Biochem. J. (1992) 288, 341-344 (Printed in Great Britain)

341

RESEARCH COMMUNICATION

Vitamin E in human low-density lipoprotein When and how this antioxida

t

becomes

a

pro-oxidant

Vincent W. BOWRY,* Keith U. INGOLDt and Roland STOCKER*$ *Biochemistry Group, Heart Research Institute, 145 Missenden Road, Sydney, New South Wales 2050, Australia, and tSteacie Institute of Molecular Science, National Research Council of Canada, Ottawa K1A OR6, Canada

Uptake of oxidatively modified low-density lipoprotein (LDL) by cells in the arterial wall is believed to be an important early event in the development of atheroscelerosis. Because vitamin E is the major antioxidant present in human lipopnoteins, it has received much attention as a suppressor of LDL lipid oxidation and as an epidemiological marker for ischaemic leart disease. However, a careful examination of lipid peroxidation in LDL induced by a steady flux of aqueous peraxyt radicals has demonstrated that, following consumption of endogenous ubiquinol-1O, the rate of peroxidation (i) declies as vitamin E is consumed, (ii) is faster in the presence of vitamin E than following:its complete consumption, (iii) is substantially accelerated by enrichment of the vitamin in LDL, either in vitro or by diet, and (iv) is virtually independent of the applied radical flux. We propose that peroxidation is propagated within lipoprotein particles by reaction of the vitamin E radical (i.e. a-tocopheroxyl radical) with polyunsaturated fatty acid moieties in. the lipid. This lipid peroxidation anism, which can readily be rationalized by the known chemistry of the a-tocopheroxyl radical and by the radicalisolating properties of fine emulsions such as LDL, explains how reagents which. reduce the a-tocopheroxyl radical (i.e. vitamin C and ubiquinol-10) strongly inhibit lipid peroxidation in vitamin E-containing LDL.

INTRODUCTION Oxidative modification of low-density lipoprotein (LDL) can lead to an increased and uncontrolled uptake of cholesterol by macrophages [1]. Formation of such high-uptake LDL in vivo is implicated as an early and perhaps crucial step in a cascade of cellular processes which leads to the formation of fatty streaks and eventually atherosclerotic lesions in the artery wall [2]. Since the formation of high-uptake LDL is generally held to be preceded by, and to some extent caused by, peroxidation of the LDL lipid, much attention has been devoted to the inhibition of lipid peroxidation in LDL by endogenous and exogenous antioxidants [3,4]. a-Tocopherol (a-TocH) is biologically and chemically the most active form of vitamin E [5]. It is also the. major lipid-soluble radical-trapping antioxidant in plasma [5] and LDL [4] and, as such, has received more attention than minor lipidsoluble antioxidants such as y-tocopherol, carotenoids, retinol and ubiquinol-10 (CoQ10H2) [4]. Generally speaking, only the last of these shows any significant antioxidant activity at physiological concentrations in LDL [6,7]. However, we have shown that after, but not before, oxidative consumption of (water-soluble) vitamin C and (LDL-associated) CoQ10H2, the peroxidation of the LDL's lipids proceeds by a free-radical chain process even in the presence of > 90 % of the original a-TocH [7]. Thus treatment of LDL with water-soluble or lipid-soluble peroxyl radicals resulted in the formation (after a lag period corresponding to the time required for the oxidation of CoQ10H2) of lipid hydroperoxides (LOOH), at a rate (RP) which was greater than the known rate at which new radicals were being generated (Rg), and having chain lengths (v) equal to Rp/Rg 2.6-8.0 [7,8]. This and other features of the a-TocH-

inhibited peroxidation of LDL [7,8] are not readily reconciled with conventional views about lipid antioxidation, which predict a v value of . 1 in the presence of endogenous a-TocH (see below). We have now examined more closely the latter a-TocHinhibited phase of LDL oxidation initiated by a steady flux of peroxyl radicals (ROO') derived by thermolysis of a watersoluble azo compound. Our findings show that for low fluxes of aqueous ROOG, a-TocH can be a strong pro-oxidant for LDL. A new mechanism for the peroxidation of a-TocH-containing LDL is proposed.

MATERIALS AND METHODS Phosphate-buffered isotonic saline (pH 7.4; 25 mm in phosphate) was prepared from Nanopure water and the highest purity reagents commercially available, and stored over Chelex100 (Bio-Rad) at 4 °C for at least 24 h to remove contaminating transition metals. The azo initiators 2,2'-azobis-(2-amidinopropane hydrochloride) (AAPH) and 2,2-azobis-(2,4-dimethylisovaleronitrile) (AMVN) (Polyscience) were diluted in freshly prepared phosphate-buffered saline and ethanol stock solutions respectively. Preparation and handling of human plasma and LDL followed procedures detailed previously [7,9]. In vitro enrichment of LDL with a-TocH [4] was achieved by adding 1-5 % (v/v) of 10 mma-TocH (Henkel Co.) in dimethyl sulphoxide to plasma, incubating the mixture at 37 °C for 5 h, and isolating the LDL by rapid ultracentrifugation [7,9]. The fresh and initially LOOHfree LDL was gel-filtered (PD- I0; Pharmacia) immediately before use to remove contaminating uric and ascorbic acids. Molar

Abbreviations used: AAPH, 2,2'-azobis-(2-amidinopropane hydrochloride); AMVN, 2,2'-azobis-(2,4-dimethylvaleronitrile); CEOOH, cholesteryl

CoQ10H2, ubiquinol-10; LDL, low-density lipoprotein; LOOH, lipid hydroperoxides; PCOOH, phosphatidylcholine hydroperoxides; PUFA, polyunsaturated fatty acids; Rg, rate of peroxyl radical generation; Ri, rate of lipid initiation; Rp, rate of LOOH accumulation;

ester hydroperoxides;

cx-TocH, ac-tocopherol; ', radical chain length.

I

To whom correspondence should be addressed.

Vol. 288

342

Research Communication

concentrations of LDL given in the Figures and Tables are calculated from the lipid analyses (based on a value of 720 cholesteryl linoleate molecules per LDL particle) and/or protein analyses. AAPH-induced peroxidations and lipid analyses were performed using the same methods as described previously [9], with incubations being carried out with 1-2 ml of LDL in foilwrapped capped glass tubes at 37 +1 °C (see Figure legends). RESULTS Incubation of LDL with the azo radical initiator AAPH resulted in the production of LOOH, which were separated into two classes by methanol/hexane extraction: (i) hydroperoxides of the polar surface lipids (principally phosphatidylcholine hydroperoxides; PCOOH), and those of the neutral core lipids [92-95 % cholesteryl ester hydroperoxides (CEOOH) and 5-8 %

Table 1. Effect of initiator concentration on peroxidation of LDL lipd LOOH (= CEOOH+PCOOH) were measured during incubation at 37 °C (see Fig. 1 legend). LDL was present at approx. 1.4 JM in apolipoprotein B and incubations initially contained 1050 JIMcholesteryl linoleate, 77 /LM-cholesteryl arachidonate, 12.1 lum-aTocH and 0.2-0.5 1sM-CoQ1OH2. R inh is the maximum rate of LOOH accumulation in the presence of a-TocH following consumption of endogenous CoQ1OH2 (cf. Fig. la and ref [7]). RpJUnih is the Rp following consumption all detected antioxidants. The effective chain length is defined as I = Rp/Rg, where the radical generation rate is taken to be Rg = 1 .3 x 10-6 [AAPH]* s-I [11]. Ri = 2d[aTocH]/dt, as defined in the text. Values in parentheses are S.D.S for an experiment performed in triplicate.

[AAPH] (mM) 0.50 2.0

10 55 12

I8

I 0 0

6

12 Il

I

0 0

0

I4-

8-i

Time (min)

Fig. 1. Peroxidation of LDL lipids induced by organic peroxyl radicals (a) LDL was incubated at 37 °C with the water-soluble radical initiator AAPH, yielding hydroperoxides of the neutral (core) lipids (predominantly CEOOH), and of the surface lipids (mostly PCOOH), which were analysed by h.p.l.c. [7] with both u.v. (234 nm) and chemiluminescence detectionr. Chemiluminescence and u.v. LOOH analyses agreed to +8 % for each data point. The peroxidation rate of core lipids was confirmed by measuring the loss of PUFA (0, same scale as LOOH) (i.e. cholesteryl linoleate and arachidonate relative to endogenous cholesteryl oleate) in the neutral lipid extract by h.p.l.c. analysis [7]. [LDL], 1.4 JpM; [AAPH], 2 mM; Rg, 2.6 nM- s-1). This experiment was performed in triplicate (S.D.S < 4 % and 5 % for CEOOH and a-TocH respectively). (b) A chloroform LDL lipid extract dissolved in t-butyl alcohol, at a concentration equal to that of LDL lipid in plasma (i.e. ,cmparable to that in a), was incubated at 37 °C with the lipophilic azo compound AMVN [10] (1 mM; Rg = 2.8 nM s-). Results are from one experiment representative of five. No detectable LOOH formation or a-TocH consumption occurred in azo-compound-free controls.

Rinh (nM s-1)

Rpunihn (nM s-1)

Pinh

12 3.6 7.8 8.4(±0.8) 3.1 8.1 (+0.6) 0.7 31 9.0 111 0.12 8.3

Puninh 5.5 3.2 2.4 1.6

R1 s-')

(nM

0.31 1.1 (+0.1) 4.5 20

triacylglycerol hydroperoxides]. LOOH were measured by h.p.l.c. with both u.v. (234 nm) and chemiluminescence detection, and peroxidation in more heavily oxidized samples was verified by measuring the loss of polyunsaturated fatty acid (PUFA) moieties from the cholesteryl esters of the LDL. In all cases the cholestesteryl ester/PUFA loss equalled CEOOH formation until a-TocH was depleted; thereafter the PUFA loss became increasingly greater than the detected CEOOH (e.g. Fig. la). CoQj0H2 is the most rapidly consumed antioxidant associated with LDL, and although. present at only one-tenth or less of the ac-TocH concentration (see Fig. 1 legend), it strongly inhibits oxidation of the lipids until it has been almost completely consumed (see [7] and below). In this work we examined the oxidation of LDL following the consumption of CoQj0H2. The minor antioxidants (i.e. carotenoids and y-tocopherol) are not considered here, as they are present at only very low concentrations (in total less than one molecule per LDL particle [4,9]) and are consumed less rapidly than are CoQ10H2 and a-TocH [5,6]. Although a-TocH is biologically the most active form of vitamin E and chemically a very efficient scavenger of peroxyl radicals [5], Fig. l(a) shows that the peroxidation rate actually decreased as ax-TocH in LDL was consumed, with the minimum rate being observed with one or less a-TocH molecules remaining per LDL particle (i.e. 0-15 % of initial concentration). After consumption of all identified antioxidants (e.g. at 450 min in Fig. 1 a), the chain length (vuninh) can be larger or smaller than in the 'ca-TocH-inhibited' phase (vinh), depending on the radical generation rate. Thus, for Rg < 2 nm s51, vinh > vuninh and vice versa for Rg > 2 nM s-s (see Table 1). Fig. 1(b), in contrast, confirms the expectation [5] that peroxidation of LDL lipid in bulk solution (induced by lipid-soluble ROO [10]) is strongly inhibited by the LDL's natural antioxidant content (vinh, 0.07; Vuninh9 1.0; note the smaller LOOH scale compared with Fig. 1(a). The fall-off in Rp at low a-TocH concentrations in LDL is almost certainly not due to inhibition by products, since the rate of a-TocH consumption remained virtually unchanged throughout (Fig. la) and since the effect is not seen in bulk-phase oxidation (e.g. Fig. lb). We are therefore forced to conclude that, under our experimental conditions, a-TocH is a pro-oxidant for.LDL. This conclusion was confirmed by demonstrating that LDL enriched with a-TocH (in vitro or by oral supplementation) was peroxidized more rapidly in the 'a-TocH-inhibited phase' than -

1992

Research Communication

343

100

160

I. 60 X 0

I

fi 40

0

-J

EI

(b)

20

15

E a-TocH

I 0 0

10

-

-i

Ee

E9~~~~~~~~~~~88(

1

CEOOH

5

0 -j

44D

PCOOH 0

60

120 Time (min)

180

2 240

Fig. 2. Peroxidation of vitamin E-enriched versus non-enriched LDL LDL (2.2 #M), isolated from plasma supplemented with a-TocH in dimethyl sulphoxide (a) [4] or from plasma supplemented with dimethyl sulphoxide alone (b), was incubated with 10 mM-AAPH (Rg = l InM s-1) and the lipids were analysed as in Fig. 1. The use of measuring CEOOH formation as an accurate index of the rate of LDL lipid peroxidation was verified by analysing the loss of PUFA. As in Fig. 1, the amounts of CEOOH formed equalled the amounts of PUFA lost from the cholesteryl esters, determined with an experimental error of +4 % in a triplicate analysis

was a non-enriched control (Fig. 2). Moreover, the acceleration of lipid peroxidation increased with the degree of a-TocH enrichment (Table 2). The effect was not caused by our method of in vitro enrichment, since in vivo (dietary) supplementation

also produced LDL which was more rapidly peroxidized than was the pre-supplementation lipoprotein. The fact that in vivoenriched LDL was peroxidized more rapidly than in vitrosupplemented LDL containing an equivalent a-TocH concentration (compare rows 3 and 4 of Table 2) may be due to dietary supplementation giving more uniform incorporation of ac-TocH. In interpreting the above results, one should note that we have defined the radical chain lengths, v, in terms of the known flux of aqueous radicals generated by the azo initiator AAPH, i.e. Rg = 1.3 x 10-6 [AAPH] s-I at 37 °C in protein-containing aqueous solutions [1]: (I) iR-N=N-R -* R [R- _ (CH3)2C -C(=NH2+)(NH2) for AAPH] R,+02 -ROO' (2) The Rg for AAPH is calculated [11] by measuring the rate of consumption of an efficient water-soluble radical scavenger (A): 2 ROO + A -- non-radical products (3) Since AAPH resides almost entirely in the water phase, and since azo compounds are not prone to induced decompositions, Rg may safely be assumed to be independent of species present in the LDL lipid. However, it is well known from work with liposomes and micelles that the rate of lipid initiation (R1) in such dispersions is only 20 60 % of the known Rg [11,12], as measured by consumption of a lipophilic radical scavenger (e.g. a-TocH). If we likewise define R, in terms of consumption of lipid antioxidants in LDL, then to a good approximation R.= -2d[a-TocH]/dt. The Ri values given in Table 2 (column 6) are comparable to data reported by Sato et al. (i.e. R1 = 6.5 nM s-I for 11.3 mM-AAPH-induced oxidation of LDL containing 11.3 ,tM-a-TocH) [8]. One can also see from Table 2 that what might be termed the efficiency of lipid initiation, Ri/Rg, is increased by a-TocH supplementation, i.e. a greater proportion of the aqueous radical flux is intercepted by lipid when more a.TocH is present in the LDL. This is not unexpected, since aTocH is by far the most reactive peroxyl radical scavenger present in LDL and, owing to its hydrogen bonding, hydroxyl groups may reside preferentially close to the lipid-water interface. Importantly, however, since Rp is virtually independent of Ri (Table 1), the observed increase in the LOOH formation rate (Rp) upon a-TocH enrichment is not due to the increased lipid radical flux (R1). In addition, LDL peroxidation initiated by lipid-soluble AMVN (cf. [8]) was also accelerated by a-TocH enrichment, but with no corresponding increase in R. (Table 2), i.e. the increase in Rp resulting from a-TocH enrichment is

Table 2. Effect of a-TocH concentration on peroxidation of LDL lipid LDL enriched in a-TocH by supplementation in vitro [4] or by diet (in vivo) [4] was compared with identically prepared non-enriched LDL with the same lipid and antioxidant composition, except for the a-TocH concentration. Experiments in vitro were run in parallel and experiments in vivo were performed both before and after the supplementation period (1-3 days). (+) and (-) denote supplemented and unsupplemented LDL respectively, and (D) denotes the dietary supplementation experiments. Lipid-soluble AMVN was added to pre-warmed LDL via an ethanolic stock (0.2 M) solution. Radical generation rates were taken to be Rg = 1.3 x 10-6 [AAPH] .s5- [11] and 2.6 x 1-0 [AMVN] * s-1 [10]. Experimental errors were + 4 % and +7 % (S.D.) for M and Ri, respectively.

Initiator AAPH AAPH AAPH AAPH

AAPH AMVN AMVN

Vol. 288

Rg (nM s-')

[LDL] CUM)

cc-TocH(+)/a-TocH(44

(JLM//zM) (ratio)

PR/v4 (ratio)

R ( )/Rj( ' (nM s-'/nM s-') (ratio)

13 13 5.3 5.2 5.2 2.4 4.9

2.2 2.2 1.4 1.4 (D) 1.4 (D) 1.4 1.4

81/17 (4.8) 121/17 (7.1) 31/9.9 (3.1) 29/8.9 (3.3) 14/9.1 (1.5) 31/9.9 (3.1) 31/9.9 (3.1)

2.0/1.1 (1.8) 2.1/0.91 (2.3) 3.2/2.1 (1.5) 5.5/2.3 (2.4) 3.4/2.5 (1.4) 0.9/0.4 (2.3) 0.5/0.22 (2.3)

9.3/5.1 (1.8) 11/4.9 (2.2) 3.9/2.2 (1.8) 3.3/2.6 (1.3) 3.3/2.7 (1.2) 0.25/0.27 (0.9) 0.49/0.51 (0.96)

Research Communication

344 clearly not a result of enhanced lipid initiation. Both of these kinetic effects, and the remarkable lack ofinitiator-concentrationdependence of R inh, are reminiscent of kinetic behaviour observed in emulsion polymerization [13], with a-TocH acting as a highly reactive chain transfer agent (rather than as an antioxidant) in the LDL dispersion. The mechanism of chain transfer is discussed below.

DISCUSSION Our present results for LDL contrast dramatically with the conventional picture of vitamin E as a chain-breaking, peroxylradical-trapping antioxidant, a picture which is derived from its behaviour in bulk lipids [5] as exemplified in Fig. 1(b). That is, a-TocH inhibits the radical chain formation defined by reactions (4) and (5): L-H + L(R)OO-+ L + L(R)OOH

L + 02 - LOO'

(4) (5)

by scavenging the lipid peroxyl radical (LOO-) or initiating peroxyl radical (ROO'): a-TocH + L(R)OO' -- a-Toc + L(R)OOH

(6)

Thus the data reported here are cdifficult to reconcile with conventional expectations of antioxidation, because in bulk solution, liposomes and micelles, inhibition 'by phenolic antioxidants such as a-TocH follows the cla-ssical kinetic laws predicted by reactions (4)-(6), i.e. Rp = constant x Rg/[Inhibitor] [5,12]. However, the anomalies we have uncovered in lipoproteins can be accounted for by one simple hypothesis, i.e. that the atocopheroxyl radical (a-Toc') formed by the reaction of a-TocH with LOO- or ROO' (reaction 6) reacts with PUFA-containing lipid (L-H) in the lipoprotein (reaction 7) to participate in a radical chain comprising, in sequence, reactions (7), (5) and (6): a-Toc' + L-H -* ac-TocH + L

(7)

By contrast, in bulk lipids the usual fate of the a-Toc' radical is to trap a second peroxyl radical (reaction 8), and thus destroy two peroxyl radicals:

a-Toc-+ L(R)OO -. non-radical products

(8)

The difference in the behaviour of vitamin E between bulk

lipids and aqueous lipoprotein dispersions resembles the wellknown differences between the polymerization of styrene in bulk and in fine aqueous emulsions [13], and can be accounted for in a similar manner. Thus the propagating radicals (including acToc') are isolated from each other by the aqueous medium and only infrequently encounter another radical (e.g. for Rg = 1 nM- s-1 and [LDL] = 1 UM, the average time between radical strikes on a particle = [LDL]/Rg = 17 min!). With so-much time

at its disposal, even a radical as unreactive as a-Toc' finds 'something to do', i.e. it participates in lipid peroxidation via reaction (7), before eventually being destroyed by capture of a second radical from the aqueous medium (i.e. via reaction 8). Indeed, e.s.r. estimates of reactivity of a-Toc' with PUFA lipid (i.e. for methyl linoleate a rate constant at 37 °C of s-' can be calculated from the data presented in [14]) k7 0.1 M-1 s suggest that the half-life of a-Toc' in LDL lipid ([L-H] 0.8 M) should be of the order of 15 s, i.e. much shorter than the above random strike interval. The potential effects of vitamin B-accelerated peroxidation in LDL (and other small lipid particles in aqueous dispersion) are far-reaching and profound. The pro-oxidant effect of a-TocH (as gauged, for example, by vinh/Vu,||h is most pronounced at lowest Rg, and radical fluxes in vivo are presumably much lower than those used in this work. Thus for prevention of lipoprotein oxidation in vivo it is essential that any x-Tor radiclsiproduced are reduced by reagents which will yield radical incapable of continuing the peroxidation chain. Under normal conditions this would appear to be achieved by two endogenous -artoxidants, i.e. vitamin C (in plasma) [6,15] and CoQ10H2 (in LDL) [7,9,16], which prevent the peroxidation of LDL containing az-TbdH-i 'by two quite distinct mechanisms. %

We thank Professor R. T. Dean for thought-provoking di'scusions. We also thank the Australian National Health & Medical Research Council for its financial support (Grant 910284 to R. S.).

REFERENCES 1. Steinbrecher, U. P., Loughneed, M., Kwan, W.-C. & Dirks, M. (1989) J. Biol. Chem. 264, 15216-15223 2. Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C. & Witztum, J. L. (1988) N. Engl. J. Med. 320, 915-924 3. Gey, K. F., Puska, P., Jordan, P. & Moser, U. K. (1991) Am. J. Clin. Nutr. 53, 326S-334S 4. Esterbauer, H., Dieber-Rotheneder, M., Striegl, G. & Waeg, G. (1991) Am. J. Clin. Nutr. 53, 314S-321S 5. Burton, G. W. & Ingold, K. U. (1986) Acc. Chem. Rev. 19, 194-201 6. Stocker, R. & Frei, B. (1991) in Oxidative Stress: Oxidants and Antioxidants (Sies, H., ed.), pp. 213-243, Academic Press, New York 7. Stocker, R., Bowry, V. W. & Frei, B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1646-1650 8. Sato, K., Niki, E. & Shimasaki, H. (1990) Arch. Biochem. Biophys. 279, 405-409 9. Bowry, V. W., Stanley, K. K. & Stocker, R. (1992) Proc. Natl. Acad. Sci. U.S.A., in the press 10. Niki, E., Saito, T., Kawakami, A. & Kamiya, Y. (1984) J. Biol. Chem. 259, 4177-4182 11. Niki, E., Saito, M., Yoshikawa, Y., Yamamoto, Y. & Kamiya, Y. (1986) Bull. Chem. Soc. Jpn. 59, 471-477 12. Castle, L. & Perkins, M. J. (1986) J. Am. Chem. Soc. 108, 6381-6382 13. Walling, C. (1957) Free Radicals in Solution, Wiley, New York 14. Nagaoka, S., Okauchi, Y., Urano, S., Nakashimna, U. & Mukai, K. (1990) J. Am. Chem. Soc. 112, 8921-8924 15. Packer, J. E., Slater, T. F. & Willson, R. L. (1979) Nature (London) 278, 737-738 16. Mohr, D., Bowry, V. W. & Stocker, R. (1992) Biochimn. Biophys. Acta 1126, 247-254

Received 25 August 1992/11 September 1992; accepted 29 September 1992

1992