NO IN LIPID OXIDATION
Shock 1998: Oxígeno, Oxido Nítrico y
Simposio Internacional, Academia Nacional de Medicina
Buenos Aires, 30 abril 1998
NITRIC OXIDE AND PEROXYNITRITE IN LIPID PEROXIDATION
Bioquímica, Facultad de Medicina, Universidad de la República,
Key words: nitric oxide, peroxynitrite, superoxide, lipid
oxidation, free radicals, antioxidants, lipids, low density
oxide (.NO) can mediate tissue protective reactions during oxidant
stress, as well as toxic and tissue prooxidant effects. Nitric oxide
regulates critical lipid membrane and lipoprotein oxidation events, by
1) contributing to the formation of more potent secondary oxidants
from superoxide (i.e. peroxynitrite) and 2) termination of lipid
radicals to possibly less reactive secondary nitrogen-containing
products (LONO, LOONO) which are in part organic peroxynitrites and
are expected to be produced in vivo. Relative rates of production and
steady state concentrations of superoxide and .NO and cellular sites
of production will profoundly influence expression of the differential
oxidant injury-enhancing and protective effects of .NO. Full
understanding of the physiological roles of .NO, coupled with detailed
insight into .NO regulation of oxygen radical-dependent reactions,
will yield a more rational basis for the use of .NO donors for
nítrico y peroxinitrito en la peroxidación lipídica. El óxido
nítrico (.NO) regula eventos críticos en procesos de
lipoperoxidación de membranas o lipoproteinas mediante 1) su
contribución a la formación de oxidantes secundarios más potentes
como el peroxinitrito, 2) por su capacidad de terminación de
reacciones de propagación lipídica con la concomitante formación de
productos no radicalares del tipo nitrosolípidos (LONO, LOONO). Las
velocidades relativas y concentraciones en el estado estacionario de
.NO y radicales libres del oxígeno, así como los sitios de
producción celular de estas especies, determinan los efectos netos
observados pro- o antioxidantes del .NO. La mejor comprensión de los
roles fisiológicos que el .NO cumple en procesos oxidativos puede dar
bases más racionales para su utilización con fines terapéuticos.
Postal address: Dr. Homero Rubbo, Departamento de Bioquímica
Facultad de Medicina, General Flores 2125, Montevideo, Uruguay 11800
Fax: 5982-9249563; E-mail: email@example.com
Nitric oxide (.NO, nitrogen monoxide) is an
endogenously-synthesized free radical first characterized as a
non-eicosanoid component of endothelial-derived relaxation factor,
(EDRF)1. Nitric oxide is produced by a variety of mammalian cells
including vascular endo-thelium, neurons, smooth muscle cells,
macrophages, neutrophils, platelets and pulmonary epithelium2. The
physiological actions of .NO range from mediating vasodilation,
neurotransmission, inhibition of platelet adherence/aggregation and
the macrophage and neutrophil killing of pathogens. Many if not all of
these effects are mediated by the activation of soluble guanylate
cyclase, synthesis of cyclic guanosine 3’,5’-mono-phosphate (cGMP)
and the activation of a family of cGMP kinases3.
Nitric oxide exerts potent actions in the regulation of cell function
and tissue viability. Chemical reaction systems, cell and animal
models and clinical studies have recently revealed an ability of .NO
to modulate reactions and pathologic processes long associated with
the excess production and biological effects of reactive oxygen
species. The focus of this review will be to discuss the observed
pro-oxidant and antioxidant reactions of .NO in the context of lipid
oxidative processes based in our own recent observations that the
protective effects of .NO can often be ascribed to its antioxidant
properties and its ability to redirect the reactivity of partially
reduced oxygen species.
Lipid reactions of .NO are an important area of focus for multiple
reasons. First, this reactive species significantly concentrates in
lipophilic cell compartments, with an n-octanol:water partition
coefficient of 6-8:1. This solvation property will further enhance the
ability of .NO to regulate oxidant-induced membrane lipid oxidation.
Second, .NO reacts with lipid alkoxyl and peroxyl radicals (LO. and
LOO.) at near diffusion-limited rates, inferring that both lipid
peroxidation processes and reactions of lipophilic antioxidants will
be influenced by local .NO concentrations4-6. Third, the central role
that .NO plays in vascular diseases includes important reactivities of
.NO both as a signal transduction mediator and toward other free
radical species i.e. superoxide (O2.-) and LOO.. The issues to be
addressed includes 1) the influences of .NO and reactive species
commonly associated with oxidant stress on lipid and lipoprotein
systems, and 2) the mechanisms accounting for the protective effects
of .NO observed in pathological events associated with excess
production of reactive oxygen species.
Nitric oxide reaction with superoxide
A critical reaction that .NO undergoes in oxygenated biologic media
is direct bimolecular reaction with O2.-, yielding peroxynitrite
(ONOO-) at almost diffusion-limited rates (6.7 x 109 M-1 s-1, ref. 7).
This rate constant is ~3.5 times faster than the enzymatic
disproportionation of O2.- catalyzed by superoxide dismutases (SOD) at
neutral pH (kSOD = 2 x 109 M-1 s-1). Thus, ONOO- formation represents
a major potential pathway of .NO reactivity which depends on both
rates of tissue .NO and O2.- production and scavenging (e.g., local
superoxide dismutase and oxyhemoglobin concentrations). Peroxynitrite
has a half-life of <1 s under physiological conditions, due to
proton-catalyzed decomposition of peroxynitrous acid (ONOOH) and
competing target molecule reactions of ONOOH8. Nitric oxide will
potentiate many aspects of O2.--mediated tissue damage via ONOO-
formation. To date, it has been shown that ONOO- is a potent oxidant
capable of a) directly oxidizing protein and non-protein sulfhydryls9,
10, b) protonating to ONOOH, which exhibits both unique and hydroxyl
radical (.OH)-like reactions via metal-independent mechanisms11, 12
and c) reaction with metal centers to yield a species with the
reactivity of nitronium cation (NO2+), an oxidizing and nitrating
intermediate13. It is noteworthy that the mechanisms and extents of
ONOO- reaction will be strongly influenced by CO2/H2CO3, which is
typically 25 mM in biological tissues and can significantly exceed
this concentration during pathologic processes14.
Nitric oxide can potentiate O2.--mediated tissue damage and leads to
ONOO- formation, representing a major potential pathway of .NO
reactivity. Peroxynitrite is now being revealed as a key contributing
reactive species in pathological events associated with stimulation of
tissue production of .NO, e.g., systemic hypotension, inhibition of
intermediary metabolism, ischemia-reperfusion injury, immune
complex-stimulated pulmonary edema, cytokine-induced oxidant lung
injury, and inflammatory cell-mediated pathogen killing/host
injury15-17. There is growing evidence that .NO-mediated production of
ONOO- readily occurs in vivo, underscoring the importance of
understanding the target molecule reactions occurring during the
coordinated production of oxygen and nitrogen-containing reactive
Antioxidant reactions of nitric oxide
Since the reaction of .NO with O2.- yields the potent oxidant
ONOO-, from a purely chemical point of view it would follow that a) an
even broader array of target molecules would become susceptible to the
toxic effects of reactive oxygen species when .NO is present and b)
.NO will potentiate the toxicity of reactive oxygen species. While
this is sometimes the case, it is evident that .NO also exerts direct
or indirect antioxidant actions in biological systems subjected to
concomitant oxidant stress from excess production of reactive oxygen
species. The following sections develop these concepts in more detail.
a) Nitric oxide reaction with lipid epoxyallylic and peroxyl
Nitric oxide has been observed to play a critical role in
regulating lipid oxidation induced by reactive oxygen and nitrogen
species and activated reticuloendothelial cells5, 6, 20. Nitric oxide
(in some conditions) will stimulate O2.- induced lipid and lipoprotein
oxidation and under other conditions mediate protective reactions in
membranes by inhibiting O2.-, copper and ONOO--induced lipid oxidation
(Figure 1). The latter actions require higher (but still biologically
relevant) rates of .NO production. This revealed that oxygen radicals
can serve critical roles as modulators of the biological reactions of
.NO. We now know that .NO reacts with radical species including O2.-
and lipid peroxyl radicals (LOO.) at almost diffusion-limited rate
Nitric oxide has been reported to have contrasting effects on low
density lipoprotein (LDL) oxidation. For both macrophage and
endothelial cell model systems, increased rates of cell .NO production
via cytokine-mediated stimulation of inducible macrophage nitric oxide
synthase gene expression and activity or exogenous addition of .NO
have been shown to inhibit cell and O2.--mediated lipoprotein
oxidation6, 21-23. In contrast to these examples, the simultaneous
production of .NO and O2.- by 1,3-morpholino-sydnonimine-HCl (SIN-1)
or the direct addition of ONOO- has been shown to oxidize lipoproteins
to potentially atherogenic forms24, 25. Peroxynitrite-dependent
tyrosine nitration reactions in areas of atherosclerotic vessel lipid
deposition has also been shown to occur during both early and chronic
stages of atherosclerotic disease18.
Nitric oxide not only stimulates O2.- induced lipid and lipoprotein
oxidation via ONOO- production, but will also inhibit O2.- and
ONOO--induced lipid oxidation at slightly higher rates of .NO
production5. The prooxidant versus antioxidant outcome of these
reactions which are sensitive to .NO regulation are extremely
dependent on relative concentrations of individual reactive species.
For example, the continuous infusion of .NO at various rates into LDL
suspensions exposed to xanthine oxidase first stimulated and then
inhibited formation of 2-thiobarbituric acid reactive products at
rates of .NO infusion greater than 3 µM.min-1 (Figure 2). Nitric
oxide only stimulated O2.--dependent lipid peroxidation in LDL when
production rates of .NO were less than or equivalent to rates of O2.-
production. Thus, there is a dynamic competition between O2.- and
lipid radicals for reaction with .NO. More investigation is required
to understand the interaction of .NO with lipid epoxyallylic radicals,
the predominant species to which lipid alkoxyl radical (LO.)
rearranges following cyclization26.
The LDL particle consists of an apolar core of cholesteryl esters and
triglycerides, surrounded by a monolayer of phospholipids,
unesterified cholesterol and one molecule of apolipoprotein B-100,
with cholesteryl esters the most abundant lipid class found in LDL and
cholesteryl linoleate the principal oxidizable lipid. Indeed, we
observed that .NO inhibited cholesteryl linoleate oxidation in LDL in
a dose-dependent manner, with the concomitant formation of
nitrogen-containing lipid adducts27. In addition, analysis of
atherosclerotic human vessel lipid extracts by liquid
chromatography-mass spectrometry analysis showed that cholesteryl
linoleate oxidation products represented more than 85% of the total
cholesteryl linoleate fraction of atherosclerotic vessels. At least
25% of the luminal cell and plaque cholesteryl linoleate fraction of
atherosclerotic vessels consisted of nitrogen-containing oxidized
lipid derivatives27. It is important to note that the products of .NO
termination of lipid radical species are unstable and may mediate a
different spectrum of as yet undefined target molecule and pathologic
b) Nitric oxide-a-tocopherol interactions in lipid oxidation.
a-Tocopherol, a lipophilic chain-breaking antioxidant in biological
membranes and lipoproteins acts by donating hydrogen atoms to
chain-propagating peroxyl radical species (LOO.) to form the
corresponding hydro-peroxide28. Since the reaction of LOO. with
a-tocopherol occurs at a rate three orders of magnitude less than for
the reaction of LOO. with .NO, .NO could act more readily than
a-tocopherol, as an antioxidant defense against oxygen radical derived
oxidized lipid species. Based on comparison of relative rate
constants, it is predicted that the termination of LOO. by .NO will be
significantly more facile than both the reaction of LOO. with
a-tocopherol (k=2.5 x 106 M-1. s-1) and the initiation of secondary
peroxidation propagation reactions by LOO. with vicinal unsaturated
lipids (k= 30 - 200 M-1 . s-1).
In support of this argument, introduction of .NO into lipid oxidation
systems containing a-tocopherol results in preferential reaction of
.NO with lipid-derived radical species and prevents oxidation of
a-tocopherol (Figure 3). One mechanism explaining the protection of
a-tocopherol from oxidation by oxidizing lipids, can be the
preferential reaction of .NO with LO. and LOO. at significantly
greater rates than a-tocopherol to yield nitrogen-containing
radical-radical termination products. Another mechanism can be the
direct reduction of a-tocopheroxyl radical (and possibly further
oxidation states of a-tocopherol) by .NO, thus regenerating reduced
a-tocopherol and limiting the net extent of apparent a-tocopherol
oxidation29. Nitric oxide is thermodyna-mically capable of inhibiting
accumulation of a-tocopherol oxidation products via one electron
reduction of a-tocopheroxyl radical, with DGo’= -5 Kcal/mol. Because
lipid radicals in the lipophilic milieu do not readily partition into
the bulk aqueous medium, we postulate that .NO can act as a reductant
of a-tocopherol in membrane and hydrophobic lipoprotein compartments,
where reducing equivalents are not readily transferred from
water-soluble reductants (eg. ascorbate, thiols, ref. 29). This
mechanism could explain the observed additive antioxidant effects of
.NO and a-tocopherol in comparison with the pair ascorbate plus
a-tocopherol (Figure 3). The mobility of a-tocopherol in the lateral
plane of the membrane and its exact positioning in the membrane may
restrict its antioxidant actions, in part explaining why .NO can be
much more facile at terminating lipid peroxyl radical species. Thus,
because of a high reactivity with other radical species, a relatively
lower reactivity of lipid radical-.NO termination products and an
ability of .NO to readily traverse membranes and lipoproteins, .NO can
effectively terminate radical species throughout all aspects of
membrane and lipoprotein microenvironments. This can help maintain
other tissue antioxidant defenses as well, during periods of oxidant
c) Nitric oxide reactions with metals
Nitric oxide can react with metal centers in proteins including
heme iron, iron-sulfur clusters and copper. Examples are the
activation of soluble guanylate cyclase, a heme-containing enzyme, via
the formation of an iron-nitrosyl complex30. It has been postulated
that .NO can exert a protective role towards metal complex and
metalloprotein-catalyzed lipid oxidation, via formation of
catalytically inactive metal iron-nitrosyl complexes, thereby
modulating the pro-oxidant effects of iron and other transition
metals31. Iron-nitrosyl complexes were detected in several proteins,
including mammalian ferritin, transferrin, myoglobin and hemoglobin,
albeit in the presence of high concentrations of .NO32. It is
important to note that the rate of .NO reaction with most metal
centers is significantly slower than for the almost diffusion-limited
reaction of .NO with either O2.- or LO. and LOO. species, critical for
propagation of radical chain reactions. It should also be noted that
.NO can exert prooxidant effects with transition metals as well, by
reducing ferric iron complexes. This can induce the release of bound
iron and indirectly substitute for other reductants in the Haber-Weiss
reaction-mediated production of .OH from H2O2.
Structural-functional studies of the catalytic site of lipoxygenase
(SLO) reveals formation of a ferrous-nitrosyl complex following enzyme
exposure to .NO. From this .NO-SLO interaction, it was proposed that
.NO inhibits SLO-dependent lipid oxidation via direct enzyme
inactivation. However, at physiological low rates of .NO production,
.NO only minimally inhibits lipoxygenase catalytic activity6. At
µM.min-1 rates of .NO production, no evidence of .NO reaction with
either Fe-EDTA or the active site of SLO was detectable by electron
spin resonance analysis6. From all of the above, it is concluded that
the inhibitory effect of .NO towards oxygen radical or SLO-dependent
oxidation of multiple lipid and lipoprotein targets, as determined by
multiple criteria, was due to termination of lipid radical chain
propagation reactions rather than .NO reaction with transition metals.
Inflammation and .NO
A number of model systems for inflammation, vascular disease
(atherogenesis, restenosis following angioplasty) and surgical
problems (ischemia-reperfusion injury, graft reanastomosis) that
include a pathogenic role for oxidant injury indicate that either
endogenous .NO biosynthesis or exogenous supplementation with sources
of .NO inhibit oxidant-dependent damage at both molecular and tissue
functional levels. Many if not all of these studies have inflammatory
injury as a common denominator.
Atherosclerosis is one example where this phenemenon occurs. The
changes which occur during atherosclerosis includes loss of the
control of vascular tone, an .NO-dependent event. Increasing the
availability of the substrate L-arginine for .NO synthesis will
restore vascular function, while inhibiting .NO synthesis is
Another early event in the atherosclerotic process is the chemical
transformation of LDL through the initiation of oxidation. Probably in
an attempt at host protection, oxidized LDL is taken up by
macrophages, resulting in lipid-laden foam cells. These cells then
become part of the problem, because of the effect of their secretory
products on other cells in the lesion and the release of pro-oxidative
enzymes such as 15-lipoxygenase. The balance between .NO and O2.-
production in the artery wall may also play a role in the oxidation of
LDL (Figure 4). Peroxynitrite oxidizes LDL, causes a rapid depletion
of several antioxidants (ascorbate, urate, protein thiols and
ubiquinol) and releases copper ions from the plasma protein
caeruloplasmin. Copper ions are powerful catalysts of LDL oxidation
which have been detected in advanced human atherosclerotic lesions.
It is interesting to note that both animal model and clinical studies
are showing that chronic administration of L-arginine improves
endothelial dependent relaxation, decreases inflammatory cell
accumulation at the vessel wall and reduces intimal hyperplasia, all
hallmarks of atherosclerotic disease36. Furthermore, balloon
angio-plasty is often used to treat atherosclerotic vasoocclusive
problems. Both administration of .NO donors as well as transfection of
constitutive nitric oxide synthase to balloon-injured vessels reduces
the intimal cell hyperplasia, often the cause for repeat angioplasty,
aortocoronary bypass graft surgery or myocardial infarction37.
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Fig. 1.- Nitric oxide inhibition of copper or SIN1-dependent low
density lipoprotein oxidation. Human LDL was incubated for 3 hr at
37oC with 10 µM cupric sulfate or the peroxynitrite donnor SIN-1 (1
mM) in the absence and presence of 3 µM.min-1 .NO production from 100
µM spermine NONOate. Lipid oxidation was assayed by thiobarbituric
acid positive material formation at 532 nm.
Fig. 2.- Pro- and antioxidant fates of nitric oxide on low density
lipoprotein oxidation. Human LDL was incubated for 3 hr at 37oC with
hypoxanthine/xanthine oxidase (3 µM.min-1 O2.- production) in the
absence and presence of .NO gas.
Low .NO / O2.- ratios increase .NO -mediated LDL oxidation via
formation of peroxynitrite.
High .NO / O2.- ratios decrease .NO -mediated LDL oxidation by
termination reaction of .NO with peroxyl radicals.
Fig. 3.- Inhibition of xanthine oxidase-induced linolenic acid
oxidation by .NO, a-tocopherol and ascorbate. Linolenic acid was
incubated for 3 hr at 37oC (control), with 50 µM hypoxanthine/ 5
mU-ml xanthine oxidase in the presence of ascorbate (50 µM),
a-tocopherol (50 µM), S-NONOate (100 µM) or a combination of both
a-tocopherol plus S-NONOate or a-tocopherol plus ascorbate.
Nitric oxide and a-tocopherol exert similar cooperative lipid
antioxidant activities than ascorbate plus a-tocopherol.
Fig. 4.- The double-edged action of nitric oxide on
superoxide-mediated lipid oxidation.