Susceptibility to onion-induced hemolysis in dogs with hereditary high erythrocyte reduced glutathione and potassium concentrations.

The hemolytic effect of onion consumption in dogs with hereditary high erythrocyte reduced glutathione and potassium concentrations (designated HK dogs) was compared with that in clinically normal dogs. Twelve hours after oral administration of boiled onions (200 g/dog), hemoglobin concentration decreased to 84.4% of the initial value in HK dogs; it decreased only to 90.5% in clinically normal dogs. At 24 hours, methemoglobin concentration was significantly (P less than 0.05) higher in HK dogs than in clinically normal dogs. The concentration of erythrocyte oxidized glutathione increased about fivefold at 10 hours in HK dogs, whereas it did not change during the experimental period in clinically normal dogs. In addition, at 12 hours, the proportion of erythrocytes containing Heinz bodies increased to 24.4% in HK dogs, but increased only to 1.2% in clinically normal dogs. Thus, results indicated that HK dogs were more susceptible to the oxidant action of onions than were clinically normal dogs.     

Hereditary high-potassium erythrocytes with high Na, K-ATPase activity in Japanese shiba dogs.

The sodium (Na) and potassium (K) concentrations and Na, K-ATPase activity were examined in erythrocytes from 24 Japanese shiba dogs and 79 dogs of 24 other breeds. Eleven of the shibas had erythrocytes with high K and low Na concentrations, together with high Na, K-ATPase activity (HK RBCs), while red cells from the remaining shibas and all of the other breeds examined showed low K and high Na concentrations, with no enzyme activity (LK RBCs). The concentration of reduced glutathione in HK RBCs was about five times that in LK RBCs. All the findings from HK shibas were in good agreement with those from HK mongrel dogs found in Japan previously. Since the shiba is a Japanese breed of dog, the results of the present study strongly suggest that the gene for HK RBCs may be inherent in dogs indigenous to Japan, particularly in shiba dogs.     

Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni

OBJECTIVE: To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni. ANIMALS: RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells). PROCEDURES: First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured. RESULTS: B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer. CONCLUSIONS: Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.     

Methemoglobin formation and reduction in canine erythrocytes with inherited high Na,K-ATPase activity

Methemoglobin formation and reduction in canine erythrocytes with inherited high Na,K-ATPase activity (HK cells) were compared with those in normal canine cells (LK cells). Nitrite-induced methemoglobin formation in hemoglobin solutions indicated that the hemoglobin from HK cells was oxidized at essentially the same rate as that of LK cells. However, methemoglobin formation in HK cells was slower due to the inhibition by high glutathione (GSH) concentration. Methemoglobin reduction was allowed to take place on nitrite-treated and washed erythrocytes in a glucose medium and was reduced more rapidly in HK cells than in LK cells. During the reduction, the amounts of lactate and pyruvate increased more rapidly in HK cells, indicating enhanced glycolysis in HK cells. It is thus evident that the hemoglobin of HK cells is more securely protected from nitrite-induced oxidation by the GSH presence in great excess and by the increase in glycolysis.     

Oxidative damage to the membrane of canine erythrocytes with inherited high Na, K-ATPase activity.

Oxidative damage to the membrane in canine erythrocytes with inherited high Na, K-ATPase activity (HK cells) was compared with that in normal canine cells (LK cells). When 30 mM beta-acetylphenylhydrazine (APH) was applied to HK and LK cells, lipid peroxidation and hemoglobin denaturation occurred. Lipid peroxidation determined from malondialdehyde (MDA) formation was significantly lower in HK than in LK cells so far as endogenous glutathione (GSH) concentration was maintained at appropriate levels. With the depletion of GSH, MDA formation was accelerated and difference between HK and LK cells was not significant. Denatured hemoglobin bound to the membrane protein was less in HK than in LK cells. During incubation with APH, osmotic fragility increased markedly in LK cells, while HK cells showed very little change. The amounts of total lipid, total and free cholesterol, glycolipid, phospholipid and fatty acids were essentially the same in both cell types. Fatty acid compositions showed very small differences. The membrane of HK cells thus appear to have greater protection against oxidative damage induced by APH, owing to the presence of excess GSH in HK cells. The capability of HK cells to withstand oxidative damage would not be due to differences in membrane lipid compositions.     

Some aspects of erythrocyte metabolism in insulin-treated diabetic dogs

Insulin-dependent diabetes mellitus (IDDM) is a common metabolic disease often complicated by a number of pathological conditions among which are haematological changes and alterations in blood cell function. Human and feline diabetes mellitus patients have been reported to be associated with oxidative stress that can lead to membrane alterations and to reduced erythrocyte life-span. Erythrocyte function in dogs affected by IDDM has been investigated during insulin therapy, paying attention to antioxidant status, membrane resistance, enzyme activities and 2,3-diphosphoglycerate (2,3DPG) concentration. Thirteen diabetic and 36 healthy dogs were bled and haematology and blood chemistry assays were performed to evaluate the degree of compensation. Osmotic fragility, the activities of the enzymes glucose-6-phosphate dehydrogenase (G6PD) and pyruvate-kinase (PK) and the concentrations of reduced glutathione (GSH) and 2,3DPG were evaluated in the erythrocytes. Diabetic dogs did not differ from controls in terms of haematological parameters, except for higher numbers of platelets. Higher values of fructosamine, glucose, protein, plasma potassium and calculated osmolality were detected in the plasma from diabetic dogs. No differences were detected in osmotic fragility, GSH concentration and PK activity between the two groups but 2,3DPG concentration and G6PD activity were statistically significantly higher in the diabetic group. The results indicate minimal alterations in erythrocyte functions occur in insulin-treated diabetic dogs. This contrasts with what has been reported for IDDM humans and cats.     

The cause of the predilection of Babesia gibsoni for reticulocytes

This study was conducted to determine why Babesia gibsoni replicates well in reticulocytes. First, B. gibsoni was cultivated in resealed erythrocyte ghosts loaded with either erythrocyte or reticulocyte lysate, and in reticulocyte ghosts loaded with either erythrocyte or reticulocyte lysate. The parasites multiplied well in the erythrocyte or reticulocyte ghosts loaded with reticulocyte lysate compared to the other resealed cells loaded with erythrocyte lysate. Second, the parasites were cultivated in erythrocytes in culture medium supplemented with either erythrocyte or reticulocyte lysate. The parasites multiplied better in reticulocyte lysate-containing cultures than in erythrocyte lysate-containing cultures. Finally, the parasites were cultivated in erythrocytes in culture medium supplemented with glutamate, aspartate, asparagine, glycine, isoleucine, proline, taurine or GSH, which were present in higher concentrations in reticulocytes than in erythrocytes. Supplementation of the culture medium with glutamate and GSH resulted in enhancement of the multiplication of the parasites, while the other amino acids did not enhance the multiplication. These results indicated that the high levels of the multiplication of B. gibsoni in reticulocytes are partly due to the high concentrations of glutamate and GSH in reticulocytes.     

互助县杂种黄牛红细胞钾型的研究

根据青海省互助县４０头杂种黄牛的红细胞钾（ＥＫ）浓度分别为高钾（ＨＫ）和低钾（ＬＫ）两种表型，其中ＬＫ型占３５头（８７．５％）为优势表型。ＬＫ型杂种黄牛的ＥＫ浓度为８．９～３０．１ｍｍｏｌ／Ｌ，ＨＫ型的为４５．８～７５．３ｍｍｏｌ／Ｌ，等位基因频率Ｋ＾ｈ＝０．３５３６，Ｋ＾Ｌ＝０．６４６４。     

Erythrocyte cation content and sodium transport in Siberian huskies

Adult mixed-breed dogs and Siberian Huskies were used to study erythrocyte cation and water contents, as well as sodium transport kinetics. Erythrocyte sodium, potassium, and water contents, as well as sodium efflux and influx kinetics, were measured. Lithium efflux studies were also performed, because previous studies reported involvement of this ion in sodium transport kinetics. Sodium, potassium, and water contents in erythrocytes from mixed-breed dogs were significantly (P less than 0.05) different from those of Siberian Huskies when cells were washed in an isotonic sodium-free medium. Lithium efflux in hypertonic choline chloride was found to be significantly (P less than 0.048) different in erythrocytes obtained from Siberian Huskies, compared with those from mixed-breed dogs. Additionally, sodium efflux in isotonic choline chloride was significantly (P less than 0.01) lower in erythrocytes isolated from Siberian Huskies. Under these experimental conditions, sodium efflux was inhibited by 10 mM LiCl in erythrocytes of both groups. Amiloride (0.2 mM) was found to be a potent inhibitor of Na+ and Li+ efflux. These data indicate that Na+ metabolism may be altered in erythrocytes of Siberian Huskies, and may provide important insight into some of the erythrocyte transport abnormalities associated with specific human diseases in which erythrocyte cation content and sodium transport kinetics are altered.     

The effects of hypocalcaemia due to a 4-hour infusion of Na2EDTA solution on various blood and urine analytes in dairy cows and a comparison of these effects between cows with high and low erythrocyte potassium concentrations.

Six HK (high erythrocyte potassium) and 7 LK (low erythrocyte potassium) dairy cows were subjected to a 4-h intravenous infusion of 4.7% Na2EDTA solution to induce and maintain hypocalcaemia. Blood samples taken immediately before infusion, hourly for 7 h, and at 24 h after commencement of infusion were subjected to determination of concentration (or count) of 16 analytes. The mean changes in concentrations (or counts) of the various blood analytes were calculated for the periods 0-4, 4-7, 7-24, and 0-24 h after commencement of the infusion for all cows combined, and then separately for the HK and LK groups of cows. Plasma Ca(PCa), plasma inorganic phosphorus (PiP) and plasma potassium (PK) showed significant decreases during the 4-h infusion period and were still below pretreatment levels 24 h later. AST, CPK, PCVs and white cell-counts (WCCs) showed significant early increases which were still significantly elevated 24 h later. Plasma magnesium (PMg) and erythrocyte Na(ENa) and K(EK) all showed delayed changes which still persisted 24 h later. Significant between-group differences were present for PCVs which increased significantly more in the LK than the HK group during the infusion period, for PCa which showed a greater increase in the HK cows than the LK cows during the 4-7 h early clinical recovery period, and for plasma bilirubin (PBil) which showed a greater increase from 0 to 24 h in the HK group than in the LK group. Urine samples, collected before infusion, 4-7 h and 24 h after commencement of the infusion, were subjected to analysis for glucose, protein, pH, 'blood' and ketones. Most cows showed increases in urinary glucose, protein and 'blood'.     

Energy metabolism in canine erythrocytes associated with inherited high Na+- and K+-stimulated adenosine triphosphatase activity

Energy metabolism in canine erythrocytes associated with inherited high Na+- and K+-stimulated adenosine triphosphatase [(Na,K)-ATPase] activity (HK cells) was compared with that in normal canine erythrocytes (LK cells). Activities of some of the glycolytic enzymes in the HK cells were significantly higher than those in LK cells. The concentrations of adenosine triphosphate (ATP) and glycolytic intermediates in HK cells were almost equal to those in LK cells. Glucose utilization and lactate production by HK cells in vitro and incorporation of [32P]orthophosphate or [14C]glucose into 2,3-diphosphoglycerate in HK cells were higher than in LK cells. Radioactivity of [32P]ATP in HK cells was lower than in LK cells, but increased to approximately that of LK cells when (Na,K)-ATPase of HK cells was completely blocked by ouabain. When HK cells and LK cells were incubated in the absence of glucose, the concentration of ATP in HK cells was decreased more than that of LK cells. Although ouabain reduced the rate of decrease in ATP in HK cells, the decrease in ATP in HK cells was still 2-fold that in LK cells. The half-life of HK cells was about one-half that of LK cells. The results indicated that glycolysis is greater in HK cells than in LK cells, and that the increased glycolysis in HK cells was stimulated by an increased rate of ATP breakdown for active cation transport by the (Na,K)-ATPase and by increased degradation of ATP for some other pathway, eg, glutathione synthesis. Thus, the increased demand for ATP in HK cells might result in shortening the lifespan of HK erythrocytes.     

The relationship between reduced glutathione level and glutathione S-transferase activity in sheep erythrocytes.

The relationship between reduced glutathione (GSH) level and glutathione S-transferase (GST) activity in erythrocytes was examined, using sheep erythrocytes, which have varying GSH concentrations, and dog erythrocytes with an inherited high concentration of GSH. There was a positive correlation (r = 0.529, p < 0.001) between the GSH level and GST activity in sheep erythrocytes. In dog erythrocytes, the GST activity in high-GSH cells was significantly (p < 0.001) higher than that in normal-GSH cells. These results indicate that the activity of GST in erythrocytes is directly correlated with the intracellular GSH level.     

Stomatocytosis of Standard Schnauzers is not associated with stomatin deficiency

Stomatocytosis resembles human overhydrated hereditary stomatocytosis (OHSt), a disease characterised by a reduced or absent stomatin expression. The objective of this report was to investigate the expression level of stomatin in erythrocytes from Standard Schnauzers with stomatocytosis. Routine haematology, intraerythrocytic Na(+)/K(+) concentration and stomatin expression were evaluated in blood from twelve Standard Schnauzers and from three controls. SDS-PAGE and Western blotting on isolated integral membrane proteins were used to investigate stomatin expression. Circulating stomatocytes, macrocytosis, anisocytosis, increased erythrocyte fragility and high intracellular sodium and potassium concentrations were found in 10/12 dogs from the same breeding line although stomatin levels were similar to those of controls. In spite of the clinico-pathological similarities between human and canine stomatocytosis, erythrocytes from affected dogs do not lack stomatin and the expression level of this protein cannot therefore be used to diagnose hereditary stomatocytosis in Standard Schnauzers.     

The effect of macrophages on the erythrocyte oxidative damage and the pathogenesis of anemia in Babesia gibsoni-infected dogs with low parasitemia

The role of macrophages in the erythrocyte membrane oxidative damage and the pathogenesis of anemia in Babesia gibsoni-infected dogs with low parasitemia were investigated. Macrophages derived from peripheral blood monocytes (PBM) from B. gibsoni-infected dogs produced significantly higher chemiluminescent responses, indicating the release of reactive oxygen intermediates, than those from non-infected dogs when the cells were subjected to non-specific stimulation with phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ), or infected dog erythrocyte membranes opsonized with infected dog serum. These results indicate that PBM of B. gibsoni-infected dogs with low parasitemia were highly activated compared to those of non-infected dogs. Furthermore, the membrane lipid peroxidation of normal dog erythrocytes incubated with PBM from B. gibsoni-infected dogs was significantly higher (p<0.05) than that of erythrocytes incubated with PBM from non-infected dogs when the PBM were stimulated with the opsonized membranes. These results suggest that the oxidative damage of erythrocytes observed in B. gibsoni-infected dogs with low parasitemia might be induced, in part, by reactive oxygen species released from the activated PBM. On the other hand, the present study also showed a significant increase (p<0.001) of IgG-bound erythrocytes in B. gibsoni-infected dogs compared with such erythrocytes in non-infected dogs. The increase of IgG-bound erythrocytes in infected dogs might reflect the increase of erythrocytes with oxidative damage induced by the infection with B. gibsoni. The results of the present study suggest that the increase of IgG-bound erythrocytes in the circulation of infected dogs induce a high degree of erythrocyte loss via immunological phagocytosis by activated macrophages, resulting in severe anemia in spite of low parasitemia.     

柴达木改良绒山羊红细胞钾型的研究

采用火焰光度法对94只柴达木改良绒山羊的红细胞钾型进行了调查研究。结果发现：（1）柴达木改良绒山羊红细胞钾浓度存在多态性，有高钾和低钾两种表型，以高钾型为优势表型（79．79％）；（2）高钾型山羊的红细胞钾浓度在37－83.2mmol/L之间，低钾型的在16－28．8mmol/L之间；（3）K^L和K^h等位基因频率分别为0．1068和0．8932，基因杂合度为0．1782。     

Pseudohyperkalemia in Akitas

Hyperkalemia was observed in a clinically normal Akita with normal ACTH stimulation test results, endogenous ACTH concentration, and SUN concentration. A diagnosis of pseudohyperkalemia caused by red cell hemolysis was made by comparing plasma potassium content from hemolyzed and non-hemolyzed specimens. Red cell potassium content was measured in 8 Akitas and 6 non-Akita control dogs. Six of 8 Akitas had high erythrocyte potassium content relative to their plasma and to that of the control dogs. Five of eight Akitas had microcytosis. Plasma from affected dogs had high potassium content after being refrigerated in contact with red cells for 4 hours or more. The increase in plasma potassium content was progressive with prolonged contact with the red cells and was accompanied by a decrease in plasma sodium content.     

Utilization of an enzyme heat stability test and erythrocyte glycolytic intermediate assays to assist in the diagnosis of canine pyruvate kinase deficiency

A macrocytic hypochromic anemia, with marked reliculocytosis and large numbers of circulating nucleated erythrocytes, was recognized in a 3 1/2-year-old male Beagle-cross dog. A presumptive diagnosis of erythrocyte pyruvate kinase (PK) deficiency was made, even though PK activity from the patient was within the range of activities measured for normal dogs, because the PK activity should have been several times normal in light of the large number of reticulocytes present. The PK activity in a hemolysate from the patient was heat-labile compared to activities in hemolysates from normal dogs, a finding consistent with results from a previous study of PK-deficient Basenji dogs. Seven phosphorylated glycolytic intermediates and 2,3-diphosphoglycerate were measured in protein-free extracts of erythrocytes from the patient. All were present in concentrations above that present in normal canine erythrocytes, further supporting the diagnosis of PK deficiency.     

Apparent pseudohyperkalemia in a Chinese Shar Pei dog

Whole blood in a serum clot tube and EDTA-anticoagulated samples from an 8-year-old spayed female Chinese Shar Pei dog were submitted by an external clinic to the diagnostic laboratory at Atlantic Veterinary College for routine biochemical and hematologic analysis prior to entropion surgery. Laboratory abnormalities included mild hyperkalemia (6.3 mmol/L, reference interval 3.6-6.0 mmol/L), mild normocytic, hypochromic, nonregenerative anemia (HCT 0.31 L/L, reference interval 0.37-0.55 L/L; MCHC 290 g/L, reference interval 320-360 g/L), and increased red cell distribution width (RDW; 26.2%, reference interval 11-14%). A small subpopulation of macrocytic, slightly hypochromic erythrocytes was noted on Wright's-Giemsa-stained blood smears. Biochemical and hematologic data obtained from this patient over the previous 7.5 years indicated that serum (and in 1 case, heparinized plasma) potassium concentration was increased (range, 6.3-10.9 mmol/L) in 5 of 8 samples (HCT ranged from 0.31-0.43 L/L, Hgb 91-124 g/L, MCHC 280-312 g/L, and RDW 18.2-26.9%). Clinical signs suggestive of hyperkalemia were not observed at any time, suggesting pseudohyperkalemia as the cause of the increased potassium concentrations. An erythrocyte lysate prepared from a heparinized blood sample had a high potassium concentration (16.8 mmol/L) compared with that of a clinically healthy, non-Shiba control dog (6.7 mmol/L). An osmotic fragility test of the patient's erythrocytes showed 50% hemolysis at 0.57% NaCl, compared with 0.48% NaCl for the control dog, indicating increased fragility. On scanning electron microscopy, a small subpopulation of erythrocytes were large, flattened, and had a tendency to fold. These findings supported the provisional diagnosis of pseudohyperkalemia due to increased intracellular RBC potassium concentration. High-potassium erythrocytes have been reported in Akitas, Shibas, Jindos, other East Asian dog breeds, and occasionally, in mixed-breed dogs. Pseudohyperkalemia should also be considered when an otherwise unexplained elevation in serum or plasma potassium concentration is observed in Chinese Shar Pei dogs, and may be accompanied by increased RDW, low MCHC, and increased osmotic fragility with or without mild anemia.     

夏洛来杂种羊红细胞钾型的初步研究

采用火焰光度法对海北州牧科所优良种畜繁殖场，９０只夏洛来杂种羊的红细胞钾型进行了研究，结果发现夏洛来杂种羊红细胞钾浓度存在多态性，存在ＨＫ和ＬＫ两种类型，高钾型羊３５只（３８．８９％），低钾型羊５５只（６１．１１％），Ｋｈ和ＫＬ等位基因频率分别为０．６２３６和０．３７６４。     

Serum from dogs Infected with Babesia gibsoni inhibits maturation of reticulocytes and erythrocyte 5'-nucleotidase activity in vitro

Erythrocyte 5'-nucleotidase is thought to be involved in the maturation of erythrocytes. In the present study, in vitro incubation of canine erythrocytes demonstrated that significant inhibition of 5'-nucleotidase activity occurred in the presence of serum from dogs infected with Babesia gibsoni, when the enzyme was assayed with cytidine 5'-monophosphate (5'-CMP) and inosine 5'-monophosphate (5'-IMP) as substrates. The multiplication of B. gibsoni in in vitro culture also resulted in a significant decrease in the enzyme activity of erythrocytes in the culture. Furthermore, the infected serum and 5'-CMP retarded the maturation of canine reticulocytes in vitro. These results suggested that nucleotides such as 5'-CMP and 5'-IMP might accumulate in young erythrocytes and/or serum in dogs infected with B. gibsoni as a result of decreased activity of erythrocyte 5'-nucleotidase, resulting in the delayed maturation of reticulocytes.