Little is known about genetic causes of congenital methemoglobinemia in dogs. Here, we report a CYB5 R3 mutation in a Pomeranian dog with congenital methemoglobinemia. A 6-year-old neutered female Pomeranian dog was investigated for cyanosis noticed during anesthesia for an orthopedic procedure. The history included lifelong mild exercise intolerance and bluish tongue. Methemoglobinemia was diagnosed using co-oximetry. The CYB5 R3 gene was analyzed by comparing the patient's genomic DNA with the reference canine sequence. Mutation functional significance was investigated using snpEff and multispecies protein homology analyses. A homozygous missense single nucleotide CYB5 R3 mutation (ATC ➔ CTC at codon 194) caused a p.Ile194Leu substitution. The pIle194 residue is highly conserved in other mammals, supporting the likely pathogenicity of the substitution. The mutation described here is identical to that associated with familial methemoglobinemia in a family of Japanese Pomeranian dogs. This observation, together with the homozygous mutation found in our case, indicates that the mutant allele may be widespread within the Pomeranian breed internationally.

A major concern during pesticide development and use is the impact on non-target species, such as raptors or domestic cats and dogs. Sodium nitrite and para-aminopropiophenone (PAPP) are two toxicants currently being studied for the control of invasive species, such as starlings and feral swine. When given to an animal these compounds oxidize hemoglobin, which renders it unable to carry oxygen resulting in methemoglobinemia. This study developed a method to estimate methemoglobin levels in mammals and birds by examining the efficacy of sodium nitrite to induce the conversion of hemoglobin to methemoglobin. Varying concentrations of sodium nitrite were added to aliquots of coyote, vole, feral swine, starling, and duck blood, collected from captive animals. The blood samples were analyzed spectrophotometrically to determine percent methemoglobin and digitally to determine red color values (RCV) associated with different methemoglobin levels. The avian and mammalian blood reached 100% methemoglobin levels at 200 mM and 15 mM sodium nitrite, respectively. All animals had similar RCV for a given percent methemoglobin. In conclusion, this study developed a procedure to quickly determine methemoglobin levels in mammals and birds. Furthermore, percent methemoglobin can be estimated with one standard curve from any animal species and an image of a blood spot. The technique will be useful during field studies, in agricultural areas, or in a veterinarian's office for the rapid diagnosis of methemoglobinemia in non-target animals that have eaten toxicants/baits or baited animals.

Methylene blue is useful for the treatment of methemoglobinemia. However, even after the patient's methemoglobin (metHb) rate has improved, careful observation is important because they could have undiagnosed congenital methemoglobinemia. In this case, a 67-year-old man underwent gastrointestinal endoscopy with the use of lidocaine for local anesthesia. During the examination, he complained of dyspnea and had low SpO2 despite normal PaO2 and SaO2. He was transferred to our department as a suspected case of acquired methemoglobinemia.

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

Dapsone (diaminodiphenyl sulfone, DDS) is currently used to treat leprosy, malaria, dermatitis herpetiformis, and other diseases. It is also used to treat pneumocystis pneumonia and Toxoplasma gondii infection in HIV-positive patients. The most common adverse effect of DDS is methemoglobinemia from oxidative stress. Ascorbic acid is an antioxidant and reducing agent that scavenges the free radicals produced by oxidative stress. The present study aimed to investigate the effect of ascorbic acid in the treatment of DDS induced methemoglobinemia.

Background and Objective: Mutations in the CYB5R3 gene cause reduced NADH-dependent cytochrome b5 reductase enzyme function and consequently lead to recessive congenital methemoglobinemia (RCM). RCM exists as RCM type I (RCM1) and RCM type II (RCM2). RCM1 leads to higher methemoglobin levels causing only cyanosis, while in RCM2, neurological complications are also present along with cyanosis. Materials and Methods: In the current study, a consanguineous Pakistani family with three individuals showing clinical manifestations of cyanosis, chest pain radiating to the left arm, dyspnea, orthopnea, and hemoptysis was studied. Following clinical assessment, a search for the causative gene was performed using whole exome sequencing (WES) and Sanger sequencing. Various variant effect prediction tools and ACMG criteria were applied to interpret the pathogenicity of the prioritized variants. Molecular dynamic simulation studies of wild and mutant systems were performed to determine the stability of the mutant CYB5R3 protein. Results: Data analysis of WES revealed a novel homozygous missense variant NM_001171660.2: c.670A > T: NP_001165131.1: p.(Ile224Phe) in exon 8 of the CYB5R3 gene located on chromosome 22q13.2. Sanger sequencing validated the segregation of the identified variant with the disease phenotype within the family. Bioinformatics prediction tools and ACMG guidelines predicted the identified variant p.(Ile224Phe) as disease-causing and likely pathogenic, respectively. Molecular dynamics study revealed that the variant p.(Ile224Phe) in the CYB5R3 resides in the NADH domain of the protein, the aberrant function of which is detrimental. Conclusions: The present study expanded the variant spectrum of the CYB5R3 gene. This will facilitate genetic counselling of the same and other similar families carrying mutations in the CYB5R3 gene.

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.

The objectives of this review are to describe the acquired and hereditary causes of methemoglobinemia, to recommend the most sensitive diagnostic tests, and to enable critical care clinicians to rapidly detect and treat methemoglobinemia. To meet these objectives, Internet search engines were queried with the keywords to select articles for review that included case reports, case series, observational, longitudinal, and surveillance studies. The most common causes of methemoglobinemia include oxidizing reactions to cocaine-derived anesthetics, such as benzocaine and lidocaine, to antibiotics, such as dapsone and other sulfonamides, and to gases, such as nitric oxide. Additionally, CO-oximetry is superior to standard pulse oximetry in detecting methemoglobinemia. Finally, effective treatments for methemoglobinemia include intravenous administration of methylene blue, ascorbic acid, and riboflavin. In this manuscript we will discuss methemoglobinemia, how it occurs, and how to treat it.

Hereditary methemoglobinemia associated with nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) deficiency is a rare autosomal recessive disorder in animals. Recently, nonsynonymous b5R gene (CYB5R3) variants have been reported to be associated with canine and feline hereditary methemoglobinemia. However, the underlying molecular mechanisms of canine and feline methemoglobinemia caused by these nonsynonymous variants have not yet been reported. Previously, we reported a Pomeranian dog family with hereditary methemoglobinemia, carrying CYB5R3 mutation of an A>C transition at codon 194 in exon 7, replacing an isoleucine residue with leucine (p.Ile194Leu). In this study, we investigated the enzymatic and structural properties of the soluble form of wild-type and Ile194Leu canine b5Rs to characterize the effects of this missense mutation. Our results showed that the kinetic properties of the mutant enzyme were not affected by this amino acid substitution. The secondary structure of the wild-type and Ile194Leu b5Rs detected by circular dichroism showed a similar pattern. However, the mutant enzyme exhibited decreased heat stability and increased susceptibility to trypsin hydrolysis. Moreover, the thermostability and unfolding measurements indicated that the mutant enzyme was more sensitive to temperature-dependent denaturation than the wild-type b5R. We concluded from these results that unstable mutant enzyme properties with normal enzymatic activity would be associated with hereditary methemoglobinemia in the Pomeranian dog family.

The protein α -synuclein is considered central to the pathogenesis of Parkinson disease (PD) on genetic and histopathological grounds. It is widely expressed in fetal life and continues to be highly expressed in adult neural tissues, red blood cells and platelets, while the remainder of adult tissues are reported to have little or no expression. Despite cellular and molecular evidence for a role in neuronal function including synaptic vesicle trafficking, neurotransmitter release, mitochondrial function, lipid metabolism, neurogenesis, neuroprotection, and neuromelanin biosynthesis, mice ablated for the gene encoding α -synuclein (Snca) have little or no neurological phenotype. Thus, nearly 20 years of intensive study have yet to reveal conclusively what the normal function of this highly abundant protein is in the nervous system. Interestingly, α -synuclein has also been shown to have enzymatic activity as a ferrireductase capable of reducing Fe+3 to Fe+2. Given its abundant expression in red blood cells, we set out to explore the role of α -synuclein in converting chemically-induced Fe+3 methemoglobin to normal Fe+2 hemoglobin. Initial in vivo experiments with the potent methemoglobin inducer, para-aminopropiophenone and its active metabolite, 4-hydroxy para-aminopropiophenone, demonstrated significantly greater and more prolonged methemoglobinemia in Snca-/- mice compared to Snca+/+ mice. In vitro experiments with red blood cells, however, and in vivo experiments in genetically engineered mouse strains that differ in their α -synuclein expression in various tissues, including the nervous system, red blood cells and liver, revealed that contrary to the initial hypothesis, a lack of expression of α -synuclein in red blood cells did not correlate with higher levels or more prolonged duration of methemoglobinemia. Instead, the greater sensitivity to chemically induced methemoglobinemia correlated with the absence of hepatic α -synuclein expression. We have uncovered a new and robust whole-animal phenotype in mice lacking α -synuclein that reflects its hitherto unrecognized role in xenobiotic detoxification.

The aim of this study was to clarify, using in vitro and in vivo approaches in the rat, the site of mediation of the inhibition of H(2)S induced arterial chemoreceptor stimulation, by hyperoxia and methemoglobinemia. We first determined the ventilatory dose-response curves during intravenous injections of H(2)S. A very high dose of NaHS, i.e. 0.4 μmol (concentration: 800 μM), was needed to stimulate breathing within 1s following i.v. injection. Above this level (and up to 2.4 μmol, with a concentration of 4800 μM), a dose-dependent effect of H(2)S injection was observed. NaHS injection into the thoracic aorta produced the same effect, suggesting that within one circulatory time, H(2)S pulmonary exchange does not dramatically reduce H(2)S concentrations in the arterial blood. The ventilatory response to H(2)S was abolished in the presence of MetHb (12.8%) and was significantly depressed in hyperoxia and, surprisingly, in 10% hypoxia. MetHb per se did not affect the ventilatory response to hypoxia or hyperoxia, but dramatically enhanced the oxidation of H(2)S in vitro, with very fast kinetics. These findings suggest that, the decrease/oxidation of exogenous H(2)S in the blood is the primary effect of MetHb in vivo. In contrast, the in vitro oxidative properties of blood for H(2)S were not affected by the level of [Formula: see text] between 23 and >760 mmHg. This suggests that the inhibition of the ventilatory response to H(2)S by hyperoxia during aortic or venous injection originates within the CB and not in the blood. The implications of these results on the role of endogenous H(2)S in the arterial chemoreflex are discussed.

In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology.

Cyanosis in an apparently healthy newborn baby may be caused by hemoglobin variants associated with the formation of methemoglobin, collectively known as M hemoglobins. They should not be confused with genetic alterations in methemoglobin reductase enzyme systems of red cells since treatment and prognosis are completely different. A newborn male child was noted to be significantly cyanotic at birth and is the basis for this report. Hemoglobin isoelectric focusing, acid and alkaline gel electrophoresis, and HBA/HBB gene sequencing were performed for the child, both parents and a sister. The newborn child was treated with methylene blue in an intensive care unit fearing that he had a defective reductase system and exposure to oxidant drugs or toxins. Newborn hemoglobin screening with high performance liquid chromatography was abnormal on the 10th and 45th days but no conclusive diagnosis was reached. Cyanosis persisted up to four years of age with no other symptoms. Hemoglobin M Iwate [alpha2 87(F8) His>Tyr, HBA2:c.262C>T] was detected. It was not present in the child's presumed mother, father, sister, and brother. The analysis of 15 short tandem repeats in the trio demonstrated a de novo mutation occurrence (p-value<1×10(-8)). The family was reassured that no further action was necessary and genetic counseling was provided. Methemoglobins should be considered for differential diagnosis of cyanosis in newborns even if no familial cases are detected. Except for cosmetic consequences, the clinical course of patients with hemoglobin M Iwate is unremarkable.

Genotype-phenotype correlations of humans and dogs with hereditary methemoglobinemia are not yet well characterized. We determined total hemoglobin and methemoglobin (MetHb) concentrations, cytochrome b5 reductase (CYB5R) enzyme activities, genotypes, and clinical signs in 30 dogs with persistent cyanosis without cardiopulmonary disease. Erythrocytic CYB5R enzyme activities were low in all dogs assayed. Owner-reported quality of life ranged from subclinical to occasional exertional syncope. Two previously reported and two novel CYB5R3 missense variants were identified among the methemoglobinemic cohort and were predicted to impair enzyme function. Two variants were recurrent: a homozygous Ile194Leu substitution was found in Pomeranians and other small dogs, and a homozygous Arg219Pro change occurred predominately in pit bull terriers. The other two variants were Thr202Ala and Gly76Ser substitutions in single dogs. Of the two common CYB5R3 genotypes, Arg219Pro was associated with a more severe metabolic phenotype. We conclude that CYB5R3 deficiency is the predominate cause of canine hereditary methemoglobinemia. Although this finding is unlikely to alter the clinical approach to hereditary methemoglobinemia in dogs, it demonstrates the possibility of how genotype-phenotype cohort analysis might facilitate precision medicine in the future in veterinary medicine.

No abstract available

Recessive hereditary methemoglobinemia (RCM) associated with severe neurological abnormalities is a very rare disorder caused by NADH- cytochrome b5 reductase (cb5r) deficiency (Type II). We report a case of 11 month old male child who had severe mental retardation, microcephaly and gross global developmental delay with methemoglobin level of 61.1%. The diagnosis of NADH-CYB5R3 deficiency was made by the demonstration of significantly reduced NADH-CYB5R3 activity in the patient and intermediate enzyme activity in both the parents. Mutation analysis of the CYB5R gene revealed a novel nine nucleotide deletion in exon 6 leading to the elimination of 3 amino acid residues (Lys173, Ser174 and Val 175). To confirm that this mutation was not an artifact, we performed PCR-RFLP analysis using the restriction enzyme Drd I. As the normal sequence has a restriction recognition site for Drd I which was eliminated by the deletion, a single band of 603-bp was seen in the presence of the homozygous mutation. Molecular modeling analysis showed a significant effect of these 3 amino acids deletion on the protein structure and stability leading to a severe clinical presentation. A novel homozygous 9 nucleotide deletion (p.K173-p.V175del3) is shown to be segregated with the disease in this family. Knowing the profile of mutations would allow us to offer prenatal diagnosis in families with severe neurological disorders associated with RCM - Type II.

The interaction between chlorhexidine (CHX) and sodium hypochlorite (NaOCl) yields a thick precipitate capable of occluding dentinal tubules. Previous studies are unclear as to the above-mentioned precipitate contains para-chloroaniline (PCA) or not. PCA is a known toxic and carcinogenic compound which may lead to methemoglobinemia in humans.

Acalypha indica is a tropical plant used as a herbal medicine in various parts of the world, including Thailand. In glucose-6-phosphate dehydrogenase (G6PD)-deficient patients, acute hemolysis has been reported following the ingestion of this plant. Methemoglobinemia was reported in the present study.

Methemoglobinemia after pesticide poisoning is associated with a mortality of 12% in Sri Lanka. Treatment is complicated by the lack of laboratory facilities. We aimed to develop and validate a low-cost bedside test for quantitative estimation of clinically significant methemoglobin to be used in settings of limited resources.

Humans are exposed to nitrate predominantly through diet with peak plasma concentrations within an hour after ingestion, but additional exposure is obtained from the environment, and minimally through de novo synthesis. Higher nitrate consumption has been associated with methemoglobinemia, spontaneous abortions, atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, amyotrophic lateral sclerosis, and neural tube defects. However, skeletal muscle development has not been examined.