Major phenolic compounds in olive oil: metabolism and health effects

It has been postulated that the components in olive oil in the Mediterranean diet, a diet which is largely vegetarian in nature, can contribute to the lower incidence of coronary heart disease and prostate and colon cancers. The Mediterranean diet includes the consumption of large amounts of olive oil. Olive oil is a source of at least 30 phenolic compounds. The major phenolic compounds in olive oil are oleuropein, hydroxytyrosol and tyrosol. Recently there has been a surge in the number of publications that has investigated their biological properties. The phenolic compounds present in olive oil are strong antioxidants and radical scavengers. Olive "waste water" also possesses compounds which are strong antioxidant and radical scavengers. Typically, hydroxytyrosol is a superior antioxidant and radical scavenger to oleuropein and tyrosol. Hydroxytyrosol and oleuropein have antimicrobial activity against ATTC bacterial strains and clinical bacterial strains. Recent syntheses of labeled and unlabelled hydroxytyrosol coupled with superior analytical techniques have enabled its absorption and metabolism to be studied. It has recently been found that hydroxytyosol is renally excreted unchanged and as the following metabolites as its glucuronide conjugate, sulfate conjugate, homovanillic acid, homovanillic alcohol, 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylacetaldehyde. Studies with tyrosol have shown that it is excreted unchanged and as its conjugates. This review summarizes the antioxidant abilities; the scavenging abilities and the biological fates of hydroxytyrosol, oleuropein and tyrosol which have been published in recent years.     

Influence of epithelial lining fluid lipids on NO(2)-induced membrane oxidation and nitration

Within the pulmonary epithelial lining layer (ELF), antioxidants such as ascorbic acid (AH(2)) and glutathione (GSH) react with inhaled nitrogen dioxide ((*)NO(2)) to produce reactive oxygen species (ROS) that induce cellular oxidation. Because the ELF contains unsaturated fatty acids (UFA), which potentially react with (*)NO(2) and/or the antioxidant-derived ROS, we studied the influence of aqueous phase model UFA [egg phosphatidylcholine (EggPC) liposomes] on exposure-induced oxidation and nitration of membranes. Our lung surface model used gas phase (*)NO(2) exposures of immobilized red cell membranes (RCM) overlaid with defined aqueous phases. Acetyl cholinesterase (AChE) activity, TBARS, and 3-nitrotyrosine (3-NT) were used to assess protein and lipid oxidation and RCM nitration, respectively. During (*)NO(2) exposure, AH(2) and GSH induced AChE loss and TBARS, which were unchanged with buffer only. Exposures of EggPC generated extensive TBARS but not AChE loss; addition of AH(2)/GSH to EggPC resulted in smaller AChE declines and fewer TBARS. 3-NT formation occurred with or without EggPC, low concentration antioxidants, SOD, catalase, or DTPA, but was inhibitable by desferrioxamine or high antioxidant concentrations. The data suggest that reaction/diffusion limitations govern (*)NO(2) distribution, that (*)NO(2) per se directly nitrates tyrosine residues within hydrophobic regions, and that the induction of secondary oxidative processes is dependent on nonlinear relationships among (*)NO(2) flux rates, antioxidant concentrations, and diffusivity of secondary reactive species.     

Effects of virgin olive oil phenolics on scavenging of reactive nitrogen species and upon nitrergic neurotransmission

The major phenolics from the polar fraction of virgin olive oil (caffeic acid, oleuropein, tyrosol and hydroxytyrosol) have well-established antioxidant activities but their effects on reactive nitrogen species and nitrergic neurotransmission have not been fully investigated. The three catechol compounds were active as scavengers of nitric oxide generated spontaneously from the decomposition of sodium nitroprusside (approximately 50% inhibition achieved at 75 microM), and had similar ability to scavenge chemically generated peroxynitrite, as determined by an alpha1-antiproteinase inactivation assay (67.2%-92.4% reduction when added at 1 mM). Tyrosol was less active in these tests, but does not possess the catechol functionality. Despite their ability to interact with chemically prepared nitric oxide, neither oleuropein nor hydroxytyrosol at 5 microM altered NO*-mediated relaxations of the nerve-stimulated rat anococcygeus preparation, but this may be because the nitrergic transmitter is protected from the effects of externally applied scavengers. In conclusion, the phenolics found in virgin olive oil possess ability to scavenge reactive oxygen and nitrogen species that are implicated in human pathologies, but their impact may be restricted to those species present in the extracellular environment.     

Postprandial LDL phenolic content and LDL oxidation are modulated by olive oil phenolic compounds in humans

Olive oil phenolic compounds are potent antioxidants in vitro, but evidence for antioxidant action in vivo is controversial. We examined the role of the phenolic compounds from olive oil on postprandial oxidative stress and LDL antioxidant content. Oral fat loads of 40 mL of similar olive oils, but with high (366 mg/kg), moderate (164 mg/kg), and low (2.7 mg/kg) phenolic content, were administered to 12 healthy male volunteers in a cross-over study design after a washout period in which a strict antioxidant diet was followed. Tyrosol and hydroxytyrosol, phenolic compounds of olive oil, were dose-dependently absorbed (p<0.001). Total phenolic compounds in LDL increased at postprandial state in a direct relationship with the phenolic compounds content of the olive oil ingested (p<0.05). Plasma concentrations of tyrosol, hydroxytyrosol, and 3-O-methyl-hydroxytyrosol directly correlated with changes in the total phenolic compounds content of the LDL after the high phenolic compounds content olive oil ingestion. A 40 mL dose of olive oil promoted a postprandial oxidative stress, the degree of LDL oxidation being lower as the phenolic content of the olive oil administered increases. In conclusion, olive oil phenolic content seems to modulate the LDL phenolic content and the postprandial oxidative stress promoted by 40 mL olive oil ingestion in humans.     

The enantioselective immunoaffinity extraction of an optically active ibuprofen-modified peptide fragment

Recent in vitro studies have demonstrated antioxidant properties of some virgin olive oil phenolic compounds. One of the prerequisites to extrapolate these data to an in vivo situation is the knowledge of their bioavailability in humans. In the present work we describe an analytical method which enables us to perform hydroxytyrosol and tyrosol quantitative determinations in human urine. This method was successfully used in bioavailability studies of both phenolic compounds after acute olive oil administration. Virgin olive oil was administered to healthy volunteers after a low phenolic diet. The dose administered of both phenolic compounds was estimated in reference to free forms of hydroxytyrosol and tyrosol present in virgin olive oil extracts before and after being submitted to hydrolytic conditions. These conditions mimic those occurring during digestion. Urine samples were collected before and after acute olive oil intake and analyzed by capillary gas chromatography-mass spectrometry. Hydroxytyrosol and tyrosol urinary recovery increased in response to olive oil administration, obtaining maximal values in the first 4 h. Our results further indicate that hydroxytyrosol and tyrosol are mainly excreted in conjugated form, since only 5.9 +/- 1.4% (hydroxytyrosol) and 13.8 +/- 5.4% (tyrosol) of the total amounts excreted in urine were in free form.     

(-)-Epicatechin elevates nitric oxide in endothelial cells via inhibition of NADPH oxidase

Dietary (-)-epicatechin is known to improve bioactivity of (*)NO in arterial endothelium of humans, but the mode of action is unclear. We used the fluorophore 4,5-diaminofluorescein diacetate to visualize the (*)NO level in living human umbilical vein endothelial cells (HUVEC). Untreated cells showed only a weak signal, whereas pretreatment with (-)-epicatechin (10 microM) or apocynin (100 microM) elevated the (*)NO level. The effects were more pronounced when the cells were treated with angiotensin II with or without preloading of the cells with (*)NO via PAPA-NONOate. While (-)-epicatechin scavenged O2(*-), its O-methylated metabolites prevented O2(*-) generation through inhibition of endothelial NADPH oxidase activity, even more strongly than apocynin. From the effect of 3,5-dinitrocatechol, an inhibitor of catechol-O-methyltransferase (COMT), on HUVEC it is concluded that (-)-epicatechin serves as 'prodrug' for conversion to apocynin-like NADPH oxidase inhibitors. These data indicate an (*)NO-preserving effect of (-)-epicatechin via suppression of O2(*-)-mediated loss of (*)NO.     

Chemoenzymatic synthesis of hydroxytyrosol monoesters and their suppression effect on nitric oxide production stimulated by lipopolysaccharides

Fatty acid monoesters of hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol] were synthesized in two steps from tyrosol (4-hydroxyphenylethanol) by successive Candida antarctica lipase B-catalyzed chemoselective acylation on the primary aliphatic hydroxy group over phenolic hydroxy group in tyrosol, and 2-iodoxybenzoic acid (IBX)-mediated hydroxylation adjacent to the remaining free phenolic hydroxy group. Examination of their suppression effects on nitric oxide production stimulated by lipopolysaccharides in RAW264.7 cells showed that hydroxytyrosol butyrate exhibited the highest inhibition (IC₅₀ 7.0 μM) among the tested compounds.     

Formation of TEMPOL-hydroxylamine during reaction between TEMPOL and hydroxyl radical: HPLC/ECD study

Nitroxyl radicals are important antioxidants that have been used to protect animal tissues from oxidative damage. Their reaction with hydroxyl radical ((*)OH) is generally accepted to be the mechanism of antioxidant function. However, the direct interaction of nitroxyl radicals with (*)OH does not always provide a satisfactory explanation in various pH, because the concentration of hydrogen ion may affect the generation of secondary (*)OH-derived radicals. In the present study, it was confirmed that the reaction between 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and (*)OH generated TEMPOL-hydroxylamine, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPON-hydroxylamine using HPLC coupled with electrochemical detection. In the absence of NADH, TEMPOL-H may be generated by the reaction with secondary (*)OH-derived radicals in acidic condition. In the presence of NADH, a large proportion of the non-paramagnetic products was TEMPOL-H. Finally, it was clarified that TEMPOL-H was generated during dopamine metabolism, which is believed to be one of the (*)OH sources in pathological processes such as Parkinson's disease.     

Use of whole cells of Pseudomonas aeruginosa for synthesis of the antioxidant hydroxytyrosol via conversion of tyrosol

For the first time, a soil bacterium, designated Pseudomonas aeruginosa, was isolated based on its ability to grow on tyrosol as a sole source of carbon and energy. During growth on tyrosol, this strain was capable of promoting the formation of a significant amount of hydroxytyrosol and trace quantities of parahydroxyphenyl acetic acid and 3,4-dihydroxyphenyl acetic acid. The products were confirmed by high-performance liquid chromatography and gas chromatography-mass spectrometry analyses. Using an optimized tyrosol concentration of 2 g liter(-1), the maximal hydroxytyrosol yield (80%) was achieved after a 7-h reaction in a growth experiment. To enhance the formation of hydroxytyrosol and prevent its degradation, a resting-cell method using P. aeruginosa was performed. The growth state of the culture utilized for biomass production, the carbon source on which the biomass was grown, the concentration of the biomass, and the amount of tyrosol that was treated were optimized. The optimal yield of hydroxytyrosol (96%) was obtained after a 7-h reaction using 4 g of tyrosol liter(-1) and 5 g of cells liter(-1) pregrown on tyrosol and harvested at the end of the exponential phase. This proposed procedure is an alternative approach to obtain hydroxytyrosol in an environmentally friendly way. In addition, the reaction is easy to perform and can be adapted to a bioreactor for industrial purposes.     

Block of cardiac ATP-sensitive K(+) channels reduces hydroxyl radicals in the rat myocardium

The present study examined whether opening of an ATP-sensitive K(+) (K(ATP)) channel can induce hydroxyl free radical ((*)OH) generation in the rat myocardium. Sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused directly through a microdialysis probe to detect the generation of (*)OH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA). Induction of cromakalim (100 microM), a K(ATP) channel opener, through the microdialysis probe significantly increased the level of 2,3-DHBA. Another K(ATP) channel opener, nicorandil, also increased the level of 2,3-DHBA. When iron(II) was administered to cromakalim-pretreated animals, a marked elevation of DHBA was observed, compared with iron(II) only-treated animals. A positive linear correlation between iron(II) and formation of (*)OH, trapped as DHBA in the dialysate, was shown (r(2) = 0.988). When corresponding experiments were performed with nicorandil-treated animals, a positive linear correlation between iron(II) and DHBA in the dialysate was shown (r(2) = 0.988). However, the presence of glibenclamide (1-50 microM) decreased the cromakalim-induced 2,3-DHBA formation in a concentration-dependent manner (IC(50) = 9.1 microM). 5-Hydroxydecanoate (5-HD; 100 microM), another K(ATP) channel antagonist, also decreased cromakalim-induced (*)OH formation. The IC(50) value for 5-HD against cromakalim-evoked increase in 2,3-DHBA was 107.2 microM. In the presence of glibenclamide (10 microM), the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, the normal elevation of 2,3-DHBA in the heart dialysate was not observed in animals pretreated with glibenclamide (10 microM). When corresponding experiments were performed with 5-HD (100 microM) pretreated animals, the same results were obtained. These results suggest that opening of cardiac K(ATP) channels may cause (*)OH generation.     

Use of Whole Cells of Pseudomonas aeruginosa for Synthesis of the Antioxidant Hydroxytyrosol via Conversion of Tyrosol

For the first time, a soil bacterium, designated Pseudomonas aeruginosa, was isolated based on its ability to grow on tyrosol as a sole source of carbon and energy. During growth on tyrosol, this strain was capable of promoting the formation of a significant amount of hydroxytyrosol and trace quantities of parahydroxyphenyl acetic acid and 3,4-dihydroxyphenyl acetic acid. The products were confirmed by high-performance liquid chromatography and gas chromatography-mass spectrometry analyses. Using an optimized tyrosol concentration of 2 g liter⁻¹, the maximal hydroxytyrosol yield (80%) was achieved after a 7-h reaction in a growth experiment. To enhance the formation of hydroxytyrosol and prevent its degradation, a resting-cell method using P. aeruginosa was performed. The growth state of the culture utilized for biomass production, the carbon source on which the biomass was grown, the concentration of the biomass, and the amount of tyrosol that was treated were optimized. The optimal yield of hydroxytyrosol (96%) was obtained after a 7-h reaction using 4 g of tyrosol liter⁻¹ and 5 g of cells liter⁻¹ pregrown on tyrosol and harvested at the end of the exponential phase. This proposed procedure is an alternative approach to obtain hydroxytyrosol in an environmentally friendly way. In addition, the reaction is easy to perform and can be adapted to a bioreactor for industrial purposes.     

Protective effect of melatonin against homocysteine-induced vasoconstriction of human umbilical artery

Hyperhomocysteinemia is a major and independent risk factor for vascular disease. Oxidative stress is a possible mechanism for homocysteine (Hcy)-induced endothelial dysfunction. Herein, we evaluated the antioxidant property of melatonin (MLT) in relation to the vasoconstrictive effect of Hcy on the human umbilical artery. In an initial experiment in a cell-free system, a micromolar concentration of iron was found to catalyze oxygen-dependent oxidation of Hcy. MLT (10 or 100 microM) did not affect oxygen-dependent oxidation of Hcy. Next, smooth muscle contraction induced by prostaglandin F(2alpha) (10 microM) was measured in arterial strips. Hcy (10 to 500 microM) increased this vascular tension in a concentration-dependent manner (P < 0.0001). Addition of Fe(2+) (10 microM) significantly potentiated the Hcy effect. Removal of endothelium (P < 0.05), pretreatment with a nitric oxide (NO) synthesis inhibitor (l-N(G)-monomethylarginine, 200 microM, P < 0.001), or pretreatment with a hydroxyl radical ((*)OH) scavenger (mannitol, 10 mM, P < 0.001) significantly attenuated contraction potentiated by Hcy plus Fe(2+). At a much lower concentration than mannitol, MLT (1 to 100 microM) significantly reduced the contractile effect of Hcy and Fe(2+) in a concentration-dependent manner. Hcy plus Fe(2+) significantly impaired calcium ionophore A 23187-induced relaxation (P < 0.0001), while MLT restored this relaxation in a concentration-dependent manner. These findings suggest that Hcy potentiates vascular tension in human umbilical artery, possibly by suppressing bioavailable NO. MLT protects against the vasoconstrictive effect of Hcy, most likely by scavenging (*)OH arising from Hcy autooxidation.     

Regulation of xanthine oxidase by nitric oxide and peroxynitrite

Xanthine oxidase (XO) is a central mechanism of oxidative injury as occurs following ischemia. During the early period of reperfusion, both nitric oxide (NO(*)) and superoxide (O-*(2)) generation are increased leading to the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the presence and nature of the interactions of NO(*) or ONOO(-) with XO and the role of this process in regulating oxidant generation. Therefore, we determined the dose-dependent effects of NO(*) and ONOO(-) on the O-*(2) generation and enzyme activity of XO, respectively, by EPR spin trapping of O-*(2) using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO(-) markedly inhibited both O-*(2) generation and XO activity in dose-dependent manner, while NO(*) from NO(*) gas in concentrations up to 200 microM had no effect. Furthermore, we observed that NO(*) donors such as NOR-1 also inhibited O-*(2) generation and XO activity; however, these effects were O-*(2)-dependent and blocked by superoxide dismutase or ONOO(-) scavengers. Finally, we found that ONOO(-) totally abolished the Mo(V) EPR spectrum. These changes were irreversible, suggesting oxidative disruption of the critical molybdenum center of the catalytic site. Thus, ONOO(-) formed in biological systems can feedback and down-regulate XO activity and O-*(2) generation, which in turn may serve to limit further ONOO(-) formation.     

Comparative effectiveness of antioxidants in protecting the bacterial plasmatic membrane from the active froms of oxygen

The effect of hydroxyl radicals OH. generated by the decomposition of H2O2 by Fe2+ ions (Fenton reaction) on the barrier properties of plasma membranes of Escherichia coli cells K-12 was studied by electroorientation spectroscopy. It was found that the administration of hydrogen peroxide led to the disturbance of the barrier properties of plasma membranes only when the cells were preincubated with Fe2+ ions and their constant concentration in the system was maintained by ascorbate or dithiotreitol (150-500 microM). The extent of the toxic action on plasma membranes depended on the concentration of reacting elements and the substance used as a reducer Fe2+. The efficiency of protection of antioxidants of different classes (enzymic, SH-containing, and phenolic compounds) against the toxic action of hydroxyl radicals on plasmatic membranes was shown.     

Application of a YHB1-GFP reporter to detect nitrosative stress in yeast

Yeast flavohemoglobin (Yhb1p) plays a pivotal role in NO(*) detoxification and has also been implicated in oxidative/reductive stress responses. In this study, we have used a YHB1-GFP reporter expressing a full-length chromosome-tagged Yhb1-GFP fusion protein to monitor changes in flavohemoglobin levels after cell treatment with oxidants, antioxidants and nitric oxide donors. Only nitric oxide donors were found to induce a dose-dependent increase in Yhb1-GFP expression while hydrogen peroxide, menadione and diamide caused a moderate diminution of YHB1-GFP fluorescence. Additionally, the levels of endogenous and hydroperoxide-induced ROS production in the Deltayhb1 mutant were comparable to those in the isogenic wild-type strain. Although peroxides increased NO(*) generation while nitrite and nitric oxide donors augmented ROS levels in yeast cells, their effects were generally not more pronounced in Deltayhb1 than in wild-type cells. Taken together, these data suggest that yeast flavohemoglobin does not contribute to cross-protection against oxidative and nitrosative stress.     

Thioredoxin, a singlet oxygen quencher and hydroxyl radical scavenger: redox independent functions

Thioredoxin is a ubiquitous small protein known to protect cells and tissues against oxidative stress. However, its exact antioxidant nature has not been elucidated. In this report, we present evidence that human thioredoxin is a powerful singlet oxygen quencher and hydroxyl radical scavenger. Human thioredoxin at 3 microM caused 50% inhibition of TEMP-(1)O(2) (TEMPO) adduct formation in a photolysis EPR study. In contrast, Escherichia coli thioredoxin caused 50% inhibition of TEMPO formation at 80 microM. Both E. coli thioredoxin and human thioredoxin inhibited (*)OH dependent DMPO-OH formation as demonstrated by EPR spectrometry. The quenching of (1)O(2) or scavenging of (*)OH was not dependent upon the redox state of thioredoxin. Using a human thioredoxin in which the structural cysteines were mutated to alanine, Trx-C3A, we show that structural cysteines that do not take part in the catalytic functions of the protein are also important for its reactive oxygen scavenging properties. In addition, using a quadruple mutant Trx-C4A, where one of the catalytic cysteines, C35 was mutated to alanine in addition to the mutated structural cysteines, we demonstrated that catalytic cysteines are also required for the scavenging action of thioredoxin. Identification of thioredoxin as a (1)O(2) quencher and (*)OH scavenger may be of significant importance in explaining various redox-related antioxidant functions of thioredoxin.     

Mechanistic studies of the oxygen-mediated oxidation of nitrosylhemoglobin

Nitrosylhemoglobin (HbFe(II)NO) has been shown to be generated in vivo from the reaction of deoxyHb with NO(*) as well as with nitrite. Despite the physiological importance attributed to this form of Hb, its reactivity has not been investigated in detail. In this study, we showed that the rate of oxidation of HbFe(II)NO by O(2) does not depend on the O(2) concentration. The reaction time courses had to be fitted to a two-exponential expression, and the obtained rates were approximately 2 x 10(-)(4) and 1 x 10(-)(4) s(-)(1), respectively. In the presence of the allosteric effector inositol hexaphosphate (IHP), the value for the fast component of the rate was significantly larger (44 x 10(-)(4) s(-)(1)) whereas that for the slow step was only slightly higher (2.5 x 10(-)(4) s(-)(1)). Moreover, we found that both in the absence and in the presence of IHP the rate of the O(2)-mediated oxidation of HbFe(II)NO is essentially identical to that of NO(*) dissociation from HbFe(II)NO, determined under analogous conditions by replacement of NO(*) with CO in the presence of an excess of dithionite. Taken together, our data show that the reaction between O(2) and HbFe(II)NO proceeds in three steps via dissociation of NO(*) (rate-determining step), binding of O(2) to deoxyHb, and NO(*)-mediated oxidation of oxyHb to metHb and nitrate.     

Effect of high-salt diet on NO release and superoxide production in rat aorta

Sprague-Dawley rats were fed either a high-salt (HS) diet (4.0% NaCl) or a low-salt (LS) diet (0.4% NaCl) for 3 days. Nitric oxide (NO) and superoxide production were assessed in the thoracic aorta by evaluating the fluorescence signal intensity from 4,5-diaminofluorescein (DAF-2DA) and dihydroethidine, respectively. Methacholine caused increased NO release in the aortas from rats on a LS but not HS diet. The SOD mimetic tempol restored methacholine-induced NO release in aortas from rats on a HS diet. Methacholine also caused superoxide production in the aortas of rats on a HS diet but not in the aortas of rats on a LS diet. Tempol and N(G)-monomethyl-l-arginine eliminated methacholine-induced superoxide production in the aortas of rats on a HS diet. Aortic rings from rats on the HS diet showed impaired methacholine-induced relaxation, which was improved by tempol. Tempol alone caused a NO-dependent relaxation of norepinephrine-precontracted aortas that was significantly greater in the aortas of rats on the HS diet than in vessels from rats on the LS diet. These data suggest that a HS diet impairs endothelium-dependent relaxation via reduced NO levels and increased superoxide production.     

Vascular effects of a common gene variant of extracellular superoxide dismutase in heart failure

A common gene variant of human extracellular superoxide dismutase (ecSOD), in approximately 5% of humans, is associated with increased risk of ischemic heart disease. The purpose of this study was to examine vascular effects of ecSOD with effects of the ecSOD variant (ecSOD(R213G)) in rats with heart failure. Seven weeks after coronary artery ligation, we studied rats with heart failure and sham-operated rats. Adenoviral vectors expressing human ecSOD, ecSOD(R213G), or a control virus were injected intravenously. In the aorta from rats with heart failure, responses to acetylcholine (69 +/- 4% relaxation, means +/- SE) and basal levels of nitric oxide (NO) (vasoconstrictor responses to a NO synthase inhibitor) were greatly impaired, and levels of superoxide and peroxynitrite were increased. Gene transfer of ecSOD restored responses to acetylcholine (92 +/- 2% relaxation) and basal levels of NO to normal and reduced levels of superoxide [from 2.3 +/- 0.2 to 0.9 +/- 0.2 relative light units per second per millimeter squared (RLU x s(-1) x mm(-2))] and peroxynitrite (from 2.4 +/- 0.2 to 0.9 +/- 0.1 RLU x s(-1) x mm(-2)) in the aorta from rats with heart failure. Gene transfer of ecSOD(R213G) produced little or no improvement. Responses to nitroprusside were not different among the groups. Expression of endogenous mRNA for SODs (CuZnSOD, MnSOD, and ecSOD) and endothelial NOS in the aorta was not different among the groups. In contrast to ecSOD, gene transfer of ecSOD(R213G) in rats with heart failure has minimal beneficial effect on oxidative stress, endothelial function, or basal bioavailability of NO. We speculate that greatly diminished efficacy of ecSOD(R213G) in protection against oxidative stress and endothelial dysfunction may contribute to increased risk of cardiovascular disease in humans with ecSOD(R213G).