At high Cu2+ concentration 175 lM,low concentrations of urate < 20 lM increases the Cu2+-induced oxidation,whereas it becomes antioxidant at higher concentrations > 30 lM.. concentration
Trang 1Anti- and pro-oxidant effects of urate in copper-induced low-density lipoprotein oxidation
Paulo Filipe1,2, Josiane Haigle3, 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, Faculdade de Medicina de Lisboa, Portugal;2Clı´nica Dermatolo´gica Universita´ria, Faculdade de Medicina de Lisboa, Hospital de Santa Maria, Lisbon, Portugal;3Laboratoire de Photobiologie, Muse´um National d’Histoire Naturelle, Paris, France;4Laboratoire de Biochimie, Universite´ de Picardie Jules Verne, CHRU Amiens, Hoˆpital Nord, Amiens, France;5INSERM U.532, Institut de Recherche sur la Peau, Hoˆpital Saint-Louis, Paris, France
We reported earlier that urate may behave as a
pro-oxidant in Cu2+-induced oxidation of diluted plasma
Thus,its effect on Cu2+-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 Cu2+
concentration,the switch between the pro- and
antioxid-ant behavior of urate occurs at different urate
concen-trations At high Cu2+concentration,in the presence of
urate,superoxide dismutase and ferricytochrome c protect
LDL from oxidation but no protection is observed at low
Cu2+concentration The use of Cu2+ or Cu+ chelators
demonstrates that both copper redox states are required
We suggest that two mechanisms occur depending on the
Cu2+ concentration Urate may reduce Cu2+ to Cu+,
which in turn contributes to O 2 formation The Cu2+ reduction is likely to produce the urate radical (UHÆ–)
It is proposed that at high Cu2+ concentration,the reaction of UHÆ– radical with O 2 generates products or intermediates,which trigger LDL oxidation At low Cu2+ 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–Cu2+ 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
Cu2+-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; UHÆ–,urate radical.
Enzyme: copper-zinc superoxide dismutase (EC 1.15.1.1).
(Received 11 July 2002,revised 4 September 2002,
accepted 10 September 2002)
Trang 2Sodium urate (Na+,UH2),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 (d¼ 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 mMphosphate buffer,pH 7.4,to remove
EDTA Then,LDL preparations were diluted to a final
concentration of 0.15 mgÆmL)1 (300 nM) To 800 lL of
these diluted LDL preparations were added 50 lL of a
stock solution of urate in pH 7.4,10 mMphosphate buffer
and 100 lL of buffer Blank LDL solutions devoid of urate
were also prepared These LDL solutions were then
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 final concentrations of Cu2+of 175 or 5 lM After
Cu2+ addition,lipid peroxidation,urate and carotenoid
consumption were measured,as described below,after a 1-h
incubation period at 37C 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 lL of the sample
were diluted 10-fold with pH 7.4,10 mMphosphate buffer
before spectrum recording The second derivative spectrum
was subtracted from the second derivative spectrum of the
matching control sample without Cu2+ 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 [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 mM
Tris/HCl and acetonitrile (30 : 70,v/v) The flow rate was
1.2 mLÆmin)1and the absorption was monitored at 270 nm
The MDA peak was identified by comparison with a
reference chromatogram of freshly prepared free MDA,
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 [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 000M )1Æcm)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
R E S U L T S
MDA production as a function of urate concentration After incubation for 15 min at 37C with various concentrations of urate,LDL solutions were exposed to either 175 lM or 5 lM of CuCl2 One hour after Cu2+ addition,the extent of LPO was estimated from MDA measurements as shown in Fig 1(A) At high Cu2+ concentration (175 lM),low concentrations of urate (< 20 lM) increases the Cu2+-induced oxidation,whereas
it becomes antioxidant at higher concentrations (> 30 lM) 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 lMare rather large as compared with those obtained at lower or higher concentrations Indeed,depending on the LDL prepar-ation,the 20 and 30 lM concentrations enhanced or lowered the LPO In other words,the 25 lMis an average threshold to switch between a pro- and antioxidant activity
of urate in Cu2+-induced LDL oxidation Hereafter,for the sake of clarity, low urate concentrations means below the average threshold whereas high urate concentrations means beyond the threshold We will also refer to these concentrations as pro- and antioxidant concentrations, respectively
At low Cu2+ concentration (5 lM) a similar pattern
is observed,as illustrated in Fig 1B At low urate
Trang 3concentrations (< 200 lM) a pro-oxidant behavior is
observed whereas the observation of antioxidant properties
of urate requires higher urate concentration (‡ 800 lM)
The switch between the pro- and antioxidant properties of
urate occurs at 400 lMurate,in the same manner as that
explained above at the high Cu2+concentration
Interest-ingly,the switch from pro- to antioxidant behavior of urate
occurs at higher urate concentrations at low Cu2+
concen-trations In other words,at low Cu2+concentration,urate
behaves as an antioxidant at much higher urate
concentra-tions than those required with high Cu2+ concentration
Interestingly too,in the absence of urate,the extent of LDL
peroxidation induced by high Cu2+concentration is only
approximately four times larger than that observed with low
Cu2+ concentration Moreover,in the presence of
pro-oxidant concentrations of urate,while the amplification of
LDL peroxidation by urate is 200% with 175 lMCu2+,it
reaches about 700% with 5 lMCu2+(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 Cu2+
Kinetic analyses may prove to be helpful in
under-standing the observed effects Cu2+-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 Cu2+ concentration (175 lM,
Fig 2A,B) and low Cu2+concentration (5 lM,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 lM for Cu2+ concentrations equal to 175 and 5 lM,
respectively,the MDA formation is fully inhibited up to
180 min of incubation with Cu2+,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 Cu2+ 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 Cu2+are rather close Interestingly there is no major difference in the maximum amount of conjugated dienes formed at low or high Cu2+ concentrations The main difference between low and high Cu2+concentrations is the less pronounced linear increase at low Cu2+concentration
At both low and high Cu2+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 Cu2+ concen-tration by a lag time longer than 180 min At low Cu2+ concentration,according to Fig 1B data,the antioxidant behavior of urate requires very high urate concentrations (‡ 800 lM) 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 Cu2+,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) 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 Cu 2+ (without or with urate) yielded nondetectable or negligible levels of MDA *50 l M urate was added 30 min after Cu2+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.
Trang 4for conjugated diene formation However,at high Cu2+
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
Cu2+,there is no urate consumption,whatever pro- or
antioxidant urate concentrations are used In the presence of
Cu2+,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 Cu2+-induced LPO in preoxidized LDL,LDL preparations were first incubated with Cu2+and then urate was added after LPO started As already shown in Fig 1,urate at high concen-tration added before Cu2+behaves as an antioxidant In contrast,when urate was added at high concentration
30 min after Cu2+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 was used with 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 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
Cu 2+ at 175 l M Cu 2+ at 5 l M Cu 2+ at 175 l M Cu 2+ at 5 l M
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 ) in 10 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
Cu2+addition Note that time zero corresponds to the shortest time after the addition of Cu2+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.
Trang 5had 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 Cu2+,the LPO
was rather low,according to data shown in Fig 2 Such a
behavior is observed either with low or high concentrations
of Cu2+ 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 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) [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 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 lM) As shown in Table 2,no effect of SOD was shown at low Cu2+concentration (5 lM) On the other hand,at high Cu2+concentration (175 lM),SOD inhibited the increase in MDA formation due to urate The involve-ment of O 2 was also evaluated using 30 lMof ferricyto-chrome c No ferricytoferricyto-chrome c reduction was observed at low Cu2+ concentration At high Cu2+ 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 Cu2+ (C,D) Experimental conditions are those of Fig 2 Urate and carotenoids were measured
at various interval after Cu2+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
Cu2+in all samples,e.g 1 min Data are expressed as a percentage of the value obtained before Cu2+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 Cu2+ LDL solution at 0.12 mgÆmL)1(240 n M ) in 10 m 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
a Data are expressed as a percentage of MDA produced in the absence of urate and SOD,and are the means ± SD of eight (Cu2+¼ 175 l M ) or six (Cu2+¼ 5 l M ) experiments performed with independent LDL preparations.bData 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.
Trang 6after Cu2+ 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 lM urate were still present 1 h after of Cu2+
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
Cu2+and Cu+,and neocuproine which selectively chelates
Cu+ [37] At low Cu2+ concentration (5 lM),no LDL
oxidation was observed in the presence of either 100 lM
EDTA or 375 lM neocuproine Upon addition of 10 lM
urate,no stimulation of LDL oxidation was observed
suggesting that the pro-oxidant activity of urate depends on
the availability of either Cu2+or Cu+(Table 3) At high
Cu2+ concentration (175 lM),no LDL oxidation was
observed in the presence of 5 mMEDTA and no stimulation
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)
D I S C U S S I O N
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 situation in relation to the potential role of LDL oxidation in pathological or prepathological situations, particularly in atherogenesis [12–15] Much attention has been devoted to the oxidation of LDL by Cu2+ ions which is widely used as a model system [39] However,the exact mechanisms relating Cu2+ 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 Cu2+and Cu+may induce LPO according to the following reactions:
Cu2þþ LOOH ! Cuþ þ LOO þ Hþ ð1Þ
Cuþþ LOOH ! Cu2þþ LOþ OH ð2Þ
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 Cu2+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 Cu2+reduction Although not demonstrated,the involvement of O 2 has been suggested [49],according to the reaction:
Cuþþ O2! Cu2þþ O 2 ð6Þ After hydrogen abstraction from at least trienic fatty acid structures (reaction 3),the chemical rearrangement of LÆ
Fig 4 Kinetic profiles of ferricytochrome c reduction during LDL
oxidation induced by 5 l M or 175 l M of Cu2+in the absence or presence
of 10 l M urate LDL solution at 0.12 mgÆmL)1(240 n M ) in 10 m M
phosphate buffer,pH 7.4,were incubated for 15 min at 37 C with
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 Cu2+in the presence and absence of urate LDL solution at 0.12 mgÆmL)1(240 n M ) in 10 m 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
Cu 2+
Trang 7radicals leads to conjugated diene radicals which further
react with O2(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
a1-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 Cu2+concentrations (5 lM
here),a rather high Cu2+concentration (175 lM) 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 lM and 800 lM with 175 and 5 lM of Cu2+,
respectively) are definitely pro-oxidant when preoxidized
LDL preparations are exposed to Cu2+,preoxidized LDL
being modeled by MM-LDL or by native LDL exposed to
Cu2+ 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 Cu2+,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 lM 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 Cu2+-induced LDL oxidation They suggested that
urate accelerates the LPO by reducing Cu2+ to Cu+,
according to:
Cu2þþ UH
2 ! Cuþþ UH þ Hþ 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 lM,respectively) while using low Cu2+concentrations (1.6 and 2.5 lM,respectively) At low Cu2+concentration (5 lM),not only did we observe this pro-oxidant effect of urate at low concentration (10 lM,data not shown),but we also observed this effect at a much higher urate concentra-tion (800 lM) corresponding to antioxidant concentration when working with native LDL (see below) Finally Bagnati
et al [23] reported that 10 lMurate introduced in the LDL solutions 30 min after Cu2+clearly stimulated the peroxi-dation only for Cu2+ to LDL ratios lower than 50 In contrast,at a higher Cu2+/LDL ratio (Cu2+¼ 175 lMand LDL¼ 0.24 lM),we found that under similar conditions, lower (10 lM) and higher (50 lM) 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 Cu2+-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 Cu2+concentration (175 lM),low Cu2+ concentra-tion (5 lM) 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 Cu2+ concentration at a much higher urate concentration than that observed with the high
Cu2+concentration Third,our data do not fully agree with those of Abuja [22] and Bagnati et al [23] who used low
Cu2+ concentrations,1.6 and 2.5 lM,respectively,and found that 20 and 10 lM,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
Trang 8level 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 lM
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 lMmuch lower than the Cu2+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 Cu2+ 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 Cu2+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 lM urate protected LDL
from the oxidation induced by 4 mM
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 Cu2+
reduction by urate,urate radicals (UHÆ–) are rapidly formed
as products of this reaction Thus,the dismutation of
UHÆ–could explain the fast urate consumption rate If such
a view is consistent with the data observed at low Cu2+
concentrations,it does not explain results obtained with
SOD and ferricytochrome c at high Cu2+concentrations
(Table 2) Both superoxide dismutase and
ferricyto-chrome c inhibited the MDA formation in the presence of
urate suggesting the involvement of O 2 In support of
the involvement of O 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 O2 (reaction 6) As a consequence of
the reduction of Cu2+by urate and of oxygen by Cu+,
UHÆ–and O 2 will be concomitantly formed Thus,O 2
could react with the simultaneously formed UHÆ–,as
recently demonstrated by pulse radiolysis [57] according to
the following reaction:
UH þ O 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
unlikely that the pro-oxidant activity of urate is due to the reaction of urate with O 2 ,as little urate is destroyed in
an O 2 -generating system [58] and O 2 reacts with urate with a rather small reaction rate constant [10] The reaction between UHÆ– and O 2 would contribute to urate con-sumption and therefore may explain the observed protec-tion of urate consumpprotec-tion by ferricytochrome c At low
Cu2+ concentrations,stationary UHÆ– and O 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 Cu2+concentrations,another mechanism,independ-ent of O 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 Cu2+by urate However,such enhanced levels would also occur at high Cu2+concentration It may
be speculated that this intriguing behavior is closely related
to complex urate–Cu2+–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 Cu2+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 Cu2+ Depending on both the Cu2+and the urate concentrations (i.e on their ratio),the reaction path may be very different With both Cu2+ 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 Cu2+ concentration,the antioxidant deterrent of urate necessitates higher urate concentrations than at high Cu2+ concentration; this observation again suggests peculiar effects due to the complex urate–Cu2+–LDL interactions
C O N C L U S I O N
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 Cu2+ 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
Trang 9A C K N O W L E D G M E N T S
This work was partly supported by grant Praxis/2/2.1/QUI/225/94
from the Fundac¸a˜o para a Cieˆncia e Tecnologia,by travel grant no 347
C0 from the Ambassade de France au Portugal,and the Instituto de
Cooperac¸a˜o Cientı´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.
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