John W. Harvey, DVM, PhD, DACVP
Erythrocytes provide vital functions of oxygen transport, carbon dioxide transport, and buffering of hydrogen ions. Although these functions do not require energy per se, energy in the form of ATP, NADH, and NADPH is needed to keep erythrocytes circulating for months in a functional state despite repeated exposures to mechanical and metabolic insults.
Nuclei are extruded during erythrocyte production; consequently, mature erythrocytes cannot synthesize nucleic acids or proteins. The loss of mitochondria during the maturation of reticulocytes results in a loss of Krebs's cycle and oxidative phosphorylation capabilities and prevents the synthesis of heme or lipids de novo in erythrocytes.
Erythrocytes utilize anaerobic glycolysis to provide ATP for maintenance of shape, maintenance of deformability, phosphorylation of membrane phospholipids and proteins, active membrane transport of various molecules, partial synthesis of purine and pyrimidine nucleotides, and synthesis of glutathione. NADH needed for the reduction of methemoglobin is also generated during anaerobic glycolysis. The pentose phosphate pathway is used to generate NADPH for the protection against oxidants. Canine erythrocytes normally contain high concentrations of 2,3-diphophoglycerate (2,3DPG), whereas feline erythrocytes have low concentrations of 2,3DPG. The oxygen affinity of hemoglobin is decreases as 2,3DPG increases in dogs.1
Disorders in ATP Generation
Deficiencies in rate-controlling enzymes in anaerobic glycolysis can result in decreased ATP generation, shortened erythrocyte life spans and anemia. Hexokinase, phosphofructokinase (PFK), and pyruvate kinase (PK) are the 3 rate-controlling enzymes in glycolysis, and inherited deficiencies in the latter two enzymes have been reported in animals.
Autosomal recessively transmitted PFK deficiency of erythrocytes and skeletal muscle occurs in English springer spaniel, American cocker spaniel and whippet dogs.2-4 Since homozygously affected dogs have a deficiency of the muscle-type subunit of PFK and the muscle subunit accounts for most of the activity in erythrocytes and skeletal muscle, total PFK activities are markedly reduced in both of these tissues.
Affected dogs have persistent compensated hemolytic anemia and sporadic episodes of intravascular hemolysis with hemoglobinuria. Erythrocyte mean cell volumes are usually between 80 fl and 90 fl. Uncorrected reticulocyte counts are generally between 10% and 30%, and hematocrit values are usually between 30% and 40%, except during hemolytic crises when the hematocrit may decrease to 15% or less. Lethargy, weakness, pale or icteric mucous membranes, mild hepatosplenomegaly, muscle wasting, and fever as high as 41°C may occur during hemolytic crises.
Physiological studies and clinical observations have demonstrated that episodes of intravascular hemolysis can result from hyperventilation-induced alkalemia. This occurs because erythrocytes in affected dogs are more alkaline fragile than in normal dogs due to a marked decrease in erythrocyte 2,3-DPG concentration.
Deficient dogs generally exhibit less evidence of myopathy than is observed in PFK-deficient humans, probably because canine skeletal muscle is less dependent on anaerobic glycolysis than is human skeletal muscle, owing to a lack of the classical fast-twitch glycolytic (type IIB) fibers in dogs. Evidence of muscle dysfunction is usually limited to exercise intolerance and slightly decreased muscle mass, but muscle cramping and severe progressive myopathy can occur. Two related whippets also exhibited congestive heart failure. Heterozygous carrier animals appear clinically normal.
Homozygous affected animals more than three months of age can easily be identified by measuring erythrocyte PFK activity. Heterozygous carrier dogs have approximately one-half normal enzyme activities in erythrocytes and skeletal muscle. A DNA test using polymerase chain reaction technology has been developed which can clearly differentiate normal and carrier animals of any age.
In contrast to PK deficiency, myelofibrosis and liver failure have not been recognized in dogs with PFK deficiency. Animals with this deficiency can have a normal life span if properly managed. Owners should avoid placing affected dogs in stressful situations or subjecting them to strenuous exercise, excitement or high environmental temperatures.
Pyruvate Kinase Deficiency
Congenital hemolytic anemia, resulting from erythrocyte pyruvate kinase (PK) deficiency, occurs in basenji, beagle, West Highland white terrier, Cairn terrier, miniature poodle, Chihuahua, pug, and American Eskimo dogs and Abyssinian, Somali, and domestic shorthair cats.2,3 The deficiency is transmitted as an autosomal recessive trait. Homozygously affected animals have decreased exercise tolerance, pale mucous membranes, tachycardia and splenomegaly. Since pyruvate kinase catalyzes an important rate-controlling, ATP-generating step in glycolysis, energy metabolism is markedly impaired in PK-deficient erythrocytes, resulting in shortened erythrocyte life spans and anemia. The bone marrow attempts to compensate by erythroid hyperplasia, with marked reticulocytosis present in peripheral blood.
Affected animals have macrocytic hypochromic anemia (hematocrit 16% to 28%), with uncorrected reticulocyte counts of 15% to 50%. Leukocyte counts are generally normal or slightly increased with a mature neutrophilia. Platelet counts are normal to slightly increased. Moderate to marked polychromasia and anisocytosis and frequent nucleated erythrocytes are recognized on stained blood films.
Diagnosis of PK deficiency can be made in cats and some dogs by measuring low total erythrocyte PK activity, but many affected dogs have normal or increased activities, because of the expression of an M2 isozyme not normally present in mature erythrocytes. Heterozygous animals have approximately 50% of normal erythrocyte PK activity.
Additional assays (an enzyme heat stability test, measurement of erythrocyte glycolytic intermediates, electrophoresis of isozymes and enzyme immunoprecipitation) may be used to reach a diagnosis of PK deficiency in dogs in which the total enzyme activity is not decreased. Breed and mutation specific DNA-based assays are required to diagnose PK deficiency in basenji, beagle, and West Highland white and Cairn terrier dogs. Unfortunately, the genetic defects are not identical in all affected dog breeds; consequently, these tests are not valid in affected animals from breeds in which the nature of the defect is different. A common molecular defect has been described in cats to date.
An unexplained feature of the disease is the progressive development of myelofibrosis and osteosclerosis. Affected animals generally die by four years of age because of bone marrow failure and/or liver disease with hemochromatosis and cirrhosis.
Glycolysis is inhibited by short-term phosphate deficiency, primarily by decreasing intracellular phosphate for the glyceraldehyde-3-phosphate dehydrogenase (GAPD) reaction.1 Decreased glycolytic rates result in decreased erythrocyte ATP concentrations and hemolytic anemia in experimental dogs made severely hypophosphatemic by hyperalimentation. Hemolytic anemia associated with hypophosphatemia has also been reported in diabetic cats and a diabetic dog following insulin therapy and in a cat with hepatic lipidosis.1 In addition to having low ATP concentrations, dog erythrocytes might hemolyze as a result of decreased 2,3DPG concentration, because dog erythrocytes with low 2,3DPG are more alkaline fragile than those of normal dogs and may hemolyze at physiologic pH values.3
Disorders in 2,3DPG Metabolism
The DPG pathway or shunt bypasses the ATP-generating phosphoglycerate kinase step in glycolysis; consequently, no net ATP is generated when glucose is metabolized through this pathway. The flow through this pathway is determined by the overall glycolytic rate. The formation of 2,3DPG is stimulated by increased phosphate concentration and increased pH. Hypoxic conditions stimulate 2,3DPG synthesis primarily by inducing hyperventilation, which results in alkalosis. Conversely, acidosis and hypophosphatemia result in decreased 2,3DPG concentrations. PFK deficiency inhibits glycolysis and results in decreased 2,3DPG concentration. The concentration of 2,3DPG can be increased by a decrease in PK activity. When PK activity is reduced relative to PFK activity, as occurs in PK deficiency, phosphorylated intermediates between the PK and GAPD reactions (including 2,3DPG) increase in concentration.1
Erythrocyte 2,3DPG increases in anemic dogs5,6 and cats.7 The resultant decrease in hemoglobin oxygen affinity would seem to be beneficial in response to anemia in the dog. 2,3DPG concentration is much lower in cat erythrocytes than in dog erythrocytes, and cat hemoglobins are generally less responsive to 2,3DPG than dog hemoglobin; consequently, the physiologic significance of this increase in cats in unclear.
Oxidants and Erythrocytes
Nature of Oxidative Injury
Oxidants may damage erythrocyte hemoglobin, enzymes (especially sulfhydryl groups), and membranes (especially polyunsaturated lipids). Methemoglobin forms when hemoglobin iron is oxidized from the +2 to the +3 state. Methemoglobin is unable to bind oxygen, but its presence alone does not result in shortened erythrocyte life span. Heinz bodies are inclusions that form within erythrocytes following the oxidative denaturation of the globin portion of hemoglobin. Heinz bodies bind to the inner surface of erythrocyte membranes, and their presence can result in premature erythrocyte phagocytosis. Oxidative membrane damage results in premature phagocytosis of injured erythrocytes. Oxidative injury to erythrocyte membranes in dogs may result in the adhesion of opposing areas of the cytoplasmic face of the erythrocyte membrane and the formation of eccentrocytes and pyknocytes.1
Sources of Oxidants
Animals are exposed to low levels of oxidants in their environment and from normal metabolic processes in the body. Cumulative injury from these exposures may account for the normal aging and removal of circulating erythrocytes. Some metabolic disorders (diabetes, hyperthyroidism, neoplasia, and inflammation) can generate oxidants in sufficient amounts to result in increased oxidant injury and shortened erythrocyte life spans, but the anemia (when present) is generally mild.1 Certain therapeutic drugs, dietary constituents, and chemicals have the potential to induce severe oxidative injury and anemia. Onions and garlic may be consumed in sufficient quantities to cause hemolytic anemia in dogs and cats. Propylene glycol has been used as a humectant in soft-moist cat and dog food in the past. At the levels included in these foods, it caused prominent Heinz body formation and shortened erythrocyte life-spans in cats, but minimal anemia. Clients may administer over-the-counter drugs, including acetaminophen and benzocaine, and veterinarians may administer prescription drugs, including methylene blue, phenazopyridine (cats), methionine (cats), and vitamin K, that induce Heinz body hemolytic anemia. Repeated propofol anesthesia has been reported to cause Heinz body formation in cats and eccentrocyte formation in dogs, but the erythrocyte injury is not severe enough to produce anemia.8 Dogs have also been reported to consume chemicals, including naphthalene and zinc containing objects (especially US pennies minted after 1982), that cause hemolytic anemia,1 and eccentrocytes have been reported in dogs with vitamin K antagonist rodenticide toxicity (before vitamin K therapy).8 Severe hemolytic anemia has been reported in a dog sprayed with skunk musk. (Zaks, Tan, et al. 2005 13742 /id)
Erythrocyte Response to Oxidants
The pentose phosphate pathway (PPP) generates NADPH, the major source of reducing equivalents in the protection of erythrocytes against oxidative injury. Normally only about 5 to 13% of glucose metabolized by erythrocytes flows through the PPP, but this flow can be accelerated markedly by oxidants.1 NADPH is utilized to reduce oxidized glutathione (GSSG) to reduced glutathione (GSH), using the glutathione reductase enzyme. GSH has an easily oxidizable sulfhydryl group that may act nonenzymatically as a free radical acceptor to counteract oxidant damage. GSH also functions as an electron donor in various reductive enzyme reactions including glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, glutathione S-transferase, and glutaredoxin. GSH concentration is reduced in erythrocytes when the oxidant stress exceeds the ability of the PPP and glutathione reductase reactions to reduce GSSG to GSH. Deficiencies in the rate limiting enzyme of the PPP, glucose-6-phosphate dehydrogenase (G6PD), and in glutathione reductase (secondary to flavin adenine dinucleotide deficiency) result in membrane damage and shortened erythrocyte life spans in horses. Similar defects have not been reported in dogs and cats.
It is estimated that 2% to 3% of erythrocyte hemoglobin is oxidized to methemoglobin each day in dogs. This formation results from spontaneous autoxidation of oxyhemoglobin, and possibly secondarily to oxidants produced in normal metabolic reactions.1 Increased methemoglobin production is induced by oxidants such as benzocaine, acetaminophen, phenazopyridine, and skunk musk. Methemoglobin can be reduced to hemoglobin within erythrocytes by the cytochrome-b5-reductase (Cb5R) or methemoglobin reductase enzyme. In the reaction, ferricytochrome b5 is first reduced enzymatically with NADH; then the resulting ferrocytochrome b5 reduces methemoglobin nonenzymatically to hemoglobin. This enzyme reaction is normally able to maintain the methemoglobin content at about 1% of total hemoglobin.
Cytochrome-b5 Reductase Deficiency
Persistent methemoglobinemia associated with erythrocyte Cb5R deficiency has been recognized in Chihuahua, borzoi, English setter, terrier-mix, Cockapoo, coonhound, poodle, corgi, Pomeranian, toy American Eskimo, cocker-toy American Eskimo, and pit bull-mix dogs and in domestic shorthair cats.9 The deficiency appears to be an inherited autosomal recessive disorder, as it is in humans.
Affected animals have cyanotic-appearing mucous membranes, and may exhibit lethargy or exercise intolerance at times, but frequently have no clinical signs of disease. Blood samples appear dark, suggesting hypoxemia, but arterial pO2 values are normal. In several cases, methemoglobinemia was recognized for the first time during routine surgery when mucous membranes and blood remained dark even when animals were given supplemental oxygen.
Methemoglobinemia may not be apparent in normally dark venous blood samples, but a spot test can be used to determine whether clinically significant levels of methemoglobin are present. One drop of blood from the patient is placed on a piece of absorbent white paper and a drop of normal control blood is placed next to it. If the methemoglobin content is 10% or greater the patient's blood should have a noticeably brown coloration, compared to a bright red color of the control blood. When assayed spectrophotometrically, methemoglobin content in deficient dogs varies from 13% to 41%. The methemoglobin content in 5 deficient domestic shorthair cats varied from 44% to 52%. There is an inverse relationship between erythrocyte enzyme activity and methemoglobin content in deficient dogs. The hematocrit is usually normal in deficient dogs, but often slightly to moderately increased in deficient cats, secondary to the chronic methemoglobinemia and resultant decreased blood oxygen content. Animals with Cb5R deficiency do not require treatment and have normal life expectancy.
1. Harvey JW. In: Clinical Biochemistry of Domestic Animals. San Diego: Academic Press; 2008:in press;
2. Giger U. In: Schalm's Veterinary Hematology. Philadelphia: Lippincott Williams & Wilkins; 2000:1020;
3. Harvey JW. Vet Clin Pathol. 2006;35:144;
4. Gerber KL, et al. Unpublished; 2008;
5. King LG, et al. J Vet Intern Med. 1992;6:264;
6. Paltrinieri S, et al. J Comp Pathol. 2000;122:25;
7. Mauk AG, et al. Science. 1974;185:447;
8. Caldin M, et al. Vet Clin Pathol. 2005;34:224;
9. Harvey JW. In: Schalm's Veterinary Hematology. Philadelphia: Lippincott Williams & Wilkins; 2000:1008.