Pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses

Deficiencies of enzymes involved in erythrocyte metabolism can have significant effects on erythrocyte function and survival. Animals with pyruvate kinase (PK) or phosphofructokinase (PFK) deficiencies have shortened erythrocyte life spans and regenerative anemia. PK-deficient dogs (but not PK-deficient cats) develop progressive myelofibrosis and osteosclerosis of bone marrow and hemochromatosis and cirrhosis of the liver. PFK-deficient dogs have sporadic episodes of hyperventilation-induced intravascular hemolysis and hemoglobinuria. Cytochrome b5 reductase (Cb5R) deficiency in dogs and cats results in persistent methemoglobinemia and cyanotic mucous membranes. Severe deficiency of glucose-6-phosphate dehydrogenase, the rate-controlling enzyme in the pentose phosphate pathway, resulted in anemia with eccentrocytosis in an American saddlebred colt. Horses with erythrocyte flavin adenine dinucleotide (FAD) deficiency have both eccentrocytosis (attributable to severe deficiency in glutathione reductase activity) and methemoglobinemia (attributable to Cb5R deficiency); the dual enzyme deficiency occurs because FAD is a required cofactor for both enzymes. Erythrocyte enzyme deficiencies do not usually shorten life expectancy, except for PK-deficient dogs and potentially PFK-deficient dogs during a hemolytic crisis. Although enzyme deficiencies are rare causes of anemia and methemoglobinemia, the ability to diagnose deficient animals allows for the possibility of eliminating these undesirable traits in future breeding. DNA-based assays are available for PK and PFK deficiencies; whereas, biochemical tests of enzyme activity are required for other deficiencies. Continued research is needed to document additional enzyme deficiencies that likely occur and to develop additional DNA-based assays to detect heterozygous animals.     

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.     

Mammalian erythrocyte glutathione reductase: kinetic constants and saturation with cofactor.

Glutathione reductase (GR) was studied in erythrocytes of horses, cats, dogs, and man. Glutathione reductase activity was measured in hemolysates with and without preincubation of hemolysates with flavinadenine dinucleotide. The percentage saturation of GR apoenzyme with cofactor (flavin-adenine dinucleotide) was lower in cats and dogs than in horses or man. The greatest amount of inactive apoenzyme was in feline erythrocytes. Total GR activity listed in order by species is cat greater than man greater than dog greater than horse. Kinetic constants for oxidized glutathione and reduced nicotinamide-adenine dinucleotide phosphate were determined in each species. Although kinetic constant (reduced nicotinamide-adenine dinucleotide phosphate) values for GR were similar, considerable species variation was observed in the kinetic constant (oxidized glutathione) for GR. The kinetic constant (oxidized glutathione) for equine GR was approximately 3 times that for human GR, with intermediate values determined for feline and canine GR.     

Interrelationships of erythrocyte glutathione peroxidase system enzymes in the fasted Beagle

Eight adult female Beagles were fasted for 21 days to investigate responses of the erythrocyte glutathione peroxidase system enzymes. Blood samples were collected once a week. Erythrocyte reduced glutathione was maintained within 10% of base-line value. Hexose monophosphate pathway dehydrogenases showed a significant inverse relationship to glutathione reductase (GR) activity. Further, active GR, as a percentage of total GR, increased as hexose monophosphate pathway dehydrogenase activity fell. A similar relationship between these enzymes has been reported in human glucose-6-phosphate dehydrogenase deficiency. Also, glutathione peroxidase showed an inverse linear response to erythrocytic reduced glutathione. Though the responses of this system are complex, they appear interrelated.     

Erythrocyte Glucose-6-phosphate Dehydrogenase and Glutathione Deficiency in Sheep

Erythrocytes of 145 sheep representing six breeds were assayed for glucose-6-phosphate dehydrogenase. All sheep had erythrocyte glucose-6-phosphate dehydrogenase values similar to glucose-6-phosphate dehydrogenase deficient erythrocytes of man. Mean erythrocyte glucose-6-phosphate dehydrogenase levels ranged from 0.65 to 1.54 micromoles of reduced nicotinamide adenine dinucleotide phosphate per gram of hemoglobin per minute. Many of these sheep also had low levels and/or unstable reduced glutathione. Sheep with low levels of erythrocyte glucose-6-phosphate dehydrogenase and reduced glutathione were given large doses of oxidizing drugs or fed fresh fava beans to determine if they would develop intravascular hemolysis. No significant hemolysis was detected as a result of drug administration or fava bean ingestion.     

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.     

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).     

Enzymes of oxidant defence system of leucocytes and erythrocytes in bovine anaplasmosis

The glutathione oxidant defence system in leucocytes and erythrocytes of six Anaplasma marginale-infected calves was examined by assaying glutathione peroxidase (GSH-px), glutathione reductase (GSSG-R), reduced glutathione (GSH) and superoxide dismutase (SOD). In addition, GSH-S-transferase and arginase levels were measured in leucocytes. There was a significant decline in the activities of leucocyte GSH-px, GSSG-R and SOD in the post-patent period; whereas arginase activity rose significantly following the patent period of anaplasmosis. The activity of erythrocyte SOD declined in the post-patent stage. The infection also caused a decline in red cell GSH (P less than 0.05). The results suggest that the glutathione oxidant defence system of peripheral blood cells is significantly influenced by the Anaplasma infection.     

Reference values for a field test to estimate inadequate glutathione peroxidase activity and selenium status in the blood of cattle.

A clinical screening procedure was developed for estimating glutathione peroxidase (GSH-Px) activity and selenium status in the blood of dairy and beef cattle. The test is based on the rate of defluorescence under long waveform UV irradiation of reduced nicotinamide adenine dinucleotide phosphate in a coupled enzyme reaction involving glutathione reductase. Defluorescence rates were significantly correlated with blood GSH-Px activity and blood selenium concentrations as determined by conventional laboratory procedures. Blood selenium concentrations and blood GSH-Px activity in several herds were compared with the selenium content of the complete ration consumed to establish reference or base-line values for these blood characteristics under field conditions. The GSH-Px screening procedure provides rapid results, is relatively inexpensive, and appears to be useful in differentiating between the reference values and grossly inadequate selenium status in cattle.     

Effects of riboflavin supplementation and selenium source on selenium metabolism in the young pig

The effect of dietary riboflavin (B2) supplementation and selenium (Se) source on the performance and Se metabolism of weanling pigs was studied. Pigs fed a B2-supplemented (10 mg/kg) casein-glucose diet for 18 d gained faster than pigs fed the B2-unsupplemented diet. Percentage active erythrocyte glutathione reductase (GR) declined rapidly when pigs were placed on the B2-unsupplemented diet and was lower (P less than .01) than that of B2-supplemented pigs after 12 d on test. Percentage active erythrocyte GR values fell below 50% before other B2 deficiency signs became evident. Supplementation of diets with 10 mg B2/kg resulted in increased kidney and muscle glutathione peroxidase (GSH-Px) activity. The Se concentration of liver and heart increased and plasma Se levels decreased with dietary B2 supplementation. Riboflavin supplementation and Se source did not alter apparent Se absorption, but B2 supplementation decreased urinary Se and thus increased Se retention. Also, there was less urinary Se excretion when selenomethionine was the dietary Se source and consequently more Se was retained than when sodium selenite was the dietary Se source. In a final trial, B2 supplementation increased kidney, muscle, heart and brain GSH-Px activity when sodium selenite was the dietary Se source, but not when selenomethionine was the dietary Se source.     

滞育发动前家蚕滞育性与非滞育性卵的谷胱甘肽氧化还原循环状态分析

为了调查滞育和非滞育性家蚕卵的谷胱甘肽氧化还原循环在滞育发动前是否存在差异,利用分光光度法检测滞育发动前(25℃中蚕卵产下后0～24 h)滞育与非滞育蚕卵的谷胱甘肽含量和相关代谢酶活性。滞育发动前,滞育性家蚕卵中的还原型谷胱甘肽(GSH)含量、氧化型谷胱甘肽(GSSG)含量、总谷胱甘肽(GSH+2GSSG)含量和GSH/GSSG比值变化不显著,谷胱甘肽S-转移酶(GST)活性变化不显著,但硫氧还蛋白过氧化物酶(TPX)活性下降23%,谷胱甘肽还原酶(GR)活性升高57%;非滞育性家蚕卵中的GSH含量及TPX和GST活性变化不显著,但GSSG含量升高61%,GSH+2GSSG含量升高41%,GSH/GSSG比值下降33%,GR活性下降16%。与非滞育性家蚕卵相比,滞育发动前滞育性家蚕卵中的GSH含量、GSSG含量和GSH+2GSSG含量较低,GSH/GSSG比值较高,TPX活性较低,GST活性无显著差异,GR活性较高。两种蚕卵中都未能检测到谷胱甘肽过氧化物酶(GPX)和硫氧还蛋白还原酶(TrxR)活性。结果表明,滞育发动前滞育性家蚕卵中较低的TPX活性和较高的GR活性共同导致较高的GSH/GSSG比值,使其谷胱甘肽氧化还原循环处于相对还原态。     

The effect of phlebotomy on canine erythrocyte metabolism.

Enzyme activity declines with erythrocyte age in most mammals. To test this concept in the dog, we decreased the PCV to less than 20 by phlebotomy. The erythrocytes were restored rapidly (1.57 per cent per day). The resulting decline in the mean erythrocyte age was accompanied by increased activity by most of the erythrocyte enzymes studied. Enzymes with lower initial enzymatic activity (hexokinase, pyruvate kinase, 6-phosphogluconate dehydrogenase and glutathione reductase) increased proportionally more than those with higher initial activity (lactate dehydrogenase, 3-phosphoglycerate kinase, glyceraldehyde-3-dehydrogenase and glucose-6-dehydrogenase). Among species, increases in enzyme activity after phlebotomy appear to be related to each species' life span. Most of the metabolites increased concomitantly with the highest reticulocyte period. Diphosphoglycerate concentrations did not change significantly during the experiment.     

Relation between erythrocyte reduced glutathione and glutamate concentrations in Korean Jindo dogs with erythrocytes possessing hereditary high activity of Na-K-ATPase and a high concentration of potassium

The concentrations of sodium, potassium, reduced glutathione (GSH) and free amino acids and Na-K-ATPase activity in erythrocytes were examined in 35 purebred Jindo dogs in Korea. The incidence of Jindo dogs with a high potassium concentration and high activity of Na-K-ATPase in erythrocytes (HK phenotype) was 25.7%. The erythrocyte GSH concentration in HK Jindo dogs varied widely, from 2.45 to 12.38 mmol/l of RBCs, and was positively correlated with the erythrocyte glutamate concentration. These results indicate that HK Jindo dogs have normal to very high levels of erythrocyte GSH, which might result from the varying quantity of Na-dependent glutamate influx in the erythrocytes.     

Blood glutathione status and activity of glutathione‐metabolizing antioxidant enzymes in erythrocytes of young trotters in basic training

The aim of this study was to evaluate response of blood glutathione status and activity of glutathione‐metabolizing antioxidant enzymes in erythrocytes of young trotters in basic training. Nine untrained trotters (aged 16–20 months) were exposed to a 4‐month training program based on exercises at low‐to‐moderate intensity. The conditioning consisted of breaking the horses and running them on distances varying from 4 to 40 km a week. The workloads were increased on a 3‐week basis. Exercise intensity was monitored by measuring heart rate and blood lactate. Blood samples were collected at rest, before (RES0) and after (RESt) the conditioning period; moreover, on the latter occasion (on day 112 of training), the blood was also taken immediately after the routine exercise (EXE0) and 60 min thereafter (EXE60). The whole blood samples were analysed for the concentration of reduced, oxidized and total glutathione (GSH, GSSG and TGSH, respectively), while the activities of glutathione peroxidase (GPX) and glutathione‐disulfide reductase (GR) were determined in haemolysates. Additionally, the erythrocytic concentrations of oxidized nicotinamide adenine dinucleotide (NAD⁺) and its phosphate (NADP⁺) were measured. All investigated parameters except NAD⁺ and reduced/oxidized glutathione ratio (GSH/GSSG) changed during the training period. Following the effortm GPX, NADP⁺ and GSH/GSSG were significantly lower (p < 0.05, p < 0.01, p < 0.001, respectively) while GSSG was markedly higher than at rest (RESt). The drop in NADP⁺, low GSH/GSSG and high GSSG concentration were sustained at EXE60. Glutathione‐disulfide reductase activity was higher after the workout but only at EXE60 the increase in activity was significant. Despite the activities of the GSH‐GSSG cycle, enzymes were considerably higher after the training period, the elevated concentration of GSSG and significantly lower GSH/GSSG ratio in the post‐exercise measurements suggest that production of reactive oxygen species possibly exceeds the capacity of antioxidative defenses of immature trotters. A more balanced diet with additional antioxidant supplementation and a revision of the basic training protocol used herein are advised.     

Acute intravascular hemolysis in the black rhinoceros: erythrocyte enzymes and metabolic intermediates

Enzymes of aerobic and anaerobic glycolysis, glutathione cycling, and nucleotide metabolism were assayed on erythrocytes from 7 healthy rhinoceroses, 2 rhinoceroses during periods of intravascular hemolysis, and 1 rhinoceros without clinical signs of illness, which was the mother of 3 offspring with intravascular hemolytic syndrome. Measurements also were made of erythrocyte concentrations of glycolytic intermediates, adenine nucleotides, and glutathione. Although comparison of results for healthy and affected rhinoceroses did not identify an enzyme abnormality as a cause for the hemolytic syndrome, the data provided information regarding the metabolic characteristics of erythrocytes from healthy rhinoceroses.     

Oxidative Stress Biomarkers and Erythrocytes Hemolysis in Well-Trained Equine Athletes Before and After Exercise

The chronic exposure to regular exercise training seems to increase resistance to oxidative stress and improves the antioxidant defense system. The purpose of the present study was to investigate the effect of an exercise test of moderate intensity on oxidative stress biomarkers, antioxidant enzymes activity, and osmotic resistance of erythrocytes in well-trained equine athletes. Eighteen middle-aged horses of Ukrainian warmblood (8.3 ± 1.6 years) and Holsteiner (7.4 ± 1.9 years) breeds were used in this study. All horses have been in regular training for several years. The exercise test induced a significant increase of erythrocyte values, hemoglobin concentration, and hematocrit in horses of both breeds. Regular training induces activation the antioxidant enzymes and thereby can reduce oxidative stress in athletic horses. Our results suggest that the exercise test in horses of both breeds attenuates oxidative stress and accompanied with a significant decrease of lipid peroxidation and oxidatively modified proteins in erythrocytes after exercise. The findings of the present study demonstrated the elevated level of erythrocytes' catalase and glutathione reductase in Ukrainian warmblood horses, as well as decreased level of superoxide dismutase and glutathione reductase in Holsteiner horses reporting changes in levels of exercise-induced oxidative stress biomarkers in horses of both breeds. Statistically significant differences in the percentage of hemolyzed erythrocytes between pre-exercise and postexercise tests were observed and thereby signifying an oxidative stress-dependent impairment of erythrocyte stability. Our data suggest that oxidative stress and enzymatic antioxidant defense biomarkers can be used for the monitoring of fitness level, health benefits, and performance of equine athletes.     

Nicotinamide-adenine dinucleotide-methemoglobin reductase activity in erythrocytes from cats

Reduced nicotinamide-adenine dinucleotide (NADH)-methemoglobin reductase activity in feline erythrocyte lysates was determined, using potassium ferricyanide as substrate. The optimum conditions for the assay were pH 8 and 37 C. Mean NADH-ferricyanide reductase activity in cats was 15.7 +/- 4.1 mumoles of substrate converted/g of hemoglobin/min. The migration of NADH-ferricyanide reductase was similar to that of the NADH-methemoglobin reductase (NADH diaphorase) on starch gel electrophoresis.     

Total glutathione and glutathione reductase in bovine erythrocytes and liver biopsy

The goal of the present study was to measure the total glutathione level and glutathione reductase activity in bovine erythrocytes and liver biopsy. Five cows were injected intraperitoneally with DL-ethionine (12.5 mg/kg B.W.), and two control cows were injected with normal saline (0.9% NaCl). Ultrasonography guided liver biopsy, and blood samples were collected at 0, 4, 7 and 10 days after injection. The hepatic total glutathione level was significantly increased on Days 7 (p<0.05) and 10 (p<0.01), and hepatic glutathione reductase activity was significantly increased on Days 4 (p<0.05), 7 (p<0.01) and 10 (p<0.01). There were insignificant changes in the erythrocytic total glutathione level. The present study demonstrated that liver biopsy is a valuable tool for detecting oxidative stress and for diagnosing hepatic dysfunction in cattle from the viewpoint of the status of glutathione and glutathione reductase.     

Heinz body hemolytic anemia with eccentrocytosis from ingestion of Chinese chive (Allium tuberosum) and garlic (Allium sativum) in a dog

A 4-year-old, intact male miniature schnauzer was presented with anorexia. The dog had ingested some Chinese steamed dumplings 2 days before, which contained Chinese chive (Allium tuberosum) and garlic (Allium sativum). Hematological examinations revealed severe Heinz body hemolytic anemia with eccentrocytosis and an increased concentration of methemoglobin, which was thought to result from oxidative damage to erythrocytes by constituents in these Allium plants. In this case, eccentrocytosis was a hallmark finding and could be detected easily, suggesting that this hematological abnormality is useful in diagnosing Allium plant-induced hemolysis.