Congenital Erythrocyte Enzyme Deficiencies

UPDATE ON CLINICAL PATHOLOGY

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CONGENITAL ERYTHROCYTE ENZYME DEFICIENCIES John W. Harvey, DVM, PhD

Mature mammalian erythrocytes do not have nuclei; consequently, they cannot synthesize nucleic acids or proteins. The loss of mitochondria during the maturation of reticulocytes prevents the synthesis of heme or lipids de novo in erythrocytes. Although metabolic demands are lower than those of other blood cell types, erythrocytes still require energy in the form of adenosine triphosphate (ATP) for maintenance of shape, deformability, active membrane transport, and limited synthetic activities such as salvage pathway synthesis of adenine nucleotides and phospholipids and the synthesis of glutathioneY Mature erythrocytes depend solely on anaerobic glycolysis for ATP generation, because the Krebs cycle and oxidative phosphorylation are present within mitochondria. Consequently, deficiencies of enzymes involved in glycolysis can have significant effects on erythrocyte function and/ or survival. Circulating erythrocytes are exposed to a variety of endogenously generated oxidants and the damage that results from these oxidants appears to play an important role in the natural aging and ultimate removal of these cells from the circulation by mononuclear phagocytes. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) generated in the pentose phosphate pathway is important in protecting against these oxidants. It is needed to maintain glutathione in the reduced state and is important in maintaining catalase in a functional form. 17 A defect in the pentose phosphate pathway can render erythrocytes susceptible to endogenous and exogenous oxidant injury. Methemoglobin differs from hemoglobin in that the iron moiety of heme groups has been oxidized to the ferric ( + 3) state, which can no

From the Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida

VETERINARY CLINICS OF NORTH AMERICA: SMALL ANIMAL PRACTICE VOLUME 26 • NUMBER 5 • SEPTEMBER 1996

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longer bind oxygen. About 3% of hemoglobin within erythrocytes is oxidized to methemoglobin each day in normal animals; however, methemoglobin usual!y accounts for less than 1% of total hemoglobin because it is constantly reduced back to hemoglobin by a reduced nicotinamide adenine dinucleotide (NADH)-dependent methemoglobin reductase (cytochrome-b5-reductase) enzyme reaction present within erythrocytes. A deficiency in this enzyme can result in persistent methemoglobinemia. DIFFERENTIAL DIAGNOSIS

Congenital erythrocyte enzyme deficiencies must be differentiated from various acquired disorders. Congenital disorders are often recognized in young animals, but in some animals erythrocyte enzyme deficiencies are not recognized for several years. Severe deficiencies of rate-controlling enzymes in glycolysis (phosphofructokinase and pyruvate kinase) result in hemolytic anemia with a regenerative bone marrow response. These deficiencies must be separated from various other causes of hemolytic anemia including autoimmune hemolytic anemia, haemobartonellosis, babesiosis, drug-induced hemolytic anemia, and microangiopathic hemolytic anemia. Enzyme-deficient animals should be Coombs' test negative, lack parasites and Heinz bodies in stained blood films, be seronegative for Babesia species, and lack evidence of disseminated coagulation, heartworm disease, or hemangiosarcoma. Deficiencies in glucose-6-phosphate dehydrogenase, the rate controlling enzyme in the pentose phosphate pathway, render erythrocytes susceptible to oxidant-induced injury. If the enzyme defect is severe, erythrocytes may be damaged by normal endogenous oxidative reactions resulting in a persistent hemolytic anemia that :g.:l:ttst be differentiated from the disorders listed above. If the defect is riot severe, anemia may only occur after administration of oxidant drugs or exposure to excessive amounts of endogenous oxidants. The presence of eccentrocytes and/ or Heinz bodies within erythrocytes indicates the presence of oxidant injury. Methemoglobinemia (methemoglobin content > 1.5%) in animals results from either increased production of methemoglobin by oxidants or decreased reduction of methemoglobin associated with a deficiency in the erythrocyte methemoglobin reductase enzyme. Both low blood oxygen tension and methemoglobinemia can result in cyanotic-appearing mucous membranes and dark-colored blood samples. Hypoxemia is documented by measuring a low Po2 of an arterial blood sample. Methemoglobinemia is suspected when arterial blood appears dark even in the face of normal or increased Po2 • The presence of clinical signs of toxicosis such as anorexia, vomiting, diarrhea, depression, rapid heart rate, rapid respiratory rate, ataxia, stupor, hemoglobinuria and/ or subcutaneous edema in an animal with

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cyanotic-appearing tongue and mucous membranes suggests drug-induced methemoglobinemia. Significant methemoglobinemia has been associated with clinical cases of benzocaine, acetaminophen, and phenazopyridine toxicities in cats and/ or dogs. These drugs can also produce Heinz body hemolytic anemiaY The presence of persistent methemoglobinemia in a nonanemic animal with minimal or no clinical signs suggests a deficiency in the methemoglobin reductase enzyme.

ERYTHROCYTE ENZYME ASSAYS

Efforts should be made to rule out acquired causes of anemia (or methemoglobinemia) before requesting special erythrocyte enzyme assays. These measurements are conducted in a limited number of veterinary laboratories. A laboratory manual written by Dr. Ernest Beutler2 is recommended for individuals wishing to conduct erythrocyte biochemical assays. Because the tests are time consuming and require special supplies and training of technologists, prior arrangements must be made with a laboratory before blood samples are submitted. Anticoagulated blood should be refrigerated and sent on wet ice (not frozen) to the laboratory so that the assay can be done within 1 day of sample collection. One or more samples from normal animals should be sent along with the patient's sample to be used as controls.

PYRUVATE KINASE DEFICIENCY

Congenital hemolytic anemia resulting from erythrocyte pyruvate kinase (PK) deficiency occurs in BasenjiP 36• 37 BeagleP· 21• 34 West Highland White Terrier,6 Cairn Terrier35 and American Eskimo (John W. Harvey, unpublished observations, 1991) dogs and Abyssinian cats.8 The deficiency is transmitted as an autosomal recessive trait. Homozygously affected animals have decreased exercise tolerance, pale mucous membranes, tachycardia, and splenomegaly. Because 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 via erythroid hyperplasia, with marked reticulocytosis present in peripheral blood. Because the defect in glycolysis occurs below the diphosphoglycerate shunt, erythrocytes from PK-deficient dogs have increased concentrations of 2,3-diphosphoglycerate (2,3DPG). As a consequence, the whole blood P50 is higher than that of normal dogs? 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 and moder-

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ate-to-marked polychromasia, anisocytosis, and frequent nucleated erythrocytes are recognized on stained blood films. Erythrocytes of affected dogs lack the normal adult R isozyme of PK but have a persistence of an M2 isozyme that normally predominates in fetal erythrocytes.4' 44 Consequently, many affected dogs have normal or increased PK activity, making diagnosis of this defect difficult based solely on total erythrocyte PK activity. In contrast, total erythrocyte PK activity is markedly reduced in cats, with no evidence of a persistent M 2 isozyme.8 Heterozygous animals have approximately 50% of normal erythrocyte PK activity. The enzyme activity in hemolysates of affected dogs is unstable and decreases rapidly when samples are kept at room temperature.40 If the M2-isozyme is unstable in vivo as it is in vitro, its rapid loss of activity would explain the dramatically shortened life span of erythrocytes with this disorder? 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 whose total enzyme activity is not decreased. 13, 25, 35 The defect in Basenji dogs is the result of a single nucleotide deletion in the R-type PK gene. 44 A DNA diagnostic test has recently been developed for Basenji dogs. 5 Thus far, this test has not been valid in other dog breeds, indicating that the defect is not identical in all breeds. An unexplained feature of the disease is the progressive development of myelofibrosis and osteosclerosis. Affected animals generally die before 4 years of age because of bone marrow failure and/ or liver disease with hemochromatosis and cirrhosis.37 • 43 PHOSPHOFRUCTOKINASE DEFICIENCY

As a rate-controlling enzyme in glycolysis, quantitative and qualitative alterations in phosphofructokinase (PFK) can result in significantly altered states of energy metabolism in tissues such as erythrocytes and intensely exercising skeletal muscle that depend heavily on glucose metabolism. 17 Autosomal recessive inherited PFK deficiency occurs in English Springer SpanieP0' 11 and American Cocker SpanieP6 dogs. Canine PFK is genetically controlled by three separate loci. They code for muscle (M)-, liver (L)-, and platelet (P)-type subunits.42 Random tetramerization of the subunits produces various isozymes. PFK in normal dog erythrocytes consists of 86% M-type, 2% L-type, and 12% P-type subunits and normal dog muscle is composed exclusively of M-type subunits.32 A point mutation is reported to occur in the M-type gene of deficient dogs, causing a loss of amino acids from the carboxyl terminus of the polypeptide. 14 Studies of brain and erythrocytes from homozygous deficient dogs indicated that native M-type subunits were not present, but small amounts of a structurally unstable truncated M-type subunit

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were found. 31• 32 As would be expected from the subunit composition of normal tissues, total erythrocyte and muscle PFK activities are markedly reduced in affected dogs. 10• 42 Changes in concentrations of glycolytic intermediates in muscle and erythrocytes reflect the block at the PFK step. 20• 24 Erythrocytes from affected dogs also exhibit altered enzyme kinetic properties due to the loss of theM-type subunit.24 Homozygously affected dogs have persistent compensated hemolytic anemia and sporadic episodes of intravascular hemolysis with hemoglobinuria. 10· 11 Erythrocyte mean cell volumes are usually between 80 and 90 fL. Uncorrected reticulocyte counts are generally between 10% and 30%, with hematocrit values 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. 10 Hemolytic crises occur secondarily to hyperventilation-induced alkalemia in vivo, and PFK-deficient dog erythrocytes are extremely alkaline-fragile in vitro.10 The greater alkaline fragility of PFK-deficient dog erythrocytes results from decreased 2,3DPG, which is formed below the PFK reaction. 28 Because 2,3DPG is the major impermeant anion in dog erythrocytes, a substantial decrease in its concentration results in a higher intracellular pH29 and thereby greater alkaline fragility than that of normal dog erythrocytes. As expected, the low 2,3DPG concentration also results in an increased oxygen affinity of Hb in affected dog erythrocytes.10 Homozygously affected animals over 3 months of age can easily be identified by measuring erythrocyte PFK activity. Heterozygous carrier dogs have approximately one-half the normal enzyme activity in their erythrocytes.26 A DNA test using polymerase chain reaction technology has been developed that can clearly differentiate normal, carrier, and affected dogs regardless of age. 15 Deficient dogs generally exhibit less evidence of myopathy than is observed in PFK-deficient people, probably because canine skeletal muscle is less dependent on anaerobic glycolysis than is human skeletal muscle, owing to a lack of the classic fast-twitch glycolytic (type liB) fibers in dogs.39 Affected dogs tire more easily than normal, and in vivo muscle studies of PFK-deficient dogs indicate altered muscle function in these animals. 5• 9 A severe progressive myopathy with associated abnormal polysaccharide deposits in skeletal muscle has been recognized in an aged PFK-deficient dog.18 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. Aspirin (10 mg/kg orally, twice daily) and/ or dipyrone (0.055 mL of 50% solution/kg subcutaneously, three times daily) are recommended

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' to treat the fever that often accompanies episodes of intravascular hemolysis and potentiates the hemolytic crises. Intravenous fluid therapy is recommended when intravascular hemolysis is severe to minimize the chance of acute renal failure. Blood transfusions are usually not needed, but should be given if the anemia becomes life threatening. GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a very common X-linked inherited defect of human erythrocytes.3 One male dog with approximately 44% of normal erythrocyte activity was found by screening several thousand dogs for G6PD activity. 38 The enzyme was partially purified and characterized and was found similar to that of normal dogs. The deficient dog was not anemic and exhibited no clinical signs; studies were not done to determine if his erythrocytes were more sensitive to oxidant damage than are normal erythrocytes. In contrast, persistent hemolytic anemia and hyperbilirubinemia has been described in an American Saddlebred Colt with less than 1% of normal G6PD activity. 41 Morphologic abnormalities of erythrocytes included eccentrocytosis, increased anisocytosis, and increased HowellJolly bodies. The presence of eccentrocytes in the absence of exposure to external oxidants indicated that the deficient erythrocytes did not have adequate metabolic capabilities to defend themselves against endogenous oxidants. Biochemical abnormalities in erythrocytes included decreased reduced glutathione, markedly reduced NADPH and an increase in the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+). Polymerase chain reaction amplification of segments of the G6PD gene of the affected colt revealed a G to A mutation, converting an arginine codon to a histidine codon.33 METHEMOGLOBIN REDUCTASE DEFICIENCY

Persistent methemoglobinemia associated with methemoglobin reductase (cytochrome-b5-reductase) deficiency has been recognized in a Chihuahua, a Borzoi, an English Setter, a Terrier mix, a Cockapoo, a Welsh corgi, Pomeranians, miniature poodles, and American Eskimo dogs1' 22' 23, 30 and recently in a domestic short-haired cat. 19 The deficiency is presumed to be a hereditary disorder, but family studies have not been reported. Except for the persistence of cyanotic-appearing tongue and mucous membranes, animals with this enzyme deficiency often show no clinical signs, although some affected animals exhibit mild lethargy and/ or exercise intolerance. The hematocrit is sometimes slightly increased secondary to chronic methemoglobinemia. Methemoglobinemia may not be apparent in normally dark venous

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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 color compared to the bright red color of the control blood. Accurate determination of methemoglobin content requires that blood rapidly be submitted to a laboratory that has this test available. Methemoglobin content in dogs with methemoglobin reductase deficiency varies from 13% to 41%. The methemoglobin content in the deficient cat was 50%. A definitive diagnosis of methemoglobin reductase deficiency is made by measuring erythrocyte methemoglobin reductase enzyme activity. Animals with methemoglobin reductase deficiency do not require treatment and have normal life expectancy.

SUMMARY

Congenital hemolytic anemias resulting from PK, PFK, and G6PD enzyme deficiencies have been reported in domestic animals. Dogs with PFK deficiency may have episodes of intravascular hemolysis with hemoglobinuria in addition to a persistent compensated hemolytic anemia. Patients with mild G6PD deficiency are not anemic but may show increased susceptibility to oxidant-induced erythrocyte injury. Persistent methemoglobinemia has been reported in dogs and cats with methemoglobin reductase enzyme deficiency. Affected animals have cyanoticappearing mucous membranes but show no or only mild clinical signs attributable to hypoxemia. Enzyme assays are usually done after acquired causes of hemolytic anemia and methemoglobinemia have been ruled out. References 1. Atkins CE, Kaneko JJ, Congdon LL: Methemoglobin reductase deficiency and methemoglobinemia in a dog. JAm Anim Hosp Assoc 17:829, 1981 2. Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods, ed 3. Orlando, Grune & Stratton, 1984 3. Beutler E: G6PD deficiency. Blood 84:3613, 1994 4. Black JA, Rittenberg MB, Standerfer RJ, et al: Hereditary persistence of fetal erythrocyte pyruvate kinase in the basenji dog. Prog Clin Bioi Res 21:275, 1978 5. Brechue WF, Gropp KE, Ameredes BT, et a!: Metabolic and work capacity of skeletal muscle of PFK-deficient dogs studied in situ. J Appl Physiol 77:2456, 1994 6. Chapman BL, Giger U: Inherited erythrocyte pyruvate kinase deficiency in the West Highland white terrier. J Small Anim Pract 31:610, 1990 7. Dhindsa DS, Black JA, Koler RD, eta!: Respiratory characteristics of blood from basenji dogs with classical erythrocyte pyruvate kinase deficiency. Respir Physiol 26:65, 1976 8. Ford S, Giger U, Duesberg C, et al: Inherited erythrocyte pyruvate kinase (PK) deficiency causing hemolytic anemia in an Abyssinian cat. J Vet Intern Med 6:123, 1992

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9. Giger U, Argov Z, Schnall M, et a!: Metabolic myopathy in canine muscle-type phosphofructokinase deficiency. Muscle Nerve 11:1260, 1988 10. Giger U, Harvey JW: Hemolysis caused by phosphofructokinase deficiency in English springer spaniels: 'Seven cases (1983-1986). JAm Vet Med Assoc 191:453, 1987 11. Giger U, Harvey JW, Yamaguchi RA, eta!: Inherited phosphofructokinase deficiency in dogs with hyperventilation-induced hemolysis: Increased in vitro and in vivo alkaline fragility of erythrocytes. Blood 65:345, 1985 12. Giger U, Mason GD, Wang P: Inherited erythrocyte pyruvate kinase deficiency in a beagle dog. Vet Clin Pathol 20:83, 1991 13. Giger U, Noble NA: Determination of erythrocyte pyruvate kinase deficiency in Basenjis with chronic hemolytic anemia. JAm Vet Med Assoc 198:1755, 1991 14. Giger U, Smith B, Griot-Wenk M, et al: Erythrocyte phosphofructokinase deficiency is caused by a point mutation and corrected by bone marrow transplantation. Blood 78(suppl. 1):365a, 1991 15. Giger U, Smith BF, Rajpurohit Y: PCR-based screening test for phosphofructokinase (PFK) deficiency: A common inherited disease in English springer spaniels. In Proceedings of the American College of Veterinary Internal Medicine Forum, 1995, p 1002 16. Giger U, Smith BF, Woods CB, et al: Inherited phosphofructokinase deficiency in an American cocker spaniel. JAm Vet Med Assoc 201:1569, 1992 17. Harvey JW: Erythrocyte metabolism. In Kaneko JJ (ed): Clinical Biochemistry of Domestic Animals, ed 4. San Diego, Academic Press, 1989, p 185 18. Harvey JW, Calderwood Mays MB, Gropp KE, eta!: Polysaccharide storage myopathy in canine phosphofructokinase deficiency (type VII glycogen storage disease). Vet Pathol 27:1, 1990 19. Harvey JW, Dahl M, High ME: Methemoglobin reductase deficiency in a cat. JAm Vet Med Assoc 205:1290, 1994 20. Harvey JW, Gropp KE, Bellah JR: Biochemical findings in phosphofructokinase-deficient canine skeletal muscle. In Ubaldi A (ed): State of Art in Animal Clinical Biochemistry, Parma, Boehringer Mannheim, 1992, p 79 21. Harvey JW, Kaneko JJ, Hudson EB: Erythrocyte pyruvate kinase deficiency in a beagle dog. Vet Clin Pathol 6:13, 1977 22. Harvey JW, King RR, Berry CR, et al: Methaemoglobin reductase deficiency in dogs. Comparative Hematology International 1:55, 1991 23. Harvey JW, Ling GV, Kaneko JJ: Methemoglobin reductase deficiency in a dog. J Am Vet Med Assoc 164:1030, 1974 24. Harvey JW, Pate MG, Mhaskar Y, et al: Characterization of phosphofructokinasedeficient canine erythrocytes. J Inherit Metab Dis 15:747, 1992 ;--"' 25. Harvey JW, Peteya DJ, Kociba GJ: Utilization of an enzyme heat stability test and erythrocyte glycolytic intermediate assays in the diagnosis of canine pyruvate kinase deficiency. Vet Clin Pathol 19:55, 1990 26. Harvey JW, Reddy GR: Postnatal hematologic development in phosphofructokinasedeficient dogs. Blood 74:2556, 1989 27. Harvey JW, Smith JE: Haematology and clinical chemistry of English springer spaniel dogs with phosphofructokinase deficiency. Comparative Hematology International 4:70, 1994 28. Harvey JW, Sussman WA, Pate MG: Effect of 2,3-diphosphoglycerate concentration on the alkaline fragility of phosphofructokinase-deficient canine erythrocytes. Comp Biochem Physiol [B] 89B:105, 1988 29. Hladky SB, Rink 1}: pH Equilibrium across the red cell membrane. In Lew VL, Ellory JC (eds): Membrane Transport in Red Cells, New York, Academic Press, 1977, p 115 30. Letchworth GJ, Bentinck-Smith J, Bolton GR, et al: Cyanosis and methemoglobinemia in two dogs due to NADH methemoglobin reductase deficiency. J Am Anim Hosp Assoc 13:75, 1977 31. Mhaskar Y, Giger U, Dunaway GA: Presence of a truncated M-type subunit and altered kinetic properties of 6-phosphofructo-1-kinase isozymes in the brain of a dog affected by glycogen storage disease type VII. Enzyme 45:137, 1991 32. Mhaskar Y, Harvey JW, Dunaway GA: Developmental changes of 6-phosphofructo-1-

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kinase subunit levels in erythrocytes from normal dogs and dogs affected by glycogen storage disease type VII. Comp Biochem Physiol [B)101B:303, 1992 Nonneman D, Stockham SL, Shibuya H, et al: A missense mutation in the glucose-6phosphate dehydrogenase gene associated with hemolytic anemia in an American saddlebred horse. Blood 82(suppl. 1):466a, 1993 Prasse KW, Crouser D, Beutler E, et al: Pyruvate kinase deficiency anemia with terminal myelofibrosis and osteosclerosis in a beagle. J Am Vet Med Assoc 166:1170, 1975 Schaer M, Harvey JW, Calderwood Mays MB, et al: Pyruvate kinase deficiency causing hemolytic anemia with secondary hemochromatosis in a cairn terrier dog. J Am Anim Hosp Assoc 28:233, 1992 Searcy GP, Miller DR, Tasker JB: Congenital hemolytic anemia in the basenji dog due to erythrocyte pyruvate kinase deficiency. Canadian Journal of Comparative Medicine 35:67, 1971 Searcy GP, Tasker JB, Miller DR: Animal model: Pyruvate kinase deficiency in dogs. Am J Physiol 94:689, 1979 Smith JE, Ryer K, Wallace L: Glucose-6-phosphate dehydrogenase deficiency in a dog. Enzyme 21:379, 1976 Snow DH, Billeter R, Mascarello F, et al: No classical type liB fibers in dog skeletal muscle. Histochemistry 75:53, 1982 Standerfer RJ, Templeton JW, Black JA: Anomalous pyruvate kinase deficiency in the basenji dog. Am J Vet Res 35:1541, 1974 Stockham SL, Harvey JW, Kinden DA: Equine glucose-6-phosphate dehydrogenase deficiency. Vet Pathol 31:518, 1994 Vora S, Giger U, Turchen S, eta!: Characterization of the enzymatic lesion in inherited phosphofructokinase deficiency in the dog: An animal analogue of human glycogen storage disease type VII. Proc Nat! Acad Sci US A 82:8109, 1985 Weiden PL, Hackman RC, Deeg J, et a!: Long-term survival and reversal of iron overload after marrow transplantation in dogs with congenital hemolytic anemia. Blood 57:66, 1981 Whitney KM, Goodman SA, Bailey EM, et a!: The molecular basis of canine pyruvate kinase deficiency. Exp Hematol 22:866, 1994 Whitney KM, Lothrop CD Jr: Genetic test for pyruvate kinase deficiency of basenjis. J Am Vet Med Assoc 207:918, 1995

Address reprint requests to John W. Harvey, DVM, PhD B2-28 BSB Department of Physiological Sciences University of Florida 1600 SW Archer Road Gainesville, FL 32610