Sulfhemoglobin under the spotlight - Detection and characterization of SHb and HbFeIII-SH.

Sulfhemoglobinemia is an incurable disease caused by an overdose of sulfur-containing drugs with oxidizing properties. Its diagnosis remains hindered due to the similarity of symptoms to other pathological state - methemoglobinemia, as well as contradictory information on the structure and characteristics of sulfhemoglobin. Herein, we present sulfhemoglobinemia model on living functional human erythrocytes, designed to recreate processes which could take place in a patient body in order to complement missing information and highlight distinctiveness of two hemoglobin (Hb) adducts formed after interaction with sulfur donors. Employed techniques, UV-Vis absorption, Raman, Fourier transformed infrared (FT-IR) and electronic circular dichroism (ECD) spectroscopies, allowed to distinguish and characterize Hb adduct with sulfur atom bounded directly to the iron ion (HbFeIII-SH), and irreversibly connected to the porphyrin ring (SHb - sulfhemoglobin). Presented herein results provided also new evidence on formation of both these hemoglobin adducts inside functional erythrocytes under oxidative conditions and during sulfur-containing drug presence, what can be further translated into future physiological studies. Moreover, we found that sulfur attachment to the porphyrin ring altered Hb structure and lead to changes in protein packing inside RBCs, eventually. Interestingly, measurement of blood drop smear by Raman spectroscopy occurred the most accurate method to differentiate HbFeIII-SH and SHb, indicating potential of this technique in sulfhemoglobinemia diagnosis.

Interpreting sulfhemoglobin and methemoglobin in patients with cyanosis: An overview of patients with M-hemoglobin variants.

INTRODUCTION: Methemoglobin (MetHb) and sulfhemoglobin (SHb) measurements are useful in the evaluation of cyanosis. When one or both values are elevated, additional analysis is important to establish the etiology of the disorder. Methemoglobinemia occurs from acquired or hereditary causes with diverse treatment considerations, while true sulfhemoglobinemia is only acquired and treatment is restricted to toxin removal. Some toxic exposures can result in a dual increase in MetHb and SHb. Hereditary conditions, such as M-Hemoglobin variants (M-Hbs), can result in increased MetHb and/or SHb values but are clinically compensated and do not require treatment if they are cyanotic but otherwise clinically well. METHODS: Herein, we report 53 hemoglobin variant cases that have associated MetHb and SHb levels measured by an adapted Evelyn-Malloy laboratory assay method. RESULTS: Our data indicate M-Hbs cause variable patterns of MetHb and SHb elevation in a fairly reproducible pattern for the particular variant. In particular, α globin chain M-Hbs can mimic acquired sulfhemoglobinemia due to an isolated increased SHb value. CONCLUSION: If the patient appears clinically well other than cyanosis, M-Hbs should be considered early in the evaluation process to differentiate from acquired conditions to avoid unnecessary testing and treatment regimens and prompt genetic counseling.

Assessment of absorbed dose of gamma rays using the simultaneous determination of inactive hemoglobin derivatives as a biological dosimeter.

Biological dosimetry based on sulfhemoglobin (SHb), methemoglobin (MetHb), and carboxyhemoglobin (HbCO) levels was evaluated. SHb, MetHb and HbCO levels were estimated in erythrocytes of mice irradiated by γ rays from a 60Co source using the method of multi-component spectrophotometric analysis developed recently. In this method, absorption measurements of diluted aqueous Hb-solution were made at λ = 500, 569, 577 and 620 nm, and using the mathematical formulas based on multi-component spectrophotometric analysis and the mathematical Gaussian elimination method for matrix calculation, the concentrations of various Hb-derivatives and total Hb in mice blood were estimated. The dose range of γ rays was from 0.5 to 8 Gy and the dose rate was 0.5 Gy min-1. Among all Hb-derivatives, MetHb, SHb and HbCO demonstrated an unambiguous dose-dependent response. For SHb and MetHb, the detection limits were about 0.5 Gy and 1 Gy, respectively. After irradiation, high levels of MetHb, SHb and HbCO persisted for at least 10 days, and the maximal increase of MetHb, SHb and HbCO occurred up to 24 h following γ irradiation. The use of this "MetHb + SHb + HbCO"-derivatives-based absorbed dose relationship showed a high accuracy. It is concluded that simultaneous determination of MetHb, SHb and HbCO, by multi-component spectrophotometry provides a quick, simple, sensitive, accurate, stable and inexpensive biological indicator for the early assessment of the absorbed dose in mice.

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.

Three-wavelength method for the optical differentiation of methemoglobin and sulfhemoglobin in oxygenated blood.

Methemoglobinemia and sulfhemoglobinemia are rare, but potentially life threatening, diseases that refer to an abnormal amount of methemoglobin or sulfhemoglobin in the blood, respectively. Unfortunately, blood samples containing abnormal quantities of methemoglobin or sulfhemoglobin have similar spectral characteristics. This makes it difficult to optically differentiate them and, hence, difficult to diagnose a patient with either disease. However, performing treatments for one of the diseases without a correct diagnosis can introduce increased risk to the patient. In this paper, we propose a method for differentiating the presence of methemoglobin and sulfhemoglobin in blood, under several conditions, using reflectance values measured at three wavelengths. In order to validate our method, we perform in silico experiments considering various levels of methemoglobin and sulfhemoglobin. These experiments employ a cell-based light interaction model, known as CLBlood, which accounts for the orientation and distribution of red blood cells. We then discuss the reflectance curves produced by the experiments and evaluate the efficacy of our method. In particular, we consider various experimental conditions by modifying the flow rate, hemolysis level and incident light direction.

[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.

The effect of γ-radiation on the hemoglobin of stored red blood cells: the involvement of oxidative stress in hemoglobin conformation.

The aim of the present work was to evaluate the redox and oligomeric effects associated with the human hemoglobin of stored red blood cells that had been previously submitted to gamma radiation. Whole blood was collected from healthy donors and irradiated with 25 Gy of γ-radiation within 24 h of collection. At days 3, 5, 7, 9, 11, 14, and 28 postirradiation, fractions were removed and centrifuged, and the levels of methehemoglobin and oxyhemoglobin were measured. Hb was isolated to measure the denaturation and UV-vis spectra. The results from electrophoresis demonstrated that there was no fragmentation or cross-linking of the hemoglobin. However, ferrous center oxidation was identified as a very significant process. This mechanism is likely through an autoxidation process of the ferrous heme center, which has a maximal intensity between 5 and 7 days of storage. Interestingly, a subsequent reduction of the oxidized heme species was observed, and after 9 days of storage, the difference between the ferric species present in the control and irradiated samples was not representative. This interesting fact suggests a type of "protective action" by the blood to control the oxidative stress generated by the gamma irradiation. The UV-vis measurements demonstrated that the oxidized species was predominantly formed by hemichrome species (bis-histidine ferric heme species), which are usually associated with Heinz bodies. After 28 days of storage, evidence from the UV-vis measurements indicated that the oxidation of the irradiated sample was much higher than that observed in the control sample. These results demonstrate that despite the minimal polypeptide changes observed in the hemoglobin of stored red blood cells after gamma irradiation, the oxidation of the heme metallic center is not irrelevant and must be controlled to improve the hematological clinical procedures associated with the storage of red blood cells.

Assessment of ruminal hydrogen sulfide or urine thiosulfate as diagnostic tools for sulfur induced polioencephalomalacia in cattle.

To determine if ruminal hydrogen sulfide, urine thiosulfate, or blood sulfhemoglobin could be used as diagnostic indicators for sulfur-induced polioencephalomalacia, 16 steers (8 cannulated, 368 ± 12 kg; 8 unmodified, 388 ± 10 kg; mean ± standard error) were fed 1 of 2 dietary treatments. Diets consisted of a low sulfate (0.24% S; control) wheat midd-based pellet or the control pellet with sodium sulfate added to achieve a high-sulfate (0.68% S) pellet. As designed, intake did not differ (P = 0.80) between treatments. At 8 hr postfeeding, ruminal hydrogen sulfide was not affected by cannulation (P = 0.35) but was greater (P < 0.01) in high S (6,005 ± 475 mg/l) than control (1,639 ± 472 mg/l) steers. Time of day of sampling affected (P = 0.01) ruminal hydrogen sulfide, with peak concentrations occurring 4-12 hr after feeding. Urine was collected prefeeding (AM) and 7-9 hr postfeeding (PM). Urine thiosulfate concentrations of high S steers sampled in the PM were greater (P > 0.01) than in the AM. However, there was no difference due to time of sampling for control. In both the AM and PM, urine thiosulfate concentrations of high S were greater (P > 0.01) than control. Although hydrogen sulfide and thiosulfate were elevated by increased dietary S intake, a concentration at which polioencephalomalacia is likely to occur could not be determined. Sampling urine for thiosulfate or rumen gas for hydrogen sulfide of nonsymptomatic pen mates 4-8 hr after feeding may be useful to assess sulfur exposure and differentiate between causes of polioencephalomalacia.

Structural determinants for the formation of sulfhemeprotein complexes.

Several hemoglobins were explored by UV-Vis and resonance Raman spectroscopy to define sulfheme complex formation. Evaluation of these proteins upon the reaction with H(2)O(2) or O(2) in the presence of H(2)S suggest: (a) the formation of the sulfheme derivate requires a HisE7 residue in the heme distal site with an adequate orientation to form an active ternary complex; (b) that the ternary complex intermediate involves the HisE7, the peroxo or ferryl species, and the H(2)S molecule. This moiety precedes and triggers the sulfheme formation.

Genotoxicity testing of sulfur dioxide (SO2) in a mouse bone marrow micronucleus test complemented with hematological endpoints.

Sulfur dioxide (SO2) is a non-flammable, non-explosive, colorless gas. It is a ubiquitous environmental pollutant and an important chemical intermediate in several industrial processes. The toxicological properties of SO2, including its genotoxic potential, have been studied extensively. The majority of the available in vitro data indicate a lack of genotoxicity of SO2, while for sulfite salts some positive results have been reported. However, recent in vivo studies, using Kunming albino mice, have pointed to in vivo clastogenicity of SO2. To re-evaluate these positive findings, a bone-marrow micronucleus test according to OECD Guideline No. 474 was performed. NMRI mice (m/f) were exposed by inhalation via whole-body exposure to 0 (clean air), 2.7, 8, 27, or 80mg/m3 (0, 1, 3, 10, or 30ppm) SO2 for 4h/day on 7 consecutive days. Animals were sacrificed 24h after start of the last exposure, and blood samples (for complementing hematology) and bone marrow smears (for analysis of micronuclei) were prepared. Under the conditions used, exposure to SO2 caused no acute toxicity, mortality, or reduction in body weight. Compared with the clean-air controls, hematological parameters such as hematocrit, hemoglobin, erythrocyte/platelet/total leukocyte counts, differential white blood cell counts, and indicators of blood formation (reticulocyte counts, ratio of polychromatic to normochromatic erythrocytes in the bone marrow) remained unchanged by SO2 treatment. Unlike the previously reported studies on micronucleus formation, SO2 did not induce micronuclei in polychromatic erythrocytes of the bone marrow, whereas the positive control cyclophosphamide (60mg/kg body weight) was quite effective in this respect. Interestingly, SO2 treatment significantly enhanced malondialdehyde levels in erythrocyte lysates (TBARS method), indicating SO2-mediated oxidative stress, but also demonstrating systemic availability of the inhaled SO2. In conclusion, the present study could not reproduce the genotoxicity findings of the previously reported studies. SO2 is thus considered non-genotoxic in polychromatic erythrocytes in the bone marrow of NMRI mice under the conditions and in the concentrations used.

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.

Identification of sulfhemoglobinemia after surgical polypectomy.

Sulfhemoglobinemia (SHb) is an uncommon cause of cyanosis that is predominantly drug-induced in adults. We report an unusual case of sodium sulfate-induced sulfhemoglobinemia in a 61-year-old woman after surgical polypectomy. Fractional hemoglobin derivates were assayed by spectrophotometry and high-performance liquid chromatography. The SHb ratio was 8.6% in the first sample and 3.77% a month later measured by spectrophotometry. In the blood hemolysate, a new peak was identified as SHb with high-performance liquid chromatography (HPLC). HPLC showed the presence of 9.37% SHb in the first sample and 4.88% a month later. After removing the suspected toxic agent the cyanosis decreased significantly. The findings underline the importance of routine SHb detection in cyanosis of unknown origin especially in emergency cases.

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.

Chemiluminescence of hemoglobin and identification of related compounds with the hemoglobin chemiluminescence in plasma.

A low level of chemiluminescence by hemoglobin (Hb) was detected in the reaction with H2O2 and hydrogen donors such as gallic acid and catechins. The photon intensity was affected by the ferric state of Hb (methemoglobin > oxyhemoglobin), and was roughly correlated with the radical-scavenging potential of catechins. We hypothesized the reversible activation reaction of Hb as the chemiluminescence mechanism of the H2O2/gallic acid/Hb system. It is indicated that the oxidized-Hb (Hb-OOH) formation was a chemiluminescence-rate-determining step and one-electron reduction by a hydrogen donor of the compound-I-type intermediate ([.XFeIV] = O) proved a chemiluminescence-specificity-determining step. Spectral analysis showed that the photon emission from the H2O2/gallic acid/Hb system was produced without singlet oxygen generation. The concentration dependence of photon intensity suggests a high consumption ratio of H2O2 leading to protection from H2O2 toxicity. Albumin was defined as a hydrogen donor by the isolation of chemiluminescent substance in plasma using this chemiluminescence system.

Sulphaemoglobin formation in fish: a comparison between the haemoglobin of the sulphide-sensitive rainbow trout (Oncorhynchus Mykiss) and of the sulphide-tolerant common carp (Cyprinus Carpio).

A method for the quantitative determination of sulphaemoglobin (SHb) in a mixture of haemoglobin derivatives by spectral deconvolution is described. SHb formation was studied in haemolysates and in red blood cells of the sulphide-sensitive rainbow trout (Oncorhynchus mykiss) and of the sulphide-tolerant common carp (Cyprinus carpio). Addition of sulphide caused the formation of SHb in haemolysates of both animals. However, haemoglobin from common carp was much less sensitive to sulphide than was trout haemoglobin. The maximal obtainable SHb fraction was approximately 30 % in trout and 10 % in carp haemolysates. In both animals, the SHb fraction increased with increasing Hb and sulphide concentrations up to 100 micromol l(-)(1) and 1 mmol l(-)(1), respectively, and was favoured by a low pH. An increase of temperature between 5 and 25 degrees C strongly increased SHb formation in trout haemolysate. In contrast, temperature changes had almost no effect on SHb production in carp. Within trout red blood cells, approximately 7 % of total haemoglobin was converted to SHb during 60 min of incubation (with 2.5 mmol l(-)(1) sulphide), inducing a 20 % loss of haemoglobin oxygen-saturation. In carp red blood cells incubated under identical conditions, SHb formation was minimal and haemoglobin oxygen-saturation was not affected.