Modulation of the Human Erythrocyte Antioxidant System by the 5- and 6-Membered Heterocycle-Based Nitroxides.

Nitroxides are stable radicals consisting of a nitroxyl group, >N-O•, which carries an unpaired electron. This group is responsible for the paramagnetic and antioxidant properties of these compounds. A recent study evaluated the effects of pyrrolidine and pyrroline derivatives of nitroxides on the antioxidant system of human red blood cells (RBCs). It showed that nitroxides caused an increase in the activity of superoxide dismutase (SOD) and the level of methemoglobin (MetHb) in cells (in pyrroline derivatives) but had no effect on the activity of catalase and lactate dehydrogenase. Nitroxides also reduced the concentration of ascorbic acid (AA) in cells but did not cause any oxidation of proteins or lipids. Interestingly, nitroxides initiated an increase in thiols in the plasma membranes and hemolysate. However, the study also revealed that nitroxides may have pro-oxidant properties. The drop in the AA concentration and the increase in the MetHb level and in SOD activity may indicate the pro-oxidant properties of nitroxides in red blood cells.

Membrane-Bound Ferric Hemoglobin in Nucleated Erythrocytes of the Black Scorpionfish Scorpaena porcus, Linnaeus 1758.

The content of membrane-bound methemoglobin (MtHb) in nucleated erythrocytes was studied in the black scorpionfish Scorpaena porcus (Linnaeus, 1758) in vitro. Spectral characteristics were determined for a whole hemolysate, a hemolysate obtained by stroma precipitation (a clarified hemolysate), and a resuspended stroma. The MtHb proportion in the erythrocyte stroma was found to exceed 80% (6.20 ± 0.59 µM). Clarified hemolysates were nearly free of MtHb (0.5 ± 0.2 µM). Membrane-bound ferric hemoglobin did not affect the erythrocyte resistance to osmotic shock. The osmotic fragility range was determined using a LaSca-TM laser microparticle analyzer (BioMedSystems, Russia) to be 102-136 mOsm/kg, much the same as in other bony fish species. A nitrite load (10 mg/L) significantly increased the MtHb content in the blood. However, the membrane-bound ferric hemoglobin content did not change significantly, amounting to 6.34 ± 1.09 µM (approximately 95%). The finding suggested a functional importance for MtHb present in the plasma membrane of nucleated erythrocytes. Membrane-bound MtHb was assumed to neutralize the external oxidative load and the toxic effect of hydrogen sulfide in bottom water layers, where the species lives.

Methemoglobin-albumin clusters for cyanide detoxification.

Sodium nitrite (NaNO2) is a universal antidote for patients with cyanide poisoning. However, its use has serious drawbacks in terms of efficacy and safety. Herein, we present a promising antidote: methemoglobin (metHb)-albumin clusters. The metHb-albumin cluster is made by a metHb core wrapped by covalently bound human serum albumin. Spectral analyses proved that the metHb-albumin clusters possessed cyanide-binding properties similar to those of naked metHb. In vitro cell experiments showed that metHb-albumin clusters prevented the cyanide-induced inhibition of cytochrome c oxidase activity, resulting in a strong cytoprotective effect. In mice subjected to cyanide poisoning, metHb-albumin clusters reduced mortality and alleviated metabolic acidosis, while maintaining the activity of cytochrome c oxidase in organs; their efficacy was better than that of NaNO2. Furthermore, the oxygen carrying capacity was maintained in poisoned mice treated with metHb-albumin clusters and was low in those treated with NaNO2. These results indicate that metHb-albumin clusters could be a more effective and safer antidote against cyanide poisoning than NaNO2.

Elucidating the binding properties of methemoglobin in red blood cell to cyanide, hydrosulfide, and azide ions using artificial red blood cell.

Methemoglobin (metHb), the oxidized form of hemoglobin, lacks the ability of reversible oxygen binding; however, it has a high binding affinity to toxic substances such as cyanide, hydrosulfide, and azide. This innate property of metHb offers the clinical option to treat patients poisoned with these toxins, by oxidizing the endogenous hemoglobin in the red blood cells (RBCs). The binding properties of naked metHb (isolated from RBC) with these toxins has been studied; however, the binding behaviors of metHb under the intracellular conditions of RBC are unclear because of the difficulty in detecting metHb status changes in RBC. This study aimed to elucidate the binding properties of metHb in RBC under physiological and poisoned conditions using artificial RBC, which was hemoglobin encapsulated in a liposome. The mimic-circumstances of metHb in RBC (metHb-V) was prepared by oxidizing the hemoglobin in artificial RBC. Spectroscopic analysis indicated that the metHb in metHb-V exhibited a binding behavior different from that of naked metHb, depending on the toxic substance: When the pH decreased, (i) the cyanide binding affinity of metHb-V remained unchanged, but that of naked metHb decreased (ii) the hydrosulfide binding affinity was increased in metHb-V but was decreased in naked metHb. (iii) Azide binding was increased in metHb-V, which was similar to that in naked metHb, irrespective of the pH change. Thus, the binding behavior of intracellular metHb in the RBC with cyanide, hydrosulfide, and azide under physiological and pathological conditions were partly elucidated using the oxidized artificial RBC.

Nitrite decreases sickle hemoglobin polymerization in vitro independently of methemoglobin formation.

The root cause of sickle cell disease (SCD) is the polymerization of sickle hemoglobin (HbS) leading to sickling of red blood cells (RBC). Earlier studies showed that in patients with SCD, high-dose nitrite inhibited sickling, an effect originally attributed to HbS oxidation to methemoglobin-S even though the anti-sickling effect did not correlate with methemoglobin-S levels. Here, we examined the effects of nitrite on HbS polymerization and on methemoglobin formation in a SCD mouse model. In vitro, at concentrations higher than physiologic (>1 µM), nitrite increased the delay time for polymerization of deoxygenated HbS independently of methemoglobin-S formation, which only occurred at much higher concentrations (>300 µM). In vitro, higher nitrite concentrations oxidized 100% of normal hemoglobin A (HbA), but only 70% of HbS. Dimethyl adipimidate, an anti-polymerization agent, increased the fraction of HbS oxidized by nitrite to 82%, suggesting that polymerized HbS partially contributed to the oxidation-resistant fraction of HbS. At low concentrations (10 µM-1 mM), nitrite did not increase the formation of reactive oxygen species but at high concentrations (10 mM) it decreased sickle RBC viability. In SCD mice, 4-week administration of nitrite yielded no significant changes in methemoglobin or nitrite levels in plasma and RBC, however, it further increased leukocytosis. Overall, these data suggest that nitrite at supra-physiologic concentrations has anti-polymerization properties in vitro and that leukocytosis is a potential nitrite toxicity in vivo. Therefore, to determine whether the anti-polymerization effect of nitrite observed in vitro underlies the decreases in sickling observed in patients with SCD, administration of higher nitrite doses is required.

Methemoglobinemia in Hemodialysis Patients due to Acute Chlorine Intoxication: A Case Series Calling Attention on an Old Problem.

INTRODUCTION: Hemodialysis uses municipal water that must be strictly purified and sterilized to be used for that procedure. Large amounts of decontaminants are often used, such as chlorine, and if these compounds are not subsequently removed they can be transferred to the blood of patients causing complications including methemoglobinemia. METHODS: In this case series study, dialysis patients in one unit were evaluated. We reviewed clinical characteristics and laboratory findings obtained on the day when the water supply was disinfected with chlorine, with the aim to quantify methemoglobin concentrations. Our objective was to characterize the clinical presentation and management of patients who presented with methemoglobinemia on a specific index day. We also reviewed reported cases in the literature regarding this underreported complication. RESULTS: Eight patients who presented with chlorine intoxication were evaluated. The methemoglobin concentrations were between 1.3% and 7.9% (reference value 0-1%). We believe this to be caused by water containing 0.78 mg/L of total chlorine. Seven patients presented with cyanosis, 4 with dizziness, 6 with dark brown blood, 4 with dyspnea, and 4 with headache and hemolytic anemia. Subjects were treated with supplemental oxygen, methylene blue, intravenous vitamin C, blood transfusions, and increased doses of erythropoietin. No patient died, and all continued with their usual hemodialysis sessions. CONCLUSION: Acute chlorine intoxication transferred by the water used during hemodialysis sessions can present with methemoglobinemia accompanied by cyanosis, oxygen desaturation, and hemolytic anemia. Chlorine levels should be carefully monitored in the water used for hemodialysis treatment.

Exploring the Anti-Hypoxaemia Effect of Hydromethylthionine: A Prospective Study of Phase 3 Clinical Trial Participants.

Methylthioninium chloride (MTC) is a standard treatment for methaemoglobinaemia. A preparation of reduced MTC has been reported to increase blood oxygen saturation (SpO2) and lower respiratory rates in patients with severe COVID-19. We have developed a stable form of reduced methylthionine (hydromethylthionine-mesylate, HMTM) having a benign safety profile in two Phase 3 trials in Alzheimer's disease. The aim of this prospective study was to determine the effects of oral HMTM on SpO2 and methaemoglobin (metHb) levels in a cohort of patients with mild hypoxaemia not due to COVID-19. Eighteen participants randomised to a single dose of 4, 75, 100 or 125 mg doses of HMTM had SpO2 levels below 94% at baseline. Patients were routinely monitored by pulse oximetry after 4 h, and after 2 and 6 weeks of twice daily dosing. Significant ~3% increases in SpO2 occurred within 4 h and were sustained over 2 and 6 weeks with no dose differences. There were small dose-dependent increases (0.060-0.162%) in metHb levels over 2 to 6 weeks. Minimum-energy computational chemistry revealed that HMT can bind within 2.10 Å of heme iron by donating a pair of electrons from the central nitrogen of HMT to d orbitals of heme iron, but with lower affinity than oxygen. In conclusion, HMTM can increase SpO2 without reducing metHb by acting as a strong displaceable field ligand for heme iron. We hypothesise that this facilitates a transition from the low oxygen affinity T-state of heme to the higher affinity R-state. HMTM has potential as an adjunctive treatment for hypoxaemia.

Hemoglobin Derivatives in Beef Irradiated with Accelerated Electrons.

The efficiency of food irradiation depends on the accuracy of the irradiation dose range that is sufficient for inhibiting microbiological growth without causing an irreversible change to the physical and chemical properties of foods. This study suggests that the concentration of hemoglobin derivatives can be used as a criterion for establishing the limit for chilled beef irradiation at which irradiation-induced oxidation becomes irreversible. The express spectrophotometry method for estimating the hemoglobin derivative concentration shows a nonlinear increase in methemoglobin concentration from 15% to 50% in beef irradiated by accelerated electrons with the doses ranging from 250 Gy to 10,000 Gy. The monitoring of the hemoglobin derivative concentration for three days after irradiation shows nonmonotonous dependencies of methemoglobin concentration in beef in the storage time since the oxidation of hemoglobin occur as a result of irradiation and biochemical processes in beef during storage. The proposed method based on the quantitative analysis of the hemoglobin derivative concentration can be used to estimate the oxidation level for irradiation of foods containing red blood cells. The study proposes a model that describes the change in hemoglobin derivative concentration in beef after irradiation considering that oxidation of hemoglobin can be triggered by the direct ionization caused by accelerated electrons, biochemical processes as a result of bacterial activity, and reactive oxygen species appearing during irradiation and storage. This research throws light on the mechanisms behind food irradiation during storage that should be taken into account for selecting the optimal parameters of irradiation.

Can We Estimate Late-Onset Sepsis by Serial Methemoglobin Levels? An Observational Study in Preterm Neonates.

Objective: To assess serial methemoglobin (MetHb) levels in preterm infants as a possible diagnostic method for late-onset sepsis (LOS). Methods: Preterm infants were assigned into two groups: those with culture-proven LOS and controls. Serial MetHb levels were measured. Results: The MetHb values of the LOS group were found to be significantly increased (p < 0.001). The cutoff value for the detection of LOS was calculated as MetHb > 1.75%, optimized for a sensitivity of 81.9% and specificity of 90%. After antimicrobial therapy, MetHb values were found to decrease significantly (p < 0.001). MetHb had an AUC of 0.810 for mortality using the calculated cutoff of >2% (p < 0.005). Conclusions: MetHb levels increase at the onset of LOS and decrease following treatment. MetHb can be added to other sepsis biomarkers as a rapid infectious process indicator for preterm neonates. MetHb > 2% is associated with LOS mortality.

Hereditary Congenital Methemoglobinemia Diagnosed at the Age of 79 Years: A Case Report.

Background: Cardiopulmonary disorders are the most common cause of central cyanosis, and methemoglobinemia is often overlooked in the differential diagnosis of patients with central cyanosis. In most cases, methemoglobinemia is acquired and hereditary congenital methemoglobinemia is rare. Only a few case reports of congenital methemoglobinemia can be found in PubMed. To date, only four cases of congenital methemoglobinemia diagnosed after the age of 50 years have been reported. Case Presentation: A 79-year-old Japanese woman presented at our hospital with the chief complaints of dyspnea and cyanosis. She exhibited cyanosis of the lips and extremities, and her SpO2 was 80%, with oxygen administration at 5 L/min. Blood gas analysis revealed a PaO2 of 325.4 mmHg and methemoglobin level of 36.9%. The SpO2 and PaO2 values were dissociated, and methemoglobin levels were markedly elevated. Genetic analysis revealed a nonsynonymous variant in the gene encoding nicotinamide adenine dinucleotide cytochrome (NADH) B5 reductase 3 (CYB5R3), and the patient was diagnosed with congenital methemoglobinemia. Conclusions: It is important to consider methemoglobinemia in the differential diagnosis of patients with central cyanosis. At 79 years of age, our patient represents the oldest patient with this diagnosis. This report indicates that it is crucial to consider the possibility of methemoglobinemia regardless of the patient's age.

Liposomal methemoglobin as a potent antidote for hydrogen sulfide poisoning.

Hydrogen sulfide (H2S) induces acute and lethal toxicity at high concentrations. However, no specific antidotes for H2S poisoning have been approved. Liposomal methemoglobin (metHb@Lipo) was developed as an antidote for cyanide poisoning. As the toxic mechanism of H2S poisoning is the same as that of cyanide poisoning, metHb@Lipo could potentially be used as an antidote for H2S poisoning. In this study, we evaluated the antidotal efficacy of metHb@Lipo against H2S poisoning. Stopped-flow rapid-scan spectrophotometry clearly showed that metHb@Lipo scavenged H2S rapidly. Additionally, metHb@Lipo showed cytoprotective effects against H2S exposure in H9c2 cells by maintaining mitochondrial function. MetHb@Lipo treatment also improved the survival rate after H2S exposure in vivo, with the maintenance of cytochrome c oxidase activity and suppression of metabolic acidosis. Moreover, metHb@Lipo therapy maintained significant antidotal efficacy even after 1-year-storage at 4-37 °C. In conclusion, metHb@Lipo is a candidate antidote for H2S poisoning.

Understanding the EKG changes in methemoglobinemia.

Methemoglobin is a form of hemoglobin that has been oxidized, changing its heme iron configuration from the ferrous to the ferric state. Unlike normal hemoglobin, methemoglobin does not bind oxygen and as a result, cannot deliver oxygen to the tissues. At the presentation in the emergency department, an electrocardiogram (EKG) is usually performed as a reflex for patients admitted for shortness of breath to rule out acute coronary syndrome. Very limited data is available on EKG abnormalities in patients with methemoglobinemia. In this study, we retrospectively analyzed the pattern of EKG changes in patients with methemoglobinemia.

Nitric oxide donor 8-bromo-cGMP suppresses cholesterol depleted RBCs band 3 dysfunction.

Red blood cells (RBCs) carry large cholesterol fractions and imbalance in them leads to several vascular complications. RBCs band 3 protein plays an important role in maintaining membrane integrity and there are many reports on cholesterol and band 3 protein interaction. Yet, RBCs band 3 protein role in regulating cholesterol homeostasis needs to be investigated. In this study, we induced cholesterol-depletion and band 3 inhibition in RBCs; both of which cause stress by decreasing band 3 channel activity with an increase in RBCs adhesion to endothelial cells (EC) by elevating band 3 phosphorylation (Tyr21), methemoglobin level and decreasing nitric oxide level. We hypothesized that nitric oxide (NO), a prominent determinant for RBC structural stability, would protect RBCs from stressors. To estimate this, we used three NO donors (SpNO, Sildenafil citrate and 8-Bromo-cGMP) and found that all 3 NO donors were able to recover, with 8-Bromo-cGMP being the most effective as it not only increased band 3 channel activity but also decreased RBC-EC adhesiveness and methemoglobin level in both stressors. Whereas NO donor's treatment did not display an ameliorative impact when both stresses were combined. Overall, these findings may shed light on the role of 8-bromo-cGMP in regulating RBC cholesterol homeostasis by maintaining band 3 function. Further studies in this direction might help identify targets for the therapeutic use of NO donors in the treatment of blood disorders.

Near-fatal pediatric methemoglobinemia secondary to intentional sodium nitrite ingestion.

Methemoglobinemia is the result of inappropriate oxidation of hemoglobin iron groups, leading to a failure of oxygen transport and delivery, resulting in a clinical state of refractory hypoxia. Methemoglobin levels above 70% are often considered fatal. Acquired methemoglobinemia can be caused by a variety of substances, including sodium nitrite, a commercially available food preservative and color fixative. This report describes a patient presenting with a methemoglobin level of 83% secondary to intentional sodium nitrite ingestion. The methemoglobin level recorded is amongst some of the highest found in surviving patients.

Pharmacokinetic Profile, Safety, and Tolerability of Topical Berdazimer Gel, 10.3% in Patients With Molluscum Contagiosum.

BACKGROUND: Berdazimer (SB206), gel 10.3% is a novel, topical, nitric oxide–releasing agent intended for molluscum contagiosum (MC) treatment. METHODS: A 12-week, open-label, multicenter trial evaluated the safety, tolerability, and pharmacokinetic (PK) parameters of berdazimer gel, 10.3% applied topically once daily for the treatment of MC. Patients were aged ≥6 months with >20 molluscum lesions. The primary endpoint was the PK profile of the hydrolyzed N-methylaminopropyl-trimethoxysilane (hMAP3) monomer and nitrate during a 2-week period of once-daily berdazimer gel, 10.3% application (PK period) under maximal use conditions. Safety and tolerability were evaluated throughout the 12-week study period. RESULTS: Half of the 34 enrolled patients (17) were female and most (97.1% [33/34]) were white. Patients were 2 to 12 years old (mean, 5.3 years) with a mean of 50 MC lesions at baseline (mean time since MC awareness, 12.4 months). No patients had quantifiable plasma hMAP3 concentrations on day 1. On day 15, 2 patients had quantifiable plasma hMAP3 concentrations; however, the maximum concentration (33.9 ng/mL) was >10-fold lower than the no observed adverse effect level (NOAEL) in an animal toxicology study. Mean nitrate concentration–time profiles were similar on days 1 and 15 and remained flat for all patients throughout the 2-week PK period. The highest plasma methemoglobin level observed was 3.2%. Application-site pain (13/34 [38.2%]) and application-site erythema (6/34 [17.6%]) were the most frequent treatment-emergent adverse events (TEAEs), and most TEAEs were mild or moderate. CONCLUSIONS: Once-daily berdazimer gel, 10.3% was well-tolerated with minimal systemic absorption. J Drugs Dermatol. 2022;21(10):1104-1110. doi:10.36849/JDD.6938.

Multi-analyte calibration and verification of a multi-parameter laser-based pulse oximeter.

Almost since its introduction pulse oximetry was known to overestimate oxygen saturation in cases of carbon monoxide poisoning or elevated methemoglobin (metHb) levels. To eliminate this dangerous behavior some manufacturers have added additional LED emitters to try to increase the number of measured hemoglobin species and to improve measurement accuracy, but have not been very successful. We hypothesized that the use of narrow-band laser light sources would make accurate and precise measurement of the four primary species of hemoglobin possible, even in cases of elevated levels of carboxyhemoglobin (COHb). Calibration and verification studies were performed on a tissue simulator that employed an artificial finger pulsating with whole human blood. This simulator allowed safe generation of 165 different combinations of the levels of oxyhemoglobin (O2Hb), COHb, metHb, and reduced hemoglobin (RHb) for calibration of the laser-based pulse oximeter. A follow-on study used 56 mixed hemoglobin levels for verification and statistical analysis of the performance of this device. This laser-based pulse oximeter measured all four species of hemoglobin accurately and precisely (ARMS ≤ 1.8%) for metHb levels in the clinically normal range. At elevated metHb levels the device continued to provide accurate and precise measurements of metHb and RHb (ARMS ≤ 1.7%). The use of monochromatic laser light sources can create a new generation of highly accurate, multi-parameter, pulse oximeters.

Alpha-Lipoic Acid and Its Enantiomers Prevent Methemoglobin Formation and DNA Damage Induced by Dapsone Hydroxylamine: Molecular Mechanism and Antioxidant Action.

Dapsone (DDS) therapy can frequently lead to hematological side effects, such as methemoglobinemia and DNA damage. In this study, we aim to evaluate the protective effect of racemic alpha lipoic acid (ALA) and its enantiomers on methemoglobin induction. The pre- and post-treatment of erythrocytes with ALA, ALA isomers, or MB (methylene blue), and treatment with DDS-NOH (apsone hydroxylamine) was performed to assess the protective and inhibiting effect on methemoglobin (MetHb) formation. Methemoglobin percentage and DNA damage caused by dapsone and its metabolites were also determined by the comet assay. We also evaluated oxidative parameters such as SOD, GSH, TEAC (Trolox equivalent antioxidant capacity) and MDA (malondialdehyde). In pretreatment, ALA showed the best protector effect in 2.5 µg/mL of DDS-NOH. ALA (1000 µM) was able to inhibit the induced MetHb formation even at the highest concentrations of DDS-NOH. All ALA tested concentrations (100 and 1000 µM) were able to inhibit ROS and CAT activity, and induced increases in GSH production. ALA also showed an effect on DNA damage induced by DDS-NOH (2.5 µg/mL). Both isomers were able to inhibit MetHb formation and the S-ALA was able to elevate GSH levels by stimulating the production of this antioxidant. In post-treatment with the R-ALA, this enantiomer inhibited MetHb formation and increased GSH levels. The pretreatment with R-ALA or S-ALA prevented the increase in SOD and decrease in TEAC, while R-ALA decreased the levels of MDA; and this pretreatment with R-ALA or S-ALA showed the effect of ALA enantiomers on DNA damage. These data show that ALA can be used in future therapies in patients who use dapsone chronically, including leprosy patients.

Antioxidant Activity of Quercetin in a H2O2-Induced Oxidative Stress Model in Red Blood Cells: Functional Role of Band 3 Protein.

During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 µM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42- uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 µM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology.

Cellular composition of the black scorpionfish (Scorpaena porcus, L 1758) blood and head kidney under short-time acute exposure to hypoxia.

In the present work, we studied the effect of short-term acute hypoxia on the cellular composition of the blood and the head kidney of the black scorpionfish. Dissolved oxygen concentration was decreased from 8.5-8.7 mg O2 l-1 (normoxia) to 3-5 mg O2 l-1 (relative normoxia), 1-3 mg O2 l-1 (moderate hypoxia), and 0-1 mg O2 l-1 (acute hypoxia) within 1.5-2 h by bubbling of water with N2. Exposure period was 4 h, water temperature was adjusted to 14-16 °C, and photoperiod was 12 h (light). Short-time acute hypoxia induced a rapid release of blast and immature cells from the head kidney into the circulating blood of the black scorpionfish, which was associated with reduction in erythropoietic reserves in 2.5 times. The number of immature erythroid cells (pronormoblasts, basophilic and polychromatophilic normoblasts) significantly increased in blood, and the simultaneously relative decrease of the number of abnormal red blood cell (RBC) and the increase of the number of RBC ghosts (lysed RBCs) in circulating blood were observed. The significant correlation between methemoglobin concentration and the number of RBC ghosts was shown (R2 = 0.640 or r = 0.800). Hypoxia induced RBC swelling on 5-6% compared to control. The number of RBC ghosts in the blood is likely involved in the stimulation of erythropoietin production under hypoxia.

[A Case of Methemoglobinemia Caused by Toluidine Revelation with Dyspnea and Cyanosis].

Toluidine is a known cause of bladder cancer, but it is less widely recognized as a cause of methemoglobinemia because methemoglobinemia is rare. We herein report a case of methemoglobinemia caused by toluidine in a 50-year-old man. A solution of toluidine overflowed from its container during transportation and adhered to the man's clothes, but he drove to his workplace 100 km away without changing his clothes or undergoing decontamination. Before arriving at his workplace, he developed dyspnea and called emergency services, and he was then transported to a local hospital. He had significant cyanosis upon arrival, and arterial blood gas analysis revealed a high methemoglobin level of 44%. He was diagnosed with toluidine-induced methemoglobinemia and was transported to our hospital, where he was admitted to the intensive care unit. Treatment for methemoglobinemia was started immediately after hospitalization, and the patient's symptoms and methemoglobin level improved. Methemoglobinemia should be considered in workers who handle toluidine and develop cyanosis and dyspnea.