Methemoglobin Production Research Articles

Close relationship between high HbA1c levels and methemoglobin generation in human erythrocytes: The enhancement of oxidative stress in the process

ObjectiveThis study aimed to investigate the effect of diabetic plasma on human red blood cells (RBCs) in order to highlight the amplification mechanisms of oxidative stress (OS) in relation to methemoglobin (metHb) production, a potential bio-indicator that could be related to diabetes disease. Research design and methodsNormal RBCs were co-incubated with the diabetic plasma of 24 patients at different HbA1c levels, for 0, 24, and 48 h in order to assess cell turbidity and hemoglobin (Hb) stability. Hb and metHb production were quantified inside and outside RBCs. Malonaldehyde (MDA) level and cell morphology were concomitantly evaluated. ResultsThe cell turbidity was significantly decreased in the group co-incubated with diabetic plasma at high HbA1c levels (0.074 ± 0.010 AU) compared to the control group (0.446 ± 0.019 AU). A significant decrease in intracellular Hb (0.390 ± 0.075 AU) and its stability (0.600 ± 0.001 AU) were revealed. Also, we found an important increase of metHb levels inside RBCs (0.186 ± 0.017 AU) and in its supernatant (0.086 ± 0.020 AU) after 48 h. Consequently, MDA absorbance increased significantly (0.320 ± 0.040 AU) in RBCs exposed to diabetic plasma with high HbA1c. ConclusionThese findings suggest that poor glycemic control in diabetes leads to metHb generation which is the main factor of the OS amplification.

Sodium nitrite poisoning; A case series of 15 fatalities

To highlight a recent surge in new drug deaths resulting from sodium nitrite (NaNO2) toxicity in which the post mortem blood nitrite (NO2-) and nitrate (NO3) concentrations were measured. The University Hospitals of Leicester (UHL) NHS Trust post-mortem toxicology service receives samples from 27% of Her Majesty's Coroners in England and Wales, analysing approximately 4000 cases per year. We report a series of 15 cases, spanning an 18-month period, in which NaNO2 was the suspected cause of death. NaNO2 is an inorganic sodium salt that has several industrial applications, the most common being preserving meat products. Excess NaNO2 ingestion has also been associated with severe toxicity/death when consumed in excess either accidentally or intentionally. Recently, suicide kits containing NaNO2, and an anti-emetic drug, to assist with ingestion of NaNO2, have become available online. Excessive nitrite ingestion results in iron oxidation within haemoglobin and subsequent production and accumulation of methaemoglobin. Methaemoglobin cannot bind or transport oxygen effectively therefore severe tissue hypoxia and death result. Nitrite is also a vasodilator and can cause coronary ischaemia and stroke as a result of hypotension, tachycardia, and hypoxia. Routine screening was performed on post mortem blood samples received as per usual laboratory protocol; including ethanol analysis by Headspace Gas Chromatography–Flame Ionisation Detection (HS GC-FID), drug screening by High Resolution Accurate Mass–Mass Spectrometry (HRAM-MS) and confirmatory drug quantitation by Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS). In this series, the majority of case histories described the presence of NaNO2 at the scene of death/purchase of a suicide kit; therefore aliquots of post mortem blood were referred to a specialist laboratory (William Harvey Research Institute) for quantitative nitrite and nitrate analysis. Measurement was based upon the gas-phase chemiluminescent reaction between nitric oxide (NO) and ozone (O 3 ); NO levels were determined using an NOA 280A, Sievers NO analyser. Fifteen post-mortem cases in which NaNO2 ingestion was considered the most probable cause of death were reported between January 2020 to July 2021; mean age 31 years (range 18 to 49), 8/15 (53%) male. In almost all cases the clinical history described mental health issues, depression, previous suicide attempts/overdose, self-harm or suicidal ideation. Two thirds of the cases listed prescribed anti-depressants/anti-psychotics; these drugs were detected in 6/15 cases. Ethanol was detected in 3/15 cases and anti-emetic drugs in 3/15 cases. Illicit drugs were present in two of the cases at concentrations suggestive of prior use. The mean nitrite and nitrate concentrations were 2106 μmol/L (min–max 1.4–8100) and 7818 μmol/L (14.5–21,100) respectively. Blood reference ranges for nitrite and nitrate are 0.1–0.4 and 20–40 μmol/L respectively. Nitrite was elevated in all but one case, nitrate was elevated in 12/15 (80%) cases. For the majority of cases (9/15) individuals were discovered deceased; 4/15 cases emergency services attended and death occurred either on route to or following hospital admission. The remaining two cases gave insufficient detail regarding circumstances of death. One post mortem report described discolouration on post mortem consistent with the presence of methaemoglobinaemia. The detection and quantitation of nitrite and nitrate during post-mortem toxicological investigations represents a significant challenge analytically and heavily relies upon circumstantial evidence. Even though it still remains a rare cause of death, it is worthwhile considering the possibility of sodium nitrite use in individuals with suicidal ideation given its apparent availability online. The provision of a quantitative nitrite/nitrate analytical service greatly assists in determining the cause of death in these cases.

Scalable manufacturing platform for the production of methemoglobin as a non-oxygen carrying control material in studies of cell-free hemoglobin solutions.

Methemoglobin (metHb) arises from the oxidation of ferrous hemoglobin (HbFe2+, Hb) to ferric hemoglobin (HbFe3+, metHb), which is unable to bind gaseous ligands such as oxygen (O2) and carbon monoxide (CO), and binds to nitric oxide (NO) significantly slower compared to Hb. Therefore, metHb does not elicit vasoconstriction and systemic hypertension in vivo due to its extremely slow NO scavenging rate in comparison to cell-free Hb, but will induce oxidative tissue injury, demonstrating the potential of using metHb as a control material when studying the toxicity of cell-free Hb. Hence, the goal of this work was to develop a novel manufacturing strategy for production of metHb that is amenable to scale-up. In this study, small scale (e.g. 1 mL reaction volume) screening experiments were initially conducted to determine the optimal molar ratio of Hb to the oxidization agents hydrogen peroxide (H2O2) or sodium nitrite (NaNO2) to achieve the highest conversion of Hb into metHb. A spectral deconvolution program was employed to determine the molar fraction of various species (hemichrome, metHb, oxyHb, metHb-, and NaNO2) in solution during the oxidation reaction. From this analysis, either a 1:1 or 1:5 molar ratio was identified as optimal molar ratios of Hb:NaNO2 (heme basis) that yielded the highest conversion of Hb into metHb with negligible amounts of side products. Hence in order to reduce the reaction time, a 1:5 molar ratio was chosen for large scale (i.e. 1.5 L reaction volume) synthesis of bovine metHb (metbHb) and human metHb (methHb). The biophysical properties of metHb were then characterized to elucidate the potential of using the synthesized metHb as a non-O2 carrying control material. The haptoglobin binding kinetics of metHb were found to be similar to Hb. Additionally, the synthesized metHb was stable in phosphate buffered saline (PBS, 50 mM, pH 7.4) at 4°C for approximately one week, indicating the high stability of the material.

Evaluation of the Anti-Sickle Cell Activity of Uvaria Chamea P. Beauv. Roots Aqueous Extract

Sickle cell disease was a very common hemoglobinosis in tropical countries. Several plants were used in traditional Beninese medicine to relieve sickle cell crises. The present work was carried out to test the efficiency of Uvaria chamea roots aqueous extract in the treatment of sickle cell crises.
 
 Methods: Uvaria chamea roots aqueous extract was preincubated at different concentrations with blood cells from SS red blood cells before or after the Emmel Test. Intra-erythrocyte oxidative stress was measured by methaemoglobin assay after incubation of the extract with hemoglobin. In vivo, the action of the extract on Hemoglobin level, Mean Corpuscular Volume, and platelet count was measured in Wistar rats.
 
 Results: The extract significantly prevented the formation of sickle cells at doses of 40 and 20 mg / ml of blood (P <0.05) and significantly reduced sickle cells to the normal biconcave form at the dose of 40 mg / ml (P <0.05). It lowered the production of methemoglobin at a dose of 10 mg / ml, suggesting antioxidant activity. Hemoglobin level, Mean Corpuscular Volume, and platelet count did not significantly increase in treated rats, indicating that the extract did not stimulate erythropoiesis or thrombopoiesis.
 
 Conclusion: Uvaria chamea extract inhibited dose-dependent sickling of red blood cells. It reduced intraerythrocyte oxidative stress but did not show hematopoietic activity. It could therefore be considered in the preventive or curative management of sickle cell crises, but not against anemia.

Transcutaneous monitoring of hemoglobin derivatives during methemoglobinemia in rats using spectral diffuse reflectance.

.Significance: Untreated methemoglobinemia may cause severe hypoxemia and even death when methemoglobin levels in the blood stream exceed 70%. Although CO-oximetry can be used to monitor the response to treatment for methemoglobinemia, it is costly and requires an invasive procedure for collecting blood samples from patients. A pulse CO-oximeter with a contact probe can be used to continuously and non-invasively measure the percentage of methemoglobin, as well as the percutaneous oxygen saturation. In terms of the prevention of infectious diseases, however, it is desirable to monitor methemoglobin and oxygen saturation levels in a non-contact manner. Diffuse reflectance spectral imaging is promising as a non-contact, non-invasive, and cost-effective clinical diagnostic tool for methemoglobinemia.Aim: To demonstrate the feasibility of visible spectral diffuse reflectance for in vivo monitoring of hemoglobin derivatives and evaluating methemoglobin production and reduction as well as hypoxemia during methemoglobinemia in rats.Approach: A new imaging approach based on the multiple regression analysis aided by Monte Carlo simulations for light transport was developed to quantify methemoglobin, oxygenated hemoglobin, and deoxygenated hemoglobin using a hyperspectral imaging system. An in vivo experiment with rats exposed to sodium nitrite () at different doses was performed to confirm the feasibility of the method for evaluating the dynamics of methemoglobin, oxygenated hemoglobin, and deoxygenated hemoglobin during methemoglobinemia. Systemic physiological parameters, including the percutaneous arterial oxygen saturation, heart rate (HR), and pulse distention, were measured by a commercially available pulse oximeter, and the results were compared to those obtained by the proposed method.Results: Both the methemoglobin concentration and methemoglobin saturation rapidly increased with a half-maximum time of . They reached their maximal values nearly 60 min after the administration of . Tissue oxygen saturation dramatically dropped to a minimum of , , , and on average for doses of 25, 37.5, 50, and 75 mg/kg, respectively. Changes in methemoglobin concentration and tissue oxygen saturation are indicative of the temporary production of methemoglobin and severe hypoxemia during methemoglobinemia. Profound increases in the HR and pulse distention implied an elevated cardiac output caused by tachycardia and the resultant increase in peripheral blood volume to compensate for the hypoxia and hypoxemia during methemoglobinemia. This was in agreement with the time course of the peripheral hemoglobin volume concentration obtained by the proposed method.Conclusions: The proposed method is capable of the in vivo non-contact simultaneous evaluation of methemoglobin levels and hypoxemia during methemoglobinemia, and that it has potential as a tool for the diagnosis and management of methemoglobinemia.

In Vivo Transcutaneous Monitoring of Hemoglobin Derivatives Using a Red-Green-Blue Camera-Based Spectral Imaging Technique.

Cyanosis is a pathological condition that is characterized by a bluish discoloration of the skin or mucous membranes. It may result from a number of medical conditions, including disorders of the respiratory system and central nervous system, cardiovascular diseases, peripheral vascular diseases, deep vein thrombosis, and regional ischemia. Cyanosis can also be elicited from methemoglobin. Therefore, a simple, rapid, and simultaneous monitoring of changes in oxygenated hemoglobin and deoxygenated hemoglobin is useful for protective strategies against organ ischemic injury. We previously developed a red-green-blue camera-based spectral imaging method for the measurements of melanin concentration, oxygenated hemoglobin concentration (CHbO), deoxygenated hemoglobin concentration (CHbR), total hemoglobin concentration (CHbT) and tissue oxygen saturation (StO2) in skin tissues. We leveraged this approach in this study and extended it to the simultaneous quantifications of methemoglobin concentration (CmetHb), CHbO, CHbR, and StO2. The aim of the study was to confirm the feasibility of the method to monitor CmetHb, CHbO, CHbR, CHbT, and StO2. We performed in vivo experiments using rat dorsal skin during methemoglobinemia induced by the administration of sodium nitrite (NaNO2) and changing the fraction of inspired oxygen (FiO2), including normoxia, hypoxia, and anoxia. Spectral diffuse reflectance images were estimated from an RGB image by the Wiener estimation method. Multiple regression analysis based on Monte Carlo simulations of light transport was used to estimate CHbO, CHbR, CmetHb, CHbT, and StO2. CmetHb rapidly increased with a half-maximum time of less than 30 min and reached maximal values nearly 60 min after the administration of NaNO2, whereas StO2 dramatically dropped after the administration of NaNO2, indicating the temporary production of methemoglobin and severe hypoxemia during methemoglobinemia. Time courses of CHbT and StO2, while changing the FiO2, coincided with well-known physiological responses to hyperoxia, normoxia, and hypoxia. The results indicated the potential of this method to evaluate changes in skin hemodynamics due to loss of tissue viability and vitality.

Relationship between effects of riociguat and levels of methemoglobin in patients with chronic thromboembolic pulmonary hypertension

Abstract Background Soluble guanyl cyclase (sGC) is a receptor for nitric oxide (NO) and plays an important role in vascular tonus. sGC stimulant is a therapeutic agent for pulmonary hypertension and an advantage of sGC stimulant over phosphodiesterase (PDE)-5 inhibitors is that sGC stimulant exerts its effect even when NO production is reduced. NO derived from vascular endothelial cells is immediately absorbed by hemoglobin (Hb), which leads to the production of methemoglobin (Met-Hb) when oxidized. Previous report has shown that the therapeutic effect of PDE-5 inhibitors was associated with levels of Met-Hb. Purpose In this study, we examined the relationship between the effect of riociguat and levels of Met-Hb in patients with chronic thromboembolic pulmonary hypertension (CTEPH). Methods The study population comprised 18 patients with CTEPH. Mean pulmonary artery pressure (mPAP) and pulmonary vascular resistance (PVR) were assessed before and after administration of riociguat, and changes in mPAP and PVR were defined as ΔmPAP and ΔPVR, respectively. Since the level of Met-Hb was obtained as the percentage of Hb (FMet-Hb), the amount of Met-Hb was calculated by following formula: Met-Hb (mg/dL) = Hb (g/dL) × FMet-Hb (%) × 10. Results The amount of Met-Hb value before administration of riociguat was significantly correlated with the degree of improvement in mPAP and PVR after administration of riocigat (R=−0.502, P<0.05 mPAP; R=−0.481, P<0.05 PVR, respectively) as shown in figures. Conclusion Our findings suggest that the level of Met-Hb before treatment may predict the therapeutic effect of sGC stimulants in patients with CTEPH. Funding Acknowledgement Type of funding source: None

DAPSONE-INDUCED METHEMOGLOBINEMIA TREATED WITH ORAL CIMETIDINE: A CASE REPORT

SESSION TITLE: Tuesday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM INTRODUCTION: Dapsone is an antibiotic with anti-inflammatory properties commonly utilized in the treatment of various immunologic skin disorders. It is known to cause methemoglobinemia, a functional anemia characterized by an increased level of methemoglobin resulting in impaired oxygen release to tissues. We report a case of dapsone-induced methemoglobinemia successfully managed with oral cimetidine. CASE PRESENTATION: A 72-year-old male presented to the emergency department with the complaint of sudden onset of shortness of breath after undergoing a cardiac ablation three days prior to presentation. His past medical history included atrial fibrillation, cardiomyopathy, and an inflammatory skin condition known as granuloma annulare (GA). Of note, he was started on dapsone 100 mg daily for GA about a week prior to presentation. Pulse oximetry on admission demonstrated an oxygen saturation of 81 percent on room air despite the arterial blood gas (ABG) demonstrating a partial pressure of oxygen of 134 mm Hg. The patient’s methemoglobin level was noted to be elevated at 7.2 percent. Physical examination revealed crackles in bilateral lung flies. In addition, interstitial pulmonary edema was demonstrated on chest X-ray. The patient was admitted and treated for heart failure with furosemide. His home medications including dapsone were continued. On hospital day two, despite showing improvement in his pulmonary edema, the patient continued to require 12 liters of oxygen to maintain an adequate oxygen saturation. On hospital day 3, a repeat ABG revealed an increase in methemoglobin levels to 14.2 percent . Given these findings, dapsone-induced methemoglobinemia was suspected. Dapsone was discontinued and oral cimetidine 400 mg three times daily was initiated. About 22 hours after cimetidine was administered, methemoglobin level decreased to 5.5 percent. Methemoglobin continued to drop to 4.9 another 24 hours later. On hospital day six, the patient’s shortness of breath had completely resolved and his oxygen saturation improved to 95 percent on room air. No side effects to cimetidine were noted. The patient was discharged to home and prescribed cimetidine to be continued for one more week after discharge. DISCUSSION: Cimetidine, a histamine-2 receptor antagonist, can mitigate dapsone-induced methemoglobinemia by inhibiting the cytochrome P450 metabolism of dapsone to its toxic hydroxylamine metabolites, which are believed to be the cause of methemoglobinemia. Cimetidine can be given to prevent the further production of methemoglobin and thus further toxicity from dapsone. In this patient, a significant decrease in methemoglobin level and clinical improvement were observed within 24 hours of initiating cimetidine. CONCLUSIONS: This case provided evidence that oral cimetidine with discontinuation of dapsone may be an effective and well-tolerated option for the acute treatment of dapsone-induced methemoglobinemia. Reference #1: Ashurst JV, et al. Pathophysiologic mechanisms, diagnosis, and management of dapsone-induced methemoglobinemia. J Am Osteopath Assoc. 2010 Jan;110(1):16-20. Reference #2: Malfara WR. Effects of H(2)-receptor antagonists on dapsone-induced methaemoglobinaemia in rats. Pharmacol Res. 2002 Apr;45(4):269-73. Reference #3: Burke P, et al. Dapsone-induced methemoglobinemia: case of the blue lady. Can Fam Physician. 2013 Sep;59(9):958-61. DISCLOSURES: Speaker/Speaker's Bureau relationship with Allergan Please note: $5001 - $20000 Added 03/20/2019 by Frances Loftus, source=Web Response, value=Honoraria Speaker/Speaker's Bureau relationship with Boerhyinger ingelheim Please note: $1001 - $5000 Added 03/20/2019 by Frances Loftus, source=Web Response, value=Honoraria No relevant relationships by Natalie Millet, source=Web Response Speaker/Speaker's Bureau relationship with Melinta Pharmaceutical Please note: $1001 - $5000 Added 03/18/2019 by Joseph Reilly, source=Web Response, value=Honoraria

Methemoglobinemia associated with massive acetaminophen ingestion: a case series

Background: Acetaminophen is a common pharmaceutical ingestion reported to US poison centers. In overdose, toxic metabolites are known to cause hepato- and nephrotoxicity. While G6PD deficiency may be a risk factor for methemoglobin production in the setting of acetaminophen overdose, it is rarely reported in patients who do not have this condition.Methods: We present two cases of methemoglobinemia following massive acetaminophen ingestion with no known history of G6PD deficiency or other substances known to induce methemoglobinemia. The two cases had peak methemoglobin measurements of 32% and 12% respectively, and both were treated with methylene blue.Discussion: A number of mechanisms may be involved in production of methemoglobin in the setting of massive acetaminophen ingestion including NAPQI-induced oxidation, depletion of glutathione stores, and production of oxidant-metabolites including paraaminophenol. While it is unlikely that the majority of acetaminophen overdoses result in any clinically significant methemoglobinemia, massive acetaminophen overdose may be complicated by development of methemoglobinemia.Conclusion: Physicians should be aware of the possibility that massive acetaminophen ingestion may be complicated by methemoglobinemia in rare instances. Further studies should aim to characterize the metabolic pathways leading to possible methemoglobinemia in humans after large acetaminophen ingestions.

Optical and photoacoustic radiofrequency spectroscopic analysis for detecting red blood cell death.

Under stress, red blood cells (RBCs) undergo programmed cell death (eryptosis). One of the signaling molecules for eryptosis, sphingomyelinase (SMase), plays an important role in monitoring the efficacy of vascular targeted cancer therapy. The high optical absorption of erythrocytes coupled with the changes of eryptotic RBCs makes RBCs ideal targets for the photoacoustic (PA) detection and characterization of vascular treatments. In this work, experiments characterizing eryptosis were performed: PA detection of high frequencies (>100 MHz) that enabled analysis at the single-cell level and of low frequencies (21 MHz) that enabled analysis at the RBC ensemble level. Ultrasound spectral analysis was performed on control and SMase-treated RBCs. Spectral unmixing was applied to quantify methemoglobin production as a by-product of RBC death. Validation was performed using a blood gas analyzer and optical spectrometry. Our results indicate that PA radiofrequency spectra could be used to differentiate the biochemically induced morphological changes as RBCs lose their native biconcave shape, and release hemoglobin into the surroundings. Spectral unmixing revealed a 7% increase in methemoglobin content for SMase-treated samples due to the oxidative stress on the RBCs. These findings suggest that PA spectral analysis of RBC death can potentially serve as a biomarker of the efficacy of vascular targeted cancer therapies.

Continuous Optical Monitoring of Red Blood Cells During a Photosensitization Reaction.

Background: An oxygen-enriched photosensitizer solution was created by the addition of red blood cells (RBCs) as an investigative tool for photosensitization reactions (PRs). Although the oxygen levels and reaction progress can be monitored using the optical characteristics of hemoglobin, previously this has only been done using intermittent measurements. An increase in methemoglobin concentration with irradiation time was reported. Objective: We constructed a continuous optical measurement system to study the dynamics of the PR in a photosensitizer solution containing RBCs. We also measured the relationship between hemolysis and methemoglobin production in the solution. Materials and methods: A 664 nm wavelength continuous laser beam at 60 mW/cm2 was used to drive the PR, and a broadband (475-650 nm) light beam was used to monitor the absorption spectra during the PR. The light sources were arranged perpendicularly to cross at a 1 × 10 mm cuvette. The sample in this cuvette was prepared from a low-hematocrit rabbit RBC suspension medium containing 30 μg/mL talaporfin sodium, a chlorine photosensitizer. The concentrations of oxygenated hemoglobin, deoxygenated hemoglobin, and methemoglobin were obtained using a multiple regression analysis of the measured spectra. Results: The oxygen saturation decreased continuously during the PR. The relationship between the degree of hemolysis and produced methemoglobin concentration was confirmed. Conclusions: We determined the dynamics of the oxidation and oxygen desorption of hemoglobin, as well as RBC hemolysis, during the PR. Our measurement system, which uses the properties of hemoglobin contained in RBCs, might be useful for continuous monitoring of PR dynamics.

Impact of Naja nigricollis Venom on the Production of Methaemoglobin.

Snakebite envenomation is an affliction currently estimated to be killing upwards of 100,000 people annually. Snakebite is associated with a diverse pathophysiology due to the magnitude of variation in venom composition that is observed worldwide. The haemolytic (i.e., lysis of red blood cells) actions of snake venoms are well documented, although the direct impact of venoms on haemoglobin is not fully understood. Here we report on the varied ability of a multitude of snake venoms to oxidise haemoglobin into methaemoglobin. Moreover, our results demonstrate that the venom of an elapid, the black necked spitting cobra, Naja nigricollis, oxidises oxyhaemoglobin (Fe2+) into methaemoglobin (Fe3+) in a time- and concentration-dependent manner that is unparalleled within the 47 viper and elapid venoms evaluated. The treatment of venom with a reducing agent, dithiothreitol (DTT) is observed to potentiate this effect at higher concentrations, and the use of denatured venom demonstrates that this effect is dependent upon the heat-sensitive proteinaceous elements of the venom. Together, our results suggest that Naja nigricollis venom appears to promote methaemoglobin production to a degree that is rare within the Elapidae family, and this activity appears to be independent of proteolytic activities of venom components on haemoglobin.

Effects of Phenyl-1-Pyridin-2yl-Ethanone (PPY)-Based Iron Chelators on the Formation of Methemoglobin

BackgroundIron chelators are used in the treatment of iron overload related diseases and are currently receiving a major attention as potential antitumor drugs. In recent studies, the antitumor activity of thiosemicarbazones-class of iron chelator, including Di-2-pyridilketone-4,4- dimethyl-3-thiosemicarbazone (Dp44mT) has been investigated in over 20 phase I and II clinical trials [1, 2,3]. Iron chelators were also considered as anti-HIV-1 agents. However, the main obstacle to using iron chelators in vivois the deleterious side effect of methemoglobinemia induced by some iron chelators that are able to scavenge electrons from the heme-bound iron in hemoglobin. In our previous studies, we developed novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators that we showed to increase IKBα expression, modulate CDK2 and CDK9 activities and inhibit HIV-1 [4].ObjectiveOur objective was to test the effect of PPYeT iron chelator for methemoglobin induction. The methemoglobin induction effect was compared with several additional iron chelators including Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and PPY analogues.MethodsFluorometric analysis was carried out in promonocytic THP-1 cells to evaluate the ability of our novel PPYeT iron chelator to reduce labile iron pool (LIP). The effect of PPYet on LIP was compared to the effect to SIH. Subsequently, spectrophotometric analysis was used to measure and quantify the production of methemoglobin in human red blood cells lysates and in isolated intact human red blood cells treated with PPYeT and various other iron chelators including DP44mT and DP4mT.ResultsPPYeT significantly reduced LIP in THP-1 cells overloaded with iron comparing to the cells treated with SIH. In RBC lysates and in intact RBC, PPYeT treatment showed notably lesser production of methemoglobin in comparison to DP44mT and DP4mT chelators. In RBC lysates, PPYeT produced about four-fold less methemoglobin than Dp44mT and ten-fold less than Dp4mT.ConclusionThe novel compound, PPYeT, shows a remarkably low ability to catalyze the formation of methemoglobin in human RBC lysates and also in intact RBCs as compared to Dp44mT. These findings indicate that PPYeT may be useful for future in vivo studies as it produces less methemoglobinemia. Further studies will evaluate the effect PPYeT as anti-cancer or anti HIV-1 inhibitor in vivo.AcknowledgmentsThis work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005, and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH.

Methemoglobin and nitric oxide therapy in Ugandan children hospitalized for febrile illness: results from a prospective cohort study and randomized double-blind placebo-controlled trial.

BackgroundExposure of red blood cells to oxidants increases production of methemoglobin (MHb) resulting in impaired oxygen delivery to tissues. There are no reliable estimates of methemoglobinemia in low resource clinical settings. Our objectives were to: i) evaluate risk factors for methemoglobinemia in Ugandan children hospitalized with fever (study 1); and ii) investigate MHb responses in critically ill Ugandan children with severe malaria treated with inhaled nitric oxide (iNO), an oxidant that induces MHb in a dose-dependent manner (study 2).MethodsTwo prospective studies were conducted at Jinja Regional Referral Hospital in Uganda between 2011 and 2013. Study 1, a prospective cohort study of children admitted to hospital with fever (fever cohort, n = 2089 children 2 months to 5 years). Study 2, a randomized double-blind placebo-controlled parallel arm trial of room air placebo vs. 80 ppm iNO as an adjunctive therapy for children with severe malaria (RCT, n = 180 children 1–10 years receiving intravenous artesunate and 72 h of study gas). The primary outcomes were: i) masimo pulse co-oximetry elevated MHb levels at admission (>2 %, fever cohort); ii) four hourly MHb levels in the RCT.ResultsIn the fever cohort, 34 % of children admitted with fever had elevated MHb at admission. Children with a history of vomiting, delayed capillary refill, elevated lactate, severe anemia, malaria, or hemoglobinopathies had increased odds of methemoglobinemia (p < 0.05 in a multivariate model). MHb levels at admission were higher in children who died (n = 89) compared to those who survived (n = 1964), p = 0.008. Among children enrolled in the iNO RCT, MHb levels typically plateaued within 12–24 h of starting study gas. MHb levels were higher in children receiving iNO compared to placebo, and MHb > 10 % occurred in 5.7 % of children receiving iNO. There were no differences in rates of study gas discontinuation between trial arms.ConclusionsHospitalized children with evidence of impaired oxygen delivery, metabolic acidosis, anemia, or malaria were at risk of methemoglobinemia. However, we demonstrated high-dose iNO could be safely administered to critically ill children with severe malaria with appropriate MHb monitoring.Trial registrationClinicalTrials.gov Identifier: NCT01255215 (Date registered: December 5, 2010).

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.

Methemoglobinemia After Isolated Lidocain Spray: A Rare Phenomenon

AbstractMethemoglobinemia is a state recognized by the increased production of met-hemoglobin, which is a form of oxidized hemoglobin, which is unable to bind oxygen. As a result the patient has a functional anemia, in which the remaining oxyhemoglobin has increased oxygen affinity, shifting the oxygen curve to left and perpetuating the impairment of oxygen delivery to tissues. Methemoglobinemia is a rare, but serious cause of hypoxemia, which can be difficult to recognize. It has been traditionally associated with the use of benzocaine class of anesthetic agents and FDA has issued warning for the use of benzocaine class of anesthetic agents for the same reason. It has been very seldom reported with the use of lidocaine class of agents alone. Through this case report we want to emphasize development of methemoglobinemia through the isolated use of lidocaine.

Protective Effects of Ascorbic Acid and α-Tocopherol on the In Vitro Oxidation of Equine Erythrocytes Caused by Extracts of Wilted Red Maple Leaves

Red maple leaf toxicosis in horses is a debilitating condition. Horses which ingest wilted leaves of red maple, as well as those of silver and sugar maple, experience depression, lethargy, pale mucous membranes, and brown discoloration of plasma and urine due to severe oxidation of erythrocytes. The objective of this study was to evaluate the protective effect of equine dietary supplementation with the antioxidants ascorbic acid and α-tocopherol on hemolysis and methemoglobin production of erythrocytes when challenged by extracts of wilted red maple leaves in bioassays. Horses were assigned to one of four groups which received either (1) no dietary supplement, (2) ascorbic acid, (3) α-tocopherol, or (4) both ascorbic ascorbic acid and α-tocopherol for 12 weeks. At weeks 1, 12, and 24 after starting supplementation, blood samples were collected from horses and incubated with either 0 or 75 μL of leaf extract. Hemolysis and methemoglobin production were determined based on spectrophotometry. After 12 weeks of ascorbic acid supplementation, percentage hemolysis and methemoglobin production were significantly reduced in blood samples incubated with 75 μL of leaf extract. In contrast, only percentage hemolysis was significantly reduced after 12 weeks of α-tocopherol supplementation. After 24 weeks, values for percentage hemolysis and methemoglobin had returned to their baseline levels after termination of supplementation at week 12. The combination of both supplements did not significantly reduce hemolysis or methemoglobin production. These results suggest that daily dietary supplements of ascorbic acid (and to a lesser degree α-tocopherol) have the potential to lessen the effects of erythrocyte oxidation which occurs during red maple leaf toxicosis.

Characterization of extra virgin olive oils produced with typical Italian varieties by their phenolic profile

Evaluation of phenolic profile, antioxidant power, and protective capacity against oxidation of red blood cells (RBCs) of olive oil phenolic extracts (OOPEs) from several Italian varieties were studied. Phenolic profiles, and quantification of seven selected bioactive compounds were performed by RP-HPLC. OOPEs exhibited high antioxidant activity, and this capacity was positively related to their phenolic amount. In particular, OOPE5 (cv Gentile di Larino, Molise region) displayed the highest phenolic and ortho-diphenolic content as well as the strongest scavenging activity determined using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) (87% DPPH inhibition). Protective capacity against stressed RBCs was investigated through the evaluation of methemoglobin (MetHb) and malondialdehyde (MDA) levels. OOPE5 was the most active against methemoglobin production (53.7% reduction), whereas OOPE1 (cv Lavagnina, Liguria region) showed the highest protection toward malondialdehyde (83.3% reduction). Overall the selected oils showed qualitative and quantitative differences in phenol composition, and this variability influenced their protective effect against oxidative damages.

Pyocyanina contributory factor in haem acquisition and virulence enhancement of Porphyromonas gingivalis in the lung [corrected

Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain production, may contribute to virulence of P. gingivalis and disease severity when co-infecting with P. aeruginosa in the lung.

Caffeine inhibits erythrocyte membrane derangement by antioxidant activity and by blocking caspase 3 activation

The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation–deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine–haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO2 thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane.Furthermore caffeine destabilizes the haeme–protein interactions within the haemoglobin molecule and triggers the production of superoxide and met-haemoglobin. However this damaging effect is almost balanced by the surprising scavenger action of the alkaloid with respect to the hydroxyl radical. These experimental findings are supported by in silico docking and molecular dynamics studies and by what we may call the “caspase silence”; in fact, there is no evidence of any caspase 3 activity enhancement; this is likely due to the promotion of positive metabolic conditions which result in an increase of the cellular reducing power.