The Scavenging Effect of Myoglobin from Meat Extracts toward Peroxynitrite Studied with a Flow Injection System Based on Electrochemical Reduction over a Screen-Printed Carbon Electrode Modified with Cobalt Phthalocyanine: Quantification and Kinetics.

The scavenging activity of myoglobin toward peroxynitrite (PON) was studied in meat extracts, using a new developed electrochemical method (based on cobalt phthalocyanine-modified screen-printed carbon electrode, SPCE/CoPc) and calculating kinetic parameters of PON decay (such as half-time and apparent rate constants). As reactive oxygen/nitrogen species (ROS/RNS) affect the food quality, the consumers can be negatively influenced. The discoloration, rancidity, and flavor of meat are altered in the presence of these species, such as PON. Our new highly thermically stable, cost-effective, rapid, and simple electrocatalytical method was combined with a flow injection analysis system to achieve high sensitivity (10.843 nA µM-1) at a nanomolar level LoD (400 nM), within a linear range of 3-180 µM. The proposed biosensor was fully characterized using SEM, FTIR, Raman spectroscopy, Cyclic Voltammetry (CV), Differential Pulse Voltammetry (DPV), and Linear Sweep Voltammetry (LSV). These achievements were obtained due to the CoPc-mediated reduction of PON at very low potentials (around 0.1 V vs. Ag/AgCl pseudoreference). We also proposed a redox mechanism involving two electrons in the reduction of peroxynitrite to nitrite and studied some important interfering species (nitrite, nitrate, hydrogen peroxide, dopamine, ascorbic acid), which showed that our method is highly selective. These features make our work relevant, as it could be further applied to study the kinetics of important oxidative processes in vivo or in vitro, as PON is usually present in the nanomolar or micromolar range in physiological conditions, and our method is sensitive enough to be applied.

Oligomerization and Nitration of the Grass Pollen Allergen Phl p 5 by Ozone, Nitrogen Dioxide, and Peroxynitrite: Reaction Products, Kinetics, and Health Effects.

The allergenic and inflammatory potential of proteins can be enhanced by chemical modification upon exposure to atmospheric or physiological oxidants. The molecular mechanisms and kinetics of such modifications, however, have not yet been fully resolved. We investigated the oligomerization and nitration of the grass pollen allergen Phl p 5 by ozone (O3), nitrogen dioxide (NO2), and peroxynitrite (ONOO-). Within several hours of exposure to atmospherically relevant concentration levels of O3 and NO2, up to 50% of Phl p 5 were converted into protein oligomers, likely by formation of dityrosine cross-links. Assuming that tyrosine residues are the preferential site of nitration, up to 10% of the 12 tyrosine residues per protein monomer were nitrated. For the reaction with peroxynitrite, the largest oligomer mass fractions (up to 50%) were found for equimolar concentrations of peroxynitrite over tyrosine residues. With excess peroxynitrite, the nitration degrees increased up to 40% whereas the oligomer mass fractions decreased to 20%. Our results suggest that protein oligomerization and nitration are competing processes, which is consistent with a two-step mechanism involving a reactive oxygen intermediate (ROI), as observed for other proteins. The modified proteins can promote pro-inflammatory cellular signaling that may contribute to chronic inflammation and allergies in response to air pollution.

Protective Role of Glutathione against Peroxynitrite-Mediated DNA Damage During Acute Inflammation.

Inflammation is an immune response to protect against various types of infections. When unchecked, acute inflammation can be life-threatening, as seen with the current coronavirus pandemic. Strong oxidants, such as peroxynitrite produced by immune cells, are major mediators of the inflammation-associated pathogenesis. Cellular thiols play important roles in mitigating inflammation-associated macromolecular damage including DNA. Herein, we have demonstrated a role of glutathione (GSH) and other thiols in neutralizing the effect of peroxynitrite-mediated DNA damage through stable GSH-DNA adduct formation. Our observation supports the use of thiol supplements as a potential therapeutic strategy against severe COVID-19 cases and a Phase II (NCT04374461) open-label clinical trial launched in early May 2020 by the Memorial Sloan Kettering Cancer Center.

Identification of sesamol byproducts produced by plasma treatment with inhibition of advanced glycation endproducts formation and ONOO- scavenging activities.

Little is known of plasma-mediated relations between major food components and their biological capacities. In the present work, the effects of dielectric barrier discharge (DBD) plasma irradiation on pure sesamol and sesame oil were investigated using spectroscopic (LC-MS, NMR) and bioassay methods. Sesamol was degraded when subjected to plasma irradiation for 40 min, and the exposed products exhibited improved anti-glycation capacities against advanced glycation end products (AGEs) formation and better ONOO- scavenging ability. Structures of newly formed compounds were determined spectroscopically. Quantitative LC-MS analysis of the major products generated in sesamol and sesame oil was achieved using isolates 1-4 of purified sesamol plasma treated for 40 min. These results indicate that the predominant chemical changes induced in sesamol and sesame oil by DBD plasma treatment might enhance biological properties.

Screening of peroxynitrite scavengers in Flos Lonicerae by using two new methods, an HPLC-DAD-CL technique and a peroxynitrite spiking test followed by HPLC-DAD analysis.

INTRODUCTION: Peroxynitrite is involved in the pathogenesis of a number of significant diseases. Peroxynitrite scavengers thus have potential application in understanding and treating these diseases. It is, therefore, important to establish screening methods able to rapidly identify peroxynitrite scavengers from herbal plants. OBJECTIVE: To develop effective and easily operable screening methods for identifying peroxynitrite scavengers in complex matrices, including Chinese herbal medicines. METHODS: Two simple and efficient screening methods have been developed for the identification of natural peroxynitrite scavengers in Flos Lonicerae Japonicae (FLJ). Method I used HPLC-DAD-(luminol-peroxynitrite)-CL techniques combined with Q-TOF MS/MS analysis, while Method II used pre-column reaction of the sample with peroxynitrite, followed by HPLC separation and Q-TOF MS/MS analysis. RESULTS: Five peroxynitrite scavengers (neochlorogenic acid, chlorogenic acid, 3,4-O-dicaffeoyl quinic acid, 3,5-O-dicaffeoyl quinic acid and 4,5-O-dicaffeoyl quinic acid) were identified in FLJ using Method I. Besides the compounds identified using Method I, three additional peroxynitrite scavengers (rutin, isoquercitrin and luteoloside) were identified using Method II. CONCLUSION: The two new methods proved to be complementary and the use of these methods should allow rapid detection of peroxynitrite-scavenging natural products from FLJ and other complex matrices.

Substituent effects on the stability and antioxidant activity of spirodiazaselenuranes.

Spirodiazaselenuranes are structurally interesting compounds and the stability of these compounds depends highly on the nature of the substituents attached to the nitrogen atoms. Aromatic substituents are known to play important roles in stabilizing the Se-N bonds in spiro compounds. In this study, several spirodiazaselenuranes are synthesized by introducing benzylic and aliphatic substituents to understand their effect on the stability of the Se-N bonds and the antioxidant activity. Replacement of phenyl substituent by benzyl/alkyl groups significantly reduces the stability of the spirodiazaselenuranes and slows down the oxidative cyclization process. The selenium centre in the spiro compounds undergoes further oxidation to produce the corresponding selenurane oxides, which are stable at room temperature. Comparison of the glutathione peroxidase (GPx) mimetic activity of the compounds showed that the diaryl selenides having heterocyclic rings are significantly more active due to the facile oxidation of the selenium centre. However, the activity is reduced significantly for compounds having aliphatic substituents. In addition to GPx activity, the compounds also inhibit peroxynitrite-mediated nitration and oxidation reaction of protein and small molecules, respectively. The experimental observations suggest that the antioxidant activity is increased considerably upon substitution of the aromatic group with the benzylic/aliphatic substituents on the nitrogen atoms.

Enhancement of antioxidant effects of naringin after atmospheric pressure dielectric barrier discharge plasma treatment.

Naringin is the natural chief bitter flavonoid found in Citrus species. Herein, bitter naringin was treated with atmospheric pressure plasma to afford two new converted flavonoids, narinplasmins A (2) and B (3), along with the known compound, 2R-naringin. The structures of the two new naringin derivatives were elucidated on the basis of spectroscopic methods. The antioxidant activity of all isolates was evaluated based on 1,1-diphenyl-2-picrylhydrazyl and peroxynitrite (ONOO(-)) scavenging assays. The new flavanone glycoside 2 containing a methoxyalkyl group exhibited significantly improved antioxidant properties in these assays relative to the parent naringin.

Simultaneous quantification and validation of new peroxynitrite scavengers from Artemisia iwayomogi.

CONTEXT: Artemisia iwayomogi Kitamura (Compositae) has been very widely used for the treatment of acute or chronic hepatitis, jaundice, and gastritis. In the course of our continuing efforts to identify and quantify peroxynitrite scavengers from Compositae plants, A. iwayomogi was used in this study. OBJECTIVE: The present study was aimed to identify and quantify the peroxynitrite scavengers of A. iwayomogi. MATERIALS AND METHODS: Silica gel and ODS were used for column chromatography. The isolated compounds were quantified using an HPLC equipped with a Capcell Pak C18 column (5 µm, 250 mm × 4.6 mm i.d.), and the method was validated for the quality control. Peroxynitrite (ONOO(-))-scavenging activities of the compounds and extracts were evaluated on the measurement of highly fluorescent rhodamine 123 converted from non-fluorescent dihydrorhodamine (DHR)-123 under the presence of peroxynitrite. RESULTS: Based on the spectroscopic evidences, a new compound, 2"-O-caffeoylrutin (2"-O-trans-caffeic acid ester of quercetin 3-O-α-L-rhamnopyranosyl(1 → 6)-ß-D-glucopyranoside) was isolated and determined together with patuletin 3-O-glucoside, scopolin, scopoletin, rutin, 3,4-dicaffeoylquinic acid, and chlorogenic acid. All of them were potent peroxynitrite scavengers (IC50 ≤ 1.88 µg/mL). DISCUSSION AND CONCLUSION: The peroxynitrite scavengers were mainly distributed in the EtOAc fraction rather than the ether and BuOH fractions. The 70% MeOH extract exhibited a high peroxynitrite-scavenging activity. Through the validation, the present HPLC method was verified to be sufficiently sensitive, accurate, precise, and stable. Therefore, this method can be used for the quality control of A. iwayomogi.

Impact of SIN-1-derived peroxynitrite flux on endothelial cell redox homeostasis and bioenergetics: protective role of diphenyl diselenide via induction of peroxiredoxins.

Increased production of reactive nitrogen (RNS) and oxygen (ROS) species and its detrimental effect to mitochondria are associated with endothelial dysfunction. This study was designed to determine the effect of a peroxynitrite flux, promoted by 1,3-morpholinosydnonimine (SIN-1), in mitochondrial function and some redox homeostasis parameters in bovine aortic endothelial cells (BAEC). Moreover, the effect of diphenyl diselenide (PhSe)2, a simple organic selenium compound, in preventing peroxynitrite-mediated cytotoxicity was also investigated. Our results showed that overnight exposure to SIN-1 (250 µM) caused a profound impairment of oxygen consumption, energy generation and reserve capacity in mitochondria of BAEC. Mitochondrial dysfunction resulted in an additional intracellular production of peroxynitrite, amplifying the phenomenon and leading to changes in redox homeostasis. Moreover, we observed an extensive decline in mitochondrial membrane potential (ΔΨm) induced by peroxynitrite and this event was associated with apoptotic-type cell death. Alternatively, the pretreatment of BAEC with (PhSe)2, hindered peroxynitrite-mediated cell damage by preserving mitochondrial and endothelial function and consequently preventing apoptosis. The protective effect of (PhSe)2 was related to its ability to improve the intracellular redox state by increasing the expression of different isoforms of peroxiredoxins (Prx-1-3), efficient enzymes in peroxynitrite detoxification.

Nitration of the birch pollen allergen Bet v 1.0101: efficiency and site-selectivity of liquid and gaseous nitrating agents.

Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO(-)), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O3/NO2). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO(-), and Y83 and Y158 for O3/NO2. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O3/NO2 was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein.

Simultaneous quantification and peroxynitrite-scavenging activities of flavonoids in Polygonum aviculare L. herb.

The plant Polygonum aviculare L. (Polygonaceae) is an annual herbaceous plant which is known to be beneficial for treating gastroduodenal ulcer, hypertension, diarrhea, hemorrhage, and hemorrhoids. Ten phenolic compounds, including nine flavonoids (myricetin, quercetin, kaempferol, myricitrin, desmanthin-1, isoquercitrin, quercitrin, avicularin, juglanin), and gallic acid were used for simultaneous HPLC quantification and peroxynitrite-scavenging assay. Simultaneous quantification of these substances were performed on five extracts (EtOH-, MeOH-, 70% MeOH-, 30% MeOH-, and H2O extracts) as well as on the three fractions (Et2O-, EtOAc-, and BuOH fractions), under the condition of a Capcell Pak C18 column (5µm, 250mm×4.6mm i.d.) and a gradient elution of 0.05% trifluoroacetic acid (TFA) and MeOHCH3CN (60:40). Of the three fractions, the EtOAc fraction displayed the highest content of flavonoids (sum, 208.9mg/g) with the strongest peroxynitrite-scavenging activity (IC50, 2.68µg/mL). The activities of the eight compounds (myricitrin, isoquercitrin, avicularin, quercitrin, myricetin, desmanthin-1, quercetin, and kaempferol) were comparable to that of the positive control (l-penicillamine; IC50: 1.03µg/mL). These results suggest that folkloric medicinal uses of P. aviculare are mainly attributed to flavonoids, such as particularly highly contained myricetin, myricitrin, and desmanthin-1.

Determination of nitration degrees for the birch pollen allergen Bet v 1.

Nitration of tyrosine residues in the major birch pollen allergen Bet v 1 may alter the allergenic potential of the protein. The kinetics and mechanism of the nitration reaction, however, have not yet been well characterized. To facilitate further investigations, an efficient method to quantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) that can be photometrically calibrated using the amino acids tyrosine (Tyr) and nitrotyrosine (NTyr) without the need for nitrated protein standards. The new method is efficient and in agreement with alternative methods based on hydrolysis and amino acid analysis of tetranitromethane (TNM)-nitrated Bet v 1 standards as well as samples from nitration experiments with peroxynitrite. The results confirm the applicability of the new method for the investigation of the reaction kinetics and mechanism of protein nitration.

Carotenoids and phenolic compounds from Solanum sessiliflorum, an unexploited Amazonian fruit, and their scavenging capacities against reactive oxygen and nitrogen species.

The composition of carotenoids and phenolic compounds from mana-cubiu (Solanum sessiliflorum), a fruit native to Amazonia, was determined by high-performance liquid chromatography coupled to diode array and mass spectrometry detectors (HPLC-DAD-MS(n)). The antioxidant capacities of the hydrophilic and carotenoid extracts against some reactive oxygen (ROO(•), H(2)O(2), HOCl, and HO(•)) and nitrogen (ONOO(-)) species were also determined. Seventeen carotenoids and three phenolic compounds were found in mana-cubiu. The major carotenoids were (all-E)-ß-carotene (7.15 µg/g of dry weight) and (all-E)-lutein (2.41 µg/g of dry weight). The 5-caffeoylquinic acid (1351 µg/g of dry weight) was the major phenolic compound, representing more than 78% (w/w) of the total phenolic compounds. Moreover, two dihydrocaffeoyl spermidines were found in the hydrophilic extract. Both mana-cubiu extracts were able to scavenge all the tested reactive species. The carotenoid extract was shown to be a potent scavenger of peroxyl radical, while the hydrophilic extract was a potent hydrogen peroxide and hypochlorous acid scavenger.

Aronia melanocarpa as a protector against nitration of fibrinogen.

Fibrinogen (Fg) also known as coagulation factor I represents about 4% of the total human plasma proteins. The main function of Fg is its involvement in last phase of blood coagulation cascade, when thrombin-induced conversion of dissolved plasma fibrinogen into an insoluble fibrin clot occurs. The reaction of fibrinogen with peroxynitrite causes both structural modifications and changes of the biological properties of this plasma glycoprotein. Recently, there is an increased interest in the screening of natural products present in fruits, vegetables and herbs for their possible antioxidative activities. Therefore, the aim of our study was to estimate the effect of extract from berries of Aronia melanocarpa against nitrative and oxidative damage induced by peroxynitrite. The extract from A. melanocarpa (0.5-50 µg/ml) added to Fg 10 min before peroxynitrite (100 µM) significantly inhibited both the formation of the high molecular weight protein aggregates and nitration of Fg molecule. The extract also abolished peroxynitrite-induced inhibition of fibrinogen polymerization (by 95% at 50 µg/ml). The obtained results indicate that natural extract from berries of A. melanocarpa has protective effects against peroxynitrite-induced nitrative damage of plasma fibrinogen, and therefore may contribute in the prevention of peroxynitrite-related cardiovascular or inflammatory diseases.

Determination of berberine in pharmaceutical preparations using acidic hydrogen peroxide-nitrite chemiluminescence system.

A stronger chemiluminescence (CL) was observed when hydrogen peroxide was mixed with nitrite and berberine in sulfuric acid solution. The stronger CL originated from peroxidation of berberine by peroxynitrous acid that was synthesized online by the mixing of acidic hydrogen peroxide solution with nitrite solution in a flow system. The emitting species was excited state oxyberberine, a peroxidized product of berberine. Based on the stronger CL, a flow injection CL method for the determination of berberine was proposed. Under optimum experimental conditions, the stronger CL intensity was linearly related to the concentration of berberine over the range of 2.0 × 10(-7) -2.0 × 10(-5) mol L(-1) . The limit of detection (s/n = 3) was 6.2 × 10(-8) mol L(-1) . The proposed method has been evaluated by analyzing berberine in pharmaceutical preparations.

Phytochemical studies of the phenolic substances in Aster glehni extract and its sedative and anticonvulsant activity.

On high performance liquid chromatography, the caffeoylquinic acid (CQ) occupying the highest proportion of the water-ethanol (7:3) extract of Aster glehni (Compositae) leaves was 3-Op-coumaroylquinic acid (46.10 ± 4.22 mg/g of dried weight) among CQs tested. The IC50 of the water-ethanol (7:3) extract was 4.23 ± 0.24 µg/mL in the peroxynitrite (ONOO(-))-scavenging assay. Phytochemical isolation from A. glehni extract yielded three kaempferol glycosides. The water-ethanol (7:3) extract and both p-coumaric acid and caffeic acid, phenylpropanoid moieties of CQs, had sedative effects in pentobarbital-induced sleeping time in mice and anticonvulsant effects in pentylenetetrazole-induced mice. Furthermore, the phenolic substance-rich W-E (7:3) extract of A. glehni could be used to treat anxiety or convulsion partly due to its peroxynitrite-scavenging mechanism.

Simultaneous quantitative determination and validation of quercetin glycosides with peroxynitrite-scavenging effects from Saussurea grandifolia.

The leaves of Saussurea grandifolia (Compositae) are used as chwinamul, a well-known Korean mountainous vegetable, or to treat hepatitis and hematuria. Since the methanolic extract of S. grandifolia leaves exhibit a potent peroxynitrite-scavenging effect, phytochemical and high-performance liquid chromatographic (HPLC) analyses were employed to identify and simultaneously quantify the active components: chlorogenic acid and 3,5-di-O-caffeoylquinic acid (3,5-DQ) as caffeoylquinic acids, and quercetin, isoquercitrin (quercetin-3-glucoside), saxifragin (quercetin-5-glucoside), and rutin (quercetin-3-rutinoside). Validation of HPLC methods was performed to verify the linearity, LOD, LOQ, intra-day and inter-day variabilities, recovery, and repeatability to ensure that this method is selective, sensitive, precise, accurate and reproducible. In particular, leaves contained saxifragin with potent peroxynitrite-scavenging activity (IC(50), 0.67 µM) as 4.65 mg/g dry weight, which is equivalent to 33.74 mg/g extract.

Beetroot betalain inhibits peroxynitrite-mediated tyrosine nitration and DNA strand cleavage.

Two major betalains, red-purple betacyanins and yellow betaxanthins, were isolated from red beetroots (Beta vulgaris L.), and their peroxynitrite (ONOO(-)) scavenging capacity was investigated. Apparent colours of the betalains were bleached by the addition of ONOO(-), and the absorbance decreases were suppressed in the presence of glutathione, a ONOO(-) scavenger. After bleaching, a new absorption maximum was observed at 350 nm in the spectrum of the resulting reaction mixture. New peaks were detected from HPLC analysis of the reaction products of betanin, a representative constituent of red beetroot betacyanins, treated with ONOO(-) monitoring at 350 nm, and the intensity of the major peak was positively correlated with ONOO(-) concentration. Betanin inhibited the ONOO(-) (0.5 mM)-dependent nitration of tyrosine (0.1 mM). Additionally, the IC(50) value of betanin (19.2 µM) was lower than that of ascorbate (79.6 µM). The presence of betanin (0.05-1.0 mM) also inhibited ONOO(-) (0.5 mM)-dependent DNA strand cleavage in a concentration-dependent manner. These results suggest that betalains can protect cells from nitrosative stress in addition to protecting them from oxidative stresses.

Clovamide-rich extract from Trifolium pallidum reduces oxidative stress-induced damage to blood platelets and plasma.

Numerous plants (including clovers) have been widely used in folk medicine for the treatment of different disorders. This in vitro study was designed to examine the antioxidative effects of the clovamide-rich fraction, obtained from aerial parts of Trifolium pallidum, in the protection of blood platelets and plasma against the nitrative and oxidative damage, caused by peroxynitrite (ONOO(-)). Carbonyl groups and 3-nitrotyrosine in blood platelet and plasma proteins were determined by ELISA tests. Thiol groups level was estimated by using 5,5'-dithio-bis(2-nitro-benzoic acid, DTNB). Plasma lipid peroxidation was measured spectrophotometrically as the production of thiobarbituric acid reactive substances. The results from our work indicate that clovamide-rich T. pallidum extract may reveal the protective properties in the prevention against oxidative stress. The presence of clovamide-rich T. pallidum extract (12.5-100 µg/ml) partly inhibited ONOO(-)-mediated protein carbonylation and nitration. All the used concentrations of T. pallidum extract reduced lipid peroxidation in plasma. The antioxidative action of the tested extract in the protection of blood platelet lipids was less effective; the extract at the lowest final concentration (12.5 µg/ml) had no protective effect against lipid peroxidation. The present results indicate that the extract from T. pallidum is likely to be a source of compounds with the antioxidative properties, useful in the prevention against the oxidative stress-related diseases.

L-Carnitine protects plasma components against oxidative alterations.

OBJECTIVE: L-Carnitine as a dietary supplement has been reported to have a beneficial effect on several cardiovascular risk parameters and exercise capacity, but the biological relevance of its activity is poorly understood. Dietary supplements (including L-carnitine) are often used to foster exercise performance; however, these may affect some pathways of human body metabolism. The aim of this study in vitro was to determine antioxidative properties of L-carnitine (0.1-100 µM) added to plasma and to assess if L-carnitine might protect plasma proteins and lipids against oxidative/nitrative damage (determined by levels of protein carbonyl groups, thiols, 3-nitrotyrosine formation and thiobarbituric-acid reactive substances generation) caused by 100 µM peroxynitrite (ONOO(-)), a strong physiologic oxidative/nitrative agent. METHODS: The level of carbonyl group generation was measured by a colorimetric method. For the estimation of 3-nitrotyrosine formation, a competition enzyme-linked immunosorbent assay was performed. Plasma lipid peroxidation was measured spectrophotometrically as the production of thiobarbituric-acid reactive substances. High-performance liquid chromatography was used to analyze total free thiol groups of plasma proteins and low-molecular-weight thiols (glutathione, cysteine, and homocysteine) in plasma. RESULTS: The L-carnitine added to plasma inhibited in vitro ONOO(-)-induced oxidation and nitration of blood plasma proteins. Incubation of plasma with peroxynitrite resulted in the decrease of protein thiols. L-Carnitine had a protective effect on peroxynitrite-induced decreased -SH level in plasma proteins. The presence of L-carnitine also prevented the decrease of low-molecular-weight thiols (glutathione, cysteine, and homocysteine) in plasma caused by peroxynitrite and protected plasma lipids against peroxidation induced by peroxynitrite. CONCLUSIONS: These results demonstrated that L-carnitine possesses antioxidative activity.