A comparison of the kinetics of low-densitylipoprotein oxidation induced by copper or by

-rays: Influence of radiation dose-rate andcopper concentration1

Abdelouahed Khalil and Tamàs Fülöp

Abstract: The oxidation of low-density lipoproteins is the first step in the complex process leading to atherosclerosis.The aim of our study was to compare the kinetics of low density lipoprotein oxidation induced by copper ions or byoxygen free radicals generated by60Co g-rays. The effects of copper concentration and irradiation dose-rate on LDLperoxidation kinetics were also studied. The oxidation of LDL was followed by the measurement of conjugated diene,hydroperoxides, and thiobarbituric acid reactive substance formation as well asa-tocopherol disappearance. In the caseof gamma irradiation, the lag-phase before the onset of lipid peroxidation was inversely correlated to the radiationdose-rate. The radiation chemical rates (v) increased with increasing dose-rate. Copper-induced LDL peroxidation fol-lowed two kinetic patterns: a slow kinetic for copper concentrations between 5–20mM, and a fast kinetic for a copperconcentration of 40mM. The concentration-dependent oxidation kinetics suggest the existence of a saturable copperbinding site on apo-B. When compared withg-rays, copper ions act as drastic and powerful oxidants only at higherconcentrations (≥40 mM).

Key words: LDL, peroxidation, kinetics, copper,g-radiolysis, dose-rate.

Khalil and FülöpRésumé: Il est maintenant bien admit que l’oxydation des lipoprotéines de faible densité constitue une première étaped’un processus très complexe menant à l’athérosclérose. Le but de cette étude était de comparer les cinétiques del’oxydation de lipoprotéines de faible densité induite par les ions cuivrique ou par les radicaux libres de l’oxygèneproduits par les rayonnementsg du 60Co. Également ont été étudiés les effets du débit de dose d’irradiation et de laconcentration en cuivre sur les cinétiques de peroxydation de LDL. L’oxydation a été suivie par la formation desdiènes conjugués, des hydroperoxydes et des SRTBA ainsi que par la disparition de l’a-tocophérol. Dans le cas del’irradiation gamma, la phase de latence, avant qu’il ait formation de produits de peroxydation lipidique, estinversement corrélée avec le débit de dose. Les vitesses de formation de produits d’oxydation et de disparition de lavitamine E [(v (x)] augmentent avec le débit de dose. La peroxydation de LDL induite par les ions cuivriques suitdeux cinétiques différentes : une cinétique lente pour les concentrations en cuivre comprises entre 5 et 20mM, unecinétique rapide pour la concentration en cuivre de 40mM. Cette différence de cinétique, en fonction de la concentra-tion, est en faveur du concept de l’existence d’un site saturable pour la fixation du cuivre au niveau de l’apo-B. Parcomparaison aux rayonnementsg, le cuivre réagit comme un oxydant drastique et très puissant seulement aux fortesconcentration (≥40 mM).

Mots clés: LDL, peroxydation, cinétique, cuivre, radiolyseg, débit de dose.

[Traduit par la Rédaction] 121

Introduction

Low-density lipoprotein (LDL) oxidation is the first stepin the complex process that leads to atherosclerotic plaqueformation (Witztum and Steinberg 1991). Oxidized LDLsare metabolized by macrophages via the scavenger receptor.This pathway is not further down-regulated and leads to the

formation of lipid-laden foam cells, the hallmark of earlyfatty streak formation (Brown and Goldstein 1983).

Oxidation of LDL is induced in vitro by various methods,including: (i) cellular oxidation. Studies in vitro have demon-strated that numerous cell types, including endothelial cells(Steinbrecher et al. 1987), smooth muscle cells (Morel et al.1984; Heinecke et al. 1986), and macrophages/monocytes

Can. J. Physiol. Pharmacol.79: 114–121 (2001) © 2001 NRC Canada

114

DOI: 10.1139/cjpp-79-2-114

Received January 5, 2000. Published on the NRC Research Press Web site on January 25, 2001.

A. Khalil 2 and T. Fülöp. Laboratoire de Bio-Gérontologie, Institut Universitaire de Gériatrie de Sherbrooke and Département deMédecine, Service de Gériatrie, Université de Sherbrooke, Sherbrooke, QC J1H 4C4, Canada.

1This paper has undergone the Journal’s usual peer review process.

2Author for correspondence at Institut Universitaire de Gériatrie de Sherbrooke, 1036 Belvédère sud, Sherbrooke, QC J1H 4C4,Canada (e-mail: akhalil@courrier.usherb.ca).

oxidatively modify LDL (Hiramatsu et al. 1987). (ii ) Chemi-cal oxidation is generally induced by incubation with metalssuch as copper or iron ions. (iii ) Radiolytic oxidation usingionizing radiationg-rays of60Co or 137Cs. The latter methodproduces quantitatively and selectively defined oxygen freeradicals (Bedwell et al. 1989; Bonnefont-Rousselot et al.1992, 1993; Khalil et al. 1998a, 1998b). The use of copperto initiate oxidation in LDL has been extensively reported(reviewed in Esterbauer et al. 1995; Ziouzenkova et al.1996). However, little progress has been made in under-standing the mechanism by which these ions actually lead tothe formation of lipid radicals or in quantifying the amountof radical species produced in the presence of copper thatare responsible for the initiation of lipid peroxidation.

Several assumptions have been advanced to explain themechanism by which copper initiates LDL peroxidation.Thus, copper in the Cu++ form would be reduced to its activeform Cu+ in the presence of traces of LOOH, producingperoxyl radicals (LOO⋅) (Thomas and Jackson 1991). An-other assumption implicates vitamin Ea-tocopherol) in thereduction of Cu++ (Proudfoot et al. 1997; Maiorino et al.1995; Yashida et al. 1994). Under these conditions,a-tocopherol would have a pro-oxidant effect. Finally, the re-duction of Cu++ could be due to the action of SH groups lo-cated on apo-B itself (Patel and Darly-Usmar 1999).

Copper ions are known to be drastic and powerful oxidants(Delattre et al. 1993; Bonnefont-Rousselot et al. 1995) as com-pared with other modes of oxidation, such as hypochlorite,azobis(2-amidinopropane), or⋅OH/O2

⋅− free radical-inducedperoxidation (Zarev et al. 1999; Yan et al. 1997). The cop-per-induced peroxidation is strongly dependent on its con-centration and until now there has been no generalconsensus on the optimum copper concentrations to be usedfor investigating the mechanisms of lipid oxidation.

Our objective was to study the effect of different radiationdose-rates as well as copper concentrations on LDLperoxidation kinetics., We compared the kinetics of LDLperoxidation induced by copper to a system (gammaradiolysis) which produces oxygen free radicals with knownradiation yields. We hoped these results will help to eluci-date the mechanism of LDL oxidation in vivo and the use ofantioxidants in protecting lipoproteins against lipid per-oxidation.

Our results demonstrate that: (i) the radiation dose-rate in-fluences the susceptibility (lag phase) and oxidizability ofLDL during oxidation; (ii ) copper concentration affects LDLperoxidation kinetics at different rates for low (5–20mM)and high copper concentrations (≥40 mM), respectively; and,(iii ) when compared withg-rays, copper ions are drastic andpowerful oxidants only at higher concentrations (≥40 mM).

Materials and methods

ReagentsAcetic acid, sulfuric acid, n-butanol, sodium phosphate,

thiobarbituric acid, methanol, and hexane were purchased fromFisher Scientific (Montréal, Que.), and 1,1,3,3-tetraethoxypropane,D-a-tocopherol, DL-a-tocopherol, and copper sulphate (CuSO4)were obtained from Sigma Chemical Co. (St. Louis, U.S.A.). Dial-ysis bags were purchased from Spectrum Medical Industries Inc.(Tex, U.S.A.).

SubjectsSera were obtained from young normolipemic male subjects af-

ter overnight fasting. They were all apparently healthy, withoutsymptoms or signs of any arterial diseases, as established by acomplete and negative clinical examination and a normal standard12-lead ECG according to WHO criteria (Rose and Blackburn1968). Blood pressure profiles were in the normal range and sub-jects were all non-smokers. Glycaemia, fibrinogen level, lipid pro-file, and coagulation profile were within the normal ranges. Thestudy was approved by the Ethics Committees of the SherbrookeGeriatric University Institute, and all subjects gave written in-formed consent.

Isolation of LDLIsolation of LDL (1.019 < d < 1.063) was performed according

to the method of Sattler et al. (1994), using the Beckman OptimaTLX ultracentrifuge equipped with a TLA 100.4 rotor, in the pres-ence of EDTA (0.4 g/L) as already described (Khalil et al. 1997).After separation, LDL samples were dialyzed overnight at 4°Cwith 10–2 M sodium phosphate buffer (pH 7). For oxidation experi-ments, the dialyzed solutions of LDL were adjusted to a concentra-tion of 100 mg/mL, expressed as total protein concentration, bydilution in the same buffer. Proteins were measured by commercialassay (Pierce method, Rockford, Ill., U.S.A.).

LDL oxidation by gamma radiolysis of waterGamma irradiations were carried out in a60Co Gamma cell 220

(Atomic Energy of Canada Ltd., Mississauga, Ont.). Irradiationswere performed as previously described (Khalil et al. 1996). Inbrief, LDL were irradiated in aerated aqueous solutions containing10–2 M sodium phosphate buffer at pH 7.0. Under these conditions,the main radical species formed are hydroxyl and superoxide anionradicals with respective yields of 2.8 × 10–7 mol·J–1 and 3.4 × 10–7

mol·J–1 (Spinks and Woods 1990). The total radiation doses werefrom 0–400 Gy. The initial dose-rate was 0.12 Gy/s as determinedby Fricke dosimetry (Fricke and Morse 1927). The dose-rate wasthereafter reduced to 0.11, 0.08, and 0.06 Gy/s by depositing leadplates of suitable thickness between the samples and the of60Cosources. In each case, dose-rate was determined by Fricke dosime-try (Fricke and Morse 1927). All LDL solutions received the sameincreased radiation doses and only the irradiation times varied as afunction of the dose-rate.

LDL oxidation by copper ionsLDL samples (100mg/mL) were incubated for indicated periods

at 37°C in 10–2 mM sodium phosphate buffer (pH 7) containingcopper sulphate. The different concentrations of CuSO4 used were5, 10, 20, and 40mM. The oxidation reaction was stopped by theaddition of 20mM butylated hydroxytoluene (BHT) and 100mMEDTA and cooled in an ice bath.

Measurement of conjugated diene, hydroperoxides,thiobarbituric acid reactive substances, and vitamin E

Different parameters were used to monitor the progress of lipidperoxidation of LDL, including conjugated diene, hydroperoxides,and thiobarbituric acid reactive substances (TBARS) formation, aswell as the disappearance of vitamin E. The conjugated diene weremeasured by differential absorbance spectra of irradiated and non-irradiated LDL samples between 200 and 700 nm on a Hitachi U-300spectrophotometer as previously described (Khalil et al. 1996). An in-crease in the differential absorbance at 234 nm may be explained bythe formation of conjugated diene,e234 = 27 000 mol–1·L·cm–1 (Pryorand Castle 1984).

Hydroperoxide formation during LDL oxidation was measuredby spectrophotometry at 365 nm, which corresponds to the conver-sion of iodide to iodine in the presence of peroxides (El Saadani et

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al. 1989). TBARS were determined as an end products of lipidperoxidation by spectrofluorometry (Yagi 1976), but without pre-cipitation by phosphotungstic acid. 1,1,3,3,-Tetraethoxypropanewas used as a standard. Endogenous vitamin E was assayed asa-tocopherol before and after irradiation of LDL samples, by reversephase HPLC, with UV detection at 292 nm coupled with electro-chemical detection, as previously described (De Leenher et al.1978; Khalil et al. 1996). All experiments were performed in tripli-cate and the data are presented as a mean ± standard deviation.

The rates of LDL oxidation were obtained from plots of the lin-ear part of curves of conjugated diene, hydroperoxide, or TBARSformation versus time for LDL oxidation by copper at differentconcentrations or by exposure to oxygen free radicals produced byg-radiolysis of water. Similarly, the rate of vitamin E disappear-ance was calculated from the initial slope of [vitamin E] vs. oxida-tion time (irradiation or copper incubation time). All LDLoxidation rates were expressed in moles of oxidation products(conjugated diene, hydroperoxides, or TBARS) formed or vitaminE disappeared per minute.

Results

Kinetics of LDL oxidation induced by oxygen freeradicals produced by gamma radiolysis of water:influence of the dose-rate

Figure 1 shows conjugated diene formation curves as afunction of irradiation for four different dose-rates. For thedose-rates of 0.12 and 0.11 Gy/s, these curves show a lagphase, followed by propagation and termination phases.

However, for the lowest dose-rates, conjugated diene forma-tion did not reach a termination phase for the dose-rate of0.08 Gy/s and was still in the onset of the propagation phasefor the dose-rate of 0.06 Gy/s. Nevertheless, for all dose-rates, the length of the lag phase was inversely proportionalto the dose-rate and was about 60 min and 10 min for thelowest (0.06 Gy/s) and highest dose-rates (0.12 Gy/s), re-spectively. Lag time was determined as the intersection ofthe initial slope, representing the initiation rate, and thepropagation slope. Lipid peroxidation rates were propor-tional to the radiation dose-rates and the values were be-tween 0.493 and 6.06mM/min for the radiation dose-rates of0.06 and 0.12 Gy/s, respectively (Table 1). At the highest ra-diation dose used, conjugated diene formation reached amaximum, which was also inversely proportional to thedose-rate. For the lowest dose-rate, 0.06 Gy/s, conjugateddiene formation was still in the propagation phase.

Figure 2 shows the decrease in endogenous vitamin E inLDL samples (aerated solutions) as a function of irradiationtime, for the four different gamma dose-rates studied (0.12,0.11, 0.08, and 0.06 Gy/s). The time required for the nearlycomplete disappearance of vitamin E ([vitamin E]≤ 0.02mM) decreased when the radiation dose-rate increased. Theinitial vitamin E (a-tocopherol) rate of disappearance duringoxidation induced by oxygen free radicals (of gammaradiolysis) was determined. The rate of vitamin E disappear-ance increased with the dose rates (Table 1) for approxi-mately the same initial concentration of endogenous vitaminE in LDL samples

TBARS formation (Fig. 3) appeared immediately in LDLwithout a lag time at dose-rates of 0.12 and 0.11 Gy/s. Thisindicates that the formation of TBARS and the disappear-ance of vitamin E were concomitant for these dose-rates.However, for the dose-rates 0.08 and 0.06 Gy/s, TBARS for-mation curves show a lag phase of about 20 and 50 min, re-spectively. Moreover, the TBARS formation rates (vTBARS)determined as the slope of the linear part of the curves, in-creased with increasing dose-rates.

Hydroperoxide (LOOH) formation in LDL during irradia-tion was also determined (data not shown). Similar to thosefor TBARS formation, the hydroperoxide curves show a lagphase only for the lowest dose-rates (0.08 and 0.06 Gy/s).Table 1 shows the hydroperoxide formation rates (vLOOH) asa function of the radiation dose-rate, which shows trendssimilar to those of TBARS formation.

Kinetics of LDL oxidation initiated by copper ions:effect of Cu++ concentrations

LDL oxidation initiated by copper ions was studied at dif-ferent Cu++ : LDL ratios. The curves of the lipid peroxida-tion data were studied for each concentration and theirkinetics were compared to those obtained during LDL oxida-tion induced by oxygen free radicals of waterg-radiolysis.

Conjugated diene formation data obtained during LDL ox-idation induced by copper ions are shown in Fig. 4. The on-set of conjugated diene formation depended on the Cu++

concentration. However, for the highest copper concentration(40 mM), the formation of conjugated dienes increasedsharply after the first minutes of incubation time andpresented an inflexion point. The conjugated diene formation

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116 Can. J. Physiol. Pharmacol. Vol. 79, 2001

Fig. 1. Conjugated diene formation in LDL as a function of irra-diation time at increasing radiation dose-rates. LDL sampleswere exposed to free radicals generated byg-radiolysis of water.The concentration of LDL was 100mg/mL in aerated aqueoussolutions containing 10–2 M sodium phosphate buffer at pH 7.Conjugated diene formation was followed by differential absorp-tion at 234 nm (e234 nm = 27 000 M–1·cm–1), pathlength =0.1 cm. The data are expressed as the means ± standard devia-tion.

rates increased slightly from copper concentrations of 5 to 20mM. However, the rate of conjugated diene formationincreased dramatically (16-fold) when copper concentrationwas increased from 20 to 40mM, (Table 1). At increased in-cubation times, conjugated diene formation reached a pla-teau that was also copper concentration-dependent. Thevalue of this plateau in the presence of 40mM Cu++ wasabout 6-times higher than its value in the presence of copperat concentrations between 5 and 20mM.

A similar relationship was observed fora-tocopherol de-pletion and copper concentrations used to initiate LDL oxida-tion. a-Tocopherol was oxidized at much lower rates in the

presence of copper concentrations between 5 and 20mM, asdetermined by the values of the initial vitamin E disappear-ance (v–vit.E). At a concentration of 40mM, Cu++ causedrapid depletion of vitamin E during the early stages of LDLoxidation with complete disappearance after 3 h of incuba-tion.

Hydroperoxide formation data were similar to those ob-tained for conjugated diene formation under the same

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Khalil and Fülöp 117

Oxidation productformationa

Gamma ray-induced peroxidation as a function ofdose rate (Gy/s)

Copper-induced peroxidation as a function ofcopper concentration (mM) product formation

0.06 0.08 0.11 0.12 5 10 20 40vDiene 0.493 1.58 2.11 6.06 0.058 0.066 0.076 1.23

vLOOH 0.016 0.087 0.166 0.424 0.075 0.075 0.12 3.80

v–vit.E 0.065 0.113 0.207 0.36 0.011 0.011 0.0208 0.64

vTBARS 0.019 0.027 0.03 0.09 nab na na 2.71

vTBARS : 0.29 0.23 0.144 0.25 na na na 4.23

v–vit.E

Note: LDL (100 mg/mL) were oxidized byg-rays or by incubation with copper in aerated aqueous solutions containing 10–2 M sodium phosphatebuffer at pH 7. For the oxidation product formation (conjugated diene CD, hydroperoxides, LOOH, and TBARS) anda-tocopherol depletion, initialoxidation yield (G) was determined as the initial slopes of the curves and as the slopes of the linear part of the curves, and expressed as the formation ofCD, LOOH, or TBARS, or ina-tocopherol decrease, in moles per oxidation time (min).

aRadiolytic rates expressed inmM?min–1.bNot available.

Table 1. Effect of radiation dose-rate and copper concentration on the oxidation products formation and vitamin E depletion yields.

Fig. 2. Endogenousa-tocopherol depletion during LDL oxidationas a function of irradiation time at increasing radiation dose-rates. LDL samples were exposed to free radicals generated byg-radiolysis of water. The concentration of LDL was 100mg/mLin aerated aqueous solutions containing 10–2 M sodium phos-phate buffer at pH 7. The data are expressed as the means ±standard deviation.

Fig. 3. TBARS formation in LDL, exposed to free radicals pro-duced byg-radiolysis of water, as a function of irradiation timeat increasing radiation dose-rates. LDL samples were exposed tofree radicals generated byg-radiolysis of water. The concentra-tion of LDL was 100mg/mL in aerated aqueous solutions con-taining 10–2 M sodium phosphate buffer at pH 7. TBARSconcentration were calculated as malondialdehyde (MDA) equiv-alents using the MDA standard curve. MDA was generated bythe hydrolysis of 1,1,3-tetraethoxypropane. The data are ex-pressed as the means ± standard deviation.

conditions (results not shown). The hydroperoxide formationrate was much greater (30-fold) in the presence of 40mMcopper as compared with 20mM copper.

TBARS formation was also determined in LDL samplesincubated with copper at different concentrations. TBARSwere formed in LDL only in tmuhe presence of copper at aconcentration 40mM.

Comparison of -radiolysis and copper ions inducedLDL peroxidation

The vitamin E disappearance rate was 10-times lower forcopper concentrations from 5 to 20mM (Fig. 5) as comparedwith results obtained with gamma radiolysis induced oxida-tion (radiation dose-rates between 0.06 and 0.12 Gy/s). Inthe presence of 40mM Cu++, the vitamin E disappearancerate was 2-times higher than that obtained using gammaradiolysis-induced oxidation at a dose-rate of 0.12 Gy/s.

TBARS formation was not detected in LDL when lipidperoxidation was initiated by copper concentrations rangingbetween 5 and 20mM. However, in the presence of a copperconcentration of 40mM, the TBARS formation rate was 30-times greater (vTBARS = 2.71 mM/min] than that obtainedwith gamma radiolysis-induced LDL oxidation at dose-rateof 0.12 Gy/s (0.09mM/min). The value of the maximum ofTBARS formed was about 3-times higher than that obtainedwith ⋅OH and O2

⋅ −oxygen free radical- induced LDL

peroxidation. Table 1 reports the main kinetic values for thetwo oxidation process.

The conjugated diene formation rates obtained usinggamma radiolysis-induced oxidation were very fast as withthose obtained in the presence of copper ions. The value ofthe maximum of conjugated diene formed at the plateau, inthe presence of copper concentrations ranging between 5and 20mM, was very low as compared with those obtainedusing gamma radiation at different dose-rates. However, themaximum value obtained in the presence of 40mM copperis about 2-times higher than its value obtained with a dose-rate of 0.12 Gy/s.

Similar to conjugated diene formation, the amount ofhydroperoxides formed in LDL in the presence of copper atconcentrations of 5, 10, and 20mM was very low as com-pared with the amount obtained using gamma radiation atdifferent dose-rates. The quantity of hydroperoxides formedin the presence of copper at a concentration 40mM was sim-ilar to that found with gamma radiation at a dose-rate of0.12 Gy/s.

Discussion

Our objective was to compare the kinetics of LDL oxi-dation induced by oxygen free radicals of gamma radiolysisand by copper ions. We also investigated the effect of

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Fig. 4. Conjugated diene formation in LDL, oxidized by copperions, as a function of incubation time at increasing copper con-centrations. LDL samples were incubated with copper and reac-tions were stopped by a cooling solution in the presence ofEDTA (100 mM) and BHT (20mM). The concentration of LDLwas 100mg/mL in aerated aqueous solutions containing 10–2 Msodium phosphate buffer at pH 7. Conjugated diene formationwas followed by differential absorption at 234 nm (e234 nm =27 000 M–1·cm–1), pathlength = 0.1 cm. The data are expressedas the means ± standard deviation.

Fig. 5. Endogenous vitamin E disappearance during LDL (mea-sured by the concentration of vitamin E in LDL) as a function ofincubation time and for increasing copper concentrations. LDLsamples were incubated with copper and reactions were stoppedby a cooling solution in the presence of EDTA (100mM) andBHT (20 mM). The concentration of LDL was 100mg/mL inaerated aqueous solutions containing 10–2 M sodium phosphatebuffer at pH 7. The data are expressed as the means ± standarddeviation.

radiation dose-rate and copper concentration on LDL oxida-tion kinetics.

For g-radiolysis-induced LDL oxidation, two radicals (⋅OHand O2

⋅− ) were selected that have known radiation yields.The characteristics of the lipid peroxidation curves were de-pendent on the radiation dose-rate. There was a dose-rate de-pendent lag period before the onset of rapid peroxidation.This lag phase was inversely correlated to the radiation dose-rate. In other words, the susceptibility of LDL to oxidationinduced by gamma radiolysis was dependent on the radiationdose-rate and was greater at the higher radiation-dose rates,as determined by the decrease in the lag phase. Esterbauer etal. (1997) demonstrated that vitamin E is responsible forabout 30% of the length of the lag phase. Indeed, for thesame initial content of vitamin E, we noted an importantvariation in the protection of LDL as a function of the dose-rate. Thus, vitamin E seems to be more efficient at low oxi-dative stress.

As we have already proposed for HDL (Bonnefont-Rousselot et al. 1997), and using our experimental conditions,we suggest the following general scheme of the kinetics ofthe action of⋅OH andO2

⋅− free radicals:

[1] ⋅OH + Vit. E →Vit. E⋅ → molecular oxidationproduct(s) from vit. E

[2] ⋅OH + LDL →R⋅→….

[3] O2⋅− + LDL →R⋅ + H2O2 →….

[4] R⋅ + O2 →RO2⋅

[5] RO2⋅ + Vit. E →Vit. E⋅ + ROOH

[6] RO2⋅ + LDL →free radicals→….

[7] Vit. E⋅ + LDL →free radicals→….

[8] O2⋅− + H+

W HO2⋅

Reactions 1 and 2 are due to the action of⋅OH free radi-cals on vitamin E and LDL, respectively. Reaction 2 is infact a sum of several reactions representing the action of⋅OH radicals on lipids, proteins, and other antioxidants thanvitamin E contained in LDL particles. Indeed,⋅OH free radi-cals are known to be very reactive and non-selective (Buxtonet al. 1988). Reaction 3 represents the initiation of lipidperoxidation induced byO2

⋅− free radicals and reaction 4 de-scribes the formation of peroxyl radicals. Reaction 5 illus-trates the action of vitamin E as a chain breakingantioxidant. Reaction 6 shows the propagation phase due tothe action of peroxyl radicals. Reaction 7 represents the pro-oxidant effect of vitamin E. This phenomenon, calledtocopherol mediated peroxidation (TMP), is very weak ornon-existent under our conditions. Indeed, Bowry andStocker (1993) demonstrated that the pro-oxidant effect ofvitamin E takes place only at very low oxidative stress (1radical produced every 17 min) which was not the case inthis work. At the high radiation dose-rates (0.12 and 0.11Gy/s) vitamin E disappearance was simultaneous to TBARSformation. However, at the low radiation dose-rates of 0.08and 0.06 Gy/s, the onset of the TBARS formation presents a

lag phase of 25 and 50 min, respectively, which corre-sponded to the nearly complete disappearance of vitamin E.Thus, from the different pathways of vitamin E oxidationrepresented in the scheme above, reaction 5 seems to be-come more favorable at the expense of reaction 1 at low ra-diation dose-rates. When the radiation dose-rate decreases,the probability that⋅OH free radicals react with vitamin Edecreases and hencev–vit.E also diminishes. Thus, our resultssuggest that at low radiation dose-rates vitamin E disappear-ance is essentially due to its natural role as a chain breakingantioxidant. In contrast, at high radiation dose-rates most ofthe vitamin E disappears by direct action of the⋅OH radicalsas schematically represented by reaction 1.

The kinetics of the copper-induced oxidation curve aredifferent from those obtained using gamma radiolysis in therange of copper concentrations studied (5 to 40mM). LDLoxidation in the presence of Cu++ cations seemed to followtwo different kinetics. (i) A slow kinetic of oxidation oc-curred at copper concentrations between 5 and 20mM, char-acterized by low conjugated diene and TBARS formationrates as well as a low rate of vitamin E disappearance. (ii ) Afast kinetic of LDL oxidation occurred at a concentration of40 mM, characterized by high conjugated and TBARS for-mation rates and also by rapid vitamin E decreases.

In agreement with the hypothesis of the existence of Cu++-binding sites on apo-B (Gieseg and Esterbauer 1994), our re-sults are in favour of the existence of a saturation thresholdfor these ions. At low concentrations, copper ions would bebonded to high affinity sites on apo-B. This Cu++-proteincomplex may form a protein-associated radical following thereduction of Cu++ (Kuzuya et al. 1992). At high concentra-tions of copper that exceedthe saturation threshold, Cu++

ions are reduced by hydroperoxides and thus lipidperoxidation rates increase.

Assuming that each conjugated diene formed is the resultof a direct radicals attack (Ziouzenkova et al. 1998), wecould estimate the flux of free radicals that strike each LDLparticle per minute and lead to conjugated diene formation.In the case of oxidation induced by gamma rays, the num-ber of free radical attacks on each LDL particle is 2.59hits/min for the lowest dose-rate (0.06 Gy/s) and 31.8hits/min for the highest dose-rate (0.12 Gy/s). In the caseof LDL oxidation induced by copper, this parameter variedbetween 0.30 hits/min for a copper concentration of 5mMto 6.47 hits/min for a concentration of 40mM. These val-ues are 5- to 9-fold lower than values obtained during oxi-dation induced by water gamma radiolysis. Thus, using ourconditions, gamma radiation produced a higher oxidativestress as compared with copper ions. However, if we com-pare the LDL oxidation curves at the termination phase forgamma radiolysis- and copper-induced LDL oxidation, thefinal concentration of conjugated diene was 2-fold higherin the presence of copper ions. This discrepancy probablyresults from the fact that⋅OH and O2

⋅ − free radicals pro-duced by gamma radiolysis action are limited to the surfaceof LDL particles. Consequently, targets would be less ac-cessible as compared to peroxidation induced by copper.These results agree with those obtained by Zarev et al.(1999).

Our data support the contention that under our conditionsthe intensity of the produced stress remains much higher

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120 Can. J. Physiol. Pharmacol. Vol. 79, 2001

than that proposed by Bowry and Stocker (1993) at whichvitamin E could have a pro-oxidant effect.

According to Sato et al. (1990), the length (or efficiency)of the peroxidation chain could be evaluated by the ratiovTBARS : vvit.E. During the oxidation initiated by oxygen freeradicals produced by gamma radiolysis, the value of this ra-tio is on average of 0.22 for the various dose-rates used.This means that for each peroxyl radical (RO2

⋅ ) reacting withvitamin E, 0.22 molecules will result in TBARS formation.For copper concentrations of 5 to 20mM, vitamin E wasable to completely inhibit TBARS formation. However, inthe presence of copper (40mM), this value was approxi-mately 20-fold higher (4.23) than that obtained with gammaradiolysis. A longer peroxidation chain length consistentlycharacterized the kinetic mechanism for the oxidation initi-ated by copper ions at high copper concentrations as com-pared withs oxidation induced by gamma radiolysis ([Cu++] =40 mM; dose-rate for gamma radiolysis: 0.06, 0.08, 0.116,and 0.12 Gy/s). Noted that in the case of the oxidation ofLDL induced by oxygen free radicals of gamma radiolysis,vitamin E disappearance is due to the simultaneous action ofRO2

⋅ and ⋅OH free radicals. Thus, the value of 0.22 for theratio vTBARS : v–vit.E would be the lower limit of the lipidperoxidation chain.

Oxidation yields for LDL peroxicdation increased with in-creasing dose-rates. These results are contradictory to thoseobtained by Hicks and Gebicki (1993), who examined withthe effect of the dose-rate on linoleic acid membranes. How-ever, LDLs are complexes of various lipids, proteins, and an-tioxidants and the study of their oxidation cannot becompared with a simple membrane or micellar system.

In summary, our results show the susceptibility of LDL tooxidation and their oxidizability increase with increasingdose-rates as well as the existence of two different kineticsfor LDL oxidation induced by copper ions. Copper ions aredrastic and powerful oxidants only at higher concentrations(≥40 mM). Further studies are needed to elucidate the ef-fects of the dose-rate on the LDL peroxidation in the pres-ence of other oxygen free radicals (O2

⋅− , HO2⋅ , and RO2

⋅ )and the effects of very low concentrations of copper ions(≤1 mM).

Acknowledgements

This work was supported in part by a grant from theNorth Atlantic Treaty Organization (NATO) (ref. no. CRG970361) and grant-in-aid from the Centre de Recherche enGérontologie et Gériatrie. This support is gratefully ac-knowledged. T.F. and A.K. are Senior and Junior 1 fellowsrespectively of Fonds de la Recherche en Santé du Quebec(FRSQ).

References

Bedwell, S., Dean, R.T., and Jessup, W. 1989. The action of de-fined oxygen-centred free radicals on human low-density lipo-protein. Biochem. J.262: 707–712.

Bonnefont-Rousselot. D., Gardes-Albert, M., Lepage, S., Delattre,J., and Ferradini, C. 1992. Effect of pH on low-density lipopro-tein oxidation byO2

⋅− / HO2⋅ free radicals produced by gamma

radiolysis. Radiat. Res.132: 228–236.Bonnefont-Rousselot, D., Gardes-Albert, M., Delattre, J., and

Ferradini, C. 1993. Oxidation of low density lipoproteins by⋅OH, and⋅OH/O2⋅− free radicals produced by gamma radiolysis.

Radiat. Res.134: 271–282.Bonnefont-Rousselot, D., Motta, C., Khalil, A., Sola, R., La Ville,

A.E., Delattre, J., and Gardes-Albert, M. 1995. Physicochemicalchanges in human high-density lipoproteins (HDL) oxidized bygamma radiolysis generated oxyradicals. Effect on their choles-terol effluxing capacity. Biochim. Biophys. Acta,1255: 23–30.

Bonnefont-Rousselot, D., Khalil, A., Delattre, J., Jore, D., andGardes-Albert, M. 1997. Oxidation of human high-densitylipoproteins by ⋅OH and ⋅OH/O2

⋅− , free radicals. Radiat. Res.147: 721–728.

Bowry, V.W., and Stocker, R. 1993. The prooxidant effect of vita-min E on the radical-initiated oxidation of human low-density li-poprotein. J. Am. Chem. Soc.115: 6019–6045.

Brown, M.S., and Goldstein, J.L. 1983. Lipoprotein metabolism inthe macrophage: implication for cholesterol deposition in ath-erosclerosis. Annu. Rev. Biochem.52: 223–261.

Buxton, G.V., Greenstock, C.L., Helamn, W.P., and Ross, A.B.1988. Critical review of rate constants for reactions of hydratedelectrons, hydrogen atoms, and hydroxyl radicals (⋅OH/O2

⋅−) inaqueous solution. J. Phys. Chem. Ref. Data,17: 513–886.

Delattre, J., Bonnefont-Rousselot, D., Khalil, A., Lepage, S.,Gardes-Albert, M., and Ferradini, C. 1993. Peroxidation of hu-man high density lipoproteins by oxygen-derived free radicals.Bull. Acad. Natl. Med. (Paris),177: 1251–1260.

De Leenheer, A.P., De Bevere, V.O., Cruyl, A.A., and Claeys, A.E.1978. Determination of serum alpha-tocopherol (vitamin E) byhigh-performance liquid chromatography. Clin. Chem.24: 585–590.

El Saadani, M., Esterbauer, H., El Sayed, M., Gother, M., Nassar,A.Y., and Jurgens, G. 1989. A spectrophotometric assay for lipidperoxides in serum lipoproteins using a commercial available re-agents. J. Lipid Res.30: 627–630.

Esterbauer, H., Gieseg, S.P., Giessauf, A., Ziouzenkova, O., andRamos, P. 1995. Role of natural antioxidants in inhibiting Cu2+

mediated oxidation of LDL.In Free radicals, lipoprotein oxida-tion and atherosclerosis.Edited byG. Bellomo, G. Finardi, E.Maggi, and C. Rice-Evans. Richelieu Press, London. 11–26.

Esterbauer, H., Schmidt, R., and Hayn, M. 1997. Relationshipsamong oxidation of low-density lipoprotein, antioxidant protec-tion, and atherosclerosis. Adv. Pharmacol.38: 425–456.

Fricke, H., and Morse, S. 1927. The chemical action of Röntgenrays on dilute ferrosulfate solutions as a measure of dose. Am. J.Roentgenol. Radiat. Ther.18: 430–432.

Gieseg, S.P., and Esterbauer, H. 1994. Low density lipoprotein issaturable by pro-oxidant copper. FEBS Lett.343: 188–194.

Heinecke, J.W., Baker, L., Rosen, H., and Chait, A. 1986.Superoxide-mediated modification of low density lipoprotein byarterial smooth muscle cells. J. Clin. Invest.77: 757–761.

Hicks, M., and Gebicki, J.M. 1993. Continuous measurement ofoxygen consumption by linoleic acid membranes exposed tofree radicals generated byg-radiation. Int. J. Radiat. Biol.64:143–148.

Hiramatsu, K., Rosen, H., Heinecke, J.W., Wolfbauer, G., andChait, A. 1987. Superoxide initiates oxidation of low density li-poprotein by human monocytes. Arteriosclerosis,7: 55–60.

Khalil, A., Wagner, J.R., Lacombe, G., Dangoisse, V., and Fulop,T., Jr. 1996. Increased susceptibility of low-density lipoprotein(LDL) to oxidation by gamma-radiolysis with age. FEBS Lett.392: 45–48.

Khalil, A., Wagner, R., and Fülöp, T. 1997. Comparative study ofthe oxidative modification of Human LDL and HDL. J. Chem.Phys.94: 365–370.

© 2001 NRC Canada

Khalil and Fülöp 121

Khalil, A., Jay-Gerin, J.P., and Fulop, T., Jr. 1998. Age-related in-creased susceptibility of high-density lipoproteins (HDL) to invitro oxidation induced by gamma-radiolysis of water. FEBSLett. 435: 153–158.

Khalil, A., Lehoux, J.G., Wagner, R.J., Lesur, O., Cruz, S., Dupont,E., Jay-Gerin, J.P., Wallach, J., and Fulop, T. 1998.Dehydroepiandrosterone protects low density lipoproteinsagainst peroxidation by free radicals produced by gamma-radiolysis of ethanol-water mixtures. Atherosclerosis,136: 99–107.

Kuzuya, M., Yamada, K., Hayashi, T., Funaki, C., Naito, M., Asai,K., and Kuzuya, F. 1992. Role of lipoprotein-copper complex incopper catalyzed-peroxidation of low-density lipoprotein.Biochim. Biophys. Acta,1123: 334–341.

Maiorino, M., Zamburlini, A., Roveri, A., and Ursini, F. 1995.Copper-induced lipid peroxidation in liposomes, micelles, andLDL: which is the role of vitamin E? Free Radical Biol. Med.18: 67–74.

Morel, D.W., DiCorleto, P.E., and Chisolm, G.M. 1984. Endothe-lial and smooth muscle cells alter low density lipoprotein in vi-tro by free radical oxidation. Arteriosclerosis,4: 357–364.

Patel, R., and Darely-Usmar, V.M. 1999. Molecular mechanisms ofthe copper dependent oxidation of low-density lipoprotein. FreeRad. Res.30: 1–9.

Proudfoot, J.M., Croft, K.D., Puddey, I.B., and Beilin, L.J. 1997.The role of copper reduction by alpha-tocopherol in low-densitylipoprotein oxidation. Free Radical Biol. Med.23: 720–728.

Pryor, W.A., and Castle, L. 1984. Chemical methods for the detec-tion of lipid hydroperoxides. Methods Enzymol.105: 293–299.

Rose, G.A., and Blackburn, H. 1968. Cardiovascular SurveyMethods, WHO Monograph series,56: 1–188.

Sato, K., Niki, E, and Shimasaki, H. 1990. Free radical-mediatedchain oxidation of low density lipoprotein and its synergistic in-hibition by vitamin E and vitamin C. Arch. Biochem. Biophys.279: 402–405.

Sattler, W., Mohr, D., and Stocker, R. 1994. Rapid isolation oflipoproteins and assessment of their peroxidation by high-

performance liquid chromatography postcolumn chemi-luminescence. Methods Enzymol.233: 469–489.

Spinks, J.W.T., and Woods, R.J. 1990. An introduction to radiationchemistry, 3rd ed. Wiley-Liss, Inc., New York. P. 574.

Steinbrecher, U.P., Witztum, J.L., Parthasarathy, S., and Steinberg,D. 1987. Decrease in reactive amino groups during oxidation orendothelial cell modification of LDL. Correlation with changesin receptor-mediated catabolism. Arteriosclerosis,7: 135–143.

Thomas, C.E., and Jackson, R.L. 1991. Lipid hydroperoxide in-volvement in copper-dependent and independent oxidation oflow density lipoproteins. J. Pharmacol. Exp. Ther.256: 1182–1188.

Witztum, J.L., and Steinberg, D. 1991. Role of oxidized low den-sity lipoprotein in atherogenesis. J. Clin. Invest.88: 1785–1792.

Yagi, K. 1976. A simple fluorometric assay for lipoperoxide inblood plasma. Biochem. Med.15: 212–216.

Yan, L.J., Lodge, J.K., Traber, M.G., Matsugo, S., and Packer, L.1997. Comparison between copper-mediated and hypochlorite-mediated modifications of human low density lipoproteins evalu-ated by protein carbonyl formation. J. Lipid Res.38: 992–1001.

Yoshida, Y., Tsuchiya, J., and Niki, E. 1994. Interaction of alpha-tocopherol with copper and its effect on lipid peroxidation.Biochim. Biophys. Acta,1200: 85–92.

Zarev, S., Therond, P., Bonnefont-Rousselot, D., Beaudeux, J.L.,Gardes-Albert, M., and Legrand, A. 1999. Major differences inoxysterol formation in human low density lipoproteins (LDLs)oxidized by ⋅OH/O2

⋅− free radicals or by copper. FEBS Lett.451: 103–108.

Ziouzenkova, O., Gieseg, S.P., Ramos, P., and Esterbauer, H. 1996.Factors affecting resistance of low density lipoproteins to oxida-tion. Lipids, Sl: 71–76.

Ziouzenkova, O., Sevanian, A., Abuja, P.M., Ramos, P., andEsterbauer, H. 1998. Copper can promote oxidation of LDL bymarkedly different mechanisms. Free Radical Biol. Med.24:607–623.