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:
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.
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