Báo cáo Y học: Anti- and pro-oxidant effects of urate in copper-induced low-density lipoprotein oxidation pdf

Anti- and pro-oxidant effects of urate in copper-induced low-density lipoprotein oxidation Paulo Filipe 1,2 , Josiane Haigle 3 , Joa ˜ o Freitas 1,2 , Afonso Fernandes 1 , Jean-Claude Mazie ` re 4 , Ce ´ cile Mazie ` re 4 , Rene ´ Santus 3,5 and Patrice Morlie ` re 3,5 1 Centro de Metabolismo e Endocrinologia, Faculdade de Medicina de Lisboa, Portugal; 2 Clı ´ nica Dermatolo ´ gica Universita ´ ria, Faculdade de Medicina de Lisboa, Hospital de Santa Maria, Lisbon, Portugal; 3 Laboratoire de Photobiologie, Muse ´ um National d’Histoire Naturelle, Paris, France; 4 Laboratoire de Biochimie, Universite ´ de Picardie Jules Verne, CHRU Amiens, Ho ˆ pital Nord, Amiens, France; 5 INSERM U.532, Institut de Recherche sur la Peau, Ho ˆ pital Saint-Louis, Paris, France We reported earlier that urate may behave as a pro- oxidant in Cu 2+ -induced oxidation of diluted plasma. Thus, its effect on Cu 2+ -induced oxidation of isolated low-density lipoprotein (LDL) was investigated by mon- itoring the formation of malondialdehyde and conjugated dienes and the consumption of urate and carotenoids. We show that urate is antioxidant at high concentration but pro-oxidant at low concentration. Depending on Cu 2+ concentration, the switch between the pro- and antioxid- ant behavior of urate occurs at different urate concen- trations. At high Cu 2+ concentration, in the presence of urate, superoxide dismutase and ferricytochrome c protect LDL from oxidation but no protection is observed at low Cu 2+ concentration. The use of Cu 2+ or Cu + chelators demonstrates that both copper redox states are required. We suggest that two mechanisms occur depending on the Cu 2+ concentration. Urate may reduce Cu 2+ to Cu + , which in turn contributes to O ÁÀ 2 formation. The Cu 2+ reduction is likely to produce the urate radical (UH Æ– ). It is proposed that at high Cu 2+ concentration, the reaction of UH Æ– radical with O ÁÀ 2 generates products or intermediates, which trigger LDL oxidation. At low Cu 2+ concentration, we suggest that the Cu + ions formed reduce lipid hydroperoxides to alkoxyl radicals, thereby facilitating the peroxidizing chain reaction. It is antici- pated that these two mechanisms are the consequence of complex LDL–urate–Cu 2+ interactions. It is also shown that urate is pro-oxidant towards slightly preoxidized LDL, whatever its concentration. We reiterate the con- clusion that the use of antioxidants may be a two-edged sword. Keywords: antioxidant; copper; low-density lipoprotein; pro-oxidant; urate. Beside ascorbate, urate is currently considered as one of the main water-soluble antioxidants of human plasma [1–4]. In this regard, under evolutionary pressure, primates have by- passed the urate catabolism pathway to elaborate other antioxidative mechanisms susceptible to cope with the loss of the capability to synthesize ascorbate. Compared with other mammals, the strong increase in urate plasma level of primates has been interpreted as a compensatory response to a low ascorbate serum concentration [5]. The protective deterrent of urate has also been associated with pathological conditions such as the Down’s syndrome, for which serum lipid resistance to oxidation was associated with an increase in serum uric acid levels [6]. The antioxidant properties of urate or its synergistic effects with other antioxidants have been attributed to its ability to scavenge hydroxyl and superoxide radicals and peroxynitrite and to chelation of transition metal ions [7–11]. Paradoxically, a lack of antioxidant activity of urate or even a pro-oxidant activity of urate have also been sometimes suggested. Atherogenesis is the major patholo- gical process leading to the most frequent cardiovascular diseases through low-density lipoprotein (LDL) oxidative modification [12–15]. Consistent epidemiological data point to the correlation of high uric acid levels with cardiovascular diseases [16–20]. These observations can be interpreted either as an antioxidant compensatory response or as a pro- oxidant effect of urate [21]. In vitro data also point out the pro-oxidant ability of urate under certain circumstances. A pro-oxidant effect of urate has been reported in the in vitro Cu 2+ -induced oxidation of preoxidized LDL [22,23]. It has been also shown that urate induces DNA stand breakage in the presence of cupric ions [24,25]. In a recent study dealing with the flavonoids and urate interplay in plasma oxidative stress, we mentioned that, in some instances, urate was pro-oxidant. In this previous work, we triggered lipid peroxidation through the exposure of diluted plasma to cupric ions [26]. Our goal here is to shed light on this observation and to study the subtle balance between antioxidant and pro-oxidant properties of urate in order to determine some of the mechanistic aspects. For this purpose, we studied copper-induced LDL oxidation, under various conditions in the presence and absence of urate. Correspondence to P. Morliere, Laboratoire de Photobiologie, INSERM U.532, Muse ´ um National d’Histoire Naturelle, 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; UH Æ– , urate radical. Enzyme: copper-zinc superoxide dismutase (EC 1.15.1.1). (Received 11 July 2002, revised 4 September 2002, accepted 10 September 2002) Eur. J. Biochem. 269, 5474–5483 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03245.x MATERIALS AND METHODS Chemicals, solvents and routine equipment Sodium urate (Na + , UH 2 – ), superoxide dismutase (SOD) from bovine kidney, catalase from bovine liver, neocupro- ine, ferricytochrome c and 1,1,3,3-tetraethoxypropane were obtained from Sigma Chemical Co. (St Louis, MI, USA). HPLC columns were purchased from Merck (Darmstadt, Germany) and HPLC grade solvents from Carlo Erba (Val de Reuil, France). All other chemicals were of the highest purity available from Sigma or Merck companies. Preparation and treatment of LDL 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.[27].Protein determination was carried out by the technique of Peterson [28]. Unless specified in the text, LDL preparations were used within 2–3 weeks. Just before experiments were carried out, LDL preparations were dialyzed twice for 8 and 16 h against 1 L of 10 m M phosphate buffer, pH 7.4, to remove EDTA. Then, LDL preparations were diluted to a final concentration of 0.15 mgÆmL )1 (300 n M ). To 800 lLof these diluted LDL preparations were added 50 lLofa stock solution of urate in pH 7.4, 10 m M phosphate buffer and 100 lL of buffer. Blank LDL solutions devoid of urate were also prepared. These LDL solutions were then incubated at 37 °C for 15 min. Lipid peroxidation (LPO) was triggered by adding 50 lL of a CuCl 2 solution in pH 7.4, 10 m M phosphate buffer preheated at 37 °Cto obtain final concentrations of Cu 2+ of 175 or 5 l M .After Cu 2+ addition, lipid peroxidation, urate and carotenoid consumption were measured, as described below, after a 1-h incubation period at 37 °C or at intervals during continuous incubation. Conjugated diene determination Conjugated diene formation was monitored by second derivative spectroscopy (220–300 nm) based on an earlier described methodology [29]. In short, 80 lLofthesample were diluted 10-fold with pH 7.4, 10 m M phosphate buffer before spectrum recording. The second derivative spectrum was subtracted from the second derivative spectrum of the matching control sample without Cu 2+ . The increase in conjugated dienes expressed in relative unit was obtained from the amplitude of the 254 nm peak. Malondialdehyde measurement The simultaneous determination of free MDA and urate was performed by HPLC using a LiChrospher100 NH 2 column [30]. After incubation, solutions were mixed with an equal volume of acetonitrile and centrifuged at 12 000 g for 5 min and frozen at )80 °C until HPLC measurement. Supernatants (200 lL) were isocratically eluted during 20 min with a mobile phase consisting of pH 7.4, 54 m M Tris/HCl and acetonitrile (30 : 70, v/v). The flow rate was 1.2 mLÆmin )1 and the absorption was monitored at 270 nm. The MDA peak was identified by comparison with a reference chromatogram of freshly prepared free MDA, obtained from the acid hydrolysis of 1,1,3,3-tetraethoxy- propane stock solution. The MDA concentration of this standard solution was determined assuming a molar absorption coefficient of 13 700 M )1 Æcm )1 at 245 nm. This solution was then diluted with pH 7.4, 54 m M Tris/HCI buffer to obtain MDA concentrations in the 1–10 l M range and then, mixed with acetonitrile (1 : 1, v/v) before HPLC. Consumption of carotenoids The basal carotenoid content of LDL preparations was spectrophotometrically determined after extraction [31]. 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 preparation. 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 an average extinction coefficient of 140 000 M )1 Æcm )1 at 448 nm based on a calculation from the four main carotenes in human plasma, a-carotene, b-carotene, b-cryptoxanthin and lycopene [32,33]. Change in carotenoid concentration during LDL oxidative treatment was monitored by second derivative absorption spectroscopy (400–550 nm) through the measure of the amplitude of the second derivative spectrum between 489 and 516 nm. Urate consumption The urate peak in HPLC chromatograms (see above) was identified by comparison with reference chromatograms of freshly prepared standard urate solutions. The concentra- tion of urate in the samples was calculated from the peak area compared with that of standard solutions. RESULTS MDA production as a function of urate concentration After incubation for 15 min at 37 °C with various concentrations of urate, LDL solutions were exposed to either 175 l M or 5 l M of CuCl 2 . One hour after Cu 2+ addition, the extent of LPO was estimated from MDA measurements as shown in Fig. 1(A). At high Cu 2+ concentration (175 l M ), low concentrations of urate (< 2 0 l M )increasestheCu 2+ -induced oxidation, whereas it becomes antioxidant at higher concentrations (> 30 l M ). Data reported in Fig. 1A, and in most other figures, were obtained from at least three experiments carried out with independent LDL preparations. It is worth noting that in Fig. 1A the standard deviations at 20 and 30 l M are rather large as compared with those obtained at lower or higher concentrations. Indeed, depending on the LDL prepar- ation, the 20 and 30 l M concentrations enhanced or lowered the LPO. In other words, the 25 l M is an average threshold to switch between a pro- and antioxidant activity of urate in Cu 2+ -induced LDL oxidation. Hereafter, for thesakeofclarity,Ôlow urate concentrationsÕ means below the average threshold whereas Ôhigh urate concentrationsÕ means beyond the threshold. We will also refer to these concentrations as pro- and antioxidant concentrations, respectively. At low Cu 2+ concentration (5 l M ) a similar pattern is observed, as illustrated in Fig. 1B. At low urate Ó FEBS 2002 Urate in copper-induced LDL oxidation (Eur. J. Biochem. 269) 5475 concentrations (<200l M ) a pro-oxidant behavior is observed whereas the observation of antioxidant properties of urate requires higher urate concentration (‡ 800 l M ). The switch between the pro- and antioxidant properties of urate occurs at $ 400 l M urate, in the same manner as that explained above at the high Cu 2+ concentration. Interest- ingly, the switch from pro- to antioxidant behavior of urate occurs at higher urate concentrations at low Cu 2+ concen- trations. In other words, at low Cu 2+ concentration, urate behaves as an antioxidant at much higher urate concentra- tions than those required with high Cu 2+ concentration. Interestingly too, in the absence of urate, the extent of LDL peroxidation induced by high Cu 2+ concentration is only approximately four times larger than that observed with low Cu 2+ concentration. Moreover, in the presence of pro- oxidant concentrations of urate, while the amplification of LDL peroxidation by urate is % 200% with 175 l M Cu 2+ , it reaches about 700% with 5 l M Cu 2+ (Fig. 1). Time courses of MDA and conjugated diene formation It must be noted that the above data deal with static measurements performed 1 h after addition of Cu 2+ . Kinetic analyses may prove to be helpful in under- standing the observed effects. Cu 2+ -induced LPO in LDL was evaluated by monitoring the formation of MDA (Fig. 2A,C) and also conjugated dienes (Fig. 2B,D) in the presence or absence of urate. The experiments, carried out with pro- and antioxidant concentrations of urate, were performed with high Cu 2+ concentration (175 l M , Fig. 2A,B) and low Cu 2+ concentration (5 l M , Fig. 2C,D). In relation to static measurements (see above), somewhat large standard deviations were sometimes observed, partic- ularly during phases of rapidly increasing or decreasing changes in the monitored concentrations. This is due to slight shifts between the onsets of the increasing or decreasing phases, because different LDL preparations we used to get data at least in triplicates. As to the MDA formation, Fig. 2A and C fully confirm the pro-oxidant activity of low urate concentrations with enhanced MDA formation. At high urate concentration, namely 50 or 800 l M for Cu 2+ concentrations equal to 175 and 5 l M , respectively, the MDA formation is fully inhibited up to 180 min of incubation with Cu 2+ , clearly illustrating the antioxidant activity of urate at such concentrations. The time courses of conjugated diene formation (Fig. 2B,D) exhibit the classical shape characterized by a lag time followed by a linear increase until a maximum followed by a slight decrease [34]. At low Cu 2+ concentra- tion, the lag time is longer and the maximum is reached after a longer incubation time though the lag times at low and high Cu 2+ are rather close. Interestingly there is no major difference in the maximum amount of conjugated dienes formed at low or high Cu 2+ concentrations. The main difference between low and high Cu 2+ concentrations is the less pronounced linear increase at low Cu 2+ concentration. AtbothlowandhighCu 2+ concentrations, the pro-oxidant activity of low urate concentrations is clearly observed, with shorter lag times. Antioxidant conditions (high urate concentration) are well characterized at high Cu 2+ concen- tration by a lag time longer than 180 min. At low Cu 2+ concentration, according to Fig. 1B data, the antioxidant behavior of urate requires very high urate concentrations (‡ 800 l M ) to be observed. Such high concentrations interfere with the differential second derivative absorption spectroscopy assay and impede accurate measurements of conjugated diene formation. However, no evident forma- tion of conjugated dienes may be suspected up to 180 min of incubation with Cu 2+ , in agreement with the lack of MDA formation during this period. Lag times for conjugated diene formation were evaluated from Fig. 2B,D and are summarized in Table 1. Time courses of urate and carotenoid consumption The consumption of urate (when present) (Fig. 3B,D) and the consumption of carotenoids were also measured (Fig. 3A,C). The latter was used as an index of the overall consumption of the LDL endogenous antioxidant. Thus, in parallel with the formation of MDA and conjugated diene, carotenoids are consumed as shown in Fig. 3A,C. The half- times of carotenoid consumption under the various experi- mental conditions can be estimated from Fig. 3B,D and are reported in Table 1. Antioxidant conditions (urate at high concentration) are characterized by longer half-consump- tion times, and are generally associated with longer lag times Fig. 1. Effect of urate on LDL oxidation induced by 175 l M (A) or 5 l M of Cu 2+ (B). In (A) and (B), LDL solution at 0.12 mgÆmL )1 (240 n M )in pH 7.4, 10 m M phosphate buffer was incubated for 15 min at 37 °C with various concentrations of urate. Then, 175 l M (A) or 5 l M (B)ofCuCl 2 were added and the mixture was further incubated at 37 °C for 1 h before MDA assay. In (A) and (B), controls in the absence of Cu 2+ (without or with urate) yielded nondetectable or negligible levels of MDA. *50 l M urate was added 30 min after Cu 2+ addition. **800 l M urate was added 30 min after Cu 2+ addition. Data are the means ± SD of at least three experiments performed with independent LDL preparations. 5476 P. Filipe et al. (Eur. J. Biochem. 269) Ó FEBS 2002 for conjugated diene formation. However, at high Cu 2+ concentration, there is no evident correlation because there is little difference in the half-time of carotenoid consumption in the absence or presence of a low concentration of urate, while a shorter lag time for conjugated diene formation is observed in the presence of urate as compared with that obtained in its absence. Indeed, pro-oxidant concentrations of urate only slightly reduce the half-time of carotenoid consumption. Finally, the time evolution of the urate concentration is shown in Fig. 3B,D. In the absence of Cu 2+ , there is no urate consumption, whatever pro- or antioxidant urate concentrations are used. In the presence of Cu 2+ , urate at pro-oxidant concentrations is rapidly consumed, while urate consumption is considerably slower at high (antioxidant) concentration. Effect of urate on copper-induced lipid peroxidation in preoxidized LDL In order to evaluate the effect of urate on the Cu 2+ -induced LPO in preoxidized LDL, LDL preparations were first incubated with Cu 2+ andthenuratewasaddedafterLPO started. As already shown in Fig. 1, urate at high concen- tration added before Cu 2+ behaves as an antioxidant. In contrast, when urate was added at high concentration 30 min after Cu 2+ addition, i.e. 30 min after the oxidation Table 1. Lag time before conjugated diene formation and half time for carotenoid consumption in Cu 2+ -treated LDL and Cu 2+ -treated MM-LDL in the absence or in the presence of high and low urate concentrations. Data in parentheses correspond to those obtained with MM-LDL. Detailed experimental conditions are those of Fig. 2. Urate at either 800 and 50 l M wasusedwithCu 2+ equal to 175 and 5 l M , respectively. Lag times before conjugated diene formation were estimated as the intercept of the linear part of diene formation kinetics with x-axis shown in Fig. 2B,D. Half-times for carotenoid consumption were obtained from the kinetics of carotenoid consumption shown in Fig. 3A,C. Conditions Lag time (min) Half time (min) Cu 2+ at 175 l M Cu 2+ at 5 l M Cu 2+ at 175 l M Cu 2+ at 5 l M No additive 24 (5) 33 (17) 27 (18) 45 (24) Urate (10 l M )12( a ) 9 (ND b ) 29 (9) 25 (ND b ) Urate (50 or 800 l M )39( a )– c () c ) > 180 (< 9) 155 (16) a Too short to be measured. b ND, not determined. c Not measurable because of high urate concentration (800 l M ) interfering in the differential second derivative spectroscopy assay. Fig. 2. Kinetic profiles of MDA (A,C) and of conjugated dienes (B,D) formation in LDL oxidation induced by 175 l M (A,B) or 5 l M of Cu 2+ (C,D). LDL solution at 0.12 mgÆmL )1 (240 n M )in10 m M phosphate buffer, pH 7.4, was incubated for 15 min at 37 °C either with or without urate. Then, 175 l M or 5 l M of CuCl 2 were added and the mixture was further incubated at 37 °C. Urate concentrations were 10 and 800 l M for LPO induction with 175 l M of Cu 2+ or 10 and 50 l M for LPO induction with 5 l M of Cu 2+ . MDA and conjugated dienes were measured at various interval after Cu 2+ addition. Note that time zero corresponds to the shortest time after the addition of Cu 2+ in all samples, e.g. 1 min. For controls, i.e. experiments in the absence of Cu 2+ , data in the presence of urate (low or high concentrations) are similar to those obtained in its absence. In (D) data for ()) were not measurable because of the high urate concentration (800 l M ) impeding the differential second derivative spectroscopy assay. Data are the means ± SD of at least three experiments performed with independent LDL preparations. Ó FEBS 2002 Urate in copper-induced LDL oxidation (Eur. J. Biochem. 269) 5477 had started, LPO was higher than that obtained in the absence of urate (Fig. 1). In other words, urate is a pro- oxidant under these conditions. It is noteworthy that at the moment of urate addition, e.g. 30 min after Cu 2+ , the LPO was rather low, according to data shown in Fig. 2. Such a behavior is observed either with low or high concentrations of Cu 2+ . With pro-oxidant instead of antioxidant concen- trations of urate, no significant increase in LPO associated with the delay in introducing urate in the reaction mixture was observed (data not shown). As a second model of slightly preoxidized LDL, we used LDL preparations that were kept in the dark at 4 °Cinthe presence of EDTA for 5–8 weeks. Such conditions are described in the literature as yielding the so-called minimally modified LDL (MM-LDL) [35,36]. Anti vs. pro-oxidant behavior of urate is observed from the time courses of conjugated diene formation and of carotenoid consumption (time courses not shown). As can be seen in Table 1, in the absence of urate, lag times for conjugated diene induction are shortened. In the presence of high urate concentration these lag times are not measurable. This definitely means that urate at high concentration is no longer an antioxidant under these conditions and behaves as a pro-oxidant. Moreover, pro-oxidant urate concentrations (low concen- tration) become more pro-oxidant. In agreement with these observations, in the presence of high urate concentrations, the carotenoid consumption is accelerated in MM-LDL as compared with native LDL (Table 1). Mechanistic approach of the pro-oxidant behavior of urate The involvement of the superoxide anion radical (O ÁÀ 2 )was tentatively probed by measuring MDA formation in experiments carried out in the absence or in the presence of SOD (15 UÆmL )1 ), using a pro-oxidant concentration of urate (10 l M ). As shown in Table 2, no effect of SOD was shown at low Cu 2+ concentration (5 l M ). On the other hand, at high Cu 2+ concentration (175 l M ), SOD inhibited the increase in MDA formation due to urate. The involve- ment of O ÁÀ 2 was also evaluated using 30 l M of ferricyto- chrome c. No ferricytochrome c reduction was observed at low Cu 2+ concentration. At high Cu 2+ concentration, about 5% reduction of ferricytochrome c was observed 1 h Fig. 3. Kinetic profiles of carotenoid (A,C) and urate (B,D) consumption in LDL oxidation induced by 175 l M (A,B) or 5 l M of Cu 2+ (C,D). Experimental conditions are those of Fig. 2. Urate and carotenoids were measured at various interval after Cu 2+ addition. For controls, shown in (A) and (C), i.e. experi- ments in the absence of Cu 2+ , data in the presence of urate (low or high concentrations) are similar to those obtained in its absence. Note that time zero in (B) and (D) corres- ponds to the shortest time after addition of Cu 2+ in all samples, e.g. 1 min. Data are expressed as a percentage of the value obtained before Cu 2+ addition and are the means ± SD of at least three experiments performed with independent LDL prepara- tions. Table 2. Effect of SOD and ferricytochrome c on the amplification by urate of LDL oxidation induced by 175 l M or 5 l M Cu 2+ . LDL solution at 0.12 mgÆmL )1 (240 n M )in10m M phosphate buffer, pH 7.4, were incubated for 15 min at 37 °C with or without 10 l M urate and with or without SOD or ferricytochrome c. Then, CuCl 2 was added and the mixture was further incubated at 37 °C for 1 h before MDA assay. Conditions SOD a Ferricytochrome c b 0UÆmL )1 15 UÆmL )1 0 l M 30 l M Cu 2+ ¼ 175 l M 207 ± 72 86 ± 58 261 ± 29 8.3 ± 1.8 Cu 2+ ¼ 5 l M 398 ± 120 418 ± 146 509 ± 60 547 ± 95 a Data are expressed as a percentage of MDA produced in the absence of urate and SOD, and are the means ± SD of eight (Cu 2+ ¼ 175 l M ) or six (Cu 2+ ¼ 5 l M ) experiments performed with independent LDL preparations. b Data are expressed as a percentage of MDA produced in the absence of urate and ferricytochrome c, and are the means ± SD of three experiments performed with inde- pendent LDL preparations. 5478 P. Filipe et al. (Eur. J. Biochem. 269) Ó FEBS 2002 after Cu 2+ addition (Fig. 4) and the increase in MDA formation due to the presence of pro-oxidant urate is abolished (see Table 2). In the presence of ferricyto- chrome c, urate is protected because 2.5 ± 0.65 of the initial 10 l M urate were still present 1 h after of Cu 2+ addition, whereas it was entirely consumed in the absence of ferricytochrome c. Finally, in order to specify the role of copper on the pro-oxidant effect of urate, experiments were performed using the copper chelators EDTA which chelates Cu 2+ and Cu + , and neocuproine which selectively chelates Cu + [37]. At low Cu 2+ concentration (5 l M ), no LDL oxidation was observed in the presence of either 100 l M EDTA or 375 l M neocuproine. Upon addition of 10 l M urate, no stimulation of LDL oxidation was observed suggesting that the pro-oxidant activity of urate depends on the availability of either Cu 2+ or Cu + (Table 3). At high Cu 2+ concentration (175 l M ), no LDL oxidation was observed in the presence of 5 m M EDTA and no stimulation of LDL oxidation occurred in the presence of 10 l M urate, suggesting the need for available Cu 2+ (Table 3). At high Cu 2+ concentration, no conclusion regarding the need for Cu + can be drawn as we observed a stimulation by neocuproine, as already reported by Bellomo et al. and Peterson [23,38] with bathocuproine. Under these condi- tions, no changes were associated with the addition of urate (Table 3). DISCUSSION The oxidation of LDL has been extensively studied during the 15 past years, and various in vitro models have been developed in an attempt to better understand the in vivo situationinrelationtothepotentialroleofLDL oxidation in pathological or prepathological situations, particularly in atherogenesis [12–15]. Much attention has been devoted to the oxidation of LDL by Cu 2+ ions which is widely used as a model system [39]. However, the exact mechanisms relating Cu 2+ redox change to the initiation of LPO in LDL are not yet clearly established [40]. In the presence of pre-existing traces of hydroperox- ides both Cu 2+ and Cu + may induce LPO according to the following reactions: Cu 2þ þ LOOH ! Cu þ þ LOO Á þ H þ ð1Þ Cu þ þ LOOH ! Cu 2þ þ LO Á þ OH À ð2Þ LOO Á þ LH ! LOOH þ L Á ð3Þ LO Á þ LH ! LOH þ L Á ð4Þ L Á þ O 2 ! LOO Á ð5Þ Reaction 1 is rather unlikely because it is thermodynami- cally unfavorable and it has been shown that the presence of pre-existing hydroperoxides is not a prerequisite for LDL oxidation. It is currently acknowledged that Cu 2+ reduction to Cu + is required for triggering LPO in LDL [41], but the nature of reductants in LDL, such as pre-existing LOOH, tryptophan residues and a-tocopherol, is still a matter of debate [37,38,42–48]. Perigini et al. [46] demonstrated that these different mechanisms are progressively recruited to promote Cu 2+ reduction. Although not demonstrated, the involvement of O ÁÀ 2 has been suggested [49], according to the reaction: Cu þ þ O 2 ! Cu 2þ þ O ÁÀ 2 ð6Þ After hydrogen abstraction from at least trienic fatty acid structures (reaction 3), the chemical rearrangement of L Æ Fig. 4. Kinetic profiles of ferricytochrome c reduction during LDL oxidation induced by 5 l M or 175 l M of Cu 2+ in the absence or presence of 10 l M urate. LDL solution at 0.12 mgÆmL )1 (240 n M )in10m M phosphate buffer, pH 7.4, were incubated for 15 min at 37 °Cwith 30 l M ferricytochrome c, either with or without urate. Then, 175 l M or 5 l M of CuCl 2 were added and the mixture was further incubated at 37 °C. Ferricytochrome c reduction was determined from the ampli- tude of the signal of the absorption second derivative spectra at 547 nm. Data are expressed as a percentage of the full reduction of ferricytochrome c and are the means ± SD of three experiments performed with independent LDL preparations. One hundred per- cent reduction was obtained with an excess of sodium dithionite as reductant. Table 3. Effect of EDTA and neocuproine on LDL oxidation induced by 175 l M or 5 l M Cu 2+ in the presence and absence of urate. LDL solution at 0.12 mgÆmL )1 (240 n M )in10m M phosphate buffer, pH 7.4, were incubated for 15 min at 37 °C with or without 10 l M urate and with or without EDTA or neocuproine. Then, CuCl 2 was added and the mixture was further incubated at 37 °C for 1 h before MDA assay. Data are the means ± SD of two experiments performed with independent LDL preparations (except *single experiment). EDTA concentrations were 0.1 or 5 m M for 5 l M or 175 l M Cu 2+ , respectively. Neocuproine concentrations were 375 or 750 l M , respectively. Conditions EDTA Neocuproine Without urate With urate Without urate With urate Cu 2+ ¼ 5 l M 0.032 ± 0.005 0 0 0.25 ± 0.30 Cu 2+ ¼ 175 l M 0.13* 0.15* 10.3 ± 1.1 11.3 ± 0.9 Ó FEBS 2002 Urate in copper-induced LDL oxidation (Eur. J. Biochem. 269) 5479 radicals leads to conjugated diene radicals which further react with O 2 (reaction 5) and finally yield hydroperoxides and cyclic endoperoxides containing the conjugated diene structure. A slow formation of conjugated diene structures occurs in the lag phase during which (endogenous) antioxi- dants are consumed, before the propagation phase corres- ponding to the chain reaction (reactions 3 and 5). The formation of conjugated dienes and the consumption of endogenous antioxidants are therefore early events of the LDL oxidation. Fragmentation of peroxides to aldehydes occurs later, including the formation of MDA from cyclic endoperoxides, whose measurement provides an overall evaluation of the peroxidation process [34]. It should be noted that carotenoids are bleached by directly reacting with lipid hydroperoxyl radicals [50]. Urate is generally considered as an antioxidant. The mechanisms of its antioxidant effect include the capability of urate to scavenge reactive species, and to chelate transition metal ions [7–11]. However, urate has been reported to enhance the oxidative stress under some circumstances. For instance, it increases the inactivation of a 1 -antiproteinase [51] and alcohol dehydrogenase [52] induced by hydroxyl radicals, and the oxidation of LDL mediated by peroxynitrite [53]. It must be pointed out that besides the commonly used low Cu 2+ concentrations (5 l M here), a rather high Cu 2+ concentration (175 l M ) was also used both here and in a previous report [26] to overcome the chelating ability of urate. Our results clearly demonstrate that a switch between anti- and pro-oxidant behavior of urate can be observed. Indeed, in contrast to native LDL and in agreement with others, antioxidant concentrations of urate (50 l M and 800 l M with 175 and 5 l M of Cu 2+ , respectively) are definitely pro-oxidant when preoxidized LDL preparations are exposed to Cu 2+ , preoxidized LDL being modeled by MM-LDL or by native LDL exposed to Cu 2+ before urate. Such a behavior has already been reported for other antioxidants. Yamanaka et al. [54,55] showed that caffeic acid (–)-epicatechin and (–)-epigallo- catechin enhanced LDL oxidation induced by Cu 2+ , when added during the propagation phase. Otero et al.[56] reported a delayed lipid peroxidation when ascorbic acid, dehydroascorbic acid, and a flavonoid extract were added to LDL suspensions at the beginning of the oxidation process induced by the addition of 2 l M copper chloride. In contrast, a pro-oxidant effect was noted when they were added at different times after the addition of copper ions [56]. In the case of urate, a similar behavior has been reported by Abuja [22] and Bagnati et al.[23]. Bagnati et al. studied the pro-oxidant effect of urate added at the end of the lag phase or during the propagation phase. They concluded that the switch between anti- and pro-oxidant activities was related to the availability of hydroperoxides formed during the early phases of the Cu 2+ -induced LDL oxidation. They suggested that urate accelerates the LPO by reducing Cu 2+ to Cu + , according to: Cu 2þ þ UH À 2 ! Cu þ þ UH ÁÀ þ H þ ð7Þ thus making more Cu + available for decomposition of lipid peroxides and propagation reactions. We may point out that both Abuja [22] and Bagnati et al. [23] observed this pro-oxidant effect of urate on preoxidized LDL with relatively low urate concentrations (20 and 10 l M , respectively) while using low Cu 2+ concentrations (1.6 and 2.5 l M , respectively). At low Cu 2+ concentration (5 l M ), not only did we observe this pro-oxidant effect of urate at low concentration (10 l M , data not shown), but we also observed this effect at a much higher urate concentra- tion (800 l M ) corresponding to antioxidant concentration when working with native LDL (see below). Finally Bagnati et al. [23] reported that 10 l M urate introduced in the LDL solutions 30 min after Cu 2+ clearly stimulated the peroxi- dation only for Cu 2+ to LDL ratios lower than 50. In contrast, at a higher Cu 2+ /LDL ratio (Cu 2+ ¼ 175 l M and LDL ¼ 0.24 l M ), we found that under similar conditions, lower (10 l M ) and higher (50 l M ) urate concentrations were still pro-oxidant. In native LDL, a switch between the pro-oxidant and antioxidant behavior of urate occurs, depending on the urate concentration. Thus, by measuring free MDA formation, we found that the Cu 2+ -induced oxidation exhibits a bell-shaped curve as a function of the urate concentration. This is fully confirmed by the kinetic studies of MDA and conjugated diene formation. Thus low urate concentrations shorten the lag time of conjugated diene formation whereas high concentrations increase it. Consis- tent with these observations, carotenoids were consumed more rapidly at low urate concentrations than in the absence of urate, but carotenoid consumption was delayed at high antioxidant urate concentration. No formation of MDA and of conjugated diene and no carotenoid con- sumption are observed at high urate concentrations because the overall antioxidant properties of urate, including scavenging of reactive species and chelation of transition metal ions, overcome its pro-oxidant action. It is quite interesting to note that urate at low concentration, i.e. at pro-oxidant concentration, is practically fully consumed during the lag phase for LPO induction. It is obvious that our data require commenting on. First, as compared with high Cu 2+ concentration (175 l M ), low Cu 2+ concentra- tion (5 l M ) used for triggering LPO of LDL paradoxically requires a much higher urate concentration in order to observe the antioxidant behavior (Fig. 1). Second, and accordingly, the switch from anti- to pro-oxidant behavior of urate occurs with low Cu 2+ concentration at a much higher urate concentration than that observed with the high Cu 2+ concentration. Third, our data do not fully agree with those of Abuja [22] and Bagnati et al. [23] who used low Cu 2+ concentrations, 1.6 and 2.5 l M , respectively, and found that 20 and 10 l M , respectively, of urate were antioxidant. At present we have no conclusive explanation for such a discrepancy (see below). From previous comments regarding the pro-oxidant behavior of urate towards preoxidized LDL, one might think that abnormally high levels of preoxidized lipid hydroperoxides could be present in our LDL prepara- tions. There are several arguments against such an assumption. First, the time courses of conjugated diene formation obtained in the absence of urate were similar to those obtained by Abuja [22] and Bagnati et al.[23] and, thus, failed to show any enhanced potential for oxidation of our LDL preparation. Second, with our LDL preparation, there is no apparent consumption of endogenous antioxidants like carotenoids, which should occur in oxidized material. However, these arguments do not allow us to rule out the fact that extremely low levels 5480 P. Filipe et al. (Eur. J. Biochem. 269) Ó FEBS 2002 of lipid hydroperoxides are present in our LDL prepa- rations as a result of our experimental procedure for their preparation. Indeed, the main experimental difference between these former studies and our study resides at the level of the LDL preparation. After LDL isolation, we removed EDTA via an extensive dialysis whereas desalt- ing columns were used in the above-mentioned studies. To rule out such a hypothesis, two sets of experiments were carried out. First, we desalted the LDL preparation through a size exclusion filtration on Bio-Rad Econo-Pac 10DG desalting columns (one or two successive filtra- tions) according to the procedure used by Abuja [22] or Bagnati et al. [23]. Second, dialysis was used, as described in the experimental section, against buffer containing 2 l M EDTA to prevent LDL oxidation during the dialysis. Then LDL preparations were diluted before experiments with buffer containing EDTA to achieve a final EDTA concen- tration of 0.2 l M much lower than the Cu 2+ concentration. Using these both experimental conditions, we still observed the pro-oxidant behavior of urate (data not shown). Thus, it may be suggested that there are no major differences in the levels of pre-existing hydroperoxides, whatever the tech- nique used for EDTA removal. As mentioned above, urate may reduce Cu 2+ to Cu + (reaction 7) providing a high concentration and rapidly reached stationary state of Cu + that accelerates LPO because of the reaction of Cu + with pre-existing traces of lipid hydroperoxides. This is in agreement with the lack of urate-enhanced LPO observed in the presence of neocup- roine as a Cu + chelator. The reduction of Cu 2+ is required to observe the urate-amplified LDL oxidation as no LPO was found in the presence of EDTA. Moreover, no amplification by urate was observed when LDL oxidation was triggered by 2,2¢-azo-bis(2-amidinopropane) hydrochlo- ride. Instead, we found that 10 l M urate protected LDL from the oxidation induced by 4 m M 2,2¢-azo-bis(2-amidi- nopropane) hydrochloride (time courses of MDA and conjugated diene formation, time courses of carotenoid consumption, data not shown). As a consequence of Cu 2+ reduction by urate, urate radicals (UH Æ– ) are rapidly formed as products of this reaction. Thus, the dismutation of UH Æ– could explain the fast urate consumption rate. If such a view is consistent with the data observed at low Cu 2+ concentrations, it does not explain results obtained with SOD and ferricytochrome c at high Cu 2+ concentrations (Table 2). Both superoxide dismutase and ferricyto- chrome c inhibited the MDA formation in the presence of urate suggesting the involvement of O ÁÀ 2 . In support of the involvement of O ÁÀ 2 , we observed the reduction of ferricytochrome c. It may be suggested that following Cu + formation, reduction of oxygen would occur, producing significant amounts of O 2 – (reaction 6). As a consequence of the reduction of Cu 2+ by urate and of oxygen by Cu + , UH Æ– and O ÁÀ 2 will be concomitantly formed. Thus, O ÁÀ 2 could react with the simultaneously formed UH Æ– , as recently demonstrated by pulse radiolysis [57] according to the following reaction: UH ÁÀ þ O ÁÀ 2 ! product(s) or intermediate(s) ð8Þ It may be suggested that the product(s) or intermediate(s) of the reaction may trigger the observed urate-amplified LDL peroxidation. The reaction between UH Æ– and O ÁÀ 2 could be considered as an activation of O ÁÀ 2 , and would explain, as emphasized in our pulse radiolysis study [57], the already observed pro-oxidant activity of urate [58]. It is unlikely that the pro-oxidant activity of urate is due to the reaction of urate with O ÁÀ 2 , as little urate is destroyed in an O ÁÀ 2 -generating system [58] and O ÁÀ 2 reacts with urate with a rather small reaction rate constant [10]. The reaction between UH Æ– and O ÁÀ 2 would contribute to urate con- sumption and therefore may explain the observed protec- tion of urate consumption by ferricytochrome c.Atlow Cu 2+ concentrations, stationary UH Æ– and O ÁÀ 2 concen- trations are expected to be much lower and therefore their bimolecular reaction becomes negligible and does not account for the urate-amplified LDL oxidation. Thus, at low Cu 2+ concentrations, another mechanism, independ- ent of O ÁÀ 2 , might be involved in the pro-oxidant action of urate. According to the data obtained in the presence of neocuproine, it may be suggested that this mechanism involves Cu + , whose levels would be increased because of the reduction of Cu 2+ by urate. However, such enhanced levels would also occur at high Cu 2+ concentration. It may be speculated that this intriguing behavior is closely related to complex urate–Cu 2+ –LDL interactions and it requires further investigation. It is rather tempting to speculate that pro- and antioxidant properties of urate will strongly depend on its binding to LDL. According to our data, the pro-oxidant activity of urate is associated with the reduc- tion of Cu 2+ to Cu + by urate. Thus, it may be supposed that the pro-oxidant activity of urate is related to its binding to LDL sites that are also able to bind Cu 2+ . Depending on both the Cu 2+ and the urate concentrations (i.e. on their ratio), the reaction path may be very different. With both Cu 2+ concentrations (high and low), the antioxidant behavior is observed by increasing urate concentration. These properties might be related to both the scavenging and chelating ability of urate. Interestingly, we demonstrated that at low Cu 2+ concentration, the antioxidant deterrent of urate necessitates higher urate concentrations than at high Cu 2+ concentration; this observation again suggests peculiar effects due to the complex urate–Cu 2+ –LDL interactions. CONCLUSION The results described here: (a) confirm a pro-oxidant behavior of high urate concentrations towards slightly oxidized LDL; (b) suggest a pro-oxidant behavior of low urate concentration towards native LDL; and (c) suggest that different mechanisms could explain the Cu 2+ concen- tration-dependent pro-oxidant effect of urate. It is accepted that in vivo, LDL oxidation proceeds in the interstitial subendothelial space in the presence of trace amounts of transition metal ions and activated macrophages. At physiological concentrations, urate might promote the atherogenic process by accelerating the peroxidation of MM-LDL in the subendothelial space and in the athero- sclerotic plaque. Finally, the present results bring new insights into the intricate relationship between the anti- and/or pro-oxidant action of antioxidants. But the detailed mechanisms of this pro-oxidant action deserve further investigation. Ó FEBS 2002 Urate in copper-induced LDL oxidation (Eur. J. Biochem. 269) 5481 ACKNOWLEDGMENTS This work was partly supported by grant Praxis/2/2.1/QUI/225/94 from the Fundac¸ a ˜ oparaaCieˆ ncia e Tecnologia, by travel grant no. 347 C0 from the Ambassade de France au Portugal, and the Instituto de Cooperac¸ a ˜ oCientı ´ fica e Tecnolo ´ gica Internacional (ICCTI), and by an exchange grant from INSERM and ICCTI. CM and J-CM thank the Universite ´ de Picardie Jules Verne and the Ministe ` re de la Recherche et de la Technologie for financial support. REFERENCES 1. Ryan, M., Grayson, L. & Clarke, D.J. (1997) The total anti- oxidant capacity of human serum measured using chemilumines- cence is almost completely accounted for by urate. Ann. Clin. 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Cu 2+ -induced oxidation of isolated low-density lipoprotein (LDL) was investigated by mon- itoring the formation of malondialdehyde and conjugated dienes and the consumption of urate and carotenoids Anti- and pro-oxidant effects of urate in copper-induced low-density lipoprotein oxidation Paulo Filipe 1,2 , Josiane Haigle 3 , Joa ˜ o Freitas 1,2 , Afonso Fernandes 1 , Jean-Claude. would explain, as emphasized in our pulse radiolysis study [57], the already observed pro-oxidant activity of urate [58]. It is unlikely that the pro-oxidant activity of urate is due to the reaction of