ROLE OF UREA IN THE HYPERAMMONEMIA OF GERM-FREE ECK FISTULA DOGS

Transcription

1 GASTROENTEROLOGY 66: , 1974 Copyright 1974 by The Williams & Wilkins Co. Vol. 66, No.1 Printed in U.S.A. ROLE OF UREA IN THE HYPERAMMONEMIA OF GERM-FREE ECK FISTULA DOGS FRANCIS C. NANCE, M.D., HENRY J. KAUFMAN, M.D., AND DAVID G. KLINE, M.D. Department of Surgery and Division of Neurosurgery, Louisiana State University School of Medicine, New Orleans, Louisiana Hyperammonemia and symptomatic hepatic encephalopathy occur in germ-free (GF) animals after creation of an Eck fistula. The role of urea as a possible substrate for ammonia production in germ-free Eck fistula dogs was assessed in this experiment. End to side portacaval shunts were performed in GF and conventional dogs. After recovery, 40 g of urea in 200 ml of water were given by orogastric tube, and blood ammonia levels measured 3 and 6 hr postingestion, both in GF and conventional Eck fistula dogs. Conventional dogs had massive increases in blood ammonia averaging 483% after urea ingestion. GF dogs had only a minimal change averaging -6,35%. Hydrolysis of urea plays no role in the hyperammonemia of GF Eck fistula dogs, in striking contrast to conventional dogs and probably patients. The experiment provides indirect evidence that there are no endogenous ureases present in the gastrointestinal tract of the dog. In a previous paper,l we reported that germ-free dogs with Eck fistulae develop hepatic encephalopathy. These animals had histologic and biochemical evidence of hyperammonemia and exhibited the clinical symptoms of hepatic coma. In a subsequent paper,2 we reported that, after ingestion of a blood meal, germ-free dogs with Eck fistulae had marked increases in blood ammonia, as great as observed in conventional Eck fistula dogs. This experiment was designed to assess the possible role of urea hydrolysis in the hyperammonemia of germ-free Eck fistula dogs. Received June 26, Accepted August 12, Presented in part at the American Gastroenterological Association, May 24, 1972, Dallas, Texas. Address requests for reprints to: Dr. Francis C. Nance, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, Louisiana Supported by United States Public Health Service Grant AI l, and by facilities Grant RR The considerable technical help of Mrs. Dianne P. Hines and Mrs. Sylvia Steckley is gratefully acknowledged. Methods Conventional dogs used in these experiments were mongrels of both sexes obtained from the Louisiana State University Animal Care Facility. All had received a single injection of penicillin and streptomycin during a 2-week period of quarantine prior to use, but no antibiotics were administered subsequently. Germ-free (GF) dogs were pure bred adult beagles of both sexes obtained from the Louisiana State University Germfree Facility. The animals were cesarean-delivered into flexible film isolators and hand-fed with sterile synthetic bitch's milk until weaning. Techniques of rearing germ-free dogs have previously been published by Heneghan et al." Weekly cultures of fresh stool specimens were obtained to monitor the bacteriologic status of germ-free animals. In addition, terminal cultures were obtained from all animals that died or were killed prior to removal from the isolators to assure continuance of their germ-free state. All animals were fed chow containing 22% protein. The chow was heat-sterilized and supplemented with filter-sterilized vitamins for administration to germ-free dogs. End to side portacaval shunts were per- 108

2 January 1974 UREA IN THE HYPERAMMONEMIA OF ECK FISTULA DOGS 109 formed in 12 germ-free and 10 conventional dogs under pentobarbital anesthesia. Conventional animals received 250 to 500 ml of normal saline intravenously, at the time of operation. Germfree animals were given 250 ml of low molecular weight dextran during the operation to prevent clotting of the anastomosis. All of the animals were allowed at least 2 weeks to recover from surgery before urea ingestion studies were performed. Urea tolerance tests were carried out in a manner similar to that employed for blood ingestion. Fasting germ-free and conventional dogs with Eck fistulae were sedated with propiopromazine 1 hr prior to study. After withdrawing a control blood sample for ammonia, an orogastric tube was passed into the stomach and 40 g of urea dissolved in 200 ml of sterile water were administered. At intervals of 3 and 6 hr after ingestion, blood samples were again ohtained and analyzed for blood ammonia levels. Blood urea nitrogen (BUN) was also measured in some samples. Animals were given water ad libitum after the ingestion of urea. Urea tolerance tests were also performed on 3 unshunted germ-free dogs. The Eck fistulae were inspected for patency in all animals at the time of death or autopsy. Seven conventional dogs survived with patent shunts for 22 to 266 days. Ten germ-free dogs survived for 16 to 166 days. All dogs surviving the surgery developed signs of portosystemic encephalopathy and had hyperammonemia. Two germ-free animals became contaminated with Staphylococcus albus. All data from these 2 animals have been excluded from the study. Blood ammonia levels were measured by the Hyland ion exchange resin technique. Results A total of 10 urea tolerance studies were performed in the 7 conventional animals with patent shunts. All developed massive rises in blood ammonia ranging from 125 to 681% of control values (table 1). Two conventional animals died in coma during the tests. A mean peak rise in blood am Illonia of 483.0% over control levels was observed. Seventeen urea tolerance tests were performed in the 10 germ-free animals with patent Eck fistulae. Changes in blood am Illonia ranged from -54% to +51% in the 6 hr after urea ingestion. The mean peak change observed in all tests was -6.35% (table 2). No significant change in blood ammonia was observed in 3 unshunted germ-free animals after urea ingestion. Blood levels of urea as measured by BUN were higher in GF animals after urea ingestion than in conventional animals, probably because no significant hydrolysis of urea occurred in the GF animals (table 3). TABLE 1. Changes in blood ammonia after urea ingestion in conventional Eck fi stula dogs Animal no. Control ammonia pg/loo ml a Mean change, Died in coma. Peak ammonia Percentage change" pg/loo ml TABLE 2. Changes in blood ammonia after urea ingestion in germ-free Eck fistula dogs Animal Control Peak Percentage no. ammonia ammonia change" pg/ looml pg/ l OOmi a Mean change

3 110 NANCE ETAL. Vol. 66, No. I T ABLE 3. Changes in blood urea nitrogen (BUN) after urea ingestion Mean Mean Group control &eak No. tests BUN UN ' g/i()()mi g/i()()mi Conventional Eck fistula GP Eck fistula GF unshunted a Peak value always occurred at 3 hr. GF, germ-free HOURS POST INGESTION -CONVENTIONAL GERMFREE FIG. 1. Blood ammonia after urea meal. Data points from each experiment are plotted. Note that fasting levels of ammonia were similar in both conventional and germ-free animals. Significant increases in blood ammonia after urea ingestion occurred only in the conventional animals. In summary, germ-free animals experienced little or no increase in blood ammonia after urea ingestion. Conventional animals experienced a massive increase. Data from all urea tolerance studies are summarized in figure 1. Discussion Our previous observations that hepatic encephalopathy could occur in the germfree Eck fistula dog have lead us to explore the possible sources of ammonia in an animal without a microbiologic flora. One obvious site of ammonia production is the kidney, but this mechanism would seem inadequate to explain the marked hyperammonemia we have repeatedly observed in germ-free Eck fistula dogs. We think there is good evidence that the gut is the major source of ammonia production in the germ-free animal (just as it is in the conventional animal): (1) hyperammonemiaonly occurs after diversion of splanchni~ blood flow to the systemic circulation 1; (2) we have consistently, observed elevated ammonia levels in blood obtained from the ileocolic vein ofgf dogs; (3) Warren and Newton 4 measured increased blood ammonia levels in the portal vein of the GF rat; (4) we have observed marked elevations in ammonia after blood ingestion in germ-free animals with portacaval shunts. 2 This experiment was undertaken to evaluate a possible substrate which might be contributing to the production of ammonia in the germ-free Eck fistula dog. Urea is generally conceded to be an important substrate for ammonia production in patients with portosystemic encephalopathy. It has been repeatedly demonstrated both in patients, 5. 6 and in conventional Eck fistula dogs, that the administration of urea is associated with rapid rises in blood ammonia. Urease inhibitors have been successfully administered in the treatment of hepatic encephalopathy. An important controversy has arisen over whether the urea is hydrolyzed only by bacterial enzymes, or whether endogenous, mucosal enzymes might also contribute to the conversion of urea to ammonia in the gastrointestinal tract. Our observations tend to support the concept that urease activity in the gastrointestinal tract is entirely due to the resident bacterial flora. The presence of ureases in the gastric mucosa of a number of vertebrates was first reported by Luck. 7 Since that time several investigators have explored the possibility that a mucosal urease was produced in the stomach or elsewhere in the gastrointestinal tract. It has long been recognized that

4 January 1974 UREA IN THE HYPERAMMONEMIA OF ECK FISTULA DOGS 111 many of the bacterial species residing in the gastrointestinal tract are capable of hydrolyzing urea; this fact has greatly complicated efforts to prove the presence of endogenous nonbacterial ureases. All investigators have agreed that the mucosal urease, if present, is located in the cells near the surface of the mucosa. The ubiquitous presence of urease-producing bacteria has made all efforts at measuring urease activity suspect. Kornberg and Davies 8 in their review, and Dintzis and Hastings 9 in their experiments, concluded, mainly on the basis of the effects of antibiotics on urease activity, that there were no mucosal ureases present in the animals they studied. Conway et al. 10 have vigorously rejected these conclusions and reported significant urease activity in the surface epithelial cells of the mouse stomach. FleshIer and Gabuzda,5 in a careful clinical study, favored the concept that a non bacterial urease was present in the stomach of patients. Belding and Kern 11 thought, on the basis of their studies in cats, that a mucosal urease must be present although they were unable to demonstrate urease activity in fetal gastric mucosa. Aoyagi et al. 6 and Summerskill and WolperP 2 in a number of clinical studies, have concluded that there are probably significant mucosal ureases in man, but they were careful to point out the possibility of bacterial contamination. In the only previous study in germ-free animals, Levenson et al. 13 administered radioactively labeled urea to rats. The urea was given subcutaneously to 4 animals and orally to 1. The breakdown rate of urea in GF animals was less than 1% that observed in conventional animals. Levenson et al. concluded that no endogenous ureases were present in the rat. On the basis of these experiments, there can be little doubt that the hydrolysis of urea plays little or no role in the hyperammonemia of germ-free Eck fistula dogs. The absence of blood ammonia changes in the GF dogs after urea ingestion contrasts strikingly with the massive (and occasionally lethal) rises in blood ammonia in the conventional animals. Even the monocontaminated animals, colonized with Staphylococcus albus carrying weak urease activity, showed no significant changes in blood ammonia. The blood urea nitrogen rose to higher levels in GF animals than in conventional animals. It seems reasonable to postulate that the difference in BUN was the result of the fact that none of the ingested urea was hydrolyzed in the GF dogs. The higher levels of BUN may account for the variability of the ammonia concentrations in the GF animals since the diuresis induced by the urea could be expected to alter water balance. Hydrolysis of urea clearly is a potential source of ammonia in the conventional Eck fistula dog. The rise in blood ammonia occurs rapidly. We have observed levels as high as 1500 /-Lg per 100 ml within 3 hr of the ingestion of urea. Although urea is hypertonic and might be expected to produce rapid intestinal transit, the early increase of ammonia suggests considerable hydrolysis and ammonia absorption may occur proximal to the colon. Although most authors seem to agree that the colon is the main site of ammonia production in patients, there have been no data presented which exclude the possibility of significant ammonia production and absorption in the small bowel. Our experiments have shown that urea hydrolysis by bacterial enzymes and ammonia absorption occur very soon after ingestion. Although the experiment reported here does not deal directly with the question of whether the dog intestine contains an endogenous urease, it does provide strong indirect evidence that such enzymes are absent. The germ-free dog appears incapable of hydrolyzing ingested urea. Our observations do not invalidate present clinical approaches to the therapy of portosystemic encephalopathy. These measures are directed mainly towards bacterial ureases and it is obvious that these may play a large role in the hyperammonemia of patients with portosystemic shunts. REFERENCES 1. Nance FC. Kline DG: Eck's fistula encephalopathy in germfree dogs. Ann Surg 174: ,1971

5 112 NANCE ETAL. Vol. 66, No.1 2. Nance FC, Batson RC, Kline DG: Ammonia production in germ-free Eck-fistula dogs. Surgery 70: , Heneghan JB, Floyd CF, Cohn I Jr: Gnotobiotic dogs for surgical research. J Surg Res 6:24-31, Warren KS, Newton WL: Portal and peripheral blood ammonia concentrations in germfree and conventional guinea pigs. Am J Physiol 197: , Fleshier B, Gabuzda GJ: Effect of ammonium chloride and urea infusions on ammonium levels and acidity of gastric juice. Gut 6: , Aoyagi T, Engstrom GW, Evans WB, et al: Gastrointestinal urease in man. I. Activity of mucosal urease. Gut 7: , Luck JM: Ammonia production by animal tissues in vitro. II. The demonstration of urease in the animal body. Biochem J 18: , Kornberg HL, Davies RE: Gastric urease. Physiol Rev 35: , Dintzis RZ, Hastings AB: The effect of antibiotics on urea breakdown in mice. Proc Nat! Acad Sci 39: , Conway EJ, Fitzgerald 0, McGeeney K, et al: The location and orgin of gastric urease. Gastroenterology 57: , Belding ME, Kern F Jr: Inhibition of urease by oxytetracycline. J Lab Clin Med 61 : , SummerskiJl WHJ, Wolpert E : Ammonia metabolism in the gut. Am J Clin Nutr 23: , Levenson SM, Crowley LV, Horowitz RE, et al: The metabolism of carbon-labeled urea in the germfree rat. Science 234: