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).

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.

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.

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.

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.

Effect of chloride on nitrite-induced methaemoglobinemia in Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus (Mitchill).

We evaluated the effects of chloride concentration on the clinical pathology in juvenile Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus (Mitchill), following semi-static exposures to 1 mg L(-1) nitrite for 96 h. In spring water naturally low in chloride (5 mg L(-1)), plasma nitrite concentrated to more than 40× environmental levels resulting in a severe methaemoglobinemia characterized by torpid behaviour, 30-fold increase in methaemoglobin fraction, anaemia, leucopenia and hyperkalaemia. Loss of intracellular water and potassium to extracellular space may have resulted in hyperkalaemia and haemodilution. Fish survived nitrite exposure, but 60% of torpid fish died following capture and tissue sampling. Fish acclimated to 10-fold higher chloride content (55 mg L(-1)) did not concentrate nitrite in the plasma above environmental levels or develop methaemoglobinemia, but did exhibit similar haematology and plasma chemistry changes. Plasma nitrite returned to preexposure levels by 14 days following nitrite exposures, but severity of clinical pathology changes persisted or increased, suggesting that Atlantic sturgeon have reduced capacity to recover from methaemoglobinemia. Fish that survive methaemoglobinemia may be susceptible to mortality from the cumulative effects of intoxication, handling and other stresses for two or more weeks following nitrite remediation. Chloride buffering in aquaculture systems reduces the toxic effects of nitrite accumulation.

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.

Nitrate and sulfate: Effective alternative hydrogen sinks for mitigation of ruminal methane production in sheep.

Twenty male crossbred Texel lambs were used in a 2 × 2 factorial design experiment to assess the effect of dietary addition of nitrate (2.6% of dry matter) and sulfate (2.6% of dry matter) on enteric methane emissions, rumen volatile fatty acid concentrations, rumen microbial composition, and the occurrence of methemoglobinemia. Lambs were gradually introduced to nitrate and sulfate in a corn silage-based diet over a period of 4 wk, and methane production was subsequently determined in respiration chambers. Diets were given at 95% of the lowest ad libitum intake observed within one block in the week before methane yield was measured to ensure equal feed intake of animals between treatments. All diets were formulated to be isonitrogenous. Methane production decreased with both supplements (nitrate: -32%, sulfate: -16%, and nitrate+sulfate: -47% relative to control). The decrease in methane production due to nitrate feeding was most pronounced in the period immediately after feeding, whereas the decrease in methane yield due to sulfate feeding was observed during the entire day. Methane-suppressing effects of nitrate and sulfate were independent and additive. The highest methemoglobin value observed in the blood of the nitrate-fed animals was 7% of hemoglobin. When nitrate was fed in combination with sulfate, methemoglobin remained below the detection limit of 2% of hemoglobin. Dietary nitrate decreased heat production (-7%), whereas supplementation with sulfate increased heat production (+3%). Feeding nitrate or sulfate had no effects on volatile fatty acid concentrations in rumen fluid samples taken 24h after feeding, except for the molar proportion of branched-chain volatile fatty acids, which was higher when sulfate was fed and lower when nitrate was fed, but not different when both products were included in the diet. The total number of rumen bacteria increased as a result of sulfate inclusion in the diet. The number of methanogens was reduced when nitrate was fed. Enhanced levels of sulfate in the diet increased the number of sulfate-reducing bacteria. The number of protozoa was not affected by nitrate or sulfate addition. Supplementation of a diet with nitrate and sulfate is an effective means for mitigating enteric methane emissions from sheep.

Evaluation of prophylactic and therapeutic effects of silymarin and N-acetylcysteine in acetaminophen-induced hepatotoxicity in cats.

Cats most commonly receive toxic amounts of acetaminophen (APAP) because owners medicate them without consulting a veterinarian. The aim of this study was to compare the hepatoprotective action of silymarin and N-acetylcysteine (NAC) against APAP poisoning. Twenty healthy cats were randomly allotted to five equal groups. Animals in group A were given APAP (single dose 150 mg/kg, p.o.); groups B and C consisted of cats that received NAC (100 mg/kg, p.o.) or silymarin (30 mg/kg, p.o.) concurrent with APAP administration respectively; groups D and E were treated like groups B and C, respectively, but 4 h after APAP administration. The serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), methemoglobin, and total and direct bilirubin were measured before APAP administration and 4, 24, and 72 h later. A single oral administration of APAP significantly elevated serum concentrations of ALT, AST, ALP, LDH, methemoglobin, and total and direct bilirubin. In both the groups receiving APAP plus NAC or silymarin, levels of serum enzyme activities, methemoglobin, and total and direct bilirubin remained within the normal values. It was concluded that silymarin as well as NAC can protect liver tissue against oxidative stress in cats with an APAP intoxication.

The role of para-aminophenol in acetaminophen-induced methemoglobinemia in dogs and cats.

Acetaminophen (APAP) overdose in most species is associated with hepatotoxicity because of the metabolite N-acetyl-p-benzoquinoneimine (NAPQI). In dogs and cats, APAP overdose primarily causes methemoglobinemia and hemolysis. Although NAPQI has been proposed as the responsible intermediate in dogs and cats, it lacks chemical or pharmacokinetic characteristics that favor methemoglobin formation. We hypothesized that para-aminophenol (PAP) rather than NAPQI induces methemoglobinemia and that deficient arylamine N-acetyltransferase (NAT) activity in dogs and cats contributes to this species-dependent methemoglobinemia. Erythrocytes from dogs, cats, mice, and rats were exposed in vitro to APAP, NAPQI, and PAP. Only PAP induced methemoglobin and it induced more methemoglobin formation in dog and cat than rat and mouse erythrocytes. PAP also induced more methemoglobin in erythrocytes from Nat1/Nat2 knockout mice than wildtype (WT) mouse erythrocytes (P < 0.05), but less than in dog and cat erythrocytes (P < 0.01). APAP and PAP toxicity were compared in vivo in WT and Nat1/Nat2 knockout mice. APAP caused no hematotoxicity while PAP induced more methemoglobin in NAT1/NAT2 knockout mice than in WT mice (P < 0.05). These results support the hypothesis that PAP is the metabolite responsible for APAP-induced methemoglobinemia and that deficient NAT activity in dogs and cats contributes to this species-dependent toxicity.

Presumptive benzocaine-induced methemoglobinemia in a slender-tailed meerkat (Suricata suricatta).

An 8-yr-old castrated male slender-tailed meerkat (Suricata suricatta) was anesthetized for physical examination and dental prophylaxis. To facilitate intubation, two short bursts of benzocaine spray were applied topically to the glottis. Shortly thereafter, the meerkat developed a muddy, blue-gray mucous membrane color and low oxygen saturation readings measured via pulse oximetry. Despite positive pressure ventilation and treatment with doxapram, the cyanosis and hypoxemia did not improve. Blood collected during the procedure was noticeably dark brown and a clinical diagnosis of methemoglobinemia was made. Because of persistent cyanosis and prolonged recovery, the meerkat was anesthetized a second time to facilitate treatment for methemoglobinemia via a slow intravenous bolus of methylene blue and subcutaneously administered dextrose. Within 20 min, the tongue and gingival color normalized. This is the first report of methemoglobinemia in this species. Although it is commonly used in small animal practice and in humans undergoing certain endoscopic procedures, and present in numerous over-the-counter preparations, the risk of topical benzocaine inducing methemoglobinemia is well described. Administration of topical benzocaine in all mammalian species, particularly small patients, should be done with caution. If it is utilized in zoo practice, clinicians are encouraged to administer benzocaine judiciously to avoid accidental overdose, and be familiar with the signs of methemoglobinemia and its treatment.

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.

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.

Methaemoglobinaemia caused by hydroxycarbamide (hydroxyurea) ingestion in a dog.

A three-year-old female neutered greyhound was presented after ingestion of its owner's hydroxycarbamide (hydroxyurea) tablets. The dog was found to be cyanosed, and methaemoglobinaemia was demonstrated by co-oximetry. Therapy included methylene blue, oxygen, packed red blood cell transfusion, N-acetylcysteine and crystalloid fluids. Methaemoglobinaemia resolved within 16 hours. Granulocyte colony-stimulating factor was administered for five days in an attempt to prevent severe neutropenia. Mild delayed transient myelotoxicity was suspected. The dog made a full recovery.

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).