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,
Trang 1Anti- 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´ Santus3,5and Patrice Morlie`re3,5
1 Centro de Metabolismo e Endocrinologia and 2 Clinı´ca Dermatolo´gica Universita´ria, Faculdade de Medicina de Lisboa, Hospital de Santa Maria, Lisbon, Portugal; 3 Muse´um National d’Histoire Naturelle, RDDM, Photobiologie – INSERM U.532, Paris, France;
4 Laboratoire de Biochimie, Universite´ de Picardie Jules Verne, U Amiens, Hoˆpital Nord, Amiens, France; 5 Institut de Recherche sur
la Peau, INSERM U.532, Hoˆpital Saint-Louis, Paris, France
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
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
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)
Trang 2It 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
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
Preparation and treatment of LDL
Serum samples were obtained from healthy volunteers
LDL (d¼ 1.024–1.050)were prepared by sequential
ultra-centrifugation according to Havel et al [38] and dialyzed
against pH 7.4, 5 mMTris buffer containing 50 mMNaCl
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 mMphosphate buffer
and/or 10 lL of a stock solution of the studied flavonoid in
pH 7.4, 10 mMphosphate 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 37C 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 37C 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 37C or at
various times during incubation
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 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 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 chromatogram of free MDA, freshly prepared by acid hydrolysis of 1,1,3,3-tetraethoxypropane stock solution The MDA concentration of this standard solution was determined assuming a molar absorbance of
13 700M )1Æcm)1at 245 nm This solution was then diluted with pH 7.4, 54 mM Tris-HCI buffer to obtain MDA concentrations in the 1–10 lMrange 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 [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
an average extinction coefficient of 140 000M )1Æcm)1 at
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
Trang 3MDA production as a function of the quercetin
concentration
After incubation for 15 min at 37C with various
concen-trations of quercetin, LDLs were exposed to 175 lM or
5 lMCuCl2 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
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 1.5 lMof 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 lMis
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
were obtained when LPO was estimated by the production
of thiobarbituric reactive substances or conjugated dienes (data not shown)
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 static conditions 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 lMurate 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 lMquercetin 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 lMquercetin 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
At low Cu2+concentration, the kinetic data in Fig 2C are in agreement with those obtained under static condi-tions in Fig 1A; that is to say 10 lM urate strongly enhances MDA production whereas 0.5 lMquercetin 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 l M (A) or 5 l M Cu2+(B) in the absence or in the presence of 10 l M urate In (A)and (B), LDL at 0.12 mgÆmL)1(240 n M )in pH 7.4, 10 m M phosphate buffer were incubated for 15 min at 37 C with various concentrations of quercetin, with or without urate Then, 175 l M (A)or 5 l M (B)of CuCl 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 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.
Trang 4up 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 lMurate as no MDA is formed
during 3 h
The same conclusions can be drawn with regard to the
formation of conjugated dienes at low Cu2+concentrations
(Fig 2D) Again, when separately added, 10 lM urate
shortens the lag time, in agreement with a pro-oxidant
behavior whereas 0.5 lMquercetin exhibits a moderate
pro-oxidant behavior When added together 0.5 lMquercetin
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 lMurate
Time courses of carotenoids and urate consumption
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 formaconcentra-tion of
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)
At low Cu2+concentration, 0.5 lMquercetin 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 lMquercetin and fully protected by 2 lMquercetin (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)
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 l M (A,B) or 5 l M (C,D) Cu2+ LDL 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 or without quercetin, and with or without
10 l M urate Then, 175 l M or 5 l M of CuCl 2 were added and the mixture was further incubated at 37 C MDA: Conjugated dienes were measured
at various intervals after Cu 2+ addition Note that time zero corresponds to the shortest time after addition of Cu 2+ in all samples, e.g 1 min Control experiments in the absence of Cu 2+ 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.
Trang 5quercetin 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
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 4C 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
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
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 (C,D) Cu2+ Experi-mental conditions are those of Fig 2 Urate and carotenoids were measured at various intervals after Cu 2+ 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 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 mean ± SD
of at least three experiments performed with independent LDL preparations.
Fig 4 Effect of quercetin on Cu2+-induced LDL oxidation in
preoxi-dized LDL CuCl 2 (175 or 5 l M )was added to LDL solutions at
0.12 mgÆmL)1(240 n M )in pH 7.4, 10 m M phosphate buffer preheated
for 15 min at 37 C, and the mixture was further incubated at 37 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 l M for Cu 2+ concentrations equal to 175 or 5 l M ,
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.
Trang 6involved 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
demonstrated that•
O2, generated by Cu2+oxidation may
be involved [60]
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 lMhere), 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
[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 antioxidants promote LPO by increasing the concen-tration of catalytic Cu+because of their ability to reduce
Cu2+ 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
antioxidant 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 lMquercetin but strongly abolished by 2 lMquercetin (Fig 2D) As to the consump-tion of endogenous carotenoids, the pro-oxidant behavior is still possibly observed at low quercetin concentration (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+
concentration,•
O2 was involved probably formed by the oxidation of Cu+by O2 We suggested that the concomitant formation of•
UH–and•
O2 could allow a reaction between these species, thus leading to some kind of•
O activation
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 l M for Cu 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 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).
Conditions
Lag time (min)Half time (min)
Cu 2+
¼ 175 l M Cu 2+
¼ 5 l M Cu 2+
¼ 175 l M Cu 2+
¼ 5 l M
Without quercetin 5 ( 24) 17 ( 33) 18 ( 27) 24 ( 45)
With quercetin ( 39) a
6 (> 120) 14 ( 50) 12 (> 150)
a
Too short to be measured.
Trang 7At low Cu2+concentration, the exact mechanism was not
elucidated, but an•
O2-independent mechanism was pro-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 antipro-oxidant behavior occurs at about 1 lMwith
quercetin and 200 lMwith 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
the couple•
QH–,H+/QH2 (0.33 vs NHE [12,67])is higher
than that of the couple•
UH–,H+/UH2 (0.59 vs NHE [34])
The reduction of Cu2+to Cu+(E 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
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 lMquercetin 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–,
H+/UH couple (0.59 V vs NHE)is higher than that of
the •
QH–,H+/QH2 couple (0.33 V vs NHE)[12,34,67] 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+ concentration, where•
O2 is believed to be involved in the pro-oxidant action of urate [37], the reaction of•
UH–with quercetin competes with its reaction with•
O2 and impedes the pro-oxidant activity of urate to occur At low Cu2+ concentration, another unidentified mechanism was sug-gested where •
O2 is not involved, but where Cu+ is necessary [37] As a consequence of the reaction 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 antipro-oxidant activity as mentioned above, whereas, in the presence of quercetin, the pro-oxidant activity of urate disappears and its antioxidant activity is revealed
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 remain un-understandable Indeed, during the •
UH– 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
It may be anticipated that the intricate relationship 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 recent 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
Trang 8This work was partly supported by an exchange grant from INSERM
and GRICES C M and J.-C M thank the Universite´ de Picardie
Jules Verne and the Ministe`re de la Recherche et de la Technologie for
financial support.
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