Pyruvate kinase deficiency anaemia in a Basenji dog

SUMMARY Pyruvate kinase deficiency anaemia was suspected in an 18-month-old male Basenji dog after other known causes of canine haemolytic anaemia had been excluded. Anaemia of moderate severity (packed cell volumes 0.20 to 0.26 1/1) and reticulocytosis (uncorrected reticulocyte counts 8 to 43%) persisted during 5 months' observation, and biopsies showed development of bone marrow fibrosis and sclerosis. The diagnosis of pyruvate kinase deficiency anaemia was presumptive because erythrocyte pyruvate kinase concentrations in the affected dog were inconclusive and related animals were not available for enzyme assay. However, the gene for pyruvate kinase deficiency is known to occur among Basenji dogs in Australia.     

Nonspherocytic haemolytic anaemia due to phosphofructokinase deficiency in an English Springer Spaniel

A five-year-old female English Springer Spaniel was evaluated for chronic episodic anaemia, pigmenturia and icterus. There was increased erythrocyte pyruvate kinase activity, increased in vitro erythrocyte alkaline fragility, decreased erythrocyte 2,3-diphosphoglycerate activity and severely decreased erythrocyte phosphofructokinase activity. The clinical features and haematological values were identical with erythrocyte phosphofructokinase deficiency and resulting nonspherocytic haemolytic anaemia recently described in this breed.     

Inherited erythrocyte Syruvate kinase eficiency in the West Highland white terrier

An eight-month-old male West Highland white terrier (WHWT) presented with mild exercise intolerance and splenomegaly caused by a severe and highly regenerative haemolytic anaemia that persisted until the dog died at two years of age. There was marked erythroid hyperplasia in the bone marrow and progressive osteosclerosis. Erythrocyte pyruvate kinase (PK) deficiency was confirmed by demonstrating a block in glycolysis at the PK step, abnormal erythrocyte PK kinetics, absence of R-type PK isoenzyme, and the presence of M₂-type PK not normally expressed in erythrocytes. From the study of family members it is concluded that erythrocyte PK deficiency in the WHWT is inherited as an autosomal recessive trait. Heterozygotes have about half-normal erythrocyte PK activity, do not express the M₂-type PK isoenzyme and are asymptomatic. This disease closely resembles erythrocyte PK deficiency in the basenji breed and should be considered as a differential diagnosis in young dogs with severe haemolytic anaemia.     

Inherited erythrocyte pyruvate kinase deficiency in a beagle dog

A 1-year-old, female Beagle dog with minimal exercise intolerance was found to have a persistent, severe, and highly regenerative anemia, splenomegaly, and progressive osteosclerosis. Despite near-normal in vitro erythrocyte pyruvate kinase (PK) activity, the authors diagnosed PK deficiency by demonstrating a glycolytic block at the PK step, the lack of normal R-type PK isoenzyme, and the presence of M(2)-type PK in the animal's erythrocytes. The dam had half-normal erythrocyte PK activity, which supports an autosomal recessive mode of inheritance. We conclude from our studies that close similarities exist between erythrocyte PK deficiency in Beagle, Basenji, and West Highland White Terrier dogs and that this form of PK deficiency may be more widespread than previously thought.     

INHERITED PYRUVATE KINASE DEFICIENCY AND NORMAL HAEMATOLOGIC VALUES IN AUSTRALIAN BASENJI DOGS

SUMMARY We have carried out a survey to determine the prevalence of carriers of pyruvate kinase deficiency haemolytic anaemia in Basenjis in Victoria. Of 186 dogs tested, 20 were found to be carriers for pyruvate kinase deficiency, a prevalence similar to that reported overseas. Of the 20 carriers found, 17 were born in Australia and were traced directly to 1 of the 3 imported carriers. Despite the relatively high frequency of carriers, no mating of 2 carriers has yet been detected and no dogs with haemolytic anaemia have been observed. The inheritance of the pyruvate kinase deficiency has been traced through up to 5 generations of dogs in Australia and is consistent with co-dominance for pyruvate kinase activity and with an autosomal recessive gene for haemolytic anemia.     

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.     

Erythrocyte pyruvate kinase deficiency in a beagle dog

A macrocytic hypochromic anemia with marked reticulocytosis was observed at each of 10 examinations during a 4 month period of a male Beagle dog that was 11 months old when first examined. The owner had noticed pale mucous membranes from the time the dog was purchased at 7 weeks of age. Based on the clinical history and negative LE, ANA and Coombs tests, an intrinsic erythrocyte defect was suspected. Erythrocyte osmotic fragility and hemoglobin electrophoresis were normal. Autohemolysis of incubated erythrocytes was marked. Hemolysis was uncorrected by glucose but partially corrected by ATP. With the exception of pyruvate kinase (PK), all of the erythrocyte enzymes measured had greater activities than those of control Beagle erythrocytes. In comparison to moderately reticulocyte-rich controls. PK activity was low in the affected Beagle. Erythrocyte phosphoenolpyruvate content was increased significantly as expected in PK deficiency. The kinetic constant (Km) for phosphoenolpyruvate was lower than that of normal control dogs, a finding in agreement with studies of PK from deficient Basenjis. On these bases a diagnosis of PK deficiency was made.     

Pyruvate kinase deficiency anemia with terminal myelofibrosis and osteosclerosis in a beagle.

A 15-month-old male Beagle with chronic hemolytic anemia was found to have erythrocytic pyruvate kinase deficiency and, terminally, myelofibrosis and osteosclerosis. The dog's erythron was studied by procedures that enabled close comparison with congenital hemolytic anemia (pyruvate kinase deficiency) of Basenji dogs. The affected dog's sire, dam, and one littermate--each clinically and hematologically normal--were found to have 50% reduction in erythrocytic pyruvate kinase (PK) activity.     

Determination of erythrocyte pyruvate kinase deficiency in Basenjis with chronic hemolytic anemia

Erythrocyte pyruvate kinase (PK) deficiency was first described in Basenjis 20 years ago. Although the approach to diagnosis had not been well established, a screening program to detect heterozygotes was thought to have eliminated PK deficiency from the Basenjis of the United States. Four not closely related Basenjis with severe chronic hemolytic anemia, from various parts of the United States, were studied. Their PCV ranged from 16 to 25% and their reticulocyte count was always above 15%. A progressive osteosclerosis was seen in all of the Basenjis and hepatic failure developed in 2 of them. The erythrocyte intermediary metabolite patterns indicated a glycolytic defect at the PK step. Erythrocyte PK activities were markedly increased in the anemic Basenjis, compared with those of a control group, but the enzyme in these Basenjis had abnormal kinetic properties and was thermolabile. An antibody against R-type PK, the regular erythrocyte PK form, did not neutralize the PK activity of affected Basenjis, and results of electrophoretic studies suggested the expression of M2-type PK, a leukocyte and fetal erythroid PK-type. Clinically healthy heterozygous Basenjis had half-normal R-type PK activity and did not express the M2-type in their erythrocytes. We conclude that severe chronic hemolytic anemia, caused by erythrocyte PK deficiency, and associated osteosclerosis still develop in Basenjis. A definitive diagnosis cannot be reached by simply measuring erythrocyte PK activity; rather, diagnosis requires measurement of glycolytic substrate accumulation and enzyme stability and immunologic or electrophoretic studies of erythrocyte PK.     

Erythrocyte pyruvate kinase deficiency in three West Highland white terriers in Ireland and the UK

Erythrocyte pyruvate kinase (PK) deficiency is described for the first time in three apparently unrelated West Highland white terriers (WHWT) from Ireland and the UK. All three dogs were diagnosed with markedly regenerative but persistent anaemia and had been treated for presumed immune-mediated haemolytic anaemia (IMHA) before hereditary erythrocyte PK-deficiency was confirmed by breed-specific DNA mutation analysis. This hereditary erythroenzymopathy causes haemolytic anaemia and affects several canine breeds with varying degrees of severity. Although eventually causing osteosclerosis, haemosiderosis and death, PK-deficient dogs can adapt to their anaemia for many years.PK-deficiency should be considered in anaemic WHWTs worldwide particularly in dogs with haemolytic anaemia where evidence for an immune-mediated, infectious or toxic underlying cause is lacking.     

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.     

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.     

Increased indices of lipid peroxidation in stress-susceptible pigs and effects of vitamin E

When stress-susceptible and stress-resistant pigs consumed diets containing 10 iu vitamin E kg-1, the stress-susceptible pigs had damaged cell membranes. This was indicated by increased plasma activities of creatine kinase and pyruvate kinase. Plasma concentrations of thiobarbituric acid reactive substances and conjugated dienes were also increased suggesting that the membrane damage was caused by free radicals. Susceptibility to free radicals was further demonstrated by an increased tendency for erythrocytes and tissue homogenates from stress-susceptible pigs to peroxidise when incubated with hydrogen peroxide and iron, respectively. Supplementation of the diets with approximately 20 times normal requirements of vitamin E decreased plasma creatine kinase and pyruvate kinase activities, and inhibited the formation of the indices of peroxidation in vivo and in vitro. It is concluded that the stress-susceptible pig has an abnormality in its antioxidant defence mechanisms. However, this abnormality was not due to a deficiency in selenium-dependent glutathione peroxidase, since activities were significantly increased in the longissimus dorsi of stress-susceptible pigs. Although the nature of the antioxidant defect is unclear, it is suggested that stress-susceptible pigs are under a sustained oxidant stress and that a decreased ability to accommodate even a normal free radical load may contribute to the rapid development of the fatal stress response.     

Identification of a 6 base pair insertion in West Highland White Terriers with erythrocyte pyruvate kinase deficiency.

OBJECTIVE: To define the disease-causing mutation in West Highland White Terriers (WHWT) with erythrocyte pyruvate kinase (R-PK) deficiency and to design a genetic test capable of recognizing affected (homozygous) and carrier (heterozygous) dogs. ANIMALS: 3 anemic WHWT littermates and 1 unaffected littermate; 16 dogs from the same kennel, including 4 unrelated, phenotypically normal dogs (control dogs), and 12 for which PK activity was not known; 2 PK-deficient Basenjis; 2 PK-deficient Beagles; 4 unaffected English Springer Spaniels; and 1 mixed-breed dog. PROCEDURES: cDNA was cloned and sequenced, and cDNA sequences were compared with the published sequence for canine R-PK cDNA to identify the putative disease-causing mutation. Genomic DNA spanning the affected region was cloned and sequenced to verify the mutation. Subsequently, polymerase chain reaction primers were designed to amplify the section of the gene containing the mutation from DNA in blood or buccal swab samples. Gel electrophoresis allowed assignment of genotypes on the basis of allele separation. RESULTS: 4 single base polymorphisms attributable to sequencing errors in the published sequence were identified, along with a 6 base pair (bp) insertion in exon 10 that was recognized as a putative disease-causing mutation. An identical insertion was found in genomic DNA. Amplification of genomic DNA yielded a 117 bp product for genotypically normal dogs and a 123 bp product for WHWT homozygous for PK deficiency. Carriers had 1 copy of each allele and variable heteroduplex structures. CONCLUSIONS AND CLINICAL RELEVANCE: A 6 bp insertion in the C domain of R-PK was identified in WHWT with PK deficiency. Affected and carrier dogs could be distinguished with a genetic test.     

Increased peroxidation of erythrocytes of stress-susceptible pigs: an improved diagnostic test for porcine stress syndrome

When incubated with solutions of hydrogen peroxide, erythrocytes of stress-susceptible pigs produced more by-products of lipid peroxidation (as measured as thiobarbituric acid-reactive substances [TBARS]) than did erythrocytes from stress-resistant pigs. Using this technique, discrimination between the 2 pig types was absolute at hydrogen peroxide concentrations of 0.9 and 1.5%. This was in contrast to other methods of identifying stress-susceptible pigs, such as osmotically induced erythrocyte lysis and the determination of plasma pyruvate kinase and creatine kinase activities, for which considerable overlap of data was observed between pig types. The increased TBARS production by erythrocytes was further evidence for the existence of an antioxidant abnormality in stress-susceptible pigs. However, because there were no discernible differences in the major blood antioxidant-related values between stress-susceptible and stress-resistant pigs, the nature of the defect remains unclear. The production of TBARS by erythrocytes when incubated with hydrogen peroxide provides an improved method for identifying stress-susceptible pigs.     

Bilirubin cholelithiasis and haemosiderosis in an anaemic pyruvate kinase-deficient Somali cat

A Somali cat was presented with recurrent anorexia, lethargy, vomiting and icterus. A macrocytic-hypochromic, regenerative haemolytic anaemia was identified and hereditary pyruvate kinase deficiency was confirmed by means of breed-specific DNA mutation analysis. The case was complicated by the presence of markedly elevated serum liver enzyme activities, hyperbilirubinaemia, coagulopathy and ultrasonographic evidence of gall bladder choleliths and extrahepatic bile duct obstruction. The choleliths consisted of 100 per cent bilirubin, likely because of chronic haemolysis and haeme degradation. In conclusion, haemosiderosis and bilirubin cholelithiasis can be a consequence of chronic haemolysis in pyruvate kinase-deficient cats, as seen in human beings with a variety of chronic haemolytic disorders.     

Pyruvate kinase deficiency in a Somali cat in Australia

A 7-year-old neutered male Somali cat, bred in Western Australia, was presented for investigation of jaundice and severe anaemia. Splenomegaly and hepatomegaly were evident on physical examination. Severe anaemia, along with leukopenia and increased liver enzymes, were present on laboratory evaluation. Clinical investigation identified cholangitis and treatment for this resolved the jaundice but failed to resolve the anaemia. Treatment for Mycoplamsa haemofelis was administered concurrently. Genetic testing was then performed and pyruvate kinase deficiency was identified, the first time this has been reported in an Australian cat. Treatment with immunosuppressive medication was not successful.     

Assessment of serum thyroid hormone concentrations in lambs with selenium deficiency myopathy

OBJECTIVE: To assess changes in serum concentrations of thyroid hormones associated with selenium deficiency myopathy in lambs. ANIMALS: 35 lambs with selenium deficiency myopathy and 30 healthy lambs. PROCEDURES: Blood samples were collected via jugular venipuncture from lambs with selenium deficiency myopathy and healthy lambs. Activities of markers of selenium deficiency myopathy (erythrocyte glutathione peroxidase [GSH-Px] and plasma creatine kinase [CK]) and serum thyroid-stimulating hormone (TSH) and total thyroxine (tT(4)) and total triiodothyronine (tT(3)) concentrations were assessed; values in affected lambs were compared with those in healthy lambs. Correlations of erythrocyte GSH-Px and plasma CK activities with serum concentrations of TSH, tT(4), and tT(3) were investigated, and the tT(3):tT(4) concentration ratio was evaluated. RESULTS: Compared with findings in healthy lambs, erythrocyte GSH-Px activity, serum tT(3) concentration, and tT(3):tT(4) concentration ratio were significantly decreased and serum concentrations of tT(4) and TSH and the activity of plasma CK were significantly increased in affected lambs. Analysis revealed a significant negative correlation in the affected group between erythrocyte GSH-Px activity and each of the following: plasma CK activity (r = -0.443), serum TSH concentration (r = -0.599), serum tT(4) concentration (r = -0.577), and serum tT(3) concentration (r = -0.621). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that notable changes in circulating amounts of thyroid hormones develop in association with selenium deficiency in lambs. Such alterations in thyroid hormone metabolism may be involved in the high incidence of disorders, such as stillbirths and neonatal deaths, in selenium-deficient flocks.     

Surface ultrastructure of pyruvate kinase-deficient erythrocytes in the Basenji dog.

The scanning electron microscope was used to study the surface morphologic features of erythrocytes from a Basenji dog with hereditary hemolytic anemia due to a pyruvate kinase-deficient erythrocytes (RBC). Cells from this dog were compared with RBC from normal dogs and from dogs with regenerative anemias unrelated to pyruvate kinase deficiency. Demonstration of spherical RBC with uniform spicules on their surface (spheroechinocytes) may provide a morphologic explanation for the shortened erythrocytic life-span previously reported in congenital hemolytic anemia of Basenji dogs. Spherical, spiculated RBC were not found in blood from normal dogs or from anemic dogs with reticulocytoses. The surface of reticulocytes from all dogs with regenerative anemia was roughened, with pronounced folding of the cell membrane.     

Comparative Study of Erythrocyte Glycolytic Enzymes in Man and in Two Species of Lagomorphae

The authors made a comparative study of red cell enzyme glycolysis in man and in two species of Lagomorphae, the pika and the rabbit. The activities of the 12 enzymes of Embden-Meyerhoff pathway and of the two dehydrogenases of pentose shunt (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined. Phosphoglycerate kinase and pyruvate kinase showed quite similar activities in pika erythrocytes and in erythrocytes from human umbilical cord. The levels of these enzymes differed significantly in the pika and in the rabbit. No differences were noted between pyruvate kinases from the rabbit and the neonatal man. The other activities gave values either identical to those found in the adult man or intermediate between the adult and the neonatal man. In the rabbit the levels of glycolitic enzymes were generally lower than in the pika except for lactate dehydrogenase and glycose-6-phosphate dehydrogenase. Some characteristics of erythrocyte metabolism specific to the pika may account for the differences observed in this species. The influence of red cell age cannot explain the variations observed for no significant reticulocytosis was observed in the circulating blood. The percentages found in the pika and the rabbit were essentially identical at determination.