Methemoglobin Production Research Articles

Core Concepts: The Biology of Hemoglobin

A consistent and organized transition from embryonic to fetal to adult hemoglobin (Hgb) occurs during human fetal development. Hgb concentrations gradually increase, averaging 18 g/dL (180 g/L) by 40 weeks' gestation. The ability to deliver oxygen to tissues in the fetus and neonate is primarily determined by the percentage of fetal versus adult Hgb and the concentration of 2,3 diphosphoglycerate (2,3-DPG). Studies continue to evaluate the relationship between Hgb concentrations and oxygen delivery in neonates to determine what Hgb concentrations best meet the needs of a wide variety of clinical situations from the critically ill extremely low-birthweight infant to the stable growing preterm infant. Biochemical interactions between nitric oxide (NO) and Hgb beyond the production of methemoglobin do occur and may be a source of deliverable NO to the microcirculation under hypoxic conditions.

Mujer de 18 años con metahemoglobinemia tras utilización de crema anestésica tópica

Methaemoglobinaemia is a very uncommon disorder, with its diagnosis being based on the appearance of high levels of methaemoglobin in the blood, both in adults and children. It is an important cause of cyanosis, and occasionally its severity of its presentation may require admission to an Intensive Care Unit. It may be acquired or hereditary; the latter being due to a mutation of the NADPH-dependent haemoglobin reductase gene. The acquired form or toxic methaemoglobinaemia is produced when red cells are exposed to oxidising chemicals that increase methaemoglobin production, overwhelming the regulatory mechanisms that function normally. A case is presented of an 18 year old woman with clinical picture of the sudden appearance of cyanosis, diagnosed as toxic methaemoglobinaemia after using EMLA ® topical anaesthetic cream (mixture of local anaesthetics, lidocaine and prilocaine).

Is threshold for treatment of methemoglobinemia the same for all? A case report and literature review

Acquired methemoglobinemia (MetHb) is a rare complication of exposure to toxic chemicals or drugs, most commonly topical anesthetic agents. This condition occurs when the rate of methemoglobin production exceeds the rate of methemoglobin reduction. Topical anesthetics have been reported to cause MetHb, but this adverse event is extremely rare and is not usually listed as one of the possible complications of transesophageal echocardiography (TEE). However, the number of published case reports of TEE-associated MetHb has recently increased. Benzocaine (ethyl aminobenzoate) is a topical anesthetic widely used for oropharyngeal anesthesia before TEE. Health care providers who are not familiar with the association of TEE and benzocaine-induced MetHb may not recognize the idiosyncratic and often nonspecific characteristics of this condition. Recognition is critical, as clinically important symptoms may occur at relatively low MetHb levels. If left untreated, MetHb can lead to cardiopulmonary compromise, severe neurologic impairment, and even death. The current report documents a case of TEE-associated MetHb from a high-volume (3000 cases per year including 300-350 TEEs per year) echo laboratory. Our patient was symptomatic and severely distressed, despite a MetHb level of only 10.8%. This case report emphasizes the importance of early recognition and treatment of MetHb, as it represents a medical emergency and can be severely symptomatic, especially in young children and the elderly, even with low MetHb levels.

Benzocaine Induced Methemoglobinemia a Potentially Life Threatening Complication after Transesophageal Echocardiogram

Methemoglobinemia is a potentially life threatening medical condition that occurs due to increased methemoglobin production or decreased elimination. Methemoglobin is a derivative of hemoglobin in which the normal ferrous (Fe2+) iron state is oxidized to the ferric (Fe3+) state. This oxidized hemoglobin is unable to bind oxygen, hence reducing he oxygen carrying capacity of blood. The clinically presentation is characterized by cyanosis, low pulse oximeter reading and normal arterial PO2. Topical use of benzocaine a local anesthetic widely used in endoscopic procedures is one of the incriminated causes of acquired methemoglobinemia.We present two cases of patients (pts) who developed methemoglobinemia after benzocaine spray for a transoesophageal echocardiogram. One pt was 50 year old male and other 30 year old female. Both had a methacilline resistant Staphylococcus Aureus bacteremia secondary to septic arthritis, they under went a transesophageal echocardiogram to rule out endocarditis. Topical local anesthesia used was benzocaine 20%. About 10–15 min after local anesthesia they desaturated to the mid 80's and they developed cynosis, they were intubated. On Blood gas analysis methemoglobin. was elevated 38.4% (male pt) and 18.9% (Female pt). They were given methylene blue at the dose of 2mg/kg intravenously over 10 minutes. Cyanosis corrected with in an hour. Methemoglobin level corrected to normal after 3 hours after administration of methylene blue. There were no adverse events with administration. The Glucose 6 Phosphate Dehydrogenase enzyme level was tested and was negative.In conclusion benzocaine induced methemoglobinemia should be considered in pts under going Transesophageal Echocardiogram when cyanosis develops that is unresponsive to oxygen. Diagnosis can be confirmed by arterial blood gas analysis. If identified early this potentially life threatening condition can be treated with methylene blue with minimal complications and good outcomes.

Ascaris suum cytochrome b5, an adult-specific secretory protein reducing oxygen-avid ferric hemoglobin

The anaerobic parasitic nematode Ascaris suum has an oxygen-avid hemoglobin in the perienteric fluid, the biological function of which remains elusive. Here, we report that Ascaris cytochrome b 5 is expressed specifically in the intestinal parasitic stage and is secreted into the perienteric fluid, thus co-localizing with Ascaris hemoglobin. We also found that cytochrome b 5 reduces Ascaris non-functioning ferric methemoglobin more efficiently than mammalian methemoglobin. Furthermore, a computer graphics model of the electron transfer complex between Ascaris cytochrome b 5 and Ascaris hemoglobin strongly suggested that these two proteins are physiological redox partners. Nitric oxide has been reported to react easily with oxygen captured in hemoglobin to form nitrate, but not toxic free radicals, which may result in production of methemoglobin for the cytochrome b 5 to regenerate functional ferrous hemoglobin. Therefore, our findings suggest that Ascaris cytochrome b 5 is a key redox partner of Ascaris hemoglobin, which acts as an antioxidant.

A novel method of measuring reduction of nitrite-induced methemoglobin applied to fetal and adult blood of humans and sheep

The reaction of nitrite with deoxyhemoglobin results in the production of nitric oxide and methemoglobin, a reaction recently proposed as an important oxygen-sensitive source of vasoactive nitric oxide during hypoxic and anoxic stress, with several animal studies suggesting that nitrite may have therapeutic potential. Accumulation of toxic levels of methemoglobin is suppressed by reductase enzymes present within the erythrocyte. Using a novel method of measuring methemoglobin reductase activity in intact erythrocytes, we compared fetal and adult sheep and human blood. After nitrite-induced production of 20% methemoglobin, the blood was equilibrated with carbon monoxide, which effectively stopped further production. Methemoglobin disappearance was first order in nature with specific rate constants (k x 1,000) of 12.9 +/- 1.3 min(-1) for fetal sheep, 5.88 +/- 0.26 min(-1) for adult sheep, 4.27 +/- 0.34 for adult humans, and 3.30 +/- 0.15 for newborn cord blood, all statistically different from one another. The effects of oxygen tensions, pH, hemolysis, and methylene blue are reported. Studies of temperature dependence indicated an activation energy of 8,620 +/- 1,060 calories/mol (2.06 kJ/mol), appreciably higher than would be characteristic of processes limited by passive membrane diffusion. In conclusion, the novel methodology permits absolute quantification of the reduction of nitrite-induced methemoglobin in whole blood.

In Vitro Activity of Tafenoquine against the Asexual Blood Stages of Plasmodium falciparum Isolates from Gabon, Senegal, and Djibouti

Antimalarial drugs, when used as monotherapies, are rapidly losing their effectiveness. One promising new drug is the antimalarial 8-aminoquinoline tafenoquine (SB-252263 [formerly WR-238605]), a new synthetic primaquine analogue codeveloped by the U.S. Army and GlaxoSmithKline, which has been shown effective not only against the liver stages, gametocytes, and sporozoites of Plasmodium falciparum (4), but also against the blood stages of the parasite (13). Tafenoquine demonstrated significant protection against P. falciparum infection in Gabon, Ghana, and Kenya (6, 7, 12). Tafenoquine has been reported to be well tolerated, with only mild gastrointestinal effects (8). Isolates were collected in 1999 from malaria patients from Libreville (Gabon, Central Africa), Dielmo and Ndiop (Senegal, West Africa), and Djibouti (East Africa). The isotopic, microdrug susceptibility test used was described previously (10). The 50% inhibitory concentration (IC50) values for tafenoquine were in the range 0.9 to 9.7 μM in Djibouti, 0.6 to 33.1 μM in Gabon, and 0.5 to 20.7 μM in Senegal. The geometric mean IC50 was 2.68 μM in Djibouti, versus 4.62 μM in Gabon and 5.06 μM in Senegal (Table ​(Table1).1). Tafenoquine was found to possess marked blood schizonticidal activity in P. falciparum in areas with high percentages of multidrug-resistant parasite populations. There was no difference in the tafenoquine mean IC50 values between Dielmo-Ndiop and Libreville, even though the levels of reduced susceptibility for chloroquine, mefloquine, cycloguanil, and pyrimethamine were different. Conversely, tafenoquine was significantly more active in Djibouti than in Gabon or Senegal (P = 0.016). The results of these in vitro tests were comparable with those reported by other authors in culture-adaptated P. falciparum clones and strains (2, 11). TABLE 1. In vitro susceptibilities and prevalence of resistance or reduced susceptibility of wild isolates of Plasmodium falciparum from Djibouti, Gabon, and Senegal to the drugs shown Published in vitro data for the blood schizonticidal activity of primaquine in P. falciparum show a range of IC50 values between 0.3 μM and 14 μM (1, 2, 13). In this study, there was no difference in the tafenoquine mean IC50 values between the three areas (P = 0.111). Tafenoquine is more active in vitro than primaquine, wherever the area. Tafenoquine exerts a blood schizonticidal activity 4 to 100 times higher than that of primaquine in the Plasmodium berghei and Plasmodium yoelii mouse model (9). Tafenoquine had a half-life that is more than 50 times longer than that of primaquine (3, 5). The difference in kinetics results in more prolonged, high concentrations of tafenoquine in the blood. These properties permit weekly dosing for prophylaxis and short-term or single-dose therapy for radical cure. Only 3.5% of the variation of response to tafenoquine is explained by response variation to primaquine. The coefficients of determination, r2, ranging from 0.001 to 0.113, are too weak to consider that cross-resistance may exist between tafenoquine and standard antimalarial drugs. Since correlation analysis provides an insight into the mode of action and cross-susceptibilities between different drugs, these data may be seen as an indication of the relative independence of tafenoquine from the susceptibility of P. falciparum to standard antimalarial drugs. In conclusion, these data permit definition of the baseline of in vitro susceptibility to tafenoquine before its use and will allow the monitoring of its resistance or its reduced susceptibility when tafenoquine will be commonly used. Given its greater schizonticidal activity, tafenoquine is a promising candidate as a short treatment for P. falciparum and Plasmodium vivax malaria. However, the potential side effects of tafenoquine, such as the production of methemoglobin and the risk of hemolysis in glucose-6-phosphate dehydrogenase-deficient patients, have to be taken into consideration (14).

Effect of wild type Escherichia coli W3110 or Escherichia coli nir-Ptac on methane emission and nitrate toxicity in nitrate-treated sheep

The effects of the two kinds of Escherichia coli ( E. coli) strains, the wild type E. coli W3110 or E. coli nir-Ptac, which has enhanced nitrite reduction activity, on methane (CH 4) emission and nitrate toxicity in nitrate-treated sheep were assessed in an open circuit respiratory system according to a 4 × 6 Youden square design. Nitrate (1.3 g NaNO 3/kg BW 0.75) and/or E. coli strains were given into the rumen through a fistula as a single dose 30 min after the morning meal. E. coli W3110 or E. coli nir-Ptac cells inoculated for sheep was 150 ml (2 × 10 10 cells/ml). The six treatments consisted of saline (0.9 g NaCl), wild type E. coli W3110, E. coli nir-Ptac, nitrate, nitrate plus E. coli W3110, and nitrate plus E. coli nir-Ptac. Methane emission from sheep was reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac by 6% ( p > 0.05) or 12% ( p < 0.05), respectively. Nitrate markedly inhibited CH 4 emission from sheep. Compared with sheep given nitrate alone, the inoculation of E. coli W3110 to nitrate-treated sheep abated ruminal and plasma toxic nitrite accumulation and blood methaemoglobin production, while keeping ruminal methanogenesis low. Ruminal and plasma toxic nitrite accumulation and blood methaemoglobin production in sheep were unaffected by the inoculation of E. coli nir-Ptac. These results suggest that ruminal methanogenesis may be reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac. The inoculation of E. coli W3110 may abate nitrate toxicity when nitrate is used to inhibit CH 4 emission from ruminants.

Effect of ruminal administration ofEscherichia coliwild type or a genetically modified strain with enhanced high nitrite reductase activity on methane emission and nitrate toxicity in nitrate-infused sheep

The effects of two kinds of Escherichia coli (E. coli) strain, wild-type E. coli W3110 and E. coli nir-Ptac, which has enhanced NO(2) reduction activity, on oral CH(4) emission and NO(3) toxicity in NO(3)-treated sheep were assessed in a respiratory hood system in a 4 x 6 Youden square design. NO(3) (1.3 g NaNO(3)/kg(0.75) body weight) and/or E. coli strains were delivered into the rumen through a fistula as a single dose 30 min after the morning meal. Escherichia coli cells were inoculated for sheep to provide an initial E. coli cell density of optical density at 660 nm of 2, which corresponded to 2 x 10(10) cells/ml. The six treatments consisted of saline, E. coli W3110, E. coli nir-Ptac, NO(3), NO(3) plus E. coli W3110, and NO(3) plus E. coli nir-Ptac. CH(4) emission from sheep was reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac by 6 % and 12 %, respectively. NO(3) markedly inhibited CH(4) emission from sheep. Compared with sheep given NO(3) alone, the inoculation of E. coli W3110 to NO(3)-infused sheep lessened ruminal and plasma toxic NO(2) accumulation and blood methaemoglobin production, while keeping ruminal methanogenesis low. Ruminal and plasma toxic NO(2) accumulation and blood methaemoglobin production in sheep were unaffected by the inoculation of E. coli nir-Ptac. These results suggest that ruminal methanogenesis may be reduced by the inoculation of E. coli W3110 or E. coli nir-Ptac. The inoculation of E. coli W3110 may abate NO(3) toxicity when NO(3) is used to inhibit CH(4) emission from ruminants.

Methemoglobinemia as a Complication of Topical Anesthesia During Bronchoscopy

Abstract:Methemoglobinemia, although uncommon, is a potentially life-threatening complication of topical anesthetics. We describe a case of a 46-year-old man developed a severe methemoglobinemia after routine bronchoscopy. After administration of 1 mg/kg intravenous methylene blue, the patient's con

Laser treatment of leg veins: Physical mechanisms and theoretical considerations

A discussion of laser treatment of leg veins is based on a review of the literature, theoretical analysis, and the clinical experiences of the authors. Theoretical computations are discussed within the context of clinical observations. A Monte Carlo model is used to examine volumetric heat production, fluence rate, and temperature profiles in blood vessels at 1,064 and 532 nm wavelengths with various beam diameters, vessel diameters, and pulse durations. Clinical observations, Monte Carlo results, and a review of the literature suggest that longer wavelengths and longer pulses durations favor vessel contraction over intraluminal thrombosis. Monte Carlo simulations show that longer wavelengths are more likely to uniformly heat the vessel compared to highly absorbing wavelengths. Methemoglobin production causes deeply penetrating wavelengths to generate more volumetric heat for the same input radiant exposure. Clinical observations and models support the role of long wavelengths and long pulses in optimal clearance of most leg telangiectasias.

Disorders of oxidised haemoglobin

Methaemoglobinaemia arises from the production of non-functional haemoglobin containing oxidised Fe 3+ which results in reduced oxygen supply to the tissues and manifests as cyanosis in the patient. It can develop by three distinct mechanisms: genetic mutation resulting in the presence of abnormal haemoglobin, a deficiency of methaemoglobin reductase enzyme and toxin-induced oxidation of haemoglobin. The normal haemoglobin fold forms a pocket to bind the haem and stabilise its complex with molecular oxygen, simultaneously preventing spontaneous oxidation of the Fe 2+ ion chelated by the haem pyrroles and the globin histidines. In the abnormal, M forms of haemoglobin (Hb Ms) amino acid substitution in or near the haem pocket creates a propensity to form methaemoglobin instead of oxyhaemoglobin in the presence of molecular oxygen. Normally, haemoglobin continually oxidises but significant accumulation of methaemoglobin is prevented by the action of a group of methaemoglobin reductase enzymes. In the autosomal recessive form of methaemoglobinaemia there is a deficiency of one of these reductase enzymes thereby allowing accumulation of oxidised Fe 3+ in methaemoglobin. Oxidising drugs and other toxic chemicals may greatly enhance the normal spontaneous rate of methaemoglobin production and if levels exceed 70% of total haemoglobin, vascular collapse occurs resulting in coma and death. Under these conditions, if the source of toxicity can be eliminated methaemoglobin levels will return to normal. Disorders of oxidised haemoglobin are relatively easily diagnosed and in most cases, except for the presence of congenitally defective haemoglobin M, can be treated successfully.

Reconstitution of bactericidal activity in chronic granulomatous disease cells by glucose-oxidase–containing liposomes

AbstractChronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by phagocytes devoid of a functioning nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The failure of CGD phagocytes to produce reactive oxygen species (ROS) results in a marked increase in the susceptibility of affected patients to life-threatening bacterial and fungal infections. This study investigated whether loading of CGD phagocytes with glucose oxidase (GO)–containing liposomes (GOLs) could restore cellular production of bactericidal ROS (eg, H2O2 and HOCl) in vitro. Results indicate that GO encapsulated in liposomes enabled NADPH oxidase-deficient phagocytes to use H2O2 for the production of highly bactericidal HOCl. The intracellular colocalization of bacteria and liposomes (or liposome-derived ferritin) was demonstrated by confocal laser microscopy and electron microscopy. After uptake of GOLs (approximately 0.2 U/mL at 1 mM total lipid concentration, size approximately 180 nm), CGD granulocytes produced HOCl levels comparable to those of normal phagocytes. Remarkably, after treatment with GOLs, CGD phagocytes killed Staphylococcus aureus as efficiently as normal granulocytes. Moreover, treated cells retained sufficient motility toward chemotactic stimuli as measured by chemotaxis assay. Side effects were evaluated by measuring the H2O2 concentrations and the production of methemoglobin in whole blood. These studies revealed that H2O2 produced by GOLs was degraded immediately by the antioxidative capacity of whole blood. Elevated methemoglobin levels were observed only after application of extremely high amounts of GOLs (2 U/mL). In summary, the application of negatively charged GOLs might provide a novel effective approach in the treatment of patients with CGD at high risk for life-threatening infections.

Acquired methemoglobinemia in anemic cattle infected with Theileriasergenti

To investigate the mechanism of anemia accompanying Japanese bovine theileriosis, we examined whether production of methemoglobin (MetHB), an indicator of erythrocyte oxidation, was associated with anemia in cattle experimentally infected with Theileria sergenti. The percentage of MetHB, which is an oxidized form of hemoglobin, increased according to the onset of anemia. During severe anemia, high levels of acquired methemoglobinemia were observed in all infected cattle. A significant correlation ( r=−0.649; P<0.01) between an increase in MetHB concentration and a decrease in packed cell volume (PCV) was observed. It was considered that hemoglobin oxidation may be one of the aggravating factors of anemia in T. sergenti infection.

Antioxidant effect of red wine anthocyanins in normal and catalase-inactive human erythrocytes

Previous studies reported that aged red wine, but not novel red wine or white wine protects human red blood cells from oxidative damage induced in vitro by H 2O 2. Here, we demonstrate that the beneficial properties of aged red wine are due, at least in part, to the presence of anthocyanins. We firstly measured the “antioxidant power” of an Italian red wine (Taurasi, Avellino) and that of its anthocyanin fractions by using Ferric Reducing Antioxidant Power Assay. Subsequently, we demonstrate that fractions containing anthocyanins lower ROS (reactive oxygen species) and methemoglobin production in human erythrocytes treated with H 2O 2. Finally, we reported that the protective effects of anthocyanins were also confirmed in an experimental model in which RBCs were deprived of catalase activity by treatment with 4 mM sodium azide. The results obtained clearly demonstrate that red wine anthocyanins protect human RBCs from oxidative stress.

Recurrent acute respiratory tract infections in areas with high nitrate concentrations in drinking water.

A review of the literature indicated an association among high nitrate ingestion, methemoglobinemia, and pathologic changes in bronchi and lung parenchyma. The present study examined a possible correlation among drinking water nitrate concentration, methemoglobin levels, cytochrome b(5) reductase activity, and acute respiratory tract infection with a history of recurrence (RRTI). Our study was conducted in five village units in the state of Rajasthan, India, with nitrate concentrations of 26, 45, 95, 222, and 459 mg NO(3) ion/L. We randomly selected 88 children. The children were up to 8 years of age, age matched, and represented 10% of the total population of these areas. We obtained detailed RRTI histories and conducted medical examinations. Methemoglobin levels and cytochrome b(5) reductase activity were estimated biochemically. The data collected were statistically analyzed using spreadsheet software on a personal computer. We observed strong interdependence between methemoglobin levels and RRTI in children up to 8 years of age. Methemoglobin levels alone explained 80% of the variation in the RRTI cases. This study indicates that methemoglobinemia, secondary to high nitrate ingestion in drinking water, causes RRTI. Increased production of methemoglobin and free radicals of nitric oxide and oxygen due to nitrate metabolism in the body lead to alveolar damage and mismatching of ventilation and perfusion, which may be the reason for high mortality in children due to RRTI.

Recurrent Acute Respiratory Tract Infections in Areas with High Nitrate Concentrations in Drinking Water

A review of the literature indicated an association among high nitrate ingestion, methemoglobinemia, and pathologic changes in bronchi and lung parenchyma. The present study examined a possible correlation among drinking water nitrate concentration, methemoglobin levels, cytochrome b(5) reductase activity, and acute respiratory tract infection with a history of recurrence (RRTI). Our study was conducted in five village units in the state of Rajasthan, India, with nitrate concentrations of 26, 45, 95, 222, and 459 mg NO(3) ion/L. We randomly selected 88 children. The children were up to 8 years of age, age matched, and represented 10% of the total population of these areas. We obtained detailed RRTI histories and conducted medical examinations. Methemoglobin levels and cytochrome b(5) reductase activity were estimated biochemically. The data collected were statistically analyzed using spreadsheet software on a personal computer. We observed strong interdependence between methemoglobin levels and RRTI in children up to 8 years of age. Methemoglobin levels alone explained 80% of the variation in the RRTI cases. This study indicates that methemoglobinemia, secondary to high nitrate ingestion in drinking water, causes RRTI. Increased production of methemoglobin and free radicals of nitric oxide and oxygen due to nitrate metabolism in the body lead to alveolar damage and mismatching of ventilation and perfusion, which may be the reason for high mortality in children due to RRTI.

Antioxidant effect of red wine polyphenols on red blood cells

The protective effect of red wine polyphenols against hydrogen peroxide (H 2O 2)-induced oxidation was investigated in normal human erythrocytes (RBCs). RBCs, preincubated with micromolar amounts of wine extract and challenged with H 2O 2, were analyzed for reactive oxygen species (ROS), hemolysis, methemoglobin production, and lipid peroxidation. All these oxidative modifications were prevented by incubating the RBCs with oak barrel aged red wine extract (SD95) containing 3.5 mM gallic acid equivalent (GAE) of phenolic compounds. The protective effect was less apparent when RBCs were incubated with wines containing lower levels of polyphenols. Furthermore, resveratrol and quercetin, well known red wine antioxidants, showed lower antioxidant properties compared with SD95, indicating that interaction between constituents may bring about effects that are not necessarily properties of the singular components. Our findings demonstrate that the nonalcoholic components of red wine, mainly polyphenols, have potent antioxidant properties, supporting the hypothesis of a beneficial effect of red wine in oxidative stress in human system. © Elsevier Science Inc. 2000

Hemoglobin autooxidation/oxidation mechanisms and methemoglobin prevention or reduction processes in the bloodstream. Literature review and outline of autooxidation reaction.

The amount of circulating methemoglobin in healthy humans is the result of a balance between methemoglobin production (from autooxidation and oxidation) and hemoglobin reduction. Hemoglobin autooxidation and oxidation are very complex and are not well understood. This article analyses the literature on hemoglobin autooxidation, oxidation and reduction and sets out a sequence of reactions for the oxidation of hemoglobin and the ways in which the percentage of methemoglobin is regulated or methemoglobin production prevented. Most of the information concerns erythrocyte hemoglobin, but plasma extracellular hemoglobin (from hemolysis or hemoglobin-based blood substitutes) is also considered where possible.

Peroxynitrite modulates tyrosine‐dependent signal transduction pathway of human erythrocyte band 3

Peroxynitrite, the product of the reaction between nitric oxide and superoxide anion, is able to nitrate protein tyrosines. If this modification occurs on phosphotyrosine kinase substrates, it can down-regulate cell signaling. We investigated the effects of peroxynitrite on band 3-mediated signal transduction of human erythrocytes. Peroxynitrite treatment induced two different responses. At low concentrations (10-100 microM) it stimulated a metabolic response, leading to 1) a reversible inhibition of phosphotyrosine phosphatase activity, 2) a rise of tyrosine phosphorylation in the 22K cytoplasmic domain of band 3, 3) the release of glyceraldehyde 3-phosphate dehydrogenase from the membrane, and 4) the enhancement of lactate production. At high concentrations (200-1000 microM), peroxynitrite induced 1) cross-linking of membrane proteins, 2) inhibition of band 3 tyrosine phosphorylation, 3) nitration of tyrosines in the 22K cytoplasmic domain of band 3, 4) binding of hemoglobin to the membrane, 5) irreversible inhibition of phosphotyrosine kinase activity, 6) massive methemoglobin production, and 7) irreversible inhibition of lactate production. Our results demonstrate that at concentrations that could conceivably be achieved in vivo (10-100 microM), peroxynitrite behaves like other oxidants, i.e., it stimulates band 3 tyrosine phosphorylation and increases glucose metabolism. Thus, one plausible physiologic effect of peroxynitrite is the up-regulation of signaling through the reversible inhibition of phosphotyrosine phosphatase activity. At high concentrations of peroxynitrite, the tyrosine phosphorylation ceases in parallel with the nitration of band 3 tyrosines, but at these concentrations phosphotyrosine kinase activity and glycolysis are also irreversibly inhibited. Thus, at least in red blood cells, the postulated down-regulation of signaling by peroxynitrite cannot merely be ascribed to the nitration of tyrosine kinase targets.