a p h y s i o l o g i cal, vitamin E concentration was required to reduce F e 3÷, p r o b a b l y o w i n g the less favourable redox potential. In l i p o s o m e s containing saturated lipids and P L P C O O H , in the presence o f Cu r÷, vitamin E consumption is stoichiometric with P L P C O O H c o n s u m p tion (Fig. 3A). On the other hand, in l i p o s o m e s containing unsaturated lipids, when vitamin E is over, P L P C O O H concentration starts rising (Fig. 3B). The apparent l o w e r c o n s u m p t i o n o f lipid h y d r o p e r o x i d e s when unsaturated l i p o s o m e s were used as substrate o f p e r o x i d a t i o n is e x p l a i n e d in terms o f the simultaneous production o f n e w h y d r o p e r o x i d e s from o x i d a b l e lipids.

2 ~ 0 "~ 0

,-1

12 50

100 150 Time, minutes

(A)

200

250

25

10

u 22.5

8

6 rn 5"

2o

~ 17.5 ¢¢

= 0 0 bd

15

2

12.5 lO 0

(B)

OQ

50

,i 100

150 200 250 Time, minutes

300

350

Fig. 3. Effect of the incubation with Cu ++ on PLPCOOH and vitamin E content in liposomes. Liposomes containing DPPC (A) or PLPC (B), PLPCOOH and vitamin E were exposed to Cu ++. PLPCOOH (.o_) and vitamin E content (-am-) were monitored by HPLC, as described under Materials and Methods, on aliquots withdrawn at different times. Control values of vitamin E (-A-) and PLPCOOH (-O-) without Cu ++ are also reported. Results represent a mean of three separate experiments. (Variation < 5%, both for vitamin E and PLPCOOH measurement).

Lipid peroxidation by Cu÷+ and vitamin E

PLOOH

Cu +÷

E

PLOO"

71

Cu +

PLOOH

E"

PLO"

ADDUCT

ADDUCT Scheme 1.

The formation of these adducts is indirectly supported by the lack of any evidence for the formation of two electron oxidation products of vitamin E (8a-hydroxyltocopherone and tocopheryl-quinone) as well as of small molecular weight phospholipid decomposition products. Reactions 4 and 5 are apparently favoured by the vicinity of phospholipids and vitamin E in the bilayer and by the similar rate of reactions 1 and 3. This concerted mechanism is illustrated in the Scheme I. The reduction of Cu +÷ in micellar dispersions of phospholipids accounts for the acceleration of peroxidation rate observed in the presence of vitamin E. z° Indeed, when PLPC, PLPCOOH, and vitamin Econtaining micelles are substrate of Cu++-induced peroxidation, the decrease of hydroperoxides observed in liposomes was not apparent, whereas vitamin E was confirmed to play a substantial pro-oxidant effect (Fig. 4). The involvement of Cu + but not cr-TOC-O" in this phenomenon is suggested by the similar rate of PLPCOOH formation during and after vitamin E depletion. This conclusion appears in contrast with the pro-oxidant effect of c~-TOC-O" reported by Bowry et al. 22'23 in experiments where peroxidation was induced by an extremely low flow of radicals, produced by thermal decomposition of a hydrosoluble diazocompound. A reasonable explanation is that, in the peculiar conditions of those experiments, initiation by a-TOCO" was kinetically more favourable than the radicalradical interaction taking place in our conditions, where a-TOC-O" was generated by Cu ++ at a fast rate. The different effect of vitamin E in liposomes or micelles undergoing peroxidation in the presence of Cu +÷, can be interpreted, again, on the light of the different physical state of the surface exposed to Cu ÷÷, which, in liposomes lead to a slower oxidation of lipids and vitamin E. This highlights further the concept that the accessibility of the oxidant species to substrates is

of fundamental importance among factors underlying oxidative resistance, Eventually, these data suggest that, although in micelles the pro-oxidant effect of vitamin E by far prevails the possible antioxidant effect, in liposomes neither the pro-oxidant nor the antioxidant effect are remarkable. Actually, due to the consumption of hydroperoxides, an antioxidant effect takes place in liposomes, although its importance in the overall length of the induction period seems not apparent (compare Figs. 1 and 3B).

LDL When L D L are exposed to Cu ÷÷, vitamin E rapidly disappears with a kinetics and a rate similar to, al100

14

< ,~. 80

,.q"

..............

40

ii................. . ~ 'i' " A........"

i

20

; .................

!

....

0 0

60

120 180 Time, minutes

240

8.4

m

5.6

°

, 2.8

t" "0

0 300

Fig. 4. Effect of vitamin E on phospholipid hydroperoxide formation in micelles incubated with Cu+÷. Micelles of PLPC, with (_._) or without (-A-) vitamin E, were incubated with Cu÷÷. PLPCOOH formation was measured by HPLC on aliquots withdrawn, at different times, as described under Materials and Methods. Time course of vitamin E depletion is also shown (-©-). Formation of PLPCOOH, taking place in the absence of Cu*÷, has been subtracted. Results represent a mean of two separateexperiments (Variation < 3%, both for vitamin E and PLPCOOH measurement).

72

M. MAIORINO et al.

15

15

,<

Table 1. Lipid Hydroperoxide Content in Aged LDL Before and After Vitamin E Depletion a Vitamin E PLOOH (nmoles/mg prot) (nmoles/mg prot)

10 ~

9

•

o_ ¢D

8.1

tj~ 3

'

ol 0

6

12 18 Time, minutes

24

30

Fig. 5. Time course of vitamin E depletion and lipid hydroperoxide formation in LDL exposed to Cu ÷+, LDL were incubated with Cu ÷÷. On aliquots, withdrawn at different times, reaction was stopped by adding EDTA and, after extraction, phospholipid hydroperoxide (-*-) and vitamin E (-II-) content was measured as described under Materials and Methods. The overall length of the lag phase, measured spectrophotometrically as described in Table 3, was 92 _+ 12 min.

though even faster than, that observed in lipid micelles. The analogy of LDL with micelles is extended to the behaviour of PLOOH, the concentration of which does not decrease--as in liposomes--during the phase when vitamin E is consumed (Fig. 5). The concentration of PLOOH, indeed, increases with the rate of 0.01 nmoles/min/mg when vitamin E is present, to 0.1 nmoles/min/mg when no more vitamin E is available. It is worth noting that this rate lasts, with a minor increase, for 92 _+ 12 min when the rapid phase of peroxidation ( > 1 nmole/min/mg) takes place. Clearly, these data argue for a minor antioxidant effect of vitamin E, taking place until the antioxidant is present, but being irrelevant to the length of the lag phase and to the overall resistance of LDL to peroxidation (see also later). The absence in LDL of the concerted mechanism of vitamin E and PLOOH disappearance during Cu ++induced peroxidation, described in liposomes (Fig. 3), has been confirmed also in LDL where the lipid hydroperoxide content was increased (Table 1). The similarity of the behaviour of LDL and micelles challenged with Cu ÷÷ expands also to the absence of the major stimulation of vitamin E consumption by PLOOH that has been observed in liposomes. The increase of LDL lipid hydroperoxide from 1 to 11 nmoles/mg indeed does not affect vitamin E consumption rate (Table 2). These data indicate that, in LDL, vitamin E is rapidly oxidised by C u t t , thus preventing the possibility of the interaction of its radical with lipid radicals,

Before incubation with Cu ++ 15 min after incubation with Cu ++

10.2

_+ 1.2

1.30 _+ 0.70

9.1

_+ 0.7

9.25 _+ 0.5

Results represent the mean _+ SD of four independent experiments. a Vitamin E and phospholipid hydroperoxide content has been determined on aged LDL (0. I mg/ml) before and after 15 rain incubation with Cu +÷ (50 #M). EDTA (1 mM) was added at the end of the incubation to stop the reaction.

which has been proposed to take place in liposomes, to account for the consensual disappearance of vitamin E and PLOOH (Fig. 3 and Scheme 1). Nevertheless, the pro-oxidant effect of vitamin E in the presence of Cu t÷ is also lacking in LDL, as shown in the experiment reported in Table 3. In these experiments, to decrease vitamin E to undetectable levels, LDL have been incubated with C u t t for 15 min, after which the reaction has been stopped with EDTA. LDL have been then dialysed and challenged again with Cu ÷t. The lag of peroxidation induced by this second pulse of Cu t+ produced a result identical to that of control LDL, processed throughout the similar procedure, but omitting the first addition of C u t t . Furthermore, the rate of conjugated diene formation was identical in the two sets of experiments (not shown). In other words, these observations suggest that in LDL, although the interaction of Cu t÷ with vitamin E (reaction 3) is apparently fast, its reaction with lipid hydroperoxides (reaction 1) is apparently much slower. This unbalance between the two reactions prevents the consensual consumption of vitamin E and PLOOH, as well as the acceleration of vitamin E consumption rate observed in liposomes containing phospholipid hydroperoxides. Our conclusion that vitamin E is oxidised by Cu ÷÷ in LDL is in agreement with the observation that vitamin E consumption rate in LDL is a function of Cu ÷+ concentration. 24 In summary, from the noted experiments on different substrates, the following conclusions, relevant to LDL-oxidative resistance in the presence of copper, can be drawn: 1. In LDL, in micelles, but not in liposomes, vitamin E is easily accessible to the oxidant (Cu÷t). 2. In liposomes, but not in micelles or LDL, the oxidation rate of vitamin E is affected by the decomposition rate of PLOOH.

73

Lipid peroxidation by Cu ÷÷ and vitamin E Table 2. Rate of Vitamin E Consumption During Cu ÷+ Induced Peroxidation of Fresh and Aged LDL"

Freshly isolated LDL Aged LDL

PLOOH (nmoles/mg prot)

Vit. E (nmoles/mg prot)

Vit. E Consumption Rate (nmoles/min/mg prot)

1.02 +_ 0.7 10.97 +_ 1.5

10.1 + 1.3 8.4 + 0.5

2,1 _+ 0.5 2.3 +_ 0.7

Results represent the mean _ SD of four independent experiments. LDL phospholipid hydroperoxide and vitamin E content in LDL were measured as described in the Methods section. Vitamin E consumption rate was measured using aliquots withdrawn every 2 min from an incubation mixture containing LDL (0.1 mg prot/ml) and Cu ÷÷ (50 #M). The consumption rate was calculated on the linear initial phase of the reaction.

3. A p r o - o x i d a n t e f f e c t o f v i t a m i n E, d u e to C u ÷~ r e d u c t i o n , t a k i n g p l a c e in m i c e l l e s , is n o t a p p a r e n t in l i p o s o m e s and L D L . 4. A n a n t i o x i d a n t e f f e c t o f v i t a m i n E, if any, takes p l a c e , s l o w i n g d o w n the v e r y early p h a s e o f L D L p e r o x i d a t i o n , and it is n e g l i g i b l e in r e l a t i o n to o v e r all l e n g t h o f the lag phase. In light o f t h e s e d a t a and the r e l a t e d d i s c u s s i o n , lag in L D L p e r o x i d a t i o n i n d u c e d by C u +÷ c a n b e h a r d l y a t t r i b u t e d to the c o n t e n t o f v i t a m i n E, w h i c h has b e e n c l a i m e d to p l a y a m a j o r a n t i o x i d a n t effect. T h e n a t u r e o f the m o l e c u l a r m e c h a n i s m u n d e r l y i n g the lag, o n the o t h e r hand, is still u n k n o w n . It is, h o w e v e r , w o r t h y o f m e n t i o n that an " i n d u c t i o n p e r i o d " takes p l a c e also in l i p o s o m e s , but not in m i c e l l e s , in the a b s e n c e o f any a n t i o x i d a n t (Fig. 1), thus s u g g e s t i n g the p r e s e n c e o f a still u n k n o w n t h r e s h o l d , p o s s i b l y r e l a t e d to a p h y s i c a l constraint, w h i c h has to b e o v e r c o m e b e f o r e m a s s i v e lipid p e r o x i d a t i o n m i g h t start. Acknowledgements - - We are grateful to Gabriele Bittolo Bon and

Table 3. Effect of Vitamin E on Lipid Peroxidation Lag Time of LDL Challenged With Cu ÷÷"

Control LDL Vitamin E-depleted LDL

Vitamin E (nmoles/mg PL)

Lag Time (min)

13 _+ 1.9 0.9 _+ 0.6

100.9 _+ 18.01 118.9 _+ 9.9

Lag time was measured by continuous monitoring of absorbance at 234 nm and quantified by determining the length of the intercept of the two lines representing respectively the slow and the fast peroxidation phases. ~ Results represent the mean _+ SD of four independent experiments. "Vitamin E depletion was obtained by incubating fresh LDL (0.1 mg/ml) with Cu ÷÷ (50 #M) for 15 rain, after which the reaction has been stopped with I mM EDTA. LDL have been then exhaustively dialysed against 10 mM Na2HPO4, 0.16 M NaCI, pH 7.4, and challenged again with Cu ÷÷ (50 #M). In control LDL, Cu ÷+ has been omitted from the first incubation.

Giuseppe Cazzolato, from the 2 Divisione Medicina Generale, Ospedale di Venezia, for the generous supply of human LDL.

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ABBREVIATIONS

a-TOC-OH--a-tocopherol a - T O C - O ' - - c h r o m a n o x y l radical D O C - - S o d i u m deoxycholate D P P C - - d i p a l m i t o y l phosphatidylcholine E D T A - - Ethylenediaminoteraacetic acid H P L C - - h i g h performance liquid chromatography L D L - - l o w density lipoproteins PL--phospholipids P L O O H - - p h o s p h o l i p i d hydroperoxides P L P C - - p a l m i t o y l linoleoyl phosphatidylcholine P L P C O O H - - p a l m i t o y l linoleoyl phosphatidylcholine 13-hydroperoxide S D S - - S o d i u m Dodecyl Sulphate