Original Contribution

CHARACTERIZATION OF THE ADDUCT FORMED FROM THE REACTIONBETWEEN HOMOCYSTEINE THIOLACTONE AND LOW-DENSITY

LIPOPROTEIN: ANTIOXIDANT IMPLICATIONS

ERIC FERGUSON,* NEIL HOGG,* WILLIAM E. ANTHOLINE,* JOY JOSEPH,*RAVINDER JIT SINGH,* SAMPATH PARTHASARATHY,† and B. KALYANARAMAN *

*Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, WI 53226,†Department of Gynecology & Obstetrics,Emory University School of Medicine, Atlanta, GA 30322

(Received14 August1998;Revised28 September1998;Accepted29 September1998)

Abstract—Homocysteine thiolactone is a cyclic thioester that is implicated in the development of atherosclerosis. Thismolecule will readily acylate primary amines, forming a homocystamide adduct, which contains a primary amine anda thiol. Here, we have characterized and evaluated the antioxidant potential of the homocystamide-low-densitylipoprotein (LDL) adduct, a product of the reaction between homocysteine thiolactone and LDL. Treatment of LDL withhomocysteine thiolactone resulted in a time-dependent increase in LDL-bound thiols that reached approximately 250nmol thiol /mg LDL protein. The thiol groups of the homocystamide-LDL adduct were labeled with the thiol-reactivenitroxide, methanethiosulfonate spin label. Using paramagnetic relaxing agents and the electron spin resonance spinlabeling technique, we determined that the homocystamide adducts were predominately exposed to the aqueous phase.The homocystamide-LDL adduct was resistant to myoglobin- and Cu21-mediated oxidation (with respect to nativeLDL), as measured by the formation of conjugated dienes and thiobarbituric acid reactive substances, and the depletionof vitamin E. This antioxidant effect was due to increased thiol content, as the effect was abolished with N-ethylmaleamide pre-treatment. We conclude that the reaction between homocysteine thiolactone and LDL generates anLDL molecule that is more resistant to oxidative modification than native LDL. The potential relationship between thehomocystamide-LDL adduct and the development of atherosclerosis is discussed. © 1999 Elsevier Science Inc.

Keywords—Low-density lipoprotein, Homocysteine, Homocysteine thiolactone, Homocystamide adduct, Electron spinresonance, Lipid peroxidation, Protein thiols, Antioxidants

INTRODUCTION

The elevation of plasma homocysteine levels (homocys-teinemia) is a well-recognized risk factor for the devel-opment of atherosclerosis [1–3]. The mechanism bywhich homocysteine exerts deleterious effects, however,remains unknown. A plethora of evidence suggests thatoxidation of low-density lipoprotein (LDL) is a key stepin atherogenesis [4–6]. The etiology of vascular diseasedue to homocysteinemia has been attributed to thiol

autoxidation, a process that generates reactive oxygenspecies such as superoxide and hydrogen peroxide. It hasbeen suggested that these damaging species are gener-ated in conditions of homocysteinemia and lead to oxi-dative damage of LDL, hence the development of ath-erosclerosis [7–9].

A number of conflicting reports have appeared in theliterature regarding the action of low-molecular weightthiol compounds during the oxidation of LDL [10–13]. Ithas now become evident that during both cell-free andcell-mediated oxidation of native LDL, low-molecular-weight thiol compounds will inhibit the rate of LDLoxidation in the absence of high levels of lipid hydroper-oxides [14–16]. In addition to low-molecular-weightthiol compounds, we have recently demonstrated thatendogenous thiols (cysteinyl residues) of apolipoproteinB-100 (apo-B) also inhibit LDL oxidation [17]. These

Address correspondence to: B. Kalyanaraman, Biophysics ResearchInstitute, Medical College of Wisconsin, 8701 Watertown Plank Road,Milwaukee, WI 53226-0509; Phone: (414) 456-4035; Fax: (414) 456-6512; E-Mail: balarama@post.its.mcw.edu

1 Rubbo, H., Batthyany, C., and Radi, R. This work was presented asan abstract and a poster presentation at the4th Annual Meeting of theOxygen Society(November, 1997 in San Francisco, CA; Abstract 3–25,104).

Free Radical Biology & Medicine, Vol. 26, Nos. 7/8, pp. 968–977, 1999Copyright © 1999 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/99/$–see front matter

PII S0891-5849(98)00287-1

968

findings strongly suggest that the proatherogenic effectsof homocysteine may be attributed to processes otherthan thiol autoxidation.

It has recently been demonstrated that homocysteinethiolactone is formed in human cells from the enzymaticconversion of homocysteine to the corresponding thio-ester [18]. Homocysteine thiolactone readily reacts withprimary amines by nucleophilic addition, and this reac-tion has been implicated to be physiologically relevant[19,20]. The homocystamide-LDL adduct, the acylationproduct of the reaction between homocysteine thiolac-tone ande-amino groups of apo-B lysyl residues, hasbeen implicated to increase the atherogenicity of LDL[2,20,21] (equation 1):

As shown in equation 1, homocystamide adduct for-mation represents the addition of a thiol at thee-amino

group of the apo-B lysyl residue. Elevated concentrationsof this adduct are thought to reflect increased homocys-teine concentrations. In principle, this could be a diag-nostic marker to detect elevated homocysteine concen-trations in biological systems. We have recentlygenerated a novel polyclonal antibody that is directedagainst the homocystamide-protein adduct [22].

In this report, we have further characterized the struc-tural aspects of the homocystamide-LDL adduct usingthe electron spin resonance (ESR) spin labeling tech-nique. Furthermore, the susceptibility of the homocysta-mide-LDL adduct to oxidative modification has beenevaluated. Contrary to earlier literature data [20,21], thepresent results reveal that homocysteine thiolactone de-creases the susceptibility of LDL to oxidative modifica-tion.

MATERIALS AND METHODS

Materials

Methanethiosulfonate spin label (MTSL) was synthe-sized as described previously [23]. Potassium chromium(III) oxalate trihydrate (CROX) and hydroxylamine hy-drochloride were purchased from Aldrich Chemical Co(Milwaukee, WI). 3,39,5,59-tetramethyl benzidine sub-strate solution (TMB), and 2,4,6-trinitrobenzenesulfonicacid (TNBS) were supplied by Pierce (Rockford, IL).Myoglobin (horse skeletal muscle), homocysteine thio-

lactone hydrochloride, N-acetyl-homocysteine thiolac-tone, Sephadex G-25, thiobarbituric acid, copper (II)sulfate, potassium bromide, diethylenetriaminepentaace-tic acid (DTPA), butylated hydroxytoluene (BHT), eth-ylenediaminetetraacetic acid (EDTA),a-tocopherol,myoglobin, and materials for phosphate-buffered saline[PBS, sodium phosphate (25 mM), sodium chloride (125mM), pH 7.4] were purchased from Sigma Chemical Co(St. Louis, MO). Heptane, methanol, and sulfuric acidwere purchased from Fisher Scientific (Itasca, IL).Chelex-100 and 15 ml disposable filtration columns wereobtained from Bio-Rad (Hercules, CA). 96-well polysty-rene ELISA plates were purchased from Corning (Horse-heads, NY). Ethanol was purchased from QuantumChemical Corp (Tuscola, IL). Bathocuproine was pur-chased from G. Fredrick Smith Chemical Company (Co-lumbus, OH). Nitrogen and argon gasses were suppliedby AIRCO Gas (Murray Hill, IL).

LDL was isolated from human plasma by sequentialultracentrifugation through a potassium bromide densitygradient [24]. LDL protein concentrations were mea-sured by the Lowry assay [25]. Freshly prepared LDLwas stored under argon at 4°C in PBS containing EDTA(1 mM). In one experiment, LDL that was stored underidentical conditions for 3 months was used and is re-ferred to as “aged” LDL. The free base of homocysteinethiolactone (Fig. 1) was prepared from the hydrochloridesalt [22]. Typically, 100–500 mg (;1–5 mmol) of thehydrochloride salt was added to a separating funnel con-taining 2 ml sodium hydroxide (1.4 N) and 3 ml meth-ylene chloride. The solution was shaken vigorously for 1minute, and the bottom organic layer was separated anddried over anhydrous sodium sulfate. This solution wasevaporated under a steady stream of nitrogen until clear,colorless oil was obtained.

Preparation of homocystamide- and N-acetyl-homo-

Fig. 1. Chemical structures. (See text for details.)

969Antioxidant properties of the homocystamide-LDL adduct

cystamide-LDL adducts. Homocystamide- and N-acetyl-homocystamide-LDL adducts were prepared by the ad-dition of LDL (;2–10 mg in PBS containing 100mMDTPA) to freshly extracted homocysteine thiolactonefree base (100–500 mg) or to a freshly prepared solutionof N-acetyl homocysteine thiolactone (in PBS containing100mM DTPA). The mixture was incubated with gentlestirring for the indicated times and passed through aSephadex G-25 column in order to separate the unreactedhomocysteine thiolactone. Protein and thiol concentra-tions were determined following this reaction.

Thiol determinations. Protein thiol groups in LDLsamples were assayed using the DTNB reagent [26]. Anextinction coefficient (e 5 11,000 M21cm21) was deter-mined from a standard curve generated using reducedcysteine and was used for all calculations. Thiol concen-trations are given in terms of nmol per mg LDL protein.

Protein Modification. Apo-B modification was as-sessed by agarose gel electrophoresis using a ParagonLipo-Gel electrophoresis apparatus [27]. LDL (5mg)was loaded onto the gel and subjected to electrophoresisfor 30 min at 150 V. Relative electrophoretic mobility(REM) was calculated as the distance migrated by thesamples and the distance migrated by native LDL.

Primary amino group determinations. The TNBS as-say was used in order to measure primary amino groupconcentrations of apo-B [28]. Results are expressed as %TNBS reactivity in which the TNBS reactivity of nativeLDL is 100%.

Thiobarbituric acid-reactive substances (TBARS)measurement. Aliquots of LDL were collected at theindicated times during Cu21-mediated oxidation, andBHT (500 mM in ethanol) and DTPA (1 mM) wereadded in order to prevent further oxidation. TBARS weremeasured by incubating LDL (;50 mg) with thiobarbi-turic acid (0.5% w/v in H2SO4, 50 mM) for 30 min at100°C [29]. Samples were centrifuged for 5 min, and thedifference in absorbance (A532 nm2A580 nm) was deter-mined. Concentrations of TBARS were calculated usingan extinction coefficient (e 5 150,000 M21cm2) aspreviously described [30].

Vitamin E measurement. The concentrations of vita-min E were determined by reverse phase HPLC as de-scribed previously [31]. Samples were extracted intoheptane, dried under nitrogen, and re-dissolved in meth-anol before injection onto a Partisil 10 ODS-3 reversephase HPLC column. The mobile phase consisted ofmethanol: water (95:5) for 10 min, a linear gradient to100 % methanol over the next 5 min, and an additional 5min of 100 % methanol. Vitamin E was monitored byfluorescence (lex 5 275 nm andlex 5 320 nm). Com-pounds were quantified using a standard curve generatedwith known concentrations of vitamin E.

Conjugated diene measurement. LDL (100mg/ml)

was incubated with myoglobin (0.75mM) in PBS at37°C. Changes in absorbance at 234 nm were monitoredcontinuously by following the formation of conjugateddienes [32].

Lipid hydroperoxide measurement. LOOH concentra-tions of native LDL and of Cu21-oxidized LDL wereassayed by the iodometric method as described previ-ously [33]. Lipid hydroperoxides are given in nmol permg LDL protein.

Copper binding to apo-B. Cu21 binding to apo-B wasdetermined using bathocuproine disulfonate [34,35].Briefly, Cu21 (200 mM) was added to LDL (1 mg/ml).The solution was gently inverted. Apo-B was obtainedusing a CHCl3:CH3OH (2:1) extraction and centrifuga-tion. The upper and lower phases were discarded. Thepellet was sonnicated in 0.9 ml SDS (0.02% in PBS). 50ml bathocuproine disulfonate (20%) and 50ml hydrox-ylamine (0.4%) were added. Following mixing and cen-trifugation, the absorbance at 486 nm was determined.Cu21 concentrations were determined from a standardcurve using CuSO4 (0–100mM) in the presence of ascor-bate (1 mM). Cu21 ion concentrations were calculatedand expressed as a % of Cu21 bound to native LDL.

ESR measurements. ESR spectra were recorded usinga Varian E-109 Century Series spectrometer. Samples forspin-labeling experiments were placed in a quartz flatcell for a TE101 cavity. Copper (II)/LDL complexes werestudied at liquid nitrogen temperature (77 K). The sam-ples were placed in quartz tubes and immediately frozenin liquid nitrogen. Samples were then placed in the ESRcavity, which contained a liquid nitrogen finger dewar.

RESULTS

Kinetics of thiol group generation in the reactionsbetween homocysteine thiolactone versus N-acetyl-ho-mocysteine thiolactone and LDL.

The reaction between homocysteine thiolactone andapo-B lysyl residues was followed by measuring thiolconcentrations. Homocysteine thiolactone free base wasadded to LDL (5 ml at 2.5 mg/mL in PBS containingDTPA 100mM). Figure 2 shows that incubation of LDLwith homocysteine thiolactone resulted in a time-depen-dent increase in LDL-bound thiol content. The reactionwas not followed after 75 min, as the mixture had begunto aggregate and precipitate at that time. If homocysteinethiolactone is removed by gel filtration before the incu-bation time exceeds 75 min, aggregation does not takeplace. Following gel filtration in order to remove anyunbound homocysteine thiolactone, mixtures containingLDL and homocysteine were treated with the thiol-blocking spin label, MTSL (Fig. 1). The addition ofMTSL completely blocked LDL-bound thiol groups,

970 FERGUSONet al.

demonstrating the accessibility of thiol groups to hydro-philic thiol blockers (Fig. 2).

It has been suggested that homocysteine thiolactoneis capable of intermolecular nucleophilic addition re-actions [21]. The N-acetylated derivative of homocys-teine thiolactone, N-acetyl homocysteine thiolactone(Fig. 1), prevents any nucleophilic reactions from oc-curring at the nitrogen atom. N-Acetyl-homocysteinethiolactone (500 mg) was incubated in the presenceand absence of LDL (5 ml at 2.5 mg/ml, DTPA 100mM). At selected time intervals, aliquots were re-moved, and thiol concentrations were immediatelydetermined. Thiol concentrations increased over a 3hour period and reached a maximum of 30169 nmolper mg LDL at 3 h. These data suggest that apo-Blysyl residues had become 83% acylated at 3 h, indi-cated by the increase in thiol concentration (assuming181 lysyl residues per apo-B molecule and a molecularweight of 500 kDa [17,36]). Figure 2 shows that theincrease in thiol content is slower in the reactionbetween N-acetyl-homocysteine thiolactone and LDLthan in the reaction between homocysteine thiolactone

and LDL. It is possible that this difference existsbecause intermolecular nucleophilic addition reactionsoccur with homocysteine thiolactone, but not N-acetylhomocysteine-thiolactone. Therefore, individual lysylresidues of LDL incubated with homocysteine thiolac-tone may contain more than 1 molecule of homocys-teine.

Locations of thiol groups in homocystamide-LDLadducts

ESR spin labeling has been used previously tocharacterize the locations of thiol groups in biologicalsamples [37]. Figure 3A shows the ESR spectrum ofnative LDL in which the 5– 6 endogenous thiol groups(cysteinyl residues) were labeled with MTSL. Thespectrum is composite of two species. The major com-ponent consists of a rotationally restricted nitroxide(denoted●). The minor component consists of a ni-troxide with relatively fast rotational mobility (denot-ed Œ). In Fig. 3B, the water-soluble paramagneticrelaxing agent, CROX (100 mM), was added to thesample in 3A. Addition of CROX broadened out the

Fig. 2. Effect of homocysteine thiolactone, N-acetyl homocysteinethiolactone, and subsequent MTSL-treatment on LDL thiol concentra-tions as measured by the DTNB reagent. LDL (5 ml at 2.5 mg/ml) wasincubated in PBS (pH 7.4, 100mM DTPA) with homocysteine thio-lactone free base freshly extracted from the hydrochloride salt (500 mghomocysteine thiolactone hydrochloride). At the indicated times, 0.75ml aliquots were removed and passed through a Sephadex G-25 col-umn. ●, Thiol concentrations were determined immediately using theDTNB reagent (e 5 11,000 M21 m21). Subsequently, samples wereincubated with MTSL (2.75 mM) overnight at 4°C. Samples werepassed through a Sephadex G-25 column in order to remove unreactedMTSL. O, Thiol concentrations were determined using the DTNBreagent.■, Thiol concentrations in LDL treated with N-acetyl homo-cysteine thiolactone (see text for details). Values represent the mean6SEM.

Fig. 3. ESR spectra of spin-labelled MTSL-modified homocystamide-LDL adduct. (a) Control, MTSL-labeled native LDL. (c) MTSL-la-beled homocystamide-LDL adduct. (b,d) As (a,c) but in the presence ofCROX (100 mM).Œ and● represent the mobilized and immobilizedcomponents of the MTSL-LDL spin adduct, respectively.�; and ■represent the mobilized and immobilized components of the MTSL-homocystamide-LDL spin adduct, respectively. ESR conditions: scanrange, 100 G; field setting, 3368 G; time constant, 0.128 sec; modula-tion amplitude, 1 G; microwave power, 5 mW; gain, 2.53 105 (a andb), 1.63 104 (c and d).

971Antioxidant properties of the homocystamide-LDL adduct

fast tumbling nitroxide (denotedŒ) but did not affectthe spectrum from the rotationally restricted nitroxide(denoted●). This indicates that apo-B thiols lie inhydrophobic environments of LDL. This result is con-sistent with our previous report in which a similar spinlabel, MAL-6, was used to determine the locations ofapo-B thiols [37]. LDL was treated with homocysteinethiolactone for 75 min and subsequently with MTSL(Fig. 3C). In contrast to Fig. 3A, the spectral intensityin Fig. 3C is much higher due to an increased level ofMTSL-labeling. Figure 3C exhibits a narrow line-shape, indicating that the spin labels are rotating morefreely in aqueous solution. This suggests that thispopulation of spin labels is on the surface of LDL. Totest this hypothesis, CROX (100 mM) was added tothe sample in Fig. 3C. As shown in Fig. 3D, the ESRspectrum was broadened by the addition of CROX,indicating that a major fraction of the spin labels wasexposed to CROX in the aqueous phase (Fig. 3C,�).A substantial percentage of spins (20% by doubleintegration), however, is not accessible to the aqueousphase, as shown by the immobilized component (Fig.3D, ■). These results are consistent with a reaction ofhomocysteine thiolactone with two populations ofapo-B lysyl residues, a major portion exposed to anaqueous environment and a minor portion exposed toa hydrophobic environment [38].

Modification of homocysteine thiolactone-treated LDL

In order to compare the net charges of the homo-cysteine thiolactone-treated LDL with native LDL,samples were subjected to agarose gel electrophoresis.The REM of homocysteine thiolactone-treated LDLwas increased for each of the thiolactone-treatedsamples (10 min homocysteine thiolactone-treatedREM 5 1.49; 45 min homocysteine thiolactone-treated REM5 2.53), indicating an increase of netnegative charge. This may be due to deprotonation ofthe thiol groups of homocystamide-LDL adduct,which is likely to occur, as the running buffer of theParagon Lipo-Gel system is pH 8.9 [11]. In these samesamples, primary amino group concentrations weremeasured using the TNBS assay. The % TNBS-reac-tivity was 1006 1.6 for native LDL, 105.16 4.7 for10 min homocysteine thiolactone-treated LDL, and104.66 5.0 for the 45 min homocysteine thiolactone-treated LDL. These values are not significantly differ-ent (p. 0.05, n5 3 for each group). This observationis consistent with the proposed reaction (equation 1) inwhich the primary amino group concentration remainsconstant.

Oxidative modification of the homocystamide-LDLadduct

In order to ascertain whether treatment of LDL withhomocysteine thiolactone caused a significant changein lipid hydroperoxide concentration, we used “aged”LDL, as it contains sufficiently high levels of lipidhydroperoxides that can be detected by the iodometricassay. “Aged” LDL was treated with homocysteinethiolactone or PBS for 10 min. Lipid hydroperoxidelevels in homocysteine thiolactone-treated and un-treated LDL were 22.46 4.0 and 23.96 1.8 nmol/mg,respectively. These values are not significantly differ-ent (P . 0.1). We sought to determine whether thehomocystamide-LDL adduct would be resistant to ox-idative modification with respect to native LDL. LDL(2.5 mg/ml in metal ion chelator-free PBS) was treatedwith homocysteine thiolactone. At 10 and 45 min,aliquots were removed and passed through a SephadexG-25 column. LDL (100mg/ml) was incubated withmyoglobin (0.75mM), a lipid hydroperoxide-depen-dent initiator of LDL oxidation, at 37°C [32]. Theformation of conjugated dienes was monitored contin-uously at 234 nm. As shown in Fig. 4, the lag time formyoglobin-mediated oxidation of the homocystamide-LDL adduct was increased with respect to native LDL.LDL containing higher levels of homocystamide ad-

Fig. 4. Oxidizability of the homocystamide-LDL adduct measured bythe formation of conjugated dienes. LDL (2.5 mg/ml) was incubated inPBS (pH 7.4, metal ion chelator-free) with homocysteine thiolactonefree base freshly extracted from the hydrochloride salt for 0 min, 10min, and 45 min. Aliquots were removed and passed through a Seph-adex G-25 column. Subsequently LDL (100mg/ml) was incubated withmyoglobin (0.75mM). The traces in A–C depict LDL treated withhomocysteine thiolactone for 0, 10, and 45 min, respectively. Theformation of conjugated dienes was monitored continuously at 234 nm.

972 FERGUSONet al.

duct exhibited a markedly increased resistance to ox-idative modification (Fig 4). This demonstrates thatthe homocystamide adduct is capable of inhibiting alipid hydroperoxide-dependent process.

We sought to determine whether the antioxidanteffect of homocysteine thiolactone could be observedusing a different oxidation initiator. LDL (2.5 mg/mlin metal ion chelator-free PBS) was treated with ho-mocysteine thiolactone. At 10 and 45 min, aliquotswere removed and passed through a Sephadex G-25column. An aliquot of the sample treated with homo-cysteine thiolactone for 10 min was incubated over-night at 4°C with slight excess of the thiol-blockingcompound NEM. LDL (100mg/ml) was incubatedwith Cu21(20 mM) at 37°C. At selected times (0 – 4 h),aliquots were removed and the reactions quenchedwith DTPA (1 mM) and BHT (0.5 mM). As shown inFig. 5, the lag time for Cu21-mediated oxidation of thehomocystamide-LDL adduct was increased with re-spect to native LDL. LDL containing higher levels ofhomocystamide adduct exhibited markedly increasedresistance to oxidation (Fig. 5). In contrast to LDLcontaining bound thiols, NEM-treated homocysta-mide-LDL adduct was more susceptible to oxidationthan native LDL. We attribute this observation to theblocking of not only homocysteine thiolactone-de-

rived thiols, but also the endogenous thiols of apo-B.This observation is consistent with our previous re-port, which demonstrates that blocking endogenousthiols (cysteinyl residues) of apo-B increases the sus-ceptibility of LDL to oxidative modification [17].

The susceptibility of the homocystamide-LDL adductto oxidative modification was also assessed by measur-ing the depletion of vitamin E during Cu21-mediatedoxidation. LDL (2.5 mg/ml) was incubated with homo-cysteine thiolactone as described in Fig. 5. At 10 min, themixture was passed through a Sephadex G-25 column,and protein concentrations were measured. LDL (100mg/ml) was incubated with Cu21 (20 mM) at 37°C. Atselected times, aliquots were removed. Reactions werequenched with DTPA (1 mM) and BHT (0.5 mM). Asshown in Fig. 6, the rate of vitamin E depletion wasgreatly inhibited in homocysteine thiolactone-treatedLDL with respect to native LDL. This further supportsthat LDL-bound thiols decrease the susceptibility ofLDL to oxidative modification.

We sought to investigate whether binding of Cu21 toLDL was altered as a result of treatment with homocys-teine thiolactone. The amount of Cu21 bound to LDLwas measured using bathocuproine disulfonate, as de-scribed in Materials and Methods. The Cu21 ion (200mM) was added to solutions of LDL (1 mg/ml) and

Fig. 5. Oxidizability of the homocystamide-LDL adduct measured by the formation of TBARS. LDL (2.5 mg/ml) was incubated in PBS(pH 7.4, metal ion chelator-free) with homocysteine thiolactone free base freshly extracted from the hydrochloride salt for the indicatedtimes:●, 0 min; ■, 10 min;E, 10 min1 subsequent NEM treatment; andŒ, 45 min. Aliquots were removed and passed through aSephadex G-25 column. Subsequently LDL (100mg/ml) was incubated with Cu21 (20 mM). At the indicated times, aliquots wereremoved and the reactions quenched with DTPA (1 mM) and BHT (0.5 mM). TBARS were then measured using an extinctioncoefficient of 150,000 M-1cm-1. Values represent the mean6 SEM.

973Antioxidant properties of the homocystamide-LDL adduct

homocystamide-LDL adduct (1 mg/ml LDL, 15 and 45minute homocysteine thiolactone-treated). Bound Cu21

was 1006 13.0%, 95.26 7.1%, and 88.26 8.7%,respectively (n5 5 for each data point). These valueswere not significantly different (P. 0.05).

In addition, the Cu21 ion was added to concentratedsolutions of native LDL and homocystamide-LDL ad-duct. Figure 7A shows the ESR spectrum of the Cu21

LDL sample at liquid nitrogen temperature. The predom-inant signal (A\ 5 165, g\ 5 2.32) is attributed to a Cu21/LDL complex. The ESR parameters indicate that muchof the Cu21 is bound to LDL, mostly through oxygenligands and possibly one nitrogen ligand. The ESR spec-trum of Cu21 bound to homocystamide-LDL adduct isshown in Fig. 7B (A\ 5 170, g\ 5 2.29). These resultsindicate that Cu21 does not bind to the homocystamide-LDL adduct via a sulfur ligand, as the resulting ESRspectrum would have a vastly different set of ESR pa-rameters (e.g., g\ 5 2.14) [38]. Based on these results, itis likely that homocysteine-derived thiols inhibit the ox-idative modification of LDL by a radical scavengingmechanism rather than Cu21 chelation by LDL-boundhomocysteine thiols.

DISCUSSION

Characterization of the homocystamide-LDL adduct

It has been suggested that the formation of homocys-teine thiolactone is physiologically relevant, as an enzy-matic mechanism has been elucidated for the formationof this cyclic thioester in human cells [18]. Oxidativemodification of LDL has been implicated in the etiology

Fig. 6. Oxidizability of the homocystamide-LDL adduct measuredby the depletion of vitamin E. LDL (2.5 mg/ml) was incubated inPBS (pH 7.4, metal ion chelator-free) with homocysteine thiolac-tone free base freshly extracted from the hydrochloride salt for theindicated times:●, 0 min.; ■, 10 min. Aliquots were removed andpassed through a Sephadex G-25 column. Subsequently LDL (100mg/ml) was incubated with Cu21 (20 mM). At the indicated times,aliquots were removed and the reactions quenched with DTPA (1mM) and BHT (0.5 mM). Samples were extracted using heptane.Vitamin E concentrations were measured by HPLC on a C18 columnusing isocratic conditions: 95% methanol and 5% H2O. Valuesrepresent the mean6 SEM.

Fig. 7. ESR spectra of Cu21 complexes observed at 77 K from incu-bations with LDL. (A) LDL (10 mg/ml in PBS) that had been treatedwith homocysteine thiolactone free base for 10 min in the presence ofCu21 (2 mM). A\ 5 165, g\ 5 2.32. ESR conditions: scan range, 1000G; field set, 2850 G; modulation amplitude, 5 G; scan time, 4 min; timeconstant, 0.128 seconds; temperature, 77 K. (B) LDL (10 mg/ml inPBS) in the presence of Cu21 (2 mM). A\ 5 170, g\ 5 2.29.

974 FERGUSONet al.

of atherosclerosis [4–6]. It has been suggested that homo-cysteine thiolactone would be capable of generating a mod-ified, proatherogenic LDLin vivo [20–21]. We have re-cently demonstrated that the homocystamide-LDL adduct isimmunogenic in rabbits [22]. Here, we describe the physi-cal and chemical properties of the homocystamide-LDLadduct.

The reaction between homocysteine thiolactone andLDL was previously investigated [39]. It was reported thata maximum of approximately 48 thiol groups could bebound to each LDL molecule. This result is in contrast toFig. 2, in which approximately 240 nmol thiol/mg LDL(corresponding to 120 nmol thiol groups per molecule ofLDL) were bound to LDL at 75 min. One explanation forthis discrepancy is that the previous study [39] was carriedout in the absence of a transition metal ion chelator. Thismay have resulted in thiol autoxidation and subsequentoxidation of LDL by superoxide and superoxide-derivedspecies in the presence of transition metal ions. Based onthe levels of LDL-bound thiol groups generated in thereaction between N-acetyl-homocysteine thiolactone andLDL, we conclude that thee-amino groups of apo-B lysylresidues had become 83% acylated after 3 h (assuming 181lysyl residues per apo-B molecule and a molecular weightof 500 kDa) [17,36]. The rate of thiol group appearance (1.3nmol/mg z min) in this reaction, however, is much slowerthan in the reaction between homocysteine thiolactone andLDL (5.9 nmol/mg z min) (Fig. 2). This suggests that thestoichiometry between apo-B lysyl residues and homocys-teine thiolactone is not 1 to 1, as shown in equation 1. It hasbeen reported that homocysteine thiolactone is capable offorming homocysteinyl-homocysteine thiolactone via inter-molecular nucleophilic addition reactions [21] (equation 2):

It is then possible that this addition product couldform under our experimental conditions. Subsequently,homocysteinyl-homocysteine thiolactone could reactwith LDL (equation 3):

This mechanism could explain the disparity in therates of LDL-bound thiol group formation in the reac-tions between LDL and either homocysteine thiolactoneor N-acetyl-homocysteine thiolactone (Fig. 2).

It has previously been reported that there were nodifferences between the oxidizability of native LDL andhomocystamide-LDL adduct [20]. In the previous report,however, the susceptibility to Cu12-mediated LDL oxi-dation was measured by TBARS formation following a5-hour oxidation [20]. As shown in Fig. 5, it is critical toperform a time-course oxidation experiment in order todetect differences in oxidizability between samples. Thelag phase for conjugated dienes and TBARS formationduring myoglobin- and Cu12-mediated oxidation, re-spectively, was increased in the homocystamide-LDLadduct with respect to native LDL (Fig. 5). Vitamin Edepletion occurs as an early event during oxidative mod-ification of LDL [28]. During Cu21-mediated oxidation,the rate of vitamin E depletion is retarded in homocys-teine thiolactone treated LDL with respect to native LDL(Fig 6). These observations are consistent with our pre-vious report on the antioxidant properties of endogenousapo-B thiols [17] (cysteinyl residues) as well as otherreports [14–16] showing the protective effects of thiolson LDL oxidation.

Apo-B contains 5–6 endogenous thiols, which wehave demonstrated to be buried in a hydrophobic regionof LDL [37]. We have previously demonstrated thatthese endogenous thiols inhibit LDL oxidation [17]. Re-cently, however, apo-B thiol radical-derived specieshave been implicated to promote Cu21-mediated LDLoxidation.1 It is well known that LDL oxidation is greatlyaffected by endogenous lipid hydroperoxides, and clearlythe pro- and antioxidant potential of thiols are dictated bythe initial concentration of lipid hydroperoxides [40].The present data indicate that LDL-bound thiols—whether endogenous (cysteinyl residues) or exogenous(homocystamide-LDL adducts)—inhibit the oxidation ofLDL.

Possible role of the homocystamide-LDL adduct inatherogenesis

It is, however, unclear whether or not the antioxidantpotential of the homocystamide-LDL adduct is relevantin vivo. The etiology of homocysteinemia-associated ath-erosclerosis remains elusive. Although elevated levels ofhomocysteine are a definite risk factor for the develop-ment of atherosclerosis, it is not clear whether homocys-teine causes or is a consequence of atherosclerosis. It ispossible that homocystamide-LDL adducts could be an-tiatherogenic by preventing oxidation of LDL. Highplasma concentrations of Lp(a) are well established riskfactors that are associated with the development of ath-

975Antioxidant properties of the homocystamide-LDL adduct

erosclerosis [41–42]. It may be possible that the thiolgroup of the homocystamide-LDL adduct could promotethe association of Lp(a) and LDL, which is known to bemediated by disulfide bond formation [43]. This pro-posed mechanism is a subject of future investigation.

In conclusion, we have demonstrated that the reactionbetween homocysteine thiolactone and LDL results in anincrease in LDL-bound thiol groups. These thiol groupsare exposed to the aqueous phases of LDL and canparticipate in radical scavenging reactions that decreasethe susceptibility of LDL to Cu21-and myoglobin-medi-ated oxidation. We are currently investigating the possi-bility that plasma homocysteine levels can be measuredusing the antibody raised by us [22] and a homocysteinethiolactone-based immunoassay. The role of homocysta-mide-LDL adducts in the development of atherosclerosisis undoubtedly a promising area for future research.

Acknowledgments— The authors thank Dr. Jimmy Feix for his criticalreading of the manuscript and continued interest in this work. Thiswork was supported by NIH Grants HL47250 and RR01008.

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ABBREVIATIONS

LDL—low-density lipoproteinTBARS—thiobarbituric reactive substancesapo-B—apolipoprotein B-100NEM—N-ethylmaleamideEDTA—ethylenediaminetetraacetic acidDTPA—diethylenetriaminepentaacetic acidBHT—butylated hydroxytolueneREM—relative electrophoretic mobilityESR—electron spin resonanceMTSL—methanethiosulfonate spin labelCROX—potassium chromium (III) oxalate trihydrateTMB—3,39,5,59-tetramethyl benzidine substrate solutionTNBS—2,4,6-trinitrobenzenesulfonic acidPBS—phosphate buffered salineELISA—enzyme-linked immunoadsorbant assay

977Antioxidant properties of the homocystamide-LDL adduct