EXPERIMENTAL AMMONIA TOXICOSIS IN THE HG That: for {In Dogma 3‘ D“. D. KICHIGAN STATE UNIVERSITY Edward J. Bicknell 1965 TREES This is to certify that the thesis entitled Experimental Ammonia Toxicosis In The Pig presented by Edward J. Bicknell has been accepted towards fulfillment of the requirements for Ph. D. degree in Patho logy Major professor meg/1% /7_/f ~ 0-169 A LIBRARY Michigan State University v—— A.r__-..oq. ._——0v 1“,“ . .. —-"mm..—__.,—v _Awmvv-,w. .A, ABSTRACT EXPERIMENTAL AMMONIA TOXICOSIS IN THE PIG by Edward J. Bicknell Three eXperiments were conducted to study acute ammonia toxicosis in the young pig and to evaluate the effects of nutritional, chemical, and surgical alteration of the liver on ammonia metabolism. In the first eXperiment, it was established that toxicosis caused by intravenous infusion of ammonium acetate, ammonium chloride, ammonium carbonate, and ammonium hydroxide depended on the rate of administra- tion per unit of body weight. The results indicated that the toxicity of these compounds was primarily due to the ammonium ion. Intraperi- toneal injection of ammonium acetate in dosages over 300 mg./lb. body weight was fatal. The signs of acute ammonia toxicosis were rapid respirations later becoming irregular and increased in depth, excessive salivation, clonic-tonic convulsions. and death. No significant gross or microscOpic lesions were found in acute ammonia toxicosis. In the second eXperiment. it was demonstrated that hepatic altera- tion by nutritional means does influence ammonia metabolism. Pigs fed a lowzprotein diet had higher blood ammonia values when intraperitoneally injected with ammonium acetate or with a protein hydrolysate, as com- pared to the control animals. The ammonia metabolism of pigs fed the lowaprotein diet and injected with carbon tetrachloride did not differ significantly from those fed Edward J. Bicknell only the low-protein diet. The amounts given apparently were not suf- ficient to cause significant hepatic changes. Clinical signs of icterus appeared within 12 hours in pigs with surgically produced biliary occlusion. Bilirubin levels generally increased up to the 5th day, then receded after this time. Following surgery, SGOT and SGPT values were increased, returning to slightly above normal levels in 48 hours. In A pigs the SGOT levels increased markedly 24 to #8 hours prior to death. These values correlated with histOpathologic findings of extensive necrosis. Pigs with biliany occlusion, when intraperitoneally injected with ammonium acetate, had blood ammonia values comparable to normal pigs. The outstanding gross lesions in pigs with biliary occlusion were enlarged, yellowishptan livers with distended bile ducts and ulcers in the asephageal portion of the stomach. These ulcers were apparently induced by the surgical ligation of the bile ducts. Hemorrhage from the ulcers caused the death of the pigs. Microscopic lesions in the liver included dilatation and proliferation of the bile ducts, necrosis, and fibrosis. Portal fibrosis and bile duct byperplasia were present in pigs with complete biliary occlusion 5 days after surgery. Interlobular fibrosis occurred after the 7th day. Necrosis did not appear to follow a chronological pattern in these pigs. Microscopically, the ulcers were characterized by necrosis, inflammation, and degenerative changes in the blood vessels of the submucosa. EXPERIMENTAL AMMONIA.TOXICOSIS IN THE PIG BY :1 Edward J-fBicknell A.THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1965 ACKNOJLEDGEMEN TS The author wishes to express appreciation to Dr. C. K. Whitehair for counsel and guidance in this research, and to Dr. S. D. Sleight for his critical reading of this manuscript. Sincere thanks to the Department of Pathology for providing the necessary facilities and to the technical staff, especially H. W. Kaczkofsky. R. A. Brooks, and J. A. Osburn, for their technical assistance. Sincere appreciation is extended to Dr. R. W. Luecke and Miss Betty Baltzer for their help in preparation of reagents and equipment for the blood ammonia determination. Thanks is offered to my guidance committee for their assistance and suggestions in the preparation of the thesis. Appreciation is expressed to the National Institutes of Health for the postdoctoral fellowship enabling the author to undertake and complete the research. Finally, to his wife Phyllis, for her assistance, encouragement, and patience, the author wishes to express his appreciation and grati tude 0 ii INTRODUCTION........ REVIEW OF LITERATURE . . . . History........ Ammonia Studies in Man Relationship of Ammonia Ammonia Metabolism in the Ruminant TABLE OF CONTENTS Page toHepaticComa............ O I O O O O O O O O I O O 0 lo AxmnoniaMetabolismintheDog................. ll AmnoniaMetabolisminO‘therAnilnalS.............. 114' Ammonia Metabolism as Influenced by Nutritional and Chemical Alterations of the Liver . . . . . . . . . . . . l7 ProteinMalnutritioninSwine................. 18 PrOtein mutrition in Other Animals 0 e e e e e e e e e e a e 20 Ammonia Metabolism as Influenced by Alteration of the Liver With Carbon Tetrachloride O O O O O O O O O O O O O O O 22 Ammonia Metabolism as Influenced by Surgically Produced ObStruCtj-on C O O 0 O O O O O O O O O 23 Extrahepatic Biliary Summary......... MATERIAISANDMETHODS ... Experimental Animal . . Clinical Observations . Chemical Analyses . . . Hematolog . . . . . . Tissue Analysis . . . . .................. 25 .................. 26 .................. 26 .................. 26 .................. 26 .................. 28 EXPERIMENT I. ACUTE AMMONIA TOXICOSIS iii Page ExperimentalProcedure.................... 30 Results...eeeeeeeeeeoeeeeeeeeeeeeee 33 EXPERIMENT II. .AMMONIA METABOLISM AS INFLUENCED BY CHEMICAL AND NUTRITIONAL ALTERATION OF THE LIVER . . . . . . . . . . . . . . 45 EJCperimentalPrOCedureeeeeeeeeeeeeeeeeeeee 45 ResultSeeeeeeeeeeeeeeeeeeeeeeeeeeee 1+7 EXPERIMENT III. AMMONIA METABOLISM AS INFLUENCED BY SURGICALLI PRODUCED EXTRAHEPATIC BILIARY OBSTRUCTION.. . . . . . . . . . . 64 ExperimentalProcedure.................... 64 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 IISTOFREFERENCES......................... 109 iv Table 10 LIST OF TABLES Effect of intravenous administration of 1.6% and 4% ammonium hydroxide on the blood buffer system of the unanBSthetizedpigeeeeeeeeeeeeeeeeeeea. Effect of intravenous administration of 4% ammonium acetate on the blood buffer system of the anesthetized andunanes'thetizedpigeoeeeeeeeeeeeeeeeee Effect of intravenous administration of 4% ammonium chloride on the blood buffer system of the unanesthetized pig 0 O O O O O C O O O O O O O O O O O O O O O O O C O O 0 Effect of intravenous administration of 4% ammonium carbonate on the blood buffer system of the unanesthe- tizedpig............o............ EDCperimentaldesignofExperimet‘ltII............ weights. hemograms, and total protein levels of test and control pigs at the beginning and end of the lowaprotein feedingperiod...........o........... Effect of intravenous administration of 4% ammonium acetate on the blood buffer system of unanesthetized pigs with.nutritional and chemical alteration of the liver .... . Effect of feeding a highpprotein diet on daily blood ammonia levels (ug./ml.) of pigs on a lowaprotein diet alone and in combination with carbon tetrachloride m3 actions 0 O O O O O O O O O O O O O O O O I O O O I O O 0 Length of survival after surgery and cause of death of pigs with a surgically produced biliary obstruction . . . . . . . Summary of the histOpathologic lesions in the livers of pigs with a surgically produced biliary obstruction . . . . . . . Page 34 35 36 37' 48 50 54 66 94 Figure 10 12 13 l4 15 l6 IJBT OF FIGURES Conway unit and pipette used in the titration of blOOdammoniaeeeeeeeoeeeeeeeeeeeee Facilities and position of a pig for intravenous infusion Blood ammonia levels as a result of intraperitoneal injection of 90 mg./lb. ammonium acetate . . . . . . . Blood ammonia levels as a result of intraperitoneal injection of 140 and 180 mg./lb. ammonium acetate . . . Blood ammonia levels as a result of intraperitoneal injection of 270 mg./lb. ammonium acetate . . . . . . . Blood ammonia levels as a result of intraperitoneal injection of 300. 345, and 420 mg./lb. ammonium acetate Signs of acute ammonia toxicosis in a pig resulting from intraperitoneal injection of 345 mg. /lb. ammonium acetate........................ Blood ammonia levels as a result of intraperitonal injection of 90 mg./lb. ammonium acetate . . . . . . . Blood ammonia levels as a result of intraperitoneal injection of 10 ml./lb. Aminisol . . . . . . . . . . . A pig fed the lowzprotein ration and a control . . . . The liver of a pig fed the lowaprotein diet and of a contr 01 O O O O O O O O O O O O O O O I O O O O O O O O ParakeratOSiS Of the tongue 0 e e e e e a e e e e e e e Page 31 31 38 39 41 1+3 52 53 56 56 57 An unidentified fungus in the parakeratotic layer illustrated inFigurelZ......o............o..o ParakeratOSis of the esophagus e e e e e e e e e e e e e e 57 58 An unidentified fungus in the parakeratotic layer illustrated 1nFigm‘914......o......o....o.... WdrOpic degeneration Of the liver 0 e e e e e e a e e e 0 vi 58 59 Figure Page 17 Hydropic degeneration of the liver . . . . . . . . . . . . 59 18 Liver of a control pig . . . . . . . . . . . . . . . . . . 60 19 Liver of a control pig . . . . . . . . . . . . . . . . . . 6O 20 Testicle of a pig fed the lowzprotein diet . . . . . . . . 62 21 Seminiferous tubule in the testicle illustrated in Figure 20 O O O O O O O C O O O O O O O O O O O I O O 0 O O 62 22 Testicle of a control pig . . . . . . . . . . . . . . . . . 63 23 Seminiferous tubule in the testicle illustrated in Figure 22 O O O O O O O O O O O O O O O O O O O I 0 O O O O 63 24 Pigs with surgically produced biliary obstruction . . . . . 67 25 Total and direct bilirubin, SGOT, and SGPT levels in pig 2 with a surgically produced biliary obstruction . . . . . 7O 26 Total and direct bilirubin, SGOT, and SGPT levels in pig 4 with surgically produced biliary obstruction . . . . . . 72 27 Total and direct bilirubin, SGOT, SGPT levels, and pro- thrombin times in pig 5 with surgically produced biliary ObSthtioneeeeeeeeeeeeeeeeeeeeeeee 7LT 28 Total and direct bilirubin, SGOT, SGPT levels, and pro- thrombin times in pig 6 with surgically produced biliary ObStruCtiOneeeeeeeeeeeeeeeeeeeeeeeo 76 29 Total and direct bilirubin, SGOT, and SGPT levels in pig 7 with surgically produced biliary obstruction . . . . . . 78 30 Total and direct bilirubin, SGOT, and SGPT levels in pigs 8 and 9 with surgically produced biliary obstruction . 8O 31 Total and direct bilirubin, SGOT, and SGPT levels in pigs 10 and 11 with surgically produced biliary obstruc- tion........................... 82 32 Total and direct bilirubin, SGOT, and SGPT levels in pigs 12 and 13 with surgically produced biliary obstruc- tion 0 O O . O C O O O O O O O O O O O O O O O O O O C O O O 8"" 33 Total and direct bilirubin, SGOT, and SGPT levels in pig 14 with surgically produced biliary obstruction . . . . . . 86 34 Total and direct bilirubin, SGOT, and SGPT levels in pig 15.85urgicalcontr01eeeeeeeeeoeeoeeeee 88 35 Blood ammonia levels as a result of intraperitoneal injec- tion of 90 mg./1b. ammonium acetate into pigs with surgically produced biliary obstruction . . . . . . . . . . . . . . . 89 vii Figure Page 36 The liver of a 5-week-old pig with surgically produced biliaryObStrUCtioneoeeeeeeeeeeeeeeeeee 91 37 A cross section of the liver from pig 7 with surgically produced biliary ObSthtion e e e e e e e e e e e e e e e 91. 38 The liver of a pig with surgically produced biliary ObStruCtioneeeeeeeeeeeeeeeeeeoeeoee 92 39 Ulcer in the esophageal region of the stomach of a pig with surgically produced biliary obstruction . . . . . . . 93 4O Ulcer in the esOphageal region of the stomach of a pig with surgically produced biliary obstruction . . . . . . . 93 41 Bilestasis........................ 95 42 Dilatation of a bile duct . . . . . . . . . . . . . . . . . 95 43 Bile duct proliferation . . . . . . . . . . . . . . . . . . 96 44 Centrolobular fatty metamorphosis . . . . . . . . . . . . . 96 45 Focal hepatic necrosis . . . . . . . . . . . . . . . . . . 97 46 Diffuse hepatic necrosis . . . . . . . . . . . . . . . . . 97 47 Bile lake . . . . . . . . . . . . . . . . . . . . . . . . . 99 48 Interlobular hepatic fibrosis . . . . . . . . . . . . . . . 99 49 Ulcer in the esophageal region of the stomach . . . . . . . 100 50 Thrombus in an artery in the submucosa underlying an ulcer in the eSOphageal region of the stomach . . . . . . . . . . 100 51 Vacuolar changes in an artery in the submucosa underlying an ulcer in the esOphageal region of the stomach . . . . . 101 52 Ryaline and vacuolar changes in an artery in the submucosa underlying an ulcer in the asephageal region of the Stomachooooeece000.00.000.00.Dee. 101 viii INTRODUCTION Ammonia and ammonium compounds are of basic biologic interest and have widespread application in medicine and agriculture. The role of ammonia in protein metabolism is well documented. The accumulation of toxic amounts of ammonia in the blood as a result of impaired metabo- lism may be a factor in the cause of certain diseases in man and animals. Urea and related compounds are used as a source of dietary nitro- gen in ruminant animals. In the ruminant, disturbances associated with urea feeding are thought to be due to the formation of toxic levels of ammonia in the blood. The use of ammonium compounds in soil improvement programs has increased in the past 20 years. It has been well established in man that elevated blood ammonia levels are associated with chronic liver disease. Likewise, in these patients neurologic symptoms are often observed terminally. Probably the most extensive studies have been on the syndrome of liver failure referred to as "hepatic coma". The exact relationship of ammonia to this condition is‘a subject of controversy. It is also well established that the liver is concerned in nitro- gen metabolism, and more specifically in the conversion of ammonia to urea. Therefore, it is possible that disturbances of the liver may interfere with ammonia metabolism. It is also known that urea is easily hydrolyzed by a number of physical and chemical-factors to pro- duce ammonia. In the ruminant, ammonia toxicosis as a result of excessive amounts of urea in the feed or accidental consumption of fertilizer is a 2 frequent occurrence. In the dog and horse there are reports suggesting disturbances associated with impaired ammonia metabolism. In the poultry industry, ammonia fumes from the litter in poorly ventilated houses are associated with corneal erosions and conjunctivitis in chickens. While there is much interest in the role of ammonia in diseases of man and animals, most of the research reports have been on the pharmacodynamic aspects of ammonia in laboratory animals. There have not been many studies on the gross and histopathologic changes resulting from ammonia toxicosis. This research was undertaken, therefore, to: (l).produce acute ammonia toxicosis in the pig and characterize the signs, biochemical, and morpholOgic changes, (2) determine the metabolism of ammonia in pigs with specifically induced chemical and nutritional alterations of the liver, and (3) determine the metabolism of ammonia in pigs with a sur- gically produced extrahepatic biliary obstruction. REVIEW OF LITERATURE Numerous reports in the literature pertain to the general aspects of ammonia metabolism, biochemistry, and use. This review is limited primarily to ammonia as associated with diseases in man and animals, with special emphasis on clinical signs, tissue, and blood alterations. gistory The theory of ammonia toxicosis was first preposed by Nencki and coaworkers (1896) from their study of Eck-fistula dogs. They observed signs of central nervous disturbance and high blood ammonia levels when these dogs were fed a meat diet. Salaskin (1898), as a result of bio- chemical studies on Eek-fistula degs, agreed with Noncki's theory that ammonia was the cause of the nervous signs observed in so-called "meat intoxication". Hawk (1908) conducted a number of experiments on Eck- fistula dogs and noted that the feeding of large amounts of meat did not always cause toxicosis but a meat extract in addition to a meat diet induced signs including ataxia, tetanus, catalepsy and paresis. Jacobson (1910) studied the chemical mechanism responsible for the tetany of parathyroidectomized animals and noted increased blood ammonia levels. Ammonium carbonate injected intravenously into dogs and eats caused tetany and the blood ammonia levels were almost identi- cal to those recorded in parathyroid tetany. She concluded that ammonia was directly responsible for the neurologic signs. Folin and Denis (1912), in studies on protein metabolism, determined that bacterial action on protein substances in the large intestine was the chief, or at least most constant, source of ammonia in the portal 3 L. blood. Matthews (1922) injected ammonium chloride intravenously into normal degs and observed a rise in the blood ammonia as well as abnor- mal nervous signs. He compared these results with the observations on Eek-fistula dogs fed a meat diet and concluded that ammonia toxicosis partly explained the signs of meat poisoning. Norvig and Iarsen (1924) observed high blood ammonia levels in epileptic patients prior to the onset of convulsions. Ammonia was studied in relation to the acid-base mechanism, and a defect in ammonia excretion was thought to be involved in the etiology of epilepsy. Luck and associates (1925), in testing Norvig's hypothesis, concluded that although high blood ammonia levels may occur in epileptic patients, there was no correlation between the high ammonia levels and onset of convulsions. They found high blood ammonia levels in ketosis due either to starvation or diabetes mellitus. Bellman and oo-workers, in a series of excellent studies on the physiology of the liver, clarified the role of the liver in ammonia metabolism. They (1924) performed hepatectomies on more than 90 dogs and observed a marked decrease in blood, tissue, and urine urea forma- tion.: This research emphasized the dependence of the animal on the liver for urea formation. In further experiments (1926), amino acids were injected into hepatectomized dogs and were recovered unchanged from the blood, urine, and tissues. No urea was formed, and the- ammonia recovered was not derived from the amino acids administered. This work clearly established the process of deaminization by the liver. The role of ammonia in.the urea cycle was further emphasized (1930) when ammonia administered to hepatectomized dogs was recovered as ammonium salts and not urea in the urine. Due to the failure of 18 ‘Heichselbaum (1935) was one of the first workers to demonstrate that rats fed a cystine-deficient diet were sluggish, icteric, cyanotic, and eventually died. On post-mortem examination there were hemorrhagic areas in the liver. He reported that either cystine or methionine prevented this condition, but once signs developed, cystine brought about recovery in most cases while methionine was without effect. Gydrgy and Goldblatt (1939) described the gross and microscOpic pathology of dietary hepatic necrosis in the rat. Fatty degeneration, hemorrhage, focal and diffuse necrosis were observed in all cases, with perilobular fibrosis occasionally noted. Daft and co-workers (1942) stated that dietary cirrhosis and hemorrhagic necrosis were separate and distinct entities. They differentiated these 2 conditions by the fact that choline prevented the development of hepatic cirrhosis and the sulfur- containing amino acids prevented the development of dietary hepatic necrosis. Radhakrishna-Rao (1948) believed that dietary factors played the major role in experimental hepatic necrosis but was unable to produce the lesion on lowbprotein diets. Gydrgy and Goldblatt (1949) and Himsworth and Lindan (1949) stated that, in addition to a protein-deficient diet, a vitamin E deficiency was required to produce dietary hepatic necrosis. grotein galgutzition in Swine Knowles (1957) fed pigs diets of protein levels ranging from h.5 to 10%. He noted that the most prominent signs of protein malnu- trition were weakness, apathy, hypochrmotrichia, anemia, and edema. The pigs fed “.5 to 6.5‘ protein diets were the most severely affected. They had a dry skin with desquamation and numerous fissures. The tota1.plasma protein and A/G ratio were low. Godwin (1957) reported 19 that uninfected pigs maintained on a lowaprotein diet from an early age (2 to 5 weeks) for 3 to 18 months developed periportal fatty infil- tration and fibrosis. Heard 91 g. (1958) fed pigs a low-protein diet and observed a syndrome resembling marasmus in children. When additional calories as carbohydrate were fed, a condition resembling kwashiorkor occurred. Barnes and his associates at Cornell University (1959) observed that weanling pigs fed a low-protein (9%) high-fat diet developed the most marked signs of protein malnutrition. They con- cluded that kwashiorkor was not readily produced eXperimentally because animals fed a lowaprotein diet ad libitum had a decreased food intake and therefore did not consume sufficient calories. In a further report from Cornell University, Loquygt.al. (1962) studied the effect of high and low caloric levels on low-protein diets in pigs and also compared this to protein quality. Kwashiorkor-like signs were most severe in the wheat gluten, high-calorie diet and least severe in the casein, lowecalorie group. British workers published a series of research papers on the biochemical and morphologic changes that occurred in swine fed lowa protein diets. McCance (1960) maintained swine on a starvation-type diet for periods of 6lmonths to 2 years. He observed the loss of hair and the crusting of skin in these pigs, which caused them to scratch constantly. Vitamin A levels in the liver were normal in the control animals and increased in the undernourished pigs. Friend gt él° (1960) reported that serum.vitamin A levels of pigs fed a low- protein diet were not dependent upon the liver stores of the vitamin but were correlated with Serum albumin. Pigs fed a lowaprotein diet had liver levels of vitamin A comparable to those fed a high-protein 20 diet, but the serum albumin and serum vitamin A levels were low. High dosages of vitamin A increased the content of vitamin A in the livers of pigs fed the loweprotein diet but had no effect on serum levels. They postulated that the low serum albumin reduced the tranSport of vitamin A from the liver. Stewart and Heard (1959) reported that the individual cells of the pancreatic islets of pigs fed loweprotein diets were reduced in size. Hypoglycemia produced by insulin was not as readily correctable in pigs fed lowaprotein diets as in the controls. Durbin and co—workers (1960), in a study of abnormal carbohydrate metabolism in pigs fed lowzprotein diets, demonstrated a lack of g1ucose-6-phoSphatase in the livers, which eXplained in part the inability to mobilize glycogen stores. English workers have reported structural changes in the central nervous system of pigs fed loweprotein diets. Godwin and Platt (1960) observed necrosis and development of cysts in the pituitary gland, and Platt and Stewart (1960) described pathologic changes in the nerve cells of the Spinal cord, which included loss of Nissl's substance, increased numbers of glial cells, and satellitosis. Eggtgin Malnutrition in Other Animals Channon and Wilkinson (1935) fed rats diets containing 90% fat with varying amounts of protein (5 to 50%) for a period of 3 weeks. The amount of hepatic fat was directly influenced by the amount of protein in the diet, irrespective of any action of choline. Rats fed a low-protein diet had higher glyceride and cholesterol ester levels in liver tissue, as compared to rats fed normal or high-protein levels. 21 Elman and Heifetz (1941) maintained dogs on a protein-deficient diet for 6 weeks. A progressive decrease in the albumin fraction of the serum proteins was noted. The outstanding histopathologic changes occurred in the liver. There was a loss of stainable cytOplasm that produced extensive vacuolization of the hepatic cord cells. Fat and glycogen stains were negative. The water content of the liver increased as the protein content decreased. In a further report, Elman and co- workers (1943) correlated the cytologic and chemical changes that occurred in the liver of dogs fed a low—protein diet. On a protein- deficient diet with adequate carbohydrate, the hepatic cells were large, and the cytoplasm was rarefied; a condition they referred to as hydrOpic degeneration. The hepatic cell of a dog fed a lowaprotein diet with low carbohydrate had a much more dense cytoplasm and a smaller nucleus. Kosterlitz (1947) studied the effect of a lowaprotein diet on rat liver from the chemical and histologic aSpect. He stated that the rarefaction of the cytoplasm of the hepatic cord cells corre- sponded with a decrease in the protein, phospholipid, and nucleic acid content, and an increase in glycogen. In a slightly different type of investigation, Lightbody and Kleinman (1939) studied changes in enzyme content of the liver in rats fed diets of varying protein content. As the protein level of the diet decreased, the arginase content per unit weight of the liver decreased. Schimke (1962) stated that the total liver content of all the urea cycle enzymes was directly prOportional to the daily con- sumption of protein. 22 Ammonia Metabolism as Influenced by Alteration of the Live; with Carbon Tetrachloride Extensive research has been conducted on carbon tetrachloride toxicosis. Reports pertinent to this research will be discussed. Meyer and Pessoa (1923) studied acute carbon tetrachloride poisoning in dogs. Histopathologic changes were present in the liver and kidneys. In the liver, fatty degeneration and necrosis were described; kidney lesions consisted of fat draplets in the epithelium of the convoluted tubules and loOps of Henle. There were active signs of regeneration in many of the liver sections. Lamson and Wing (1926) reported on chronic carbon tetrachloride toxicosis in the dOg as pro- duced from the administration of the chemical over extended periods of time. They described the following microscOpic changes in theliver: distortion of lobulation, necrosis, fibrosis, accumulations of inflamma- tory cells, fatty degeneration, and areas of bile duct proliferation. Cameron and Karunaratne (1936) induced hepatic lesions in rats by administering carbon tetrachloride at short intervals over long periods. They stated that when time was allowed for recovery after each dose, carbon tetrachloride could be given indefinitely without producing permanent hepatic change. White (1939) injected carbon tetrachloride at the rate of 0.6 ml./kg. body weight subcutaneously into young pigs as a single dose without causing death. Centrolobu- lar necrosis was observed histologically, within 24 hours. In about 1 week the liver was normal. Repeated administration of 0.7 ml./kg. body weight at 4- to 5-day intervals resulted in the development of cirrhosis. I" i? 23 Miller and Whipple (1940) demonstrated that dogs fed a low-protein diet were more susceptible to the toxic effects of chloroform than dogs fed optimum protein. In further work, they (1942) administered methionine to protein-depleted dogs a short time before chloroform anesthesia and completely protected them from hepatotoxic effects. Along this same line, Campbell and Kosterlitz (1948) observed that rats fed a protein-deficient diet for a prolonged time were more highly susceptible to the hepatotoxic effects of carbon tetrachloride. Ammonia Metabolism as Influenced by Surgically Produced Extzahepatic Biliagy Obstruction. The technic to produce extrahepatic biliary obstruction by surgi- cal means has been established by numerous workers. Literature basic to this research is reviewed. Richardson (1911) reported the clinical and pathologic changes in rabbits when the common bile duct was surgically ligated. In all rabbits, jaundice appeared within 24 hours after ligation. Hepatic necrosis, fibrosis, and bile duct proliferation were the outstanding histOpathologic changes. He stated that the primary injury to the liver parenchyma was due to the escape of dammed bile from rupture of bile capillaries. MacMahon gtugl. (1929) reviewed the early work on experi- mental biliary obstruction and concluded that prolonged bile stasis resulted in definite hepatic changes. The rapidity of production and the characteristics of the histopathologic changes varied in different species thMahon and Mallory (1929) studied 30 cases of uncomplicated biliary cirrhosis in man and reported that, in addition to the usual microscopic lesions, hepatic infarcts and organizing bile thrombi were present. Cameron and Oakley (1932) conducted research on experimental 24 biliary occlusion in the rat, guinea pig and rabbit. They noted that the focal areas of necrosis which resulted from ligation of the common bile duct healed by regeneration of hepatic cells. Their work indi- cated that in the absence of infection fibrosis did not occur as a result of biliary occlusion. Lieber and Stewart (1934) and Shorter and Baggenstoss (1959) stated, however, that extensive fibrosis could occur in the liver of man with extrahepatic obstruction in the absence of demonstrable infection. An interesting study of the abnormal conditions related to experi- mental bile fistulas in dogs was described by Hawkins and Whipple (1935). They mentioned that acute intestinal disturbances, spontaneous bleeding, generalized osteOporosis, cholelithiasis, and duodenal ulcers were often complicating factors in dogs with bile fistulas. Rarely were all conditions present in a single animal. Hawkins-and Brinkhous (1936) discovered that the bleeding tendency which developed in bile- fistula dogs was due to a prothrombin deficiency. Further work to explain this phenomenon was conducted by Smith and coaworkers (1938), who stated that faulty absorption of vitamin K from the intestine was an important causative factor of the low prothrombin levels noted by Hawkins (1936). They demonstrated that the feeding of bile and vitamin K concentrate caused a rapid rise in prothrombin levels of these dogs. Brinkhous and warner (1941) described a muscular dystrOphy in bile- fistula dogs which was similar to a vitamin E deficiency. Faulty- absorption of vitamin 8 due to the absence of bile in the intestine was given as the causative factor. Cameron 23,31. (1957) induced a complete biliary obstruction in rabbits by filling the lower end of the common bile duct with a quickly 25 setting plastic material. They observed focal necrotic lesions in the liver, which they believed due to a cytotoxic effect of bile accumula- tion within the hepatic cells. Cornelius 23,31. (1965) surgically pro- duced extrahepatic biliary obstruction in 2 horses. This resulted in a marked bilirubinemia, portal fibrosis, coma, and death. Summggy Numerous papers have appeared and considerable interest exists on the metabolism of ammonia in man and animals. In addition, a number of reports have attempted to establish the role of ammonia in specific disease conditions. An exact cause and effect relationship is still in dispute. Only a few reports are available concerning ammonia metabo- lism as affected by specific lesions in the liver. The literature supports the view that specific alterations of the liver of animals can be produced by lowbprotein diets, carbon tetrachloride poisoning, and surgically produced extrahepatic biliary obstruction. MATERIALS AND METHODS This research was started in the fall of 1964 and was continued through the summer of 1965. The research was conducted primarily in Giltner Hall, Michigan State University. Experimental Animal The young pig was used in this research because (1) it was of a size that would.provide ample tissue for analysis and microscopic study, (2) clinical observations could be made, (3) it was readily available, and (4) it is a suitable experimental animal to evaluate basic medical problems (Bustad, 1965). The pigs were a Yorkshire-Hampshire cross obtained from local farms. They were allotted to the various treatments in accepted experi- mental design and littermates were used as controls. A standard growing type ration was purchased from the Department of Animal Husbandry, Michigan State University, and fed 5g,libi§um. Clinical Obsegzations All experimental animals were Observed at least twice daily. weight changes, signs, and other pertinent information were recorded during the course of the experiments. emi ses Blood Ammonia Samples of whole blood were analyzed for ammonia nitrOgen by the microdiffusion technic of Conway (1947). A reagent blank and a standard 26 27 ammonium sulfate control were used with each group of unknown determi- nations. The error of this technic was less than 10% on recovery of ammonia from the standard ammonium sulfate solution. All results were recorded in micrograms of ammonia nitrogen per milliliter of blood (ug./ml.). The blood was collected in a vacuum-sealed test tube (Vacutainer, Becton-Dickinson), with sodium heparin as the anticoagu- lant. Immediately after collection, the tubes were placed in an ice- water bath (as suggested by Merchant gt_gl., 1960) and were left there until analyzed. Experiments conducted during the early phase of this research confirmed Merchant's work, since blood held in an icewater bath had no increase in ammonia level after 3 hours. The Conway unit and the pipette used for the titration are shown (Figure 1). C02 Combining Power Samples of serum were analyzed for carbon dioxide combining power using the colorimetric technic as described by Exton gtflgl. (1941). Venous blood samples were obtained from the anterior vena cava, placed into glass tubes, and stoppered. Care was taken to avoid exposing the blood to the atmosphere during the subsequent procedures. All results were recorded in volumes percent (Vol.$). Serum Bicarbonate Serum samples were analyzed for bicarbonate content using the tech- nic described by Henry (1964). Blood was obtained at the same time as for 002 combining power. The same precaution of avoiding exposure of blood to the atmosphere was taken. Values were recorded in milliequiva- lents per liter (mEq./liter). 28 Sodium and Potassium Sodium and potassium were determined in blood serum using the Model 21 Coleman Flame Photometer (Operating directions for the Coleman Model 21 Flame Photometer D-248A, 1957). Lab-trol (Dade Reagents) was used for the control and Harleco (Hartman—Leddon Company) for standards. Values were recorded in mEq./1iter. Bilirubin Blood serum samples were analyzed for bilirubin content according to the spectrophotometric method of Biordano-Pestrud, modified as described by Levinson and MacFate (1961). Direct readings were made at l- and lO-minute intervals. Values were recorded in mg./100 m1. Transaminase and Prothrombin Time Determinations The serum glutamic oxaloacetic and serum glutamic pyruvic transamin- ases were determined according to the method of Reitman-Frankel, modified (1957). Values were recorded in Sigma-Frankel (S.F.) units. Prothrombin time was determined on plasma using the 1-stage technic described by Quick (1957). Total Protein Blood serum samples were analyzed for total protein content by the spectrOphotometric method of Wolfson gt a1. (1948). Values were recorded in gm. /100 m1. Hematology Blood samples were obtained from the anterior vena cava, using heparin as the anticoagulant. Hemoglobin was determined by the cyan- methemoglobin method. Packed cell volume values were determined by the micro method (capillary tube). 29 Tissue Analysis At the termination of each specific experiment, or death of any of the pigs, a necropsy was performed. Routinely, the following tissues 'were collected for microscOpic study: liver, stomach, pancreas, jejunum, kidney, spleen, lung, and heart. They were preserved in 10% neutral' formalin and stained with Sudan IV for examination of fat content. Some sections of liver were preserved in Carnoy's fixative and stained 'with Best's carmine technic for examination of the glycogen content. Other special stains were employed when indicated. The histologic procedures followed were those suggested by the Armed Forces Institute of Pathology Manual of fiistologic ggd Spgcial Staining Technics (1957). EXPERIMENT I. ACUTE AMMONIA TOXICOSIS Thirty-one 4—week-old pigs were used to determine the dose rates (mEq. NHu/kg./min.), signs, blood ammonia levels and influence on the blood buffer system of 4 ammonium compounds administered intravenously and 1 compound intraperitoneally. ggerimental Procedure Ammonium acetate, ammonium carbonate, and ammonium chloride were used at a concentration of 4% in sterile 5% dextrose in water (Abbott). Ammonium hydroxide was used at concentrations of 1.6 and 4% in the same diluent. Commercial ammonium carbonate consists of equal amounts of ammonium carbamate and ammonium carbonate. This compound was selected for intravenous infusion to determine the toxic effect of the carbamate ion. The ammonium carbonate was infused immediately after preparation, since carbamate is unstable in solution for any extended period of time. The unanesthetized pig was positioned as shown (Figure 2). A blood sample for ammonia, carbon dioxide combining power, and bicarbo- nate was obtained from the anterior vena cava prior to the experimental infusion. The syringe was then removed from the needle and the intra- venous tubing inserted. The blood samples were put in appropriate tubes. A stopwatch was used to time the rate of infusion. At numerous times during the infusion the number of drOps in 30 seconds was counted and recorded. At the time respirations ceased, the infusion was stopped and blood samples were obtained by intracardiac puncture for ammonia, carbon dioxide combining power, bicarbonate, sodium, and potassium. 3O Figure 1. Conway unit (above) and ipette used in the titration of blood amonia (below . . acilities and position of a pig for “wens infusion. 32 To determine the influence of anesthesia on the toxicosis, 2 anesthe— tized (pentobarbital sodium) pigs were intravenously infused with 4% ammonium acetate following the above procedure. The rate of infusion per minute was determined by obtaining an average of the number of drops per 30 seconds and multiplying by 2. The number of drops in 1 ml. of each solution was determined and the ml. of NH“ compound per minute were calculated. To assess the influence of the anion and pH of the ammonium com- pound, control pigs were intravenously infused with the following solutions for the times indicated:‘ 1. 1% sodium carbonate in 5% dextrose in water - pH 10.2 e 6.5 minutes. 2. .003% hydrochloric acid in physiological saline - pH 5.3 - 9 minutes. 3. .M sodium hydroxide in 5% dextrose in water - pH 10.5 - 15 minutes. 4. 4% sodium acetate in 5% dextrose in water - pH 7.5 - 11 minutes. Each control solution correSponded to l of the ammonium compounds used with respect to the anion and pH of the final solution. The rates of infusion are given in the results. The length of time for the infusion of each control was the average of the time for death to occur in pigs given each corresponding compound. Blood samples for ammonia, bicarbonate, carbon dioxide combining power, and sodium and potassium were obtained at the end of the infusion. The pig was then euthanatized. Four percent ammonium acetate in 5% dextrose in water was used for the intraperitoneal injection. The dosages administered were 90, 140, 33 180, 270, 300, 345, and 420 mg. of ammonium acetate per pound body weight. The entire volume was given, by syringe, over a period of 2 to 3 minutes. Samples for determination of blood ammonia were obtained from the anterior vena cava just prior to and at 15, 30, 45, 60, and 90 minutes after injection. Clinical signs of toxicosis were recorded. esults The dose rates, carbon dioxide combining power, bicarbonate sodium, potassium and blood ammonia levels resulting from the intra- venous infusion of 4 ammonium compounds are given (TABLES 1, 2, 3, and 4). There was a reciprocal relationship between the rate of administra- tion of the ammonium compound and the time of death. The results indicated that anesthetized pigs required larger amounts of ammonium compound to produce death in the same period of time than did the unanesthetized pigs (TABLE 2). The carbon dioxide combining power and serum bicarbonate levels decreased as a result of the intravenous infusion of 4% ammonium ace- tate,4% ammonium chloride, and 1.6% ammonium hydroxide. A much smaller decrease in carbon dioxide combining power occurred with the infusion of 4% ammonium hydroxide and 4% ammonium carbonate. Infusion of these 2 compounds caused little or no change in serum bicarbonate levels. The serum sodium values were normal or slightly decreased. The serum potassium levels, on the other hand, were elevated. All blood ammonia values increased as a result of the intravenous infusion of ammonium compounds. The results of the intraperitoneal administration of ammonium ace- tate are illustrated (Figures 3, 4, 5, and 6). Pigs intraperitoneally injected with 90 mg./1b. ammonium acetate had the highest blood ammonia 34 TABLE 1. Effect of intravenous administration of 1.6% and 4% ammonium hydroxide on the blood buffer system of the unanesthetized pig. Pig No. 1 2 3 4 5 6 and Item Conc. 1.6% 1.6% 1.6% 4% 4% Control* Weight (kg.) 6.3 9.3 9.0 6.5 5.0 9.0 Dose Rate of Ammonium Compound ml./min. (avg.) 6.0 9.3 12.0 10.0 9.6 12.3 mqu NHu/mine (‘vge) le6 2.5 3.2 7eo 6e“ -9- mEq. NH4/kg./min. (avg.) .25 .27 .35 1,07 1,3 --- Time for Death to Occur (Min.) 26.0 20.0 15.0 6.5 5.0 --- Serum Carbon Dioxide Combining Power (vol.%) Before Infusion 58.0 58,0 55.0 64.0 --- 48.0 After Infusion 39.0 43.0 45.0 58.0 --- 67.0 Serum Bicarbonate mEq./liter Before Infusion 21.0 20.0 18.0 18.0 --- 15.0 After Infusion 11.0 12.0 14.0 18.0 --- 18.0 Serum Electrolytes mEq./liter After Infusion NA 128.0 144.0 138.0 138.0 --- 154.0 K+ 7.6 7.1 7.5 4.2 --- 5.2 Blood.Ammonia ug./mlo Before Infusion .31 .41 .41 .41 --- .41 After Infusion 77.9 69.0 73.0 121.7 --- .61 *Pig infused with .ou$ sodium hydroxide in 5% dextrose in water - pH 10.5 - and euthanatized. 35 TABLE 2. Effect of intravenous administration of 4% ammonium acetate on the blood buffer system of the anesthetized and unanesthetized pis- W Pig No. 1 2 3 4 5 6 Item Unan. Unan. Unan. An. . An. Control* Weight (kg.) 7.2 6.8 5.0 6.8 5.0 9.5 Dose Rate of Ammonium Compound m1./min. (avg.) 8.8 8.4 4.0 10.8 14.0 8.3 mEq. NHu/min. (avg.) 4.4 4.3 2.1 5.5 7.1 --- mEq. NHulkg./min. (avg.) .61 .63 .42 .84 1.4 --- Time for Death to Occur (Min.) 10.0 11.0 14.0 11.5 8.5 --- Serum Carbon Dioxide Combining waer (vol.%) Before Infusion 54.0 71.0 --- 50.0 44.0 45.0 After Infusion 36.0 36.0 --- 22.0 26.0 48.0 Serum Bicarbonate A mEq./liter Before Infusion 21.0 19.0 --- 15.0 15.0 15.0 After Infusion 10.0 4.0 --- 5.0 9.0 16.0 Serum Electrolytes mEq./1iter After Infusion NA+ 146.0 142.0 --- --- --- 148.0 K+« 7.4 6.9 --- --- -—- 5.4 Blood.Ammonia ug./m1. Before Infusion .40 .61 --- .80 .80 .40 After Infusion 89.2 101.4 --- 202.8 113.6 .40 *Pig infused with 44 sodium acetate in 5t dextrose in water - pH 7.5 - and euthanatized. Bio: 36 TABLE 3. Effect of intravenous administration of 4% ammonium chloride on the blood buffer system of the unanesthetized pig. Item Control* ‘Weight (kg.) 5.2 6.4 5.0 7.5 9.0 Dose Rate of Ammonium Compound ml./min. (avg.) 10.0 10.0 5.0 5.2 13.6 mEq. NH4/min. (avg.) A 7.5 7.5 3.7 3.8 --- mEq. NHs/kg./nin. (avg.) 1.4 1.2 .74 .50 --- Time for Death to occur (Min.) 5.0 6.0 11.0 12.0 --- Serum Carbon Dioxide Combining Power (vol.%) Before Infusion 64.0 71.0 51.0 --~ 64.0 After Infusion 18.0 26.0 41.0 --- 57.0 Serum Bicarbonate mEq./1iter Before Infusion 18.0 19.0 19.0 --- 15.0 After IflfllSlOn 300 300 5.0 --- 14.0 Serum Electrolytes mEq./1iter After Infusion NA+ --- --- 143.0 --- 145.0 n+1 --- --- 7.7 --- 4.5 Blood.Ammonia ug./ml. Before Infusion --- .31 .41 --- .51 After Infusion --- 242.0 141.0 --- .41 *Pig infused with .003% hydrochloric acid in physiological saline - pH 5.3 - and euthanatized. am .5511. II Item Weight Dose E Amen: Occur Serum Coati- Aft Sermn ast/ Bef Aft Serum “go/n Bet Aft PH 1E 37 TABLE 4. Effect of intravenous administration of 4% ammonium carbonate on the blood buffer system of the unanesthetized pig. Pig No. l 2 3 4 Item Control* Weight (kg.) 5.9 5.0 5.0 5.9 Dose Rate of Ammonium Compound ml./min. (avg.) 5.6 11.6 12.5 14.4 mEq. NHu/min. (avg.) 3.7 7.7 8.3 --- mEq. NHu/kg./min. (avg.) .66 1.5 1.7 --- Time for Death to Occur (Min.) 8.0 6.0 4.5 --- Serum Carbon Dioxide Combining Power (vol.%) Before Infusion 63.0 65.0 62.0 52.0 After Infusion 60.0 49.0 59.0 65.0 Serum Bicarbonate msq . / liter Before Infusion 21.0 15.0 17.0 14.0 After Infusion 20.0 15.0 15.0 15.0 Serum Electrolytes mEq./1iter After Infusion NA+ 148.0 141.0 --- 140.0 1* 5.4 7.6 --- 6.0 Blood Ammonia ug./ml. Before Infusion .61 .89 .81 .40 After Infusion 121.0 93.2 130.0 .60 I"Pig infused with 1% sodium carbonate in 5% dextrose in water - pH 10.2 - and euthanatized. 38 .memeoom Esflcozzw .oa\.m£ om mo compoonsa Hmocopaoodmapca mo panama m we mHo>oa mecoses sooam .m madman noose? a can. one f" odes om .. 42.4 .DH\.mE on I I odds om .. I oxootxxoxndmmfl CD r4 .HE\.MS zummz 39 .mpmpooe €5HCOSEm .DH\.me owa mom oda mo coeeoomce Hmocoefleeamnpcw mo badmmm m we mamsma mflcossm wooam .3 mmdmam mops—mas 5 08.2. one on so we on me o f 1 , .oaxme 8H .. I renxms sea . Artie oxootxxoxnemmfi O H .HE\.wd zummz 4O .oemeoom ssflcoeem .bH\.me 0mm mo compomhce Hmmcopfloommepca mo paommo m mm mameoa memosem mooam .m chewem noses? 5. sea 84 om 8 me on 3 o i d ‘ it. . . [I . eHE\emH~ Bios cam .. I .. em odes RN .. olo . mm 41 .momeoom adecocrm .ba\.me owe mom .mam .oom m someomMCfl Hmocoeeemosepce go paomep w mm mamsma meeoesm mooam .m ensued sources 5 sea. cud cm so we on me o d d w a a u a .eH\.ms com - .vuuaw . odes men .. Ola . odds on: .. I L. .. 0H ON on on cm on ow om 00H Odd on a one odd one .He\.md zummz 42 values in 15 to 30 minutes, returning to normal in 60 minutes. At 140 mg. /1b. and 180 mg./1b. dosage, peak ammonia levels were reached at 15 and 30 minutes, respectively, returning to normal in 90 minutes. At 270 mg./1b., peak ammonia levels occurred in 30 minutes, returning to normal in 120 minutes. The highest nonfatal dosage of ammonium ace tate given intraperitoneally was 300 mg./ 1b. The blood ammonia level reached a maximum at 45 minutes and had not returned to normal in 120 minutes. Dosages of 345 mg./lb. and 420 mg./1b. caused death in 13 and 16 minutes, respectively. Following the intravenous administration of ammonium compounds, these signs were most consistently observed: (1) increased respira- tions, later becoming irregular and increased in depth: (2) increased salivation; (3) vomition: (4) twitching of muscles: (5) clonic-tonic convulsions: (6) respiratory arrest: and (7) death. The onset of the above signs occurred in 1% to 3 minutes, depending upon the rate of infusion. There was grinding of the teeth by some Pigs, along with the increased salivation. After respiration had ceased, cardiac pulsations continued for 15 to 30 seconds. The inten- sity of the signs in the anesthetized pigs given intravenous infusions of ammonium acetate was greatly decreased. The signs in pigs intraperitoneally injected with ammonium acetate, t1'10“»gh more prolonged, were essentially the same as those given for intrawnous infusions.(1|‘igure 7). Those given 90, 140 and 180 mg. /1b. am°nium acetate exhibited no toxicosis. At the 270 and .300 mg./1b. Mounts. most of the signs appeared, but death did not occur. Signs were most pronounced in intensity between 15 and 30 minutes after in- jecticm, at the time blood ammonia levels were the highest. 43 Figure 7. Signs of acute ammonia toxicosis in a pig resulting from intraperitoneal injection of 345 mg./1b. ammonium acetate. (A) Before injection; (8) ataxia, increased rCSpirations; (C) posterior weakness. respiration irregularity; (D) vomition and increased salivation; (E) tonic convulsion; (F) death. 44 There were no clearly significant gross or hist0pathologic lesions in either the experimental or control pigs as a result of acute ammonia toxicosis. EXPERIMENT II. AMMONIA METABOLISM AS INFLUENCED BY CHEMICAL AND NUTRITIONAL ALTERATION (1" THE LIVER Sixteen 2%dweek-old pigs were used to study the effect of liver alteration, produced by nutritional and chemical means, on ammonia metabolism. gaperimental Procedure The pigs were weighed at the beginning of the experiment. The ears were notched and a blood sample was taken from the anterior vena cava for hemoglobin, packed cell volume, and total protein determina- tion. Twelve pigs were fed a low-protein diet for 5 weeks, composed of the following ingredients: ground shell corn, 91.75 1b.: beet sugar. 5 1b.: limestone, 1 1b.: dicalcium phosphate, 0.75 lb.: high zinc trace mineral salt, 0.5 lb.: vitamin antibiotic trace mineral mix, 1 1b. This ration contained 8.3% protein of poor quality but was otherwise nutritionally adequate for pigs. Four of the 12 pigs fed the low-protein diet were given a series of subcutaneous injections of carbon tetrachloride (C.P.) at a dosage of 0.2 ml. per pound body . 'weight. The injections were administered at 5-day intervals during the 4th and 5th weeks of the feeding period. Four pigs were used as controls and fed the MSU growing ration. I At the end of the 5-week feeding period, blood samples were taken for hemoglobin, packed cell volume, and total protein determinations. The experimental design is given (TABLE 5). 45 46 TABDI 5. EXperimental design of Experiment II. Group No. and Treatment after Number the 5dweek of Feeding Period Pigs Liver Disturbance Produced by: 1 Ammonium acetate, 4 Low Protein Low Protein intravenous infusion Low Protein + CCLQ Control* 2 Ammonium acetate, 4 Low Protein Low Protein intraperitoneal injection Low Protein + 0CD“ Control* 3 Aminisol, intra- 4 Low Protein Low Protein peritoneal . injection Low Protein + COL” Control* 4 High Protein 4 Low Protein Low Protein feeding . Control* Low Protein + CCLh 'Fed MSU ration and maintained separately. 47 To assess the effect on ammonia metabolism, ammonium acetate (4%) in 5% dextrose in water was administered intravenously and intraperi- toneally to treatment groups 1 and 2, reapectively. The intraperi- toneal dosage was 90 mg./lb. The experimental procedure was the same as described in Experiment I. Aminisol (Abbott), a protein hydrolysate, was given intraperitoneally to treatment group 3 at a dosage of 10 ml./lb. body weight. Blood samples were taken for ammonia determination just prior to and at 4 l-hour intervals after injection. A high-protein diet composed of equal parts vitamin-free casein and MSU growing ration was fed to treatment group 4 for,an 8-day period at a level of 50 gm. of feed/lb. body weight/day. Blood samples for ammonia determination were taken.daily. Pigs that died or were euthanatized at the end of each specific treatment were necropsied and tissues collected for microscopic study. fiesults The details on the effects of feeding a low-protein diet and injections of carbon tetrachloride are given (TABLE 6). The hemoglobin, packed cell volume, and total protein values in the pigs fed the lowbprotein ration decreased. These values remained the same in the control pigs. The average weight gain, for the deeek feeding period, of the pigs fed the lowbprotein ration was 0.36 kg., as compared to 3.5 kg. for the controls. The results of the intravenous infusion of ammonium acetate into the pigs fed the lowbprotein ration and the control pig are given (TABLE 7). A comparison of dose rates in relation to the time for death to occur between the pigs on the lowbprotein ration and the control pig 48 TABLE 6. weights, hemograms, and total protein levels of test and control pigs at the beginning and end of the low-protein feeding period. Total Protein Pig NO. and Wt. Hb. Hot. (gm/100 m1.) Diet “(3.) (gm/100101.) (3%) Be innin of Low- otei eedin eriod 1 1.?“ + CCLL, 5.7 9.9 34.0 8.1 2 LP 4.7 10.7 35.0 6.6 3 LP 5.0 10.4 35.0 5.8 4 LP + CCLh 5.0 8.2 28.0 5.8 5 LP 4.3 10.7 35.0 4.8 6 LP 5.2 10.4 34.5 5-5 7 LP + COL“ 5.0 10.4 34.0 3.3 8 LP 5.0 10.2 33.0 7.7 9 LP + CCLu 4.5 10.4 35.0 5.1 10 LP 4.5 12.3 39.0 6.9 11 LP 4.5 11.6 41.0 6.8 12 LP 4.5 10.7 37.0 5.7 13** Growmng ration 4.1 10.4 34.0 6.7 14** Growing ration 4.5 10.2 33.0 5.1 15'“I Growing ration 4.5 9.9 30.0 4.3 16'“I Growing ration 4.7 10.7 37.0 4.? End of ngefizotein Eeeding Period 1 LP + COL“ 6.4 8.9 30.0 3.7 2 LP 4.? 9.4 29.0 4.0 3 LP 5.0 9.9 31.5 3.9 4 LP + COL“ 5.4 8.6 29.0 4.0 5 LP 5.0 8.9 28.0 3.7 49 TABLE 6.--continued "Hm Pig No. and Wt. Hb. Hct. Total Protein Diet (kg.) (gm./100 ml.) ($) (gm./lOO m1.) 6 LP 5.7 10.7 33.0 4.2 7 LP + act“ 5.7 10.7 36.0 4.7 8 LP 5.2 7.7 32.0 4.3 9 LP + cot“ 5.0 10.2 33.0 4.0 10 LP 4.5 9.4 29.0 4.6 11 LP 5.0 9.9 30.0 4.6 12 LP 4.7 9.6 37.0 4.3 13""I Growing ration 6.7 10.2 33.0 5.5 14** Growing ration 8.6 9.9 31.0 5.3 15“ Growing ration 9.8 10.4 32.0 3.9 16'“I Growing ration 6.4 11.6 44.0 4.6 A_ *LP - Low-protein ration. I"""Control. 50 TABLE 7. Effect of intravenous administration of 4% ammonium acetate on the blood buffer system of unanesthetized pigs with nutritional and chemical alteration of the liver. Pig No. 1 2 3 4 and . _ Low Prot. & Control Item Diet Low Prot. Low Prot. Carbon Tet. Growing Ration weight (kg.) 5.0 5.5 5.0 6.3 Dose Rate of Ammonium Compound ml./min. (an.) 7.7 10.8 11.6 12.8 mm. nut/min. (avg.) 3.9 5.5 5.9 6.5 mEq. Hag/kg./min. (avg.) .79 1.1 1.2 1.0 Time for Death to Occur (Min.) 6.5 5.5 6.0 7.0 Serum Carbon Dioxide Combining Power (vol.$) Before Infusion 56.0 49.0 51.0 60.0 After Infusion 29.0 29.0 27.0 27.0 Serum Bicarbonate mEq./liter Before Infusion 19.0 17.0 15.0 19.0 After Infusion 8.0 6.0 4.0 6.0 Serum Electrolytes mEq./1iter After Infusion NA+ 130.0 118.0 114.0 144.0 K+ 9.2 8.8 8.7 5.4 Blood Ammonia ug./ml. Before Infusion .61 .81 .81 .41 After Infusion 246.2 311.2 270.0 211.4 51 indicates a slight difference. The changes in the carbon dioxide com- bining power and bicarbonate of the pigs on the low-protein diet did not differ markedly from those of the control. The serum sodium levels of the pigs fed the low-protein diet were lower than those of the con- trol: however, the potassium levels were higher. The blood ammonia values were essentially the same. There were no essential differences in the results between the pig fed the low-protein diet and given carbon tetrachloride and those fed the low-protein diet alone. The results of the intraperitoneal injection of ammonium acetate into the pigs fed the low-protein diet and the control are illustrated (Figure 8). The blood levels of ammonia in the pigs fed the low-protein diet were much higher than those of the control and returned to normal in 90 minutes. The blood ammonia levels of the pig injected with carbon tetrachloride on the low-protein diet were only slightly ele- vated and returned to normal in 60 minutes. The contro1 pig had blood ammonia.values essentially the same as those recorded in.RXperiment I. The details of intraperitoneal injection of 10 ml. Aminisol/lb. body weight in the pigs fed the lowbprotein diet and the control are illustrated (Figure 9). The blood.ammonia values resulting from this injection were slightly higher in the pigs fed the low-protein diet than in the control. The blood ammonia levels in the pig fed the low-protein diet and given carbon tetrachloride injections did not markedly differ from those of the pigs fed the lowbprotein diet. Blood ammonia values of pigs fed the lowbprotein diet were increased as a result of feeding a high-protein ration (TABLE 8). -However, the blood.ammonia values of the control pig were elevated to almost the same extent, with the exception of pig 1. Pig 1 had the most consistent elevation of blood ammonia as compared to the control. The differences .mpmpmom mBHuoEEm .94.. .me om mo soapomhca. Amocopfimmammpfi mo pgmmm a no mama/ma masonaa Booam .m snowflm newsman: ca osfl. 52 ONH cm 7 H N m s m w m .Hs\mws m 2! m2 0 0H Hoaucpo AYIL'. HH pone 2888.33 I NH +300 3.3 page :Hopoamlzog a 0'0 . ma no.3 camped-.33 .. I .. .3” 53 .Honecnea QOHpomnqw Hammopflammmapca mo pasmmu m we mam>ma masoEEd vooam mason a“ cede .8H\.Hs OH mo .m onemnm pone gnoooeonzog - Hoapooo n 2400 moan node mfiouonouzog u peso oaoooeouzoq - IIII eHE\emHH z-mmz 54 TABLE 8. Effect of feeding a high-protein diet on daily blood ammonia levels (ug./ml.) of pigs on a low-protein diet alone and in combination with carbon tetrachloride injections. Days of Eeeding a High-Protein Diet Pig No. and Diet 0 l 2 3 4 5 6 7 8 1 Low protein 0.81 --- 5.6 3.7 2.8 2.8 10.0 3.7 2.6 2 Low protein 0.81 --- 2.0 2.8 2.0 2.0 2.0 2.4 3.2 3 Low protein + 0.81 -- 2.0 2.4 2.0 2.4 2.4 2.8 3.6 Carbon tet. 1+ CODtT‘Ol 0.81 ..- 196 le6 1.6 2.8 1.6 le8 1.6 55 in levels of this pig and the control on days 4 and 8 are slight. Pigs 2 and 3 had an increase in blood ammonia values on day 8. Signs of toxicosis were apparent sooner in pigs fed the low- protein diet when intravenously infused with ammonium acetate than in the control. No other differences were observed. Rapid breathing, excessive salivation. and vomition occurred in the pigs fed the loweprotein diet and intraperitoneally injected with 90 mg./1b. ammonium acetate. No toxicosis develOped in the control pig. Toxicosis was not observed in any of the pigs when given Aminisol or fed the high-protein diet. The outstanding gross change was the difference in size between the pigs on the lowa and normal-protein rations (Figure 10). Pigs on the low-protein ration had a dry, scaly skin. The epithelium of the anterior third of the dorsum of the tongue appeared thickened and rough. The livers of pigs fed the low-protein diet were smaller (Figure 11), and there was accentuation of the lobular pattern as compared to the controls. The testicles of the male pigs in this group were smaller than the controls. Microscopically, there was parakeratosis of the tongue in the pigs fed the lowzprotein diet (Figure 12). In some cases fungi were observed in the parakeratotic layers (Figure 13). Parakeratosis was also present in the mucosa of the esOphagus (Figure 14). There were also fungi in the superficial parakeratotic layers (Figure 15). The hepatic cells of the pigs on the lowaprotein ration had vacuolar changes in excess of any observed in the control pigs (Figures 16, 17, 18, and 19). Since sections of liver from.these pigs were negative for excess fat content and were only'slightly positive for glycogen, the vacuolar changes probably represent hydrOpic 56 Figure 10. A pig fed the lowzprotein ration (left) and a control. -1 _..‘- -/ Figure 11. The liver of a pig fed the low-protein diet (right) and of a control (left). Figure 12. Parakeratosis of the tongue. Pig was fed the low-protein diet. Hematoxylin and eosin. x 187. Figure 13. An unidentified fungus (arrow) in the parakeratotic layer illustrated in Figure 12. Hematoxylin and eosin. x 750. 58 Figure 14. Parakeratosis of the esophagus. Pig was fed the low-protein ration. Hematoxylin and eosin. x 187. 1’71 .’. , ._ e 15. An unidentified fungus (arrow) in the para- Hanatcuquin and Figur keratotic layer illustrated in Figure 14. eosin. x 750. Figure 16. Wdropic degeneration of the liver. Pig fed the low-protein diet. Hematoxylin and eosin. x 187. Figure 17. I-hrdrOpic degeneration of the liver. Higher power of Figure 16. Renatoawlin and eosin. x 750. Figure 18. Liver of a control pig. Hematoaquin and eosin. x 187. ' 1 e 19. Liver of a control pig. Higher power of Figure 1-8. Hematoxylin and eosin. x 750. 61 degeneration. Sections of liver from the control pigs were negative for fat and.positive for glycogen. Many cells of the seminiferous tubules of pigs fed the low-protein diet had vacuolar changes in the cytoplasm which were negative for fat and glycogen (Figures 20 and 21). These spaces also probably represent hydropic degeneration and were not present in the control pigs (Figures 22 and 23). There were no differences in the extent or severity of the microscopic lesions of pigs fed the lowbprotein diet and given carbon tetrachloride injec- tions and those fed the lowbprotein diet. Figure 20. Testicle of a pig fed the low-protein diet. Note the numerous vacuoles. Hanatoxylin and eosin. x 187. Figure 21. Seminiferous tubule in the testicle illustrated in Figure 20. Hematoxylin and eosin. x 750. . -t: " h A. - “HQ .. 1' . - h ’1‘ 7‘\ " ' '9 - o\ b ‘ WC: ~ “3 ' “rt. ‘2 r; ’V‘ I ‘5'.” ‘ e «5". "' I '~*1 f. &‘a'.‘#“ I * -'3 ~93 vim!" I .’ ‘~ . ... 3‘ I ‘ . ' ‘ ‘ .O ' ’u 1‘” ’ -"~.*‘ 2 Figure 22. Testicle of a control pig. Hanatoxylin and eosin. x 187. ’ Seninifercus tubule in the testicle Hematoxylin and eosin. x 750. Figure 23. illustrated in Figure 22. EXPERIMENT III. AMMONIA METABOLISM.AS INFLUENCED BY SURGICALLY PRODUCED EXTRAHEPATIC BILIARY OBSTRUCTICN Alteration of the liver of 13 pigs was produced by ligation of the hepatic, common, and cystic bile ducts. The effect of this procedure on ammonia metabolism was studied. In addition, other biochemical and histopathologic changes were evaluated. Experimental Procedure Fifteen 2%-to-4~week-old pigs from 2 farms were used for this experiment. They were fed the MSU growing ration. Feed was withheld 12 hours prior to surgery. A laparotomy was performed to expose the liver and extrahepatic biliary system. Care was taken in the dissec- tion of the bile duct from the surrounding tissue. The hepatic, cystic, and common bile ducts were each ligated with umbilical tape. Bile was aspirated from the distended gallbladder. Closure of the abdomen ‘was made with an interrupted pattern of nylon sutures. Aseptic tech- niques were followed throughout. Blood samples for bilirubin, serum glutamic oxaloacetic transamin- ase (SGOT) and serum glutamic pyruvic transmdnase (SGPT) were taken just prior to and just after surgery. Additional blood samples were collected at 6, 12, 24, 48, 72, 96, and 120 hours after surgery. Following this, in most cases, blood samples were obtained every 48 hours until the pig died or was euthanatized. Prothrombin determina- tions were made when an abnormal clotting time was noted. Two control pigs were given the same surgical treatment, except for ligation of the bile ducts. 65 Three of these pigs, approximately 7 to 8 days after surgery, were used to determine the metabolism of ammonium acetate. They were injected intraperitoneally with 90 mg./lb. of 4% ammonium acetate in 5% dextrose in water. The procedure followed was the same as described in Experiment I. All pigs were observed at least 4 times daily. When a pig was obviously distressed, blood samples for analyses were obtained and euthanasia performed, using pentobarbital sodium intravenously. A necropsy was performed and tissues collected for microscopic study. fiesults The survival time and cause of death of the pigs with a surgically produced biliary obstruction are summarized (TABLE 9). Twelve of 13 pigs developed clinical signs of icterus in 8 to 12 hours. Pig number 1 had a partial obstruction and icterus was observed 40 days after surgery. The intensity of the icterus increased up to the 7th post- surgical day in most of the pigs, decreasing slightly after this time. In pig number 6, however, all the signs of icterus disappeared between the 5th and 7th day after surgery. At the 9th day, icterus was again Observed and remained until the animal died. These pigs did not appear otherwise abnormal, as they were active, alert, and made weight gains (Figure 24). However, at about the 9th day after surgery (some as early as the 4th day and as late as the 23rd day), they suddenly developed signs of anorexia, weakness, ataxia, and died within 24 hours. All pigs with a complete biliary obstruction had a large volume of blood in the gastrointestinal tract, resulting from a bleeding gastric ulcer. This lesion is discussed in detail later. This complication interfered with the maintenance of the pigs for as long a period of time as was originally planned. 66 TABLE 9. Length of survival after surgery and cause of death of pigs with a surgically produced biliary obstruction. Length of Survival Pig No. after Surgery (days) Cause of Death 1* 48 Euthanasia 2 9 Hemorrhage from gastric ulcer 3'”I 42 Euthanasia 4 l7 Hemorrhage from gastric ulcer 5 21 Hemorrhage from gastric ulcer 6 24 Hemorrhage from gastric ulcer 7 ll Hemorrhage from gastric ulcer 8 6 Hemorrhage from gastric ulcer 9 7 Hemorrhage from gastric ulcer 10 6 Hemorrhage from gastric ulcer 11 8 Hemorrhage from gastric ulcer 12 ll Hemorrhage from gastric ulcer 13 5 Hemorrhage from gastric ulcer l4 9 Hemorrhage’from gastric ulcer 15*** 10 Euthanasia 'Partial ligation. ‘*Surgical control. ***Surgical control. 6? Figure 24. Pigs with surgically produced biliary obstruction. Pig on the left 18 days after surgery and pig on the right 20 days after surgery. 68 Information on the bilirubin, SGOT, SGPT, and prothrombin determi- nations of the pigs with biliary obstruction and the control is illustrated (Figures 25 through 34). The total bilirubin levels increased after surgery, reaching a maximum level in 3 to 7 days. In pig number 5, how- ever, the total bilirubin increased until day 13, after which there was a slight decline. The total bilirubin values in pig number 6 correlated with the clinical observations. The direct bilirubin levels paralleled the total bilirubin in almost all instances. The bilirubin values of the control pig were within normal limits. The SGOT values increased 6 hours after surgery in all pigs, including the control. _There were marked quantitative differences in these values in the 48—hour period after surgery. There was only a slight variation in the values after 96 hours, except in pigs number 5, 7, l3, and 14. In these pigs a sharp rise in the level was noted 24 to 48 hours prior to death. The SGPT values of the pigs with biliary obstruction also fluctu- ated, but to a lesser degree than the SGOT values. Abnormal prothrombin times were noted in pigs number 5 and 6“ increased values beginning on days 14 and 19, respectively. Pig number 7 had a prothrombin time of 37.5 seconds just prior to death. The blood ammonia values in the 3 pigs with biliary obstruction and given ammonium acetate at a dosage of 90 mg./1b. are illustrated (Figure 35). The blood ammonia values were essentially the same as those recorded for normal pigs in Experiment I. There were no signs of toxicosis. The outstanding gross lesion in the pigs with biliary obstruction was generalized icterus. The liver was enlarged, having an average 69 Figure 25. Total and direct bilirubin, SGOT, and SGPT levels in pig 2 with surgically produced biliary obstruction. One division on the ordinate corresponds to 1 mg./lOO ml. total and direct bilirubin, and 100 Sigma-Frankel units SGOT and SGPT. 70 MH NH dd 0H eaofiflfl 823 .58 .88 fiofiaflo .33 mm oedema hhomuom means when m m a. m m is m m one 9.0 OH NH Baum .aoom .cdnou Iddfim accede one Hopes 71 . Figure 26. Total and direct bilirubin, SGOT, and SGPI levels in pig 4 with surgically produced biliary obstruction. One division on the ordinate correSponds to l mg./100 ml. total and direct bilirubin and 100 Sigma- Frankel units SGOT and SGPT. mm oedema romeo» menu.- when 72 NN HN ON mH wH Ba OH 0H 3H NH NH HH OH 0 m m. w n 3 n N H o u A. u u d 1 J _ I q q . a a u A a c 1 a n r ..... .- ...... I II ' U I """ 9 “““ ? 'l‘ < I H . “““ ' """ ‘\O l N ' 8" " I ‘ ‘ ‘.‘ -.. '8 ' ' .‘ ‘\‘ .m A d .n .w Baum .808 a.nansnaaam 1 p023 Us devour i m .m 5353 poets - e--e S .88 .. 0:0 . 800m I I HA fiflzdfldfl Hen—.08 I I n ._ NH 73 Figure 27. Total and direct bilirubin, SGOT, SGPT levels and prothrombin times of pig 5 with surgically produced biliary obstruction. One division on the ordinate corresponds to 1 mg. /100 ml. total and direct bilirubin, 100 Sigma- Frankel units SGOT, SGPT. and 5 seconds prothrombin time. 74 am onsmem knew-3» mound amen smnwwwflommmmafiflmasanafifloamoanmen«Ho O ||||| .l'll'l. ||||| *lllll’ IIIIIIII 00‘ IIIIIII .- llll‘UIPOO‘I‘IOOOOO’I . I" H e\ N s s \ M l \ X .1 a \ .\ sss n m . x 2.3 :3 r1 \..-./ X-.. --.--.. i mE-mmemwwmm I ..I X. -- ..... .\ 52238 (x .-.-----.i-----\\-- . a 888 o5 38. . m A m . S q q - 83 58853. . ele . fiestas 888 .. ele . d .58 _- 0:0 .38 II 4 NH flash-no H38 . olo 75 Figure 28. Total and direct bilirubin. SGOT, SGPT levels and prothrombin times of pig 6 with surgically produced biliary obstruction. One division on the ordinate corresponds to 1 mg./lOO ml. total and direct bilirubin, 100 Sigma- Frankel units SGOT, SGPT and 5 seconds prothrombin time. mm opdmflm hummusm poems when 3N mm mm IN om ma ma ma ea ma :H «I NH Ha OH 0 m n w v a m N H o J.‘ a d 4 q u A 1 d d u - a a d 4 d d a u d n d q ----.'I--' ‘0’, I OIIIIIIQIIIIIOI II . . IIIIoIII \\ 47:? YIIIIIII. \ I x s I .a - s I . - a - - . a n I . - I .u I . I x I s a \ \K II s \ I \ \‘n‘l'luli ‘ I If \”I \\ / \\ OI \ It I, \ .\\ III \\ \\ .‘ Ox ofifip canroumpoum I nYIAu cans-H.340» poops I .II. whom I OIIO Boom I nYIlI cwpsufiawp Hmuoe I nYIAu ('\ .T. 4 0 mafia CHDHOhLMOhm .- on \- rr (I... o .p_mm .cwnanflafim c noonwn mflm Hmuob (7\ «H Ha 77 Figure 29. Total and direct bilirubin, SGOT, and SGPT levels in pig 7 with surgically produced biliary obstruction. One division on the ordinate corresponds to 1 mg. /100 m1. total and direct bilirubin and 100 Sigma- Frankel units SGOT and SGPT. mm mnSMIm bow-3n your“: 905 mafimamafioamwnonammao -‘ d d a 4 4 a d d 4 a a d 4 1 Baum .908 .53?" . m lad—"m poo-3n 6nd 309 ..oa finfian I255 .88 .. .88 .. 53.33 H38. 112! fi— '1 l‘ki' nigh 79 Figure 30. Total and direct bilirubin, SGOT and SGPT levels in pigs 8 (left) and 9 (right) with surgically produced biliary obstruction. One division on the ordinate corresponds to 1 mg. /100 ml. total and direct bilirubin and 100 Sigma- Frankel units SGOT and SGPT. om mafia hhowudm scams mhmo N. omsmm H o 80 1 q q a d a d 0! II I. IOIII 0|. 0” IQ\* . H ~ 0 \ o\ L N m 3 m m hmwm d .eoom .cHnsr -HHHm oodeo Ugo Hmpoe m m 0H firEHHB 823 ...-.0 .Hanm I OIIO I. HH 909m.I-YI1I . NH fins-"HHS H38 - olo L NH l 81 Figure 31. Total and direct bilirubin. SGOT, and SGPT levels in pigs 10 (left) and 11 (right) with surgi— cally produced biliary obstruction. One division on the ordinate correSponds to 1 mg. [100 ml. total and direct bilirubin and 100 Sigma- Frankel units SGOT and SGPT. Hm eeeMHe know-5m hop-«d when 0H m m N. w n é m N H o N. 0 m d m N H o 4 j d 1 d + d d 1 q d d WWII“, d 4 01 J. . Illi‘o'lou-- ‘|I\ '6‘---‘ -000! 9|! IIOII I OII Ilo . Ion I9. e H ‘0 . H s s s ~ N 0‘ _ N s s _ \ r m .e m \ s ‘ C \\ III \ . 3 \ \\K 1 3 .II \\ C ’1’ ...-‘0“ ll \ x n .\ 4 h o\\ III \ L N. . d .58 . . doom 63?" I m Idem pOOhfiQ I m 65 1009 . I m I o I 1 OH I OH ... HH EBay-«Han vow-«.3 ICII. L HH :8 IOIIO L NH 806m II .4 NH find-Han flinch. IOIIO 83 Figure 32. Total and direct bilirubin. SGOT and SGPT levels in pigs 12 (left) and 13 (right) with surgi- cally produced biliary Obstruction. One division on the ordinate corresponds to 1 mg. /100 ml. total and direct bilirubin and 100 Sigma- Frankel units SGOT and SGPT. mm 8&3 hpmmpSm pmpmw when mHNHHHOHmmdmmemmHo a I d J 1 4 d d 1 1 d d J - ' ---II'I\\I-'II'-°I||°I/I \ \I. .II I \0 ‘ H III xx . II// x. N ’I’ \ elloz \ m I’, .\\‘ ... . _ e m 4 . . w . Edam d .eomm .cHnee . . .. IHHHm pee-Sh wcm Hmuop 1 m I m rBEHHB 888 I IIO I OH ER .96 ._ OH .88 I I I HH .BEHHE H38 I OIO . HH 1 85 Figure 33. Total and direct bilirubin, SGOT, and SGPT levels in pig 14 with surgically produced biliary obstruction. One division on the ordinate corresponds to l mg./lOO ml. total and direct bilirubin and 100 Sigma- Frankel units SGOT and SGPT. mm eeeNHd bow-Ham non-«d when NH H OH 0 m x. w n d m N H o 4 J1! J W 1a! 1 fl d a q a d 0’ O ’ \ I ._ O'III'O/ \OIIIII'0\ .‘s H II x x / x x N // \ ‘ o . I . gm x I- . . II x 4 d I e I \o I .... . . m I I .\ I \\ w I j RHW I \\1\ I“ A N. . l m I m I. OH fine-"HHS pee-"8 I o... ammmw_l nvziu I .HH 900m I I finfiIHHHHD Hdpa. I 0'0 - I NH . $8 .88 £33 ..HHHN 323 e5 Hera VI!- 87 Figure 34. Total and direct bilirubin, SGOT and SGPT levels in pig 15, a surgical control. One division on the ordinate correSponds to 1 mg. [100 ml. total and direct bilirubin and 100 Sigma- Frankel units SGOT and SGPT. 3m mudwwm row-Ham hop-Ha when nHNHHHOHmmnwndMNHo d ‘1 I .I'Ild--'.'--""r'-"'-'rl-lfl-" sans-HHHHD poo-Ha IOII. swam I OIIO 900m II nun-PHHHHD. H.309 I 0'0 «.IIIHIIYI. Baum .998 .59g IHHHm vegan was Haven. .cOHpoSNQon zhmHHHn moons Iona mHHmonhdm news mmHQ opCH mpmpmoa Edficosrm .QH\.mE om mo coapomnca Hmmcopflpommnpcfl mo pHSmoh m mm mHm>oH chosrm cOOHm .mm omfimHm caused: s.“ 05H. ONH om G d ‘ . Chs CO D~»‘() U\ 43' G\ (V r4 I .HE\.M.9 - 0H 2&2 #H w: I I I HH NH NE I Olo I NH 3 an I xlx - H I 3H I ux r4 90 weight of 4.5% of the body weight, in contrast to the livers of the con- trol pigs weighing 3.0% of the body weight. It was yellowish-tan with a distinct lobular pattern and cut with increased resistance (Figure 36). In a pigs, necrosis was diffusely distributed throughout the liver (Figure 37). The bile ducts were greatly distended (Figure 38) and on cutting appeared thickened. The color and consistency of the bile, in periods up to 11 days, was dark green and mucoid, and from 17 days on was light yellow and watery. There were ulcers in the esophageal region of the stomach in all of the pigs with ligation of the bile ducts (Figure 39). These measured h x 3 cm., on an average, and had distinct margins, especially where bordering the glandular region of the stomach. The surface, to which blood clots adhered, was usually rough and in some'Cases indurated (Figure #0). An ulcer 1 cm. in diameter was found in the mucosa of the duodenum in pig number 7. The outstanding histopathologic lesions in the livers of pigs with surgically produced biliary obstruction were bile stasis, dilata- tion and proliferation of the bile ducts, necrosis, and fibrosis. A summary of these lesions is given (ThBLE 10). Bile stasis was conspicuous in the bile canaliculi and ducts (Figure #1). Bileastained hepatic cells were mainly centrolobular in distribution. The bile ducts in the portal triads were greatly dilated (Figure 42), with bile duct proliferation in the portal and interlobu- lar areas (Figure 43). Fatty metamorphosis, as shown by positive Sudan IV stain, was present in the centrolobular portion of the liver (Figure hh). Necrosis, both focal and diffuse, was present (Figures #5 and #6). Lesions related to necrosis were noticed in the periphery 91 Figure 36. The liver of a 5-week—old pig with surgically produced biliary obstruction. Note the promi- nent lobular pattern. Figure 37. A cross section of the liver from pig 7 with surgically produced biliary obstruction. Note the light gray areas of necrosis. 92 Figure 38. The liver of a pig with surgically pro- duced biliary obstruction. Note the dilated bile duct arrow . 93 I \ Figure 39. Ulcer in the esophageal region of the stomach of a pig with surgically produced biliary obstruc- tion. Figure #0. Ulcer in the esophageal region of the stomach of a pig with surgically reduced biliary obstruc- tion. Closebup of Figure 39. (A Esophagus. (B) Blood clots adhering to ulcerated surface. 99 TABLE 10. Summary of the histopathologic lesions in the livers of pigs with a surgically produced biliary obstruction. Item Days after Fibrosis Bile Duct Necro- Fatty Pig No. Surgery Portal Interlob. Prolif. sis Meta. l #8 + - - - - 2 , 9 + + + - - 3C* #2 - a - - -~ h 17 + + + - + 5 21 + + + + - 6 2b + + + - + 7 11 + + + + - 8 6 + - + - + 9 7 + - + - - 10 6 + — + - - ll 7 + + + - - 12 12 + + + - + 13 5 + — + + + 1n 9 + + + + + 15C* 10 - - - - — + - Present. - - Absent. * - Surgical control. Figure 1+1. Bile stasis. Bile ducts in a portal area. Pig with surgically produced biliary obstruction. Note inflanmatory cells in bile and the portal fibrosis. Hema- toxylin and eosin. x 75. Figure 1+2. Dilatation of a bile duct. Pig with surgically produced biliary obstruction. Hematoaquin and eosin. x 75. 96 4.’ I 2’13.“ l .‘ 2' . .l'VI “ I» were EM..- ; >~ run-1.33.1... I ‘ Figure ’43. Bile duct proliferation. Pig with surgically produced biliary obstruction. Hanatoxylin and eosin. x 187. Figure M. Centrolobular fatty metamorphosis. Pig with surgically produced biliary obstruction. Hema- toxylin and eosin. x 187. :fififi Figure 45. Focal hepatic necrosis. Pig with surgically produced biliary obstruction. Hematoxylin and eosin. x 187. u ' Figure #6. Diffuse hepatic necrosis. Pig with surgically produced biliary obstruction. Hepatic cords (arrow). Hematoxylin and eosin. x 75. 98 of the lobule. They have been termed bile lakes by Obel (1957) and apparently resulted from areas of necrosis becoming filled with an inspis~ sated bile admixed with inflammatory cells. These lesions incited only a slight inflammatory reSponse (Figure 47). Increased fibroblastic activity in the portal area occurred relatively early in experimental biliary obstruction (Figure bl). Inflammatory cells were scattered in the fibrous tissue. Fibrosis in the interlobular areas appeared later (Figure #8). Hist0pathologic examination of the gastric ulcers disclosed numerous interesting features. The lesion usually involved only the stratified squamous epithelium (Figure #9). The surface of the ulcer had a layer of amorphous eosinOphilic staining material heavily infil- trated with nuclear debris. In a few cases fungi were observed in this layer. The inflammatory response in the submucosa included polymorphonuclear eosinophilic and neutrophilic leukocytes, lympho- cytes, macrophages, and fibroblastic proliferation. Degenerative changes, as well as thrombosis, were present in some of the arteries in the submucosa (Figures 50, 51, and 52). Edema in the submucosa was a constant finding. Figure 47. Bile lake. Note inflammatory cells admixed within the strands of bile substance. Hematoxylin‘ and eosin. x 187. Figure 48. Interlobular hepatic fibrosis. Pig with surgically produced biliary obstruction. Hematoxylin and eosin. x 187. 100 \V‘fif 787; W ' Q .14” -§ fig, Figure #9. Ulcer in the esophageal region of the stomach. Pig with surgically produced biliary obstruction. (A) Area of ulceration, (B) Stratified squamous epithelium, (C) Glandular mucosa of the cardiac region of the stomach. Hematoxylin and eosin. x 75. ‘ ‘ J . . ‘ ‘fi: . r: .' _9. O. ‘ _ l / , ’1’, .' f . . . Y - ” ' I *~.\ ‘afi 7‘ _ I ’4 i ‘A 7 3": ".‘g Figure 50. Thrombus in an artery in the submucosa underlying an ulcer in the esophageal region of the stomach. Pig with surgically produced biliary obstruction. Note extensive inflammation in this area. Hematoxylin and eosin. x 187. Figure 51. Vacuolar changes in an artery in the sub- mucosa underlying an ulcer in the asephageal region of the stomach. Pig with surgically produced biliary obstruction. Hematoxylin and eosin. x 750. Figure 52. maline and vacuolar changes in an artery in the submucosa underlying an ulcer in the esophageal region of the stomach. Pig with a surgically produced biliary obstruction. Hematoxylin and eosin. x 750. DISCUSSION These experiments demonstrate that ammonia toxicosis can be pro- duced in the pig. The signs of ammonia toxicosis in the pig, namely, increased respiratory rate, excessive salivation. clonic-tonic con» vulsions and death, are essentially the same as observed in the dog and rat (Karr and Hendricks, l9h9; Warren, 1958). The results of the intravenous infusion of ammonium compounds clearly indicate that toxicosis depends primarily upon the rate of administration per unit of body weight. These results agree with those of Karr and Hendricks (1949) in their study of ammonia toxicosis in the dog. This research further indicates that the toxicity of these compounds is due to the ammonium ion, since the carbon dioxide combining power, bicarbonate, sodium and potassium values remained at levels which are compatible with life (Hoffman, 1964). The decreases in carbon dioxide combining power and bicarbonate were related to the increased respiratory rate of the pigs. There has been considerable interest and research work to determine whether ammonia alone is the cause of this increased respiratory exchange (vanamee g§_§l., 1956). The lesser effects of the infusion of 4% ammonium hydroxide and ammonium carbonate on the carbon dioxide combining power.and bicarbonate are explained by the alkaline nature of these compounds, which tends to counteract the effect of the overbreathing. The intravenous infusion of 1.6$ ammonium hydroxide, however, resulted in a decrease in carbon dioxide combining power and bicarbonate almost comparable to the 102 103 neutral and acidifying ammonium compounds. This is due to the lower concentration of the ammonium hydroxide and the longer infusion time allowing a greater loss of carbon dioxide. The changes in the serum electrolytes compare with those reported in experimental ammonia toxicosis in the dog (Roberts gtflgl., 1956). The toxicosis resulting from the intravenous infusion of ammonium carbonate did not differ from that of the other alkaline ammonium compound. This would suggest that carbamate is not a factor in the toxicity of ammonium carbonate. Ammonium acetate in excess of 300 mg./lb. body weight injected intraperitoneally resulted in death. The signs of toxicosis from intraperitoneal injection were essentially the same as those from the intravenous infusions. No gross or microscopic lesions were found in pigs due to ammonia toxicosis. This finding might be expected, since this was an acute toxicosis with primarily biochemical rather than morphologic change. However, the use of electron microscopy may disclose ultrastructural changes. This research does suggest that hepatic damage resulting from feeding a low-protein diet.and characterized by hydrOpic degeneration may influence the metabolism of ammonium compounds. This was shown by the intraperitoneal infusion of ammonium ace- tate resulting in higher blood ammonia values and signs of toxicosis in pigs fed the lowbprotein diet as compared to pigs fed an optimum protein level, The former also had higher blood ammonia values when a protein hydrolysate was administered. The absence of weight gains and decrease in the serum protein levels of pigs fed the low-protein diet were to be expected. 104 Carbon tetrachloride injections did not increase the severity of hepatic alterations of the pigs fed the low-protein diet as evaluated by changes in ammonia metabolism or microsc0pic lesions. The amount of carbon tetrachloride given apparently was insufficient to produce severe hepatic damage. The pigs with surgically produced biliary obstruction used for the intraperitoneal injection of ammonium acetate apparently had little hepatic change, as evidenced by a normal ammonia metabolism. The origi- nal intention of this eXperiment was to surgically produce a complete biliary occlusion in pigs and maintain them a sufficient period of time for extensive fibrosis and, possibly, collateral portal circula- tion to occur. However, a complication in the form of gastric ulcers made this impossible. The bilirubin values increased after bile duct ligation, reaching maximum values in 5 to 7 days. Thereafter, a gradual decrease occurred. Cornelius gt,§l. (1955) reported essentially the same findings in surgically produced biliary obstruction in the horse. Increased SGOT values correlated with hepatic necrosis in pigs with complete biliary obstruction. Slight increases in SGPT values were noted but did not specifically correlate with necrosis. These results agree with the work of Cornelius gt 3;. (1959), who found that SGOT values increased during hepatic necrosis in the horse, cow, pig, and dog, while elevations of SGPT values were limited to the dog. The prolonged prothrombin times recorded in the pigs with complete biliary obstruction may be explained by a disturbed vitamin K metabolism and/or hepatic dysfunction. Dilatation and proliferation of the bile ducts, necrosis, and fibrosis were the outstanding histOpathologic lesions observed. These 105 findings agree with those of several workers (Richardson, 1911; Mac Mahon.gt_gl., 1929; and Cameron and Oakley, 1932). Portal fibrosis and bile duct proliferation were present in the pigs 5 days after ligation of the bile ducts. Interlobular fibrosis developed 7 to 9 days after surgery. Hepatic necrosis did not seem to follow any chronological pattern. An important finding in this study was the high incidence of gastric ulcers apparently related to the ligation of the bile ducts. Elman and co-workers (1940) found duodenal but not gastric ulcers in dags with surgically produced bile fistulas. Obel (1951) observed ulcers in the esophageal portion of the stomachs of 22% of the pigs in which toxic liver dystrOphy was diagnosed. She was of the Opinion that the ulcers resulted from the detrimental action of peroxides on the gastric mucosa formed from unsaturated fatty acids in the absence of vitamin 8. Several possibilities concerning gastric ulcers in relation to biliary occlusion should be discussed. The absorption of fat soluble vitamins may be impaired, since there is an absence of bile in the intestine. Thus, the theory, proposed by Curtin gtflgl. (1963) that a subclinical vitamin A deficiency may be a factor in gastric ulcers of swine, may have merit. The possible role of vitamin B deficiency in the development of gastric ulcers has been previously stated. The degenerative changes and thrombosis of the blood vessels in the submucosa underlying the ulcer may be important in the pathogenesis of this process. It must be determined, however, that the thrombosis is a precursor to the ulcer and not merely the result of an extension of the inflammatory process occurring in.the submucosal layer. 106 Fungi are likely secondary to the ulcerative process. Perhaps the use of the gnotobiote would clarify this point. The pig appears to be a very useful research animal in studies on ammonia metabolism. This research indicates that in the pig ammonia toxicosis can occur and is influenced by alterations of the liver. The results of the experiment on surgically produced biliary obstruction provide a technique for further studies on the pathOgenesis of gastric ulcers. In addition, it is a technique that may be used to do additional basic research on diseases of the liver. SUMMARY Three experiments were conducted to study acute ammonia toxicosis in the young pig and to evaluate the effects of nutritional, chemical and surgical alteration of the liver on ammonia metabolimn. In the first experiment, it was established that toxicosis caused by intravenous infusion of ammonium acetate, ammonium chloride, ammonium carbonate, and ammonium hydroxide depended on the rate of administra- tion per unit of body weight. The results indicated that the toxicity of these compounds was primarily due to the ammonium ion. Intraperia toneal injection of ammonium acetate in dosages over 300 mg./lb. body weight was fatal. The signs of acute ammonia toxicosis were rapidI respirations later becoming irregular and increased in depth, excessive salivation. clonic-tonic convulsions, and death. No significant gross or microscopic lesions were found in acute ammonia toxicosis. In the second experiment, it was denonstrated that hepatic alteraI- tion by nutritional means does influence ammonia metabolism. Pigs fed a lowbprotein diet had higher blood ammonia values when intraperitoneally injected with ammonium acetate or with a protein hydrolysate, as coma pared to the control animals. The ammonia metabolism of pigs fed the lowuprotein diet and injected with carbon tetrachloride did not differ significantly from those fed only the loweprotein diet. The amounts given apparently were not suf= ficient to cause significant hepatic changes. Clinical signs of icterus appeared within 12 hours in pigs with surgically produced biliary occlusion. Bilirubin levels generally 107 108 increased up to the 5th day, then receded after this time. Following surgery. SGOT and SGPT values were increased, returning to slightly above normal levels in 48 hours. In # pigs the SGOT levels increased markedly 2# to #8 hours prior to death. These values correlated with histopathologic findings of extensive necrosis. Pigs with biliary occlusion, when intraperitoneally injected with ammonium acetate, had blood ammonia values comparable to normal pigs. The outstanding gross lesions in pigs with biliary occlusion were enlarged, yellowiSh-tan livers with distended bile ducts and ulcers in the eSOphageal portion of the stomach. These ulcers were apparently induced by the surgical ligation of the bile ducts. Hemorrhage from the ulcers caused the death of the pigs. 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