Methemoglobin Formation Research Articles

시안화이온의 pentafluorobenzyl bromide (PFBBr)에 의한 유도체화 후 GC/MS 분석에 의한 차량화재 변사체 사인규명에 관한 사례연구

시안화수소는 ABS (acrylonitrile-butadiene-styrene) 등의 플라스틱류 화재시에 발생될 수 있는 유독성 기체이기에 시안화수소가 함유된 연기를 흡입하였을 경우 변사자의 혈액에서도 검출될 수 있다. 따라서 열기로 혈액 등의 생체시료의 손상정도가 강하여 일산화탄소-헤모글로빈 농도의 측정이 어려운 경우 사인규명을 위한 지표로 활용될 수 있으리라 사료된다. 본 연구의 사례는 유서와 함께 화재가 발생한 차량내에서 발견된 변사체의 사인규명과 관련이 있다. 화재사의 경우 일산화탄소-헤모글로빈 농도가 결정적인 정보를 제공하지만 열기로 심하게 손상되어 이에 대한 측정이 어려워 시안화이온을 분석하고자 하였다. 시안화이온을 양이온계면활성제와 함께 pentafluorobenzyl bromide로 유도체화 시킨 다음 기체크로마토그래피/질량분석법으로 scan mode와 SIM mode로 분석하였다. 변사체 심혈과 뇌혈에서 시안화이온이 정상 흡연자 14인의 평균값(0.041 µg/mL)보다 높은 0.36 µg/mL 및 1.20 µg/mL 확인되었다. Hydrogen cyanide (HCN) is an extremely toxic gas frequently produced during the incineration of plastics, such as acrylonitrile-butadiene-styrene (ABS). A victim of a fire who has inhaled smoke could have cyanide in the blood. Therefore, cyanide could be a good marker for a post-mortem examination of a fire as well as carboxyhemoglobine (COHb) test of blood samples. For a particular fire case, a burned body with a suicide note was found inside a burned vehicle. Even though the COHb value is conclusive evidence, measuring the COHb for denatured blood might be difficult due to severe thermal denaturation or the formation of methemoglobin (MetHb). To overcome this difficulty, cyanide could be used as an indicator when investigating the death of a fire victim. In this study, gas chromatography/mass spectrometry (GC/MS) was adopted to measure the levels of cyanide in the blood through derivatization with pentafluorobenzyl bromide (PFBBr) under cation surfactant by scan and SIM mode. The concentration of cyanide in the blood of heart blood and brain of the victim was found to be 0.36 µg/mL and 1.20 µg/mL respectively, which was higher than the average value (0.041 µg/mL) found in the blood of 14 people who smoked.

The effect of environmental hypercapnia and size on nitrite toxicity in the striped catfish (Pangasianodon hypophthalmus)

Striped catfish (Pangasianodon hypophthalmus) are farmed intensively at high stocking densities in Vietnam where they are likely to encounter environmental hypercapnia as well as occasional high levels of aquatic nitrite. Nitrite competes with Cl− for uptake at the branchial HCO3−/Cl− exchanger, causing a drastic reduction in the blood oxygen carrying capacity through the formation of methaemoglobin and nitrosylhaemoglobin. Environmental hypercapnia induces a respiratory acidosis where the branchial HCO3−/Cl− exchange activity is reduced in order to retain HCO3− for pH recovery, which should lead to a reduced nitrite uptake. To assess the effect of hypercapnia on nitrite uptake, fish were cannulated in the dorsal aorta, allowing repeated blood sampling for measurements of haemoglobin derivatives, plasma ions and acid-base status during exposure to 0.9mM nitrite alone and in combination with acute and 48h acclimated hypercapnia over a period of 72h. Nitrite uptake was initially reduced during the hypercapnia-induced acidosis, but after pH recovery the situation was reversed, resulting in higher plasma nitrite concentrations and lower functional haemoglobin levels that eventually caused mortality. This suggests that branchial HCO3−/Cl− exchange activity is reduced only during the initial acid-base compensation, but subsequently increases with the greater availability of internal HCO3− counter-ions as pH is compensated. The data further suggest that branchial Na+/H+ exchange plays a significant role in the initial phase of acid-base compensation. Overall, longer term environmental hypercapnia does not protect against nitrite uptake in P. hypophthalmus, but instead enhances it. In addition, we observed a significant size effect in nitrite accumulation, where large fish attained plasma [nitrite] above the ambient concentration, while small fish did not. Small P. hypophthalmus instead had significantly higher plasma [nitrate], and haemoglobin concentrations, revealing greater capacity for detoxifying nitrite by oxidising it to nitrate.

Protective effects of resveratrol against hypoxanthine-xanthine oxidase-induced toxicity on human erythrocytes

An in vitro model of erythrocyte damage induced by hypoxanthine-xanthine oxidase (HX-XOD) was developed. The protective effects of resveratrol, a main component of red wine, were evaluated using this model. The results showed that resveratrol treatments maintained erythrocyte morphological and structural integrity, inhibited the formation of methaemoglobin, and suppressed the release of lactate dehydrogenase. Further investigation found that resveratrol attenuated the HX-XOD-induced reactive oxygen species generation, preserved erythrocyte antioxidant enzymes (SOD, CAT and GSH-Px) activities and increased GSH level. Also, resveratrol regulated the pyruvate kinase activity and pyruvate concentration, preserving the normal functioning of erythrocyte membrane ATPases (Na+/K+-ATPase and Ca2+/Mg2+-ATPase). This study established an ideal cellular model for the research of erythrocyte antioxidant system, and revealed the protective mechanisms of resveratrol on human erythrocyte. The results of this study provided novel evidences for further researches on the utilization of resveratrol as a protector on human erythrocyte.

Quercetin Potentiates Toxicity Of Hemolytic Drugs In G6PD Deficient Human Erythrocytes

Quercetin, a flavonoid, is widely distributed in plants and a prominent constituent of dietary supplements. Quercetin-based supplements are recommended for prevention of cancer, improvement of cardiovascular functions and mental health. However, these effects have not been validated clinically. Except for limited interactions with some drugs (fluoroquinolone antibiotics and taxol/paclitaxel), there is little evidence of safety concerns, and with oral use, quercetin-containing preparations have been generally well tolerated. Quercetin inhibits NRH: quinone oxidoreductase 2 (NQO2), an enzyme involved in quinone metabolism and detoxification. Individuals with genetic deficiency of glucose 6-phosphate dehydrogenase (G6PD) are highly susceptible to hemolytic toxicity of certain oxidant drugs. Treatment of G6PD deficient human erythrocytes in vitro with quercetin caused significant increase in methemoglobin formation and selective depletion of reduced glutathione (GSH) levels. Further, when tested in combination with 5, 6-orthoquinone PQ [6] and dapsone hydroxylamine [3], the hemotoxic metabolites of primaquine [4] and dapsone [2] respectively, caused significant potentiation of their hemotoxic effect as determined through an increase in methemoglobin formation and depletion of GSH. Quercetin also augmented the hemotoxic effect of 2-amino-4-chlorophenol in vitro. The results warrant further investigation of quercetin regarding its toxic interaction with drugs clinically known to cause hemolytic toxicity.

Hemolytic and antimalarial effects of tight-binding glyoxalase 1 inhibitors on the host-parasite unit of erythrocytes infected with Plasmodium falciparum

Glyoxalases prevent the formation of advanced glycation end products by converting glycolysis-derived methylglyoxal to d-lactate with the help of glutathione. Vander Jagt and colleagues previously showed that erythrocytes release about thirty times more d-lactate after infection with the human malaria parasite Plasmodium falciparum. Functional glyoxalases in the host-parasite unit might therefore be crucial for parasite survival. Here, we determined the antimalarial and hemolytic activity of two tight-binding glyoxalase inhibitors using infected and uninfected erythrocytes. In addition, we synthesized and analyzed a set of diester derivates of both tight-binding inhibitors resulting in up to threefold lower IC50 values and an altered methemoglobin formation and hemolytic activity depending on the type of ester. Inhibitor treatments of uninfected erythrocytes revealed an extremely slow inactivation of the host cell glyoxalase, irrespective of inhibitor modifications, and a potential dispensability of the host cell enzyme for parasite survival. Our study highlights the benefits and drawbacks of different esterifications of glutathione-derived inhibitors and demonstrates the suitability of glyoxalase inhibitors as a tool for deciphering the relevance and mode of action of different glyoxalase systems in a host-parasite unit.

Nitric oxide donation by the binuclear tetranitrosyl iron complexes in the presence of erythrocytes

The kinetic regularities of nitric oxide donation in the presence of erythrocytes by representatives of a new class of synthetic nitric oxide donors, binuclear tetranitrosyl iron complexes (B-TNIC) [Fe2(SR)2(NO)4] with thiol-containing ligands, where R is pyrimidin-2-yl, 1-methylimidazol-2-yl, benzothiazol-2-yl, and penicillamine residue, were established. The NO-donating ability of B-TNIC was estimated from the apparent rate constants of the first order for the formation of intraerythrocyte methemoglobin with the variation of the initial concentration of the complex. During the standard experimental time (15—17 min), three of the four complexes released in the solution no more than a quarter of available NO groups. Their NO-donating ability turned out to be variable increasing with an increase in the initial concentration of the complex. At the same time, the complex bearing penicillamine residues as ligands donated almost all NO groups within approximately 1 min. Features of NO donation by the B-TNIC in the presence of erythrocytes are related to the formation in the system of an additional equilibrium pool of the membrane-associated complex, which is characterized by a reduced rate of hydrolytic dissociation with NO releasing to the solution. The NO-donating ability of the B-TNIC in the presence of erythrocytes is determined by the ratio of volumes of the free and membrane-associated pools of the complex.

Methemoglobin Formation and Characterization of Hemoglobin Adducts of Carcinogenic Aromatic Amines and Heterocyclic Aromatic Amines.

Arylamines (AAs) and heterocyclic aromatic amines (HAAs) are structurally related carcinogens formed during the combustion of tobacco or cooking of meat. They undergo cytochrome P450 mediated N-hydroxylation to form metabolites which bind to DNA and lead to mutations. The N-hydroxylated metabolites of many AAs also can undergo a co-oxidation reaction with oxy-hemolgobin (HbO2) to form methemoglobin (met-Hb) and the arylnitroso intermediates, which react with the β-Cys(93) chain of Hb to form Hb-arylsulfinamide adducts. The biochemistry of arylamine metabolism has been exploited to biomonitor certain AAs through their Hb arylsulfinamide adducts in humans. We examined the reactivity of HbO2 with the N-hydroxylated metabolites of 4-aminobiphenyl (ABP, HONH-ABP), aniline (ANL, HONH-ANL), and the HAAs 2-amino-9H-pyrido[2,3-b]indole (AαC, HONH-AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, HONH-PhIP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx, HONH-MeIQx). HONH-ABP, HO-ANL, and HONH-AαC induced methemoglobinemia and formed Hb sulfinamide adducts. However, HONH-MeIQx and HONH-PhIP did not react with the oxy-heme complex, and met-Hb formation and chemical modification of the β-Cys(93) residue were negligible. Molecular modeling studies showed that the distances between the H-ON-AA or H-ON-HAA substrates and the oxy-heme complex of HbO2 were too far away to induce methemoglobinemia. Different conformational changes in flexible helical and loop regions around the heme pocket induced by the H-ON-AA or H-ON-HAAs may explain the different proclivities of these chemicals to induce methemoglobinemia. Hb-Cys(93β) sulfinamide and sulfonamide adducts of ABP, ANL, and AαC were identified, by Orbitrap MS, following the proteolysis of Hb with trypsin, Glu-C, or Lys-C. Hb sulfinamide and sulfonamide adducts of ABP were identified in the blood of mice exposed to ABP, by Orbitrap MS. This is the first report of the identification of intact Hb sulfinamide adducts of carcinogenic AAs in vivo. The high reactivity of HONH-AαC with HbO2 suggests that the Hb sulfinamide adduct of AαC may be a promising biomarker of exposure to this HAA in humans.

The modification of structural and functional properties of human hemoglobin induced by nitroglycerin under different oxygen regime conditions

Human oxyhemoglobin exhibits high resistance to nitroglycerin during incubation of the protein with this compound for 0.3-3 h. Prolonged exposure (24 h) leads to activation of methemoglobin production. In the presence of nitroglycerin hemoglobin molecules undergo rapid oxidation during deoxygenation with formation of methemoglobin as the terminal product of human oxyhemoglobin interaction with nitroglycerin. The scheme of interaction processes of oxyhemoglobin with nitroglycerin in different conditions of oxygen regime is proposed. Partially deliganded hemoglobin plays the leading role in the initiation of hemoglobin oxidation processes.

The effect of antioxidants on in vivo and in vitro methemoglobin formation in erytrocytes of patients with Parkinson`s disease

Methemoglobin formation was examined in erytrocytes of 16 patients with Parkinson`s disease (PD) (stage 3-4 by the Hoehn and Yahr scale). The patients receiving levodopa-containing drugs (madopar, nakom) were also treated with intramuscular injections of mexidol (daily dose 100 mg/day) for 14 days. Control group included 12 clinically healthy persons. The erythrocyte methemoglobin content was determined by electronic paramagnetic resonance (EPR) using the EPR signal intensity with g-factor 6.0. The methemoglobin content was significantly higher in erythrocytes of PD patients than in healthy donors. The complex therapy with mexidol normalized the methemoglobin content in erythrocytes of PD patients. Incubation in vitro of erythrocytes of donors and PD patients with acrolein increased the methemoglobin content, while incubation with carnosine normalized the methemoglobin content in erythrocytes of PD patients. Prophylactic (i.e. before acrolein addition) and therapeutic administration of carnosine to the incubation system with acrolein decreased the methemoglobin content to its initial level. Results of this study suggest that inclusion of the antioxidants mexidol and carnosine in the scheme of basic therapy of PD may reduce side effects associated with methemoglobinemia.

Nanoformulations of anticancer thiosemicarbazones to reduce methemoglobin formation and improve anticancer activity

Triapine and two derivatives were encapsulated into polymeric nanoparticles as well as liposomes. The most stable formulation showed strongly reduced methemoglobin formation and improved anticancer activity.

Novel Mechanism of Cytotoxicity for the Selective Selenosemicarbazone, 2-Acetylpyridine 4,4-Dimethyl-3-selenosemicarbazone (Ap44mSe): Lysosomal Membrane Permeabilization.

Selenosemicarbazones show marked antitumor activity. However, their mechanism of action remains unknown. We examined the medicinal chemistry of the selenosemicarbazone, 2-acetylpyridine 4,4-dimethyl-3-selenosemicarbazone (Ap44mSe), and its iron and copper complexes to elucidate its mechanisms of action. Ap44mSe demonstrated a pronounced improvement in selectivity toward neoplastic relative to normal cells compared to its parent thiosemicarbazone. It also effectively depleted cellular Fe, resulting in transferrin receptor-1 up-regulation, ferritin down-regulation, and increased expression of the potent metastasis suppressor, N-myc downstream regulated gene-1. Significantly, Ap44mSe limited deleterious methemoglobin formation, highlighting its usefulness in overcoming toxicities of clinically relevant thiosemicarbazones. Furthermore, Cu-Ap44mSe mediated intracellular reactive oxygen species generation, which was attenuated by the antioxidant, N-acetyl-L-cysteine, or Cu sequestration. Notably, Ap44mSe forms redox active Cu complexes that target the lysosome to induce lysosomal membrane permeabilization. This investigation highlights novel structure-activity relationships for future chemotherapeutic design and underlines the potential of Ap44mSe as a selective anticancer/antimetastatic agent.

Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice.

Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications.

Protective Effect of Annurca Apple Extract Against Oxidative Damage in Human Erythrocytes

Apple is one of the most worldwide-consumed fruits and a number of cultivars, differing in organo-leptic and nutritional characteristics are available for the market. Annurca apple is a Campania region variety (Southern Italy). This fruit is known for crispness, special taste and long shelf life. The object of this study is to evaluate the capacity of polyphenols extracted from Annurca apple (APE) to protect red blood cells (RBCs) from oxidative stress. The in vitro oxidative stress on human RBCs was induced by t-butyl hydroperoxide. Pre-treatment of cells with 10 µM APE provides a remarkable protection against oxidative damage, particularly decrease ROS production (-35%), lipid perossidation (-81%) and methemoglobin formation (-13%). Moreover, APE incubation decreased the RBC morphological alterations (shrunken cytoplasm and the number of echinocytes) caused by t-BHP. APE protective effect seems to be associated with the antioxidant activity of polyphenolic compounds. Our results indicate that Annurca polyphenols may directly influence erythrocyte properties. Our data confirm higher antioxidant power of Annurca apple, that may therefore represent accessible sources of natural antioxidants with potential application in nutritional/pharmaceutical fields, as preventive or therapeutic agents in diseases in which free radicals are implicated. Keywords: Annurca, apple, antioxidant, erythrocytes, oxidative stress, polyphenols.

In Vitro Protective Effect and Antioxidant Mechanism of Resveratrol Induced by Dapsone Hydroxylamine in Human Cells.

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDS-NHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10–1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.

Comparative study of the effect of BPA and its selected analogues on hemoglobin oxidation, morphological alterations and hemolytic changes in human erythrocytes

Bisphenol A (BPA) has been shown to provoke many deleterious impacts on human health, and thus it is now successively substituted by BPA analogues, whose effects have been poorly investigated. Up to now, only one study has been realized to assess the effect of BPA on human erythrocytes, which showed its significant hemolytic and oxidative potential. Moreover, no study has been conducted to evaluate the effect of BPA analogues on red blood cells. The purpose of the present study was to compare the impact of BPA and its selected analogues such as bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF) on hemolytic and morphological changes and hemoglobin oxidation (methemoglobin formation) of human erythrocytes. The erythrocytes were incubated with different bisphenols concentrations ranging from 0.5 to 500μg/ml for 1, 4 and 24h. The compounds examined caused hemolysis in human erythrocytes with BPAF exhibiting the strongest effect. All bisphenols examined caused methemoglobin formation with BPA inducing the strongest oxidative potential. Flow cytometry analysis showed that all bisphenols (excluding BPS) induced significant changes in erythrocytes size. Changes in red blood cells shape were conducted using phase contrast microscopy. It was noticed that BPA and BPAF induced echinocytosis, BPF caused stomatocytosis, while BPS did not provoke significant changes in shape of red blood cells. Generally, the results showed that BPS, which is the main substituent of bisphenol A in polymers and thermal paper production, exhibited significantly lower disturbance of erythrocyte functions than BPA.

Oxidative stress-induced methemoglobinemia is the silent killer during snakebite: a novel and strategic neutralization by melatonin.

Oxidative stress-induced methemoglobinemia remained an untouched area in venom pharmacology till date. This study for the first time explored the potential of animal venoms to oxidize hemoglobin to methemoglobin. In in vitro whole-blood assay, methemoglobin forming ability of venoms varied as Naja naja > Ophiophagus hannah > Echis carinatus > Daboia russellii > Apis mellifera > Mesobuthus tamulus > Hippasa partita. Being highly potential, N. naja venom was further studied to observe methemoglobin formation in RBCs and in combinations with PMNs and PBMCs, where maximum effect was observed in RBCs + PMNs combination. Naja naja venom/externally added methemoglobin-induced methemoglobin formation was in parallel with ROS generation in whole blood/RBCs/RBCs + PMNs/RBCs + PBMCs. In in vivo studies, the lethal dose (1 mg/kg body weight, i.p.) of N. naja venom readily induced methemoglobin formation, ROS generation, expression of inflammatory markers, and hypoxia-inducible factor-3α. Although the mice administered with three effective doses of antivenom recorded zero mortality; the methemoglobin and ROS levels remained high. However, one effective dose of antivenom when administered along with melatonin (1:50; venom/melatonin, w/w), not only offered 100% survival of experimental mice, but also significantly reduced methemoglobin level, and oxidative stress markers including hypoxia-inducible factor-3α. This study provides strong drive that, complementing melatonin would not only reduce the antivenom load, but for sure greatly increase the success rate of antivenom therapy and drastically minimize the global incidence of snakebite deaths. However, further detailed investigations are needed before translating the combined therapy towards the bed side.

The effect of feeding frequency and dose rate of nitrate supplements on blood haemoglobin fractions in Bos indicus cattle fed Flinders grass (Iseilemia spp.) hay

Twelve Bos indicus steers (liveweight ± s.d., 317.8 ± 28.5) kg were used in an experiment to examine two factors: daily nitrate dose (0, 30, 40 or 50 g of nitrate/day) and feeding frequency (once or twice a day) on methaemoglobin concentration, daily peak methaemoglobin concentration, rate of incline for methaemoglobin concentration, carboxyhaemoglobin concentration, oxyhaemoglobin concentration, total haemoglobin concentration, haematocrit and dry matter intake of Flinders grass hay. Increasing the dose rate of nitrate increased the fraction of methaemoglobin in the blood of steers (P = 0.014). A highly significant effect was demonstrated for the interaction of dose rate × day (P < 0.001). For once a day intake of nitrate, the dose rates of 40 and 50 g per day showed a greater increase in mean methaemoglobin values than for the 0 and 30 g of nitrate per day. Increasing the dose rate of nitrate also increased the daily peak methaemoglobin fraction and the rate of incline to peak methaemoglobin values for both once and twice a day feeding of the nitrate supplements. However, increasing the dose of nitrate had no significant overall effect on total haemoglobin, deoxyhaemoglobin, carboxyhaemoglobin, haematocrit or dry matter intake. Twice a day feeding of nitrate decreased the formation of methaemoglobin in the blood of Bos indicus steers. This study demonstrates that caution should be exercised when feeding nitrates as a non-protein nitrogen source to cattle grazing low quality pastures in northern Australia.

Antagonism of Acute Sulfide Poisoning in Mice by Nitrite Anion without Methemoglobinemia.

There are currently no FDA-approved antidotes for H2S/sulfide intoxication. Sodium nitrite, if given prophylactically to Swiss Webster mice, was shown to be highly protective against the acute toxic effects of sodium hydrosulfide (∼LD40 dose) with both agents administered by intraperitoneal injections. However, sodium nitrite administered after the toxicant dose did not detectably ameliorate sulfide toxicity in this fast-delivery, single-shot experimental paradigm. Nitrite anion was shown to rapidly produce NO in the bloodstream, as judged by the appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin, together amounting to less than 5% of the total hemoglobin present. Sulfide-intoxicated mice were neither helped by the supplemental administration of 100% oxygen nor were there any detrimental effects. Compared to cyanide-intoxicated mice, animals surviving sulfide intoxication exhibited very short knockdown times (if any) and full recovery was extremely fast (∼15 min) irrespective of whether sodium nitrite was administered. Behavioral experiments testing the ability of mice to maintain balance on a rotating cylinder showed no motor impairment up to 24 h post sulfide exposure. It is argued that antagonism of sulfide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity of nitrite rather than formation of methemoglobin.

Acute visual loss following dapsone-induced methemoglobinemia and hemolysis

Objective. While methemoglobinemia is a possible complication of chronic dapsone therapy or of acute overdose, serious adverse manifestations related to methemoglobin formation remain rare. We present an unusual case with severe ischemic retinal injury. Case report. A 30-year-old African woman presented with a sudden decrease of visual acuity secondary to retinal ischemia. She was chronically treated with dapsone (50 mg/day) for a dermatologic disease and denied any drug overdose. However, the determination of serum dapsone level on admission revealed a largely supratherapeutic concentration (20,044 μg/ml compared with 1–3.5 ± 0.5 μg/ml for therapeutic levels). The methemoglobin level at admission was 32% (sulfhemoglobin 1.2%), with hemoglobin level, 7.4 g/dl, schistocytes count, 2–5%, lactate dehydrogenase level, 580 IU/l, and haptoglobin level, < 10 mg/dl. The patient had both alpha-thalassemia and sickle cell trait. She was treated with methylene blue, vitamin C, and exchange transfusion. There was no improvement in visual symptoms over time. Conclusions. In a patient with supratherapeutic serum levels of dapsone, the severity of visual injury was associated with dapsone-induced methemoglobinemia and hemolysis, and perhaps also with some hematologic predisposing factors.

Effect of quinone reductase 2 (NQO2) inhibitor melatonin on primaquine induced hemolysis

The 8-aminoquinoline anti-malarial drug, primaquine (PQ) is the drug of choice for treatment for some forms of malaria. It has antihypnozoite activity against Plasmodium vivax and Plasmodium ovale and gametocytocidal activity against Plasmodium falciparum. Clinical use of PQ is limited due its hemolytic toxicity, especially in glucose-6-phosphate dehydrogenase (G6PD) deficient individuals. Human erythrocytic NQO2 is the only protein target identified for PQ, and redox cycling of quinone metabolites has been implicated in hemotoxicity of PQ. Melatonin, a selective and potent inhibitor of NQO2, was tested for its effect on the hemolytic response to 5-hydroxy PQ (5-HPQ) and 6-methoxy-8-hydroxylaminoquinoline (MHQ), which are among the putative hemotoxic metabolites of PQ. An in vitro assay using normal and G6PD deficient human erythrocytes with accumulation of methemoglobin and generation of oxidative stress, as the markers of hemolysis, was employed. 5HPQ is a highly unstable compound, which is spontaneously converted to 5, 6-orthoquinone (5, 6-OQPQ). Melatonin synergistically increased the methemoglobin formation and reactive oxygen species (ROS) generation induced by 5-HPQ (5, 6-OQPQ) and MHQ. These results suggest a protective role of NQO2 in hemolytic toxicity of PQ. The precise interactions of these metabolites and others, along with PQ itself, with NQO2 should be further explored.