Báo cáo khoa học: Anti- and pro-oxidant effects of quercetin in copper-induced low density lipoprotein oxidation Quercetin as an effective antioxidant against pro-oxidant effects of urate

doi:10.1111/j.1432-1033.2004.04111.x

Eur. J. Biochem. 271, 1991–1999 (2004) (cid:1) FEBS 2004

Anti- and pro-oxidant effects of quercetin in copper-induced low density lipoprotein oxidation Quercetin as an effective antioxidant against pro-oxidant effects of urate

Paulo Filipe1,2, Josiane Haigle3, Joa˜ o Nuno Silva1,2, Joa˜ o Freitas1,2, Afonso Fernandes1, Jean-Claude Mazie` re4, Ce´ cile Mazie` re4, Rene´ Santus3,5 and Patrice Morlie` re3,5 1Centro de Metabolismo e Endocrinologia and 2Clinı´ca Dermatolo´gica Universita´ria, Faculdade de Medicina de Lisboa, Hospital de Santa Maria, Lisbon, Portugal; 3Muse´um National d’Histoire Naturelle, RDDM, Photobiologie – INSERM U.532, Paris, France; 4Laboratoire de Biochimie, Universite´ de Picardie Jules Verne, U Amiens, Hoˆpital Nord, Amiens, France; 5Institut de Recherche sur la Peau, INSERM U.532, Hoˆpital Saint-Louis, Paris, France

and Cu+ with an expected yield larger than that obtained with urate owing to a more favorable redox potential. It is also shown that the pro-oxidant activity of urate can be inhibited by quercetin. An electron transfer between quercetin and semioxidized urate leading to the repair of urate could account for this observation as suggested by recently published pulse radiolysis data. It is anticipated that the interactions between quercetin–Cu2+–LDL and urate, which are tightly controlled by their respective concentra- tion, determine the balance between the pro- and antioxidant behaviors. Moreover, as already observed with other anti- oxidants, it is demonstrated that quercetin alone behaves as a pro-oxidant towards preoxidized LDL.

Keywords: antioxidant; copper; flavonoid; low-density lipo- protein; oxidative stress; pro-oxidant; urate.

We recently reported that, depending on its concentration, urate is either a pro- or an antioxidant in Cu2+-induced low- density lipoprotein (LDL) oxidation. We also previously demonstrated an antioxidant synergy between urate and some flavonoids in the Cu2+-induced oxidation of diluted serum. As a result, the effect of the flavonoid quercetin on the Cu2+-induced oxidation of isolated LDL has been studied either in the presence or absence of urate. We demonstrate that, like urate, quercetin alone, at low concentration, exhibits a pro-oxidant activity. The pro-oxidant behavior depends on the Cu2+ concentration but it is not observed at high Cu2+ concentration. When compared with urate, the switch between the pro- and the antioxidant activities occurs at much lower quercetin concentrations. As for urate, the pro-oxidant character of quercetin is related to its ability to reduce Cu2+ with the formation of semioxidized quercetin

It is generally accepted that diets rich in fruit and vegetables protect against cardiovascular diseases [1,2], certain types of cancer [3], and perhaps against other pathological condi- tions. This protection has been attributed to the anti- oxidants present in plants including various phenolic compounds such as flavonoids. The biological, preventive, and therapeutic properties of flavonoids have been exten- sively studied. It has recently been shown that some flavonoids can cross the intestinal barrier into the blood- stream [4,5]. However, detected levels are extremely low in relation to the ingested amounts.

Low-density lipoprotein (LDL) oxidative modification is currently thought to be a key process in the atherogenesis [6–9]. The in vitro inhibitory effects of flavonoids on LDL peroxidation induced by copper(II) ions, azo derivatives and macrophages are well established. The radical scaven- ging properties of phenolic compounds depend on their redox potentials [10–12]. In addition, the antioxidant action is related to their ability to chelate transition metal ions [13–16]. The flavonoid antioxidant efficacy in biological systems also depends on the partition coefficient between the lipophilic and the aqueous phases [17,18], the binding to macromolecules [19,20], and the interaction with other antioxidants [21–28].

In most reports, the concentration of phenolic com- pounds to produce half of the maximum inhibition lies in the 1–10 lM range. With the exception of ascorbate, the antioxidant synergy between flavonoids and other extra- cellular antioxidants has not been fully explored. The interaction of flavonoids with ascorbate was first des- cribed in 1936 [29,30], when it was found that the extracts of Hungarian red pepper contained an ascorbate-protect- ive factor, named vitamin P. Later on, this factor was identified as a mixture of flavonoids, and its effect was interpreted as the result of the antioxidant action of these compounds.

Correspondence to P. Morlie` re, Muse´ um National d’Histoire Naturelle, RDDM, Photobiologie – INSERM U.532, Case Postale 26, 43 rue Cuvier, 75231 Paris cedex 05, France. Fax: + 33 1 40793716, Tel.: + 33 1 40793884, E-mail: morliere@mnhn.fr Abbreviations: LDL, low-density lipoprotein; LPO, lipid peroxidation; MDA, malondialdehyde; MM-LDL, minimally modified low-density lipoprotein; SOD, superoxide dismutase; TBARS, thiobarbituric acid-reactive substances. (Received 30 December 2003, revised 3 March 2004, accepted 25 March 2004)

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Conjugated diene determination

Conjugated diene formation was monitored by second derivative spectroscopy (220–300 nm) based on an earlier described methodology [40]. In short, 80 lL of the sample were diluted 10-fold with pH 7.4, 10 mM phosphate buffer before spectrum recording. The second derivative spectrum was subtracted from the second derivative spectrum of the matching control sample without cop- per(II). The increase in conjugated dienes expressed in relative unit was obtained from the amplitude of the 254 nm peak.

Malondialdehyde measurement

It is well known that urate, one of the most important plasma antioxidants resulting from the breakdown of purines, inhibits LDL peroxidation [31]. The mechanisms of this effect include the ability of urate to scavenge reactive oxygen and nitrogen species, and to chelate catalytic metal ions [32–34] (reviewed in [35]). However, data are lacking on the interaction of urate with other antioxidants. As a consequence we have investigated the effect of dietary flavonoids, associated or not with urate, on the oxidation of human LDL induced by copper(II) ions. This investigation appears sensible as it was recently demonstrated [36] that, at low concentration, urate may behave as a pro-oxidant. Moreover, in an earlier report it was suggested that flavonoids and urate could act in synergy to protect plasma lipoproteins against the oxidative stress [37]. The main goal of this work was therefore to evaluate whether and how urate and flavonoids interact during the Cu2+-induced LDL oxidation. Because of its largely recognized anti- oxidative properties, quercetin was chosen as a model flavonoid in this study.

Materials and methods

Materials, solvents and routine equipment

free MDA,

1,1,3,3-tetraethoxypropane

this

Quercetin dihydrate and sodium urate were obtained from Sigma Chemical Co. (Saint-Louis, MO, USA). The high- performance liquid chromatography (HPLC) columns were supplied by Merck (Darmstadt, Germany) and HPLC grade solvents were purchased from Carlo Erba (Val de Reuil, France). All other chemicals were of the highest purity available from Sigma or Merck companies.

The simultaneous determination of free MDA and urate was performed by HPLC using a LiChrospher100 NH2 column [41]. After incubation, solutions were mixed with an equal volume of acetonitrile, centrifuged at 12 000 g for 5 min and frozen at )80 (cid:2)C until HPLC measurement. The supernatants were isocratically eluted during 25 min with a mobile phase consisting of pH 7.4, 54 mM Tris-HCI and acetonitrile (30 : 70, v/v). The flow rate was 1.0 mLÆmin)1 and the absorption was monitored at 270 nm. The MDA peak was identified by comparison with a reference freshly prepared by chromatogram of acid hydrolysis of stock solution. The MDA concentration of standard solution was determined assuming a molar absorbance of )1Æcm)1 at 245 nm. This solution was then diluted 13 700 M with pH 7.4, 54 mM Tris-HCI buffer to obtain MDA concentrations in the 1–10 lM range and then, mixed with acetonitrile (1 : 1, v/v) before HPLC.

Preparation and treatment of LDL

Consumption of carotenoids

The basal carotenoid content of LDL preparations was spectrophotometrically determined after extraction [42]. To this end, 0.25 mL of water, 1 mL of ethanol and 2 mL of hexane were added to 0.25 mL of LDL. The hexane upper phase (2 mL) was collected and the visible absorption spectrum (350–600 nm) was recorded. The concentration of total carotenoids was determined using )1Æcm)1 at an average extinction coefficient of 140 000 M 448 nm based on a calculation from the four main carotenoids in human plasma, a-carotene, b-carotene, b-cryptoxanthin and lycopene [43,44]. Change in caro- tenoid concentration during LDL oxidative treatment was monitored by second derivative absorption spectros- copy (400–550 nm) through the measure of the amplitude of the second derivative spectrum between 489 and 516 nm.

Urate consumption

As mentioned above, urate was determined by HPLC, simultaneously with MDA. The urate peak in chromato- grams was identified by comparison with reference chro- matograms of freshly prepared standard urate solutions in the 1–20 lM range. The concentration of urate in the samples was calculated from the peak area compared to that of standard solutions.

Serum samples were obtained from healthy volunteers. LDL (d ¼ 1.024–1.050) were prepared by sequential ultra- centrifugation according to Havel et al. [38] and dialyzed against pH 7.4, 5 mM Tris buffer containing 50 mM NaCl and 0.02% EDTA. Protein determination was carried out by the technique of Peterson [39]. Unless specifically stated in the text, these LDL preparations were used within 2–3 weeks. Just before experiments, LDL were dialyzed twice for 8 and then 16 h against 1 L of pH 7.4, 10 mM phosphate buffer to remove ETDA. Then, LDL were diluted to a final concentration of 0.15 mgÆmL)1 (300 nM). To 800 lL of these LDL solutions, were added 50 lL of a stock solution of urate in pH 7.4, 10 mM phosphate buffer and/or 10 lL of a stock solution of the studied flavonoid in pH 7.4, 10 mM phosphate buffer or ethanol. LDL solutions without urate and/or flavonoid but containing matching solvent volumes were similarly prepared. Then, all these LDL solutions were diluted to a final volume of 950 lL with phosphate buffer and were incubated at 37 (cid:2)C for 15 min. Lipid peroxidation (LPO) was triggered by adding 50 lL of a CuCl2 solution in pH 7.4, 10 mM phosphate buffer preheated at 37 (cid:2)C to obtain a final concentration of Cu2+ of 5 or 175 lM. After Cu2+ addition, the formation of conjugated dienes and malondialdehyde (MDA) and the consumption of urate and carotenoids were measured, as described below, either after a 1 h incubation at 37 (cid:2)C or at various times during incubation.

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Results

were obtained when LPO was estimated by the production of thiobarbituric reactive substances or conjugated dienes (data not shown).

MDA production as a function of the quercetin concentration

Time courses of MDA and conjugated diene formation

The data presented in Fig. 1 have been obtained at a constant time (1 h) after the addition of Cu2+. Kinetic analyses may prove to be helpful in understanding the effects observed under (cid:2)static conditions(cid:3). Our attention has been focused on the effect of low concentrations of quercetin. Depending on the Cu2+ concentration this flavonoid may be pro-oxidant in the absence of urate or may inhibit the pro-oxidant effect of urate. The Cu2+- induced LPO in LDL was evaluated by monitoring the formation of MDA (Fig. 2A,C) and of conjugated dienes (Fig. 2B,D) in the presence and absence of 10 lM urate at high (175 lM) (Fig. 2A,B) and low (5 lM) (Fig. 2C,D) Cu2+ concentrations. With regard to the MDA formation at high Cu2+ concentration, Fig. 2A fully confirms the pro- oxidant activity of low urate concentrations with enhanced MDA formation and the moderate protection brought by 0.5 lM quercetin at short times after Cu2+ addition. In the presence of both urate and quercetin, the MDA formation is strongly slowed down and occurs about 90 min after Cu2+ addition. Similar effects are observed with the formation of conjugated dienes (Fig. 2B). Thus, the time course of their formation (Fig. 2B,D) exhibits the classical shape charac- terized by a lag time followed by a linear increase until a maximum is reached. Then they slowly decay [45]. When separately added, 10 lM urate decreases the lag-time whereas 0.5 lM quercetin slightly increases it. On the other hand, when simultaneously added, quercetin and urate have a strong antioxidant activity with a lag-time for conjugated diene formation lasting for about 2 h.

After incubation for 15 min at 37 (cid:2)C with various concen- trations of quercetin, LDLs were exposed to 175 lM or 5 lM CuCl2. One hour after Cu2+ addition, the extent of LPO was estimated from MDA measurements as shown in Fig. 1. At high Cu2+ concentration (175 lM), the MDA production decreases with increasing quercetin concentra- tion (Fig. 1A). It is worth noting that quercetin concentra- tions as low as 0.5 lM already significantly decreases the MDA production. At low Cu2+ concentration (5 lM) a pro-oxidant effect is observed at low quercetin concentra- tions (< 2 lM) whereas quercetin becomes antioxidant at higher concentration (‡ 2 lM). The switch between the pro- and antioxidant activities occurs at (cid:2) 1.5 lM of quercetin. These experiments were also performed in the presence of urate. They were carried out as described above, except that LDL were simultaneously incubated with various concen- trations of quercetin and 10 lM urate prior to Cu2+ addition. As can be seen in Fig. 1, urate alone at 10 lM is pro-oxidant, with an amplification of about 200% at high Cu2+ concentration (175 lM) and about 600% at low Cu2+ concentration (5 lM) in agreement with reference [36]. At high Cu2+ concentration and concentrations of quercetin as low as 0.5 lM, the production of MDA drops to negligible values much lower than those obtained in the absence of both urate and quercetin. At low Cu2+ concentration, quercetin also decreases the MDA level but the full inhibition requires larger concentrations than those needed at high Cu2+ concentration. Interestingly, we may note that the pro-oxidant effect observed with low concentrations of quercetin alone did not add to the pro-oxidant effect of 10 lM urate. On the contrary, such low concentrations lowered the MDA production. For example, 0.75 lM quercetin or 10 lM urate alone enhances the MDA production by factors of about 250 and 600%, respectively. When added together they significantly decrease the MDA level to 60%. Data profiles similar to those shown in Fig. 1

At low Cu2+ concentration, the kinetic data in Fig. 2C are in agreement with those obtained under (cid:2)static condi- in Fig. 1A; that is to say 10 lM urate strongly tions(cid:3) enhances MDA production whereas 0.5 lM quercetin also increases it but to a lesser extent. Higher quercetin concentrations, namely 2 lM, inhibit the MDA formation

Fig. 1. Effect of quercetin on LDL oxidation induced by 175 lM (A) or 5 lM Cu2+ (B) in the absence or in the presence of 10 lM urate. In (A) and (B), LDL at 0.12 mgÆmL)1 (240 nM) in pH 7.4, 10 mM phosphate buffer were incubated for 15 min at 37 (cid:2)C with various concentrations of quercetin, with or without urate. Then, 175 lM (A) or 5 lM (B) of CuCl2 were added and the mixture was further incubated at 37 (cid:2)C for 1 h before MDA assay. In (A) and (B), controls in the absence of Cu2+ yielded non-detectable or negligible levels of MDA (data not shown). Data were normalized to those obtained in the absence of quercetin and urate (taken as 100%) and are the mean ± SD of at least three experiments performed with independent LDL preparations.

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conjugated dienes (Fig. 2B) or of MDA (Fig. 1A), 0.5 lM quercetin behaves as an antioxidant and induces a delay in the carotenoid consumption (Fig. 3A).

up to about 2 h. Finally, as shown in Fig. 2(C), slightly pro-oxidant concentrations of quercetin (0.5 lM) protect from the pro-oxidant effect of 10 lM urate. Larger quercetin concentrations (e.g. 2 lM) completely inhibit the pro-oxidant effect of 10 lM urate as no MDA is formed during 3 h.

The same conclusions can be drawn with regard to the formation of conjugated dienes at low Cu 2+ concentrations (Fig. 2D). Again, when separately added, 10 lM urate shortens the lag time, in agreement with a pro-oxidant behavior whereas 0.5 lM quercetin exhibits a moderate pro- oxidant behavior. When added together 0.5 lM quercetin partly inhibits the pro-oxidant effect of 10 lM urate. At higher concentration (2 lM), quercetin strongly delays the appearance of the propagation phase when alone but completely abolishes the pro-oxidant effect of 10 lM urate.

Time courses of carotenoids and urate consumption

At low Cu2+ concentration, 0.5 lM quercetin becomes pro-oxidant with enhanced formation of MDA and conju- gated diene, as presented above. Accordingly no delay in the carotenoid consumption is observed (Fig. 3C). However, the protective effect of quercetin is recovered at higher concentrations, namely 2 lM (Fig. 3C). In the presence of a pro-oxidant concentration of urate (e.g. 10 lM), 0.5 lM quercetin, which is pro-oxidant in the absence of urate, markedly slows down the carotenoid consumption (Fig. 3C). Increasing the quercetin concentration to 2 lM leads to strong protection of the carotenoids, this protection being even better than with quercetin alone (Fig. 3C). Interestingly, at this low Cu2+ concentration, urate, which is rapidly consumed when added alone, is slightly protected by 0.5 lM quercetin and fully protected by 2 lM quercetin (Fig. 3D). At high Cu2+ concentration, 0.5 lM quercetin delays the carotenoid consumption more efficiently that quercetin alone (Fig. 3A) and also protects urate (Fig. 3B).

The consumption of carotenoids was measured as an index of the overall consumption of the endogenous antioxidants of LDL during their Cu2+-induced oxidation. As shown in Fig. 3(A,C), carotenoids are consumed in parallel to MDA and conjugated diene formation. As already reported [36], at low Cu2+ concentration, 10 lM urate accelerates the carotenoid consumption (Fig. 3C). At high Cu2+ concen- tration, the consumption of carotenoids is practically unchanged by the presence of this pro-oxidant concentra- tion of urate (Fig. 3A). As observed with the formation of

Effect of quercetin on the copper-induced lipid peroxidation in preoxidized LDL In order to evaluate the effect of quercetin on the Cu2+- induced LPO in pre-oxidized LDL, LDL were first incuba- ted with Cu2+ and then quercetin was added after LPO started. Quercetin concentrations were chosen such as

Fig. 2. Kinetic profiles of MDA (A,C) and conjugated diene (B,D) formation in LDL oxidation induced by 175 lM (A,B) or 5 lM (C,D) Cu2+. LDL at 0.12 mgÆmL)1 (240 nM) in pH 7.4, 10 mM phosphate buffer was incubated for 15 min at 37 (cid:2)C with or without quercetin, and with or without 10 lM urate. Then, 175 lM or 5 lM of CuCl2 were added and the mixture was further incubated at 37 (cid:2)C. MDA: Conjugated dienes were measured at various intervals after Cu2+ addition. Note that time zero corresponds to the shortest time after addition of Cu2+ in all samples, e.g. 1 min. Control experiments in the absence of Cu2+ yielded non detectable or negligible levels of MDA and of conjugated dienes (data not shown). Data are the mean ± SD of at least three experiments performed with independent LDL preparations.

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30 min after the oxidation started, LPO was higher than that obtained in the absence of quercetin (Fig. 4). In other words, under these conditions quercetin becomes pro- oxidant. As a second model of slightly preoxidized LDL, we used LDLs that were kept in the dark at 4 (cid:2)C in the presence of EDTA for 5–8 weeks. Such conditions are described in the literature as yielding the so-called minimally modified LDL (MM-LDL) [46,47]. As can be seen in Table 1, in the presence of quercetin, lag times for conjugated diene formation are shorter than those measured in its absence. In addition, quercetin accelerates the consumption of carotenoids in MM-LDL treated with Cu2+ (Table 1). This definitely means that under these conditions quercetin is no longer an antioxidant but behaves as a pro-oxidant.

Fig. 3. Kinetic profiles of carotenoid (A,C) and urate (B,D) consumption in LDL oxidation induced by 175 lM (A,B) or 5 lM (C,D) Cu2+. Experi- mental conditions are those of Fig. 2. Urate and carotenoids were measured at various intervals after Cu2+ addition. Control experiments in the absence of Cu2+ (·), yielded no significant loss of carotenoids or urate. Note that time zero in (B) and (D) corresponds to the shortest time after addition of Cu2+ in all samples, e.g. 1 min. Data are expressed as a percentage of the value obtained before Cu2+ addition and are the mean ± SD of at least three experiments performed with independent LDL preparations.

Discussion

Over the 15 past years, the oxidation of LDL has been widely studied in order to understand better the role of LDL oxidation in vivo in pathological situations, such as ather- ogenesis [6–9]. For this purpose, in vitro models have been developed including the oxidation of LDL by Cu2+ to which much attention has been paid [48]. However, the exact mechanisms relating Cu2+ redox change to the LPO in LDL are not yet clearly established [49]. In the presence of pre-existing traces of hydroperoxides (LOOH), Cu2+ and Cu+ may be either reduced or oxidized, generating hydroperoxyl (LOO•) or alkoxyl (LO•) radicals that in turn can trigger the LPO propagation phase. It is currently admitted that Cu2+ reduction to Cu+ is required for triggering LPO in LDL [50], but the exact nature of the

quercetin behaves as an antioxidant in un-oxidized LDL, i.e. 0.75 lM and 2 lM for high and low Cu2+ concentra- tions, respectively. By contrast, when quercetin is added

Fig. 4. Effect of quercetin on Cu2+-induced LDL oxidation in preoxi- dized LDL. CuCl2 (175 or 5 lM) was added to LDL solutions at 0.12 mgÆmL)1 (240 nM) in pH 7.4, 10 mM phosphate buffer preheated for 15 min at 37 (cid:2)C, and the mixture was further incubated at 37 (cid:2)C for 1 h before MDA assay. Quercetin was added either 15 min before Cu2+ addition or 30 min after Cu2+ addition. Quercetin concentra- tions were 0.75 or 2 lM for Cu2+ concentrations equal to 175 or 5 lM, respectively (see text). Data were normalized to those obtained in the absence of quercetin (taken as 100%) and are the mean ± SD of at three experiments performed with independent LDL preparations.

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Table 1. Effect of quercetin on the lag time for conjugated diene formation and on the half time for carotenoid consumption in Cu2+-treated MM-LDL. Data in parentheses correspond to those obtained with native LDL. Detailed experimental conditions are those of Figs 2 and 3. Quercetin concentrations were 0.75 and 2 lM for Cu2+ equal to 175 and 5 lM, respectively. Lag times before conjugated diene formation were estimated as the intercept of the linear part of the kinetics of diene formation shown in Fig. 2(B,D) with x-axis. Half times for carotenoid consumption were obtained from the kinetic of carotenoid consumption shown in Fig. 3(A,C).

Lag time (min) Half time (min)

Conditions Cu2+ ¼ 175 lM Cu2+ ¼ 5 lM Cu2+ ¼ 175 lM Cu2+ ¼ 5 lM

a Too short to be measured.

involved reductants in LDL, such as pre-existing LOOH, tryptophan residues or even a-tocopherol, is still questioned [51–59]. Their progressive involvement has been suggested by Perugini et al. [57]. It has also been suggested but never –, generated by Cu2+ oxidation may demonstrated that •O2 be involved [60].

[64], ascorbate [63] or urate [36,65,66]. This pro-oxidant behavior was described when the antioxidant is added at various times after LPO started, i.e. when some lipid peroxides are already formed. Accordingly, this pro-oxidant behavior is also encountered in Cu2+-induced oxidation of slightly oxidized LDL. We observed that quercetin also exhibits this pro-oxidant capacity. Indeed, the production of an exacerbated amount of MDA (Fig. 4) demonstrates that quercetin is antioxidant when present before LPO started but becomes pro-oxidant when introduced 30 min after addition of Cu2+. It is also illustrated in Table 1 reporting that, as opposed to native LDL, quercetin accelerates the formation of conjugated dienes (shortened lag time) and the consumption of endogenous carotenoids (shortened half time) in MM–LDL. It is accepted that this pro-oxidant behavior is related to the availability of hydroperoxides and that (cid:2)antioxidants(cid:3) promote LPO by increasing the concen- tration of catalytic Cu+ because of their ability to reduce Cu2+.

Our main goal was to investigate the interplay between urate and flavonoids in the Cu2+-induced oxidation of LDL. Such a work was undertaken because we recently reported, in mimicking oxidative stress in diluted plasma [37], that flavonoids could act in synergy with urate, one of the major plasma antioxidant. Antioxidant properties of flavonoids have been widely studied within the last 20 past years. A peculiar attention has been paid to the antioxidant effect of flavonoids towards the oxidation of LDL whose oxidative modification is thought to be a key process in atherogenesis. The antioxidant protection conferred by phenolic compounds is believed to be caused by a combi- nation of their binding to critical sites on LDL, their metal chelation properties and their free radical scavenging activities. Their overall antioxidant efficacy in biological systems also depends on their partition between lipophilic and aqueous phases, their binding to other biomolecules and their interaction with other antioxidants. Quercetin was chosen as a representative flavonoid in this study as it is rather universally found in plants and it has been the subject of numerous studies. As a result, we first characterized its antioxidant properties in our LDL oxidation model. It must be pointed out that besides the commonly used low Cu2+ concentration (5 lM here), we also performed experiments with a physiologically unrealistic high Cu2+ concentration (175 lM). Such a high Cu2+ concentration was used to overcome the chelating ability of quercetin. Moreover the use of both low and high Cu2+ concentrations allows discussing the present data in the light of our earlier reports [36,37]. It must be underlined that the quercetin concentra- tions that were used here (0.25–2 lM) are biologically realistic. Indeed, it has been shown that the concentration of quercetin derivatives in plasma reached about 0.4 [4], 0.6 [5] and 0.8 lM [61], 2–3 h after the ingestion of a quercetin-rich meal (about 50–90 mg of ingested quercetin). These values are about an order of magnitude higher than the baseline concentration.

In addition to their well established antioxidant proper- ties, flavonoids, as several other antioxidants, to be pro- oxidant under certain circumstances. In the Cu2+-induced LDL oxidation model, catechins [62] and a flavonoid extract [63] share this pro-oxidant ability with caffeic acid

More importantly, our data demonstrate that a pro- oxidant behavior of quercetin can be observed in the absence of pre-existing hydroperoxides, i.e. working with native LDL with quercetin present before LPO started. The presence of abnormally high levels of hydroperoxides in our LDL preparations, that could explain the data reported above, has been ruled out as discussed in [36,] This is observed at low Cu2+ concentration and it is illustrated on Figs 1 and 2C in terms of MDA production and on Fig. 2D in terms of conjugated diene formation. Interestingly, this pro-oxidant behavior is observed at low concentration of (cid:2)antioxidant(cid:3). In view of the MDA formation profile on Fig. 1, the switch from the pro-oxidant behavior to the classical antioxidant properties occurs with 1–2 lM querce- tin. This is confirmed with the conjugated diene formation, which is slightly promoted by 0.5 lM quercetin but strongly abolished by 2 lM quercetin (Fig. 2D). As to the consump- tion of endogenous carotenoids, the pro-oxidant behavior is low quercetin concentration still possibly observed at (0.5 lM) while 2 lM quercetin definitely induces a strong protective effect. We recently reported such a behavior with another well-known physiological antioxidant, e.g. urate [36]. We suggested that this pro-oxidant behavior was related to the ability of urate to reduce Cu2+, leading to •UH– and Cu+. We brought evidence that, at high Cu2+ – was involved probably formed by the concentration, •O2 oxidation of Cu+ by O2. We suggested that the concomitant – could allow a reaction between formation of •UH– and •O2 – activation. these species, thus leading to some kind of •O2

Without quercetin With quercetin (cid:2) 5 ((cid:2) 24) ((cid:2) 39)a (cid:2) 17 ((cid:2) 33) (cid:2) 6 (> 120) (cid:2) 18 ((cid:2) 27) (cid:2) 14 ((cid:2) 50) (cid:2) 24 ((cid:2) 45) (cid:2) 12 (> 150)

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– couple (0.33 V vs. NHE) [12,34,67] the •QH–,H+/QH2 and allows the reaction of quercetin with •UH–. Using pulse radiolysis, we recently provided good evidence for this reaction [68]. We showed, upon addition of quercetin, an increase in the decay rate of the transient absorption of •UH– accompanied by a growth of the transient absorp- tion of the semireduced quercetin (•QH–) demonstrating the regeneration of urate by quercetin. At high Cu2+ – is believed to be involved in the concentration, where •O2 pro-oxidant action of urate [37], the reaction of •UH– with – and impedes quercetin competes with its reaction with •O2 the pro-oxidant activity of urate to occur. At low Cu2+ concentration, another unidentified mechanism was sug- – is not involved, but where Cu+ is gested where •O2 the reaction of necessary [37]. As a consequence of quercetin with •UH–, it turns out that •UH– would also be involved in the pro-oxidant action of urate. Interestingly, at high Cu2+ concentration, in the presence of 0.5 lM quercetin and 10 lM urate, the LDL oxidation measured in terms of MDA and conjugated diene formation drops to a level below that observed in the absence of urate (Figs 1A and 2A,B). The same concept applies to the carotenoid consumption shown in Fig. 3A. Though we have no definite interpretation for such an observation, we may suppose that in the absence of quercetin the pro- oxidant activity of urate masks its antioxidant activity as mentioned above, whereas, in the presence of quercetin, the pro-oxidant activity of urate disappears and its antioxidant activity is revealed.

At low Cu2+ concentration, the exact mechanism was not –-independent mechanism was pro- elucidated, but an •O2 posed, still involving the reduction of Cu2+ by urate. This intriguing behavior has to be related to complex urate– Cu2+–LDL interactions, which are governed by their respective concentrations. In the view of the analogous behaviors observed here with quercetin and previously with urate, it may be hypothesized that similar mechanisms are involved. We already provided evidence that quercetin was able to reduce Cu2+ [37]. When comparing the present data with those obtained with urate, it is interesting to note that at low Cu2+ concentration the switch between the pro- oxidant and antioxidant behavior occurs at about 1 lM with quercetin and 200 lM with urate [36]. As we pointed out in our earlier report dealing with urate, the pro-oxidant mechanism observed at low concentration is still operative at high concentration but is no longer observed because, at high concentration, the antioxidant activity (chelation, radical scavenging) prevails. Thus, the lower concentration for switching between pro- and antioxidant properties for quercetin as compared to urate may reflect the better overall antioxidant activity of quercetin. Conversely, it may also reflect a pro-oxidant ability larger for quercetin than for urate, thus requiring lower concentrations to be observable. As a matter of fact, at pH 7, the standard redox potential of – (0.33 vs. NHE [12,67]) is higher the couple •QH–,H+/QH2 – (0.59 vs. NHE [34]). than that of the couple •UH–,H+/UH2 The reduction of Cu2+ to Cu+ (E(cid:2) ¼ 0.167 vs. NHE) is thermodynamically unfavorable but less unfavorable for quercetin than for urate. Thus the production of equal amounts of catalytic Cu+ from urate or quercetin will require lower quercetin concentrations.

recent

Conclusions The results presented here on the Cu2+-induced oxida- tion of LDL show that quercetin, as other oxidants, can be pro-oxidant towards slightly oxidized LDL and, at low concentration, can behave like urate as a pro-oxidant towards native LDL. This study also demonstrates that quercetin at low and pro-oxidant concentrations is no longer pro-oxidant in the presence of pro-oxidant urate concentrations. Under these conditions, it even protects against the pro-oxidant activity of urate. As to the mechanism, it is suggested that quercetin, because of its appropriate redox potential can regenerate urate by reducing the •UH– radical. Accordingly, quercetin inhibits the pro-oxidant activity of urate associated with the presence of the •UH– radical. However, some points still Indeed, during the •UH– remain un-understandable. repair, •QH– would be generated, which may be associ- ated with the pro-oxidant activity of quercetin at low concentration. It is quite obvious that a detailed know- ledge of these mechanisms deserves further investigations. the intricate relationship It may be anticipated that between antioxidant and pro-oxidant action of antioxi- dants, such as quercetin or urate, is closely related to the spatial and temporal interactions of the various actors, as emphasized by our study on the repair of semioxidized urate by quercetin bound to HSA [69]. In other words, the interactions between quercetin, Cu2+, LDL, and urate when present, which are closely controlled by their respective concentrations, may be determinant in the balance between the pro- and antioxidant behaviors.

Finally, as mentioned in the introduction, we studied the interaction of urate and quercetin in the Cu2+-induced LDL oxidation model. As stated above, urate at moder- ately low concentrations behaves as a pro-oxidant at low and high Cu2+ concentrations. This is illustrated with 10 lM urate in Figs 1 and 2(B,C) showing an overpro- duction of MDA. It is also clearly shown with an accelerated conjugated diene formation (Fig. 2B,D) and carotenoid consumption especially at low Cu2+ concen- tration (Fig. 3C). At high Cu2+ concentration, the addi- tion of 0.5 lM quercetin, which, alone, is moderately antioxidant, not only inhibits the pro-oxidant effect of urate, but exerts an overall protective effect on the LDL oxidation larger than that obtained with quercetin alone. Under these conditions, the urate destruction is slowed down by the presence of quercetin (Fig. 3C). At low Cu2+ concentration, a somewhat similar behavior is observed. Thus, 0.5 lM quercetin which, in this case, is pro-oxidant when alone, partly inhibits the pro-oxidant action of urate (Figs 1B, 2C,D and 3C). Larger quercetin concentrations, namely 2 lM, completely inhibit the pro-oxidant action of urate leading to full overall protection. It is interesting to mention that 2 lM quercetin inhibits the urate consump- tion up to 3 h after Cu2+ addition (Fig. 3D). This powerful antioxidant interaction of quercetin and urate can be partially explained by the interception of reactive species, chelation of transition metal ions and/or regener- ation of urate from its radical form. The recycling of urate is possible as at pH 7, the redox potential of the •UH–, – couple (0.59 V vs. NHE) is higher than that of H+/UH2

1998 P. Filipe et al. (Eur. J. Biochem. 271)

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Acknowledgements

16. Yoshino, M. & Murakami, K. (1998) Interaction of iron with polyphenolic compounds: application to antioxidant character- ization. Anal. Biochem. 257, 40–44.

This work was partly supported by an exchange grant from INSERM and GRICES. C. M. and J.-C. M. thank the (cid:2)Universite´ de Picardie Jules Verne(cid:3) and the (cid:2)Ministe` re de la Recherche et de la Technologie(cid:3) for financial support. 17. Chimi, R., Cillard, J., Cillard, P. & Rahmani, M. (1991) Peroxyl and hydroxyl radical scavenging activity of some natural phenolic antioxidants. J. Am. Oil Chemists Soc. 68, 307–312.

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