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B‘Vvfig 4.wEEJF.§-.ifixh§rgfl W13 ‘-——\-_.._7 .h ._ _ ‘4 Mural? .- g * HUAB & $0"? = 800K BINDFRY INC. 'I‘1F‘ARY BY" er5 ‘E ‘ ‘c: BINDING BY ~ -r'w"'"t!fi1‘3 ABSTRACT IRON TOLERANCE IN THE YOUNG PIG By Roger W. Cook Sixteen 7- to 9-day—old pigs from 4 litters were used in an experiment to evaluate the pathologic changes following a large intra- peritoneal dosage of iron. The dams had been fed a 5% cod liver oil (CLO), corn, soybean meal ration during lactation and late pregnancy. Eight pigs were injected with iron dextran (750 mg Fe/kg body weight). The good clinical tolerance of such a high dosage of this compound, despite marked increases in serum iron levels, was attributed to con— tinued binding of iron to the dextran. Two pigs injected with ferrous sulfate (500 mg Fe/kg body weight) died within 4 hours, while the 6 control pigs were unaffected by the intraperitoneal injection of 0.92 sodium chloride solution. Iron injections produced two- to threefold elevations of serum calcium and inorganic phosphorus levels and increased serum magnesium levels. These increases persisted along with high serum iron levels in the iron dextran-treated group during the 2-day observation period. Two of the 8 pigs injected with iron dextran had bilateral macro— scopic skeletal myodegeneration when autopsied 2 days later. These Roger W. Cook lesions, without associated clinical abnormality, appeared to have been iron-induced although the possibility that they were due to the dams' CLO diet could not be completely eliminated. A significant finding was that red and white fiber types could be clearly identified by histochemical methods in pigs of this age. In the muscle lesions of the 2 affected pigs, the aerobic, red fibers, centrally located in muscle fasciculi, were initially involved as had previously been described in vitamin E—selenium—responsive myopathy in weanling pigs. IRON TOLERANCE IN THE YOUNG PIG By Roger W. Cook A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1974 Dedicated, with love, to my parents John Wallace and Mollie Anne Cook 11 ACKNOWLEDGEMENTS I am most grateful to Dr. Robert L. Michel, my major professor, for support during this period of study and advice in the preparation of this thesis. The counsel of Dr. C. Kenneth Whitehair and the generous assistance of Dr. Elwyn R. Miller on many occasions during this project are greatly appreciated. I am indebted to Mrs. Barbara A. Wheaton for her patient coopera- tion in the histochemical studies and to Dr. Pao K. Ku for his help with the atomic absorption spectrophotometry. My special thanks also go to Mrs. Elaine M. Dunlap for her technical assistance. To the New South Wales Department of Agriculture in Australia, I wish to express my appreciation for allowing this period of graduate study. My thanks also go to the faculty and to the staff of the Department of Pathology at Michigan State University for the training I have received during this period. Finally, I acknowledge the understanding, encouragement and many sacrifices of my wife, Ruth. iii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . l LITERATIJRE REVIEW 0 O 0 O O O O O O O O 0 O O O O O 0 O O O O O N Iron Metabolism " . . . . . . . . . . . . . . . . . . . General. . . . . . . . . . . . . . . . . Iron supplementation in swine. . . . . . . . . Iron supplementation in man. . . . . . . . . . . Acute Iron Toxicosis in Man and Experimental Animals. Acute iron toxicosis in man. . . . . . . . . . . Acute iron toxicosis in experimental animals . Toxicity and iron formulation. . . . . . . . . . Pathology. . . . . . . . . . . . . . . . . . . . Acute Iron Toxicosis with Myodegeneration . . . . . . Swine. . . . . . . . . . . . . . . . . . . . . Mice . . . . . . . . . . . . . . . . . . . . . . 12 Calcium Mobilization Associated with Iron Injection . . 13 Histochemistry of Skeletal Muscle . . . . . . . . . . . 14 General. . . . . . . . . . . . . . . . . . . . . l4 Swine. . . . . . . . . . . . . . . . . . . . . . 18 Myodegeneration in acute iron toxicosis of swine. . . . . . . . . . . . . . . . . . . 19 Calciphylaxis. . . . . . . . . . . . . . . . . . 19 Vitamin E—selenium—responsive myopathy in swine. . . . . . . . . . . . . . . . . . . l9 \DOQO‘U‘IUIUIWWNN MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . . 22 General Experimental Plan . . . . . . . . . . . . . . . 22 Histological Techniques . . . . . . . . . . . . . . . . 25 Analytical Procedures . . . . . . . . . . . . . . . . . 26 Hematology . . . . . . . . . . . . . . . . . . . 26 Serum collection . . . . . . . . . . . . . . . . 27 Serum aspartate aminotransferase (glutamic oxaloacetic transaminase, SGOT) . . . . . . . 27 Blood urea nitrogen (BUN). . . . . . . . . . . . 27 Serum electrolytes . . . . . . . . . . . . . . . 27 Statistical Analyses. . . . . . . . . . . . . . . . . . 28 RESUI‘TS O O O O O O C O O O O O O O O C O O O I O O O O O O O O 29 iv Page Clinical Signs. . . . . . . . . . . . . . . . . . . . . 29 Analytical Procedures . . . . . . . . . . . . . . . . . 29 Hematology . . . . . . . . . . . . . . 30 Blood urea nitrogen (BUN) , , , , , , , , 30 Serum aspartate aminotransferase (SCOT). . . . . 33 Serum electrolytes . . . . . . . . . . . . . . . 33 Pathology . . . . . . . . . . . . . . . . . . . . 37 Gross findings . . . . . . . . . . . . . . . . . 37 Histopathology . . . . . . . . . . . . . . . . . 42 Histochemistry . . . . . . . . . . . . . . . . . 45 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Serum Iron and Toxicosis. . . . . . . . . . . . . . . . 55 Blood Urea Nitrogen (BUN) . . . . . . . . . . . . 56 Serum Aspartate Aminotransferase (SCOT) . . . . . . . . 56 Serum Sodium and Potassium. . . . . . . . . . . . . . 57 Serum Calcium, Inorganic Phosphorus and Magnesium . . . 58 HistOChmis try 0 O O O O O O O O O O O O O O O O O O O O 60 SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . 64 VITA O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 73 LIST OF TABLES Table Page 1 Classification of mammalian skeletal muscle fibers [after Gauthier (1974) and Peter et al. (1972)] . . . . 16 2 Composition of diets. . . . . . . . . . . . . . . . . . 23 3 Experimental design . . . . . . . . . . . . . . . . . . 24 4 Packed cell volumes (2) . . . . . . . . . . . . . . . . 31 5 Blood urea nitrogen concentrations (mg/d1). . . . . . . 32 6 Serum aspartate aminotransferase (SGOT) activities (Sigma-Frankel units) . . . . . . . . . . . . . . . . . 34 7 Serum iron concentrations (pg/d1) . . . . . . . . . . . 35 8 Serum magnesium concentrations (mg/d1). . . . . . . . . 36 9 Serum calcium concentrations (mg/d1). . . . . . . . . . 38 10 Serum inorganic phosphorus concentrations (mg/d1) . . . 39 11 Serum sodium concentrations (mEq/l) . . . . . . . . . . 40 12 Serum potassium concentrations (mEq/l). . . . . . . . . 41 vi Figure LIST OF FIGURES Page Iron distribution in the young pig following iron dextran injection . . . . . . . . . . . . . . . 44 Serial frozen cross sections of normal areas of semitendinosus muscle of iron dextran-treated pig #34. Eight histochemical methods demonstrate the 3 general fiber types. The aerobic, red fibers in central clumps within muscle fasciculi are surrounded by anaerobic, white fibers with a zone of inter- mediate fibers interposed (x 120) . . . . . . . . . . . 47 Serial frozen cross sections of an affected area of semitendinosus muscle of iron dextran—treated pig #34. Fiber degeneration and increased cellularity involve the central areas of muscle fasciculi where red fibers are located. White fibers at the periphery of fasciculi remain intact (x 120). . . . . . . . . . . 50 Serial frozen cross sections of normal (A, C, E, G) and abnormal (B, D, F, H) areas of semitendinosus muscle of iron dextranrtreated pig #34 stained by 4 histochemical methods. The degenerative process does not involve the peripherally located white fibers preferentially stained by the last 3 methods (x 250). . 52 Serial frozen cross sections of normal (A, C, E, G) and abnormal (B, D, F, H) areas of semitendinosus muscle of iron dextran—treated pig #34. Degeneration clearly involves the centrally located red fibers stained by these 4 histochemical methods (x 250). . . . S4 vii INTRODUCTION Acute toxicosis with skeletal myodegeneration following oral or parenteral iron administration has been described in the young pig (Brag, 1958; Arpi and Tollerz, 1965). Field and experimental obser- vations indicated that pigs from sows fed diets high in unsaturated fats and low in vitamin E were more susceptible to this form of iron toxicosis (Lannek et aZ., 1962). Treatment of susceptible pigs with vitamin E or synthetic antioxidants prior to iron administration prevented the toxicosis (Tollerz and Lannek, 1964). Patterson et al. (1967b, 1969, 1971), demonstrating an increase in muscle tissue peroxides, suggested that the initial effect of iron on muscle was potentiation of membrane lipid peroxidation. Histochemical studies by Ruth and Van Vleet (1974) of vitamin E—selenium—responsive skeletal myodegeneration in weanling pigs revealed a preferential susceptibility of aerobic, red (type I) fibers. The present research investigated the effect of a single large intraperitoneal dosage of iron dextran on 7- to 9-day-old pigs from sows fed a vitamin E-selenium—deficient ration. Treatment was delayed until this age to permit histochemical differentiation of muscle fibers and identification of susceptibility of a particular fiber type to any iron-induced myopathy. LITERATURE REVIEW Iron Metabolism General Iron is present in trace amounts in the animal body. Most, 65%, is present in hemoglobin, 3 to 5% is in myoglobin, and a small amount is found in enzyme systems and plasma (Bothwell, 1972). The remaining 25- 30Z is in the storage forms, ferritin and hemosiderin, in liver, spleen, bone marrow and other tissues. Ferritin (20% iron) consists of a central core of ferric hydroxidephosphate [(FeOOH)8'(FeO:PO H2)] surrounded by apoferritin 3 (molecular weight approximately 400,000), a protein shell comprising 20 or 24 subunits that may be identical (Linder and Munro, 1973). Hemosiderin (35% iron) is less well defined chemically but is possibly an aggregated degradation product of ferritin. It is recognized by its variable protein content, which is lower than that of ferritin, and its insolubility, which is probably due to the fact that most or all of its protein is denatured (Trump et aZ., 1973). Ferritin is the predominant iron storage form under normal conditions, while the prOportion of iron stored as hemosiderin rises as iron storage increases (Bothwell and Finch, 1962). Transferrin (molecular weight approximately 90,000) is a plasma glycoprotein which binds most plasma iron. The normal level of plasma 2 3 iron is approximately 100 ug Fe/dl. This represents 33% of the total iron binding capacity (TIBC) of transferrin present. While most iron released from degradative processes is recycled within the body, small but significant amounts are lost from the gut, urinary tract and skin (Bothwell, 1972). Finely controlled iron absorption from the gut replaces these obligatory losses and maintains a relatively constant body iron level in the adult animal. The 2 most common causes of iron-deficiency anemia following depletion of body iron stores are chronic blood loss and inadequate dietary iron. The latter condition is a problem particularly in rapidly growing, milk-fed neonates. Iron supplementation in swine. The pig is born with approximately 40 mg of iron incorporated in hemoglobin (Hfigberg et aZ., 1968) and low body iron stores. The latter are soon depleted as the young pig grows quickly and its iron needs are not met by the sow's milk. Anemia develops in 2 to 3 weeks if the pig does not receive additional iron (Ullrey et aZ., 1960). As the newborn pig's iron stores cannot be increased by supplementation of the sow during pregnancy, prophy- lactic treatment of pigs at 2 to 3 days of age by intramuscular injection of 100 to 200 mg iron as iron dextran is.standard husbandry practice. Iron supplementation in man. Milkrfed human babies require iron supplementation to prevent anemia after a period of months.‘ This delayed onset of iron deficiency compared with that of the pig is due to the larger iron stores at birth and slower growth rate of the human baby. 4 Bothwell (1972) attributed the tropical belt of severe human iron deficiency to an interplay of several factors, including extended periods of breast feeding followed by a predominantly cereal diet which is maintained through childhood into adult life. Superimposed on this limited supply of dietary iron were the iron drain of menstru- ation and repeated pregnancies in females, the chronic blood loss of hookworm infestations, and limitation of dietary iron uptake due to gastrointestinal disease and, in some societies, clay eating. An optimal western diet provides a daily maximum of 3 to 4 mg of body iron, exceeding the daily requirements of 1 mg and 1.5 mg, respectively, in the adult male and menstruating female. However, in the second and third trimesters of pregnancy the daily requirement rises to 5 mg or more, which cannot be satisfied by the normal dietary intake (Bothwell, 1972). Prophylactic iron supplementation during human pregnancy is a routine practice to avoid depletion of body iron stores and possible iron deficiency in the pregnant female. Oral ferrous sulfate is the most common form of iron supplementa— tion in man, although other ferrous salts prescribed include the carbonate, chloride, fumarate, gluconate, lactate and glutamate (Gleason et al., 1969). Ferric salts such as ferric ammonium citrate and ferric hydroxide, and chelated compounds including iron choline citrate, have also been used. Parenteral therapy with iron dextran is reserved for the small number of human patients for which oral therapy is unsuitable (Bothwell, 1972). 5 Acute Iron Toxicosis in Man and Experimental Animals Iron toxicosis is usually caused by iron compounds used for the iron supplementation commonly practised in man and swine. Acute iron toxicosis in man. Acute iron toxicosis has been well documented in man (Duffy and Diehl, 1952; Brown and Gray, 1955; Hoppe et al., 1955; Amerman et al., 1958; Wallerstein and Mettier, 1958; Covey, 1964; Gleason et al., 1969). Usually involving children that have ingested an overdosage of iron tablets, the number of reported cases has steadily grown and a 50% mortality rate has been estimated (Aldrich, 1958; Cann and Verhulst, 1960). Reissmann et al. (1955) reported that 3 to 10 grams of ferrous sulfate were usually fatal in young children. Aldrich (1958) characterized several distinct phases of iron poisoning. Epigastric pain and vomiting within 10 to 60 minutes were followed during the next 6 to 8 hours by shock, coma and death (Lindquist, 1949; Smith et aZ., 1950; Swift et aZ., 1952; Charney, 1961). Survival of this phase resulted in recovery or relapse within 12 hours into fata1,secondary shock (Prain, 1949; Aldrich, 1958). Autopsy findings usually included periportal hepatic necrosis and hemorrhage (Large, 1961; Luongo and Bjornson, 1954), and local effects on gastric mucosa which have caused pyloric stenosis in survivors (Brown and Gray, 1955). Acute iron toxicosis in experimental animals. The species, the degree of ionization of iron within the preparation, and the route of administration affect the toxic iron dosage. 6 Reissmann et al. (1955), using dissociable iron salts, found that no dog or rabbit survived a dosage of more than 200 mg Fe/kg body weight given orally or as an enema. Somers (1947) determined the median lethal oral toxic dosage (LDSO) of a number of iron salts to be 300, 600 and 900 mg Fe/kg body weight in guinea pigs, rabbits and mice, respectively. Brown and Gray (1955) found that for rabbits 9.3 mg Fe/kg body weight was the minimum lethal intravenous dosage of ferrous sulfate. The corresponding dosage in guinea pigs of 6.1 mg Fe/kg had been determined by Edge and Somers (1948). Patterson et al. (1971) pro- duced death in rabbits within hours of intraperitoneal administration of 47 mg Fe/kg body weight using ferric ammonium citrate with dextrose. Campbell (1961) produced iron toxicosis in 17 pigs ranging from 2 to 10 days of age using oral ferrous sulfate at a dosage of 600 mg Fe/kg body weight. Vomiting within 30 minutes was followed by shivering, hyperpnea and incoordination within an hour. After several hours most developed diarrhea and tetanic convulsions, generally dying at 6 hours, although one survived for 40 hours. Gross lesions were not mentioned and histological examination of only the gastric mucosa was detailed. Changes were slight in those dying acutely and, in those surviving 24 hours, consisted of edema of the gastric wall with mucosal necrosis and inflammatory cell infiltration. Changes in pigs dying most acutely were insufficient to account for death. Toxicity and iron formulation. The toxicity of parenteral iron prepa- rations was reduced by decreasing the amount of dissociable iron they contained. Earlier modifications included colloidal ferric hydroxide, 7 saccharated iron oxide and EDTArchelated iron compounds. Martin et a1. (1955), testing a series of compounds, obtained the best results with a combination of iron with a low molecular weight dextran as a macromolecule which appeared to act as a protective lyophilic colloid. The LD after intravenous injection of mice with the iron 50 dextran and saccharated iron oxide was 1013 mg Fe/kg and 231 mg Fe/kg body weight, respectively. The same compounds were administered intravenously to rabbits. Iron dextran at 500 mg Fe/kg and saccharated iron oxide at 150 mg Fe/kg caused death at 9 days and within 24 hours, respectively. Iron dextran at 150 mg Fe/kg was not lethal. Rabbits survived iron dextran at 690 mg Fe/kg intramuscularly and mice tolerated 450 mg Fe/kg (it was not practicable to give a higher intramuscular dose of iron dextran to mice). Iron dextran was very stable and disappeared slowly from the circulation after intravenous injection. Peak serum levels of more than 1,000,000 ug Fe/dl following an intravenous rabbit dosage of 500 mg Fe/kg corresponded to the theoretical value based on uniform distribution of the dose throughout the plasma. These levels were maintained for a day and then began to fall slowly. Iron dextran complexes proved useful for intramuscular treatment of iron deficiency pig anemia (Martin et al., 1955; Barber et aZ., 1955; Brownlie, 1955). Preparations containing iron dextrin complex (Brag, 1957), hydrogenated iron dextran (Herin, 1962), dextrin—ferric oxide complex (Linkenheimer et al., 1960), polysaccharide-iron complex (Zuschek et al., 1960), a complex of iron, dextrin, sorbitol, citric and lactic 8 acids (Hngerg et al., 1968) and a complex of iron and a sorbitol- gluconic acid polymer (Carlsson at aZ., 1974) have also been reported as useful in treating or preventing anemia in young pigs. Pairs of 3-day-old pigs tolerated the last preparation at 150, 300, 450 and 600 mg Fe/kg body weight intramuscularly. Pathology. The mechanism of iron toxicosis is not understood in man, nor in a number of species that have been the subjects of experimentation. Working with rabbits and dogs, Reissmann and Coleman (1955) and Reissmann et a1. (1955) established that a rapid absorption of toxic doses of iron from the small and large intestine was followed by profound metabolic acidosis. This suggested that the cause of death was more than the local necrotizing effect of iron on the gut, result- ing in shock from hemorrhage and fluid loss, as had previously been thought (Forbes, 1947; Smith et al, 1950). Consistent lesions severe enough to account for acute deaths have only been seen in the rabbit and the vitamin E-deficient pig and mouse. Heavy iron accumulation in the liver particularly involves the Kupffer cells in all species. In the rabbit marked hepatic parenchymal iron accumulation and necrosis have been described (Luongo and Bjornson, 1954; Brown and Gray, 1955) with death attributed to hepatic failure (Patterson et al., 1971). Witzleben (1966) studied ultrastructural changes in rabbit liver following intravenous administration of ferrous sulfate at approximately 10 mg Fe/kg body weight. Within 2 hours parenchymal cell mitochondria appeared damaged. At 4 hours damage was extensive 9 with flocculent densities present between mitochondrial cristae. By 8 hours widespread hepatic necrosis was maximal and readily evident by light microscopy. Cells with minimal mitochondrial injury had increased endoplasmic reticulum. In a review of iron toxicosis, Trump et a1. (1973) stated that it was evident from the observed toxic effects of iron that there was interaction of iron with membranes of the mitochondria and cell surface. They noted that membrane lipid peroxidation was a possible means of expression of iron toxicity. Acute Iron Toxicosis with Myodegeneration Swing. Brag (1957, 1958) described acute iron toxicosis in 2-week- old pigs reared on concrete and exposed to soil sprayed with ferrous sulfate. Within hours they developed pallor, ataxia, dyspnea and convulsions with front limb paddling. Whole litters often died within 24 hours of treatment. Autopsy findings included carcass pallor, waxy degeneration of skeletal and cardiac muscles, and gastroenteritis occasionally accompanied by mucosal ulceration. Arpi and Tollerz (1965) cited observations by Lannek and Tollerz (1962), Tollerz (1962), Henriksson (1962), Ludvigsen (1962) and Guarda (1963) of waxy degeneration of skeletal muscles in young pigs dying within a few hours or days of oral or parenteral iron administration. Hydropericardium was commonly seen. Nilsson (1960) examined 10 young pigs that died within 12 hours of intramuscular injection with the normal 1 to 2 m1 dose of iron dextrin. He observed focal hydropic myocardial degeneration with glycogen 10 accumulation, hydropericardium and hydrothorax, without waxy degenera- tion of muscle. Lannek et a1. (1962) induced hypersensitivity to normal intra- muscular iron dosages of 150 mg Fe, by feeding the sows a vitamin E- deficient diet during the final 6 to 27 days of gestation and during lactation. The diet consisted of 76.3% ground grain (equal parts of oats and barley) and 4.6% cottonseed oil mixed and heated to 100 C for 30 hours under a continuous flow of air, before adding 16.8% skim milk powder and 2.3% minerals (CaHPO4 and Na HPO 2 4’ pigs died following iron treatment at 10 to 27 days of age. Mean 3:1). Nine of 17 survival time was 2.5 days. Autopsy findings included hydropericardium, hydrothorax, and extensive waxy degeneration of skeletal but not cardiac muscles. Plasma aspartate aminotransferase (GOT) levels were markedly elevated following iron treatment, returning to normal within a week in survivors. Arpi and Tollerz (1965) reported autopsy findings in 78 pigs aged 3 to 13 days and mainly from sows fed experimental diets similar to Lannek's. Intramuscular iron dextran and iron dextrin and oral ferrous fumarate and sulfatewer-e given to 62, 3, 6 and 7 pigs, respectively. Dosages ranged from 80 to 1500 mg iron with most receiving 600 to 1000 mg. During the 5 days following treatment, there were respectively 59, 11, 3, 3 and 2 deaths. They did not observe the transudation into body cavities described in Lannek's experimental work and earlier field reports. The main lesion was skeletal myodegeneration which was visible macroscopically only in pigs surviving 3 days or longer. A11 pigs receiving oral ferrous sulfate had catarrhal to necrotizing gastroenteritis. The absence 11 of muscle lesions in 4 of these pigs suggested another mechanism, possibly similar to that causing the conventional form of iron toxicosis (Campbell, 1961). Tollerz and Lannek (1964) protected 3- to 8-day-old pigs by treatment with vitamin E (dl-a-tocopherol acetate) 1 day or more before intramuscular iron administration of 40 to 375 mg Fe/kg in the form of an organic iron compound containing 75 mg Fe/ml. This experiment involved 90 pigs from 11 litters whose dams had been fed the vitamin E-deficient ration during late pregnancy. The synthetic antioxidants NN'—diphenyl—p-phenylenediamine (DPPD) and 1,2—dihydro- 6-ethoxyh2,2,4etrimethquuinoline (EQ) were protective when given at the same time as iron injections. Selenium as sodium selenite was not protective when similarly given, although it did increase the iron tolerance of mice (Arpi and Tollerz, 1965). Using 2-day-old pigs from sows fed Lannek's diet during the last month of pregnancy, Patterson et al. (1967a, 1969, 1971) produced acute coagulative necrosis of skeletal muscle and death within 5 hours of intraperitoneal injection of a commercial or an experimental iron dextrose preparation at a dosage of 47 mg Fe/kg body weight. The severe muscle lesions were not visible grossly and were associated with marked elevations of plasma aspartate aminotransferase levels. Hyperglycemia and hyperkalemia were also observed, the latter reaching lethal levels of 11 mEq/liter or greater. Complete eversion of the T wave in the electrocardiogram prior to cardiac arrest suggested that hyperkalemia was the cause. 12 Patterson et al. (1967b, 1969) provided initial evidence of iron-induced muscle lipid peroxidation using the indirect method of estimating malondialdehyde content of muscle with the thiobarbituric acid (TBA) reaction. These observations were later substantiated with improved sampling and microiodometric estimation of muscle lipid peroxides (Patterson et al., 1971). With a similar sow diet and using an experimental iron formula- tion of 20% ferric ammonium citrate and 9% dextrose, Patterson et a1. (1971) were unable to produce toxicosis in 8-day-old pigs with an intraperitoneal dosage of 47 mg Fe/kg body weight. Nine of 16 2-day-old pigs similarly treated were affected with diarrhea and vomiting, and 2 died. They observed, particularly in 8-day-old pigs (all clinically unaffected), 2- to 3-fold elevations of plasma calcium, inorganic phosphorus, and less marked elevations of magnesium levels. Hyperphosphatemia occurred in a few affected 2-day—old pigs, but in unaffected pigs both calcium and phosphorus levels were raised. Patterson proposed a protective function of the hypercalcemia against iron toxicosis in the older pigs, citing Wills' (1969) in vitro studies of calcium protection against tissue peroxidation. Mice. Lannek et a1. (1962) and Tollerz and Lannek (1964) demonstrated an intraperitoneal LD of iron dextran to be 810 mg Fe and 1000 mg 50 Fe, respectively, for mice fed for 3 weeks a vitamin E-deficient ration similar to that of the sows. Tollerz and Lannek (1964) decreased the LD to 100 mg Fe by feeding the ration for 6 weeks. 50 On normal rations the LD50 was 1800 mg Fe. This was raised to >4000 13 mg Fe for mice on the deficient ration, by a-tocopherol acetate injections twice weekly for 3 weeks prior to iron treatment. Arpi and Tollerz (1965) described iron toxicosis with skeletal muscle lesions in 8 mice fed a similar ration for an unspecified period prior to iron dextran treatment. Four mice also had slight acute waxy myocardial degeneration. Calcium Mobilization Associated with Iron Injection Calciphylaxis is an experimental condition of induced systemic laypersensitivity in which tissues respond to appropriate challenging eagents with calcification (Selye, 1962). The components of this 1>henomenon were described by Gabbiani and Selye (1963) as a systemic (:alcifying agent or sensitizer [parathormone, vitamin D compounds and ciihydrotachysterol (DHT)] and an inorganic or organic challenger (iron or other metal salts, and serotonin). Rats sensitized with IIHT'subcutaneously and challenged with subcutaneous serotonin, snibcutaneous iron dextran or egg albumen, or intravenous iron denotran with mastocyte discharger (polymyxin) developed myositis, massive cutaneous calcification or dermatomyositis with calcification, respectively (Selye et al., 1962, 1961; Selye, 1962). Canal at al. (1969) confirmed Selye's observation that the myositis induced by serotonin was not characterized by calcium deposition microscopically. Calciphylaxis was the suspected cause of death in 10 and illness in 38 pigs of a group of 150 which had been given iron galactan and vitamin D3 at 3 days of age. One hundred twenty milligrams of iron and 125,000 IU of vitamin D were administered intramuscularly in 3 separate legs. Effects were observed over a period of 3 weeks 14 following treatment (Ablett et al., 1969). The vitamin D3 dosage greatly exceeded that of up to 2000 IU normally given to pigs of this age. Within 48 hours the owner noticed swelling at the site of iron injection, and calcification was observed histologically. Two of the sick pigs killed at 6 weeks had slight calcification at the injection sites and in the kidneys, aortas and muscular arteries. Penn (1970) observed localized calcification judged macro- scopically at the sites of injection with one of several iron preparations, including iron galactan, in rats previously or simul— taneously injected with large dosages of vitamin D3. This seemed to support the view of Ablett et a1. (1969) that the pigs had died after developing the so-called calciphylactic syndrome. Marked mobilization in young pigs of calcium and inorganic phosphorus, presumably from bone following intraperitoneal ferric ammonium citrate injection was not associated with vitamin D3 treat- ment (Patterson and Allen, 1970; Patterson et al., 1971). The fact that it occurred in all 8-day—old pigs but in only a few 2—day-olds suggested that this effect may have been age-dependent. Histochemistry of Skeletal Muscle General. Ranvier (1873) first observed that fibers of slowly con- tracting, fatigue-resistant, red skeletal muscle were smaller and Imore granular than those of rapidly contracting, white muscle. Early ‘workers (Needham, 1926; Denny—Brown, 1929) differentiated red fibers by their granular lipid staining which identified dark triglyceride 15 droplets and lighter staining phospholipid components of the numerous mitochondria (Gauthier and Padykula, 1966). Most skeletal muscles are heterogeneous, containing a mixture of at least 3 fiber types that have recently been identified by ultra- structural, histochemical and electrophysiological methods (Table l). The various systems of fiber classification are not completely compatible (Yellin and Guth, 1970) and none is generally accepted. The red fibers are equipped for aerobic metabolism with larger and more mito- chondria, more lipid, oxidative enzymes and capillaries than the larger, anaerobic, white fibers which, with higher phosphorylase and actomyosin adenosine triphosphatase (ATPase) activity, also contain more glycogen. The so—called intermediate fibers share properties with both red and white fibers. Edstrdm and Kugelberg (1968) and Burke et al. (1971) directly measured several physiological parameters of histochemically uniform fiber populations by stimulating individual motor units in the rat and cat, respectively. The 3 groups of physiological responses (S, FR and FF) corresponded with each of the43 basic histochemical profiles of motor units (see Table 1). This confirmed the correlation of histochemical characteristics with function of individual fibers. The aerobic, slowly contracting, fatigue-resistant, red fibers were adapted for sustained activity while the more rapidly contracting, easily fatigued, white fibers provided short term, intense contractile power from anaerobic metabolism. In most species, fibers of newborn animals are histochemically and functionally red, many transforming to white type after birth 16 Table 1. Classification of mammalian skeletal muscle fibers [after Gauthier (1974) and Peter et a1. (1972)] Reference Basis of identification Terminology Structure RED WHITE Ranvier (1873) Color of muscle red white Fiber morphology small dark large light granular clear INTER- RED MEDIATE WHITE Padykula and Mitochondrial content high inter- low Gauthier (1967) mediate Gauthier Z line width wide inter- narrow (1969) mediate Histochemistry I II Dubowitz and Ratio of phosphorylase low high Pearse to succinic dehydrogenase, (1960a,b) cytochrome oxidase Engel (1962) Actomyosin (myosin, myo- low high fibrillar) ATPase C B A Stein and Succinic dehydrogenase high inter- low Padykula mediate (1962) II(DE) III(FGH) I(ABC) Romanul (1964) Phosphorylase inter— low high mediate Yellin and Actomyosin ATPase pH 8 a8 a Guth (1970) lability (species variation) Brooke and Same I IIA IIB Kaiser (1970) Ashmore and Same BRed aRed aWhite Doerr (1971a,b) (BR) (GR) (0W) 17 Table 1 (continued) Reference Basis of identification Terminology Physiology» Barnard et a1. Physiological characteristics slow fast fast (1971) of whole muscle twitch twitch twitch inter- red white mediate (F-TR) (F-TW) (S-TI) Peter et al. Physiological characteristics 810W fast fast (1972) of whole muscle twitch twitch twitch oxida- oxida- glyco- tive tive lytic (SO) glyco- (FG) lytic (FOG) Burke et a1. Physiological characteristics slow fast fast (1971) of individual motor units con- con— con- tract- tract- tract- ing ing ing fatigue fatigue fast resis- resis- fatigue tant tant (FF) (S) (FR) 18 (Ashmore et al., 1972). Qualitative changes in histochemical profiles of skeletal muscle have also been produced by altered innervation (Guth et al., 1970) and exercise (Edgerton et al., 1969). Guth and Yellin (1971) therefore proposed that muscle cells could undergo continual alteration throughout life in adaptation to changing func- tional demands and that the histochemical fiber types merely reflected the fiber's constitution at a particular time. The fiber classification system of Ashmore at al. (1971a,b) par— tially reconciled Guth and Yellin's view with that of true fiber‘ heterogeneity. They identified a and B fibers on the basis of acto- myosin ATPase activity (Guth and Samaha, 1969, 1970), noting that B fibers were always red (BR) while a fibers could transform from red (oR) to white (oW) as judged by other histochemical methods. Ashmore et al. (1973) presented evidence that a fibers corresponded to the secondary myotubes which formed around the groups of primary myotubes (8 fibers) during fetal development. §gigg, There is a unique distribution of histochemical fiber types in mature porcine skeletal muscle. Each fasciculus, surrounded by perimysium, contains one or more central aggregates of red fibers surrounded by white fibers (Davies, 1972; Van Den Hende et aZ., 1972) with a zone of fibers of overlapping (intermediate) staining reac— tions interposed (Moody and Cassens, 1968). This is consistent with maintenance throughout life in the pig of the fetal fiber orientation with central groups of B fibers (always red, BR) surrounded by a fibers, which are red (aR) at birth but gradually transform from the Periphery to form white (aW) during postnatal development (Ashmore 19 et al., 1972). This transformation, with increase in fiber size and loss of oxidative enzyme activity, was obvious at 13 days of age (Cassens et al., 1968) or earlier (Van Den Hende et al., 1972). Myodegeneration in acute iron toxicosis of swine. Patterson (1969) reported clumping and partial disruption of centers, pre- sumably mitochondria, of succinic dehydrogenase (SDH) activity, and loss of phosphorylase activity in affected skeletal muscle fibers of 2-day—old pigs following experimental iron toxicosis. Although histochemical fiber differentiation would not be marked at this age, in a figure illustrating the SDH clumping in their study, the fiber involved was at the center of a muscle fasciculus. Calciphylaxis. Canal at al. (1969) observed in calciphylactic myopathy of rats, condensation and margination of oxidative enzyme activity (succinic dehydrogenase and reduced nicotinamide adenine dinucleotide-tetrazolium reductase) in the red fibers, suggesting mitochondrial damage. They attributed a decrease in muscle glycogen and sarcoplasmic phosphorylase activity to extramitochondrial damage, with preliminary electron microscopic studies indicating more marked structural change in the sarcoplasmic reticulum than in mitochondria. Vitamin E-selenium—responsive myopathy in swine. Ruth and Van Vleet (1974) found. that aerobic, red skeletal muscle fibers were preferen- tially affected in weanling pigs fed a vitamin E-selenium-deficient ration. Mitochondrial membranes rich in unsaturated fatty acids were susceptible to peroxidative damage especially when isolated from vitamin E-deficient rats (Tappel and Zalkin, 1959), and the first morphologic 20 alterations of skeletal muscle were in the inner mitochondrial membranes of vitamin E-deficient rabbits (Van Vleet et aZ., 1968) and rats (Howes et al., 1964) and vitamin E-selenium-deficient chicks (Cheville, 1966). Ruth and Van Vleet (1974) considered that increased susceptibility of the aerobic,red muscle fibers due to their high mitochondrial content was consistent with an antioxidant func- tion of vitamin E and selenium. Sarcoplasmic phosphorylase activity of white fibers was also decreased in affected pigs. As histochemical phosphorylase activity depends on cellular glycogen levels (Martin and Engel, 1972), Ruth and Van Vleet (1974) suggested that decreased phosphorylase activity may have been associated with white fiber glycogen depletion follow- ing red fiber destruction. No evidence of glycogen depletion was presented. Rotruck et a1. (1972, 1973) proposed that vitamin E prevented fatty acid hydroperoxide formation mainly within membranes. 0n the other hand, the sulfur-containing amino acids, as precursors of glutathione, and selenium as an integral part of the enzyme gluta- thione peroxidase (4 gram atoms of selenium per mole of enzyme), were involved in endogenous peroxide destruction. The glutathione- dependent system also protected nonmembranous proteins (e.g., hemoglobin) from oxidative damage. Scott and Noguchi (1972) and Noguchi et a1. (1973) hypothesized similarly on the interaction of vitamin E and selenium in the pathogenesis of exudative diathesis and nutritional muscular dystrophy in chicks. Vitamin E deficiency also affects synthesis of heme compounds, which include peroxidase 21 enzymes, at the level of either 6 amino levulinic acid synthetase or hydratase activity (Murty et aZ., 1970). Iron salts (mainly ferrous) are among the compounds used for in vitro lipid peroxidation (Noguchi, 1973). It would be logical to expect that skeletal myodegeneration following iron-induced peroxidative membrane damage in pigs would also preferentially affect the aerobic, red fibers. MATERIALS AND METHODS General Experimental Plan Sixteen Yorkshire or Yorkshire-Hampshire crossbred pigs from 4 litters were used in this study. The Michigan State University gestation ration (Table 2), normally low in vitamin E and selenium, was modified (Table 2) to include 5% cod liver oil (CLO) during the final 4 or 6 weeks of gestation. Modification of the dams' diet was an attempt to increase the vitamin E and selenium requirements of the resulting litters and consequently their susceptibility to iron toxicosis. Litter sizes, ages and treatments are detailed in Table 3. Litters were earmarked and tail docked at 3 days but were not given their usual iron dextran injection. On the day of treatment (day 9 for Litters l and 2, day 7 for Litters 3 and 4), each pig was Iveighed and bled from the anterior vena cava with a sterilized 18-gauge needle and 20-m1 syringe. Pigs were separated from their dams 3 hours before each bleeding Procedure to avoid lipemia. A heparinized microhematocrit tube was filled for packed cell ‘Halume (PCV) and total protein (TP) estimations. The remainder of theblood was allowed to clot in acid washed test tubes for electro- lyte and enzyme determinations. 22 23 Table 2. Composition of diets Ingredient MSU gestation ration1 (%) Modified ration (%) 2 Cod liver oil 5.0 Ground shelled corn 85.0 80.0 Soybean meal (49% protein) 11.5 11.5 Ground limestone 1.0 1.0 Dicalcium phosphate 1.5 1.5 Salt 3 0.5 0.5 MSU VTM premix 0.5 0.5 Total 100.0 100.0 1Low vitamin E-selenium: average values of previous analyses - d—a—tocopherol, 5—10 IU/kg, Se, 0.05 ppm. 2Whitcod 600D-1500A, Whitmoyer Laboratories, Inc., Myerstown, Pennsylvania 17067. Each 0.46 kg contains 272,400 International Chick Units of vitamin D3 and 681,000 USP Units of vitamin A. 3Vitamin-Trace Mineral premix: vitamin A, 3.0 million IU; vitamin D2, 0.6 million IU; riboflavin, 3.0 gm; nicotinic acid, 16.0 gm; d-pantothenic acid, 12.0 gm; choline chloride, 100.0 gm; vitamin B12, 18.0 mg; zinc, 68.0 gm; manganese, 34.0 gm; iodine, 2.5 gm; copper, 9.0 gm; iron, 54.0 gm; antioxidant,4 45.0 gm; carrier (ground yellow corn) to bring to 4.6 kg. 4Butylated hydroxyanisole (BHA) and/or butylated hydroxytoluene 24 Table 3. Experimental design Dam Weeks pre— Treatment parturition Litter Pigs/Treatment on 5% Age 1 Ferrous 4 Litter CLO diet (days) Size Dextran sulfate Control #15 gilt 4 9 6/10 2 2 2 #25 sow 4 9 5/10 3 2 #3 gilt 6 7 4/4 2 2 #4 sow 6 7 1/16 1 1Live pigs at time of treatment/original litter size. 2ArmidextranR, Bradley Products Company, Omaha, Nebraska 68103, Division of Armour Pharmaceutical Company: each m1 contains 100 mg elemental iron as ferric hydroxide complexed with a low molecular weight dextran fraction. Dosage: 750 mg Fe/kg body weight i.e., 7.5 ml/kg intraperitoneally (I/P) 320% ferrous sulfate solution Dosage: 4 500 mg Fe/kg i.e., 12.5 m1/kg I/P 0.9% sodium chloride solution Dosage: 7.5 ml/kg I/P 5Pigs in Litters #1 and #2 received a daily oral supplement of 10 ml CLO from 4 days of age. Pigs in Litter #1 were also moved to another sow on the 5% CLO diet at this time as their dam had ulcerated teats and was not feeding them. 25 Following intraperitoneal injection of the various treatments, the pigs were observed for several hours and then returned to their dams. Additional blood samples were taken at 9, 21 and 45 hours from most pigs. Histological Techniques Surviving pigs were killed by exsanguination at 21 or 45 hours. All pigs were autopsied. All pigs born in Litter 4 and which died prior to the experiment were autopsied to detect the possible presence of nutritional myopathy. Multiple tissues were taken for histological examination and fixed in 10% buffered formalin and Zenker's fixative. These tissues were later retrimmed, paraffin embedded, sectioned and stained with hematoxylin and eosin and Prussian blue stains. Pro- cedures were those recommended by the Manual of'Histologic Staining Methods of’the Armed Forces Institute of'Pathology edited by Luna (1968). Immediately after exsanguination, samples of longissimus dorsi and any grossly affected skeletal muscle were rolled in talcum powder and immersed in isopentane (a-methyl-butane) for approximately 60 seconds. The isopentane was precooled to -140 to -l85 C by immersing the container in liquid nitrogen. The frozen muscle was stored in individual sealed containers at -80 C. Later 10 mm thick sandwich blocks were cut with a precooled microtome knife from the midsection of each muscle sample thawed to —20 C. These smaller blocks were immediately mounted on chucks with 5% gum tragacanth. Serial cross sections 10 pm thick were then prepared using a rotary microtome 26 * cryostat. The sections were mounted on glass coverslips and air dried. The following histochemical procedures were applied to the serial sections from each muscle sample: hematoxylin and eosin (H&E) (Luna, 1968); Gomori's trichrome (Engel and Cunningham, 1963); periodic acid-Schiff (PAS) (Lillie, 1954); PAS-diastase (Luna, 1968); o-glycerophosphate dehydrogenase (aGPD) (Engel and Brooke, 1966); actomyosin adenosine triphosphatase with acid or alkaline preincubation [ATPase (acid), ATPase (alk.)] (Guth and Samaha, 1970); oil red 0 (Preece, 1972); reduced nicotinamide adenine dinucleotide—tetrazolium reductase (-diaphorase) (NADH—TR) (Engel and Brooke, 1966); succinic dehydrogenase (SDH) (Barka and Anderson, 1963). Tissue morphology and staining reactions were evaluated by light microscopy. Analytical Procedures Hematology Packed cell volume¥(PCV). Hematocrit values were determined by centrifuging the heparinized microhematocrit tubes for 5 minutes in an International model MB microhematocrit centrifuge. Total p1asma_protein (TP). Total plasma protein concentrations ** wereaestimated with a total solids (TS) refractometer using plasma from the microhematocrit tube. * International-Harris Microtome-Crystat, Model CTI, Inter- national Equipment Company (IEC), Needham Heights, Massachusetts. ** American Optical TS meter. 27 Serum collection. Following coagulation in acid washed tubes, the blood samples were centrifuged for 15 minutes. The serum was then pipetted into acid-washed tubes and stored at -4 C until analyzed. Serum aspartate aminotransferase (glutamic oxaloacetic transaminase, * SGOT). The colorimetric Sigma Frankel procedure was used for this determination. Blood urea nitrogen (BUN). This parameter was measured in serum ** using the Beckman Blood-Urea—Nitrogen (BUN) Analyzer. Serum electrolytes. Serum iron, magnesium and calcium were determined by atomic absorption spectrophotometry using a model IL453 Atomic Absorption Emission Spectrophotometer*** and an acetylene flame. The method of Olson and Hamlin (1969) for iron determination in- volved protein precipitation with trichloroacetic acid and incubation for 15 minutes at 90 C before centrifugation and measurement of iron in the supernate. A wave length of 248.3 nm was used. Serums from pigs that had received iron injections were diluted 1:100 with deionized distilled water prior to the iron procedure. Calcium was determined on a trichloracetic acid supernate after dilution with a strontium chloride solution to provide a final * Sigma Technical Bulletin 505 (9-64), Sigma Chemical Company, St. Louis, Missouri 63118. ** Beckman Instruments, Inc., Fullerton, California 92634. *** Instrumentation Laboratory, Inc., 113 Hartwell Ave., Lexington, Massachusetts 02173. 28 strontium concentration of 10,000 ppm to overcome phosphate inter- ference. Calcium absorption was measured at 422.7 nm. Magnesium absorption was measured at 285.2 nm on serum diluted 1:100 with a 10,000 ppm strontium solution. Inorganic phosphorus was determined by the spectrophotometric method of Gomori (1942). Serum sodium and potassium determinations were made by flame emission spectrophotometry using the Beckman.KLiNa Flame Photometer.* This instrument uses propane fuel and a lithium standard solution as an internal reference. Statistical Analyses Mean values with standard errors of each of the blood parameters were determined for control and treatment groups at each sampling. Using the Student's t-test, the means of each group were compared with that group's pretreatment mean. Iron dextran and control group means were also compared at each sampling. * Beckman Instruments, Inc., Fullerton, California 92634. RESULTS Clinical Signs The control pigs,which received intraperitoneal saline injec- tions,appeared normal throughout the observation period. Pig #32 of this group died with massive hemothorax following the 9-hour bleeding while the remainder were killed at 21 or 45 hours. Pigs injected with iron dextran were initially lethargic, tend- ing to lie down during the several hours following injection. Within an hour their skins developed a brown color (Figure 1A) which persisted throughout the experiment. They suckled when returned to their dams and remained active and alert until killed. Pigs #11 and #17 in the iron dextran group had diarrhea at the 9-hour sampling. Their feces were gray and orange, respectively. Both had pelleted feces when autopsied at 21 and 45 hours, respectively. One of the pigs injected with ferrous sulfate vomited after an hour, when both were reluctant to stand. They became progressively more depressed and died 3 and 4 hours, respectively, after injection. Analytical Procedures Blood from the iron dextran-treated pigs was dark and clotted poorly. The serum and plasma were dark brown. Serum from the ferrous sulfate treated pigs had a distinctly brownish tinge. 29 Hematology Packed cell volume (PCV). Initial hematocrit values varied (Table 4) and were lowest in the heaviest animals (#15 and #17). Values for individual pigs were usually lower at subsequent samplings, although only the iron dextran group mean at 21 hours was signifi- cantly lower than the pretreatment mean. The only post-treatment value recorded in the ferrous sulfate-treated group was for pig #9, whose elevated terminal PCV value indicated hemoconcentration. Total plasma_protein (TP). Low pretreatment TP values in pigs from Litter l (#4, #6, #7, #8, #9, #10), ranging from 3.9 to 5.0 gm/dl, indicated failure of these pigs to obtain adequate colostrum. Pretreatment values for the other litters ranged from 5.6 to 6.7 gm/dl. The T? values of individuals within the control group were relatively unchanged at repeated samplings. Increases of approximately 1 to 2 gm/dl Observed following iron dextran treatment were sugges- tive of hemoconcentration; however, hemolysis and plasma iron dextran levels probably contributed to these increases, which were associated with blurring of the refractometer reading. Rises of 0.5 to 1.0 gm/dl were seen in plasma blanks with which iron dextran was mixed to give iron levels similar to those in this study. Blood urea nitrogen (BUN). There were small, significant increases in mean BUN levels of the iron dextran-treated group at the 3 post- treatment samplings (Table 5). Pig #12 within the control group had unexplained, markedly increased BUN levels at all samplings despite an absence of any sign of illness. 31 Table 4. Packed cell volumes (%) Pre- Weight treat— Time after treatment (hours) Treatment Pig (gm) ment 9 21 45 Control 4 1570 23 15 6 1990 32 29 27 12 1285 31 26 26 15 3780 15 l6 14 15 32 2050 31 26 33 2200 32 24 22 27:81 24:3 21:3 18:4 Dextran 7 2140 29 26 17 10 1830 29 21 11 2100 23 22 22 20 16 2360 24 27 23 26 17 2400 20 15 22 31 2120 34 25 23 23 34 1960 32 27 22 23 35 1240 29 19 19 26 27:2 23:2 zliiaaa 24:; Terminal sample Ferrous 8 1950 29 sulfate 9 1840 32 42(4 hours) 3012 42 1Meanistandard error. aaaSignificantly lower than Pretreatment value (P