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L". ‘ I‘ ‘I“ “' ' '3, £1" ""L “’V W371. “AI" 33““ 3%“ ‘h‘i MA- 3“ I: \ ' _.~—. ‘1‘ __-'— THESI$ “v‘J_—.iL-_~a f’-. ) i - ‘6‘ 1' 21,133,»; a 3' *- Michigan Scam University «cow '"F "if m "by," .,_ i This is to certify that the thesis entitled The Relationship of Serum Iron, Erythropoietin and Erythrocyte Survival to Anemia of Inflammatory Disease presented by Douglas J. Weiss has been accepted towards fulfillment of the requirements for Ph. D. Aggree in Pathology click a ll; Major professor Date May 18, 1981 0-7639 ’l! I» -m..- . w. .- r‘ _ A '. - ; -5‘,"" *7. Wirrfizt r’ u _,a (w run} “a ‘3 :9.- . ‘ \ -t“ ; ~“‘\ ‘K vi A'im \ ' I III I” O”/ ‘ OVERDUE FINES: 25¢ per day per item RETURNIE LIQRARY MATERIALS: Place in book return to remove ' charge from circulation records THE RELATIONSHIP OF SERUM IRON, ERYTHROPOIETIN AND ERYTHROCYTE SURVIVAL TO ANEMIA OF INFLAMMATORY DISEASE BY Douglas J. Weiss A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1981 ABSTRACT THE RELATIONSHIP OF SERUM IRON, ERYTHROPOIETIN AND ERYTHROCYTE SURVIVAL TO ANEMIA OF INFLAMMATORY DISEASE BY Douglas J. Weiss Anemia frequently accompanies chronic infections and neoplastic diseases in humans and a variety of animal species. Induced sterile abscesses in cats provided an experimental model for the investigation of the complex pathogenesis of this disorder. The role of serum iron, erythropoietin and erythrocyte survival in anemia of inflammatory disease was investigated. Maintenance of increased serum iron concentrations during the sterile abscess did not prevent but did allow the bone marrow to respond to the anemia. Daily cobalt injections in cats with abscesses produced a mild polycythemia, but the magnitude of the polycythemia was considerably less than in control cats. A significant reduction in erythrocyte sur- vival was also observed in animals with sterile abscess. The major conclusions of this study were: 1) the primary cause of anemia in the early stages of inflammatory disease is reduced erythrocyte lifespan; 2) low serum iron levels prevented the bone marrow from responding to the Douglas J. Weiss anemia; and 3) serum iron concentrations, in addition to erythrOpoietin, were involved in the control of maximal erythrOpoiesis. TABLE OF CONTENTS INTRODUCTION. LITERATURE REVIEW . Iron Metabolism. . . . . . Transferrin . . . . . . Ferritin. Hemosiderin . Lactoferrin . Cellular Uptake and Release of. Iron ErythrOpoietin . Chemical Properties Biosynthesis and Control .of Secretion . Erythrocyte Lifespan Anemia of Inflammatory Disease Clinical Description. Impaired Bone Marrow Response to. the Anemia . Impaired Iron Release from Mononuclear Phagocytes Erythrocyte Survival. MATERIALS AND METHODS . Experimental Animals Experimental Design. Experiment I. Experiment II Experiment III. Experiment IV . Experiment V. Experiment VI . Experiment VII. Experiment VIII Procedures . . . Complete Blood Count. Plasma Protein. Reticulocyte Count. . . . . . . Serum Iron and Total Iron Binding Capacity . . Erythrocyte Survival. Serum Erythropoietin. ii Page Statistical Analysis . . . . . . . . . . . . . 32 RESULTS . . . . . . . . . . . . . . . . . . . . . . . 33 Experiment I . . . . . . . . . . . . . . . . . 33 Experiment II. . . . . . . . . . . . . . . . . 33 Experiment III . . . . . . . . . . . . . . . . 37 Experiment IV. . . . . . . . . . . . . . . . . 39 Experiment V . . . . . . . . . . . . . . . . . 39 Experiment VI. . . . . . . . . . . . . . . . . 41 Experiment VII . . . . . . . . . . . . . . . . 44 Experiment VIII. . . . . . . . . . . . . . . . 44 DISCUSSION. . . . . . . . . . . . . . . . . . . . . . 48 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 63 REFERENCES. . . . . . . . . . . . . . . . . . . . . . 64 APPENDICES. . . . . . . . . . . . . . . . . . . . . . 73 A - INDIVIDUAL ANIMAL DATA FOR EXPERIMENTS I THROUGH VII. . . . . . . . . . . . . 73 B - EFFECTS OF INOSINE, HYPOXANTHINE AND ALLOPURINOL ON SERUM IRON AND URIC ACID CONCENTRATIONS . . . . . . . . . . . . . . 104 iii Table 10 11 LIST OF TABLES Tests performed in Experiments I through VII Experimental design for study of anemia of inflammatory disease Selected data from control cats (Experiment I) Erythropoietin concentrations in control cats, cats with sterile abscesses, cats treated with cobalt and cats with abscesses treated with cobalt . . . . . . . . . Selected data from cats with sterile abscesses (Experiment II). Selected data from cats treated with cobalt (Experiment III) . . . . . The effects of cobalt on the hematologic alterations caused by a sterile abscess (Experiment IV). . . . . . . . . . . . . Selected data from cats treated with intra- venous iron (Experiment V) The effects of intravenous iron on hemato- logic alterations caused by a sterile abscess (Experiment VI). . The effects of cobalt and intravenous iron on hematologic alterations caused by a sterile abscess (Experiment VII) Erythrocyte survival in normal cats and cats with abscesses induced on day 10 Appendix A Individual animal data for Experiment I. Individual animal data for Experiment II Individual animal data for Experiment III. iv Page 24 29 34 35 36 38 4O 42 43 45 47 74 84 Table Page A-4 Individual animal data for Experiment IV . . . 89 A-S Individual animal data for Experiment V. . . . 94 A-6 Individual animal data for Experiment VI . . . 96 A-7 Individual animal data for Experiment VII. . . 100 Appendix B B-l The effect of inosine injection on serum iron concentrations (ug/dl) in cats . . . . . . . . 105 B-Z The effects of hypoxanthine injection on serum iron (ug/dl) and serum uric acid (mg/d1) con- centrations in dogs. . . . . . . ._. . . . . . 106 B-3 The effects of hypoxanthine injeCtion on serum iron (ug/dl) and serum uric acid (mg/d1) con- centrations in anesthetized dogs . . . . . . 107 B-4 The effects of allopurinol injection on serum iron (ug/dl) and serum uric acid (mg/d1) con- centrations in dogs. . . . . . . . . . . . . . 108 LIST OF FIGURES Figure Page 1 Sequential mean packed cell volumes in the sterile abscess group (Group II), cobalt treatment group (Group III), and combined abscess and cobalt treatment group (Group IV). 50 2 Sequential mean packed cell volumes in the sterile abscess group (Group II), cobalt treatment group (Group III), and combined abscess and iron treatment group (Group VI). . 52 3 Sequential mean packed cell volumes in the sterile abscess group (Group II), cobalt treatment group (Group III), and combined abscess, cobalt and iron treatment group (Group VII). . . . . . . . . . . . . . . . . . S3 4 Sequential mean serum iron concentrations in the sterile abscess group (Group II), cobalt treatment group (Group III) and combined abscess and cobalt treatment group (Group IV). 54 5 Sequential mean type I reticulocytes in the sterile abscess group (Group II), combined abscess and iron treatment group (Group VI) and combined abscess, cobalt and iron treatment group (Group VII). . . . . . . . . . . . . . . SS 6 Sequential mean type II and type III reticulo- cytes in the sterile abscess group (Group II), cobalt treatment group (Group III), combined abscess and iron treatment group (Group VI) and combined abscess, cobalt and iron treatment group (Group VII). . . . . . . . . . . . . . . 56 7 Mean 51Cr erythrocyte survival in normal cats and cats with sterile abscesses induced on day 10 . . . . . . . . . . . . . . . . . . . . 60 vi INTRODUCTION Anemia frequently accompanies chronic infections and neoplastic diseases in man and a variety of animal species. The term "anemia of chronic disorders" has been the most accepted name for this anemia. However, since inflammation has been found to be the common denominator in all cases, anemia of inflammatory disease would be a more precise and descriptive name. Anemia of inflammatory disease has been clinically defined as a mild to moderate, poorly regenerative anemia with altered iron metabolism characterized by low serum iron and total iron binding capacity and normal to increased tissue iron. The pathogenesis of the anemia has been found to be complex. At least 3 factors have been implicated: 1) im- paired marrow response to the anemia, 2) impaired release of iron from mononuclear phagocytes, and 3) shortened erythrocyte survival. The relative importance of these factors and their underlying cause have not been established. The investigation of these problems will not only provide further knowledge of the pathogenesis of anemia of inflam- matory disease but will help to define the interrelationship of the several mechanisms which control erythrocyte produc- tion and destruction. 2 Induced sterile abscesses have provided a simple experimental model for study of the pathogenesis of anemia of inflammatory diseases. This model will be used to explore 1) the mechanism of reduced erythropoiesis, 2) the mechanism of iron sequestration, and 3) the role of erythro- cyte survival in anemia of inflammatory disease. LITERATURE REVIEW This literature review will be divided into 4 parts: 1) iron metabolism--transferrin, ferritin, hemosiderin, lactoferrin and control of cellular uptake and release of iron; 2) erythropoietin--chemical prOperties, biosynthesis and control of secretion; 3) erythrocyte survival; and 4) anemia of inflammatory disease--clinical description, impaired marrow response to anemia, impaired iron release from mononuclear phagocytes and erythrocyte survival. Iron Metabolism Transferrin Transferrin is the major plasma iron transport protein. The physical and chemical prOperties have been reviewed (Morgan, 1974; Harrison and Huehns, 1979). The molecule is a glycoprotein of approximately 76,000 molecular weight and 5.3% carbohydrate. Each of 2 lobes has a prolate ellipsoid shape with maximum overall dimensions of 10 x 50 x 40 Ang- stroms. A single iron binding site is present on each lobe. These 2 sites are identical in structure and have a very high affinity for iron. The interaction of ferric iron with the binding sites was found to involve 3 tyrosyl residues, 4 2 histidyl residues and the concurrent binding of 1 bicar- bonate ion (Rogers, et al., 1977). The relative availability of iron from the 2 binding sites to various tissues has been disputed. Fletcher and Huens, (1968) reported that iron from the 2 iron binding sites was not equally available for hemoglobin synthesis. They postulated that this functional dissimilarity regula- ted iron distribution within the body. Harrison and Aisen (1975), however, found no functional difference in the availability of iron from the 2 sites. In addition, they found that iron from diferric and monoferric transferrin was equally available to body tissues. Ferritin Ferritin is known to be the major iron storage protein (Harrison, et al., 1974). The physical chemical properties of ferritin have been reviewed (Harrison, et al., 1974; Richter, 1978; Munro and Linder, 1928). Ferritin was found to be a water-soluble macromolecule consisting of subunits arranged in a octahedral fashion around a central core of iron. The subunits have been described as acidic proteins of approximately 18,500 daltons with 60% of the molecule in an alpha helical conformation (Harrison and Huehns, 1979). Harrison, et al., (1974) reported that the crystalline core of ferritin contained up to 4500 atoms of iron stored as a colloidal hydrous ferric oxide-phosphate complex. 5 Administration of iron to guinea pigs was found to cause rapid stimulation of apoferritin synthesis in the liver (Findberg and Greenberg, 1955). The first detectable product was an iron-poor apoferritin. The subunits of apoferritin were postulated to assemble spontaneously into a hollow shell which rapidly accumulated free iron (Harrison et al., 1974). Munro and Linder (1978) have postulated that apoferritin controls its own translation by preventing binding of messenger RNA to the endoplasmic reticulum. In their model increased cytosolic iron causes aggregation and re- distribution of apoferritin, thus removing the block for the binding of messenger RNA. Since ferritin messenger RNA is known to be very stable, this theory would provide a mechanism for rapid uptake and release of iron by ferritin. This function has been considered important to iron homeo- stasis in that free serum iron is toxic and cannot be rapidly excreted by the body. Hemosiderin Hemosiderin has been characterized by: 1) Prussian blue staining, 2) insolubility in water, and 3) molecular heterogeneity (Munro and Linder, 1978). Ultrastructural examination has suggested that hemosiderin was a degraded form of ferritin which resulted from partial loss of the protein shell (Munro and Linder, 1978). This transformation may occur in secondary lyososomes (Monro and Linder, 1978). Munro and Linder (1978) conclude that hemosiderin as presently defined includes several intracellular forms of 6 iron. These forms include both loosely chelated iron and dense storage granules. Lactoferrin Lactoferrin, an iron binding protein of 76,000 molecu- lar weight, has been found in high concentrations in external body secretions and in the specific granules of neutrophilic granulocytes (Karle et al., 1979). Lactoferrin was found to share several biochemical properties with transferrin, including a closely related amino acid composition and similar iron binding sites (Karle et al., 1979). The mole- cules differed immunologically and in their affinity for iron (Morgan, 1974; Van Snick et al., 1974). Cellular Uptake and Release of Iron Three cell types have been found to play unique roles in iron homeostasis: l) mononuclear phagocytes, 2) reticulo- cytes, and 3) intestinal mucosa. Intestinal iron absorption has been reviewed and will not be further discussed (Turnbull, 1974). Iron can be taken up by mononuclear phagocytes in several forms, including 1) uptake of intact erythrocytes, 2) uptake of iron from transferrin, 3) uptake of protein- bound hemoglobin and heme, and 4) uptake of serum ferritin (Munro and Linder, 1978). Destruction of senescent erythrocytes by the mononu- clear phagocyte system has been considered a normal physio- logic mechanism for recycling iron and amino acids. The erythrocytes were found to be taken up by phagocytosis and 7 to be digested in secondary lysosomes (Munro and Linder, 1978). Hepatocytes have been shown to have receptors for hemoglobin-haptoglobin complexes and for heme-hemopexin complexes. These complexes can be taken up by phagocytosis (Munro and Linder, 1978). The mechanism by which transferrin interacts with cells has been primarily investigated using reticulocytes. 125I-Iabeled Hemmaplardh and Morgan (1977) have shown that transferrin first attached to a specific cell surface receptor and then entered the cell by endocytosis to become free in the cytoplasm. Fielder and Speyer (1974) suggested that transferrin did not enter the cell but gave up its iron at the cell surface. Whatever the mechanism, a large part of the transferrin was found to recirculate (Munro and Linder, 1978). Factors affecting the rate of reticulocyte iron uptake have been reviewed by Morgan (1974). Increased transferrin saturation accelerated iron uptake. Chelating agents did not block uptake, which suggested that iron was not present in ionic form at any time during the absorption process. Anoxia and cell lysis were found to inhibit uptake, sug- gesting that uptake was an energy-requiring process dependent on cellular metabolism and cellular integrity. Mazur et a1. (1960) investigated the incorporation of transferrin-bound iron into ferritin in liver slices. They found that adenosine triphosphate (ATP) and ascorbic acid were essential to iron uptake. They concluded 8 that ascorbic acid was necessary for the reduction of ferric iron bound to transferrin. Egyed (1974) reported that ATP was directly involved in iron uptake by reticulo- cytes and proposed that ATP was bound to the cell membrane. Carver and Frieden (1972) reported that polyphosphate, adenosine diphosphate (ADP) and ATP accelerated iron release from transferrin by acting as intracellular chelators of iron. Graham and Bates (1974) proposed the following sequence of events in iron release from transferrin: 1) chelation of ferric iron with ATP or other cellular Chelating agents, 2) reduction of ferric iron by reducing agents including ascorbic acid, 3) disruption of the bond between amino acids and the iron molecule, 4) allosteric alterations in the transferrin molecule, and S) weakening of the anion thermodynamic linkage. Munro and Linder (1978) proposed that iron from both transferrin and secondary lysosomes contributed to a chelat- able iron pool in the cytosol. They postulated that this pool was in equilibrium with ferric iron in ferritin and that the size of the pool regulated synthesis of apoferritin through changes in the availability of messenger RNA for ferritin synthesis. The chelatable iron has also been shown to stabilize ferritin precursors and iron-poor ferritin (Drysdale and Munro, 1966). Steps in the deposition of ferritin iron have been proposed by Crichton and Collet-Cossart (1977). These steps include: 1) ferrous iron binding to apoferritin, 2) formation of a dioxygen molecule with 2 ferrous ions, 9 3) reduction of ferrous iron to a peroxo-complex, and 4) hydrolysis of the peroxo-complex and migration of the ferric oxyhydroxide to the interior of the protein shell. Possible mechanisms involved in the release of iron from ferritin have been reviewed by Harrison et a1. (1974). Release may be regulated by the level of reducing agents and/or Chelating agents within the cell. Ascorbic acid deficiency has been found to result in increased hepatic ferritin levels (Lipschitz et al., 1971). Mazur, Carleton and Carlsen (1961) have provided evidence that the release of iron from ferritin was regu- lated by the state of oxidative metabolism within tissue. This theory was supported by the in vitro observation by Baker et a1. (1977) that liver hypoxia resulted in release of iron from hepatocytes while hyperoxia caused decreased iron release. They proposed that decreased oxygen acted directly on an NADH-linked ferric reductase. Crichton and Collet—Cassart (1977) reported the existence of a ferro- reductase which required FMN as a coenzyme and NADH or NADPH as a source of electrons. Mazur et a1. (1958) provided both in vitro and in vivo evidence that xanthine oxidase reduces ferritin iron. Intravenous injection of substrates of xanthine oxidase into dogs, guinea pigs and rabbits resulted in prompt increase in serum iron. They proposed that iron mobiliza- tion from ferritin was regulated by the plasma level of xanthine oxidase substrates. Tissue hypoxia from anemia or other causes would increase the release of xanthine 10 oxidase substrates, thus stimulating release of ferritin iron. However, lowering of xanthine oxidase levels by feeding sodium tungstate and by treatment with a110purinol resulted in no accumulation of iron in the liver of rats (Kinney et al., 1961; Emmerson, 1966). The formation of hemosiderin from ferritin has been reviewed by Matioli and Bates (1963). They proposed that ferritin initially undergoes oxidative denaturation, which causes the cell to sequester the denatured molecules in vacuoles. Conditions within the vacuoles favored aggrega- tion of the denatured ferritin into granules. These granules, however, still maintained the ferritinic substructure. They further proposed that proteolytic degradation of this type of hemosiderin resulted in formation of hemosiderin in a nonferritinic form. Banerjee and Chakrabarty (1965) reported that deposits of hemosiderin were greater than normal in liver of ascorbic acid deficient guinea pigs, while the soluble fraction was reduced. This led to the proposal that ascorbic acid pre- vented oxidation of ferritin. Ferritin has been generally accepted as a readily mobilized iron storage pool, while hemosiderin was considered a more stable storage form (Richter, 1978). However, Shoden et a1. (1963) reported that iron mobilized from tissue was derived from both hemosiderin and ferritin. They concluded that the 2 types of storage iron were functionally indistinguishable. 11 Erythropoietin Chemical Properties Sheep plasma erythropoietin and human urinary erythro- poietin have been found to be acidic glycoproteins which were heterogeneous on isoelectric focusing (Wintrobe et al., 1974). The molecule was found to contain approximately 29% carbohydrate. The molecular weight has been estimated to be between 27,000 and 60,000 daltons (Goldwasser and Kung, 1971). Goldwasser and Kung (1968) reported that removal of terminal sialic acid residues resulted in loss of in vivo biological activity but retention of in vitro biological activity. Lukowsky and Painter (1972) found that oxidation of asialo-erythropoietin with galactose oxidase restored biological activity in vivo. They concluded that, as with other proteins, exposure of the penultimate galactose residue resulted in rapid degradation of erythropoietin in vivo. Biosynthesis and Control of Secretion The site of production of erythropoietin has been con~ sidered to be the kidney based on the association of nephrectomy and anemia (Wintrobe et al., 1974a). However, following bilateral nephrectomy in people erythropoietin could still be detected (Krantz and Jacobson, 1970). Contrera and Gordon (1966) isolated a factor from the light mitochondrial fraction of rat kidneys which generated biologically active erythropoietin after 12 incubation with a plasma factor. It has been termed renal erythropoietic factor, or erythrogenin. Renal erythro- poietic factor and plasma factor were both found to be immunochemically distinct from erythropoietin. Alterna- tively, Sherwood and Goldwasser (1978) found that extracts of rat, ox, dog and rabbit kidney contained biologically active erythropoietin and not a precursor hormone. Rogers, Fisher and George (1974) reported that hypoxia and synthetic prostaglandin E1 induced a renal protein kinase which activated renal erythropoietic factor by phosphorylation in the presence of cyclic AMP. The effects of cobalt on erythropoiesis were first discovered by Waltner and Waltner (1926). They found that cobalt produced a polycythemia in experimental animals. Barron and Barron (1936) reported that daily subcutaneous injections of .01 gm of cobalt sulfate produced a polycy- themia in rabbits within 6 or 7 days. The polycythemia was accompanied by reticulocytosis and nucleated erythrocytes in the peripheral blood and erythroid hyperplasia in the bone marrow. Based on in vitro culture of erythrocytes with cobalt, they concluded that cobalt acted directly by producing an anoxic condition within the bone marrow. How- ever, Warren, Schubmehl and Wood (1944) were unable to reproduce these results. Goldwasser et a1. (1958) reported that cobalt caused an increase in plasma erythropoietin within 4 hours after injection. They postulated that cobalt acted by producing an anoxic state in the kidney. 13 Erythrocyte Lifespan Numerous methods have been used to study erythrocyte lifespan (Wintrobe et al., 1974b). These have been divided into cohort methods in which tracers are incorporated into newly formed cells in the marrow and random methods in which the tracers bind to erythrocytes in the circulation regard- less of age. Two methods for determination of in vivo erythrocyte survival were reviewed and standardized by the International Committee for Standardization in Hematology (1971). Tracers 51 for these methods were Cr sodium chromate and radioactive diisopropyl fluorophosphate. Grey and Sterling (1950) first reported that erythro- cytes mixed with radioactive sodium chromate rapidly took 51 up chromium. Disappearance of Cr from the circulation was studied by Ebaugh, Emmerson and Ross (1953). They found that 51 Cr eluted from erythrocytes and correction for this elution could be made. Erythrocyte lifespan has been studied in cats by Kreier et a1. (1970) and by 611115 and Mitchell (1974). Kreier et a1. (1970) labeled erythrocytes concurrently with diiso- prOpyl fluorOphosphate and 51 Cr sodium chromate. They found that rapid elution of 51Cr occurred at a rate of 5.88 i .90% per day. Erythrocyte lifespan was found to be 70 days. l4 Anemia of Inflammatory Disease Clinical Description Anemia frequently accompanies chronic infectious and ne0plastic diseases in man and a variety of animal species (Cartwright, 1966; Mahaffey and Smith, 1978). Finch (1978) suggested that inflammation was the common denominator among various causes of this anemia. In humans, the anemia deve10ped slowly over the first month of illness but thereafter plateaued (Cartwright, 1966). Similar anemias deve10ped in dogs, cats and rats with experimental septic or nonseptic sterile abscesses (Lukens et al., 1967; Cartwright et al., 1946; Mahaffey and Smith, 1978). In rats and cats, the anemia was reported to develop more rapidly with significant decrease in hematocrit by 3 and 5 days, respectively (Mahaffey and Smith, 1978; Lukens et al., 1967). Morphologically the anemia was usually normocytic, normochromic but normocytic, hypochromic or microcytic, hypochromic anemias were also observed (Cartwright, I966). Reticulocyte counts were normal to decreased with minimal or no alterations in bone marrow cytology (Mahaffey and Smith, 1968; Cartwright, 1966). Biochemically the anemia was characterized by: 1) de- creased plasma iron, 2) decreased total iron binding capacity, 3) decreased saturation of transferrin, 4) decreased bone marrow sideroblasts, 5) normal or increased mononuclear phagocyte iron, 6) increased plasma copper, and 7) increased 15 free erythrocyte protoporphyrin (Cartwright, 1966). Cart- wright and Lee (1971) stated that these changes were unique to anemia of inflammatory disease. At least 3 factors have been implicated in the patho- genesis of the anemia: l) impaired bone marrow response to the anemia, 2) impaired release of iron from mononuclear phagocytes, and 3) shortened erythrocyte survival. Each of these factors will be discussed separately. Impaired Bone Marrow ResEpnse to the Anemia Cartwright and Lee (1971) implicated 2 factors which may have limited erythrOpoiesis: 1) inadequate iron supply to the marrow and 2) impaired erythropoietin release. Prolonged iron deficiency has been clearly found to result in anemia (Wintrobe et al., 1974c). However, the role of iron supply in control of erythropoiesis in the non-iron-deficient state has been less well established. Hillman and Henderson (1969) investigated marrow production reSponse of people to daily phlebotomies over a 3- to 4-week period. In response to sudden reduction of hemato- crit to 32 to 37 volume %, marrow production increased over a lO-day period and reached a plateau at 1.8 to 3.5 times normal. When iron was provided as a single intravenous injection of iron dextran, marrow production increased 4.5 to 5.5 times normal. When iron was provided by infusion of nonviable erythrocytes, marrow production increased 6.6 to 7.8 times normal. They concluded that marrow iron supply played an important role in the control of erythropoiesis. 16 The role of erythropoietin in the pathogenesis of anemia of inflammatory disease has been disputed. This has been largely due to the relative insensitivity of the bioassay procedure at normal and low serum levels of erythropoietin. Zucker et al. (1974) reported that serum levels of erythropoietin were decreased relative to the degree of anemia. This conclusion was based on comparison of erythropoietin-hemoglobin ratios in people with chronic inflammatory disease and normal peOple. However, this relative decrease was not observed in cancer patients. Lukens (1973) determined erythropoietin levels in adjuvent- induced chronic inflammation in rats. Serum levels were only slightly lower than normal controls; however, erythro- poietin concentrations following exposure to hypobaric conditions were markedly lower. These changes were interpreted as a relative failure in the production of bio- logically active erythropoietin. Douglas and Adamson (1975) studied anemia of inflammatory disease in people and reported that reduced erythropoietin concentrations were not a uniform finding. They concluded that marrow proliferation was primarily limited by the relative unavaila- bility of iron. Ward et a1. (1971) reported that erythro- poietin levels in people with chronic infections and malig- nancies were significantly lower than in people with iron deficiency and hemolytic diseases. Reissmann and Udupa (1978) reported that inflammation in mice caused a longlasting reduction in the number of erythroid colony forming units (CFU-E) in the bone marrow. 17 They attributed this effect to a blood-borne mediator which inhibited proliferation of CPU-E. However, they also found that CPU-E and hematopoietic stem cells (CFU-S) in the spleen were markedly increased. Whitcomb et a1. (1965) reported a serum inhibitor of erythropoiesis in anemia of inflammatory disease. Wallner et a1. (1976) reported a similar inhibitor of erythropoiesis in anemia of chronic renal disease but were unable to demon- strate a serum inhibitor in anemia of inflammatory disease. Rinehart et a1. (1978) reported that peripheral blood monocytes and tissue macrOphages could regulate erythro- poiesis in vitro. They postulated that proliferation of monocytes during inflammation suppressed erythropoiesis. Despite these conflicting reports, anemia of inflam- matory disease has been shown to reSpond to the administra- tion of purified erythropoietin, cobalt and hypoxia (Gutnesky and Van Dyke, 1963; Wintrobe et al., 1947). However, it was found that with all 3 stimuli the final hematocrits were not as great in rats with sterile abscesses as they were in the control groups. Thus, these stimuli did not completely abolish the defect in erythropoiesis produced by the inflammation (Cartwright, 1966). This discrepancy could be explained by: 1) decreased erythro- cyte survival, 2) unavailability of iron for erythropoiesis, or 3) a serum inhibitor of erythropoietin or erythropoiesis. 18 Impaired Iron Release from Mononuclear‘Phaggcytes Impaired release of iron from mononuclear phagocytes was first reported by Freireich et a1. (1957) and has since been confirmed by others (Haurani et al., 1965; Quastel and Ross, 1966; Hershko et al., 1974). The impaired release has been demonstrated by infusion of nonviable erythrocytes which were labeled with radioiron. In anemia of inflamma- tory disease less than 40% of the radioiron was reutilized for hemoglobin synthesis as compared to 55 to 70% in normal subjects. Several mechanisms for the iron sequestration have been proposed. They include: 1) increased ferritin synthe- sis (Konejn and Hershko, 1977), 2) oxidation of ferritin to hemosiderin (Feldman and Kaneko, 1978), 3) excessive lacto- ferrin release from neutrophils (Van Snick et al., 1974), and 4) increased oxidative metabolism in mononuclear phago- cytes (Mazur et al., 1961). Konijn and Hershko (1977) studied plasma iron turnover and ferritin synthesis in the liver and spleen of rats with sterile abscesses. They found that an increased rate of ferritin synthesis preceded, by several hours, changes in plasma iron turnover. They suggested that increased ferritin synthesis was responsible for the sequestration of iron in mononuclear phagocytes. They concluded by drawing an analogy between ferritin and "acute-phase” proteins and proposed that increased ferritin synthesis was part of a primary non-specific response to inflammation. l9 Kampschmidt and Upchurch (1969) reported that injec- tion of leukocyte extracts resulted in a marked decrease in serum iron concentrations in rats. The extract has been termed leukocyte endogenous mediator and has been found to also have antimicrobial activity (Kamschmidt and Pulliam, 1975). A later report suggested that leukocyte endogenous mediator and leukocyte endogenous pyrogen were the same molecule (Merriman et al., 1977). Bornstein and Walsh (1978) injected endotoxin-free endogenous pyrogen into rabbits and found that it produced both an "acute-phase" reaction and hypoferremia. Van Snick et a1. (1974) reported that neutrophils released iron-free lactoferrin during phagocytosis in vitro. This lactoferrin was able to remove iron from trans- ferrin at a pH of 7.0 or in the presence of a high concen- tration of citrate. Intravenous injection of human apolac- toferrin into rats caused a marked decrease in plasma iron levels which could be retarded by mononuclear phagocyte blockade. Immunofluorescence studies indicated that lacto- ferrin was bound to and ingested by monocytes and that the iron was transferred to ferritin. They concluded that lactoferrin may mediate the hypoferremia associated with inflammation. The turnover of radioiodine-labeled human lactoferrin in rabbits was studied by Karle et a1. (1979). The half- 1ife was found to be approximately 25 hours and most of the lactoferrin accumulated in the liver. The rate of synthesis in normal humans was estimated to be about 25 mg per day. 20 It was concluded that this form of iron transport was insignificant in the normal state. Although this pathway was increased in inflammatory disease, it also appeared to be insignificant when compared to toal iron turnover rate. However, this experiment did not take into account lactoferrin, which was metabolized by macrOphages in the extravascular space and thus not entering the plasma pool. Mazur et al. (1961) reported that increased oxidative metabolism in rat liver slices resulted in increased incorporation of transferrin-bound radioiron into tissue. They concluded that stimulation of ATP synthesis favored movement of serum iron into liver and spleen ferritin, whereas hypoxia favored iron release. They postulated that oxidative metabolism in mononuclear phagocytes during inflammation resulted in iron sequestration. Feldman and Kaneko (1979) reported that hepatic super- oxide dismutase activity was increased during anemia of inflammatory disease in dogs. They suggested that increased superoxide dismutase activity generated enough hydrogen peroxide to denature ferritin miscelles, producing deposits of less readily mobilized iron in the form of hemosiderin. Erythrocyte Survival Ferrokinetic studies have been performed on humans and experimental animals with anemia of inflammatory disease (Freireich et al., 1957; Quastel and Ross, 1966). In 21 general, the plasma iron turnover and erythrocyte iron turnover were normal to increased and the fraction of cells renewed daily was increased. These data suggested that the rate of erythrOpoiesis was normal or slightly increased and therefore that the rate of erythrocyte destruction must be increased. In addition, the rapidity with which the anemia deve10ped in cats with turpentine abscesses could not be accounted for by decreased erythropoiesis alone (Mahaffey and Smith, 1978). Erythrocyte survival has been determined in humans and experimental animals (Richmond et al., 1961; Rigby et al., 1962; Hyman, 1963; Karle, 19683). In general, a modest shortening of erythrocyte survival has been demonstrated (Cartwright and Lee, 1971). However, Waggener et al. (1958) studied erythrocyte survival in patients with leu- kemia and lymphoma and found that erythrocyte survival was only 50% of normal in many patients. Normal erythrocytes transfused into patients with anemia of inflammatory disease have been found to have a decreased erythrocyte survival, whereas erythrocytes from patients with anemia of inflammatory disease have a normal survival when transfused into normal patients. These studies have been interpreted as indicating an extracor- puscular hemolytic factor (Cartwright, 1966). The mechanism of the decreased erythrocyte survival has not been defined. The 2 most commonly expressed theories include 1) a hemolytic factor elaborated from the site of the inflammation and 2) a hyperplastic mononuclear phagocyte 22 system which is overactive in destroying erythrocytes (Jacobs et al., 196; Cartwright, 1966). Decreased erythro- cyte survival associated with splenic hyperplasia has been observed in patients with infectious and neoplastic diseases and in animals injected with particulate material (Ultmann, 1958; Jacobs et al., 1963). Conversely, splenic atrophy has been observed in animals following repeated bleeding (DeLangen, 1943). Jacobs et al. (1963) proposed that the size of the mononuclear phagocyte system in various organs is regulated by the total particulate workload presented to it. Ultmann (1958) demonstrated that splenic sequestration of erythrocytes was an important cause of anemia in patients with chronic lymphocytic leukemia and lymphosarcoma. Evidence for a hemolytic factor in anemia of malignancy has been reviewed by Hyman (1963). Wasserman et al. (1955) proposed that non-specific antibodies may be produced by neoplastic tissue which cross react with erythrocyte anti- gens. Barry and Crosby (1957) reviewed 10 cases of hemolytic anemia related to ovarian cysts and teratomas. Many of these patients had a positive direct Coombs test. The anemia responded to removal of the cyst or tumor but not to splenectomy. MATERIALS AND METHODS Eight major experiments will be described. These have been designated Experiments I through VIII (Table 2). Experi- ment V was divided into 5 subgroups designated Experiments VA through VE. The relationship of hematocrit, reticulo- cyte count, serum iron concentration, serum erythropoietin concentration and erythrocyte survival to experimental turpentine abscesses in cats was determined. Experimental Animals Adult domestic cats were used in Experiments I through VIII. Adult Beagle dogs were used in Experiments VB, VC and VD. All animals were random source animals and were obtained from the Laboratory Animal Care Service at Michigan State University. The cats were preconditioned for 3 to 4 weeks by the Laboratory Animal Care Service. The condition- ing program consisted of vaccinations for feline panleuko- penia, calicivirus and herpesvirus, deworming and dusting for ectoparasites. The cats and dogs were housed in the Veterinary Clinical Center in masonry cages with steel doors except for cats in Experiment VIII, which were housed in Laboratory Animal Care Service quarters. Cages were cleaned and animals fed twice daily. 24 Experimental Design Experiment I The objective of this experiment was to establish normal baseline values for tests performed in other experi— ments and to assess the effect of serial bleeding on hematologic parameters. Five cats were used in the experiment. Daily, 1 ml of blood was aspirated from a cephalic vein into a sterile disposable syringe. Leaving the needle in the vein, an additional 0.5 m1 of blood was aspirated into a second syringe containing 15 ul cfif ethylenediamine- tetraacetate (EDTA). Tests performed were those listed in Table 1. Table l. Tests performed in Experiments I through VII Hemoglobin Reticulocyte count PCV Plasma protein RBC count Serum iron MCV Total iron binding capacity MCHC Serum erythropoietin Total leukocyte count Expgriment II The objective of this experiment was to assess the effects of a sterile abscess on the hematocrit, reticulocyte count, serum iron and serum erythropoietin. These results were used for comparison with later experiments in which 25 various treatments were employed to modify the effects of the sterile abscess. Five cats were anesthetized with ketamine hydrochloride and .75 ml of filter-sterilized USP turpentine was injected subcutaneously in the gluteal region. Blood samples were drawn daily for the first 8 days and on alternate days for the next 6 days. The procedure for drawing the blood was that described in Experiment I, and the tests performed were those listed in Table 1. Experiment III The objective of this experiment was to assess the effects of cobalt injection on the hematocrit, reticulocyte count, serum iron and serum erythropoietin. These data were necessary to establish whether or not the effects of cobalt in cats were similar to those reported in other species. Five cats received daily injections of 0.5 ml of cobalt chloride (50 mg/ml) in sterile water. Blood samples were drawn on alternate days for 2 weeks. The procedure for drawing blood samples was that described in Experiment I, and tests performed were those listed in Table l. Expgriment IV The objective of this experiment was to assess the capacity of cobalt to modify the hematologic effects of a sterile abscess. Five cats were anesthetized with ketamine hydrochloride and 0.75 ml of sterile USP turpentine was injected subcutaneously into the gluteal region. A cobalt 26 injection (0.5 m1 of 50 mg/ml cobalt chloride) was given at the same time and repeated daily thereafter until the abscess opened. Blood samples were drawn daily for 8 days and on alternate days for the next 6 days. The procedure for drawing the blood samples was that described in Experiment I, and the tests performed were those listed in Table 1. Experiment V The objective of this experiment was to find a method for increasing serum iron concentration in cats and to assess the effects of elevated serum iron on normal cats. The most acceptable method would then be used to evaluate the hemato- logic effects of elevated serum iron during a sterile abscess. In Experiment VA, inosine (50 mg/kg) in 3 divided doses was injected into 4 cats by jugular catheter. The injections were given at 15-minute intervals. Serum iron concentrations were determined at hourly or bi-hourly intervals. Because serum iron concentrations failed to increase in cats, hypoxanthine (50 mg/kg) in 3 divided doses was injected into 5 dogs via a cephalic vein. Serum iron concentrations and uric acid concentrations were determined at hourly inter- vals for 3 hours. In Experiment VC, the dogs were placed under barbituate anesthesia in order to reproduce conditions of previous studies. The experiment was otherwise similar to Experiment VB. In Experiment VD, allopurinol (300 mg/day) was orally administered to 2 dogs for 7 consecutive days. Serum iron and uric acid concentrations were determined daily. 27 In Experiment VE, a solution containing ferric chloride was administered at a rate of 1 mg per hour by continuous intravenous drip to 2 cats over a 36-hour period. The solu- tion was prepared by adding 25 mg ferric chloride and 250 mg of trisodium citrate to 30 ml of distilled water. The solution was filter-sterilized and added to 220 ml lactated Ringer's solution. The rate of administration was approxi- mately 10 ml per hour. Blood samples were drawn twice daily. The procedure for drawing blood was that described in Experiment I, and tests performed were those listed in Table 1. Experiment VI The objective of this experiment was to assess the effects of continuous intravenous iron administration on the hematologic alterations associated with sterile abscesses. These data, when compared with Experiment II, provided a means of assessing the relative contribution of serum iron concentrations to the pathogenesis of anemia of inflamma- tory disease. Four cats were anesthetized and sterile abscesses were induced as described in Experiment II. An indwelling catheter was placed in a jugular or cephalic vein and an intravenous drip of the ferric chloride solution described in Experiment V was administered. Blood samples were drawn twice daily for the first 5 days, daily for the next 3 days, and every other day for the next 6 days. Tests performed were those listed in Table l. 28 Experiment VII The objective of this experiment was to assess the effects of combined intravenous iron and cobalt on the hematologic alterations associated with a sterile abscess. The procedure was similar to Experiment VI, except that daily cobalt injections (25 mg) were administered. Experiment VIII The objective of this experiment was to determine in vivo erythrocyte survival in control cats and in cats with sterile abscesses. Six cats were anesthetized and indwelling catheters were placed in a cephalic vein as previously described. Five milliliters of blood was withdrawn into a syringe containing 1 ml acid citrate-dextrose solution. The blood was placed in a sterile 10 ml tube. Fifty micro- curies of sterile sodium chromate (NaZCr51 04) was added to each blood sample and the blood was incubated for 1 hour at 37 C. Ascorbic acid (30 mg) was added and the blood was promptly reinjected into the cats. Blood samples were drawn 1 hour following reinjection and daily thereafter for 17 days. Sterile abscesses were induced in 4 cats on day 10 (Table 2). 29 Table 2. Experimental design for study of anemia of inflam- matory disease Experiment Animals/Experiment Treatments I 5 Normal control II 5 Abscess III 5 Cobalt in normal IV 5 Cobalt in abscess V 3 Intravenous iron in normal VI 4 Abscess + intravenous iron VII 4 Abscess + cobalt + intravenous iron VIII 4 RBC survival in normal and abscess Procedures Complete Blood Count The packed cell volume was determined by the micro- hematocrit method (McInroy, 1954). Hemoglobin, red cell count, MCV, MCHC and total leukocyte count were determined by standard methods with an electronic cell counter8 (Schalm et al., 1975a). 3Coulter Counter, Model S, Coulter Electronics, Inc., Hialeah, Florida. 30 Plasma Protein Total plasma proteins were determined by refractive . b 1ndex as measured by a refractometer. Reticulocyte Count Reticulocytes were stained with new methylene blue according to the method of Brecher (1949). These cells were classified as Type I, II or III reticulocytes, as described by Schalm et al. (1975b). The relative number of each type was enumerated by counting 1000 red blood cells. Type I reticulocytes were those with isolated foci of reticulum. Type II reticulocytes were those with 1 to several filaments in addition to the isolated foci. Type III reticulocytes were those with an interwoven mass of reticulum which occupied a large portion of the cell. Serum Iron and Total Iron Binding»Capacity Serum iron and total iron binding capacity were deter- mined by a modification of Goodwin's method (Stookey, 1970). The reagents were purchased as a kitC and samples were analyzed on a centrifugal clinical chemistry analyzer.d Preliminary evaluation of the kit revealed that color deve10pment was not stable. Color intensity continued to develop with time, giving significantly higher serum iron bGoldberg Refractometer, American Optical Co., Buffalo, New York. CGemini Serum Iron and TIBC Kit, Electro-Nucleonics, Inc., Fairfield, New Jersey. dGemini Centrifugal Analyzer, Electro-Nucleonics, Inc., Fairfield, New Jersey. 31 concentrations. Good precision was obtained by allowing exactly 4 minutes between the end of the blank run and the beginning of the test run. With each batch of samples a 500 ug/dl iron standard, normal feline control and commercial human control serum8 were determined. Results were accepted only if all control sample values were within 2 standard deviations of their mean. Epythrogyte Survival Radiolabeled whole blood (0.5 ml) was placed in a counting tube and lysed with distilled water (0.5 m1). Counting tubes were placed in a well-type scintillation counter. A minimum of 10,000 counts were accumulated on each sample by varying the counting time between 1 and 5 minutes. Raw counts were corrected for background and decay as described by Gillis and Mitchel (1974) and the percent activity remaining was plotted on semilog paper. Serum Erythroppietin Serum_ erythropoietin concentrations were determined by Dr. R. D. Lange by an in vitro fetal mouse liver cell assay (Dunn, Lange and Jones, 1979). This assay has been shown to detect elevated erythropoietin concentrations in the serum of cats and dogs following exposure to hypobaric conditions and phlebotomy (Dunn, Lange and Jones, 1979; eMonotrol I and II, Dade Division American Hospital Supply Corporation, Miami, Florida. 32 Dunn and Legendre, 1980). A purified human urinary standard and feline control serum were used. A highly significant correlation ( r=.6, p=.001) was found between the in vivo and the in vitro bioassay procedures. The index of pre- cision for the procedure was .05-.15. Statistical Analysis Means, standard deviations and standard errors of the mean were calculated for the data in each experiment. The significance of daily changes within gruops I through VII was analyzed by analysis of variance. The significance of day to day intergroup variation was analyzed either by analysis of variance (PCV) or by the nonparametric rank sum test (serum iron, reticulocytes-types I II and III and erythpopoietin). Bartlet's test was used to decide which of the two statist- ical tools was appropiate for each set of data. In Experiment VIII, mean percent radioactivity remain- ing was plotted against time on semilog graph paper. A line of best fit was determined for days 1 through 9 and days 13 through 17 for both control and abscess groups. Lines of best fit were calculated by regression analysis following log transformation of the data. Slopes for each of the 4 regression lines were calculated and compaired by the Student's t-test. RESULTS Experiment I Experiment I assessed hematologic alterations caused by the bleeding protocol. Total blood withdrawn over the 2-week period was approximately 16.5 ml. Results have been tabulated in Appendix Table A-1 and summarized in Table 3. Significant changes in the tests were not observed except for type II reticulocytes, which increased during the experiment. The packed cell volume increased slightly, which indicated that the bleeding protocol by itself did not cause anemia. Serum samples for erythropoietin determination were drawn on day 1 of the experiment. The mean serum erythro- poietin concentration was found to be 22.5 1 2.1 mU/ml (Table 4). Experiment II Experiment II assessed the hematologic changes caused by a sterile abscess. Results have been listed in Table 5 and Appendix Table A-2. The packed cell volumes were increased in all cats on day 2. Following day 2 the packed cell volume decreased 33 34 Table 3. Selected data from control cats (Experiment I) Days 1 2 _4 6 8 12 PCV (Vol.%) 34 35 35 35 37 36 +73 :2 :2 :7 :2 :2 Serum iron 103 102 110 96 105 105 (pg/d1) :30 :9 :10 :13 :17 :9 Retic Type I 366 418 431 457 627 481 (x103/u1) :133 :110 :69 :58 :108 :49 Retic Type II 11 15 10 15 106b 79 (x103/u1) :7 :6 :7 :7 :28 :17 Retic Type III 0 0 0 0 4.3 0 (x103/u1) ‘ :3 a Values represent mean : standard error for 5 cats. bSignificantly different from day 1 (p<.05). 35 Table 4. Erythropoietin concentrations in control cats, cats with sterile abscesses, cats treated with cobalt and cats with abscesses treated with cobalt Control Abscess* Cobalt* Abscess and Cobalt* 19.6 32.1 16.2 28.5 31.6 29.3 15.7 16.4 19.0 29.3 38.2 16.3 20.7 33.3 49.1 15.6 22.7 31.2 31.0 31.6 22.0 16.8 15.9 29.8 Mean 22.5 31.0' 30.0 21.7 SD 6.1 1.8 14.4 7.7 SE 2.1 .8 6.4 3.5 * Samples drawn on day 5 after initiation of the abscess and/or cobalt. 36 Table 5. Selected data from cats with sterile abscesses (Experiment II) Days 1 *2 I4 6 8 12 PCV (Vol.%) 32 39 31 24b 28 29 :2a :1 :2 . :2 :2 :2 Serum iron 81 23C 38C 16C 64 80 (ug/dl) :5 :8 :14 :4 :30 :17 Retic Type I 355 508 164 95b 163 530 (x103/u1) :68 :147 :78 :38 :48 :154 Retic Type II 26 47 22 39 67 224b (x103/ul) :13 :36 :14 :21 :35 :104 Retic Type III .5 « 3 O 0 7 27b (x103/ul) :.S :3 :4 :12 a Values represent mean : standard error for 5 cats. bSignificantly different from day 1 (p<.05). CSignificantly different from day 1 (p<.01). 37 progressively in all cats until the abscesses opened. This decrease was statistically significant (p<.05) by day 6 with an average decrease of 8 volume percent. Serum iron concentrations were decreased in all cats by day 2 (p<.01). Iron concentrations remained low until the abscesses opened (days 6 to 8). Type I reticulocytes decreased progressively between days 2 and 7. The decrease was statistically significant by day 5 (p<.05). Significant decreases in type II and type III reticulocytes were not observed. Serum erythropoietin concentrations were determined on the fifth day after induction of the abscess. Mean serum erythropoietin concentrations were higher than the control group, but the change was not significant (p=.1). Experiment III Experiment III assessed hematOIOgic changes caused by cobalt administration. The data have been listed in Table 6 and Appendix Table A-3. The packed cell volumes in all cats increased progres- sively during the experiment. An average increase of 13 volume percent occurred during the 2-week period of the experiment. A significant increase in type I and type II reticulocytes also occurred in all cats. Mean serum erythropoietin concentrations (Table 4) varied considerably. Three of the cats had significantly increased concentrations, while 2 cats had values slightly lower than the normal group. 38 Table 6. Selected data from cats treated with cobalt (Experiment III) Days 1 2 *4 ET 8 PCV (Vol.%) 36 39 44 45 47 :2a :2 :2C :2C :3C Serum iron 71 116b --- 137b 105b (ug/dl) :15 :17 :24 :15 Retic Type I 876 1624C 1396C 1656C 1708C (x103/u1) :85 :325 :352 :335 :227 Retic Type II 15 46b 101C 173C 211C (x103/u1) :6 :32 :77 :117 :115 Retic Type III 1.5 21 67 69 47 (x103/u1) :1.5 :11 :44 :31 :24 a Values represent mean : standard error for 5 cats. bSignificantly different from day 1 (p<.05). CSignificantly different from day 1 (p<.01). 39 Experiment IV Experiment IV assessed the effects of cobalt on the hematologic changes caused by a sterile abscess. The data have been presented in Table 7 and Appendix Table A-3. The packed cell volumes of all cats incresaed signifi- cantly by day 2 (p<.01). The mean packed cell volume con- tinued to increase until day 5 but thereafter decreased. The mean packed cell volume was significantly less than than in Experiment III by day 7 (p<.01). Serum iron concentrations decreased slightly during the course of the abscess but remained significantly higher than in Experiment II (p<.01). Types 1, II and III reticulocytes increased significantly (p<.01) during the course of the abscess. By day 3 this change was significantly different from Experiment II, in which reticulocyte numbers decreased (p<.01). As in group III, erythropoietin concentrations varied considerably (Table 4). The mean concentration was essentially the same as in the control group. Experiment V This group assessed the effects of elevated serum iron concentrations on control cats. Initially, attempts were made to increase serum iron by releasing storage iron. Inosine was injected into cats and hypoxanthine was injected into anesthetized and nonanesthetized dogs (Appendix Tables B-l through B-4). All experiments were uniformly unsuccessful in increasing serum iron concentrations. However, uric acid levels were increased. 40 Table 7. The effects of cobalt on the hematologic alterations caused by a sterile abscess (Experiment IV) Bars. 1 2 4 6 8 10 12 PCV (Vol.%) 32 39b 42 41 38 36 35 :1a :1 :2C :3 :3 :4 :2 Serum iron 117 89 67C 100 120 99 81 (pg/d1) :17 :18 :8 :14 :35 :15 :13 Retic Type I 688 1241 1376b 1100 771 890 961 (x103/ul) :242 :493 :316 :188 :107 :88 :57 Retic Type II 5 11 86b 43 42 27 108 (x103/u1) :s :7 21 :19 :17 :13 :28 Retic Type III 0 7 41C 44C 16 21 0 (x103/u1) . :4 :22 :29 +9 :16 a Values represent mean : bSignificantly different CSignificantly different standard error from day l (p<. from day l (p<. 01). 05). for 5 cats. 41 Alternatively, cats were given ferric chloride by slow intravenous drip at a rate of 1 mg/hr. It was technically difficult to maintain jugular and cephalic catheters in normal cats, as the cats could remove them by biting or scratching. Thus, the experiment was limited to 3 days. During this time serum iron levels were maintained between 400 and 550 ug/dl. The mean packed cell volume increased from 33 to 37.5 (Tables 8 and Appendix Table A-5). Reticulocyte counts did not increase. Experiment VI Experiment VI assessed the effects of intravenous iron on the hematologic alterations caused by a sterile abscess. Serum iron levels were maintained between 109 and 249 ug/dl. As in Experiment 11, the mean packed cell volume decreased significantly by day 6 (p<.05) and increased after the abscesses opened (Table 9 and Appendix Table A-6). The net decrease in PCV during the abscess was essentially the same as in Experiment II. Reticulocyte counts increased during the abscess rather than decreasing as they did in group II (Table 9 and Appendix Table A-6). Type I reticulo- cytes were significantly greater than in Experiment II by day 4 (p<.05) and types II and IIIreticulocytes were greater by day 6 (p<.01). 42 Table 8. Selected data from cats treated with intravenous iron (Experiment V) Days I 2 3 PCV (Vol.%) 33 35 36 :1a :1 :.5 Serum iron (pg/d1) 90 471 388 :7 :31 :10 Retic Type I 892 957 782 (x103/u1) :237 :272 :112 Retic Type II 24 29 12 (x103/ul) :8 :12 :12 Retic Type III 0 0 0 (x103/ul) a Values represent mean + standard error for 3 cats. 43 Table 9. The effects of intravenous iron on hematologic alterations caused by a sterile abscess ‘(Experiment VI) Days 1 2 4 6 8 10 12 PCV (Vol.%) 37 35 34 29b 30 37 33 :2a :1 :2 :2 :3 :2 :2 Serum iron 131 236 223 195 125 108 112 (pg/d1) :19 :34 :58 :35 :49 :56 :56 Retic Type I 737 485 718 628 537 583 985 (x103/u1) :192 :239 :195 :276 :147 :72 :200 Retic Type II 34 8 91 168b 368C 415C 262C (x103/u1) :23 :8 :23 :32 :61 :74 :54 Retic Type III 0 o 8 30 77 57 58 (x103/ul) :5 :8b :24C :33b :34b aValues represent mean : standard error for 4 cats. bSignificantly different from day l (p<.05). CSignificantly different from day l (p<.01). 44 Experiment VII Experiment VII assessed the effects of intravenous iron and cobalt on the hematologic alterations caused by a sterile abscess. Serum iron concentrations were maintained between 125 and 350 ug/dl, except for cat B, which removed its catheter on day 3. After 3 hours without iron supple- mentation, the serum iron had decreased to 51 pg/dl (Table 10 and Appendix Table A-7). The changes in mean packed cell volume in this group generally paralleled Experiment IV, with an increase in the packed cell volume over the first several days followed by a drop after day 6 (Appendix Tables A-7 and A-8). In this experiment, however, the decrease was transient, followed by a progressive increase on days 8 and 10. Reticulocyte counts increased markedly during the abscess. Both type I and type II reticulocytes were sig- nificantly increased by day 2 (p<.01 and p<.05). Type III reticulocytes were significantly increased by day 3 (p<.05). Type II and type III reticulocytes increased progressively during the abscess, while type I reticulocytes decreased on days 5 and 6. All 3 types of reticulocytes were signifi- cantly greater than reticulocytes in all other experiments (p<.01). Experiment VIII Experiment VIII assessed changes in erythrocyte survival during a sterile abscess. In the control group the slope of days 1 through 9 was .037 : .004, while the slope for days Table 10. 45 The effects of cobalt and intravenous iron on hematologic alterations caused by a sterile abscess (Experiment VII) PCV (Vol.%) Serum iron (us/d1) Retic Type (x103/p1) Retic Type (x103/u1) Retic Type (x103/ul) Days 1 2’ 4’ 6 8 10 12 30 37 38 40 39 45 44 : a :2b :3b :5C :4b :6C :4C 87 236 220 269 92 52 56 :11 :39 :15 :31 :2 :8 :3 I 817 1426C 1258C 774 986 1295C 2096C :97 :137 :162 :151 :135 :60 :307 II 93 341 311 659 760 605 495 :30 :65b :80b :71C :104C :127C :141b III 3 5 268C 515C 278C 7 31 :3 :5 :57 :171 :53 :7 :20 a Values represent mean : standard error for 4 cats. bSignificantly different than day 1 (p<.05). CSignificantly different than day 1 (p<.01). 46 13 through 17 was .031 : .007 (Table 11, Figure 7). In the sterile abscess group the SIOpe for days 1 through 9 (pre- abscess) was .035::.001, while the slope during the abscess was .056 : .005 (Figure 7). Pre-abscess and abscess slopes were significantly different (p<.01). These data indicate a significant decrease in erythrocyte survival during the abscess. 47 Table 11. Erythrocyte survival in normal cats and cats with abscesses induced on day 10 Days Control Group Abscess Group 2 10,660 : 2,3313 10,791 : 281 3 8,694 : 390 10,151 : 367 4 8,110 : 1,241 9,909 : 543 5 7,465 : 1,517 8,219 : 485 6 7,345 : 1,993 7,566 : 444 7 6,542 : 1,399 7,031 : 388 8 5,865 : 905 6,972 : 418 9 5,653 : 1,078 6,264 : 279 10 5,880 : 1,284 6,387 : 463 11 6,332 : 830 6,147 : 290 12 4,838 : 333 6,122 : 339 13 4,240 : 746 4,526 : 424 14 4,104 : 509 4,054 : 469 15 3,827 : 73 3,552 : 316 16 3,684 : 427 2,913 : 263 17 3,524 : 243 2,587 : 293 a Values represent mean : standard error for radio- activity in counts per minute/0.5 m1 whole blood. DISCUSSION The 8 experiments discussed herein investigated the pathogenesis of anemia of inflammatory disease. Although contemporary controls were not used, related experiments were conducted successively and all experiments were completed within 4 months. Environmental conditions within the kennel were carefully controlled throughout the study. In the control group (Experiment I), significant changes were not observed except for type II reticulocytes which increased. The results indicated that the bleeding protocol by itself did not cause anemia. ErythrOpoietin concentra- tions (22.5 : 2 mU/ml) were lower than normal values reported by the in Vivo exhypoxic mouse assay (50 H- .03 mU/ml) and the in vitro fetal mouse liver cell assay (30 H- .01 mU/ml) (Dunn and Legendre, 1980). Although results of these 2 assays have been shown to correlate (r=.6, p<.001), the difference in mean normal values appeared to represent real differences in the amount of erythropoiesis-stimulating substances detected (Dunn, Lange and Jones, 1979). In vitro assays were shown to detect desilated erythropoietin, whereas in vivo assays did not (Dunn, Lange and Jones, 1979). Also, serum regulatory proteins, which modify the activity of erythro- poietin, were reported to have more effect on in vitro assays 48 49 (Dunn, Lange and Jones, 1979). Thus, erythropoietin concen- trations determined by present assay techniques should be considered only approximations of the true serum activity. Cats with induced sterile abscesses (Experiment II) developed a hematologic disorder generally consistent with anemia of inflammatory disease as described in humans, dogs, rats and mice (Cartwright, 1966). A similar disorder described as "depression anemia" was reported in cats (Mahaffey and Smith, 1978). The anemia in all species in these reports was characterized by: 1) poorly regenerative, normocytic, normochromic or normocytic, hypochromic anemia, 2) low plasma iron, 3) decreased total iron binding capacity, 4) decreased saturation of transferrin, 5) increaSed storage iron, 6) decreased bone marrow sideroblasts, 7) increased plasma copper, 8) increased free erythrocyte protoporphyrin, and 9) shortened erythrocyte survival. In these experiments detectable anemia deve10ped by the fifth day after induction of the abscess in all cats (Figure 1). In rats with sterile abscesses, anemia was found to develop by the third day. However, in humans and dogs anemia deve10ped only after 2 to 3 weeks (Lauritsen et al., 1946; Lukens et al., 1967; Cartwright and Lee, 1971). This species difference in onset time of anemia may relate to the nor- mally shorter erythrocyte lifespans in cats and rats. Serum iron concentrations decreased rapidly within the first 24 hours and remained low until the abscess opened (Figure 4). The rapid and consistent decrease in serum iron 50 E; o/ ‘\x m 0 a n a 301—- b :3 2 a 20- 1011111111111111 1234567891011121314 Days Figure l. Sequential mean packed cell volumes in the sterile abscess group (Group II H), cobalt treatment group (Group III<3--743), and combined abscess and cobalt treatment group (Group IVX——-)( ). aSignificantly different from the mean of group IV; p<.01; b-p<.05; ns - not significantly different. 51 has been considered a hallmark of anemia of inflammatory disease (Cartwright, 1966). Type I reticulocytes decreased during the sterile abscess (Figure 5). This failure of the bone marrow to respond to the anemia was consistent with hypoproliferative nature of the anemia of inflammatory disease. Administration of cobalt to normal cats (Experiment III) resulted in detectable polycythemia, reticulocytosis and hyperferremia by day 2. Normally a 3- to 5-day lag period is present between stimulation of the marrow and increased erythrocyte output (Schalm, 1975). Thus, other factors such as splenic contraction or subclinical hemoconcentration may have been involved in the early phase of the polycythemia (Mahaffey and Smith, 1978). ErythrOpoietin concentrations in cobalt-treated cats' varied (Table 4). Three cats had increased concentrations while 2 had concentrations in the low—normal range. These results are in contrast to those of Goldwasser et a1. (1958). They reported that cobalt injection into rats caused a rapid increase in erythropoietin activity in serum. ErythrOpoietin concentrations in their study peaked about 10 hours after injection and then drOpped rapidly over the next 10 hours. A maximum increase of 2-6 times baseline levels occurred at various doses of cobalt. The discrepancy in this study may have related to the time of sampling. Serum samples were collected 24 hours after the last cobalt injection. Thus, the erythrOpoietin peak may have been missed. 52 50 Ilia 40b . .\° F ,l ‘ in E .3 //x 5'30... ‘ n n M/ I 2 I 20— ,0 l 1 l l l l 1 1 1 1 l 1 1 1 1234567891011121314 Days Figure 2. Sequential mean packed cell volumes in the sterile abscess group (Group 11 H), cobalt treatment group (Group III ()——4D), and combined abscess and iron treatment group (Group VI X—X ). ns - Not significantly different from the mean group II at the = 0.05 level. 50— 40- PCU (Vol °/.) 8 I 20 10 11111111111111 2 4567891011121314 Days Figure 3. Sequential mean packed cell volumes in the sterile abscess group (Group II H ), cobalt treatment group (Group III C>-62 om.w mH.: o~.: e:.a mo.m em.m ew.a as.“ ~a\ asses .eHx noose see m.ea a.~a :.~H e.~a a.~H m.mH m.NH m.~a He\m eanefimeee: o.He e.mm o.em o.mm o.em o.:m o.mm o.em afie> >oe < ea, In; :1, e m a n N 1H1 muse: aeaaeeaeneaea see when 1 , . -w:< \ ~ H ucoewpomxm sow sump Hmeficm Hmscw>flwcm .H-< ofinmb 75 V? # GOMHMHDHNW :Hhhowmcmph o.maH ~e\ma umae o.~a o.ee o.ae c.~a o.mm He\ma eon“ eaten a o o o o e o o Ha\ .on HHH ease o.H~H a.~ea e.a e.ae e.ma e.w ”.me o.- o H:\ . aosx HH ease H.HNN :.mm~ e.~aa m.ama m.eem a.aee m.-e N.:AN 8.:a Ha\ V .on H ease H.~em m.eam N.~wa a.osa a.aee o.eme m.Hoa N.oom e.:a H:\ Hence aoax "mouxuonowuom H.a a.e :.A e.a e.e H.s m.e o.e H.a He\m ewes -ohm mammfim A.NH H.: a.mH a.~H m.OH m.o~ m.~s N.HH a.NH Ha noose -oxzoa Hanoe n.4m :.em a.em n.4m o.mm a.em m.em e.em o.mm He\m ozoz o.Ne o.~e o.~e o.~e o.~e o.~e o.me e.me o.~e Hm >uz no.5 mm.a me.a ~.m em.a ee.s ::.a ma.e N~.a H=\ nHHeU .oax needs no: H.NH e.HH N.eH o.~H a.os :.oH e.HH :.H~ o.~H He\w ewbeameEe: c.8m o.em o.oe o.em e.mm o.mm o.mm e.mm o.Nm aae> >ua m Isa NH: OH, lull .51. e m. n» n N H. mesa: eeaoaeaeneeea are mxma -w:< \ ~ fleeseaaeoov H-< asses 76 0Q * COMHMHDHmm :whhmmmcmhh o.omm Hu\m; umHe o.wNH o.mmH o.Hm o.om o.om c.5m o.Hc~ o.moa ~u\m: can“ espom o o.mH o o o o o o Hn\ ncflx HHH oaxa N.OOH c.ou~ m.mm H.~H o.vH o o o H=\ ”aflx HH maze o.em¢ m.ouv m.oom «.mom m.qom m.OHm ¢.wmm ¢.Hom H:\ ncHx H maze N.¢mm o.vmc H.0mm m.mHm m.w~m m.o~m «.mmm V.Hom H:\ Hmuoe A ”omx ”mouxuoHsufiuoz m.o m.o ”.0 m.o m.o wu.o m.c m.o o.h Hu\m cam“ -ona «Emma; m.NH o.~H o.h . m.o~ o.NH o.mH H.5H v.0H m.oH H: mouxu uOXDMH HMHOH o.mm m.¢m H.mm m.¢m m.mm m.mm H.em m.cm ~.om Hc\m one: o.e¢ o.vq o.m¢ o.m¢ o.~¢ o.~e o.me o.~¢ o.Hv Hm >02 mm.o em.o «H.o mm.o Ho.“ um.c H~.o mm.o mm.o H;\ mHHou ocfix woofia com N.oH o.o~ m.a o.m c.o~ m.m H.m m.o~ m.o~ Hu\m zwnofimosoz o.m~ o.m~ 0.5N o.n~ o.m~ c.m~ o.om o.m~ o.m~ *Ho> >um O NH ml *5 c m e m N \H:, mafia: sowpaaflauoumn Has mum: 1. -«:< Awoscfigcouv H-< ofinmw 77 ON ov a fiOHHMHSHmm cfihhowmcmhh. o.wem o.Hmm Hu\w: umHH o.¢NH o.HOH o.HH o.mmH o.¢vH o.HcH Hc\ma :oHH sshom o H.m o o a.vH o m.mH o o H:\ mon HHH QQHH m.om v.mNH H.HH ~.m¢ w.mm H.NN o.om m.m~ m.om H;\ . ”OHx HH waxy m.oon H.me m.HOH H.oqo m.mom mimmm o.Hmo m.voo H.cHo H:\ . n2x H maze N.mwm m.HHon.-H m.mmo m.mvo m.H~o m.~ou m.~mc m.cvo H:\ Hmuoe . n3x ”mouxuonuHuom m.o m.o m.o 0.0 o.H o.H o.H m.o o.H Hw\m :HmH -OHQ mammHm m.m H.H a.mH H.mH w.mH N.HH o.a m.HH m.mH H: mouzu now—30H HmHOH o.¢m «.mm o.em c.em m.mm m.mm H.mm m.mm H.vm Hw\m uzuz o.Hm c.Hm c.om o.om o.~m o.Hm o.~m N.Hm o.Hm Hm >02 No.H ma.m Hm.H mm.H we.H no.5 Ho.H Ho.H mo.H H=\ mHHou .cHx wooHa gem «.mH w.mH o.NH m.NH o.~H o.MH m.mH o.mH H.mH Hu\m :HnonoEmz o.mm c.~v o.mm o.mm c.5m c.wm c.Hm o.Hm o.wm “Ho> >um a HH NH oH m 51, o m, H n N H muHca coHumcHsumHoa Hus aka: -H:< \ y HmoscHHcouV H-< oHan 78 GM * :OMHQHfl—Hmm :whhommnmhh o.mmm HVNN: umHH o.Nm o.NNH o.NNH c.mmH o.mmH o.NNH o.mNH o.NmH o.mmH Hu\ma :oHH ssHom o o o o o o o o a H=\ fi3x HHH waxy H.HN c.NH o.m m.N o o H.0H N.NN m.mN H:\ n2x HH oHHH N.mmm m.qoo N.mmm o.omm o.mem N.HNm N.amm N.Nwo H.NON H:\ n2x H «HHH o.Hmm oqNNo N.NON m.mmm c.mHm N,HNm o.mmm o.oHN «.mmN H;\ Hmuoe . Hoax “mouzuoasuHuom H.N H.N m.N H.N N.N N.N N.N N.N H.N Hu\m :HmH noun mammam ¢.m H.o m.N H.w v.0 N.c H.m H.o m.o H; moHHu :OMNHmH HMHOH w.mm N.¢m m.mm m.¢m ¢.em N.¢H a.mm o.¢m H.Hm Hc\m uzuz o.m¢ o.m¢ o.mq o.mv o.a< o.m¢ c.w¢ o.NH o.w¢ HH >02 NV.» NH.” cm.w mN.N om.m No.m NH.N NN.N we.» H:\ mHHm.U mcHx cooHn com c.mH o.mH m.mH N.HH o.HH N.mH N.mH H.HH o.mH Hu\m :HnoHNoEQ: o.mm o.mm o.mm o.o¢ o.ov o.mm o.mm o.mm o.mm HHo> , >um m NH NH oH m N o m N n N ““1 muHaa :oHHacHEHoHon Has mzwn N _ -H:< \ HcmscHucouV H-< oHnmw 79 mm co mm NH Nm NH we H :oHHmHsHam cwhhmmmcmhh o.oqN o.OON o.omm o.mNH o.me o.omH o.NON o.omH Hu\Nn umHH o.NHH o.Ha o.mNH o.HH o.oH o.mc o.mm o.¢m o.aN o.HN Hc\m: :oHH ssHom o o o o o o o o o o H=\ non HHH QHHH o o o o o o o o o o H;\ non HH oaHH o.qu N.HH o N.aN H.NH o.Nm m.om 0.0NH H.NmN H.NmN H:\ i non H maxe c.oHH H.HH o N.mN ¢.NH o.Nm m.¢w c.0NH H.NmN H.NmN H:\ HmHoH . Hoax ”mouxuoHSUHuom o.N N.o H.N w.o 0.0 N.N N.N m.c m.o m.o Hc\N chH -opn mammHm N.N H.NH o.NH o.NH m.NH H.HH m.NH o.NH m.mN 0.x H1 moHHu IOMSQH awn-OH. H.HH H.HH o.mm m.¢m o.mm H.HH N.mm N.mm m.mm H.HH H.HH Hc\m 0:02 o.Nv c.0v o.ov o.ce o.NH c.0e o.wq o.NH o.¢¢ o.om o.w¢ HH >uz mm.o mo.N Hm.o mm.o NN.N ON.o Ho.” mo.m om.N mN.N Ho.N H=\ mHH8 ucHx cooHn com H.HH H.HH H.HH o.oH N.HH N.oH o.mH H.HH N.NH N.HH H.HH Hc\m =HnoHNoso: o.mN o.NN o.mN o.mN o.oN o.HN o.mm c.mm H.HH o.o¢ o.Nm HHo> >um < NH cH N . bl. o m H m N H‘: muHsa :oHHaaHaHmHoa Has mks: -H:< ~\ HH acmEHHoaxm How «Haw HmEHcm Hmst>chH .~-< oHan 80 NN NN NN NH a mN cc H :oHHmhsuam :Hahommcmhh o.NmH o.NNH o.HcH o.NoH c.HoH c.0NH o.HNH Hu\Nn UNHH o.NN o.ve o.em o.oH o.om o.HN o.NH o.om o.mN HNNN= aoHH ssHom o o o o o o o o o o o H:\ non HHH maze 0.0H o o H.HH o o o o o o o H:\ . ,on HH «HHH N.Hmm o m.NN H.HNN o o.NH ¢.NN N.mv N.om N.Nom m.NNm H:\ n2x H «HHH o.HHOH o N.NN N.mom o o.NH N.NN N.me N.om N.Ncm m.NNm. H:\ HmHoH ncHx "mouxuoHauHuom m.c N.o m.o v.0 m.c m.o N.o m.o H.o m.o v.0 Hu\N =HoH _ -oum mammHm N.HH N.mH N.mH N.oH N.NH N.NH m.NH N.NN o.NN N.HH H: moHHu -oxsmH Hmuos H.HH o.mm N.mm o.¢m m.om N.NH m.mm N.¢N N.em H.HH Hm\m uzuz o.¢e o.Ne o.NN o.m¢ o.H¢ o.m¢ o.ev o.m¢ o.m¢ o.q¢ Hm >02 mN.N No.N No.N mo.N . Nm.o mq.o em.N Nm.N NN.N NN.o H:\ mHHmu o3x nooHn wag N.NH N.¢H o.oH N.oH m.m m.m H.HH H.HH m.NH H.¢H H3N =HnoHNoeo: o.Nm o.mN o.HN o.NN o.NH o.oN o.mN o.om o.mN o.mm o.NN NHo> >um m \NH NH OH nmr, N o m, .H H N HI, mHHaa coHHmcHsgoHon Has mzma .. . -H:< \ ~ HwoacHHcouH N-< oHan 81 em av mm OH «H mm c » :OMHwHSme Gwhhmmmcmhb o.NON o.NNN o.mNN o.NHN o.NNH c.HNH o.NoH Hv\N: UNHH o.Na o.HHH o.om o.NN o.ON o.mm c.OH o.mN Hc\N= :oHH esHmm H.mm N.NN H.HH H.N o o c.HH o o o c H:\ mcHx HHH waxy N.omN o.HON N.NQH H.HNH N.No N.NH o.NN H.HH o H.@ H.cH H:\ non HH QQHH H.HHN N.HNH N.HmH m.cNH N.mm o.NN H.HNH m;moH o.mN N.NHN o.m9N H:\ y mon H QHHH m.oom m.mov m.oo¢ o.omn o.mNH H.HNH H.HHN N.NNH c.mN m.NNN H.HNN H;\ HmHoH _ ncHx ”mouxuonquom N.N o.N N.N m.N H.N H.N N.N o.N m.N N.N m.N Hc\m :HoH -opm mammHm o.NH m.HH N.NH N.NH H.NH N.NH m.mH H.oH N.NH o.NH c.NH H: moHHu -oxsoH Nance c.em N.NH N.NH o.¢m H.HH N.NH N.NH c.em H.HH H.HH H.HH Hc\m 0:02 o.o¢ o.oe o.NH c.mv o.vN H.HH o.mq c.mv o.ov o.NN H.HH Hm >02 NN.N Nm.N Nc.N NH.N Nm.o om.N om.N mN.m m.N H.m ON.N H=\ .mHHmU o3x cooHn mom N.NH N.HH m.HH N.NH N.OH H.HH N.HH N.N H.HH N.NH N.NH Hu\m anoHNoeo: o.qm o.mm o.¢m o.mm o.mN o.cm o.mN o.mN c.Hm H.HH c.0m HHo> >UH u \LNH NH oH m N o m N m N H11 muHca aoHHacHsHoHoa Has mxmo . -H:< \ N HcoacHHcouV N-< oHan 82 NN mN N o N :oHHmpsumm Gwhhmmmcmhh o.mNN o.NmH o.NNH o.HmH Hu\mn umHH o.oN o.Nm o.OH o.oH o.HH o.OH o.m o.oo Hu\N: coHH esHom N.om N.NH o N.N c o o o o o o H:\ nH.Hx HHH oHHH N.NNH o.NHm N.NNN N.NN m.NoH m.mHH o.aN N.NN N.Nm c.wm o.mc H:\ mon HH oHNH N.NON N.NHN o.NON o.oNH H.Nmm H.NoN H.HHN H.HHH c.NmH H.qu o.HmH H:\ HOHx H waxy H.quHo.maNHm.HNo H.HHN o.H¢N o.NHm o.omN m.emH c.maH N.omm o.on H:\ Hmuoe _ HOHx ”mouxuoHsquom N.o o.N o.N N.o m.o m.N N.N N.N H.N o.N o.N Hw\N :HoH noun mammHm N.HN N.o H.HH N.oH N.mH m.NH o.mH H.HH N.mH N.NH N.NH H: moHHu -oxsma NmHOH o.HN c.mm N.NH N.NH N.NH N.NH o.mm N.NH N.NH o.vm N.Nm Hu\m 0:0: o.mv o.NH o.mq o.me o.me c.NN o.mq o.NH c.m¢ o.NN o.NH HH >02 oe.w mm.N NH.N N.N mm.N NN.N mN.N NN.N NN.N Nc.m N.N H:\ mHHou oon cooHn com N.NH N.NH N.oH a.oH N.oH m.OH m.OH H.HH N.NH N.NH c.NH Hu\N :HnoHNoEm: c.Nm o.mm o.HN c.Hm N.NN o.mN o.mN o.Nm c.qm o.oe o.mm HHo> >um a xNH NH oH .m‘ HI, 0 m. N in N H1: muHaa coHHacHsHouoo Hus mxmn -N:< \ N HwoscHHcouH N-< oHan 83 ma em HH «H OH ma m c » nequHSHQm :Hupommcmhe o.mNN o.HNN o.HNN o.NNN o.Nom o.NNN o.mmm o.cmm Hc\N: umHH o.om o.No c.HN o.¢m o.mN c.0v o.NH o.mH o.Nm Hw\N: :oHH esHom N.mm N.Nm mm.H m.HN o.oN c o o N.NH H.NH o.N H:\ mH.HH HHH oaHH N.NHm N.Noq N.om m.mv N.HH N.om H.HH H.HN N.©NH m.NNH o.NN H:\ _ n3x HH oHHH N.oNN N.NON N.mNH o.NmH N.NH N.NHH H.HNH N,HNN N.chw.cmoHHimaN H:\ .w HOHx H QHHH N.NmN N.NcN o.NNN m.moN o.mHH H.omH N.NNN N.Nem H.HON_N.QHNH‘N.NHN H:\ HmHoH . mon “mouxuonuHuoz N.N m.N m.o m.o N.m N.m v.0 m.o H.N N.N N.o Hc\N :HmH -oum mammHm N.©H o.NH o.vm o.mm N.NN o.Nm H.HN N.NH H.HN m.Hm o.mH H1 moHNu IOXSmH HmHOH o.Nm N.NN H.Hm m.om N.NH H.HH N.NH H.Nm o.Nm o.Nm H.HH Hw\m uzuz o.NH o.a¢ c.0H o.NN o.mH o.m¢ o.vq c.0e o.ce o.v¢ o.mv HH >02 Nm.m HH.o HN.H ae.m NN.m mH.o mo.N HN.N HH.m mm.N Nm.o H:\ mHHmu m.HHHHH wooHn com m.N N.N m.o o.N N.N H.m H.HH o.HH N.NH N.NH m.m Hu\N :HnoHNoeoz o.NN o.mN m.NH c.NN o.NN o.mN o.Nm o.Nm o.mm o.mm N.NN NHo> >um m «H NH oH {NW N o m N m N ,w; mHHca goHHmcHaHonn Has mxmn . -H:< HamsaHHcouv N-< oHnmw 84 w Sownpmhzumw thhmmmcmhh. HN\N: oNHN N.NNH N.NN N.NNH o.NNH o.mNH N.NN N.NoH Hc\mn :oHH esHom N.NNH N.NNH N.NNH N.NH o N.NN N.N H:\ n3x HHH maze N.NHN N.NHN m.ooo N.Nmo N.Nmm N.NNH m.om H:\ . ‘ non HH oQNN o.HNN N.NNN N.NNN N.NNN N-NNm N.NHN N.NNO H:\ . mH.Hx H NHNN o.ONoH o.HNoH N.NmoH N.NNNH N NHN H.NNoHo.HNN H:\ HNHON . NOHx ”mouxuoHsuHumm o.N o.N N.N N.o N.o N.N m.o Hc\m cHOH -oua mammHm N.NH N.NH N.NH N.NH N.NH oN.o m.HH H: mmuxu -oxon Hmuoe o.mm c.mm N.Nm N.Nm N.Nm N.NN N.NN HN\N uzuz o.Hm N.NH N.NH o.Hm o.om N.Nm o.Hm HH >02 Nm.N HN.N mm.N NH.N Nm.N om.N No.N H:\ mHHou Non NooHa Nam H.HH N.NH N.NH N.NH N.NH H.mH N.NH HN\N :HnoHNosw: N.NN N.NN N.NN o.HN N.NN N.NN N.Nm HHo> >0; < lNH \bH 1m{[ nix o N N Hun muHca aoHumanHouoa Hme mxmn .. -H:< ~\ HHH HCOEwHQme how mumfi Hmecm Hm3©w>mw~HH .m-< omnmb » :oNHMNsumm 85 :whhmwmfimhh HN\Nn UNHN N.NNH N.NHH N.NNH N.NNH o.ONH o.mN HN\N: :oHH sapom o o N.NN N.NN N.N c o H:\ "NHx HHH maze N.NNH N N.Nm N.NN N.N o o H:\ n3x HH mmxe N.NomN N.NHNNN.NHNN N.NNNH N.NHNH N.NNNHN.HHHH H:\ . n3x H omxh N.NHNN N.NHNNN.NNNN N.NNNH NNNmNH N.NNNHo.HHHH H:\ HNHoN . ”OHx “mouzuoasufiuom N.N N.N N.N N.N N.N N.N m.N HN\N cHoH -ogg mammHm N.NH N.N N.NH H.NH N.mH N.HN N.mH H: mouxu . -oxsoH HNHoN N.NN N.Nm N.NN N.Nm N.Nm N.NN N.Nm HN\N uzuz o.mN c.mN N.NN N.NN N.NN N.NN N.NN HH >02 N.NH N.NH N.NH N.HH N.HH N.HH NN.N H:\ mHHou .on NooHn Nam N.NN N.NN N.NH N.NH m.cH N.NH N.NH HN\N :HnoHNoso: o.mm c.mm N.Nm N.Nm N.NN N.NN N.NN NHo> >um N NH NH oH rm N N m N N N H:. mHHsa :oHumcHsHoHon HNE mxma -H:< \ ~ Amozcwucouv m-< mHnme « :oNumusumm 86 :whhmmmcmhh HN\N: UNHN N.NN N.NN N.NN N.HHH N.NN HN\N: :oHH saHmm o o N.NNH N.NNH N.NH o H:\ mNHx HHH oaks N.NN N.NN N.NN N.NN N.NH N.NH H:\ a2x HH «axe N.NNNH N.NNNH N.NNNN N.NNHN N.NNNNN.HNNH H:\ . ncHx H NHNN N.NNNH N.NNNH N.NmmN N NNNN N.NNNNN.NNNH H1\ HNHoN Non ”mouxuoH30Huom H.N N.N H.N m.N N.N N.N N.N HN\N :HmH -oum «EmmHm N.NH N.NH N.NN N.NH N.NN N.NH H1 mouzu uoxsmfi HNHOH N.NN N.HN N.Nm N.Hm N.Hm N.Nm HN\N uzuz N.NN N.HN N.NN N.NN N.NN N.NN HH >02 N.HH N.HH N.HH N.HH N.NH NN.N H:\ mHHou oon NooHs Noz N.NH N.NH N.NH H.mH N.NH N.NH HNNN =HnoHNoso: N.Nm N.Nm N.NN N.NN N.NN N.NN N.Nm NHo> >0; u [NH NH NH 11ml N .N ‘1 N NJ: mNHca coHHNNHsHOHma HNs mxmn -Nc< \ HNoschcouH m-< oHan 87 » :oNumuaumm thhmwmcmhb HN\N; QNHN N.HHH N.NNH N.NN N.HN NNH HN\N: :oHH asgom o o H.NH H.NH N.NN o H:\ mon HHH QQHN o.mmH N.NN N.NNH N.Nm N.NH o H:\ m3x HH waxy m.HmNH N.NNNHN.NNNN N.NNNN N.NHNNN.NmN H:\ mcHx H NNHN m.NNON N.NmNHo.mmmN m.oomN H.NNNNN.NmN H:\ HNHoN . mon "mouxuofisuHuom N.N N.N N.N N.N N.N N.N N.N HN\N :HoH -oum «EmmHm N.NH N.NH N.NN N.NN N.Nm N.NN H: moHHu -oxsmH HNHOH N.Nm H.mm H.NN N.NN N.Nm N.HN HN\N 0:02 N.NN N.NN o.mN N.NN N.NN N.NN Hm >02 mN.N NN.N mo.m No.m Ho.N NN.N H:\ mHHou o3x nooHa Nam N.NH N.NH N.NH H.NH N.HH N.NH HN\N =Hnonoso: N.NN N.NN N.NN N.NN N.NN N.Nm N.NN NHo> >um a NH Na ca lml. o v N H mews: coaumcwshmumn HmE mxmn «J: -N:< All! \ s HNochHcouV N-N QHNNN 88 mN » :oNumuzumm Gmhhommcwhfi N.NNN HN\N: umHN N.NHH N.HHH N.NNH o.mNH N.NNH HEN1 :oNH snpom m.m N.Nm N.Nm N.Nm H.NN o o H:\ mon HHH NQHN N.mm N.NN N.NmH H.NN N.NN N.NH N.mN H:\ aNHx HH waxy m.NNNH N.NomHN.NNNH N.NmoH m4NmoH N.NNN H.NoN H=\ nNHx H maze N.NNmH N.NHNHN.NNmH N.NNHH N.mmHH H.oom N.NNN H:\ HNHoN . Noax "mouxuoNzuHuom N.N N.N N.N N.N m.N N.N N.N HN\N chH -OHQ «Emma; N.NH N.NH N.NH N.HH N.NH H.HH N.N H: moHNu IOMSQH munch. N.NN N.NN N.NN N.NN N.Nm N.Nm H.NN HN\N onus N.NN N.NN N.NN N.Nm N.NN N.NN N.NN HH >oz Nm.m Nm.m NN.N HN.N HN.N NN.N mN.N H:\ mHHou oon NooH; NON N.NH N.NH N.NH H.NH N.NH N.NH N.NH HN\N =HnoHNoEO= o.mN N.mN N.NN N.HN N.NN N.HN N.NN NHo>, >um m NH NH NH In, N N m N N.N m .HA: mHHcs :oHumcHeNmHmn Has mzmn a . . -Nc< ~\ HmoscHucouv m-< oHnNN 89 NN NN mN HN ON Nm mN HH NH HH N :oHpmuaumm :HHhommcmHH N.HNH N.NNH N.NNH N.NNH N.NNH N.NNH N.NNN N.HHm o.omm N.Hom HN\N: UNHN N.NN N.NN N.NN N.NN N.Nm N.NN N.NN c.mm N.No o.mm HNNN: moHH ssHom o o o N.NN m.mH N.NmH N.NH N.NNH N.NH o o H:\ mcHx HHH maze m.oN N.NH N.NN N.NN N.NN m.moH N.Hm N.NmH N.Nm m.NH o H:\ .NHx HH maze m.NNHHo.oNNHN.NNN N.NmN N.NNN N.Nmo N.NoN N:mNN NH.om N.HNN N.NOH H:\ , nH.Hx H «axe N.NHNHN.omoHN.NNN N.NNN N.NNN N.NNN N.NmN N.NmN N.Nmm N.NNN N.NOH H:\ HNHoN . ncHx ”mmuxuoNsuNumz N.N N.N N.N N.N N.N N.N N.m N.N N.m N.m N.m HN\N :Hop scum «EmmHm m.Nm N.NN N.NN N.NN N.NN N.NN m.mm H.NN N.NN N.HN N.NH H: moHHu . -oxsmH NmNOH N.NN N.Hm N.Hm N.HN N.Hm N.HN N.Nm N.Hm N.Nm N.Hm N.NN HN\N ozuz N.NN N.NN N.NN N.NN N.NN c.mN N.NN N.NN o.mN N.NN N.NN HH >u2 mN.N NH.N NN.N NN.N NN.N NN.N om.N NN.N mN.N cN.N NN.N H:\ mHHmu NcHx nooHn Nam N.N N.N N.N m.oH N.NH N.NH H.NH N.HH N.NH N.HH N.NH HN\N =HaoHNoso: N.NN N.NN N.NN N.NN N.NN N.Nm N.NN N.NN N.HN c.mm N.HN NHo> >um < NH NH NH 1m >1 o \m, N m N H|. mHHaa aoHumcHsHoHoa HNe mxwa -w:< ~ >H unmENHoaxm How mumw Hmewcm Hmsww>fiwcH .N-< mflnme 90 NN NH NN NN NN NN NN NN NH NN N :oHNNHNNNm :mHHGMmcth N.NNN N.NNN N.NNN N.NNN N.NNN N.HNN N.NNN N.NHN N.NNN N.NNN HN\N: NNHN N.NN N.NN N.NNH N.NNH N.NNH N.NNH N.NN N.NN N.NN N.NNH HN\N: :oaH aauom N N N N N N N.NH N.NN H.N N N H=\ nNHx HHH NNNN N.HNH H.NN N N N.NN N.NH N.NH N.NN H.N H.NN N H=\ aNHx HH NNNN N.NNHHN.HNN N.HHN N.NNNN.NNNHN.NNNHN.HNNHN.NNNHN.NHNHN.HNNNN.NNNH H:\ nNHx H NNNN N.NNNHN.NNN N.HHN N.NNNszNNHN.NNNHN.NNNHN.NNNHN.NNNHN.NHNNN.NNNH H:\ HNNNN NcHx ”mouxuoHsuHuom N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N HNNN NHNN -oun mammHm N.NH N.NN H.NH H.NH H.NH N.HN N.NH N.NH N.HN N.NN N.NH H: mouzu noxsmH HNHOH N.NN N.HN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN HN\N ozoz N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN Hm >02 NN.N HN.N NN.N NN.N NN.N NN.N HN.N NN.N NH.N NN.N N.N H;\ mHHou NNHx NooHN Nam N.HH N.HH N.NH H.NH H.NH H.NH N.NH N.NH N.NH N.NH N.HH HN\N :HNNHNNEN: N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN NHo> >NN N NH NH NH in, NI, N m ‘N ‘N N Hi: NNHNN NNHNNNHENNNNN HNe mxmn -N:< \ N HNNNNHNNNUN N-N NHNNN 91 mm Hm NN mm NN mm mm NN Nm NN N :oHamhsumm Qwhhvmmcmhfi N.NNN N.NNN N.NNN N.NNN N.NNN N.HNN N.NNN N.NNN N.HNN N.NNN HN\N: NNHN N.NNH N.HN N.NN N.NNN N.NNH N.NHH N.NHH N.NN N.NN N.NNH N.NNH HN\N= :oNH ashom N N N N N N.NH N.NNH N.NH N N.NH N H:\ nNHx HHH NNNN N.NNH N.NNH N.NN N.NN N.NNH N.NN N.NNN N.HN N.NH N N.NN HN\ mNHx HH NNNN N.NNNN.NNNHN.NNNHN.HNN N.HNNN.NNNHN.NNNHHNNHHNN.NNNHN.HHNNN.NNN H:\ . nNHx H NNNN H.HNNN.NNNHN.NHHHH.NNNN1NNHHN.HNNHN.NNNHN.NNNNN.HHNHN.NNHNN.NNN HNN HNHoN . Noax “mouxoonuNuom N.N N.N N.N N.N N.N H.N N.N N.N N.N N.N N.N HNNN NHNH -oum mEmmHm N.NH N.HN H.NN N.NH N.NH H.HN N.NH N.NH N.NH N.NH H.NH H: mouxu IOMSQH HmHOH N.NN N.NN N.NN H.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN HN\N Nzuz N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.Hm N.NN N.HN Hm >Nz NN.N NN.N HN.N NN.N NN.N NN.N NN.N NH.N NN.N NN.N NH.N HNN NHHNU oNHx NooHN NON H.NH N.NH N.NH N.NH H.NH N.NH N.NH N.NH N.NH N.NH N.NH HN\N NHNNHNNEN: N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN NJHN NHo> >NN N NH NH NH m; Nit N N N .N N H NNHNN :oHNNNHeNNHNN HNE NNNN . . -HNN 3 \ N HNNNNHNNOUN N-N NHNNN 92 HN Hm NN NN NN NN NN NN NN N :OHONONONO nflhhmwmcmhe N.NNN N.HNN N.NNN N.NNN N.NHN N.NHN N.NNN N.NNN N.NNN HN\N: NNHN N.NNH N.NNH N.NNH N.NNH N.NNH N.NNH N.NHH N.NN N.NN N.NN N.NNH HN\N: NOOH ENNON N N H.NH N.NH N N.NN N N.NN N.NN N N H:\ nNHx HHH ONNN N.NN N.HNH N N.NN N N.NN N N.NN N N N HN\ nNHx HH ONNN N.NNNH N.NNNNNHNHHNNN N.NNNNNHNHN.NNNNNNHHNNNN N.NNN N.NNN HE . nNHx H ONNN N.NNNHNHNNHHNNNH N.NNN N.NNNNNNNHN.NNNNNNNHNNNN N.NNN N.NNN HE H38 . Noax ”mouxuoHsuNuom N.N N.N N.N H.N N.N N.N N.N N.N N.N N.N N.N HN\N :HON -opm mammHm N.NH N.NH N.NH N.NH N.NH N.N N.N N.NH N.NH N.NH N.N H: NONNO noxzmfi Hmuoh. N.NN N.NN N.NN H.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN HNNN 0:02 N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN Hm >Nz NN.N NN.N NN.N HN.N NN.N HN.HH N.HH N.HH NN.HH NN.N HN.N H=\ mHHOO mNHx NOOHN NON N.NH H.NH N.NH N.NH N.NH N.NH N.NH N.NH N.NH N.NH N.NH HN\N :HNOHNOEO: N.HN N.HN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN NHO> >NN N NH NH NH N NI, N m N N N H NOHNN NOHNNNHNNOOON HNE mxmn -N:< \ ~ HNONNHONOON N-N OHNNN 93 HN om mm 0H ca ma 0H mH ca Hm cm » :owumhaumm :whhowmcmhk N.NNN N.NNN N.NNN N.NNN N.NNN N.NNN N.NNN.N.NHN N.NNN N.NNN N.NNN HNNN; NNHN N.NN N.NN N.NNH N.NN N.NN N.NN N.NN N.NN N.NN N.HN N.NNH HNNNN :ONH asNON N N N.NN N.NH N N N.NH N N.NN N.NH N HNN nNHN HHH ONNN N.NNH N.NNH H.NN N.NH N N N N.NN N.NN N N HNN nNHN HH ONNN N.NNNHN.NNN H.NNN H.NNN N.NNN N.NNN N.NNN N;NHN H.NNN N.NNN N.HNN HNN . nNHx H ONNN N.NNNHH.NNN N.NNN N.NNN N.NNN N.NNN N.NNN N.NNN N.NNN N.NNN N.HNN HNN HNNON . NoNx “mouxuoHsuNuom N.N N.N H.N N.N N.N N.N N.N N.N N.N N.N N.N HNNN :HON -oum mamwam N.NH N.N N.N N.NH N.NH N.NH N.N N.N N.N H.N N.N H: NOONO -oxsmH Nance N.NN N.HN N.NN N.NN N.NN N.NN N.NN N.NN H.NN N.NN N.NN HNNN Nzuz N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN Hm >N2 NH.N NN.N NN.N NH.N NN.N NN.N NN.N NN.N NN.N NH.N NN.N HNN mHHOO oNHN NOOHN NON N.HH H.NH N.N N.N N.NH N.HH N.NH N.NH N.NH N.NH N.NH HNNN :HNOHNONO: N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.HN NHO> >NN N NH NH NH ‘Nn‘ N N m N ‘N N H71. NOHNN :OHHNNHENONON HNE mxma . . -N:< \ NNONNHNNOON N-N OHNNN 94 NN-NN NNH-NN NN N NOHONNNONN :NNNommcmNH NNN-NNNNNN-NNN NHN_ NNNNN NNHN NNNNNN NNNNNN NNN-NN HNNNN 5.: NEON N N N HNN nNHN HHH ONNN N N.NH N.NN HNN “NHN HH ONNN N.NNNN.NNNH N.NNHH HNN nNHN H ONNN N.NNNN.NNNH H.HNHH HNN HNNON Noax “mouxuoHsuNumm N.N N.N N.N HNNN :HON -oym mammam N.NN H.NN N.NN H: NONNO noxnod Hmuop. N.NN N.NN N.NN HNNN 0:02 N.NN N.NN N.NN Hm >Nz NN.N NN.N NH.N HNN mHHOO oNHN NOOHN NON H.HH N.HH H.HH HNNN NHNOHNOEO: NN-NN NN-NN NN-NN NHo> >NN N NH NH om N m N. H mafia: :oNumzwsuoqu Hue -HN< .I \ N > acoENNoaxm Now mumw Nmemcm Hmzww>wwcm .m-< mNnmN 95 N :oNumNzumm :NuumwmcmNh HNNNN NNHN NNNNNN NNNNNN NNNNN HNNN: NEH NEON N N N HNN nNHN HHH ONNN N.NN N.NN N.NH HNN aNHN HH ONNN N.NNN N.NNN N.NNN HNN mNHN H ONNN N.NNN N.NNN N.NNN HNN HNNON . NoNx “mmuxuoasowuom N.N N.N N.N HNNN :HOO -oun mammam N.NH N.NH N.NH H: NONNO soxsoH HNHOH N.NN N.NN N.NN HNNN umoz N.NN N.NN N.NN HN >uz NH.N NH.N NH.N HNN NHHOO NNHN NOOHN NON N.HH H.HH N.HH HNNN :HNOHNOeO: NN-NN NN-NN NN-NN NHO> >NN N ELNH NH NH N N N N H NNNNN :OHNNNHNNONON HNE mxmn -N:< \ N NNONNHONOON N-< OHNNN 96 NN NN HN NN NN NNH NN NN HN NN NN N NOHNNNNNNN awhhmmmflmhfi NNN NNN NNN NNN NNN NNN NNN HNN HHN NNN NNN HNNN; NNHN NHN NNN NNN NNN NHN NNN NNN-NNNNNN-NNNNNN NNN NNNr#fiHHN\N: NONN ENNON N N.NNH N.NNH N.NNH N.NN N.NH N N.NH N N N HNN MNHN HHH ONNN N.NNN H.NNN N.NNN N.NNN N.NNH N.NNH N.NN N.NN N.HN N N.HN HNN mNHx HH ONNN N.NNHHN.NNN N.NNN H.NNN N.NHN N.NNN N.NNN N;NHN N.NNN H.NNH N.NNN HNN . nNHN H ONNN N.NNNHN.NNHHN.NNNN.NNHHN.NNN N.NNN N.NNN N.HNN N.NNN H.NNH H.NNN H.HN HNOON . Noax ”mouxuoHsuNuom N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N HNNN :HON -oun mammNm N.NH N.NH N.NH N.N N.N N.N N.N N.N H.N N.N N.N H: NONNO IOMSQH Hmuoh. N.NN N.NN N.NN H.NN N.NN N.NN N.HN N.NN N.NN H.NN N.NN HNNN use: N.NN N.NN N.NN N.HN N.HN N.NN N.NN N.NN N.NN N.NN N.NN Hm >Nz NN.N NN.N NN.N NN.N NN.N NN.N NN.N NN.N NN.N NN.N NN.N HNN mHHOO uNHx NOOHN NON N.NH N.HH N.HH H.HH N.NH N.N N.NH N.NH N.NH N.NH N.NH HNNN :HNOHNOsO: N.NN N.NN N.NN N.HN N.NN N.NN N.NN N.NN N.NN N.NN N.NN NHO> >NN < NH NH NH |N« N N N N N N H11. NOHNN :OHNNNHENONON HNN mxmn -N:< , \ H> acoENNomxm Now mumw Hmswcm Hmzwfl>wncH .o-< vague 97 NN NN NN NN NN NN NN NN NN NN N :OHNNNNNNN GMHHQWmcmHF NNN HNN NNN NHN NHN NNN NNN NNN NNN NNN HNNN: NNHN NN NN NN NNH NHH NNH NNN-NNN NHN-NHNHNN NNN NNN-NNH HNNN: :ONH ENNON N.NN N N N.NN N.NN N.HN N N N N N HNN nNHN HHH ONNN H.NNH N.NNH N.NNN N.NNN N.NHN N.NNN N.NNN N.NN N.NH N.NN N.NN HNN nNHx HH ONNN N.NNNHN.NNN N.NNN N.NNN N.NNNN;NNNHH.NNNHN.NNNHN.NNHHN.NNHHN.NNNH HNN . nNHN H ONNN N.NNNHN.NNN N.NNNH.NNNHN.NNHHN.NNNHN.NNNHN.NNNHN.NNNHN.NNNHN.NNNH HNN HNNON . NoNx “mmuxuoHsuNuom N.N N.N N.N N.N N.N N.N N.N N.N N.N H.N N.N HNNN NHON -oum NENNNN N.NH N.N N.N N.N H.N N.N H.N N.N N.N N.N N.NH H: NONNO aoxfimH HmHOH N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN H.NN N.NN HNNN NNNz N.NN N.NN N.NN N.HN N.NN N.HN N.NN N.NN N.HN N.NN N.NN Hm >Nz NN.N NN.N NN.N NN.N NN.N NN.N NN.N NN.N NN.N NN.N NN.N HNN NHHOO NNHN NOOHN NON N.HH N.NH N.NH N.NH N.NH N.NH N.HH N.NH N.HH N.NH N.NH HNNN NHNOHNOEO: N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN NHO> >NN N NH NH NH IN. N N mi! N N N, er NNHNN :OHNNNHENONON HNE . mxmn -N:< \ N NNONNHNNOON N-< OHNNN 98 HN HN NH NH NN NN NN HN HN NN NN N :OHNNNNONN thhmmmcmhh HHN NNN NNN NNN NNN NNN NNN NNH HNH HNN NNN HNNNN NNHN NN NN NN NN HHH NNNNNN-NHNNNH-NNHNNH-NNHHNH NNN-NNH NNNN: :ONH NNNON N.NN N.NN H.NH N.NN H.NN N.HN N.NN N N N N HNN flNHx HHH ONNN N.NNN N.NNN N.NNN N.NNN N.NNN H.NNH N.HN N.NN N.NN N N HNN . nNHN HH ONNN N.NNNHN.NNNHN.NNN H.HNN N.NNN N.NNN N.NNN NrNNN N.NNN N.NNN N.NNN HNN : NNHN H ONNN H.NHNHN.NNNHN.NNNHN.NNN H.NNN N.NNN N.NNN N.HNN N.NNN N.NNN N.NNN HNN HNNON . NOHx “mouzuoNsuNuwm N.N N.N N.N N.N N.N N.N H.N N.N N.N N.N N.N HNNN :NON -ONQ «ENNNN N.N N.N N.NH N.NH N.NH N.N N.N N.N N.N N.N N.NH H: NONNO noxnmfi HQHOH N.HN N.HN N.NN N.HN N.NN N.NN N.NN N.NN N.NN N.NN N.NN HNNN NNN: N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.HN N.HN N.NN N.HN Hm >N2 NN.N NN.N NN.N NN.N HN.N NH.N NN.N NN.N NN.N NN.N NN.N HNN NHHOO NNHN NOOHN NON N.N N.N N.N N.N N.N N.N N.N N.NH N.NH N.NH N.NH HNNN :HNOHNOEO: N.NN N.NN N.NN N.NN N.HN N.NN N.NN N.NN N.NN N.NN N.HN NHO> >NN N LNH NH NH aNW N17 N N N N N NA: NNHNN :OHNNNHENONON HNN NNN: -N:< ‘8'." n \ .. NNONNHNNOON N-< OHNNN 99 um am Nm Nm an on ms 55 cm on w cowuwhsuwm :mhhommamhh NNN NNN NNN NNN NNH HNN NNH NNH NNN NNN HNNN: NNHN NN NN NN NN 9NNNN-NNHNNN-NNHNNH-NNHNNH-NNHNNH NNN-NN NNNN: :ONH ENNON H.NN N.NN N.NN N.HN N.NNH N.NN H.NH N.NH N N N HNN MNHx HHH ONNN N.NNN N.NNH N.NNN N.NNH N.NNN N.NNH N.NN N.NNH H.NNH N N HNN NNHx HH ONNN N.NNN N.NHN N.NNN H.HNN N.NNN N.NNN N.NNN N:NNN N.NNN N.NHN N.HNN HNN _ mNHN H ONNN N.NNNHN.NNHHN.NNHHN.NNN N.NNN N.NNN N.NHN N.NNN N.NNN N.NHN N.HNN HNN HNNON . Noax "mouzuoNsuNuom N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N HNNN NHNN -oun «ENNHN N.N N.NH N.N N.HH H.N N.N N.N N.NH N.N N.NH H: NONNO noxsmH HMHOH N.NN N.NN N.HN N.NN N.NN N.NN N.NN N.NN H.NN N.NN HNNN NNN: N.NN N.NN N.HN N.NN N.NN N.NN N.HN N.HN N.NN N.HN Hm >Nz NN.N HN.N NN.N HN.N HH.N NN.N NN.N NN.N NN.N NN.N HNN mHHOO oNHx NOOHN NON N.HH N.HH N.N N.HH N.N N.HH N.NH N.NH N.HH N.NH HNNN :HNOHNOEO: N.NN N.NN N.NN N.NN N.NN N.NN N.HN N.NN N.NN N.NN N.NN NHo> >NN N NH NH NH IN NI, N N N 1N N H NNNNN NOHNNNHENONON HNN mxma ., _ -N:< \ N NNONNHNNOON N-N OHNNN 100 NN NN NN NN NN NN NN N :OHNNNNNNN :Nhhommcmpe NNN NNN NNN NNN NNH NNN NNN HNNNN NNHN NN NN NNN NNN NHN NNNNHN.NNHNNN.NNNNNN.NNHN\N1 NONH ENNON N N H.HNN H.NNN N.NNN N.NNN N.NNN N H.NH N HNN nNHN HHH ONNN N.NNN N.NNN H.NNN N.HNN N.NNN N.NNN H.NNN N.HNN N.NNN N.NH HNN aNHN HH ONNN N.HNNNN .NNNHHNHNH N.NNN N.NNHHNNNNHNNNNNHN.NNNHN.NNNH N.NNN HNN . nNHN H ONNN N.NNNNN.NNNNN.HNNNN.NNNHN.NNNNN.NNNNN.NNHNN.NNNHN.NNHNN.NNN HNN HNNON . Noax “mmuxuoNsuNuom N.N N.N N.N N.N N.N N.N N.N N.N H.N N.N HNNN NHON -ona NENNNN N.NN N.NN N.NN N.NH N.NH N.NH N.NN H.NN H.NH N.HH H: NONNO nOMSmH Han—OH N.NN N.NN N.NN N.HN N.HN N.NN N.HN N.HN N.HN N.HN HNNN NNN: N.NN N.NN N.NN N.NN N.NN N.NN N.HN N.NN N.NN N.NN Hm >N: NN.NH NN.HH NN.N NN.N NN.N NN.N NN.N NN.N NN.N NN.N HNN NHHOO NNHx NOOHN NON N.NH N.NH N.NH N.NH N.NH N.NH N.NH N.NH H.NH N.NH HNNN :HNOHNOEO: N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.HN NHO> >NN N NH NH NH ml, >1, N N N N N Nzr. NNNNN NOHNNNHENONON HNN mxma . . -N:< N HN> acmENNomxm Now mama Nmewcm Nm=©N>chH .N-< oNnNN 101 NH NN NN mN NN NH «N on H :oHngsumm :wHHonGMHH NNH HNN NNN NNN NNN NNN NNN NNN Hu\un umHe 3 3H NNN HHN NNH NNNHN mN~-mNN~NN-NN HEN; co; 23% N.NN N.Nmm N.NNN N.NHN N.NNN N.Nmm N.NN N N H:\ nNHx HHH onxp N.NNN N.NNNHH.NNN N.NNN N.NNN H.NNN N.NNN N.NHN N.NNH H:\ mNHx HH «NNN N.HNNH N.NNHHHH.NNN.N.NNNHN.NHNHH.NNNH~.NNNHN.NmmHNN.NHNH H;\ nNHx H ”NNN N.NNNN H.mmmNN.NHNNN.Nmm~m.mNNNN.mme~o.NNNHN.NmNHH.HNHH H:\ Hmpoe _ Non ammuxooasuwuom N.N N.N N.N H.N N.N N.N N.N N.N N.N Hc\m :Hop . -oum mammflm N.NN N.Nm N.NN N.NN m.NN N.NN N.NN N.NN N.NH H1 mmuxu -oxsmfl HmHOH N.NH N.NN N.NN N.NN N.NN N.NN N.Hm N.NN N.NN Hc\m uzuz N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN HH >02 NN.N NN.N HN.N NN.N NN.N HN.N NN.N NN.N NN.N H:\ mHHou mNHx wooHN com N.NH H.NH H.NH N.NH N.NH N.NH N.NH N.NH N.NH Hu\m aHnoner: N.NN N.HN N.NN N.NN N.NN N.NN N.Nm N.NN N.NN NHo> >um N NH ~H[, NH 1m sl[ 0 m H m N HA], muHca :oHuacHeHopoa Has aka: -fi:< \ ~ meschcouN N-< NHNNN 102 mm mm ow » :OMuNHSumm :whhmwmcmhb NHN NNN HNN HENN UNHN mm . NNH NNH NNN_NNH-NNHNNH-NNHNNH-NN HN\N: :oHH sshom N.NH N.HHN N.NHN N.NNN H.NNN N.NNN N.Nm N N H=\ nNHNH HHH NNNN N.NNH N.NNN N.HNN N.HNN H.NNN N.NNN N.NHN N.NNH N.NNH H;\ .. nNHNH HH NNNN N.NNNH N.NNN N.NNN H.NmmN.NHNHN.NNNHH.NNHHN.NNHHN.NNN H:\ .. nNHx H NNNN N.NNNH N.NNNHN.NNNHN.NNNHN.NNNNN.HNmHN.mNHNN.NNmHN.NNN HN\ Hmuoe . Non ”mouxuonoHuom N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N N.N Hc\m :Hou -oum «EmmHm N.NH N.NH N.NH N.NH N.NH N.NH H.NH N.HH N.NH H: moHNu uoxzmfl HNHOH N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN Hc\m 0:02 N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN Hm >02 NN.N HN.N NN.N NN.N NH.N NN.N NN.N NN.N NN.N H:\ mHHou mNHx NooHN NON N.HH N.NH N.NH N.NH N.NH H.NH N.NH N.HH N.NH HN\N :HnoHNoeo: N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.NN N.HN NHo> >uN 0 NH NH NH XINI' N N m N m N |HJ1 muHca coHchHeNonN Has mxmn -H:< '..- [‘0‘ . S N HcoaaHucouN N-< NHNNN 103 mm mH mH Nm mm mm Nm mm mm N cowumusumm :wnuommcmhb mmm mmm Hem mmm mmm HcN ch mmm mmm Hw\m: umHh NHH mm NN mm HmH NNN NNN meNN~-NoNHcN-NNmmmm-HHH Hv\m: :ONN Eshom o w.m~ ~.mH N.NNH N.NoH c.HHH N.HmH N.mHH N.No o m.mH H:\ NNHx HHH maze N.aoH o.mNN o.mNN N.Nmm m.oNN N.onm m.wo¢ N.mHH m.nmN o.on N.Hw H:\ . nNHx HH NNNN N.NQQHN.mONHo.mmNHH.HHo N.Nmo o.H~N m.mow o.~mmHiNhNHo.wNNHo.me H:\ MNHx H NNNN N.NmHNw.womHN.oNNHH.ommHN.mmHHo.moHHm.NNmHN.aHNHw.ommHo.mmmHo.mmm H:\ Hmuoe . . Noax “mouxuonuwuom o.m .N.w m.m m.w o.N m.N N.N N.N 3.x N.N N.N Hv\m :Hop -oua memaHL o.MH N.NH N.HH w.a N.N m.N N.N m.N c.mH m.N N.N H: mouxu -oxsoH Hmuoe N.Nm N.Nm N.Hm o.~m H.Nm H.Nm o.Hm m.~m N.Nm N.Hm N.Nm Hu\w 0:92 N.NN o.NN o.mN o.wN o.NN o.mN o.mN c.mN o.mN c.mN o.mN Hm >u2 mo.N 0N.o No.o mn.o om.m o~.o mm.o mo.N HN.N om.m NN.N H:\ mHHou Non vooHs com N.HH o.oH m.oH N.NH N.N H.m m.m H.NH N.HH o.NH m.m Hvxw :Nnoamoao: o.Nm o.Nm o.Nm o.om o.o~ N.NN N.NN o.om o.~m 0.0m o.mN NHo> >om 9 NH NH ca m s o m e m m HI, mafia: :oHamcwshouoo Has mxma -N:< I \ "\" al. \ s HnoscHucouN N-< o35 APPENDIX B EFFECTS OF INOSINE, HYPOXANTHINE AND ALLOPURINOL ON SERUM IRON AND URIC ACID CONCENTRATIONS 104 105 Table B-1. The effect of inosine injection on serum iron concentrations (pg/d1) in cats Hours Animal ‘U 1 —7 4 6 A 77 78 78 78 121 B 138 140 138 145 129 C 150 138 129 140 D 111 108 100 84 106 Table B-2. The effects of hypoxanthine injection on serum iron (ug/dl) and serum uric acid (mg/d1) con- centrations in dogs Hours 4___ Animal Determination 0 l 2 3 A iron 75.0 67.0 69.0 69.0 uric acid .4 2.3 .82 B iron 138.0 142.0 146.0 139.0 uric acid .5 .8 .5 C iron 51.0 34.0 56.0 74.0 uric acid .4 .8 D iron 151.0 158.0 128.0 135.0 uric acid .6 2.1 .6 .7 E iron 165.0 164.0 167.0 165.0 uric acid .5 .8 .5 107 Table B-3. The effects of hypoxanthine injection on serum iron (pg/d1) and serum uric acid (mg/d1) con- centrations in anesthetized dogs Hours Animal Determination 0 T5” 1’ 2 A iron 111.0 112.0 118.0 117.0 uric acid 1.0 2.3 2.7 1.7 B iron 151.0 145.0 142.0 149.0 uric acid 1.2 3.0 2.6 1.8 C iron 122.0 172.0 124.0 170.0 uric acid 1.0 3.1 2.8 1.5 108 N. m. c. N. o. N. o. m. wwom UNN: o.NwH o.~om o.NwH o.NNH o.NNH o.moa o.mHN o.omH :ONH m N. m. N. n. n. a. wwum UNA: o.HNH o.mmN o.omH o.omH c.~m o.moa :ONH < N w m m N H 4b, :oHumcmENouom Hmsflc< wNmn mmov cw mcoNumNucoocoo Aaw\wav wflum oHN: Espom mam fiHm\w:V :ONH sapom :o :oHuooncfl HocwhsaoHHm mo mpoomwo 0:9 .N-m manmb