Antioxidative Pseudoenzymatic Mechanism of NAD(P)H Coexisting with Oxyhemoglobin for Suppressed Methemoglobin Formation.

Oxyhemoglobin (HbO2) coexisting with equimolar NADH retards autoxidation and oxidant-induced metHb formation based on the pseudocatalase (CAT) and pseudosuperoxide dismutase (SOD) activities. In this work, we compared the effects of NADH with those of NADPH and estimated the binding site of NAD(P)H to HbO2 to elucidate the antioxidative mechanisms. The results clarified that pseudo-CAT and pseudo-SOD activities of HbO2 coexisting with NADPH were similar to activities obtained with NADH. Prompt MetHb formation (<40 min) facilitated by oxidants (H2O2, NO, and NaNO2) was hindered by NADPH. These effects were similar to those of NADH. However, we found that NADPH is thermally unstable compared to NADH and that NADPH cannot sustain antioxidative effects for a long period of autoxidation to metHb such as 24 h. Lineweaver-Burk plots clarified that the Michaelis constants of these pseudoenzymatic activities are in the millimolar range. Addition of inositol hexaphosphate (IHP) and 2,3-diphosphoglycerate (DPG), which are known to bind not only with deoxyHb but also weakly with HbO2, showed competitive inhibition of pseudoenzymatic activities. These results suggest that the binding site of NADH and NADPH on HbO2 is the same as those of IHP and DPG. 31P nuclear magnetic resonance definitively showed 1:1 stoichiometric binding of NADH to HbO2. High-performance liquid chromatography analysis showed that NADH preferentially inhibited autoxidation of α-subunit heme. Docking simulations also predicted that the binding site of relaxed-state HbO2 with NAD(P)H is the same as those with IHP and DPG. Collectively, the pseudoenzymatic activities of HbO2 coexisting with NAD(P)H are induced by the 1:1 stoichiometric binding of NAD(P)H to HbO2.

[Evolution of the carboxyhemoglobinemia and methemoglobinemia during endoscopic resection: An observational study]. / Évolution de la carboxyhémoglobinémie et de la méthémoglobinémie lors de résections endoscopiques : une étude observationnelle.

AIM OF THE STUDY: Combustion of organic tissues due to endoscopic resection could induce methemoglobin (MetHb) and carboxyhemoglobin (COHb) formation. The aim of this study is to evaluate MetHb and COHb formation in patients undergoing prostatic or bladder endoscopic procedures. METHODS: COHb and MetHb measurements were performed in 44 patients at the beginning and end of the procedure. A third measurement was done in patients who stayed more than one hour in the recovery room. Means were compared using Student t-test, simple regressions were used for quantitative variables and ANOVA for categorical variables. Multiple linear regressions were used for multivariate analysis. RESULTS: COHb increased by 0.5±0.9 % (95 % CI: 0.2 to 0.7 % P=0.001). MetHb increase was 0.0±0.4 % (95 % CI: -0.1 to 0.2 % P=0.552). In univariate analysis, the variables associated with COHb increase are the length of surgery, the amount of irrigation fluid and location (prostate or bladder) of the procedure. In the multivariate model, COHb increase is associated with the amount of liquid and the location. CONCLUSION: MetHb did not increase during endoscopic surgery. In contrast, COHb increases, and can, in some patients, exceed 2-4 %. This could be responsible for a decreased angina threshold in patients with ischemic heart disease. LEVEL OF EVIDENCE: 4.

Computational Study on the Effect of Exocyclic Substituents on the Ionization Potential of Primaquine: Insights into the Design of Primaquine-Based Antimalarial Drugs with Less Methemoglobin Generation.

The effect of an exocyclic substituent on the ionization potential of primaquine, an important antimalarial drug, was investigated using density functional theory methods. It was found that an electron-donating group (EDG) makes the ionization potential decrease. In contrast, an electron-withdrawing group (EWG) makes the ionization potential increase. Among all the exocyclic positions, a substituent at the 5- or 7-position has the largest effect. This can be explained by the contribution of the atomic orbitals at those positions to the highest occupied molecular orbital (HOMO). In addition, a substituent at the N8-position has a considerably large effect on the ionization potential because this atom makes the second largest contribution to the HOMO. These findings have potential implications for the design of less hemotoxic antimalarial drugs. We suggest that it is worth considering placement of an EWG at the 5-, 7-, or N8-positions of primaquine in future drug discovery attempts.

Synthesis and methemoglobinemia-inducing properties of benzocaine isosteres designed as humane rodenticides.

A number of isosteres (oxadiazoles, thiadiazoles, tetrazoles and diazines) of benzocaine were prepared and evaluated for their capacity to induce methemoglobinemia-with a view to their possible application as humane pest control agents. It was found that an optimal lipophilicity for the formation of methemoglobin (metHb) in vitro existed within each series, with 1,2,4-oxadiazole 3 (metHb%=61.0±3.6) and 1,3,4-oxadiazole 10 (metHb%=52.4±0.9) demonstrating the greatest activity. Of the 5 candidates (compounds 3, 10, 11, 13 and 23) evaluated in vivo, failure to induce a lethal end-point at doses of 120mg/kg was observed in all cases. Inadequate metabolic stability, particularly towards hepatic enzymes such as the CYPs, was postulated as one reason for their failure.

Membrane peroxidation and methemoglobin formation are both necessary for band 3 clustering: mechanistic insights into human erythrocyte senescence.

Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120 day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor methemoglobin (MetHb) formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that binding of MetHb to the cytoplasmic domain of band 3 in peroxidized, but not in untreated, erythrocyte membranes induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density gradient procedure, have allowed us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ∼0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced nicotinamide adenine dinucleotide-dependent reductase activity and accumulated oxidative membrane damage. These findings have allowed us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably because of its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from band 3, allowing the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.

Alkyl substituted 2'-benzoylpyridine thiosemicarbazone chelators with potent and selective anti-neoplastic activity: novel ligands that limit methemoglobin formation.

Thiosemicarbazone chelators, including the 2'-benzoylpyridine thiosemicarbazones (BpT) class, show marked potential as anticancer agents. Importantly, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) has been investigated in >20 phase I and II clinical trials. However, side effects associated with 3-AP administration include methemoglobinemia. Considering this problem, novel BpT analogues were designed bearing hydrophobic, electron-donating substituents at the para position of the phenyl group (RBpT). Their Fe(III/II) redox potentials were all within the range accessible to cellular oxidants and reductants, suggesting they can redox cycle. These RBpT ligands exhibited potent and selective antiproliferative activity, which was comparable or exceeded their BpT counterparts. Major findings include that methemoglobin formation mediated by the lipophilic t-BuBpT series was significantly (p < 0.05-0.001) decreased in comparison to 3-AP in intact red blood cells and were generally comparable to the control. These data indicate the t-BuBpT ligands may minimize methemoglobinemia, which is a marked advantage over 3-AP and other potent thiosemicarbazones.

[Effect of selenium on resistance of hemoglobin to photooxidative processes].

On an example of a guinea pig it is shown that exogenous selenium (0.5 mg Na2SeO3 per 1 kg of the animal weight) during 2-hour exposition in the animal organism increases the resistance to the photo-induced oxidation of haemoglobin in erythrocyte lysates without additional stimulation of glutathione peroxidase mechanism of haemoglobin protection by exogenous selenium. It is shown that the saturation of haemoglobin fractions by selenium hampers the oxidative modification of haemoglobin. Using pregnancy of women as a natural model of selenium-deficiency condition, it has been shown that physiological debilitation of saturation erythrocytes with selenium, including haemoglobin fractions of lysates erythrocytes caused debilitation of resistance of haemoglobin to photooxidative destruction. Under these conditions not only activity of enzyme glutathione peroxidise in erythrocyte lysates, but also the peroxidase activity of haemoglobin (in the presence of glutathione) were decreased. It is more characteristic of erythrocyte lysates with a less content of selenium, i.e. for the erythrocytes of women on late terms of pregnancy that testifies to the presence of certain relation between haemoglobin saturation with selenium and its peroxidase activity (in the presence of glutathione).

Understanding the mechanisms for metabolism-linked hemolytic toxicity of primaquine against glucose 6-phosphate dehydrogenase deficient human erythrocytes: evaluation of eryptotic pathway.

Therapeutic utility of primaquine, an 8-aminoquinoline antimalarial drug, has been limited due to its hemolytic toxicity in population with glucose 6-phosphate dehydrogenase deficiency. Recent investigations at our lab have shown that the metabolites generated through cytochrome P(450)-dependent metabolic reactions are responsible for hemotoxic effects of primaquine, which could be monitored with accumulation of methemoglobin and increased oxidative stress. The molecular markers for succeeding cascade of events associated with early clearance of the erythrocytes from the circulation were evaluated for understanding the mechanism for hemolytic toxicity of primaquine. Primaquine alone though did not induce noticeable methemoglobin accumulation, but produced significant oxidative stress, which was higher in G6PD-deficient than in normal erythrocytes. Primaquine, presumably through redox active hemotoxic metabolites generated in situ in human liver microsomal metabolism-linked assay, induced a dose-dependent methemoglobin accumulation and oxidative stress, which were almost similar in normal and G6PD-deficient erythrocytes. Primaquine alone or in presence of pooled human liver microsomes neither produced significant effect on intraerythrocytic calcium levels nor affected the phosphatidyl serine asymmetry of the normal and G6PD-deficient human erythrocytes as monitored flowcytometrically with Annexin V binding assay. The studies suggest that eryptosis mechanisms are not involved in accelerated removal of erythrocytes due to hemolytic toxicity of primaquine.

In vitro suppression of drug-induced methaemoglobin formation by Intralipid(®) in whole human blood: observations relevant to the 'lipid sink theory'.

To provide further evidence for the lipid sink theory, we have developed an in vitro model to assess the effect of Intralipid® 20% on methaemoglobin formation by drugs of varying lipid solubility. Progressively increasing Intralipid concentrations from 4 to 24 mg.ml⁻¹ suppressed methaemoglobin formation by the lipid soluble drug glyceryl trinitrate in a dose-dependent manner (p < 0.001). Both dose and timing of administration of Intralipid to blood previously incubated with glyceryl trinitrate for 10 and 40 min resulted in significant suppression of methaemoglobin formation (p < 0.0001 and p < 0.05, respectively). Mathematical modelling demonstrated that the entire process of methaemoglobin formation by glyceryl trinitrate was slowed down in the presence of Intralipid. Intralipid did not significantly suppress methaemoglobin formation induced by 2-amino-5-hydroxytoluene (partially lipid soluble) or sodium nitrite (lipid insoluble; both p > 0.5). This work may assist determination of the suitability of drugs taken in overdose for which Intralipid might be deployed.

A hemoglobin variant associated with neonatal cyanosis and anemia.

Globin-gene mutations are a rare but important cause of cyanosis. We identified a missense mutation in the fetal Gγ-globin gene (HBG2) in a father and daughter with transient neonatal cyanosis and anemia. This new mutation modifies the ligand-binding pocket of fetal hemoglobin by means of two mechanisms. First, the relatively large side chain of methionine decreases both the affinity of oxygen for binding to the mutant hemoglobin subunit and the rate at which it does so. Second, the mutant methionine is converted to aspartic acid post-translationally, probably through oxidative mechanisms. The presence of this polar amino acid in the heme pocket is predicted to enhance hemoglobin denaturation, causing anemia.

Synthesis, antiprotozoal, antimicrobial, ß-hematin inhibition, cytotoxicity and methemoglobin (MetHb) formation activities of bis(8-aminoquinolines).

In continuing our search of potent antimalarials based on 8-aminoquinoline structural framework, three series of novel bis(8-aminoquinolines) using convenient one to four steps synthetic procedures were synthesized. The bisquinolines were evaluated for in vitro antimalarial (Plasmodiumfalciparum), antileishmanial (Leishmaniadonovani), antimicrobial (a panel of pathogenic bacteria and fungi), cytotoxicity, ß-hematin inhibitory and methemoglobin (MetHb) formation activities. Several compounds exhibited superior antimalarial activities compared to parent drug primaquine. Selected compounds (44, 61 and 79) when tested for in vivo blood-schizontocidal antimalarial activity (Plasmodiumberghei) displayed potent blood-schizontocial activities. The bisquinolines showed negligible MetHb formation (0.2-1.2%) underlining their potential in the treatment of glucose-6-phosphate dehydrogenase deficient patients. The bisquinoline analogues (36, 73 and 79) also exhibited promising in vitro antileishmanial activity, and antimicrobial activities (43, 44 and 76) against a panel of pathogenic bacteria and fungi. The results of this study provide evidence that bis(8-aminoquinolines), like their bis(4-aminoquinolines) and artemisinin dimers counterparts, are a promising class of antimalarial agents.

Disnea y cianosis acra en paciente con dermatitis herpetiforme / No disponible

No disponible

Methemoglobinemia in critically ill patients during extended hemodialysis and simultaneous disinfection of the hospital water supply.

INTRODUCTION: To evaluate the cause of methemoglobinemia in patients undergoing extended daily hemodialysis/hemodiafiltration we analyzed the relationship between methemoglobinemia and the water disinfection schedule of the hospital. METHODS: We reviewed all arterial blood gas analyses, obtained over a one-year period, in patients undergoing extended hemodialysis/hemodiafiltration, and compared the methemoglobin concentrations obtained on the days when the water supply was disinfected, using a hydrogen peroxide/silver ion preparation, with data measured on disinfection-free days. RESULTS: The evaluation of 706 measurements revealed a maximum methemoglobin fraction of 1.0 (0.8; 1.2) % (median and 25th; 75th percentiles) during hemodialysis/hemodiafiltration on the disinfection-free days. The methemoglobin fraction increased to 5.9 (1.3; 8.4) % with a maximal value of 12.2% on the days of water disinfection (P < 0.001 compared to disinfection-free days). Spot checks on hydrogen peroxide concentrations in the water supply, the permeate, and the dialysate, using a semi-quantitative test, demonstrated levels between 10 and 25 mg/l during water disinfection. CONCLUSIONS: Our results demonstrate that even a regular hospital water disinfection technique can be associated with significant methemoglobinemia during extended hemodialysis. Clinicians should be aware of this potential hazard.

[Rasburicase therapy may cause hydrogen peroxide shock]. / Az uratoxidaz- (rasburicas-) terápia lehetséges szövôdménye a hidrogen-peroxid okozta sokk.

Hyperuricemia contributes to the pathomechanism of diseases such as renal failure, gout, tumor lysis syndrome and metabolic syndrome. Tumor lysis syndrome is a complication of malignancies caused by massive tumor cell lysis due to either spontaneous tumor cell lysis or to different therapies and it may cause hyperuricemia. Recently, for treatment of hyperuricemia the recombinant urate oxidase (rasburicase) therapy has been used. This enzyme converts uric acid with high affinity into soluble allantoin which is eliminated by the kidneys. In this reaction high concentration of hydrogen peroxide is generated. This hydrogen peroxide could cause hemolysis and especially methemoglobin formation, in case of glucose-6-phosphate-dehydrogenase and catalase deficiencies. Therefore it is recommended that these enzymes are determined before therapy. For monitoring of rasburicase therapy the determination of serum uric acid concentration is used. More than 95 per cent of Hungarian clinical laboratories are using the uricate oxidase/peroxidase reactions and hydrogen peroxide measurements in the uric acid assays. These assays may be interfered by ascorbic acid and hydrogen peroxide which is generated by rasburicase either in vivo or in vitro.

Operant leverpressing and wheelrunning were differentially reduced by PAPP (p-aminopropiophenone)-induced methemoglobinemia.

Cyanide is a potent toxin that binds to cytochrome oxidase blocking electron transfer and the synthesis of adenosine triphosphate (ATP). Many antidotes to cyanide poisoning oxidize hemoglobin to methemoglobin (metHb), which serves as a scavenger of the cyanide anion. However, sufficiently high levels of metHb can be toxic because metHb cannot bind O(2) until it is reduced. The purpose of the proposed study was twofold: (1) Characterize the time course of metHb formation for different doses of p-aminopropiophenone (PAPP), a drug that oxidizes hemoglobin and can be used as an antidote to cyanide intoxication; and (2) Determine whether the effort of an operant response affects the behavioral toxicity of metHb, since more effortful responses presumably are more energetically demanding. In Experiment I, the oral metHb kinetics of p-aminopropiophenone (PAPP) were studied; four doses of PAPP (1, 5, 10, and 20 mg/kg) or the vehicle, polyethylene glycol 200 (PEG200), were delivered via a gavage tube to separate groups of rats. In Experiment II, rats were trained to press a lever or run in an activity wheel at any time during a 12-hour light/dark cycle for their entire daily food intake; five presses or turns were required for the delivery of each food pellet. The same doses of PAPP were delivered p.o. shortly before the onset of darkness, 2100 h. Results from Exp I showed that PAPP induced a dose-dependent rapid increase and relatively slower exponential-like decline in metHb concentration. In Exp. II, the same doses of PAPP induced a dose-dependent reduction in hourly outputs of leverpresses and wheelturns however; wheelturns were reduced significantly more than leverpresses. When the best-fitting metHb curves from Experiment I were superimposed on the time scale for outputs of wheelturns and leverpresses, reduction of output was inversely related to the kinetics of metHb formation. These findings are consistent with the conclusion that PAPP-induced metHb formation reduced the output of wheelrunning more than leverpressing because the more energetically demanding response of wheelrunning was more affected by metHb induced hypoxemia. Furthermore, these data suggest that although certain longacting metHb formers might be useful prophylactics for warfighters, it will be critical to determine the energetic loads of required battlefield activities because even low (10%) therapeutic metHb levels might impair the performance of those activities.

Hemoglobin autoxidation and regulation of endogenous H2O2 levels in erythrocytes.

Red cells from mice deficient in glutathione peroxidase-1 were used to estimate the hemoglobin autoxidation rate and the endogenous level of H2O2 and superoxide. Methemoglobin and the rate of catalase inactivation by 3-amino-2,4,5-triazole (3-AT) were determined. In contrast with iodoacetamide-treated red cells, catalase was not inactivated by 3-AT in glutathione peroxidase-deficient erythrocytes. Kinetic models incorporating reactions known to involve H2O2 and superoxide in the erythrocyte were used to estimate H2O2, superoxide, and methemoglobin levels. The experimental data could not be modeled unless the intraerythrocytic concentration of Compound I is very low. Two additional models were tested. In one, it was assumed that a rearranged Compound I, termed Compound II*, does not react with 3-AT. However, experiments with an NADPH-generating system provided evidence that this mechanism does not occur. A second model that explicitly includes peroxiredoxin II can fit the experimental findings. Insertion of the data into the model predicted a hemoglobin autoxidation rate constant of 4.5 x 10(-7) s(-1) and an endogenous H2O2 and superoxide concentrations of 5 x 10(-11) and 5 x 10(-13) M, respectively, lower than previous estimates.

Effect of dietary fat on selected parameters of toxicity following 1- or 3-month exposure of rats to toluidine isomers.

The aim of the study was to investigate the effect of the dietary fat on selected parameters of toluidines toxicity in rats during subchronic exposure. Three isomers of toluidine (ortho, meta, and para) were administered to rats in the diet for 1 and 3 months at levels 40, 80, 160 mg/kg/day in two kinds of diet containing either 4% or 14% fat. All doses of toluidine isomers produced a 1.5- to 9.8-fold increase in methemoglobin (MetHb) level during both treatment periods. A distinct dose-response relationship was observed, especially for o- and m-toluidine; the effect was generally greater in rats fed high-fat diet. Reduced glutathione level in liver was increased in all treated groups, 1.5- to 5.1-fold, irrespective of the kind of diet. An increase in hepatic lipid peroxidation (thiobarbituric acid reactive substances; TBARS), 1.5- to 4.5-fold, was noticed in the majority of the treated groups. Generally, there was no consistent effect of diet except for p-toluidine where the level of hepatic TBARS was lower in rats fed high-fat diet. Blood urea nitrogen (BUN) level in animals treated with all doses of o- and m-toluidine was 1.3- to 5.0-fold higher in comparison with respective controls. No clear relationship between BUN level and the kind of diet was found. No effect of toluidines on the activity of serum aspartate aminotransferase (AST) and sorbitol dehydrogenase (SDH) were observed. In the majority of groups treated for 30 and 90 days the amount of toluidines in 24-h urine was lower in rats fed high-fat diet. Final body weight gain in rats treated with o- and p-toluidine (80 and 160 mg/kg body weight [b.w.]) was lower as compared to controls. In summary the high-fat diet stimulated methemoglobin formation in rats treated with o- and m-toluidine and cause the decrease in the amount of toluidines in 24-h urine. The high content of fat did not affect consistently the other parameters tested.

Role of thiamine thiol form in nitric oxide metabolism.

In alkaline media the thiamine cyclic form is converted into a thiol form (pK(a) 9.2) with an opened thiazole ring. The thiamine thiol form releases nitric oxide from S-nitrosoglutathione (GSNO). Thiamine disulfide, mixed thiamine disulfide with glutathione, and nitric oxide are produced in the reaction. Free glutathione was recorded in small amounts. The concentration of formed nitric oxide agreed well with the concentration of degraded GSNO. The concentration of released nitric oxide was determined under anaerobic conditions spectrophotometrically by production of nitrosohemoglobin. In air, the release of nitric oxide was recorded by the production of nitrite or the oxidation of oxyhemoglobin to methemoglobin. The concentration of the thiol form in the body under physiological pH values (7.2-7.4) did not exceed 1.5-2.0%. We believe that due to the exchange reactions between the thiamine thiol form and S-nitrosocysteine protein residues, nitric oxide can be released and mixed thiamine-protein disulfides are formed. The mixed thiamine disulfides (including thiamine ester disulfides) as well as the thiamine disulfide form are quite easily reduced by low molecular weight thiols to form the thiamine cyclic form with a closed thiazole ring. A possible role of the thiamine thiol form in releasing deposited nitric oxide from low-molecular-weight S-nitrosothiols and protein S-nitrosothiols and in regulation of blood flow in the vascular bed is discussed.

Aniline derivative-induced methemoglobin in rats.

Methemoglobinemia and hemolysis are the most prominent side-effects of exposure to a wide variety of arylamine drugs, including agricultural and industrial chemicals. Recent studies with aniline and dapsone have identified N-hydroxyl metabolites as the red blood cell (RBC) mediators. This study examines the time-course methemoglobinemic potential of several halogenated aniline phenylhydroxylamines. Symptoms of aniline poisoning include headache, fatigue, dizziness, respiratory and cardiac arrest, and possibly death. Initial studies indicated that the parent compounds are converted to their toxic metabolites (N-hydroxylamine), which enter the RBC and react with oxyhemoglobin. Consequent reduction of molecular oxygen to active oxygen species occurs, leading to RBC damage. Our laboratory is investigating the role of redox cycling and an alternative hypothesis--that a "hydroxylamine-centered" radical formed during arylhydroxylamine-oxyhemoglobin reaction results in RBC injury. The methemoglobinemic capacities of several structurally related N-hydroxy derivatives of aniline--phenylhydroxylamine (PHA), p-fluoro-, p-chloro-, p-bromo-, and p-iodo-PHA--were studied spectrophotometri-cally by treating washed rat RBC at concentrations ranging from 30 to 300 microM of the test compounds for up to 240 minutes. The results showed dose- and time-dependent changes in the induction of methemoglobin (MetHb) by aniline derivatives. The MetHb levels peaked to as high as 75% and remained elevated up to 240 minutes, depending on the electronegativity of halogenated phenylhydroxylamine aniline. This study supports the previous findings that there may be several aniline-derived metabolites other than PHA that are capable of inducing MetHb. The minimum dose required to induce this effect and duration of the MetHb may vary with the test agent.

Mechanistic studies of the oxidation of oxyhemoglobin by peroxynitrite.

The strong oxidizing and nitrating agent peroxynitrite has been shown to diffuse into erythrocytes and oxidize oxyhemoglobin (oxyHb) to metHb. Because the value of the second-order rate constant for this reaction is on the order of 10(4) M(-)(1) s(-)(1) and the oxyHb concentration is about 20 mM (expressed per heme), this process is rather fast and oxyHb is considered a sink for peroxynitrite. In this work, we showed that the reaction of oxyHb with peroxynitrite, both in the presence and absence of CO(2), proceeds via the formation of oxoiron(iv)hemoglobin (ferrylHb), which in a second step is reduced to metHb and nitrate by its reaction with NO(2)(*). In the presence of physiological relevant amounts of CO(2), ferrylHb is generated by the reaction of NO(2)(*) with the coordinated superoxide of oxyHb (HbFe(III)O(2)(*)(-)). This reaction proceeds via formation of a peroxynitrato-metHb complex (HbFe(III)OONO(2)), which decomposes to generate the one-electron oxidized form of ferrylHb, the oxoiron(iv) form of hemoglobin with a radical localized on the globin. CO(3)(*)(-), the second radical formed from the reaction of peroxynitrite with CO(2), is also scavenged efficiently by oxyHb, in a reaction that finally leads to metHb production. Taken together, our results indicate that oxyHb not only scavenges peroxynitrite but also the radicals produced by its decomposition.