EPR detection of sulfanyl radical during sulfhemoglobin formation - Influence of catalase.

Hemoglobin in its ferryl form oxidizes hydrogen sulfide and is transformed to sulfhemoglobin, where the sulfur is inserted covalently at the heme edge. Shown here is evidence that-as previously proposed by others-this process involves oxidation of hydrogen sulfide to a sulfanyl radical detectable by spin-trapping in electron paramagnetic resonance (EPR) spectroscopy. The yields and rates of formation of sulfhemoglobin as well as of the sulfanyl radical are affected by the same factors that affect the reactivity of hemoglobin ferryl, in bovine hemoglobin and in phytoglobins as well. A freely-diffusing sulfanyl radical is thus proposed to be involved in sulfhemoglobin formation. Catalase is shown to accelerate this process due to a previously described hydrogen sulfide oxidase activity, within which EPR evidence for sulfanyl generation is shown here for the first time. The reaction of preformed ferryl with hydrogen sulfide-in absence of hydrogen peroxide-is studied by stopped-flow at several pH values and explained in light of reactivity and redox potential control.

[Influence of sodium nitrite exposure on sulfhemoglobin and hydroxyl radicals in mice].

Objective: To investigate the influence of sodium nitrite exposure on sulfhemoglobin and hydroxyl radicals in mice. Methods: A total of 60 mice were randomly divided into low-, middle-, and high-dose groups (the concentrations of sodium nitrite were 0.055 mg/ml, 0.110 mg/ml, and 0.220 mg/ml, respectively) and control group (treated with distilled water) , with 15 mice in each group (male/female ratio=1: 1) . A free-drink model was applied and the duration of exposure was 2 weeks. The body weight of all mice was recorded before exposure and at weeks 1 and 2 of exposure. At the end of exposure, the mice were treated with intraperitoneally injected sodium salicylate to capture the hydroxyl radicals and produce 2, 5-dihydroxybenzoic acid and 2, 3-dihydroxybenzoic acid, and high-performance liquid chromatography was used to measure their content. Spectrophotometry was used to measure the relative content of sulfhemoglobin. Results: At week 2 of exposure, the low-, middle-, and high-dose groups had significantly lower body weight than the control group (22.8±2.8 g/21.6±2.8 g/21.2±3.0 g vs 25.6±2.2 g, P<0.05) . The low-, middle-, and high-dose groups had a significantly higher total content of hydroxyl radicals than the control group[ (0.015 3±0.006 5) µg/ml, (0.016 4±0.017 2) µg/ml, and (0.062 7±0.091 0) µg/ml vs (0.009 ±0.007 3) µg/ml, P<0.05]. The relative content of sulfhemoglobin was 1.54%±0.73%, 2.22%±0.44%, and 2.80%±0.69%, respectively, in the low-, middle-, and high-dose groups, and the middle- and high-dose groups had a significant increase in the relative content of sulfhemoglobin compared with the control group (2.22%±0.44%/2.80%±0.69% vs 1.76%±0.60%, P<0.05) . The content of hydroxyl radicals was positively correlated with the relative content of sulfhemoglobin (r=0.837, P<0.05) . Conclusion: Sodium nitrite exposure can increase the content of sulfhemoglobin and hydroxyl radicals in blood, and there is a positive correlation between them.

Effects of long-term exposure to hydrogen sulfide on human red blood cells.

BACKGROUND: Hydrogen sulfide (H2S) exhibits both physiological and toxicological roles in the biological systems. Acute exposure to high levels of H2S is life threatening while long-term exposure to ambient levels of H2S elicits human health effects. OBJECTIVE: To study the harmful effects of long-term exposure to low levels of H2S on human blood cells. METHODS: 110 adult workers from Iran who were occupationally exposed to 0-90 ppb H2S for 1-30 years were studied. The participants aged between 18 and 60 years and were exposed directly or indirectly to sulfur compounds (exposed group). The origin of H2S was natural gas processing plants. A control group consisting of 110 males who were not in contact with H2S was also studied. For all participants, hematological profile including total hemoglobin and red blood cell count and sulfhemoglobin, methemoglobin levels were measured. RESULTS: Among all parameters evaluated in this study the mean methemoglobin and sulfhemoglobin levels were significantly higher among workers who were exposed to sulfur compounds than the control group. Major differences throughout the study period for sulfhemoglobinemia among exposed groups were observed. CONCLUSION: Long-term exposure to even low levels of H2S in workplaces may have potential harmful effects on human health.

Pregnancy loss and maternal methemoglobin levels: an indirect explanation of the association of environmental toxics and their adverse effects on the mother and the fetus.

The aim of this epidemiologic study was to point out a relationship between the exposure to products of coal combustion, and complications in pregnancy where one third of causes of stillbirth are still unknown. In the town of Labin (Croatia) a coal-powered thermoelectric power plant is the single major air polluter. We compared the records of miscarriages, premature births and stillbirths in two periods: the control and the exposure period. Data on reproductive loss was based on the records of pregnant women visiting for regular monthly pregnancy checkups. At the time of the epidemiological prospective study, 260 women (n = 138 in the clean period and n = 122 in the dirty period) were considered representative. The data were processed using Chi square and correlation tests. The frequencies of miscarriages and stillbirths were significantly lower in the control than in the exposure period (p < 0.05). Methemoglobinemia and stillbirths recorded over the "exposure" period are significantly higher than in the "control" period (p = 0.0205). The level of methemoglobin in the bloodstream is an worthy biomarker, predictor and precursor of environmental toxics' adverse effects on the mother and fetus, and can indirectly explain the unrecognized level of fetal methemoglobin. Methemoglobin and heme, having prooxidant properties, also cause the early and late endothelial dysfunction of vital organs. Despite our retrospective epidemiological study findings, we emphasize that the rate of reproductive loss represents a hypothetical risk, which needs to be confirmed with further fetal clinical and anatomopatholgical researches about the effects of methemoglobin catabolism products on the fetal CNS.

The origin of hydrogen sulfide in a newborn with sulfhaemoglobin induced cyanosis.

This report investigated the origin of H(2)S in a newborn boy with sulfhaemoglobin induced cyanosis, who died because of multiple organ failure. Frozen material was collected and studied after death. The results suggest that enzymes had been released from deteriorating organs into the blood and abdominal fluid, and that the reaction of one of these enzymes with sulfur containing amino acids might have resulted in increased H(2)S concentrations. It is hypothesised that this release of enzymes resulted from a haemolysin produced by an invasive haemolytic Escherichia coli that was found in the blood and organs of this patient.

[Study of hemoglobin system components and antioxidation enzymes in cadmium intoxication]. / Vyvchennia komponentiv systemy hemohlobinu ta antyoksydantnykh fermentiv za kadmiievoï intoksykatsiï.

Cadmium intoxication has been defined to make material changes in hemoglobin system by elevation of sulfhemoglobin and methhemoglobin levels on the back-cloth of lowering of general hemoglobin. Alongside with the latter there is observed a violation of antioxydation protection including the activity of erythrocytic catalase, ceruloplasmin, ferrum saturation of transferrine.

Sulfhemoglobin formation in human erythrocytes by cystalysin, an L-cysteine desulfhydrase from Treponema denticola.

Cystalysin, isolated from the oral pathogen Treponema denticola, is an L-cysteine desulfhydrase (producing pyruvate, ammonia and hydrogen sulfide from cysteine) that can modify hemoglobin and has hemolytic activity. Here, we show that enzymatic activity of recombinant cystalysin depends upon stochiometric pyridoxal phosphate. The enzyme was not functional as an L-alanine transaminase, and had a strong preference for L-cysteine over D-cysteine. Cystalysin preferred small alpha-L-amino acids as substrates or inhibitors and was far more active towards L-cysteine than towards the other standard amino acids that undergo pyridoxal phosphate-dependent beta-elimination reactions (serine, threonine, tryptophan and tyrosine). Cystalysin tolerated small modifications to the carboxylate of L-cysteine (i.e., the methyl and ethyl esters of L-cysteine were good substrates), but the smallest possible peptide with an N-terminal cysteine, L-cysteinylglycine, was a very poor substrate. These results, combined with the implicit requirement for a free amine for pyridoxal phosphate-dependent reactions, imply that cystalysin cannot catabolize cysteine residues located within peptides. Cystalysin has Michaelis-Menten kinetics towards L-cysteine, and there was little or no inhibition by ammonia, H2S, pyruvate and acetate. Human erythrocytes incubated with H2S or with cystalysin and cysteine primarily accumulated sulfhemoglobin and methemoglobin, along with minor amounts of choleglobin and protein aggregates. Erythrocytes retained the ability to reduce methemoglobin in the presence of H2S. Cystalysin could not modify hemoglobin when beta-chloroalanine was the substrate, indicating an absolute requirement for H2S production. Cystalysin appears to be an unregulated L-cysteine catabolizing enzyme, with the resulting H2S production being essential to the atypical hemolytic activity.

Hemoglobin oxidation products extract phospholipids from the membrane of human erythrocytes.

Hydrogen peroxide oxidation of human erythrocytes induces a transfer of phospholipid from the membrane into the cytosol [Brunauer, L.S., Moxness, M.S., & Huestis, W.H. (1994) Biochemistry 33, 4527-4532]. The current study examines the mechanism of lipid reorganization in oxidized cells. Exogenous phosphatidylserine was introduced into the inner monolayer of erythrocytes, and its distribution was monitored by microscopy and radioisotopic labeling. Pretreatment of cells with carbon monoxide prevented both hemoglobin oxidation and the transfer of phosphatidyserine into the cytosolic compartment. The roles of the various hemoglobin oxidation products in lipid extraction were investigated using selective oxidants. Nitrite treatment of intact cells produced almost complete conversion to methemoglobin, but no detectable lipid extraction. Treatments designed to produce the green hemoglobin derivatives, sulfhemoglobin and choleglobin, resulted in cytosolic extraction of phosphatidylserine. Ion exchange and size exclusion chromatography of oxidized cytosolic components revealed a lipid-hemoglobin complex. The interaction between lipid and hemoglobin oxidation products was verified in a model system. Purified hemoglobin, enriched in sulfhemoglobin and choleglobin by treatment with H2O2, H2S, or ascorbate, extracted phospholipid from small unilamellar phospholipid vesicles. Electron paramagnetic resonance studies demonstrated that hemoglobin oxidation products also adsorb fatty acids from solution. This newly described activity of hemoglobin may play a role in the clearance of oxidatively damaged and senescent cells from circulation.

Topical anesthetic-induced methemoglobinemia and sulfhemoglobinemia in macaques: a comparison of benzocaine and lidocaine.

Benzocaine (BNZ) and lidocaine (LC) are commonly used topical (spray) anesthetics approved for use in humans. Benzocaine has structural similarities to methemoglobin (MHb)-forming drugs that are current candidates for cyanide prophylaxis, while LC has been reported to increase MHb in man. In this study, we compared MHb and sulfhemoglobin (SHb) production in three groups of Macaques (Chinese rhesus and Indian rhesus (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina)) after exposure to BNZ and LC. Formation of SHb, unlike MHb, is not thought to be reversible and therefore is considered to be of greater toxic significance. Both MHb and SHb levels were measured periodically on a CO-Oximeter. All rhesus macaques (n = 8) were administered an intratracheal/intranasal) dose of 56 mg (low dose) or 280 mg (high dose) of BNZ or 40 mg of LC in a randomized cross-over design (all animals received all three treatments). Pig-tailed macaques (n = 6) were given an intranasal dose of 56 mg of BNZ and 40 mg of LC. As no differences in the peak MHb or time to peak (mean +/- SD) were observed among the three macaque subspecies, the data were pooled. Lidocaine did not cause MHb or SHb formation above baseline in any monkey. In contrast, all monkeys (n = 14) had a significant elevation in peak MHb formation after 56 mg of BNZ, which ranged from 4.0% to 19.4% with an average of 8.6 +/- 4.0% (mean +/- SD), with peak MHb levels reached at 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

The incorporation of sulphaem into recombinant adult human haemoglobin produced in a yeast expression system.

The production of adult human haemoglobin in a yeast expression system has been shown to lead to the formation of functional oxygen-binding tetrameric proteins with the incorporation of endogenously synthesized haem. Adachi et al. [(1992) Protein Engng, 5, 807-810] identified two partially resolvable forms of the expressed haemoglobin, one of which showed higher oxygen affinity and lower cooperativity than normal. We show that in contrast to the previously expressed view that the abnormal form is due to abnormal protein folding, that it represents tetrameric haemoglobin containing incorporated sulphaem. Furthermore, the incorporation of sulphaem is shown to be a time-dependent process, with no detectable sulphaem being incorporated prior to 16 h post-induction. Numerical simulation based on our analysis of sulphaem composition gives an excellent fit to oxygen binding data previously reported for samples containing mixtures of normal haemoglobin and sulphaemoglobin.

Evaluation of the methemoglobinemia associated with sulofenur.

A new class of antineoplastic agents, the diarylsulfonylureas entered clinical trials with the testing of Sulofenur (LY186641). Phase I trials and preclinical studies showed the dose limiting toxicity to be methemoglobinemia. We studied the incidence of methemoglobinemia, sulfhemoglobinemia and cytochrome b5 reductase deficiency in nine consecutive patients enrolled in a phase II trials using Sulofenur. The specific Malloy method as well as clinically standard co-oximeter measurements were used to determine methemoglobin levels and marked discrepancies were noted. One patient with symptomatic methemoglobinemia had enzyme levels and family history consistent with a heterozygous state for a cytochrome b5 reductase deficiency. We conclude that the clinical incidence of methemoglobinemia will be overestimated by co-oximeter measurements but that Sulofenur does produce clinically significant methemoglobinemia in cytochrome b5 reductase deficient patients.

Management of dapsone poisoning complicated by methaemoglobinaemia.

The currently recommended dosage regimen for methylene blue (intermittent bolus dose) in the treatment of methaemoglobinaemia caused by dapsone is often inadequate. This is due to the long half-life of dapsone which provides a continuing oxidative stress that can cause a recurrence of clinically significant methaemoglobinaemia. Methylene blue infusion is effective, as demonstrated in an illustrative case report, and should be supported by repeated doses of activated charcoal to enhance dapsone elimination. The principles of treatment of methaemoglobinaemia due to dapsone can be applied to methaemoglobinaemia due to any agent producing prolonged oxidative stress.

Sulfhemoglobin. Properties of partially sulfurated tetramers.

The properties of sulfhemoglobin (sulfHb) were investigated using disc gel isoelectric focusing and optical spectrophotometry. Laboratory-prepared samples, which contained a high yield of sulfHb (70-85%), and a patient-derived sample, which contained a low yield (12%), contain a tetrameric population that reflects a random distribution of modified (sulfurated) subunits. Hybrid tetramers, i.e. those containing sulfurated and unmodified subunits, were resolved from fully sulfurated and unmodified hemoglobin upon electrofocusing of ferric or ferrous sulHb samples. The electrophoretic differences between ferric sulfHb and metHb arise from a difference in the pK for the met to hydroxymet conversion of sulfurated subunits (Carrico, R., Peisach, J., and Alben, J. O. (1978) J. Biol. Chem. 253, 2386-2391). Both partially and fully sulfurated tetramers, when in the deoxy form and when in the fully ligated CO form, co-focus with their unmodified deoxy-Hb and Hb CO counterparts. Thus, these sulfurated tetramers exhibit normal ligand-dependent ionization of Bohr protons. In air-equilibrated samples, hybrid tetramers are partially ligated as a result of the reduced O2 affinity of the sulfurated subunits. These partially ligated tetramers exhibit a pI intermediate between oxy-Hb and deoxy-Hb due to the fractional ionization of Bohr protons. The partially sulfurated, partially ligated tetramers as well as deoxy-sulfHb tetramers were found to bind 2,3-diphosphoglycerate in disc gels. Despite the preservation of these heterotropic effects, which are confirmed in CO binding studies in solution, fully sulfurated tetramers recovered using preparative isoelectric focusing exhibit little or no cooperativity.

Oxido-reductive reactions of sulfhemoglobin with various reagents associated with changes in conformation of the protein.

The optical peaks at Soret region and near at 620 nm of sulfhemoglobin (sulfHb) shifted from 419.5 nm to 423 nm and from 623 nm to 619 nm, respectively, according to the decrease in oxygen concentrations of the sulfHb solution (1 atm to zero atm). This shift of optical peaks was strengthened by the addition of inositol hexaphosphate (P6-inositol), a strong allosteric effector of Hb. Thus the shift of the optical peaks seems to reflects the changes in conformation of sulfHb. In order to clarify the relationship between the conformation of sulfHb and the reactivity of the protein with various reagents, we studied the oxidation and reduction reaction of the protein with ascorbic acid, ferricyanide and nitrite under aerobic and anaerobic conditions. The results suggested that the rate of oxido-reductive reactions of ferrous and ferric sulf Hbs with these compounds are associated with the conformation of the protein.

Studies on ligand binding to sulphaemoglobin.

Equilibrium studies show that, at low protein concentrations, sulphaemoglobin, in the ferrous form, binds carbon monoxide at pH 6.0 in a non co-operative manner with a Hill coefficient of 1.15 and an affinity constant of 8 . 10(-7) M. At both pH 6.0 and pH 9.0 the kinetics of CO binding show the presence of a simple mono-exponential process with a second-order rate constant of 8 . 10(3) M-1 . s-1. The rate of dissociation of CO from sulphaemoglobin is approx. 0.01 s-1. The activation energy of the binding process is calculated as 40 kJ . mol-1. A comparison is presented between the CO binding properties of sulphaemoglobin, myoglobin and haemoglobin and a mechanism whereby the CO binding parameters of sulphaemoglobin are modified is proposed.

[Uremic plasma as cause of metabolic changes in red blood cells]. / Das urämische Plasma als Ursache für Stoffwechselveränderungen roter Blutzellen.

Under the influence of the uraemic plasma a number of changes of the metabolism of red blood cells develop. By means of cross experiments with the help of filtrability, of sulf-haemoglobin formation and the 125-iodine-hippuran intake the influence of the uraemic plasma and of the plasma of healthy persons on the red blood cells of children who undergo a chronic haemodialysis as well as of healthy adults (cross experiments) was examined. The red blood cells of the control persons after an incubation at 37 degrees and 60 or 180 minutes showed approximately the same changes of filtrability, formation of sulf-haemoglobin and 125-iodine-hippuran as the red blood cells of the patients with incubation in the autologous plasma. Disturbances of the filtrability and the formation of sulf-haemoglobin could be proved in the uraemic red blood cells still after reincubation in the normal plasma, though clearly reduced. Only in 125-iodine-hippuran a normalisation could be got. Filtrability and formation of sulf-haemoglobin apparently comprise more complex processes which essentially influence the life span of the red blood cells, whereas the 125-iodine-hippuran value characterizes only a part of the metabolism of the red blood cells, the passive transport of anions.