©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Prevention of Tocopherol-mediated Peroxidation in Ubiquinol-10-free Human Low Density Lipoprotein (*)

(Received for publication, August 29, 1994; and in revised form, December 6, 1994)

Vincent W. Bowry (§) Detlef Mohr Janelle Cleary Roland Stocker (¶)

From the Biochemistry Group, The Heart Research Institute, Camperdown, Sydney, New South Wales 2050, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
FOOTNOTES
REFERENCES

ABSTRACT

Oxidation of low density lipoprotein (LDL) may be involved in the development of atherosclerosis. It has recently been shown that alpha-tocopherol (alpha-TOH) can act either as an antioxidant or prooxidant for isolated low density lipoprotein (LDL). In the absence of an effective co-antioxidant, alpha-TOH is a prooxidant and this activity is evidently due to reaction of the alpha-tocopheroxyl radical (alpha-TObullet) with the LDL's polyunsaturated lipids (Bowry, V. B., and Stocker, R.(1993) J. Am. Chem. Soc. 115, 6029-6045). Herein we examined the effectiveness of selected natural and synthetic radical scavengers as co-antioxidants for inhibiting peroxyl radical-induced peroxidation in LDL that is devoid of ubiquinol-10 (an effective endogenous co-antioxidant) but still contains most of its natural complement of alpha-TOH. Various quinols, catechols, and aminophenols, as well as ascorbate, 6-palmityl ascorbate, and bilirubin, were very effective co-antioxidants under our test conditions, whereas ordinary phenolic antioxidants, including short-tailed alpha-TOH homologues, were less effective. Reduced glutathione, urate, and Probucol were ineffective. These findings confirm that the prooxidant activity of alpha-TOH in LDL relies heavily on the segregation of water-insoluble radicals (particularly alpha-TObullet) into individual LDL particles, since it was those compounds that are expected to either irreversibly reduce alpha-TObullet or accelerate the diffusion of radicals between particles which most effectively inhibited the tocopherol-mediated phase of peroxidation. Theoretical and practical implications of these findings are discussed, as is their relevance to the ``LDL oxidation'' hypothesis of atherogenesis.


INTRODUCTION

The oxidation of human low-density lipoprotein (LDL) (^1)in the artery wall is thought to be an initiator of atherosclerosis(1) . Evidence from in vitro, in vivo, and epidemiological studies (2, 3, 4, 5, 6) suggests that ``oxidatively modified'' LDL is recognized and scavenged by macrophages, leading to transformation of the latter into lipid laden cells, which in turn contribute to the formation of fatty streaks and eventually plaque in the artery wall. If LDL oxidation helps to initiate atherosclerosis, it follows that increasing the resistance of LDL to oxidation will mitigate or even prevent atherosclerosis. This idea is supported by studies showing that supplementation with antioxidants can attenuate atherosclerosis in animal models (7, 8, 9) and possibly in humans(10, 11) .

Free radical-mediated aerobic oxidation (peroxidation) of LDL has thus become a widely studied model for atherosclerosis research(12, 13, 14) . A commonly used method for oxidatively modifying LDL is to incubate the lipoprotein with a redox catalyst (e.g. copper) or with cells in a transition metal-containing cell culture medium(13, 14) . With relatively high concentrations of a strong redox oxidant, such as copper ions, little lipid hydroperoxide (LOOH) is formed until alpha-tocopherol (alpha-TOH) and the minor antioxidants have been depleted. Following this apparent ``inhibited'' phase lipid peroxidation may enter a rapid ``uninhibited'' phase, toward the end of which the epitope of LDL's apolipoprotein B-100 may become ``modified,'' i.e. recognizable to the scavenger or oxidized LDL receptors of monocyte/macrophages(1, 15, 16) . The ``susceptibility'' of LDL toward oxidative modification is gauged by the duration of the inhibited phase of peroxidation, the rate of peroxidation during the uninhibited phase, and the maximal amount of peroxidized lipids accumulated(14) .

The relatively low rate of LOOH formation in the inhibited phase of copper-initiated LDL oxidation has led to the conclusion that alpha-TOH (the major and most reactive peroxyl radical scavenger in LDL) is an effective antioxidant for LDL in vitro and that it protects LDL from oxidation in vivo(14) . In view of this, we were surprised to find recently that the type of activity exhibited by alpha-TOH in LDL actually depended on the time taken for the alpha-TOH to be consumed. Thus, a rapid flux of peroxyl radicals from an azo initiator produced relatively little LOOH in LDL in the brief period of alpha-TOH consumption, whereas a comparatively mild flux of initiating peroxyl radicals could led to LOOH formation that was faster during than following the period of alpha-TOH consumption. Likewise, whereas the rapid oxidation of LDL with high concentrations of copper ions produces little LOOH in the brief (0.4 h) period of alpha-TOH consumption(14) , the low concentrations of copper and iron ions in the mildly oxidizing F-10 cell culture medium afford a faster maximal rate of LOOH formation during than following the period (30 h) of alpha-TOH consumption(17, 18) . Remarkably, in the latter case up to 50% of the polyunsaturated fatty acids in the LDL lipid were oxidized before alpha-TOH was depleted(17) , clearly demonstrating that substantial LOOH formation can occur in alpha-TOH-containing LDL.

These and other unusual findings about LDL peroxidation in the presence of alpha-TOH (17, 19, 20, 21) have led us to conclude that alpha-TOH in LDL can have prooxidant activity arising from a reaction between the alpha-tocopheroxyl radical (alpha-TObullet) and the active bisallylic methylene groups of polyunsaturated fatty acids (LH) of the LDL.

We have deduced from kinetics measurements that this normally slow reaction (k approx 0.1 M s) can lead to fairly rapid peroxidation in LDL because in the absence of an effective co-antioxidant the highly water-insoluble alpha-TObullet radicals are isolated from each other by the aqueous medium of the LDL suspension (see below)(21) .

Herein we examined the effect of selected biological and synthetic radical scavengers on the rate of LOOH formation in the alpha-TOH-containing phase of LDL peroxidation induced by lipid- and/or water-soluble organic peroxyl radicals (ROObullet). The dramatic co-antioxidant effectiveness of ascorbate (AscH), quinols, and certain other radical scavengers in this study is shown to arise neither by sparing of alpha-TOH nor by competitive scavenging of peroxyl radicals, but by attenuation of the prooxidant activity of alpha-TOH, most likely through elimination of alpha-TObullet from LDL particles.


EXPERIMENTAL PROCEDURES

Materials

The co-antioxidants were obtained from Sigma, except 2AP, 3AP, L-AscH, bilirubin (BR), DBHA, TBHQ, and Trolox®, which were from Aldrich. L-Adrenalin and pyrroloquinoline quinone were obtained from Fluka (Buchs, Switzerland), Probucol was a gift from Marion Merell Dow Inc. (Cincinnati, OH), BR-DT was purchased from Porphyrin Products (Logan, UT), and desferrioxamine B from Ciba Geigy (Basel, Switzerland). The reduced spin trap TDPHI was a generous gift from Dr. V. A. Roginsky (Russian Academy of Sciences, Moscow, Russia). The alpha-TOH homologues C1OH, HC1OH, C5OH, and C6OH, and C15OH were kindly provided by Drs. C. Suarna (Heart Research Institute) and P. Southwell-Keely (University of New South Wales, Sydney, Australia), whereas C11OH and C13OH were provided by Dr. P. Aryha (National Research Council of Canada, Ottawa). DBH2 was synthesized from C1OH (22, 23) . Human serum albumin (fraction V, fatty acid-free) and Cu,Zn-superoxide dismutase from bovine erythrocytes were obtained from Sigma. Buffers, HPLC eluents, and other reagents, including the radical initiators AAPH and AMVN, were all as described previously(17, 19, 24) . Aqueous solutions were stored over Chelex-100 to remove trace metal contaminations before use. The efficacy of this treatment was tested routinely by Buettner's AscH autoxidation method (25) and found to decrease the AscH autoxidation rate from 4 to 0.6%/20 min, indicating successful demetalization(25) .

LDL Isolation and Oxidation

LDL was isolated by ultracentrifugation (24) of freshly prepared heparinized plasma obtained from healthy male or female donors (23-38 years). The LDL (1-2 mg of protein/ml) was stored under air at 4 °C for 16-36 h before use, resulting in oxidation of most of the endogenous ubiquinol-10 (CoQH(2)). This enhanced the comparability and reproducibility of the results. (^2)When used, LDL contained < 0.15 µM cholesteryl linoleate hydroperoxide (Ch18:2OOH), the single major lipid oxidation product formed during the initial stages of peroxyl radical-mediated LDL oxidation, and < 0.1 µM CoQH(2).

The methods used for oxidizing LDL and analyzing the oxidized and non-oxidized lipids have been described(17, 19, 20, 24) . Most of the non-water-soluble antioxidants were added in methanol (<1%, v/v) to LDL and pre-incubated for 10 min before adding the azo-compound, a procedure that caused no turbidity or precipitation of the protein at the concentrations specified in the tables and figures. Incorporation of these antioxidants into the LDL was almost instantaneous, as judged by disappearance of turbidity In contrast, incorporation of the highly lipophilic compounds alpha-TOH, C13OH, and C15OH required preincubation in plasma, with the extent of incorporation being determined by HPLC following ultracentrifugal isolation of the LDL and gel filtration to remove any non-incorporated material (see (17) for alpha-TOH).

We defined the ``effectiveness'' of a co-antioxidant by the relative amount of Ch18:2OOH formed with versus without the added inhibitor in the presence of alpha-TOH but in the absence of CoQH(2), which is itself an effective co-antioxidant for native LDL(19, 26) . The amount of inhibitor added was [inhibitor](0) = [alpha-TOH](0) = 10 µM, and the amounts of Ch18:2OOH formed was determined at a time point corresponding to 20% consumption of alpha-TOH in the ``without'' sample (^3)(see ``Results''). Lipids from the core and surface regions of the LDL particle oxidize in a uniform manner, with LH groups in the phospholipids being consumed at about the same fractional rate (-d[LH]/dt)/[LH]) as LH groups from the lipid core(17) . The rate of conversion of Ch18:2 (which contains 50% of LDL's total LH groups) into Ch18:2OOH thus gauges the total rate of LDL lipid peroxidation (noting that [LOOH] approx 2 times [Ch18:2OOH]; (17) ). An LDL sample supplemented with butylated hydroxytoluene (BHT) at a concentration equimolar to that of endogenous alpha-TOH was included as internal control sample in each set of experiments to check for the effect of potential (inadvertent) variations in the lipoprotein composition and oxidation conditions. The variation in Ch18:2OOH formation was rate BHT/rate = 15 ± 5% (mean value ± S.D. of 13 different experiments with LDL from 13 different donors) for AAPH-induced oxidation and 4.8 ± 2% (n = 9, with 9 different LDL preparations) for AMVN-induced oxidation. This is comparable to the random analytical uncertainties, rate BHT/rate = 16 ± 2% for a triplicate analysis of one sample. At the time points indicated, aliquots of the LDL samples were withdrawn, extracted, and analyzed for antioxidants and Ch18:2OOH(18, 24) .


RESULTS

Peroxyl Radical-induced Oxidation of Native LDL

This study concerns the effect of added radical scavengers on the early phase of lipid peroxidation in LDL exposed to a steady flux of organic peroxyl radicals (ROObullet) thermally generated by a water- or lipid-soluble azo-compound (AAPH or AMVN, respectively).

For AAPH, Rbullet = (CN(2)H(5))(CH(3))(2)Cbullet; for AMVN, Rbullet = (Me(2)CHCH(2))(CH(3))(CN)Cbullet. In the absence of an added co-antioxidant, the ensuing LDL peroxidation exhibits three quite distinct phases in which the rate of LOOH formation: (i) is very slow until the endogenous CoQH(2) present in freshly prepared LDL is consumed, (ii) accelerates markedly (20-40-fold) to a relatively high value immediately after CoQH(2) consumption before decreasing to second minimum just as the last few percent of the alpha-TOH are being depleted, and (iii) accelerates again following consumption of all recognized antioxidants in the LDL (see, e.g., the control in Fig. 1A). Under all but the mildest oxidation conditions, the first phase is relatively brief because the amount of CoQH(2) in LDL is small (i.e. CoQH(2)/alpha-TOH 0.05) even when special precautions are taken to prevent its autoxidation during LDL isolation.^2 Extensive LOOH formation occurs during the phase of alpha-TOH consumption if a low concentration of AAPH or AMVN, or F-10 medium, is used to induce LDL lipid peroxidation(17, 18, 19, 20) . The amount of LOOH formed and the maximal rate of phase ii lipid peroxidation are greater in alpha-TOH-enriched than normal LDL. These and other observations have led us to conclude that alpha-TOH can act as a pro-oxidant for LDL, and that the mechanism of LDL peroxidation is quite different in the presence of alpha-TOH than in its absence (see below).


Figure 1: Inhibition of AAPH-initiated peroxidation in LDL. LDL containing 6.6 µM alpha-TOH, 800 µM Ch18:2, < 0.1 µM CoQH2, 0.4 µM Ch18:2OOH, and 4 mM AAPH was supplemented with the specified amount of antioxidant (e.g. 10 BHT = +10 µM BHT), and its peroxidation at 37 °C was monitored by HPLC(20, 24) . Panel A shows alpha-TOH consumption (squares) and Ch18:2OOH formation (circles) during the inhibited and uninhibited phases of LDL oxidation in the absence (brokenlines) and presence of 2 µM BHT (solidlines). PanelB represents an expanded view of the early Ch18:2OOH formation rates during the inhibited period (see Table 1for structures). The results shown are representative of 3-15 separate experiments.





Antioxidation of CoQH(2)-free LDL

Fig. 1illustrates our method for assessing the efficacy of co-antioxidants. Fig. 1A shows the overall effect of BHT on LDL peroxidation, whereas Fig. 1B gives representative LOOH formation rates for LDL still containing > 60% of its initial endogenous alpha-TOH. Fig. 1B shows that the quinol TBHQ was far superior to the phenols BHT and DBHA for inhibiting LOOH formation in LDL.

The fact that strong co-antioxidants like 3HAA or 2AP (Fig. 2) were often consumed more rapidly than alpha-TOH, and that the peroxidation rate accelerated after their disappearance, meant that apparent stoichiometric factors (n) for some of the compounds could be estimated from the inhibition period, t, before onset of the more rapid tocopherol-mediated peroxidation (TMP). Defining n = R(i)t/[inhibitor], where R(i) is the radical initiation rate measured via the rate of alpha-TOH consumption in native LDL (assuming n = 2.0 for alpha-TOH; (27) ), we calculated n approx 2.8, 2.2, 3.0, and 2.8 for 2AP, 3HAA, BHT, and TBHQ, respectively. The relatively large n values for 2AP and 3HAA are consistent with our earlier observations(28) . n values give a practical index for LDL protection; they assess the relative capacities of co-antioxidants to delay the onset of untrammeled phase ii peroxidation in LDL, which may be quite different to their contribution to the total radical trapping capacity (as defined in (29) ) of the supplemented lipoprotein.


Figure 2: Antioxidation of LDL by the aminophenols 2AP and 3HAA. Conditions were as per Fig. 1with LDL containing 16 µM alpha-TOH, 1800 µM Ch18:2, < 0.1 µM CoQH, 0.2 µM Ch18:2OOH, and 5 mM AAPH. alpha-TOH consumption (squares) and Ch18:2-OOH formation (circles) were monitored in the absence (brokenlines) and presence (solidlines) of 10 µM either 3HAA or 2AP. Effective inhibition times (t) (see text), estimated from the maximum slope tangents to the plots, are indicated by arrows. The results shown are representative of three separate experiments.



From the above and other studies, it became evident that the rate-lowering effect of a co-antioxidant ([Ch18: 2OOH]/[Ch18:2OOH]) was independent of initiator concentration and incubation period, provided that neither alpha-TOH nor the co-antioxidant was substantially depleted. We therefore examined a wider selection of co-antioxidants using carefully matched and internally controlled conditions, described under ``Experimental Procedures.'' Under this protocol, the effectiveness of the antioxidants depended very little on whether water-soluble AAPH or lipid-soluble AMVN was used to initiate LDL oxidation (Table 1). Thus, by segregating compounds according to their reactive functional groups, we were able to describe a number of general and clearcut trends in the data, as enumerated below.

Ascorbate

Ascorbate (AscH) virtually eliminated LOOH formation in the presence of LDL's endogenous alpha-TOH. The oil-soluble 6-palmityl ascorbate (6PAH(2)) was just as effective as AscH in spite of its C16 fatty acid tail, suggesting that the antioxidant location and mobility were not critical for this type of co-antioxidant.

Substituted Small Molecular Weight Phenols

Substituted small molecular phenols were moderately effective inhibitors of LDL peroxidation, whereas the bulkier, more lipophilic estradiol (ED) and Probucol had no appreciable co-antioxidant activity even though they have similar reactivity toward peroxyl radical as BHT (see (27) and below).

Quinols, Catechols, and Aminophenols

Quinols, catechols, and aminophenols used in this study were clearly superior to the monoprotic phenols for inhibiting azo-initiated oxidation of LDL. For example, TBHQ, a commercial antioxidant used in some edible oils, was over 30-fold more effective than BHT, which is often used as an antioxidant for the storage of LDL. Likewise the catecholic estradiol catabolite, 2-hydroxyestradiol (2HED), was a remarkably effective co-antioxidant for LDL compared with its monoprotic precursor ED. Of the aminophenols, the synthetic 2AP was as effective as the structurally similar, natural tryptophan metabolite 3HAA, which has been shown to be an efficient (peroxyl) radical scavenger(30) . The low activity of 3AP compared to 2AP probably arises from the fact that the aroxylradical in former cannot resonance stabilize to the same extent as in 2AP.

Bilirubin

Bilirubin was an effective inhibitor of LDL peroxidation even when present as a complex (HSA-BR) with its natural carrier protein, human serum albumin, which on its own had no effect on LDL oxidation(17, 31) . Free BR and BR-DT, a model for water-soluble, ``conjugated BR,'' were more effective than HSA-BR.

Desferrioxamine

Desferrioxamine at 10 µM did not inhibit LDL oxidation. However, when present at 3 mM (a much higher concentrations than that used in our standard test), 71% inhibition was observed (data not shown). This antioxidant activity was not due to metal-chelation, as comparable high concentrations of EDTA or diethylenetriaminepentaacetic acid had no effect on the rate of azo-initiated LDL lipid peroxidation (data not shown). Desferrioxamine has peroxyl radical scavenging activity in a lipid dispersion (32) and reduction of alpha-TObullet and/or LOObullet by the hydroxamic ``NOH'' moieties of desferrioxamine was probably responsible for its antioxidant activity. NOH-containing compounds can be very efficient LDL antioxidants, as demonstrated clearly by the reduced spin trap TDPHI. Although 10 µM hydroxylamine hydrochloride were almost ineffective (Table 1), low millimolar amounts showed significant inhibitory activity (not shown).

Glutathione

Glutathione (GSH), even at millimolar concentrations, had little effect on AMVN-initiated peroxidation of LDL (data not shown). This is consistent with GSH failing to protect alpha-TOH-containing liposomes against lipid-soluble ROObullet (33) , the very slow reaction of GSH with alpha-TObullet in micelles (k approx 25 M s), the sparing of protein thiols by alpha-TOH in AMVN-initiated red blood cell membrane(34) , and the unfavorable equilibrium for the reaction alpha-TObullet + RSH alpha-TOH + RSbullet(35) .

Superoxide Radical

Superoxide radical (O(2)) appeared not to participate in the AMVN-induced peroxidation of CoQH(2)-depleted LDL, with or without GSH, since the inclusion of superoxide dismutase (up to 10 µM) had no measurable effect on the rates of LOOH formation and alpha-TOH consumption (data not shown).

alpha-TOH Homologues

Various compounds containing the antioxidant active ``head'' group of alpha-TOH but with shorter ``tails'' were tested in AMVN-initiated LDL. Our results (Table 2) showed that the effectiveness of such compounds decreased markedly as progressively longer tails were attached to the head group. Since the length of the tail has little if any effect on the reactivity of the chromanoxyl group (36) in homogeneous solutions, the observed trend in reactivity in LDL needs to be discussed in terms of the special physical properties of the lipoprotein dispersion (see below).



Mechanisms and Kinetics of LDL Antioxidation

According to the generally accepted mechanism of uninhibited lipid peroxidation, LH reacts with O(2) in a radical chain process

The highly active phenolic antioxidant alpha-TOH can interrupt this radical chain by reacting with peroxyl radicals to afford LOOH and the relatively unreactive alpha-TObullet.

The latter eventually combines with a second radical to afford non-radical products (NRP),

thereby destroying two radical species and terminating two potential radical chains. This scheme (Reactions 3-6) adequately explains the products and kinetics of inhibited peroxidation for homogeneous solutions of lipids containing low concentrations of a phenolic or quinolic antioxidant(27) . The effectiveness of a radical scavenger for lowering the rate of LOOH formation via this mechanism is determined solely by its peroxyl radical reactivity.

Most compounds in Table 1are kinetically much weaker radical scavengers than alpha-TOH(27) . The fact that many of these compounds strongly inhibited peroxidation in alpha-TOH-containing LDL is thus contrary to expectations for a peroxyl radical-scavenging mechanism (37) . That is, (a) the most reactive hydrogen donor should be consumed first, and (b) the total radical ``trapping rate'' of the mixture should simply be the sum of the component trapping rates. In contrast, the addition of micromolar amounts of BHT to LDL markedly slowed the rate of both alpha-TOH consumption and LOOH formation (Fig. 1), even though BHT is 200-fold less reactive than alpha-TOH toward peroxyl radicals and alpha-TOH is consumed before BHT in a homogeneous system(38) . The fact that AscH caused such a large reduction in LOOH formation for AMVN-initiated LDL (Table 1) is even more at odds with conventional paradigms because AscH (in the aqueous phase of a lipid dispersion) is a very weak inhibitor of LOOH formation initiated by lipophilic peroxyl radicals in liposomes and micelles(39, 40) .

The problems revealed above are eliminated if it is assumed that the main LOOH forming mechanism in LDL is not a peroxyl radical chain at all but, rather, the radical chain sequence based on , which we have termed TMP (see Fig. 3). In essence, peroxidation is initiated in an LDL ``particle'' (17) when an initiating radical, ROObullet, reacts with alpha-TOH to generate a single alpha-TObullet, which, being too hydrophobic to escape the LDL particle readily, must ``wait around'' for a second ``initiating'' radical (ROObullet) to strike the particle. This ``waiting period'' may be many min under our conditions of low rates of radical generation(20, 21) , thus providing the alpha-TObullet with ample time to generate several molecules of LOOH via and (Fig. 3)(17, 20, 21) .


Figure 3: Tocopherol-mediated peroxidation (TMP) in LDL. A TMP chain is initiated in an LDL particle by ROObullet (e.g. from ) and continues until a second ROObullet reacts with the particle (see text). Rate constants shown are those reported for organic solutions (17, 21) . Note however that lower values, k approx 8 times 10^4M s and k approx 3 times 10^8M s are reported for liposomes(37) .



Effective inhibition of the early stages of LDL oxidation must therefore depend on elimination of alpha-TObullet from the LDL particle before this radical can react with LH. AscH inhibits LOOH formation in LDL because it reacts rapidly with alpha-TObullet to regenerate alpha-TOH(41) , thereby ``exporting'' the radical center into the aqueous medium (21) (Fig. R7).


Figure R7: Reaction 7.



The charge and thermodynamic stability of the ascorbyl radical (Asc ensure that it would terminate in the aqueous phase (e.g.k 3 times 10^5M s) (42) rather than back-reacting with alpha-TOH to re-initiate TMP. Since the antioxidation results from a simple competition between and Fig. R7, the kinetic chain-length (R(p)/R(i)) in LDL initiated with lipophilic peroxyl radicals is given by ,

where [LH] is the concentration of LH groups within the LDL particle (cf.(17) ). and our AMVN data therefore give a lower limit for k(7) under the test conditions; i.e.R(p)/R(i) leq (0.03 pM/s)/(0.15 pM/s) approx 0.2 implies k(7) > 5 times 10^4M s (based on k[LH] 0.1 s; (21) ). In liposomes k = 2 times 10^5 M s has been measured (37) .

In contrast to AscH, data from homogeneous phase studies indicate that most phenolic antioxidant radicals (Xbullet) (whether they are produced directly via reaction with aqueous peroxyl radicals or indirectly via reaction of alpha-TObullet with the antioxidant XH, Fig. R9), would react rapidly with alpha-TOH to (re)generate alpha-TObullet (Fig. R9). XH would therefore have little effect on the LDL oxidation rate unless Xbullet can escape the particle (Fig. R10) before back-reacting with the alpha-TOH ( Fig. R9and Fig. R10).


Figure R9: Reaction 9.




Figure R10: Reaction 10.



Even if radical export does take place, however, it will not lead to a reduction in the peroxidation rate unless the diffusing radical reacts with a peroxidizing particle (Fig. R12) rather than with a non-peroxidizing particle (Fig. R11).


Figure R12: Reaction 12.




Figure R11: Reaction 11.



This ``radical-shuttling'' mechanism neatly explains the trends seen in the Table 1and Table 2since the antioxidant effectiveness of XH depends on (i) its reactivity toward alpha-TObullet, (ii) the rapidity with which Xbullet escapes the particle (ampiphilicity), and (iii) the relative rate at which an ``escaped'' radical, (Xbullet), reacts with non-oxidizing versus oxidizing particles (see ``Appendix'' for a more detailed analysis).


DISCUSSION

This study concerns the inhibition of free radical peroxidation (LOOH formation) in LDL that is devoid of CoQH(2) but which still contains normal amounts of alpha-TOH. Although our list of co-antioxidants is by no means exhaustive, it is clear from the selected examples that the antioxidation of LDL exposed to a slow flux of peroxyl radicals follows definite trends, which can be readily explained by mechanisms that inhibit TMP (Fig. 3) via the elimination of alpha-TObullet (Reactions 7-12). Our experimental findings and kinetic analysis for ``alpha-TObullet-inhibited'' LDL peroxidation suggest that the reactivity, size, and water solubility of the antioxidant and its radical are all relevant for suppressing TMP (see above and ``Appendix''). On this basis, we note the following.

(i) Among the low molecular weight phenolic antioxidants, those with the highest peroxyl radical reactivity tended to be the most active co-antioxidants for LDL. This is because phenols with high peroxyl radical reactivities usually also have high reactivities toward phenoxy radicals, including alpha-TObullet (see (43) ), thereby favoring Fig. R10. Accordingly, the activating paramethoxy group in DBHA makes it both a more active radical scavenger and 2-3-fold more effective than BHT in preventing radical-induced LOOH formation in LDL. Pyrroloquinoline quinone was inactive in our test because it lacks a good hydrogen donor group (weak N-H or O-H bond), whereas the reduced nitroxide TDPHI is extremely active, probably because of the exceptional weakness of its NO-H bond (i.e. from bond strength data, DeltaH(r)(alkyl(2)NO-H + alpha-TObullet) approx -8 kcal/mol).

(ii) Compounds that react rapidly with alpha-TObullet are not expected to inhibit TMP unless the radical formed can escape the LDL particle (Fig. R10) before back-reacting with alpha-TOH (Fig. R9). A rapid rate of radical escape accelerates the ``shuttling'' of radicals between particles and hastens radical termination (via Fig. R11). Accordingly, low molecular weight ampiphilic compounds had greater antioxidant activity for LDL than did bulkier, hydrophobic species of similar type and radical reactivity. The stronger antioxidation observed by the ``short-tailed'' compared to the ``long-tailed'' homologues of alpha-TOH (Table 2) demonstrates that we are dealing with a diffusion effect and not with the replacement of LH-reactive alpha-TObullet with a less LH-reactive antioxidant radical since the reactivity of a homologue radical would not differ appreciably from that of alpha-TObullet. The importance of inter-particle diffusion is clearly manifested in the activities of the alpha-tocopherol homologues, with the effectiveness order HC1OH > C1OH > C5OH > C11OH > C13OH > C15OH alpha-TOH (``C16OH'') (Table 2) being the same order that one would predict for their relative water solubilities, and with the least water-soluble species (C15OH and alpha-TOH) actually having a net pro-oxidant effect on AMVN-initiated LDL oxidation (cf. (17) ). The fact that HC1OH (R = CH(2)OH, Table 2) is significantly more effective than the lower molecular weight C1OH (R = CH(3)) confirms that inter-particle diffusion is the key factor for this chromanol series rather than intra-particle mobility, as has been suggested by Niki and co-workers(44) , especially since hydration of the extra hydroxy group on HC1OH is likely to compound any (intra-particle) ``mobility'' difference between C1OH and HC1OH.

(iii) An antioxidant radical that escapes an LDL particle may either react with alpha-TOH (to re-initiate TMP) or with alpha-TObullet (to destroy two radicals and thereby terminating two TMP chains). The marked superiority of quinols and aminophenols over similarly constituted monoprotic phenols (Table 1) might thus be due to a high selectivity by semiquinone-type radicals for the latter terminating reaction (Fig. R10) since semiquinone radicals are strongly reducing species that can undergo rapid termination reactions of the type ``QHbullet + Ybullet Q + YH''. Any tendency for the semiquinone radical to deprotonate and/or to react with O(2) would probably enhance this effect because the product radicals (Q or O(2)) are likely to be both more water-soluble and more reducing than the parent (QHbullet). In order to distinguish deprotonation from a direct termination selectivity, we examined DBH(2) (the ``dibenzyl dimer'' of C1OH), since its radical (DBH) very rapidly reacts with alpha-TObullet, (^4)but is unlikely to deprotonate or react with oxygen. The observed high effectiveness of DBH(2), relative to say C1OH, confirmed that a rapid cross termination rate enhances anti-TMP activity.

Ascorbic acid, either as water-soluble AscH or as the oil-soluble palmityl ester (6PAH(2)), exhibited remarkable anti-TMP activity. This is consistent with the observation that in human plasma the combination of AscH (10-50 µM) and LDL-associated alpha-TOH fully protect the lipoprotein's lipids from oxidation by lipid-and water-soluble peroxyl radicals(45) . We deduce from our data and from bulk phase studies (46) that AscH prevented lipid peroxidation in LDL by rapidly and irreversibly converting alpha-TObullet into a more inert radical that either reacts with oxygen (to produce O(2)) or undergoes radical-radical termination in the aqueous medium ()(35, 39, 41) . (^5)Although AscH may scavenge some or even most water-soluble ROObullet radicals from AAPH in the aqueous phase before they initiate LDL peroxidation(47) , the contribution of this to its LDL antioxidant activity cannot be deduced from the data because AscH (or 6PAH(2)) eliminated virtually all LOOH formation in AAPH- and AMVN-initiated LDL under the standard test conditions. In any event, our previous results with aqueous peroxyl radical scavenger urate have shown that the scavenging of even a large proportion of initiating radicals in itself is not necessarily sufficient to slow the rate of LOOH formation in LDL (see the ``urate paradox'' in (17) ).

Strong inhibition of LOOH formation in AMVN-initiated LDL by AscH implies that TMP was virtually the only peroxidation pathway operating under our conditions. That is, the fact that AscH eliminated the LOOH formation induced by a lipid-soluble ROObullet indicates the LOOHs must have been formed via direct reaction of alpha-TObullet (which can be eliminated by AscH(2)/AscH; (39) and (41) ) rather than via reactions of the lipid with ROObullet or LOObullet (which are not eliminated by AscH(2)/AscH; (39) ).

Under our test conditions, Probucol neither inhibited LOOH formation nor spared alpha-TOH in the ``TOH-inhibited'' phase of AAPH- or AMVN-initiated peroxidation. This is consistent with recent data by Gotoh et al.(48) , which clearly show that Probucol had almost no effect on the rate of LOOH formation in AAPH or AMVN-induced peroxidation as long as alpha-TOH was present. This is hardly surprising according to the TMP model since Probucol is a relatively weak antioxidant for homogeneous lipids (48) and, perhaps more importantly, it is very insoluble in water (which makes its radical less likely than low molecular weight phenoxyls to diffuse from an LDL particle before back-reacting with alpha-TOH). Probucol does, however, inhibit the post-alpha-TOH phase of oxidation, thereby increasing LDL's resistance toward oxidative modification in a copper oxidation test(48) . Whether this activity explains Probucol's effectiveness as anti-atherosclerotic drug independent of its cholesterol-lowering activity (10) remains unclear (see, e.g., (49) ). We speculate that the 4,4`-bisphenolic oxidation product (50) would have greater anti-TMP activity than the parent Probucol. As the bisphenol is present at relatively high concentration in LDL of Probucol-treated rabbits, it may contribute to the anti-atherogenic activity.

Bilirubin is less effective than alpha-TOH in preventing azo-initiated peroxidation in a lipid solution or liposome dispersion under air(51) . It therefore seems likely that a combination of alpha-TObullet reactivity (31) and perhaps high termination selectivity (see iii on previous page) makes this pigment an effective co-antioxidant for LDL (see (31) for detailed discussion). Since BR is present throughout most of the body and is constantly being replenished via the breakdown of heme, BR could be an important LDL antioxidant in vivo(50, 52) . This is consistent with a recent study suggesting low bilirubin levels to be associated with an increased risk for development of atherosclerosis(53) .

With regard to the LDL oxidation theory for atherosclerosis(1) , the inhibition of LOOH formation in the early phase of LDL peroxidation may well be critical to its overall antioxidation since circulating LDL is virtually LOOH-free(19, 54) , and small amounts of LOOH can cause rapid initiation and damaging oxidation of LDL in the presence of redox catalysts(55) . Moreover, analysis of human atherosclerotic lesion material for its antioxidant content has revealed the presence of comparatively (to human plasma) high concentrations of alpha-TOH, whereas CoQH(2) could not be detected. (^6)Thus, the CoQH(2)-free LDL used for the antioxidation studies here may be biologically relevant, particularly if in vivo oxidation was indeed initiated by very mild flux of free radicals or weak redox oxidant(s).

In conclusion, our TMP model of LDL oxidation and antioxidation presents a novel concept of how lipids in alpha-TOHcontaining LDL can be converted into their hydroperoxides and how small amounts of an effective co-antioxidant would inhibit such oxidation. In light of the oxidation theory of atherosclerosis, our model may therefore provide the basis for the rational design of an efficient LDL antioxidant and, hence, potential anti-atherosclerotic drug.


APPENDIX

The kinetics of the LDL peroxidation inhibited via reactions 9-12 can be analyzed if it is assumed that: (a) diffusion is relatively ``free,'' (b) critical reinitiating and terminating reactions take place within the LDL's lipid compartment, (c) all diffusing radicals are formed by Fig. R10, and (d) the only significant terminating reaction is between X and alpha-TObullet radicals (since neither alpha-TObullet nor LOObullet diffuse readily between particles; (17) ). In this situation the concentration of X is defined by the equilibrium of Fig. R9.

Since the flux of diffusing radicals R = k[X] (Fig. R10), we find

The rate of termination will then depend on the proportion of these diffusing Xbullet, which react with alpha-TObullet (Fig. R12) rather than with alpha-TOH (Fig. R11), i.e.:

Steady-state radical concentrations require R = R(i), so becomes

Combining with R(p)inh = k[LH][alpha-TObullet] (see ), we finally obtain the inhibited rate of lipid peroxidation:

in which

A co-antioxidant effectiveness factor C is thus defined that depends on the forward rate of Fig. R9, the size and water solubility of Xbullet relative to the tendency for Xbullet to back-react with alpha-TOH (Fig. R9), and the rate of Fig. R12relative to Fig. R11(which might be estimated from the corresponding homogeneous phase rate ratio). implies that the degree of inhibition will fall off as the square root of the inhibitor concentration. The predicted ``R(p) bullet ([initiator]/[inhibitor])'' relationship of / has been experimentally observed for BHT (17) and HSA-BR (31) in AAPH-initiated LDL and has now been verified for TBHQ, 3HAA, and 2AP (data not shown).


FOOTNOTES

*
This research was supported by National Health and Medical Research Council of Australia Grants 910284 and 940915 (to R. S.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Research School of Chemistry, Australian National University, Canberra, Australian Capitol Territory 0200, Australia.

To whom correspondence should be addressed: Biochemistry Group, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, New South Wales 2050, Australia. Tel.: 61-2-550-3560; Fax: 61-2-550-3302.

(^1)
The abbreviations used are: LDL, low density lipoprotein; AAPH, 2,2`-azobis(2-amidinopropane hydrochloride); 2AP, 2-aminophenol; AMVN, 2,2`-azobis(2,4-dimethylvaleronitrile); Asc, ascorbyl radical; AscH, ascorbate; BHA, tert-butyl-4-hydroxyanisole; BHT, 2,6-di-tert-butyl-4-methylphenol; BR, bilirubin; BR-DT, bilirubin-ditaurate; C1OH, HC1OH, C5OH, C6OH, C11OH, C13OH, and C15OH, 2-methyl-, 2-hydroxymethyl-, 2-(penten-1-yl)-, 2-hexyl-, 2-undecyl-, 2-tridecyl- and 2-pentadecyl-2,5,7,8-tetramethylchroman-6-ol; Ch18:2, cholesteryl linoleate; Ch18:2OOH, cholesteryl linoleate hydroperoxide(s); CoQH(2), ubiquinol-10; DBH(2), 1,2-bis(2,5,7,8-tetramethyl-6-chromanol-5)ethane; DBHA, 2,6-di-tert-butyl-4-hydroxyanisole; ED, 17beta-estradiol; 3HAA, 3-hydroxyanthranilic acid; HSA-BR, human albumin-bound bilirubin; LH, active bisallylic methylene groups of polyunsaturated fatty acids; LOObullet, lipid peroxyl radical; LOOH, lipid hydroperoxide(s); 6PAH(2), 6-palmityl ascorbate; R(i), rate of radical initiation; R(p) = -d[LH]/dt d[LOOH]/dt, rate of lipid peroxidation; ROObullet, alkyl peroxyl radical; TBHQ, tert-butylhydroquinone; TDPHI, 2,2,5,5-tetramethyl-3-phenyl-4N-oxo-3,4-dehydro-1-hydroxyimidazolin; alpha-TOH, alpha-tocopherol; alpha-TObullet, alpha-tocopheroxyl radical; Xbullet, phenoxy radical; HPLC, high performance liquid chromatography; TMP, tocopherol-mediated peroxidation.

(^2)
Although the LDL isolation method used in this work (namely rapid isolation from heparin-plasma; see ``Experimental Procedures'') yielded higher redox ratios (ubiquinol-10/ubiquinone-10) and lower amounts of LOOH of any other LDL isolation procedure we have tested, exactly the same pattern of post-CoQH(2) peroxidation has been observed with EDTA as the blood anti-coagulant and with LDL prepared by larger scale procedures.

(^3)
Experiments with 3HAA, 2AP, TBHQ, DBHA, and BR indicated that using up to 3-fold shorter or longer incubation times gave the same relative effectiveness values.

(^4)
V. W. Bowry and K. U. Ingold, unpublished observation.

(^5)
The hydrophobic palmityl group in 6PAH(2) makes radical export and termination in the manner described for AscH less likely. The very high antioxidant activity of 6PAH(2) in LDL might thus be due to reaction of its radical with oxygen (cf. LDL-associated 7CoQH(2); (17) and (21) ) as has recently been proposed to explain the high antioxidant activity of 6PAH(2) in lipid micelles(56) .

(^6)
C. Suarna, R. T. Dean, and R. Stocker, unpublished data.


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