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

-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+

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

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

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
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

-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+

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

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

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+

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

(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.

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

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

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

), 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

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

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+
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

,
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

, 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+

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

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

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

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-

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

fica e Tecnolo
´
gica Internacional (ICCTI), and by an
exchange grant from INSERM and ICCTI. CM and J-CM thank the
Universite
´
de Picardie Jules Verne and the Ministe
`
re de la Recherche et
de la Technologie for financial support.
REFERENCES
1. Ryan, M., Grayson, L. & Clarke, D.J. (1997) The total anti-
oxidant capacity of human serum measured using chemilumines-
cence is almost completely accounted for by urate. Ann. Clin.
Biochem. 34, 688–689.
2. Aldini, G., Yeum, K J., Russell, R.M. & Krinsky, N.I. (2001) A
method to measure the oxidizability of both the aqueous and lipid
compartments of plasma. Free Radic. Biol. Med. 31, 1043–1050.
3. Chevion, S., Berry, E.M., Kitossky, N. & Kohen, R. (1997)
Evaluation of plasma low molecular weight antioxidant capacity
by cyclic voltametry. Free Radic. Biol. Med. 22, 411–421.
4. Nyssonen, K., Porkkala-Sarataho, E., Kaikkonen, J. & Salonen,
J.T. (1997) Ascorbate and urate are the strongest determinants of
plasma antioxidative capacity and serum lipid resistance to oxi-
dation in Finnish men. Atherosclerosis 130, 223–233.
5. Ames, B.N., Cathcart, R., Schwiers, E. & Hochstein, P. (1981)
Uric acid provides an antioxidant defense in humans against
oxidant- and radical-caused aging and cancer: a hypothesis. Proc.
Natl Acad. Sci. USA 78, 6858–6862.
6. Nagyova, A., Sustrova, M. & Raslova, K. (2000) Serum lipid

15. Witzum, J.L. (1994) The oxidation hypothesis of atherosclerosis.
Lancet 344, 793–795.
16. Fang, J. & Alderman, M.H. (2000) Serum uric acid and cardio-
vascular mortality. The NHANES I epidemiologic follow-up
study, 1971–92. J.Am.Med.Assoc.283, 2404–2410.
17. Culleton, B.F., Larson, M.G., Kannel, W.B. & Levy, D. (1997)
Serum uric acid and risk for cardiovascular disease and death: the
Framington Heart Study. Heart 78, 147–153.
18. Bickel, C.H.J.R., Blankenberg, S., Rippin, G., Hafner, G.,
Daunhauer, A., Hofman, K.P. & Meyer, J. (2002) Serum uric acid
as an independent predictor of mortality in patients with angio-
graphically proven coronary artery disease. Am. J. Cardiol. 89, 12–
17.
19. Moriarity, J.T., Folsom, A.R., Iribarren, C., Nieto, F.J. &
Rosamond, W.D. (2000) Serum uric acid and risk of coronary
disease: Atherosclerosis Risk in Communities (ARIC) Study. Ann.
Epidemiol. 10, 136–143.
20. Puig, J.G. & Ruilope, L.M. (1999) Uric acid as a cardiovascular
risk factor in arterial hypertension. J. Hypertens. 17, 689–872.
21. Nieto, F.J., Iribarren, C., Gross, M.D., Comstock, G.W. &
Cutler, R.G. (2000) Uric acid and serum antioxidant: a reaction to
atherosclerosis? Atherosclerosis 148, 131–139.
22. Abuja, P.M. (1999) Ascorbate prevents prooxidant effects of urate
in oxidation of human low density lipoprotein. FEBS Lett. 446,
305–308.
23. Bagnati, M., Perugini, C., Cau, C., Bordone, R., Albano, E. &
Bellomo, G. (1999) When and why a water-soluble antioxidant
becomes pro-oxidant during copper-induced low-density lipo-
protein oxidation: a study using uric acid. Biochem. J. 340, 143–
152.

chromatography assay of retinol, a-tocopherol, b-carotene,
a-carotene, lycopene and b-cryptoxanthin in plasma, with toco-
pherol acetate as internal standard. Clin. Chem. 34, 377–381.
33. Windholz, M. (1976) The Merck Index. An Encyclopedia of
Chemicals and Drugs, 9th edn. Merck, Inc, Rahway.
34. Patel, R.P. & Darley-Usmar, V.M. (1999) Molecular mechanisms
of the copper dependent oxidation of low-density lipoprotein. Free
Radic. Res. 30, 1–9.
35. Berliner, J.A., Territo, M.C., Sevanian, A., Ramin, S., Kim, J.A.,
Bamshad, B., Esterson, M. & Fogelman, A.M. (1990) Minimally
modified low density lipoproteins stimulates monocyte endothelial
interactions. J. Clin. Invest. 85, 1260–1266.
36. Carrero, P., Ortega, H., Martı
´
nez-Botas, J., Go
´
mez-Coronado, D.
&Laruscio
´
n, M.A. (1998) Flavonoid-induced ability of minimally
modified low-density lipoproteins to support lymphocyte pro-
liferation. Biochem. Pharmacol. 55, 1125–1129.
37. Proudfoot, J.M., Croft, K.D., Puddey, I.B. & Belli, L.J. (1997)
The role of copper reduction by a-tocopherol in low-density
lipoprotein oxidation. Free Radic. Biol. Med. 23, 720–728.
5482 P. Filipe et al. (Eur. J. Biochem. 269) Ó FEBS 2002
38. Perugini, C., Seccia, M., Albano, E. & Bellomo, G. (1997) The
dynamic reduction of Cu(II) to Cu(I) and not Cu(I) availability is
a sufficient trigger for low density lipoprotein oxidation. Biochim.
Biophys. Acta 1347, 191–198.

iron, by human low density lipoprotein. J. Biol. Chem. 270, 5158–
5163.
48. Patel, R., Svistunenko, D., Wilson, M.T. & Darley-Usmar, V.
(1997) Reduction of Cu(II) by lipid hydroperoxides: inplications
for the copper-induced oxidation of low-density lipoprotein. Bio-
chem. J. 322, 425–433.
49. Pecci, L., Montefoschi, G. & Cavallini, D. (1997) Some new details
on the copper–hydrogen peroxide interaction. Biochem. Biophys.
Res. Commun. 235, 264–267.
50. Krinsky, N.I. (1992) Mechanism of action of biological antioxi-
dants. Proc.Soc.Exp.Biol.Medical200, 248–254.
51. Aruoma, O.I. & Halliwell, B. (1989) Inactivation of a
1
-antipro-
teinase by hydroxyl radicals. The effect of uric acid. FEBS Lett.
244, 76–80.
52. Kittridge, K.J. & Willson, R.L. (1984) Uric acid substantially
enhances the free radical-induced inactivation of alcohol dehy-
drogenase. FEBS Lett. 170, 162–164.
53. Santos, C.X.C., Anjos, E.I. & Augusto, O. (1999) Uric acid oxi-
dation by peroxynitrite: multiple reactions, free radicals forma-
tion, and amplification of lipid oxidation. Arch. Biochem. Biophys.
372, 285–292.
54. Yamanaka, N., Oda, O. & Nagao, S. (1997) Green tea catechins
such as (–)-epicatechin and (–)-epigallocatechin accelerate Cu
2+
-
induced low density lipoprotein in propagation phase. FEBS Lett.
401, 230–234.
55. Yamanaka, N., Oda, O. & Nagao, S. (1997) Prooxidant activity