Chem. Res. Toxicol. 1999, 12, 1173-1181
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Lipid Oxidation in Human Low-Density Lipoprotein Induced by Metmyoglobin/H2O2: Involvement of r-Tocopheroxyl and Phosphatidylcholine Alkoxyl Radicals Paul K. Witting,† Celeste A. Willhite,† Michael J. Davies,‡ and Roland Stocker*,† Biochemistry and EPR Groups, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, New South Wales 2050, Australia Received March 19, 1999
Metmyoglobin (metMb) and H2O2 can oxidize low-density lipoprotein (LDL) in vitro, and oxidized LDL may be atherogenic. The role of R-tocopherol (R-TOH) in LDL oxidation by peroxidases such as metMb is unclear. Herein, we show that during metMb/H2O2-induced oxidation of native LDL, R-tocopheroxyl radical (R-TO•) and hydroperoxides and alcohols of cholesteryl esters [CE-O(O)H] and phosphatidylcholine [PC-O(O)H] accumulate concomitantly with R-TOH consumption. The ratio of accumulating CE-O(O)H to PC-O(O)H remains constant as long as R-TOH is present. Accumulation of CE-O(O)H is dependent on, and correlates with, LDL’s R-TOH content, yet does not require preformed lipid hydroperoxides or H2O2. This indicates that in native LDL R-TOH can act as a phase-transfer agent and R-TO• as a chaintransfer agent propagating LDL lipid peroxidation via tocopherol-mediated peroxidation (TMP). After R-TOH depletion, CE-O(O)H continues to accumulate, albeit at a slower rate than in the presence of R-TOH. This second phase of LDL oxidation is accompanied by depletion of PCOOH, a rapid increase in the CE-O(O)H/PC-O(O)H ratio, formation of lipid-derived alkoxyl radicals and phosphatidylcholine hydroxides (PC-OH), and accumulation of a second organic radical, characterized by a broad singlet EPR signal. The latter persists for several hours at 37 °C. We conclude that metMb/H2O2-induced peroxidation of LDL lipids occurs initially via TMP. After R-TOH depletion, cholesteryl esters peroxidize at higher fractional rates than surface phospholipids, and this appears to be mediated at least in part via reactions involving alkoxyl radicals derived from the peroxidatic activity of metMb on PC-OOH.
Introduction Oxidative modification of the lipid and protein components of low-density lipoprotein (LDL)1 are implicated as early events in atherogenesis (1, 2), though the precise mechanism(s) by which LDL becomes oxidized in vivo remains unclear. As a result, interest has focused on the mechanisms of LDL oxidation initiated by a variety of oxidants. Studies in which strongly oxidizing conditions were employed to initiate LDL lipid peroxidation have indicated that R-tocopherol (R-TOH) is an efficient antioxidant for LDL (3). Thus, when LDL encounters initiating species with high frequency (e.g., at Cu2+ to LDL * To whom correspondence should be addressed. Phone: +61 (2) 8595 0237. Fax: +61 (2) 9550 3302. E-mail:
[email protected]. † Biochemistry Group. ‡ EPR Group. 1 Abbreviations: AAPH, 2,2′-azobis(2-amidinopropane); apoB, apolipoprotein B-100; apoB•, apoB-derived radical(s); CE, cholesteryl esters; CE-O(O)H, cholesteryl ester hydroperoxides and alcohols; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTPA, diethylenetriaminepentaacetic acid; EPR, electron paramagnetic resonance; globin•, myoglobin-derived protein radical(s); GSH, reduced glutathione; H2O2, hydrogen peroxide; LDL, low-density lipoprotein; LOO•, lipid peroxyl radicals; LH, bisallylic hydrogen-containing lipid; metMb, metmyoglobin; PC, phosphatidylcholine; PC-OOH, phosphatidylcholine hydroperoxides; PC-OH, phosphatidylcholine alcohols; Ri, rate of initiation of lipid peroxidation; Rp, rate of lipid peroxidation; Rpmax, maximal rate of lipid peroxidation; R-TOH, R-tocopherol; TMP, tocopherol-mediated peroxidation; R-TO•, R-tocopheroxyl radical.
ratios of at least 10-16:1), relatively low amounts of lipid hydroperoxides accumulate in the presence of R-TOH, and upon consumption of R-TOH, lipid peroxidation enters a rapid, “uninhibited” phase (3, 4). Enrichment with vitamin E can increase the resistance of LDL lipids toward such strongly oxidizing conditions (5). Under mild oxidizing conditions, however (e.g., at Cu2+ to LDL ratios of no greater than 3:1 or when LDL encounters a low frequency of initiating hydroxyl or peroxyl radicals), LDL’s R-TOH acts as a pro-oxidant in the initial stages of lipid peroxidation of ubiquinol-10free, isolated LDL (3, 6-10). That is, lipid peroxidation proceeds via a radical chain in the presence of R-TOH, is enhanced by enrichment of LDL with R-TOH, and is markedly suppressed in LDL deficient in R-TOH. Importantly, LDL lipid peroxidation under both strong and weak oxidizing conditions can be explained by tocopherolmediated peroxidation (TMP) (6). In this model, R-tocopheroxyl radical (R-TO•), formed as a result of the oxidant assault, either initiates and propagates LDL lipid peroxidation (mild oxidizing conditions) or participates in radical-radical termination reactions (strong oxidizing conditions) which eliminate the chain-carrying R-TO•. Treatment of LDL with horseradish peroxidase and hydrogen peroxide (HRP/H2O2) results in rapid accumulation of cholesteryl ester hydroperoxides and alcohols [CE-O(O)H] concomitant with consumption of R-TOH and
10.1021/tx9900472 CCC: $18.00 © 1999 American Chemical Society Published on Web 11/03/1999
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Chem. Res. Toxicol., Vol. 12, No. 12, 1999
formation of R-TO• (10). In the absence of active HRP, LDL particles containing R-TO• continue to accumulate CE-O(O)H (10), indicating that, under these oxidizing conditions, R-TO• acts as the lipid peroxidation chaincarrying species. At protein:H2O2 molar ratios of g1:1, reaction of metmyoglobin (metMb) with H2O2 yields ferryl [Fe(IV)oxo] myoglobin and protein radicals (globin•). The latter is capable of oxidizing a variety of biomolecules (11). The globin radical is localized on tyrosine (12, 13) and/or tryptophan residues (14, 15). As myoglobin is released under some pathological situations (15) and H2O2 is produced continuously in vivo (17), metMb/H2O2 may be a physiologically relevant oxidant. MetMb/H2O2 has previously been shown to be capable of oxidizing LDL, although the underlying mechanism of this process remains unclear (18, 19). Both globin• [reacting directly with LDL’s lipids (18)] and ferryl myoglobin [reacting with preformed lipid hydroperoxides (19)] have been proposed to be responsible for the initiation of LDL lipid peroxidation. Hydroxyl radicals do not appear to be involved (18). In light of these conflicting reports about the likely initiating species, we have further studied metMb/H2O2-induced LDL lipid peroxidation by combining biochemical analyses with electron paramagnetic resonance (EPR) spectroscopy.
Experimental Procedures Materials. Phosphate buffer (pH 7.4, 50 mM) was prepared from nanopure water or deuterium oxide (D2O buffer). All reagents that were employed were of the highest purity available. Buffers were stored over Chelex-100 (Bio-Rad) at 4 °C for g24 h, to remove contaminating transition metals. 2,2′-Azobis(2-amidinopropane) (AAPH) was obtained from Polysciences (Warrington, PA). Solutions of metMb (2 mM, Sigma) were prepared using phosphate buffer and stored at 4 °C. R-TOH (96% pure) was obtained as a gift (Henkel Corp.) and prepared as an 18 mM stock solution in DMSO. D2O (99.9% D), 5,5′dithiobis(2-nitrobenzoic acid) (DTNB), diethylenetriaminepentaacetic acid (DTPA), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were obtained from Sigma. DMPO solutions (1 mM in phosphate buffer) were purified by stirring with activated charcoal for 30 min in the dark, filtering the solution, and storing it at -20 °C prior to use. Cetyltrimethylammonium chloride micelles (100 mM) and dispersions of R-TOH in such micelles were prepared as described previously (20). Standards of cholesteryl linoleate hydroperoxides (used as a standard for CE-OOH) and phosphatidylcholine hydroperoxides (PC-OOH) were prepared (21) and stored in ethanol at -20 °C. Phosphatidylcholine alcohols (PC-OH) were produced from PC-OOH by borohydride reduction; standards were quantified by spectroscopy using an 234 ∼29500 M-1 cm-1. Caution: AAPH is toxic and is an irritant; use care in handling this compound to avoid exposure to skin and eyes. Preparation of Native and D2O- and R-TOH-Enriched, -Depleted, and -Replenished LDL. Blood was obtained from nonfasted healthy donors (male and female who were 28-41 years of age), drawn into heparin-containing vacutainers, and LDL was isolated by ultracentrifugation (22). The obtained LDL (∼0.25-0.5 mg of protein/mL) was stored at 4 °C for 16 h before use; this results in the oxidation of the endogenous ubiquinol10 without detectable accumulation of lipid hydroperoxides (detection limit of
