Comparison of low-density lipoprotein modification by myeloperoxidase-derived hypochlorous andhypobromous acids

Free Radical Biology & Medicine, Vol. 31, No. 1, pp. 62–72, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter

PII S0891-5849(01)00552-4

Original Contribution COMPARISON OF LOW-DENSITY LIPOPROTEIN MODIFICATION BY MYELOPEROXIDASE-DERIVED HYPOCHLOROUS AND HYPOBROMOUS ACIDS ANITRA C. CARR,* ERIC A. DECKER,† YOUNGJOON PARK,†

and

BALZ FREI*

*Linus Pauling Institute, Oregon State University, Corvallis, OR, USA; and †Department of Food Science, Chenoweth Laboratory, University of Massachusetts, Amherst, MA, USA (Received 7 December 2000; Accepted 27 March 2001)

Abstract—Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, catalyzes the oxidation of halides to hypohalous acids. At plasma concentrations of halides, hypochlorous acid (HOCl) is the major strong oxidant produced. In contrast, the related enzyme eosinophil peroxidase preferentially generates hypobromous acid (HOBr). Since reagent and MPO-derived HOCl converts low-density lipoprotein (LDL) to a potentially atherogenic form, we investigated the effects of HOBr on LDL modification. Compared to HOCl, HOBr caused 2–3-fold greater oxidation of tryptophan and cysteine residues of the protein moiety (apoB) of LDL and 4-fold greater formation of fatty acid halohydrins from the lipids in LDL. In contrast, HOBr was 2-fold less reactive than HOCl with lysine residues and caused little formation of N-bromamines. Nevertheless, HOBr caused an equivalent increase in the relative electrophoretic mobility of LDL as HOCl, which was not reversed upon subsequent incubation with ascorbate, in contrast to the shift in mobility caused by HOCl. Similar apoB modifications were observed with HOBr generated by MPO/H2O2/ Br⫺. In the presence of equivalent concentrations of Cl⫺ and Br⫺, modifications of LDL by MPO resembled those seen in the presence of Br⫺ alone. Interestingly, even at physiological concentrations of the two halides (100 mM Cl⫺, 100 ␮M Br⫺), MPO utilized a portion of the Br⫺ to oxidize apoB cysteine residues. MPO also utilized the pseudohalide thiocyanate to oxidize apoB cysteine residues. Our data show that even though HOBr has different reactivities than HOCl with apoB, it is able to alter the charge of LDL, converting it into a potentially atherogenic particle. © 2001 Elsevier Science Inc. Keywords—Ascorbate, Hypobromous acid, Hypochlorous acid, Low-density lipoprotein, Myeloperoxidase, Thiocyanate, Free radicals

INTRODUCTION

enhanced recruitment and retention of leukocytes [2], enhanced uptake of the modified LDL by macrophages, resulting in the formation of lipid-laden “foam” cells [2], and cytotoxic and proliferative effects towards endothelial and smooth muscle cells, respectively [3]. All of these effects can potentially contribute to the development and progression of atherosclerotic lesions. Leukocytes and vascular cells contain a number of enzymes capable of generating reactive species that can oxidatively modify LDL [4]. These enzymes include NAD(P)H oxidases, which generate superoxide and, via spontaneous dismutation, hydrogen peroxide [5] and nitric oxide synthase isozymes, which generate nitric oxide that can react with superoxide to form peroxynitrite [6]. In addition, leukocytes release heme peroxidases such as myeloperoxidase (MPO) from neutrophils and mono-

Development of atherosclerotic lesions is a complicated, multi-step process [1]. Although the initiating steps involved are not fully understood, oxidative modification of low-density lipoprotein (LDL) is thought to be an early event in lesion development [2]. LDL trapped within the subendothelial space can be oxidized by leukocytes and vascular cells, including neutrophils, monocytes, macrophages, endothelial cells, and smooth muscle cells [2]. Oxidatively modified LDL has a number of potentially atherogenic effects on these cells, including Address correspondence to: Anitra Carr, Ph.D., Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 973316512, USA; Tel: (541) 737-5085; Fax: (541) 737-5077; E-Mail: [email protected]. 62

Modification of LDL by HOCl and HOBr

cytes [5,7], and the related enzyme eosinophil peroxidase from eosinophils [8]. In the presence of hydrogen peroxide, these peroxidases oxidize halides to their respective hypohalous acids. At plasma concentrations of halides (i.e., 100 –140 mM Cl⫺, 20 –100 ␮M Br⫺, 100 –500 nM I⫺), MPO primarily oxidizes Cl⫺ to the strong oxidant hypochlorous acid (HOCl) [7]. In contrast, eosinophil peroxidase preferentially oxidizes Br⫺ to hypobromous acid (HOBr) [8]. HOCl and HOBr are relatively strong oxidants that can react with a wide variety of biological targets including thiols [9], amines [10,11], and aromatic [12,13] and olefinic [14,15] compounds. The pseudohalide thiocyanate (SCN⫺; 20 –120 ␮M in plasma) is also a substrate for both enzymes, resulting in the formation of hypothiocyanous acid (HOSCN) [16, 17]. HOSCN, in contrast to HOCl and HOBr, is a relatively mild oxidant, which reacts primarily with thiols [17]. LDL modified by reagent and MPO-derived HOCl has a number of potentially atherogenic properties. HOCl reacts primarily with the protein component of LDL (apolipoprotein B-100; apoB), resulting in enhanced uptake of HOCl-modified LDL by macrophages [18,19]. HOCl-modified LDL also causes release of reactive oxygen species, enzymes and cytokines from leukocytes [20 –22], adhesion of leukocytes to endothelial cells, and endothelial dysfunction [20,23]. Furthermore, catalytically active MPO has been detected in human atherosclerotic lesions [24], as have epitopes of HOCl-modified proteins [25], and these were both found to colocalize with monocyte/macrophages, endothelial cells and the extracellular matrix [26]. 3-Chlorotyrosine, a specific product marker of HOCl, has also been detected in lesions [27]. Thus, MPO-derived HOCl appears to play a role in atherogenesis. Because MPO can generate halogenated oxidants such as HOBr and HOSCN in addition to HOCl, we investigated the relative reactivities of these oxidants with LDL. HOBr and HOCl exhibited different reactivities with specific amino acid residues and unsaturated fatty acids. HOSCN showed intermediate reactivity with apoB thiols. Interestingly, the same increase in relative electrophoretic mobility of LDL, indicative of a change in net charge, was observed with both HOBr and HOCl. Our data suggest that HOBr may be disrupting LDL structure to expose more negatively charged residues, rather than modifying positively charged residues, as is the case with HOCl. MATERIALS AND METHODS

Materials Human leukocyte myeloperoxidase (MPO) and HOCl were from Calbiochem (San Diego, CA, USA) and

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Aldrich (Milwaukee, WI, USA), respectively. 7-Fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) and tris(2-carboxyethyl)phosphine (TCEP) were from Molecular Probes (Eugene, OR, USA). All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA). Thionitrobenzoic acid (TNB) was prepared from 5,5⬘-dithiobis(2-nitrobenzoic acid) by alkaline hydrolysis with subsequent neutralization [28]. Phosphate-buffered saline (PBS) was composed of 10 mM sodium phosphate, 140 mM NaCl, pH 7.4. For chloride-free experiments 10 mM sodium phosphate buffer, pH 7.4, was used. All buffers contained 100 ␮M of the metal chelator diethylenetriaminepentaacetic acid (DTPA).

Isolation of LDL Peripheral blood was drawn from healthy volunteers into vacutainer tubes containing EDTA as an anticoagulant. The blood was centrifuged at 1125 ⫻ g for 20 min at 4°C to obtain plasma. LDL was isolated from the plasma by a sequential density ultracentrifugation method [29], modified as described previously [30]. The isolated LDL (1.019 ⬍ d ⬍ 1.063 g/ml fraction) was desalted by two passages through PD-10 gel filtration columns (Pharmacia Biotech, Uppsala, Sweden) using phosphate buffer, pH 7.4. The total protein content was determined using the Lowry Micro Method Kit (Sigma P5656).

Preparation of HOCl and HOBr HOCl was standardized at pH 12 (␧292 ⫽ 350 M⫺1cm⫺1) [31]. Standardized HOCl was freshly diluted in phosphate buffer, pH 7.4; the pKa of HOCl is 7.5 [31], therefore the solution will contain both HOCl and OCl⫺. HOBr was prepared by adding HOCl to a small excess of NaBr in water and standardizing at pH 12 (␧329 ⫽ 332 M⫺1cm⫺1) [32]. Standardized HOBr was freshly diluted in phosphate buffer, pH 7.4; the pKa of HOBr is 8.7 [33], therefore the solution will contain predominantly HOBr.

Treatment of LDL with HOCl and HOBr Freshly prepared HOCl and HOBr (25– 400 ␮M each) were added with rapid, gentle mixing to LDL, which was subsequently incubated for 30 min at 37°C. Ascorbate was used in some experiments and was freshly diluted in PBS containing 100 ␮M DTPA and standardized at 265 nm (␧ ⫽ 15,000 M⫺1cm⫺1) [34]. Ascorbate (200 ␮M) was added to LDL during (coincubation) or after (postincubation, 15 min at 37°C) exposure to HOBr.

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Incubation of LDL with MPO/H2O2/halide MPO (50 nM) was incubated with LDL for 30 min at 37°C in the presence of H2O2 (25– 400 ␮M) and either bromide or chloride, or both together. In some experiments the pseudo-halide thiocyanate (SCN⫺) was also used. H2O2 was freshly diluted in phosphate buffer, pH 7.4, and standardized at 240 nm (␧ ⫽ 43.6 M⫺1cm⫺1) [35], and was added to the LDL samples in aliquots of 25–50 ␮M over the 30 min incubation period. Under these conditions all of the added H2O2 was converted into HOCl as determined by TNB oxidation using 10 mM taurine as a trap [28]. Quantification of apoB modifications Tryptophan residues were measured directly by fluorescence (excitation ⫽ 280 nm, emission ⫽ 335 nm) [18]. Lysine residues were measured following fluorescamine derivatization (excitation ⫽ 390 nm, emission ⫽ 475 nm) [36]. Free cysteine residues were measured following ABD-F derivatization (excitation ⫽ 280 nm, emission ⫽ 335 nm) and total thiols were determined using ABD-F following reduction of disulfides with TCEP, as described previously [30]. N-Haloamines were determined using TNB oxidation (␧412 ⫽ 14,100 M⫺1cm⫺1) [28]. Carbonyls were measured following 2,4-dinitrophenylhydrazine derivatization (␧450 ⫽ 22,000 M⫺1cm⫺1) [37]. The change in the relative electrophoretic mobility (REM) of LDL was determined by agarose gel electrophoresis using a Paragon Lipoprotein Electrophoresis Kit (Beckman Coulter, Fullerton, CA, USA). Preparation of lipid samples Lipids from HOCl- and HOBr-treated LDL (0.5 mg protein/ml) were extracted by the method of Folch [38], following addition of 500 ␮M heptadecanoic acid (C17:0) as an internal standard. Oleic acid chlorohydrin and bromohydrin standards were prepared by a modified method of Maerker [39]. Briefly, cis-9,10-epoxystearic acid, in acetonitrile:isopropanol (1:1, v/v), was treated with a 10-fold molar excess of HCl or HBr, respectively. After mixing for 30 min at ambient temperature, the samples were extracted twice with dichloromethane following addition of an equal volume of water. LDL lipid samples and the fatty acid chlorohydrin and bromohydrin standards were methylated as previously described [40], and the fatty acid methyl esters were subsequently converted into trimethylsilyl derivatives [15]. Measurement of halogenated lipids Gas chromatography (GC) analyses were performed with a Hewlett-Packard HP Model 5890A gas chromato-

graph, equipped with a split/splitless capillary inlet system and a flame ionization detector (FID), and interfaced to a HP 3396 series II Integrator (Hewlett-Packard, Avondale, PA, USA). A EC-1 fused-silica capillary column (Alltech, Deerfield, IL, USA; dimensions of 30 m ⫻ 0.25 mm I.D., and 0.25 ␮m film thickness) was used. The inlet pressure of helium as the carrier gas was 150 kPa. Samples were injected in the splitless injection mode. The oven temperature was held at 120°C for 2 min and subsequently increased at a rate of 30°C/min to 180°C followed by an increase of 5°C/min to 280°C. The injector and detector temperatures were 260°C and 280°C, respectively. Peaks were identified by comparison of retention times with standards, previously characterized using GC-MS [14,15], and were quantified based on the internal standard (C17:0). Statistical analyses Statistical analyses were carried out using ANOVA with Fisher’s post hoc analysis using StatView software (SAS Institute, Cary, NC, USA). Statistical significance was set at p ⬍ .05. RESULTS

Modification of apoB by HOBr and HOCl We [30] and others [18,41] have shown previously that HOCl readily modifies specific amino acid residues of apoB. HOCl-mediated modifications include oxidation of tryptophan, cysteine, and lysine residues, as well as formation of N-chloramines and a concomitant increase in the anodic electrophoretic mobility of LDL due to neutralization of the positive charge of the lysines’ ␧-amino group. Compared to HOCl, equivalent concentrations of HOBr significantly increased oxidation of tryptophan and cysteine residues of LDL (Fig. 1). Only half the concentration of HOBr was required to cause equivalent oxidation of the tryptophan residues as that observed for HOCl (IC50 for HOBr ⫽ 158 ␮M, IC50 for HOCl ⫽ 326 ␮M) (Fig. 1A). Similarly, equivalent oxidation of cysteine residues occurred at 3-fold lower concentrations of HOBr than HOCl (IC50 for HOBr ⫽ 14 ␮M, IC50 for HOCl ⫽ 42 ␮M) (Fig. 1B). In contrast, HOBr was significantly less reactive than HOCl with apoB lysine residues, i.e., about twice as much HOBr was required to cause equivalent modification of lysine residues (Fig. 2A). Similarly, no significant formation of N-bromamines was observed with increasing concentrations of HOBr (Fig. 2A). It is possible that N-bromamines were formed transiently. For example, Hazell et al. [37] showed that a small percentage of N-chloramines break down to form carbonyls. In confir-

Modification of LDL by HOCl and HOBr

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Fig. 1. HOBr- and HOCl-dependent oxidation of amino acid residues. LDL (0.5 mg protein/ml) was incubated for 30 min at 37°C with 12.5– 400 ␮M HOBr (F) or HOCl (E). Oxidation of (a) tryptophan and (b) cysteine residues was determined as described in Materials and Methods. Data represent mean ⫾ SD, n ⫽ 3. HOX ⫽ HOBr or HOCl.

mation of these data, we found that approximately 5% of the added HOCl was accounted for by the formation of carbonyls (data not shown). However, even fewer carbonyls were detected using HOBr (data not shown). Thus, conversion of N-bromamines to carbonyls cannot account for the lack of N-bromamine formation in HOBr-treated LDL. Interestingly, although HOBr, compared to HOCl,

caused significantly less oxidation of apoB lysine residues and very little formation of N-haloamines (Fig. 2A), an identical increase in REM was observed for LDL incubated with HOBr or HOCl (Fig. 2B). Since an increase in REM of LDL is due to either a decrease in positive charge or an increase in negative charge, it is possible that, rather than modifying lysine residues, HOBr modifies the structure of apoB and thus exposes

Fig. 2. HOBr- and HOCl-dependent modifications of apoB. LDL (0.5 mg protein/ml) was incubated for 30 min at 37°C with 12.5– 400 ␮M HOBr (F ) or HOCl (E ƒ). (A) Modification of lysine residues (F E) and increase in N-haloamines (RNHX;  ƒ), and (B) increase in relative electrophoretic mobility (REM) were determined as described in Materials and Methods. Percent N-haloamines was estimated using 356 lysine residues per LDL [60]. Data represent mean ⫾ SD, n ⫽ 3. HOX ⫽ HOBr or HOCl.

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A. C. CARR et al. Table 1. Effects of Ascorbate Co-incubation and Post-incubation on HOBr-dependent Modifications of apoB of LDL

Tryptophan Lysine Cysteine

RNHBr REM

HOBr (␮M)

No ascorbate (%)

0 100 200 0 100 200 0 25 50 100 200 0 100 200 0 100 200

100 ⫾ 0 67 ⫾ 3 37 ⫾ 3 100 ⫾ 0 94 ⫾ 2 85 ⫾ 6 100 ⫾ 0 11 ⫾ 1 6⫾5 7⫾2 12 ⫾ 2 0⫾0 1⫾1 2⫾1 0⫾0 22 ⫾ 6 68 ⫾ 10

Ascorbate co-incubation (%)

Ascorbate post-incubation (%)

90 ⫾ 9* 83 ⫾ 4*

68 ⫾ 5 39 ⫾ 3

98 ⫾ 4 94 ⫾ 1

93 ⫾ 2 92 ⫾ 5

86 ⫾ 3* 68 ⫾ 10* 18 ⫾ 3* 8 ⫾ 1*

14 ⫾ 2 4⫾1 6⫾1 15 ⫾ 1*

0⫾1 0⫾1

1⫾1 2⫾1

0 ⫾ 0* 5 ⫾ 1*

20 ⫾ 3 68 ⫾ 1

LDL (0.5 mg protein/ml) was treated with HOBr (25–200 ␮M) in the absence or presence (co-incubation) of ascorbate (200 ␮M). HOBr-treated LDL was also incubated with ascorbate (200 ␮M, post-incubation). ApoB modifications were determined as described in Materials and Methods. Data represent mean ⫾ SD, n ⫽ 3. * Indicates significant difference from “No ascorbate” values, p ⬍ .05, as determined by ANOVA with Fisher’s post-hoc analysis. RNHBr ⫽ N-bromamines; REM ⫽ relative electrophoretic mobility.

more negatively charged residues. In support of this notion, at concentrations of HOBr ⱖ 200 ␮M, there was an apparent increase in cysteine residues (see Fig. 1B). This finding could be due to disruption of the apoB structure resulting in either enhanced access of the thiolderivatizing reagent to previously buried cysteine residues [42], or a change in the fluorescent properties of the probe due to a change in its microenvironment. Since HOBr is significantly more reactive than HOCl with thiol groups (see Fig. 1B), disulfide residues in apoB [43] were reduced in order to determine whether or not HOBr was also more reactive with disulfides, thus possibly disrupting the tertiary structure of apoB. However, there was no significant difference between the number of apoB disulfides detected in LDL exposed to HOCl or HOBr (data not shown).

with thiols compared with ascorbate. Ascorbate did not reverse the oxidation of tryptophan and cysteine residues induced by either HOBr (Table 1), or HOCl [30]. Because there was only limited modification of lysine residues and practically no formation of N-bromamines with HOBr concentrations up to 200 ␮M (Table 1), no significant protection or reversal of these changes was observed with ascorbate coincubation or postincubation, respectively (Table 1). Interestingly, 200 ␮M ascorbate significantly protected against the HOBr-dependent increase in REM of LDL (Table 1), most likely due to direct scavenging of the oxidant. However, ascorbate could not reverse the increased REM of HOBr-treated LDL (Table 1), in contrast to that of HOCl-treated LDL [30]. These data confirm that the increase in REM induced by HOBr is not due to conversion of lysine residues to N-bromamines.

Effects of ascorbate on HOBr-dependent apoB modifications

Modification of LDL lipids by HOBr and HOCl

Previously, we have shown that ascorbate not only protects against the majority of HOCl-dependent modifications of apoB, but also reverses some of these changes, in particular modification of lysine residues, formation of N-chloramines, and increase in REM of LDL [30]. Similarly, ascorbate significantly protected against modification of tryptophan and cysteine residues by HOBr (Table 1). However, ascorbate was significantly less efficient at protecting the cysteine residues from oxidation by HOBr (Table 1) compared to HOCl [30] suggesting that HOBr is more reactive than HOCl

Although LDL lipids are a minor target for HOCl [37], phospholipid chlorohydrins have been detected in LDL using electrospray ionization mass spectrometry [44]. In the present study, treatment of LDL with HOBr resulted in the formation of significantly higher levels (nearly 4-fold) of fatty acid bromohydrins compared with fatty acid chlorohydrins in HOCl-treated LDL (Fig. 3). These data compare well with those of an earlier study using phosphatidyl ethanolamine lipoposomes [45], in which fatty acid bromohydrins were detected at 3- to 6-fold lower levels of oxidant than fatty acid

Modification of LDL by HOCl and HOBr

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acid bromohydrins and chlorohydrins were also formed with other unsaturated fatty acids [14,15], and possibly also cholesterol [46,47]. Modification of apoB by MPO/H2O2/halide

Fig. 3. HOBr- and HOCl-dependent modification of LDL lipids. LDL (0.5 mg protein/ml) was incubated for 30 min at 37°C with 200 – 800 ␮M HOBr (F) or HOCl (E). LDL lipids were extracted and derivatized for analysis by gas chromatography as described in Materials and Methods. Data represent mean ⫾ SD, n ⫽ 3. HOX ⫽ HOBr or HOCl.

chlorohydrins. Interestingly, the fatty acid halohydrins were detected at physiologically relevant concentrations of the hypohalous acids (i.e., 200 ␮M; Fig. 3) and at levels of oxidant at which apoB was only partially oxidized (see Figs. 1 and 2). Although halohydrin derivatives of only oleic acid were measured in the present study (Fig. 3), it is likely that equivalent ratios of fatty

In the presence of H2O2, MPO converts halides into their respective hypohalous acids [5]. Incubation of LDL with MPO and increasing concentrations of H2O2 (25– 400 ␮M) in the presence of Cl⫺ (100 mM) resulted in almost equivalent oxidation of amino acid residues (Fig. 4), and formation of N-chloramines and increase in REM (Fig. 5), as observed with reagent HOCl (see Figs. 1 and 2). Similarly, incubation of LDL with MPO, H2O2, and Br⫺ (100 mM) resulted in comparable modifications of apoB as caused by reagent HOBr (Figs. 4 and 5 vs. Figs. 1 and 2). Slightly greater modification of lysine and cysteine residues occurred with the MPO/H2O2/halide systems than with the individual oxidants (Figs. 4 and 5 vs. Figs. 1 and 2). This enhanced specificity could be due to association of MPO with the LDL particle resulting in site-directed oxidation of specific residues, as has been proposed previously [48]. Interestingly, when both Cl⫺ and Br⫺ were present together at equivalent concentrations (100 ␮M each), modifications of apoB resembled those seen in the presence of Br⫺ alone (Figs. 4 and 5). These data confirm that MPO oxidizes Br⫺ in preference to Cl⫺ [49]. However, the in vivo concentration of Br⫺ in plasma is approximately 1000-fold lower than that of Cl⫺ [49]. Due to the different extents of oxidation of cysteine

Fig. 4. MPO/H2O2/halide-dependent oxidation of amino acid residues. LDL (0.5 mg protein/ml) was incubated for 30 min at 37°C with MPO (50 nM) and 12.5– 400 ␮M H2O2 in the presence of 100 mM Br⫺ (F), Cl⫺ (E), or the two together (). Oxidation of (A) tryptophan and (B) cysteine residues was determined as described in Materials and Methods. Data represent mean ⫾ SD, n ⫽ 3.

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Fig. 5. MPO/H2O2/halide-dependent modifications of apoB. LDL (0.5 mg protein/ml) was incubated for 30 min at 37°C with MPO (50 nM) and 12.5– 400 ␮M H2O2 in the presence of 100 mM Br⫺ (F 䊐), Cl⫺ (E ■), or the two together ( ƒ). (A) Modification of lysine residues (F E ) and increase in N-haloamines (RNHX; ■ 䊐 ƒ), and (B) increase in relative electrophoretic mobility (REM) were determined as described in Materials and Methods. Percent N-haloamines was estimated using 356 lysine residues per LDL [60]. Data represent mean ⫾ SD, n ⫽ 3.

residues by HOCl and HOBr (see Fig. 4B), it is possible to distinguish between oxidation of thiols by these two oxidants. Incubation of LDL with MPO, H2O2 (25 ␮M) and Cl⫺ (100 mM) in the presence of decreasing concentrations of Br⫺ showed that even at physiological concentrations of Br⫺ (100 ␮M), MPO appeared able to utilize a portion of the Br⫺ to preferentially oxidize cysteine residues (Fig. 6A). However, because nonenzymatic oxidation of Br⫺ by MPO-derived HOCl is possible, the effect of Br⫺ on oxidation of apoB cysteine residues by reagent HOCl was also investigated. Enhanced oxidation of these residues by HOCl was observed in the presence of Br⫺ concentrations ⱖ 1 mM (Fig. 6B). This result suggests that unphysiologically high concentrations of Br⫺ can compete with LDL for HOCl, although the levels required are at least 10-fold higher than the level required to cause enhanced oxidation of cysteine residues by the MPO system (compare Figs. 6B and 6A, respectively). To further confirm that oxidation of apoB cysteine residues by MPO (Fig. 6A) was due to enzymatic, rather than nonenzymatic oxidation of Br⫺ to HOBr, taurine was included in the incubations. Recent work has shown that taurine can inhibit HOCl-mediated, but not HOBrmediated reactions [50]. Coincubation of taurine (10 mM) with LDL had no significant effect on oxidation of apoB cysteine residues by the MPO system (Fig. 6A), but significantly inhibited oxidation of these residues by reagent HOCl (Fig. 6B). Thus, these results confirm that

MPO is directly oxidizing a portion of the added Br⫺ to form HOBr, even in the presence of a vast excess of Cl⫺. Because the pseudo-halide SCN⫺ is also a substrate for MPO [16] and can oxidize thiols [51], the oxidation of cysteine residues in LDL exposed to MPO, H2O2, and SCN⫺ (100 mM) was compared to that seen in the presence of equivalent concentrations of Br⫺ and Cl⫺. At 25 ␮M H2O2, SCN⫺ caused modification of 74 ⫾ 9% cysteine residues, compared with 90 ⫾ 8% for Br⫺ and 29 ⫾ 2% for Cl⫺. Thus, HOSCN is less reactive with apoB thiols than HOBr, but more reactive than HOCl. In the presence of both Cl⫺ (100 mM) and SCN⫺ (100 mM) oxidation of 71 ⫾ 1% of thiols was observed, and in the presence of the physiologically relevant concentration of 100 ␮M SCN⫺ 76 ⫾ 1%, indicating that even at these low concentrations MPO utilizes SCN⫺ in preference to Cl⫺. DISCUSSION

This is the first study to report on the reaction of HOBr with LDL. We found that HOBr, compared to HOCl, caused significantly less modification of apoB lysine residues. Nevertheless, HOBr-modified LDL exhibited significantly increased mobility on agarose gel electrophoresis, indicating a change in the net charge of the LDL particle. Oxidative modification of lysine residues by HOCl has been associated with increased electrophoretic mobility of LDL, as well as enhanced uptake of the HOCl-modified LDL by macrophages [18,19].

Modification of LDL by HOCl and HOBr

Fig. 6. Effect of Br⫺ on MPO- and HOCl-dependent modification of cysteine residues. LDL (0.5 mg protein/ml) was incubated for 30 min at 37°C with (A) MPO (50 nM) and 25 ␮M H2O2 or (B) 25 ␮M HOCl in the presence of Cl⫺ (100 mM) and decreasing concentrations of Br⫺ (10 – 0.05 mM). Taurine (10 mM) was included in some experiments (light gray bars). Oxidation of cysteine residues was determined as described in Materials and Methods. * Indicates significant difference from “0” Br⫺ values, p ⬍ .05, as determined by ANOVA with Fisher’s post-hoc analysis. Data represent mean ⫾ SD, n ⫽ 3.

Thus, it is possible that HOBr, rather than decreasing the positive charge of LDL by modifying apoB lysine residues, is instead exposing more negatively charged residues on LDL, perhaps by disrupting its structure. In support of this notion, we observed an apparent increase in apoB cysteine residues when LDL was exposed to increasing concentrations of HOBr. This finding could be due to enhanced access of the thiol-derivatizing reagent

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to previously buried cysteine residues [42] as a consequence of altered apoB structure, or a change in the fluorescent properties of the probe due to a change in its microenvironment. There are several possible mechanisms for the HOBrdependent increase in the electrophoretic mobility of LDL. We have shown previously that HOBr reacts readily with proteins and lipids, both in isolation and in whole cells [15,45,47]. Modification of unsaturated fatty acids and phospholipids by HOBr results in the formation of ␣,␤-bromohydrin derivatives (R™CH¢CH™R ⫹ HOBr 3 R™CH(OH)™CH(Br)™R) [15,45]. Lipid halohydrins are more bulky and polar than their parent lipids and have been implicated in liposome disruption by MPO/H2O2/halide systems [52]. We have also shown that incorporation of preformed lipid halohydrins into red blood cells causes hemolysis [53] and incorporation into cultured endothelial cells causes enhanced cell permeability [54]. Since reagent and MPO-derived HOBr are more efficient at halogenating unsaturated lipids than the equivalent systems using HOCl (this study and [14, 15,45]), it is possible that the formation of greater amounts of lipid bromohydrins than chlorohydrins is disrupting LDL structure. This could result directly in exposure of more negatively charged apoB residues, or affect apoB folding with subsequent exposure of these residues. HOBr reacts readily with primary amines to form N-bromamine derivatives (RNH2 ⫹ HOBr 3 RNHBr ⫹ H2O) [11]. However, the levels of N-bromamines detected in the presence of other substrates are very low (this study and [8,55]). This finding could be due to lower reactivity of HOBr, compared to HOCl, with amines [55], or to transient formation of N-bromamine intermediates, which subsequently react with other susceptible targets. Although H2O2 added with MPO could reduce both HOBr and N-bromamines [11,55], this mechanism is unlikely to account for the lack of Nbromamine formation in LDL, because these are relatively minor reactions and similar results were observed with reagent HOBr in the absence of H2O2. N-bromamines, like N-chloramines, can react with thiols and thioethers such as methionine residues [45]. Furthermore, we [45] and others [13] have shown that N-bromamines, unlike N-chloramines, can also cause halogenation of unsaturated lipids [45] and aromatic compounds such as tyrosine residues [13]. Thus, either direct modification of LDL lipids or specific apoB residues by HOBr, or indirect modification by HOBr-derived N-bromamine intermediates could result in disruption of apoB structure. It is of interest to note that treatment of red blood cells with HOBr results in 10-fold faster hemolysis than treatment of red blood cells with HOCl [47]. Although lipid

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bromohydrins were detected at 10-fold lower levels of oxidant than chlorohydrins, hemolysis was more closely correlated with protein oxidation, in particular protein cross-linking and clustering of specific proteins within the plasma membrane [47]. HOCl has been shown to cause aggregation of LDL particles via protein rather than lipid oxidation [37]. Although the exact mechanism(s) for protein cross-linking have not yet been elucidated [37,47], HOBr could be causing enhanced crosslinking of LDL. Reagent HOBr caused significantly greater oxidation of apoB cysteine residues than HOCl. Furthermore, in the MPO system containing equivalent concentrations of Br⫺ and Cl⫺, oxidation of thiols resembled that of the system containing Br⫺ alone, suggesting that Br⫺ is the preferred substrate. This finding is expected based on the oxidation potentials (⫺0.93 to ⫺1.07 V vs. ⫺1.08 to ⫺1.36 V) and the second order rate constants (1.1 ⫻ 106 M⫺1s⫺1 vs. 2.5 ⫻ 104 M⫺1s⫺1) for Br⫺ and Cl⫺ with MPO, respectively [49]. At physiological plasma concentrations of the two halides, i.e., at Cl⫺ concentrations approximately 1000-fold higher than Br⫺ concentrations, production of HOCl predominates [7]. However, even with these disparate concentrations of halides, we observed enhanced oxidation of apoB thiols, suggesting that MPO-dependent oxidation of Br⫺ occurs to a certain extent. Others have also observed a mixture of oxidants formed by MPO [11,16] and it should be noted that the halogenating activity of MPO is not saturated at plasma concentrations of Cl⫺ [16]. Although HOBr could be formed by nonenzymatic oxidation of Br⫺ by MPOderived HOCl, this mechanism was discounted in the present study because taurine was able to inhibit HOClmediated, but not HOBr-mediated reactions [50]. The pseudohalide SCN⫺ is a physiologically relevant substrate for both MPO [16] and EPO [17]. Since thiols are a major target of SCN⫺-derived oxidants [17,51], we investigated oxidation of apoB cysteine residues by MPO/H2O2/SCN⫺. This system caused intermediate oxidation of apoB thiols compared with the equivalent systems using Br⫺ or Cl⫺. Interestingly, even in the presence of 1000-fold higher concentrations of Cl⫺, SCN⫺ caused the same degree of thiol oxidation as SCN⫺ alone, suggesting that SCN⫺ is the preferred substrate [49]. Since thiols are the primary target of SCN⫺-derived oxidants, it is perhaps surprising that not more oxidation of this target was observed with MPO/ H2O2/SCN⫺. However, since more of apoB’s cysteine residues are buried than are exposed [42], it is possible that the differences in reactivities between the three halide systems may not only be due to the availability of alternative targets, but may also depend on the pKa of the three oxidants. HOBr, HOCl, and HOSCN have pKas of 8.7, 7.5, and 5.3, respectively [31,33,56]. Thus, the major

forms of each at physiological pH will be HOBr, HOCl/ OCl⫺, and OSCN⫺, respectively; hypohalite anions may be less likely to react with buried thiol groups in LDL. EPO, in contrast to MPO, utilizes Br⫺ in preference to ⫺ Cl , even in the presence of a greater than 1000-fold excess of Cl⫺ [8,57]. Eosinophils, however, are not directly associated with the development and progression of atherosclerosis, in contrast to MPO and MPO-containing leukocytes [1,2]. Nevertheless, formation of MPOderived HOBr may be relevant at sites of inflammation, such as in the artery wall, especially if concentrations of Br⫺ are increased, such as during Br⫺ therapy [58]. Interestingly, Steele and Woody [58] showed a positive correlation between increased Br⫺ levels and increased production of neutrophil-derived reactive oxygen species, as assessed by chemiluminescence. Finally, we have previously shown that the watersoluble antioxidant ascorbate, which is present in atherosclerotic lesions [59], can effectively scavenge HOCl and thus protect LDL from oxidative modification [60]. In the present study, we observed relatively less protection by ascorbate against certain HOBr-induced apoB modifications, in particular thiol oxidation, and reversal of increased REM. Thus, although HOBr may be formed in vivo in lower amounts than HOCl, it may be more damaging to LDL. The relevance of these findings to the initiation and progression of atherosclerosis remains to be investigated. Acknowledgements — This work was supported by grants from the American Heart Association Northwest Affiliate (9920420Z to A.C.) and the U.S. National Institutes of Health (HL-56170 and AT-00066 to B.F.). REFERENCES [1] Ross, R. Atherosclerosis: an inflammatory disease. N. Engl. J. Med. 340:115–126; 1999. [2] Berliner, J. A.; Heinecke, J. W. The role of oxidized lipoproteins in atherogenesis. Free Radic. Biol. Med. 20:707–727; 1996. [3] Chisolm, G. M.; Chai, Y. Regulation of cell growth by oxidized LDL. Free Radic. Biol. Med. 28:1697–1707; 2000. [4] Carr, A. C.; McCall, M. R.; Frei, B. Oxidation of LDL by myeloperoxidase and reactive nitrogen species. Reaction pathways and antioxidant protection. Arterioscler. Thromb. Vasc. Biol. 20:1716 –1723; 2000. [5] Weiss, S. J. Tissue destruction by neutrophils. N. Engl. J. Med. 320:365–376; 1989. [6] Moncada, S.; Higgs, A. The L-arginine-nitric oxide pathway. N. Engl. J. Med. 329:2002–2012; 1993. [7] Kettle, A. J.; Winterbourn, C. C. Myeloperoxidase: a key regulator of neutrophil oxidant production. Redox Rep. 3:3–15; 1997. [8] Weiss, S. J.; Test, S. T.; Eckmann, C. M.; Roos, D.; Regiani, S. Brominating oxidants generated by human eosinophils. Science 234:200 –203; 1986. [9] Winterbourn, C. C. Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to hypochlorite. Biochim. Biophys. Acta 840:204 –210; 1985. [10] Weiss, S. J.; Lampert, M. B.; Test, S. T. Long-lived oxidants generated by human neutrophils: characterization and bioactivity. Science 222:625– 628; 1983.

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ABBREVIATIONS

ABD-F—7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide DTPA— diethylenetriaminepentaacetic acid EPO— eosinophil peroxidase MPO—myeloperoxidase PBS—phosphate-buffered saline (10 mM phosphate, 140 mM NaCl, pH 7.4) TCEP—tris-(2-carboxyethyl)phosphine TNB—thionitrobenzoic acid