Archives of Biochemistry and Biophysics 423 (2004) 302–308

ABB
www.elsevier.com/locate/yabbi

Nitric oxide inhibits prooxidant actions of uric acid during

copper-mediated LDL oxidation
Silvia M. Sanguinetti,a Carlos Batthy

any,b Andr
es Trostchansky,b Horacio Botti,b

pez, Regina L.W. Wikinski,a Homero Rubbo,b and Laura E. Schreiera,*
Graciela I. Lo a

a
Laboratorio de L
ıpidos y Lipoprote
ınas, Departamento de Bioqu
ımica Cl
ınica, Facultad de Farmacia y Bioqu
ımica,
Universidad de Buenos Aires, Buenos Aires, Argentina
b
Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Rep
ublica, Montevideo, Uruguay
Received 15 September 2003, and in revised form 23 December 2003

Abstract

Interactions between uric acid and physiologically relevant fluxes of nitric oxide ( NO) during copper-mediated low-density li-
poprotein (LDL) oxidation were evaluated. In the absence of  NO, a dual pro- and antioxidant action of uric acid was evident: low
concentrations of uric acid enhanced lipid oxidation and a-tocopherol consumption, while its protective role was observed at higher
concentrations. The prooxidant effects of uric acid were mostly related to its copper-reducing ability to form Cuþ , an initiator of
lipid oxidation processes. While the prooxidant action of uric acid was completely inhibited by  NO, the antioxidant action of  NO
was slightly counterbalanced by uric acid. Enhancement of a-tocopherol consumption by uric acid was inhibited in the presence of

NO while additive antioxidant effects between  NO and uric acid were observed in conditions where uric acid spared a-tocopherol.
Altogether, these results suggest that in the artery wall, the  NO/uric acid pair may exert antioxidant actions on LDL, even if in-
creased amounts of redox active copper were available at conditions favoring prooxidant activities of uric acid.
Ó 2004 Elsevier Inc. All rights reserved.

Keywords: Nitric oxide; Uric acid; Copper; Free radical; Antioxidant; LDL oxidation

Low-density lipoprotein (LDL) oxidation plays a key (LOOH), a-tocopherol (a-TOH), and amino acid resi-
role in the development of atherosclerosis. Among the dues [5,7–9]. The early phase of copper reduction is
wide variety of mechanisms claimed to be involved in mostly dependent on a-TOH concentration and takes
promoting LDL oxidation, considerable attention has place during the lag phase of LDL oxidation [10].
been focused on the role played by transition metals, Several antioxidants present in LDL, as well as in
such as copper and iron [1–4]. Moreover, copper-in- plasma, preserve the integrity of circulating LDL [11].
duced oxidation represents a highly reproducible meth- Uric acid is considered the major low-molecular weight
od, frequently used to assess LDL susceptibility to plasma antioxidant because of its relatively high con-
oxidation as a risk factor for atherosclerosis as well as centration (150–500 lM) [12]. In addition to its capacity
for performing well-controlled kinetic studies [5,6]. The to inhibit LDL oxidation by copper [13], uric acid has
mechanisms underlying copper-mediated LDL oxida- peroxynitrite (ONOO
), aqueous peroxyl radicals
tion involve Cu2þ binding to apolipoprotein B-100 (apo (ROO ), and hydroxyl radical ( OH) scavenging activity
B)1 followed by its reduction by endogenous compo- [14–16]. On the other hand, uric acid may behave as
nents in LDL such as preformed lipid hydroperoxides prooxidant during LDL oxidation promoted by copper
or hydrophilic ROO [17–20]. This effect requires the
*
Corresponding author. Fax: +11-54-11-5950-8694.
E-mail address: lipids@ffyb.uba.ar (L.E. Schreier). monioethyl)amino]diazen-1-ium-1,2-diolate]; BC, bathocuproine di-
1
Abbreviations used:  NO, nitric oxide; ROO , aqueous peroxyl sulfonate; Apo B, apolipoprotein B-100; ONOO
, peroxynitrite anion;

radical; LOO , lipid peroxyl radical; LO , alkoxyl radical; LOOH, lipid OH, hydroxyl radical; a-TOH, a-tocopherol; a-TO , a-tocopheroxyl
hydroperoxide; DETA NONOate, (Z)-1-[2-(2-aminoethyl)-N -(2-am radical.

0003-9861/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2003.12.034
 S.M. Sanguinetti et al. / Archives of Biochemistry and Biophysics 423 (2004) 302–308 303

presence of copper either as Cu2þ or Cuþ , as well as low teristics of typical LDL particles [6]. The isolated LDL
Cu2þ /LDL molar ratios [17,19]. Prooxidant actions of fraction was stored at 4 °C in the dark until used (within
uric acid became more evident when added late during 3 days). HPLC-gel filtered LDL was immediately used
the lag or propagatory phases of LDL oxidation for oxidation experiments.
[17,18,20]. Recently, uric acid was found as the main
determinant of the pro- and antioxidant properties of LDL oxidation
human serum ultrafiltrates, a model for the low-molec-
ular weight components of the interstitial fluid [21]. Purified LDL was incubated with Cu2þ (cupric
Nitric oxide ( NO) is an endogenously generated free chloride) in 50 mM potassium phosphate buffer, pH 7.4,
radical, which may be able to modulate processes asso- at 37 °C. Oxidation assays were performed at a Cu2þ /
ciated with the excess production of reactive oxygen LDL molar ratio of 25, assuming that 100 lg LDL
species in vivo, due to its ability to redirect the reactivity protein/ml ¼ 0.2 lM [32]. Some experiments were per-
of partially reduced oxygen and nitrogen species [22–24]. formed varying the Cu2þ /LDL ratio from 12/1 to 300/1.
In fact,  NO exerts strong antioxidant actions during Solutions of uric acid were prepared in 50 mM potas-
LDL oxidation by scavenging lipid radicals involved in sium phosphate buffer, pH 7.4. Stock solutions of
oxidative chain propagation reactions and by its capac- DETA NONOate (half-life of 20 h [22]) were prepared
ity to spare a-TOH [25,26]. The ability of  NO to readily in 20 mM potassium phosphate buffer, pH 9.5. Nitric
traverse the LDL surface accessing the hydrophobic lipid oxide production rates were measured spectrophoto-
core of the particle affirms a role for endothelial-derived metrically at k ¼ 250 nm [33]. Additions of uric acid and

NO as a major lipophilic antioxidant in LDL [27]. DETA NONOate to LDL dilutions were made just
In the initial stages of atherosclerosis, LDL enters the prior of Cu2þ . LDL dilutions showed no changes in pH
intimal subendothelium of the arterial wall, where it can after uric acid addition (1–600 lM).
be subsequently oxidized. Plasma uric acid may be
present in the interstitial fluid within this compartment, Biochemical analyses
in addition to its high production in endothelial cells
[28]. On the other hand, variable fluxes of  NO are Lipid oxidation was assessed by conjugated diene
continuously released from vascular cells in response to formation [32], which was continuously monitored by the
diverse stimuli [29]. Therefore, within the intima, accu- increase in absorbance at 234 nm (e ¼ 29,500 M
1 cm
1 )
mulated LDL may co-exist with uric acid, as well as at 37 °C, in a SHIMADZU UV-2401PC 6 cuvette spec-
endothelial-derived  NO, giving rise to a scenario where trophotometer (Tokyo, Japan). Lag times were obtained
different potential interactions could take place. from the intercept of the linear least square slope of the
The aim of our study was to gain further insight into curve with the x axis and expressed in minutes.
the controversial pro- and antioxidant actions of uric Low-density lipoprotein a-TOH was determined by
acid during copper-induced LDL lipid and a-TOH ox- reverse phase HPLC with fluorometric detection [34].
idation and how this is modulated by physiologically Samples (50 ll) were mixed with 450 ll methanol and
relevant fluxes of  NO. vortexed for 10 s twice, and centrifuged at 10,000g for
10 min at 4 °C and 50 ll was injected in the HPLC.
Analyses were performed on a Supelcosil LC-18 column
Materials and methods (25  0.46 cm, 5 lm), isocratic mode, 1 ml/min, with a
Model 122 Gilson fluorometric detector (kex ¼ 295,
Materials kem ¼ 330 nm). Quantitative analysis was performed by
comparing peak areas with corresponding standard
(Z)-1-[2-(2-Aminoethyl)-N -(2-ammonioethyl)amino] curves [34].
diazen-1-ium-1,2-diolate] (DETA NONOate) was from Cuprous ion formation was measured spectrophoto-
Cayman Chemical (Ann Arbor, MI). All other reagents metrically in the presence of 10-fold excess bathocupr-
were from Sigma (St. Louis, MO). oine disulfonate (BC) at 480 nm [9,10]. An integral rate
approach was used to determine the pseudo-first-order
LDL isolation rate constants of Cuþ formation in the presence of excess
urate and 0.2 lM LDL. BC was added to LDL solutions
Human LDL was obtained from plasma of healthy in the presence or absence of uric acid before triggering
normolipidemic donors and purified by ultracentrifu- the reaction with Cu2þ using a stopped-flow rapid-mixing
gation followed by HPLC-gel filtration as before [9]. equipment (Applied Photophysics). Concentrations of
Purity of LDL was tested by agarose gel electrophoresis Cuþ were calculated from the absorption coefficient of the
[30] while the protein content was determined by a BC–Cuþ complex (e ¼ 9053 M
1 cm
1 ).
modification of the Lowry method [31]. Chemical Initial rates of oxygen consumption were determined
composition of the LDL preparation exhibited charac- using a Clark Model YSI 5300 electrode (Yellow Spring
304 S.M. Sanguinetti et al. / Archives of Biochemistry and Biophysics 423 (2004) 302–308

Instruments) in a 1.6-ml water-jacketed chamber main- enhanced by low uric acid levels (5–125 lM), as indi-
tained at 37 °C as before [25,35]. cated by the shortening of lag phases from 150 min (in
Data shown are representative of at least three inde- the absence of urate) to 52 min, at 125 lM urate. In
pendent experiments. contrast, an increased lag time was observed at a higher
uric acid concentration (600 lM), with no propagation
observed up to 340 min (Fig. 1A). Experiments were also
Results performed varying the Cu2þ /LDL ratio from 12/1 to
300/1, where the dual pro- and antioxidant actions of
Human LDL was incubated with Cu2þ at a Cu2þ / uric acid were also observed (data not shown).
LDL molar ratio of 25, in the absence or presence of To further characterize the dual role of uric acid during
uric acid and time courses of conjugated diene forma- copper-induced LDL oxidation, consumption of a-TOH
tion were determined (Fig. 1A). Lipid oxidation was was also assessed. While uric acid at low prooxidant
concentrations (5 lM) slightly enhanced a-TOH con-
sumption, at higher concentrations (125 lM) uric acid
spared a-TOH (Fig. 1B), although it still diminished the
lag phase of conjugated diene formation (Fig. 1A). In
addition, there was a complete protection of a-TOH
consumption at higher uric acid concentrations (600 lM)
(Fig. 1B), in parallel with an effective lag phase prolon-
gation (Fig. 1A).
Next, Cu2þ reduction was determined within a
prooxidant as well as an antioxidant range of uric acid
concentrations (Fig. 2). In the absence of uric acid, LDL-
dependent Cuþ formation reached a maximum within
10 min. In the presence of uric acid, Cuþ formation was
highly stimulated (Fig. 2A). Moreover, the pseudo-first-
order rate constants of Cu2þ reduction in the presence of
LDL increased linearly with uric acid concentrations
(Fig. 2B). Interestingly, the second-order rate constant of
the reaction of Cu2þ with uric acid in the absence of LDL
was twofold higher than in the presence of LDL
(Fig. 2B). Urate-derived chain-propagating species (i.e.,
aminocarbonyl radical) capable of enhancing lipid oxi-
dation are formed during ONOO
-mediated uric acid
oxidation [14]. The potential formation of these radicals
during copper-mediated uric acid oxidation was explored,
to determine if this mechanism could account in part for
the prooxidant activity of uric acid. For this, oxygen
consumption was determined as an indicator of free
radical formation. It was observed that prooxidant levels
of uric acid on lipid oxidation (5–125 lM) were unable to
induce oxygen consumption in the presence of Cu2þ and
Cu2þ –BC (not shown). As a positive control, significant
yields of oxygen consumption were obtained by bolus
addition of ONOO
(not shown).
Fig. 1. Pro- and antioxidant effects of uric acid on copper-mediated Interactions between physiologically relevant fluxes
LDL oxidation (A) and a-tocopherol consumption (B). (A) LDL of  NO and uric acid were evaluated at concentrations of
(0.2 lM) was incubated in 50 mM potassium phosphate buffer, pH 7.4,
uric acid promoting lipid oxidation (Figs. 3 and 4). In-
at 37 °C, with 5 lM Cu2þ either in the absence (square) or presence of
5 lM (circle), 35 lM (triangle), 125 lM (diamond), and 600 lM (cross) cubations were performed at a Cu2þ /LDL molar ratio of
uric acid. Lipid oxidation was monitored by measuring conjugated 25, in the absence or presence of uric acid (5–125 lM)
diene production at 234 nm. One experiment typical of five is shown. and a  NO flux of 10 nm/min (Fig. 3). As expected,
(B) LDL (0.2 lM) was incubated as in (A), either in the absence 
NO prolonged LDL lag phases even in the presence of
(square) or presence of 5 lM (circle), 125 lM (diamond), and 600
prooxidant concentrations of uric acid. However, in the
(cross) lM uric acid. a-Tocopherol consumption was determined by
RP-HPLC with fluorometric detection. Results are expressed as per- presence of  NO, rising concentrations of uric acid
centage of remaining a-tocopherol. One experiment typical of three is within the prooxidant range increasingly diminished the
shown. lag times of conjugated diene formation, indicating that
 S.M. Sanguinetti et al. / Archives of Biochemistry and Biophysics 423 (2004) 302–308 305

Fig. 2. Uric acid promotes Cu2þ reduction during LDL oxidation. (A) LDL (0.2 lM) was oxidized by 5 lM Cu2þ as before, either in the absence
(square) or presence of 1 lM (circle), 2.5 lM (triangle), 5 lM (diamond), and 10 lM (cross) uric acid. Cuþ –bathocuproine complex was measured at
480 nm. (B) Observed rate constants of Cuþ –bathocuproine complex formation after mixing equal volumes of Cu2þ (5 lM) with uric acid (51–
255 lM) and bathocuproine (50 lM), either in the absence (open squares) or presence (closed squares) of LDL (0.2 lM). Inset: Time-course of Cuþ
formation in the presence of 153 lM uric acid and LDL. One experiment of three is shown.

Fig. 3. Conjugated diene formation in the presence of uric acid and Fig. 4. Effect of uric acid on a-tocopherol consumption in the presence
nitric oxide. LDL was incubated as before, either in the absence (open of nitric oxide. LDL was oxidized as in Fig. 3, either in the absence
symbols) or presence of DETA NONOate (10 lM, closed symbols) (open symbols) or presence of DETA NONOate (10 lM, closed sym-
and 0 lM (square), 5 lM (circle), 35 lM (triangle), and 125 lM (dia- bols) and 0 lM (square), 5 lM (circle), 35 lM (triangle), and 125 lM
mond) uric acid. Oxidation was monitored by measuring conjugated (diamond) uric acid. One experiment typical of three is shown.
dienes at 234 nm. One experiment typical of three is shown.

In addition, interactions between prooxidant concen-

the antioxidant action of  NO was slightly counterbal- trations of uric acid and physiologically relevant  NO
anced by uric acid (Fig. 3). fluxes were investigated.
Finally, the ability of  NO to spare a-TOH during Previous studies are controversial with respect to the
copper-mediated LDL oxidation in the presence of effects of uric acid on copper-mediated LDL oxidation
prooxidant uric acid concentrations was analyzed [13,17–19,21,32]. Although uric acid has been found to
(Fig. 4). Nitric oxide inhibited the prooxidant effects of inhibit lipid oxidation even at extremely low doses
low concentrations of uric acid (5, 35 lM) on a-TOH [13,32], dual pro- and antioxidant actions of uric acid
consumption and further enhanced the a-TOH sparing have also been reported [17–19,21]. Several factors, such
effects exerted by 125 lM uric acid (Fig. 4). as a high Cu2þ /LDL molar ratio, may influence a
shifting from pro- to antioxidant behavior of uric acid
[17]. In the present study, under a 25/1 Cu2þ /LDL ratio,
Discussion concentrations of uric acid between 5 and 125 lM
revealed a prooxidant behavior whereas 600 lM uric
Herein, the dual pro- and antioxidant actions of uric acid acted as antioxidant (Fig. 1A). These results are
acid on copper-induced LDL oxidation were analyzed. consistent with a previous report in which a shift from
306 S.M. Sanguinetti et al. / Archives of Biochemistry and Biophysics 423 (2004) 302–308

pro- to antioxidant activity of uric acid was observed at port a rate constant of 4.5  103 M
1 s
1 for this process
200–400 lM uric acid concentrations [19]. Our results (Fig. 2B). These results support that uric acid competes
show that the promotion of lipid oxidation by low uric with a-TOH for LDL-bound Cu2þ reduction while in-
acid concentrations (5 lM) was paralleled by a slight creasing the availability of Cuþ . On the other hand,
increase in a-TOH consumption (Fig. 1B). The en- Filipe et al. [19] reported that throughout the whole
hancement of Cu2þ reduction by uric acid shown herein prooxidant concentration range, uric acid increased ca-
(Fig. 2) highlights the central role of this mechanism rotenoid consumption during LDL oxidation. This
regarding its prooxidant behavior. Uric acid is directly suggests that unlike a-TOH, more lipophilic reductants
oxidized by Cu2þ with the generation of urate anion free in LDL may not be relevant for Cuþ formation. Taken
radical along with Cuþ and a series of oxidized end together, our results indicate that the net effect of uric
products, including allantoin, oxenic/oxaluric acid, and acid on LDL oxidation may be determined by a balance
parabanic acid [28]. In a reaction analogous to FentonÕs, between its pro- and antioxidant activities, which are
the generation of Cuþ by uric acid may greatly facilitate favored at low and high uric acid concentrations, re-
the decomposition of preformed LOOH in LDL, giving spectively. At low uric acid concentrations, the increased
rise to lipid alkoxyl radicals (LO ) capable of initiating availability of Cuþ favoring oxidation prevails upon the
oxidative processes in LDL [36]. In agreement, the time- sparing of a-TOH involved in Cu2þ reduction by uric
point of urate addition seems to be crucial to make ev- acid. On the other hand, at higher uric acid concentra-
ident the prooxidant activity of uric acid, since some tions additional antioxidant reactions may contribute to
authors only found it when the additions were made the antioxidant behavior of uric acid until effectively
10 min after the addition of copper, after a critical overcoming the prooxidant activity.
concentration of LOOH had already been formed Among the reactions potentially involved in the an-
[17,18,20,21]. In contrast, we and others [19] made the tioxidant activity of uric acid during LDL oxidation, the
addition of uric acid and copper at the same time and reduction of a-TO to regenerate a-TOH should be re-
yet detected the prooxidant behavior of uric acid. This considered. Previous studies have shown that uric acid,
discrepancy may be explained by different initial con- in contrast to ascorbate, failed to inhibit lipid oxidation
centrations of LOOH in the LDL preparation, i.e., as well as a-TOH consumption during LDL oxidation
higher proportion of minimally modified hydroperox- started by a lipophilic initiator [38]. This report suggests
ide-rich LDL [37]. Additionally, lipid oxidation initia- that whereas accessibility to LDL lipid core for both
tion by apo B-tryptophanyl radicals derived from Cu2þ scavengers is too low, only ascorbate can reduce a-TO
reduction may be involved in LOOH formation early at the LDL–water interface to achieve inhibition of
during the lag phase [9]. LDL lipid core peroxidation. Moreover, in examining
Recently, the aminocarbonyl radical, a urate-derived reactions with the vitamin E analog Trolox C by pulse
free radical, has been demonstrated as the species radiolysis, it was demonstrated that Trolox C phenoxyl
responsible for the effects of urate in amplifying radical is not repaired by uric acid or at least the reac-
ONOO
-mediated LDL oxidation [14]. Therefore, we tion is not fast enough to be determined by this tech-
investigated the formation of urate-derived radicals, as nique [39]. Nevertheless, we cannot rule out the
an additional prooxidant mechanism. However, a lack regeneration of a-TOH in our experimental conditions,
of oxygen consumption in solutions of uric acid plus where higher than previously used uric acid to a-TOH
Cu2þ suggests that urate-derived radicals may have not ratios have been assayed. Finally, an alternative anti-
been formed in our experimental conditions. Moreover, oxidant pathway may be due to a peroxidase-like ac-
oxygen consumption was not observed after the addi- tivity reported for certain protein/copper/urate
tion of BC either, thus emphasizing that not even in interactions [40]. Proudfoot et al. proposed that albu-
conditions favoring Cuþ formation was uric acid oxi- min-bound Cu2þ resembles the Fe3þ complex of por-
dized by oxygen radical-dependent mechanisms. phyrin protein in peroxidase enzymes. In the presence of
The copper-reducing ability of uric acid not only reducing agents such as uric acid, this complex is ca-
explains its prooxidant behavior but also can explain, in pable of breaking down lipid hydroperoxides [41]. Fur-
part, its antioxidant activity. In the present study, high thermore, other authors have proposed that uric acid is
uric acid concentrations inhibiting lipid oxidation a true substrate for several peroxidase enzymes [42].
(600 lM) were able to completely block a-TOH con- Therefore, since in the present work LDL preparations
sumption (Fig. 1B). Interestingly, intermediate uric acid were albumin-free, we speculate that an apo B–Cu2þ
concentrations (125 lM) inhibited a-TOH consumption complex in the presence of uric acid might have exhib-
although shortened the lag phases (Fig. 1A and B). Fi- ited a peroxidase-like activity, explaining in part the
nally, low uric acid concentrations (5–35 lM) enhanced antioxidant activity of uric acid.
both lipid and a-TOH oxidation. The rate of reduction Nitric oxide exerts a potent antioxidant action on
of apo B-bound Cu2þ by uric acid in the presence of BC copper-induced LDL oxidation [43,44]. Herein, the
linearly depends on uric acid concentration and we re- prooxidant action of uric acid was inhibited by the
 S.M. Sanguinetti et al. / Archives of Biochemistry and Biophysics 423 (2004) 302–308 307

presence of low fluxes of  NO (Fig. 3). This can be linked atherosclerotic lesions, transition metal ions may en-
to its ability to scavenge lipid radicals at diffusion-lim- hance the oxidation of LDL previously modified by
ited rates, giving rise to nitrogen-containing lipid prod- other systems such as oxidant enzymes [46]. On the
ucts [22–27,45]. Interestingly, a slight decrease in the other hand, plasma urate has free access to the intimal
antioxidant efficacy of  NO in the presence of uric acid subendothelium, raising the possibility of interactions
was observed (Fig. 3). This effect is a consequence of the between uric acid, copper, and LDL in this com-
higher rates of Cuþ formation observed at increasing partment (Fig. 5). Hypouricemia, defined as uric acid
uric acid concentrations, promoting, in turn, higher levels below 120 lM, can be seen in severe liver dis-
rates of peroxidation initiation. Therefore, the increase ease with a decreased synthesis of purines, hereditary
in rates of initiation cannot be completely counterbal- deficiency of xanthine oxidase, administration of al-
anced by a fixed  NO flux (10 nM/min). lopurinol or a defect in renal tubular reabsorption of
As regards a-TOH, it was observed that  NO inhib- uric acid, such as FanconiÕs syndrome [47]. These
ited urate-stimulated a-TOH consumption and potenti- clinical conditions represent a situation where uric
ated urate-mediated sparing of a-TOH (Fig. 4). This can acid may act as prooxidant. Nevertheless, our results
be explained in part by competition between  NO and a- indicate that physiological  NO fluxes may effectively
TOH for lipid radicals [26]. It is noteworthy, however, inhibit prooxidant actions of low-dose uric acid during
that the extent of a-TOH sparing by  NO is not as copper-mediated LDL oxidation, which highlights the
strong as the inhibition of lipid oxidation (Figs. 3 and 4). potential antioxidant impact associated with the  NO/
This may be due to direct oxidation of a-TOH by Cu2þ , urate pair (Fig. 5).
which is not affected by  NO. In summary, our results show that physiologically
Despite the wide variety of oxidants potentially relevant  NO fluxes inhibit the prooxidant action of uric
involved in LDL oxidation in vivo, the relative con- acid and spare a-TOH during copper-mediated LDL
tribution of each one is still controversial. In advanced oxidation. These results suggest that within the

α 
  α 

















α  α 
 




  



Fig. 5. Proposed mechanism for the prooxidant activity of uric acid and its inhibition by  NO during copper-mediated LDL oxidation. Low uric acid
(UA) concentrations promote apo B-bound Cu2þ reduction to Cuþ (1) and preformed LOOH react with Cuþ to form lipid alkoxyl radicals (LO ) (2),
which initiate lipid peroxidation by reacting with a fatty acyl residue (LH) (3). Lipid peroxyl radicals (LOO ) propagate lipid peroxidation by reaction
with an additional LH (4), whereas a-tocopherol (a-TOH) inhibits lipid oxidation by scavenging chain-propagating species (5). Nitric oxide inhibits
lipid oxidation and spares a-TOH by its faster termination reaction with LO and LOO with the formation of nitrogen-containing lipid products (6).
308 S.M. Sanguinetti et al. / Archives of Biochemistry and Biophysics 423 (2004) 302–308

subendothelial space of arterial intima, the  NO/uric [19] P. Filipe, J. Haigle, J. Freitas, A. Fernandes, J.C. Maziere, C.
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This work was supported by grants from University
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