Methemoglobin Research Articles

A new approach to detect the imbalance of hemoglobin and methemoglobin levels in human plasma based on a fluorescent modified nanocellulose chemosensor

This work has developed a new, rapid, highly sensitive, and precise approach to identify the imbalance levels of hemoglobin and methemoglobin through fluorescence emission spectroscopy. A study on a new Schiff base-modified nanocellulose (PY-PDA-DANC) applied as a fluorescent chemosensor is reported. To prepare the chemosensor, nanocellulose (NC) was first converted into 2,3-dialdehyde nanocellulose (DANC), and then modified by grafting o-phenylenediamine (PDA), followed by 1-pyrenecarboxaldehyde giving rise to the new blue-fluorescent complex PY-PDA-DANC. All the synthesized products were characterized using FTIR, 1H NMR, XRD, EDX and UV–vis spectroscopy. The fluorescence response of PY-PDA-DANC against various metal ions (Co2+, Cr3+, Cu2+, Fe2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+) in DMSO/H2O media was evaluated using PL spectroscopy. The fluorescent sensor showed high and selective quenching response to both Fe2+ and Fe3+ ions with low limit of detection (LOD). PY-PDA-DANC was also successfully applied for sensing of heme–iron coordinated to hemoglobin (HB) and methemoglobin (MHB), displaying a LOD of 28.45 nM and 65.36 nM, respectively. Subsequently, it was applied for the analysis of [Fe2+@HB] and [Fe3+@MHB] in human plasma sample with good recoveries.

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.

Comparison of the effects of gasoline burn and chromic acid burn on different internal organs and immune functions in rats.

Burn as physical injury ranks as the fourth most prevalent trauma across the world. In this study, we aimed to compare the impact of gasoline burn and chromic acid burn on the internal organs and immune functions in rats. The results showed that the levels of methemoglobin (MHb) to total hemoglobin (Hb) as well as the Cr6+ content showed significant elevation in the chromic acid burn group relative to the gasoline burn group. HE staining was used to evaluate the histological changes in the injured tissues as well as the tissues excised from internal organs. We found that chromic acid burn-induced more severe damage to rat tissues. Gasoline burn showed no significant impact on the intestinal tissues of rats, while the chromic acid burn-induced increased cell death in rat intestines. Moreover, the results of HE staining also revealed that gasoline burn and chromic acid burn showed no evident impact on rat hearts. Gasoline burn also showed no significant effects on the liver, lungs and kidneys of rats, while the chromic acid burn caused injuries to such internal organs in comparison with the control and gasoline burn groups. In addition, the MPO activity was higher in the liver, intestine, lungs and kidneys of rats with chromic acid burn. Furthermore, the expression of inflammation response cytokines was examined in the serum of rats. The results demonstrated that the levels of IL-6, IL-1β and TNF-α showed a significant increase in both the gasoline burn and chromic acid burn groups of rats relative to the control, and the levels were higher in the chromic acid burn group in comparison with the gasoline burn group. In conclusion, the chromic acid burn-induced more severe organ injury, inflammation and immune response compared with the gasoline burn, which may provide reference data for the clinical treatment of patients with different burn injuries.

Endogenously produced H2O2 is intimately involved in iron metabolism in Streptococcus pneumoniae.

Heme degradation provides pathogens with growth essential iron, leveraging on the host heme reservoir. Bacteria typically import and degrade heme enzymatically, and here, we demonstrated a significant deviation from this dogma. We found that Streptococcus pneumoniae liberates iron from met-hemoglobin extracellularly, in a hydrogen peroxide (H2O2)- and cell-dependent manner; this activity serves as a major iron acquisition mechanism for S. pneumoniae. Inhabiting oxygen-rich environments is a major part of pneumococcal biology, and hence, H2O2-mediated heme degradation likely supplies iron during infection. Moreover, H2O2 reaction with ferrous hemoglobin but not with met-hemoglobin is known to result in heme breakdown. Therefore, the ability of pneumococci to degrade heme from met-hemoglobin is a new paradigm. Lastly, this study will inform other research as it demonstrates that extracellular degradation must be considered in the interpretations of experiments in which H2O2-producing bacteria are given heme or hemoproteins as an iron source.

The Potential of Leucomethylene Blue in Methemoglobinemia Treatment: A New Hope for Patients with G6PD?

Methylene blue (MB) has been routinely used to treat methemoglobinemia. In the body, MB is reduced to leucomethylene blue (LMB) by NADPH-dependent methemoglobin (MetHB) reductase, and then LMB reduces Fe3+ to Fe2+. In glucose-6-phosphate dehydrogenase (G6PD) deficiency, NADPH is not produced sufficiently to protect erythrocytes against oxidative stress and to take part in relevant biochemical reactions. Since MB is an oxidative agent, its administration in individuals with G6PD deficiency leads to an increased risk of hemolysis through oxidative stress and even death. Therefore, its administration has been prohibited from treating methemoglobinemia in G6PD patients. As an antioxidant and direct reducing agent for Fe3+, LMB may be prescribed for treating MetHB in patients with G6PD deficiency. Considering the biochemical process of turning MB into LBM and the reducing nature of LMB, it seems LMB is a safer drug than MB in treating methemoglobinemia. LMB can even be administrated in other treatments without any concern about increasing oxidative stress, exacerbating the inflammation. Proof-of-concept experimental and clinical trials could substantiate this hypothesis.

Silk Fibroin Particles as Carriers in the Development of Hemoglobin-Based Oxygen Carriers.

Oxygen therapeutics have a range of applications in transfusion medicine and disease treatment. Synthetic molecules and all-natural or semi-synthetic hemoglobin-based oxygen carriers (HBOCs) have seen success as potential circulating oxygen carriers. However, many early HBOC products stalled in development due to side effects from excess hemoglobin in the blood stream and hemoglobin entering the tissue. To overcome these issues, research has focused on increasing the molecular diameter of hemoglobin by polymerizing hemoglobin molecules or encapsulating hemoglobin in liposomal carriers. This work leverages the properties of silk fibroin, a cytocompatible and non-thrombogenic biopolymer, known to entrap protein-based cargo, to engineer a fully protein-based oxygen carrier. Herein, an all-aqueous solvent evaporation technique was used to form silk particles via phase separation from a bulk polyvinyl alcohol phase (PVA). Particles size was tuned, and particles were formed with and without hemoglobin. The encapsulation efficiency and ferrous state of hemoglobin were analyzed, resulting in 60% encapsulation efficiency and a maximum of 20% ferric hemoglobin, yielding 100 µg/mL active hemoglobin in certain sfHBOC formulations. The system did not elicit a strong inflammation response in vitro, demonstrating the potential for this particle system to serve as an injectable HBOC.

Pharmaceutical Integrity of Lyophilized Methemoglobin-Albumin Clusters after Reconstitution.

Covalent attachment of a ferric hemoglobin (metHb) core to three human serum albumin molecules to form metHb-albumin clusters has previously been used to develop an antidote for hydrogen sulfide poisoning. Lyophilization is one of the most effective approaches to preserve protein pharmaceuticals with minimum contamination and decomposition. However, there is concern that lyophilized proteins may undergo pharmaceutical alteration on reconstitution. This study investigated the pharmaceutical integrity of metHb-albumin clusters on lyophilization and reconstitution with three clinically available reconstitution fluids, (i) sterile water for injection, (ii) 0.9% sodium chloride injection, and (iii) 5% dextrose injection. The metHb-albumin clusters retained their physicochemical properties and structural integrity on lyophilization and reconstitution with sterile water for injection or 0.9% sodium chloride injection, along with comparable hydrogen sulfide scavenging ability compared to non-lyophilized metHb-albumin clusters. The reconstituted protein completely rescued lethal hydrogen sulfide poisoning in mice. On the other hand, lyophilized metHb-albumin clusters reconstituted with 5% dextrose injection showed physicochemical changes and a higher mortality rate in mice subjected to lethal hydrogen sulfide poisoning. In conclusion, lyophilization represents a potent preservation method for metHb-albumin clusters if either sterile water for injection or 0.9% sodium chloride injection is used for reconstitution.

Detail study on the interaction between perfluorooctanoic acid (PFOA) with human hemoglobin (Hb)

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are often referred to as legacy perfluoroalkyl substances (PFAS). Human exposure to PFAS leads to severe negative health impacts including cancers, infertility, and dysfunction in the kidneys. Steady-state absorbance, fluorescence, and circular dichroism (CD) methods were used to study the interactions between PFOA and Hb. The results demonstrate the presence of multiple PFOA binding sites on the Hb protein. The detailed analysis of the ferric hemoglobin protein (met Hb) absorbance data as a function of PFOA concentration indicates the presence of at least two binding sites with equilibrium dissociation constants of 0.8 ± (0.2) × 10−6 M and 63 ± (15) × 10−5 M. A competitive binding study with 1,8-ANS showed PFOA can bind to the same binding site as 1,8-ANS on the Hb protein. The titration curve for PFOA binding to Hb in its CO bound form (CO-Hb) yields a single equilibrium dissociation constant of 139 ± (20) × 10−6 M. PFOA binding at low concentrations occurs at the high-affinity sites leading to the destabilization of the protein structure as reflected by changes in the CD spectrum. PFOA interactions with Hb also interfere with the kinetics of CO association to this protein. The rate for CO association to Hb increases at low PFOA concentrations, whereas at elevated PFOA concentrations, the ligand association is biphasic as a new kinetic process with a different rate constant was observed. Overall, this study provides a detailed explanation of PFOA-induced structural and conformational changes to the Hb protein based on the spectroscopy data.

A Cascade Nanoreactor of Metal-Protein-Polyphenol Capsule for Oxygen-Mediated Synergistic Tumor Starvation and Chemodynamic Therapy.

Starvation therapy kills tumor cells via consuming glucose to cut off their energy supply. However, since glucose oxidase (GOx)-mediated glycolysis is oxygen-dependent, the cascade reaction based on GOx faces the challenge of a hypoxic tumor microenvironment. By decomposition of glycolysis production of H2 O2 into O2 , starvation therapy can be enhanced, but chemodynamic therapy is limited. Here, a close-loop strategy for on demand H2 O2 and O2 delivery, release, and recycling is proposed. The nanoreactor (metal-protein-polyphenol capsule) is designed by incorporating two native proteins, GOx and hemoglobin (Hb), in polyphenol networks with zeolitic imidazolate framework as sacrificial templates. Glycolysis occurs in the presence of GOx with O2 consumption and the produced H2 O2 reacts with Hb to produce highly cytotoxic hydroxyl radicals (•OH) and methemoglobin (MHb) (Fenton reaction). Benefiting from the different oxygen carrying capacities of Hb and MHb, oxygen on Hb is rapidly released to supplement its consumption during glycolysis. Glycolysis and Fenton reactions are mutually reinforced by oxygen supply, consuming more glucose and producing more hydroxyl radicals and ultimately enhancing both starvation therapy and chemodynamic therapy. This cascade nanoreactor exhibits high efficiency for tumor suppression and provides an effective strategy for oxygen-mediated synergistic starvation therapy and chemodynamic therapy.

The Balancing of Peroxynitrite Detoxification between Ferric Heme-Proteins and CO2: The Case of Zebrafish Nitrobindin.

Nitrobindins (Nbs) are all-β-barrel heme proteins and are present in prokaryotes and eukaryotes. Although their function(s) is still obscure, Nbs trap NO and inactivate peroxynitrite. Here, the kinetics of peroxynitrite scavenging by ferric Danio rerio Nb (Dr-Nb(III)) in the absence and presence of CO2 is reported. The Dr-Nb(III)-catalyzed scavenging of peroxynitrite is facilitated by a low pH, indicating that the heme protein interacts preferentially with peroxynitrous acid, leading to the formation of nitrate (~91%) and nitrite (~9%). The physiological levels of CO2 dramatically facilitate the spontaneous decay of peroxynitrite, overwhelming the scavenging activity of Dr-Nb(III). The effect of Dr-Nb(III) on the peroxynitrite-induced nitration of L-tyrosine was also investigated. Dr-Nb(III) inhibits the peroxynitrite-mediated nitration of free L-tyrosine, while, in the presence of CO2, Dr-Nb(III) does not impair nitro-L-tyrosine formation. The comparative analysis of the present results with data reported in the literature indicates that, to act as efficient peroxynitrite scavengers in vivo, i.e., in the presence of physiological levels of CO2, the ferric heme protein concentration must be higher than 10−4 M. Thus, only the circulating ferric hemoglobin levels appear to be high enough to efficiently compete with CO2/HCO3− in peroxynitrite inactivation. The present results are of the utmost importance for tissues, like the eye retina in fish, where blood circulation is critical for adaptation to diving conditions.

Ultraviolet light oxidation of fresh hemoglobin eliminates aggregate formation seen in commercially sourced hemoglobin

Elevated levels of circulating cell-free hemoglobin (CFH) are an integral feature of several clinical conditions including sickle cell anemia, sepsis, hemodialysis and cardiopulmonary bypass. Oxidized (Fe3+, ferric) hemoglobin contributes to the pathophysiology of these disease states and is therefore widely studied in experimental models, many of which use commercially sourced CFH. In this study, we treated human endothelial cells with commercially sourced ferric hemoglobin and observed the appearance of dense cytoplasmic aggregates (CAgg) over time. These CAgg were intensely autofluorescent, altered intracellular structures (such as mitochondria), formed in multiple cell types and with different media composition, and formed regardless of the presence or absence of cells. An in-depth chemical analysis of these CAgg revealed that they contain inorganic components and are not pure hemoglobin. To oxidize freshly isolated hemoglobin without addition of an oxidizing agent, we developed a novel method to convert ferrous CFH to ferric CFH using ultraviolet light without the need for additional redox agents. Unlike commercial ferric hemoglobin, treatment of cells with the fresh ferric hemoglobin did not lead to CAgg formation. These studies suggest that commercially sourced CFH may contain stabilizers and additives which contribute to CAgg formation.

Food‐induced methemoglobinemia: A systematic review

AbstractMethemoglobinemia is a sporadic, potentially fatal disease of poor tissue oxygenation in which ferrous hemoglobin (Fe2+) is oxidized to the ferric (Fe3+) state, rendering it incapable of binding oxygen (O2). Fortunately, it is diagnosable and treatable. Here, we present a systematic review of food‐induced methemoglobinemia. PubMed and Embase databases were searched using the term “methemoglobin*,” for articles up to December 31, 2020. Inclusion criteria were confirmed or probable cases of methemoglobinemia with an oxidant confirmed in food or body tissue samples, or the oxidant likely to have come from food. We found 97 articles describing 568 cases. Median age was 6 years (range: 2 weeks to 80 years). Median methemoglobin fraction was 30% (n = 142). Oxidizing agents were predominantly nitrites and nitrates. The commonest type of presentation was children eating vegetables (30%), followed by accidental ingestions (27%), and meat curing misadventures (22%). Favism was found to result in mild methemoglobinemia, highest fraction reported was 15.8%. Of the 35 deaths, 32 were from accidental ingestions (91%). In some fatal cases, diagnosis was likely delayed or missed, and antidote was not administered. The majority of cases survived, even with severe methemoglobin levels of up to 89%, provided that methylene blue was administered. Treatment with methylene blue alone resulted in an average methemoglobin drop of 39.1% (n = 22). Methemoglobinemia cases continue to occur due to accidental exposure, meat curing misadventures, and babies ingesting nitrate‐rich vegetables which have been inappropriately stored. Early recognition of the toxidrome, instituting antidote treatment, and notifying public health authorities are key to improved outcomes.

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.

A rare case of congenital methemoglobinemia with severe mitral stenosis: An anesthetic and surgical challenge

Methaemoglobinemia is a condition that occurs when red blood cell contains higher than 1% methaemoglobin (MHb). It can be congenital or acquired. The condition is generally asymptomatic until the serum MHb level reaches as high as 40%. Dyspnea, acidosis, cardiac dysrhythmias, heart failure, seizures, and coma may occur at levels exceeding 45%, and a high mortality rate is seen with levels above 70%. We report a rare association of a case of rheumatic heart disease with severe mitral stenosis with congenital methemoglobinemia diagnosed incidentally during mitral valve replacement procedure under cardiopulmonary bypass.

Ferrous hemoglobin and hemoglobin-based oxygen carriers acting as a peroxidase can inhibit oxidative damage to endothelial cells caused by hydrogen peroxide.

Oxidative damage caused by the ferryl hemoglobin is one of the major clinical adverse reactions of hemoglobin-based oxygen carriers (HBOCs), while the production of reactive oxygen species in a pathological state can oxidize hemoglobin (HbFe2+ ) to ferryl Hb, which can then enter the pseudoperoxidase cycle, making hemoglobin highly toxic. In this study, we found that ferrous hemoglobin and polymerized porcine hemoglobin (one of the HBOCs) have the peroxidase activity different from the pseudoperoxidase activity of ferric hemoglobin. Ferrous hemoglobin can catalyze the reaction of tyrosine (Tyr) with hydrogen peroxide. In addition, the results also indicated that ferrous hemoglobin and pPolyHb have a strong inhibitory effect on the pseudoperoxidase activity of ferric hemoglobin. Therefore, hydrogen peroxide was consumed in a large amount, which greatly prevented hemoglobin from becoming oxidized and entering the pseudoperoxidase cycle, thus inhibiting ferryl Hb toxicity. We further cultured human umbilical vein endothelial cells and monitored cell morphology, viability, cell cycle, apoptosis, lactate dehydrogenase (LDH) release, and malondialdehydes (MDAs) formation when incubated with H2 O2 , Tyr, and HbFe2+ . HbFe2+ and pPolyHb reduced cell cycle arrest, apoptosis, LDH release, and MDA formation. These results showed that reducing oxidative damage induced by H2 O2 and converted hemoglobin from a molecule that is toxic to one that inhibits oxidative damage, suggesting a new strategy for development of a safer HBOCs.

A106 BENZOCAINE-INDUCED METHEMOGLOBINEMIA: A RARE BUT POTENTIALLY FATAL COMPLICATION OF TOPICAL ANESTHETIC USE IN ESOPHAGOGASTRODUODENOSCOPY

Abstract Background Methemoglobinemia is an uncommon and potentially lethal condition arising when the iron moiety of heme is oxidized from the ferrous (Fe2+) to ferric (Fe3+) state, causing a disruption of O2 binding and impairment of O2 delivery to tissue. There are two forms of methemoglobinemia: inherited and acquired. Acquired methemoglobinemia is caused by medications, including topical anesthetics (TAs) used in esophagogastroduodenoscopy (EGD). Aims To raise awareness of this serious complication of TA use. Methods A 66-year old woman underwent EGD for dysphagia and globus sensation. Her past medical history included gastroesophageal reflux disease, paraoesophageal hernia, coronary artery disease, and hypertension. Prior to EGD, her oropharynx was anesthetized with benzocaine spray and she was sedated with fentanyl 100 mcg and midazolam 3 mg. EGD was unremarkable. At completion she required no supplemental oxygen (SpO2 96%). In recovery, she became drowsy and cyanotic. Vital signs revealed an SpO2 of 86% that did not improve with 4L supplementary O2 via nasal prongs. Aside from cyanosis, physical examination was normal. ECG, chest X-ray, and CT scan with pulmonary embolism protocol did not identify a cause for hypoxia. Arterial blood gas (ABG) revealed dark brown blood with an PaO2 of 397 mmHg. Methemoglobinemia was suspected and confirmed with a serum methemoglobin (MHg) level of 18% (ref <3%). Results Intravenous methylene blue (1g/kg) was administered with complete resolution of symptoms. She was admitted for monitoring and discharged the next day with no sequelae. Conclusions Methemoglobinemia is a rare condition (incidence 1/7000 procedures) of increased MHg due to the oxidation of iron in heme, compromising oxygen binding/offloading and delivery to tissues. Normal MHg levels are <3%, which is maintained by NADH-MHg reductase. This enzyme reduces Fe3+ to Fe2+. TAs cause elevated methemoglobinemia by oxidizing iron (Fe2+ to Fe3+) at a rate 100-1000x faster than NADH-MHg reductase can reduce it. Benzocaine has been associated with a 3.7- fold higher risk of methemoglobinemia than other topical anesthetics, and the risk is not dose-dependent. Presentation varies with degree of MHg. Hallmark signs of cyanosis, impaired SpO2 with normal ABG, and “chocolate-colored” blood occur at levels>10%. Death/coma occurs at levels >50%. Incidence is idiosyncratic; however, those with smoking history or underlying cardiac/lung disease are at elevated risk for poor outcomes. The activity of NADH-MHg reductase is increased by methylene blue. When administered at doses of 1-2g/kg, it rapidly reduces MHg, restoring the physiological equilibrium. TAs are used routinely in EGD. Gastroenterologists who use TAs in procedures need to be aware of the association between TAs and methemoglobinemia. Prompt recognition/treatment will prevent morbidity/mortality. Funding Agencies None

Nitrate-nitrite toxicosis associated with duckweed (Portulaca Oleracea L.) (Portulacaceae) consumption in a herd of sheep in Kenya

Background: Nitrate-nitrite toxicosis associated with the consumption of duckweed (Portulaca oleracea L.) (Portulacaceae) was diagnosed in a small herd comprising of 11 sheep in Korayo Sub Location, Rangwe Sub County of Homabay County in Kenya. Methods: The clinical signs were dyspnea, froth from the mouth, labored breathing, incoordination, tachycardia, increased urination, neck, fore and hind limb distension, aggressive movements, bloat, convulsions, and coma, before death. Results: All the 11 sheep died of intoxication before institution of therapy. Brownish mucous membranes and chocolate colored and poorly clotted blood were the significant necropsy observations. Mild pulmonary edema was characterized by the presence of limited froth in the trachea and bronchioles. At postmortem, the liver of the sheep was grossly congested, and the cardiac pericardium was inflamed. There was gastritis, enlarged, and congested kidneys with pin-point hemorrhages. Diphenylamine tests carried out on the fed duckweed and on ruminal duckweed contents were positive for nitrates. Duckweed (Portulaca oleracea) fed on sheep had 7.4% nitrate on dry matter basis (DM), whereas ruminal contents had 5.38% nitrate on DM basis. Chocolate-colored blood collected during postmortem had a methemoglobin fraction of 86%. Nitrate levels in pastures of 0.5% and above have been shown to be potentially dangerous, with acute poisoning likely to occur at times when the levels are above 1%. Death in sheep can occur when blood methemoglobin levels are in the range of 67%–90%. Conclusion: The observations from the present study are indicative of death in a herd of sheep due to nitrate-nitrite poisoning associated with the consumption of duckweed (Portulaca oleracea).

Deficiência da glicose-6-fosfato desidrogenase (G6PD) em recém-nascidos, numa maternidade pública da cidade de Campina Grande, Estado da Paraíba, Brasil

Nos eritrócitos deficientes de glicose 6 fosfato desidrogenase (G6PD), a diminuição da redução de NADP em NADPH, leva a um baixo potencial redutor que interfere na capacidade metabólica oxidativa do organismo, ficando vulnerável a hemólise, com oxidação da membrana do glóbulo, podendo levar a crise hemolíticas. As manifestações da deficiência de G6PD são desencadeadas por fatores ambientais como drogas, fava, infecções. As manifestações clínicas consistem de crise hemolítica e icterícia neonatal. Estudos indicam que no Brasil a prevalência de G6PD está em torno de 1 a 10%, sendo os maiores índices em populações com ancestralidade africana. O objetivo foi a pesquisa da deficiência da G6PD em recém-nascidos numa maternidade pública na cidade de Campina Grande. Trata-se de um estudo transversal, onde foram coletados sangue do cordão umbilical de 147 recém-nascidos. O diagnóstico da deficiência da G6PD será realizado através do teste da redução da metahemoglobina (teste de Brewer). A cidade de Campina Grande, bem como todo o Compartimento da Borborema não apresentam nenhum dado relativo a prevalência da deficiência da G6PD. O Ministério da Saúde fortalece através do SUS a coleta de informações relativas a esta doença na perspectiva de implantação de um programa de atenção à saúde dos doentes detectados, promovendo melhoras na sua qualidade de vida.

Comparison of methemoglobin levels in food protein-induced enterocolitis syndrome and other gastrointestinal diseases in neonates

Introduction and objectivesMethemoglobinemia has been reported to be associated with severe food protein-induced enterocolitis syndrome (FPIES). However, no reports have evaluated methemoglobin (MHb) levels in FPIES without symptomatic methemoglobinemia or the usefulness of MHb measurement for the diagnostic prediction of FPIES. To evaluate the MHb levels of patients with neonatal-onset FPIES and determine whether MHb levels are higher in FPIES than in other gastrointestinal diseases. Patients and methodsEleven neonates with severe acute FPIES (FPIES group) and 139 neonates with other gastrointestinal diseases (non-FPIES group) were included in this study. Patient characteristics, symptoms, and venous blood test values (MHb, pH, HCO3−, and C-reactive protein) were evaluated. ResultsThe median age at onset was 16 days vs. 1 day; males comprised 64% vs. 46%, the median gestational age was 38 weeks vs. 38 weeks, the median birth weight was 2710g vs. 2880g, and the median hospitalization duration was 31 days vs. 6 days for the FPIES vs. non-FPIES groups, respectively. MHb (%) was higher in the FPIES group than in the non-FPIES group [median (range), 1.1 (0.6–10.9) and 0.6 (0.3–1.2), respectively, p<0.001]. There were no differences in terms of pH, HCO3−, and C-reactive protein (p>0.05). In the receiver operating characteristic analysis for FPIES diagnosis based on MHb (%), the area under the curve was 0.885, specificity was 97.1%, and sensitivity was 72.7% at a MHb cutoff of 1.0. ConclusionHigh MHb levels may help diagnose severe acute FPIES in neonates, but careful evaluation is needed.

Oxidized Hemoglobin Causes Human Lung Microvascular Endothelial Barrier Dysfunction

Cell‐free hemoglobin is released into the circulation during conditions in which red blood cell lysis occurs such as hemolytic disorders and sepsis. In septic patients, increased levels of circulating cell‐free hemoglobin are independently associated with mortality and contribute to organ dysfunction, including acute respiratory distress syndrome (ARDS). Furthermore, during inflammatory conditions in which reactive oxygen species are high, cell‐free hemoglobin can be converted to higher oxidation states which enhances its damaging capacity. We previously showed that hemoglobin contributes to tissue edema in an ex vivo perfused human lung and microvascular endothelial barrier dysfunction in vitro; hemoprotein reductants (ascorbate and acetaminophen) protected against this phenomenon. Therefore, we hypothesized that higher oxidation states of hemoglobin are more injurious to the barrier function of lung microvascular endothelium. Utilizing primary human lung microvascular endothelial cells (HLMVEC), we found that stimulation with 0.5 mg/mL ferric (Hb3+; −380.2 Ω, p&lt;0.001 vs. control) but not ferrous (Hb2+; +278.3 Ω, p=0.9893 vs. control) hemoglobin resulted in a drop in transendothelial electrical resistance (Figure ). The barrier‐disrupting effects of Hb3+ were reduced with haptoglobin (binds free hemoglobin; +208.5 Ω, p&lt;0.0001 vs. Hb3+) and hemopexin (binds free heme), but not the iron chelator deferoxamine. The mechanism of Hb3+‐mediated microvascular endothelial barrier dysfunction involved disruption of adherens junction protein VE‐cadherin at cell‐cell borders and rearrangement of the actin cytoskeleton with intercellular gap formation. These data suggest that oxidized cell‐free hemoglobin causes microvascular endothelial barrier dysfunction to a greater degree than its non‐oxidized form.Support or Funding InformationThis work is funded by NIH HL135849, HL103836, and HL094296.Figure 1