Methemoglobinemia, Increased Deformability and Reduced Membrane Stability of Red Blood Cells in a Cat with a CYB5R3 Splice Defect.

Methemoglobinemia is an acquired or inherited condition resulting from oxidative stress or dysfunction of the NADH-cytochrome b5 reductase or associated pathways. This study describes the clinical, pathophysiological, and molecular genetic features of a cat with hereditary methemoglobinemia. Whole genome sequencing and mRNA transcript analyses were performed in affected and control cats. Co-oximetry, ektacytometry, Ellman's assay for reduced glutathione concentrations, and CYB5R activity were assessed. A young adult European domestic shorthair cat decompensated at induction of anesthesia and was found to have persistent methemoglobinemia of 39 ± 8% (reference range < 3%) of total hemoglobin which could be reversed upon intravenous methylene blue injection. The erythrocytic CYB5R activity was 20 ± 6% of normal. Genetic analyses revealed a single homozygous base exchange at the beginning of intron 3 of the CYB5R3 gene, c.226+5G>A. Subsequent mRNA studies confirmed a splice defect and demonstrated expression of two mutant CYB5R3 transcripts. Erythrocytic glutathione levels were twice that of controls. Mild microcytosis, echinocytes, and multiple Ca2+-filled vesicles were found in the affected cat. Erythrocytes were unstable at high osmolarities although highly deformable as follows from the changes in elongation index and maximal-tolerated osmolarity. Clinicopathological presentation of this cat was similar to other cats with CYB5R3 deficiency. We found that methemoglobinemia is associated with an increase in red blood cell fragility and deformability, glutathione overload, and morphological alterations typical for stress erythropoiesis.

Emergency treatment with intravenous infusion of methylene blue followed by oral administration in a cat presented with severe recurrent methemoglobinemia.

OBJECTIVE: To describe the use of IV infusion followed by oral administration of methylene blue (MB) to successfully treat recurrent methemoglobinemia (MetHb) in a young cat. CASE SUMMARY: A 6-month-old male Ragdoll cat presented with recurrent episodes of severe MetHb and was successfully managed with IV infusion of MB followed by a course of oral MB. Although the definitive cause of the patient's MetHb remains unknown, the cat made a full recovery following treatment without developing any significant side effects secondary to therapy and at the time of writing not had any further recurrences. Follow-up at 6 months found the patient in good health and without any long-term consequences. NEW INFORMATION PROVIDED: To the authors' knowledge, this is the first report of a cat presented with severe MetHb quantitatively assessed via co-oximetry and successfully treated with both IV and oral administration of MB.

Monitoring methaemoglobinaemia in birds using 5 µL of whole blood.

Methaemoglobin (MetHb) forming compounds such as para-aminopropiophenone (PAPP) and sodium nitrite (NaNO2) have recently been adopted for the lethal control of a range of invasive carnivores and mustelids. Determining the relative hazard of these compounds to non-target bird species is an important component of ecological risks evaluation. Problematically, some potential non-target bird species may be as small as 10 g in body mass, thus placing limitations on blood volumes that can be routinely sampled. Accordingly, we developed methods to quantify markers of increasing methaemoglobinaemia at their point of collection that required only 5 µL of whole blood. A 3 µL blood aliquot is pipetted into a plastic micro-cuvette and placed in a custom made holder optically coupled to the Ocean Optics spectrometer, enabling absorbance for oxyhaemoglobin (HbO: 575 nm) and MetHb (630 nm) to be determined. Haemoglobin (HbFe2+), packed cell volume (PCV) and lactate (LAC) data were generated from the remaining 2 µL aliquot apportioned to biosensor strips for the Cera-Check® and Lactate Scout® point-of-care devices. After oral doses of PAPP, a methaemoglobinaemia absorbance index (MAI = absorbance 575 nm-absorbance 630 nm) was strongly and significantly associated with dose-dependent declines in HbFe2+ in 9 bird species. Quantifying dose-dependent responses to MetHb-forming agents at the point of sample collection avoids analytical and storage artifacts arising from sample degradation that appears to be a much greater problem in avian blood compared to mammalian blood.

Inflammatory phenotype, clinicopathologic variables, and effects of long-term methylene blue in dogs with hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency.

OBJECTIVE: To determine whether dogs with cytochrome b5 reductase (CYB5R) deficiency have a constitutive proinflammatory phenotype, characterize hematologic and serum chemistry results, and describe changes in methemoglobin (MetHb) levels and serum C-reactive protein (CRP) concentrations after long-term per os (PO) methylene blue (MB) therapy. ANIMALS: 21 client-owned dogs (CYB5R deficient, n = 10; healthy controls, 11). PROCEDURES: In this prospective, case-control study, methemoglobin levels were measured using a blood gas analyzer with co-oximetry. Plasma tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) concentrations were measured using a canine-specific multiplex bead-based assay. Serum CRP concentrations were measured with a canine-specific commercial ELISA kit. Serum CRP concentration and MetHb levels were measured in 6 dogs with CYB5R deficiency after ≥ 60 days of PO MB therapy. RESULTS: As expected, MetHb levels were higher in dogs with CYB5R deficiency compared to controls (P < .001). Plasma TNF-α, IL-6, IL-10, and serum CRP concentrations were no different between CYB5R-deficient and control dogs. Dogs with CYB5R deficiency had lower absolute lymphocyte (P = .005) and eosinophil counts (P = .04) and higher alanine transaminase (P = .04) and alkaline phosphatase activity (P = .02) than controls, but these changes were not clinically relevant. Methemoglobin levels decreased after PO MB therapy (P = .03). CLINICAL RELEVANCE: These results suggest that otherwise healthy dogs with CYB5R deficiency do not have a constitutive proinflammatory phenotype and clinically relevant abnormalities in hematologic and serum chemistry panels are not expected. Dogs with decreased quality of life attributed to methemoglobinemia from CYB5R deficiency might benefit from PO MB therapy.

Oral Methylene Blue Treatment in A Dog with Cytochrome B5 Reductase Deficiency And 78, XX Testicular Disorder of Sex Development.

A 6-month-old mixed breed dog was referred for evaluation of a potential disorder of sex development (DSD) and lower than expected energy level. Genitourinary examination revealed ambiguous external genitalia, hypospadias, and a subtle pouch of skin that resembled an empty scrotum. Corrective surgery was planned and subsequently aborted after cyanosis was identified preoperatively and an arterial blood gas analysis by co-oximetry identified increased methemoglobin (MetHb) concentration (35%, normal <2%) with normal arterial oxygen tension. Ensuing investigations confirmed hereditary methemoglobinemia caused by cytochrome b5 reductase (CYB5R) deficiency via molecular genetic (Arg219Pro homozygous variant in CYB5R3 gene) and biochemical (cytochrome b5 reductase enzyme activity of 8% [normal, 100% activity] testing. Karyotyping and molecular analysis of sex chromosomes revealed the dog was genetically female with a normal female karyotype (78,XX), and was negative for the Y-linked SRY gene and positive for the X-linked androgen receptor gene. Methylene blue (MB, 3.3 mg/kg per os [PO] q24 h) was administered and the MetHb concentration decreased to 9% within 14 days. Urogenital revision surgery proceeded without complication and the dog was maintained on MB (3-4 mg/kg PO q24 h) long-term without adverse effects. This is the first report to describe the use of PO MB to decrease MetHb concentrations in a dog with CYB5R deficiency in preparation for anesthesia and highlights its potential as a viable alternative to the intravenous formulation for elective procedures. In addition, this report describes the clinical, molecular, imaging, surgical, and macroscopic and microscopic pathological features of a dog with SRY-negative, 78,XX testicular DSD.

Characterization of a novel nicotinamide adenine dinucleotide-cytochrome b5 reductase mutation associated with canine hereditary methemoglobinemia.

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.

Clinical, metabolic, and genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in cats.

Two non-pedigreed male castrated cats had persistent cyanosis over a 3-year observation period. Clinical cardiopulmonary evaluations did not reveal abnormalities, but the blood remained dark after exposure to air. Erythrocytic methemoglobin concentrations were high (~40% of hemoglobin) and cytochrome b5 reductase (CYB5R) activities in erythrocytes were low (≤15% of control). One cat remained intolerant of exertion, and the other cat developed anemia and died due to an unidentified comorbidity. Whole-genome sequencing revealed a homozygous c.625G>A missense variant (B4:137967506) and a c.232-1G>C splice acceptor variant (B4:137970815) in CYB5R3, respectively, which were absent in 193 unaffected additional cats. The p.Gly209Ser missense variant likely disrupts a nicotinamide adenine dinucleotide (NADH)-binding domain, while the splicing error occurs at the acceptor site for exon 4, which likely affects downstream translation of the protein. The 2 novel CYB5R3 variants were associated with methemoglobinemia using clinical, biochemical, genomics, and in silico protein studies. The variant prevalence is unknown in the cat population.

The effects of dietary nitrate on plasma glucose and insulin sensitivity in sheep.

Nitrate (NO3 ¯ ) is an effective non-protein nitrogen source for gut microbes and reduces enteric methane (CH4 ) production in ruminants. Nitrate is reduced to ammonia by rumen bacteria with nitrite (NO2 ¯ ) produced as an intermediate. The absorption of NO2 ¯ can cause methaemoglobinaemia in ruminants. Metabolism of NO3 ¯ and NO2 ¯ in blood and animal tissues forms nitric oxide (NO) which has profound physiological effects in ruminants and has been shown to increase glucose uptake and insulin secretion in rodents and humans. We hypothesized that absorption of small quantities of NO2 ¯ resulting from a low-risk dose of dietary NO3 ¯ will increase insulin sensitivity (SI ) and glucose uptake in sheep. We evaluated the effect of feeding sheep with a diet supplemented with 18 g NO3 ¯ /kg DM or urea (Ur) isonitrogenously to NO3 ¯ , on insulin and glucose dynamics. A glucose tolerance test using an intravenous bolus of 1 ml/kg LW of 24% (w/v) glucose was conducted in twenty sheep, with 10 sheep receiving 1.8% supplementary NO3 ¯ and 10 receiving supplementary urea isonitrogenously to NO3 ¯ . The MINMOD model used plasma glucose and insulin concentrations to estimate basal plasma insulin (Ib ) and basal glucose concentration (Gb ), insulin sensitivity (SI ), glucose effectiveness (SG ), acute insulin response (AIRg) and disposition index (DI). Nitrate supplementation had no effect on Ib (p > .05). The decrease in blood glucose occurred at the same rate in both dietary treatments (SG ; p = .60), and there was no effect of NO3 ¯ on either Gb , SI , AIRg or DI. This experiment found that the insulin dynamics assessed using the MINMOD model were not affected by NO3 ¯ administered to fasted sheep at a low dose of 1.8% NO3 ¯ in the diet.

Hereditary methemoglobinemia in a cyanotic cat presented for ovariohysterectomy.

A 1-year-old, female, domestic shorthair cat with a history of cyanotic mucous membranes for several months was referred for ovariohysterectomy. Blood samples exhibited a noticeably brownish discoloration, while laboratory screening revealed mild-to-moderate erythrocytosis and near normal partial arterial oxygen pressure. Blood methemoglobin content was 41% of total hemoglobin concentration, and erythrocytic methemoglobin reductase activity was < 1% compared with control samples. A diagnosis of hereditary methemoglobinemia was established. After an intravenous injection of methylene blue, the cat's mucous membranes became transiently pink, and the ovariohysterectomy was uneventful. Methylene blue may have improved safety during anesthesia and surgery. Hereditary methemoglobinemia should be considered in persistently cyanotic cats with normal partial arterial oxygen pressure and lack of evidence of cardiopulmonary disease, anemia, or toxin exposure.

Méthémoglobinémie héréditaire chez une chatte cyanotique présentée pour une ovariohystérectomie. Une chatte domestique âgée de 1 an avec une anamnèse de muqueuses cyanotiques pendant plusieurs mois a été recommandée pour l'ovariohystérectomie. Des prélèvements sanguins présentaient une décoloration brune manifeste tandis que les tests de laboratoire ont révélé une érythrocytose de légère à modérée et une pression d'oxygène artérielle partielle presque normale. Le contenu de méthémoglobine sanguine était de 41 % de la concentration totale des hémoglobines et l'activité de la réductase de la méthémoglobine érythrocytaire était < 1 % comparativement aux prélèvements témoins. Un diagnostic de méthémoglobinémie héréditaire a été posé. Après une injection intraveineuse de bleu de méthylène, les muqueuses du chat sont devenues provisoirement roses et l'ovariohystérectomie a été réalisée sans complications. Le bleu de méthylène peut avoir amélioré l'innocuité durant l'anesthésie et la chirurgie.(Traduit par Isabelle Vallières).

Genetic cause for congenital methemoglobinemia in an Australian Pomeranian dog.

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.

Severe Heinz body anemia and methemoglobinemia in a kitten with chronic diarrhea.

INTRODUCTION: A 2-month-old kitten was referred for depression and partial anorexia since 3 days and chronic diarrhea lasting for over 3 weeks. General physical examination showed pale and cyanotic mucous membranes. Blood sample was of brownish appearance. Venous blood gas analysis and complete blood count showed 16% methemoglobin level and severe regenerative anemia with Heinz bodies in about 40% of the erythrocytes, respectively. The kitten was transfused with fresh whole blood and treated with supportive care, antimicrobial and antioxidant agents. The kitten totally recovered. To the authors' knowledge, this represents the first case report of severe Heinz body hemolytic anemia and methemoglobinemia with concurrent chronic diarrhea in a young kitten. Diarrhea resolution coincided with Heinz bodies and methemoglobin disappearance. The possibility that diarrhea might have stimulated an inflammatory state causing release of oxygen radicals and prolonged erythrocytes oxidative damage has been discussed.

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH-Cytochrome B5 Reductase Gene.

BACKGROUND: 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. OBJECTIVES: To analyze the NADH-cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency. ANIMALS: Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls. METHODS: Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing. RESULTS: Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu). CONCLUSIONS AND CLINICAL IMPORTANCE: This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH-binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.

Long-term Treatment with Methylene Blue in a Dog with Hereditary Methemoglobinemia Caused by Cytochrome b5 Reductase Deficiency.

A juvenile male mixed breed dog was presented for lethargy, exercise intolerance, and aggression when touched on the head. Cyanosis, tachycardia, and tachypnea were observed and persisted during oxygen supplementation. Arterial blood gas analysis by co-oximetry identified an increased methemoglobin concentration (27%; normal, <2%) with normal arterial oxygen tension. The methemoglobinemia and associated clinical signs resolved after administration of methylene blue (1 mg/kg) IV, and the dog was discharged. The affected dog's whole-genome sequence contained 2 potentially causal heterozygous CYB5R3 missense mutations suggesting that cytochrome b5 reductase deficiency was responsible for the methemoglobinemia. This hypothesis was confirmed by enzyme analysis that identified cytochrome b5 reductase activity in the affected dog's erythrocytes to only approximately 6% of that in a control sample. Clinical signs recurred 11 days after discharge but normalized and the methemoglobin concentration decreased with methylene blue administration PO (1.5 mg/kg, initially daily and then every other day).

Detecting Methemoglobinemia in Animals with a Drop of Blood.

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.

Oxidant-induced damage to equine erythrocytes from exposure to Pistacia atlantica, Pistacia terebinthus, and Pistacia chinensis.

Two horses were referred for methemoglobinemia and hemolytic anemia following 5 acute deaths in their herd from an unidentified toxin source. Horses have a greater risk than other mammalian species of developing methemoglobinemia and hemolytic anemia following ingestion of oxidizing toxins, due to deficiencies in the mechanisms that protect against oxidative damage in erythrocytes. Their susceptibility to oxidative erythrocyte damage is evident in the numerous cases of red maple (Acer rubrum) toxicosis. The suspected toxins causing A. rubrum toxicosis are tannic acid, gallic acid, and a metabolite of gallic acid, pyrogallol. These compounds can be found in a variety of plants, posing a risk to equine health. In order to quickly identify toxin sources, 2 rapid in vitro assays were developed to screen plant extracts for the ability to induce methemoglobin formation or cause hemolysis in healthy equine donor erythrocytes. The plant extract screening focused on 3 species of the genus Pistacia: P. atlantica, P. terebinthus, and P. chinensis, which were located in the horse pasture. Extracts of the seeds and leaves of each species induced methemoglobin formation and resulted in hemolysis, with seed extracts having greater potency. The in vitro assays used in the current study provide a useful diagnostic method for the rapid identification of oxidizing agents from unidentified sources. There is no effective treatment for oxidative erythrocyte damage in horses, making rapid identification and removal of the source essential for the prevention of poisoning.

Identification of protoxins and a microbial basis for red maple (Acer rubrum) toxicosis in equines.

The leaves of Acer rubrum (red maple), especially when wilted in the fall, cause severe oxidative damage to equine erythrocytes, leading to potentially fatal methemoglobinemia and hemolytic anemia. Gallic acid and tannins from A. rubrum leaves have been implicated as the toxic compounds responsible for red maple toxicosis, but the mechanism of action and toxic principle(s) have not been elucidated to date. In order to investigate further how red maple toxicosis occurs, aqueous solutions of gallic acid, tannic acid, and ground dried A. rubrum leaves were incubated with contents of equine ileum, jejunum, cecum, colon, and liver, and then analyzed for the metabolite pyrogallol, as pyrogallol is a more potent oxidizing agent. Gallic acid was observed to be metabolized to pyrogallol maximally in equine ileum contents in the first 24 hr. Incubation of tannic acid and A. rubrum leaves, individually with ileum contents, produced gallic acid and, subsequently, pyrogallol. Ileum suspensions, when passed through a filter to exclude microbes but not enzymes, formed no pyrogallol, suggesting a microbial basis to the pathway. Bacteria isolated from ileum capable of pyrogallol formation were identified as Klebsiella pneumoniae and Enterobacter cloacae. Therefore, gallotannins and free gallic acid are present in A. rubrum leaves and can be metabolized by K. pneumoniae and E. cloacae found in the equine ileum to form pyrogallol either directly or through a gallic acid intermediate (gallotannins). Identification of these compounds and their physiological effects is necessary for the development of effective treatments for red maple toxicosis in equines.