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Comparative ammonia toxicity
Comp. Biochem. Physiol., 1968, Vol. 25, pp. 295 to 301. Pergamon Press. Printed in Great Britain
COMPARATIVE A M M O N I A T O X I C I T Y * R. P. W I L S O N , M. E. M U H R E R and R. A. B L O O M F I E L D Department of Agricultural Chemistry, University of Missouri, Columbia (Received 29 August 1967) A b s t r a c t - - 1 . Intravenous and intraperitoneal LDs0 and LD,,.. values for
ammonium acetate were determined in a uricotelic species (Gallus domesticus) and in a ureotelie species (Mus muscu/us). 2. The intravenous LVs0and LD,., values were 2"72 +0.101 and 4"87 _+0"101 m-mole/kg body weight for chicks and 5"64 _+0.083 and 7.67 _+0.083 m-mole/kg body weight for mice. 3. The intraperitoneal LDs0and I.Dga.gvalues were 10.44 _+0.405 and 26.2 _+ 0.405 m-mole/kg body weight for chicks and 10.84_+0.203 and 18.0_+0.203 m-mole/kg body weight for mice. 4. Intravenously, the ammonium acetate was twice as toxic in the chick as in the mouse, indicating in the chick a critical organ more sensitive to the ammonium ion. 5. Intraperitoneally, the ammonium acetate did not differ significantly in tolerance by mice and chicks, indicating that in the avian liver some other pathway may be as efficient for detoxifing exogenous ammonia as the urea cycle in the mouse liver. INTRODUCTION IN VERTEBRATESexisting in a water environment, the problem of detoxiiication and disposal of ammonia is handled by a simple diffusion of ammonia into the surrounding environment. T h e adaptation of the higher vertebrates to a terrestrial environment requires the organism to excrete excess ammonia in a non-toxic form, as either urea or uric acid. T h e various species may be divided into three classes according to the manner in which they excrete excess nitrogen or detoxify ammonia: the ammoniotelic, uricotelic and ureotelic species. Since the relationship between liver disease in man and ammonia toxicity has been established (McDermott, 1957; Brown et al., 1963 ; Dastur et al., 1963 ; Egense, 1963; Zuidema et al., 1963a, b), most of the studies on ammonia toxicity have been limited to the mammalian vertebrate or ureotelic species (Greenstein et al., 1956; Warren, 1958; Warren & Schenker, 1960, 1962; Navazio et al., 1961; Salvatore & Bocchini, 1961 ; Salvatore et al., 1964). In the mammalian vertebrate the collective actions of glutamic dehydrogenase, glutamine synthetase and carbamyl phosphate synthetase have been suggested as being responsible for the extremely low concentrations of ammonia in animal tissue (Brown et al., 1957; K a m i n & Handler, 1957; D u d a & Handler, 1958). However, as one attempts to differentiate the relative * Journal Series No. 5240, approved by the Director of the Missouri Agricultural Experimental Station. 295
296
R. P. WILSON,M. E. MUHRERANDR. A. BLOOMFIELD
importance of each of the various detoxification pathways for ammonia, it seems appropriate to study this toxicity in a uricotelic species which lacks one of the major pathways essential to urea formation, that of carbamyl phosphate synthetase (Bowers & Grisolia, 1962; Tamir &Ratner, 1963a, b; Mora et al., 1965). Snetsinger & Scott (1961) reported the intraperitoneal LDs0 and LDgg.9 values for ammonium sulfate in the chick of 8-3 and 14-2 m-mole/kg body weight, respectively. These values corresponded closely to those reported by Underhill & Kapsinow (1922) in the rat. This investigation was designed to compare the toxicity of exogenous ammonia in a uricotelic species (Gallus domesticus) to that in a ureotelic species (Mus
musculus). MATERIALS AND METHODS Young female albino mice (NLW) (Mus rnusculus) with an average weight of 20 g, and 3- to 4-week-old White Leghorn (Gallus domesticus) male chicks with an average weight of 260 and 346 g were used for the intravenous and intraperitoneal studies. A total of 200 chicks and 180 mice were used in this investigation. One-half to 1.5 ml of isotonic saline containing ammonium acetate (reagent grade) in varying concentrations was injected rapidly into the wing vein of the chicks and into a tail vein of the mice for the intravenous studies and into the peritoneal cavity for the intraperitoneal studies. The lethal effect was determined in ten animals for each of eight to eleven concentration levels. The animals were observed continually until all signs of acute toxicity disappeared and the survivors counted after 1 hr. The animals were maintained on a practical ration and fasted for 24 hr prior to the injection studies. The curves relating per cent mortality and administered dose were computed by the probit method (Cornfield, 1954) with the aid of a computer. The LDs0, LDso,LD,0 and LDg0 values along with the 95 per cent confidence intervals were obtained and plotted on semi-log paper (Figs. 1, 2). Thus the ammonium acetate LDs0value for chicks in Fig. 1 would be 2.72 m-mole/kg but the two dashed lines indicate that it might be as low as 2.53 m-mole or as high as 2.89 m-mole/kg. The standard deviations of tolerated doses were computed according to Gullino et al., 1956. RESULTS When the ammonium acetate was administered in a single rapid intravenous dose, a reaction began immediately in both species, characterized by hyperventilation and clonic convulsions. This was followed by a fatal tonic extensor convulsion among the non-survivors. However, in mice and chicks which survived the reaction differed. The mice which survived showed signs of hyperventilation and hyperirritability but did not go into a coma, whereas the chicks exhibited clonic convulsions followed by a gradual onset of coma. The chicks remained comatose for 20-45 min with signs of clonic convulsions but completely recovered in 50-60 min.
COMPARATIVE
AMMONIA
297
TOXICITY
The reaction to the intraperitoneal dose was quite similar in the two species. It began 10-15 min after injection followed by clonic convulsions accompanied by a gradual onset of coma. The animals then either exhibited a fatal tonic extensor convulsion or recovered in 50-60 rain. ,
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Dosage. m - m o l e s / k g b o ~ weight FIG. 1 . The dose response curves for ammonium acetate administered intravenously to mice (BB l l ) and chicks (@ @). The doses which gave 0 per cent and 100 per cent observed mortality are indicated as (~) and (~), respectively. The dashed lines indicate the 95 per cent confidence intervals.
There was a significant difference in the intravenous LDs0 values for the two species as can be seen in Fig. 1. The intravenous LDso values were 2"72 + 0-101 and 5.64_+0.083 m-mole/kg body weight for chicks and mice, respectively. The intravenous LDag.9 values were 4"87 _+0"101 and 7.67 _+0.083 m-mole/kg body weight for chicks and mice, respectively. However, the intraperitoneal LDs0 values were not significantly different (Fig. 2), being 10.44+0.405 and 10.84 _+0.203 m-mole/kg body weight for chicks and mice, respectively. The intraperitoneal L%9.a values were 26"2 + 0"405 and 18.0 _+0.203 m-mole/kg body weight for chicks and mice, respectively. These data indicate that ammonium acetate, w h e n a d m i n i s t e r e d i n t r a v e n o u s l y , w a s twice as t o x i c to the c h i c k s as to t h e mice.
However, the results show the intraperitoneal toxicity to be about the same in the t w o species.
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FIo. 2. The dose response curves for ammonium acetate administered intraperitoneally to mice ( • i ) and chicks (O • ) . The doses which gave 0 per cent and 100 per cent observed mortality are indicated as (~) and (~), respectively. The dashed lines indicate the 95 per cent confidence intervals. DISCUSSION In the mammalian vertebrate the collective actions of glutamic dehydrogenase, glutamine synthetase and carbarnyl phosphate synthetase have been suggested as being responsible for the extremely low concentrations of ammonia in animal tissue (Brown et a/.,' 1957; Kamin & Handler, 1957), indicating that these enzyme systems are utilized in the detoxification of exogenous ammonia. D u d a & Handler
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(1958), utilizing Nlb-NHs, reported that in the rat glutamine synthesis was the major fate of exogenous ammonia, followed in order of importance by carbamyl phosphate synthetase and glutamic dehydrogenase. Work in our laboratories has also shown glutamine synthesis to be the immediate fate of exogenous ammonia in rats, chicks and sheep (Wilson a al., 1966). Earlier studies by GuUino et al. (1956) and Greenstein et al. (1956) have demonstrated that under certain conditions arginine protects rats treated with a lethal intraperitoneal dose of various L-amino acids or ammonium acetate. An appropriate dose of an ornithine-aspartate mixture was shown to have a similar effect on rats injected intraperitoneally with a LD~0 dose of ammonium acetate (Salvatore & Bocchini, 1961). The protective treatments lowered the free blood ammonia level and increased the amount of plasma urea. Therefore it was assumed that both arginine and the ornithine-aspartate combination stimulated urea formation in vivo from the toxic ammonia. Snetsinger & Scott (1961) have reported that the chick's need for glycine and arginine is greatly accentuated when excess amino acids are incorporated in the ration. They postulated that glycine and arginine function in overcoming the amino acid toxicities by enhancing the excretion of excess nitrogen via the uric acid and urea cycles, respectively. However, recent investigators have been unable to detect carbamyl phosphate formation in the avian liver (Tamir & R a t n e r , 1963a, b; Bowers & Grisolia, 1962; Mora et al., 1965) and attribute the urea present to the action of arginase on dietary arginine (Brown, 1966). Salvatore et al. (1964) have reported evidence that arginine protects against ammonia toxicity through some mechanism other than the one involved in urea synthesis. Therefore, the protective effect indicated by Snetsinger & Scott (1961) may be attributed to a similar nonurea synthesis mechanism in the chick. Previous ammonia toxicity investigations conducted on dogs and sheep in our laboratories indicate that the animal dies from a cardiac death (Wilson et al., 1964). Cardiac effects were observed in the electrocardiograms immediately after a toxic dose of the ammonium salts was injected, and terminated in ventricular fibrillation. Based on these results it is not surprising that the intravenous administration is much more toxic than the intraperitoneal route because of the high blood levels of ammonia resulting immediately. However, it was surprising to note that ammonium acetate was twice as toxic to the chick as to the mouse. This possibly indicates that the heart of the chick may be more sensitive to the ammonium ion than the heart of the mouse. There was no significant difference between the intraperitoneal LDs0 values for the mouse and chick. In this case, one must consider the distribution of the ammonia after administration. Following intraperitoneal injection ofthe ammonium acetate, the ammonia is absorbed via the portal circulation system and passes through the liver before it enters the systemic circulatory system. Thus, this route of administration provides an index of the detoxification capabilities of the animal. As the capabilities of the detoxification enzyme systems are surpassed, systemic blood levels increase to a level which becomes toxic to a critical organ, perhaps the
300
R. P. WILSON, M. E. MUHaER AND R. A. BLOOMFIELD
heart. Therefore, the findings of this study indicate that the avian liver is able to detoxify exogenous ammonia as readily as the mouse liver, even in the absence of one of the major detoxification pathways functional in the mouse, namely carbamyl phosphate synthesis. Probably in the chick some other pathway, possibly the uric acid pathway, may be as efficient in detoxification as the urea cycle in the mouse. Investigations utilizing N15-NHs are being carried out in our laboratories to determine the relative kinetics of these ammonia detoxification enzyme systems in the rat and chick.
Acknowledgement--This work was supported in part by grant GM-12540 from the National Institute of Health. REFERENCES BowERs M. D. & GRISOLIAS. (1962) Biosynthesis of carbamyl aspartate in pigeon and rat tissues. Comp. Biochem. Physiol. 5, 1-16. BROWN G. W. (1966) Studies in comparative biochemistry and evolution--I. Avian liver arginase. Archs Biochem. Biophys. 114, 184-194. BROWNH., BROWNJ. & YAsI-mtrri S. (1963) Hepatic coma and ammonia metabolism in dogs. ft. Am. Med. Assoc. 183, 335-338. BROWN R. H., DUOA G. D., KOaKES S. & HANDLERP. (1957) A colorimetric micromethod for determination of ammonia; the ammonia content of rat tissues and human plasma. Archs Biochem. Biophys. 66, 301-309. CORNEmLDJ. (1954) Statistics and Mathematics in Biology. Iowa State College Press, Ames, Iowa. DASTIm D. K., SESHADRIR. & TALAGERIV. R. (1963) Liver-brain relationship in hepatic coma. Archs intern. Med. 112, 899-916. DUDA G. D. & HANDLERP. (1958) Kinetics of ammonia metabolism in vivo. ft. biol. Chem. 232, 303-314. EGENSEJ. (1963) Ammonia and hepatic coma. Acta med. scand. 173, 7-17. GREENSTEINJ. P., WlNITZ M., GULLINOP., BIRNBAUMS. M. & OTEY M. C. (1956) Studies on the metabolism of amino acids and related compounds in v/vo--III. Prevention of ammonia toxicity by arginine and related compounds. Archs Biochem. Biophys. 64, 342-345. GULLINOP., WINITZ M., BIRNBAUMS. M., CORNFIELDJ., OTEY M. C. & GREENSTEINJ. P. (1956) Studies on the metabolism of amino acids, individually and in mixtures, and the protective effect of L-arginine. Archs Biochem. Biophys. 64, 319-332. KAMIN H. & HANDLERP. (1957) Amino acid and protein metabolism. Ann. Rev. Biochem. 26, 419--490. McDERMOTT W. V. (1957) Metabolism and toxicity of ammonia. New Engl..7. Med. 257, 1076-1081. MORA J., MARTUSCELLIJ., ORTIZPINEDAJ. & SOBERONG. (1965) The regulation of ureabiosynthesis enzymes in vertebrates. Biochem..7. 96, 28-35. NAVAZIOF., GERRITSENT. & WRIGHTG. J. (1961) Relationship of ammonia intoxication to convulsions and coma in rats. ~. Neurochem. 8, 146-151. SALVATORE F. & BOCCHINI V. (1961) Prevention of ammonia toxicity by amino-acids concerned in the biosynthesis of urea. Nature, Lond. 191, 705-706. SALVATORE F., CIMINO F., D'AYELLo-CARACCILOLOi . & CITTADINI D. (1964) Mechanism of the protection by L-omithine-L-aspartate mixture and by L-arginine in ammonia intoxication. Archs Biochem. Biophys. 107, 499-503. SCnENKER S. & WARREN K. S. (1962) Effect of temperature variation on toxicity and metabolism of ammonia in mice. J. Lab. din. Med. 60, 291-301.
COMPARATIVE AMMONIA TOXICITY
301
SNETSINGERD. C. & SCOTTH. M. (1961) The relative toxicity of intraperitoneally injected amino acids and the effect of glycine and arginine thereon. Poultry Sci. 40, 1681-1687. TAMm H. & RAa~m~ S. (1963a) Enzymes of arginine metabolism in chicks. Archs Biochem. Biophys. 102, 249-258. TAMIR H. & RATr,ma S. (1963b) A study of ornithine, citrulline and arginine synthesis in growing chicks. Archs Biochem. Biophys. 102, 259-269. UNDERHILL F. P. & KAPSINOWR. (1922) The comparative toxicity of ammonium salts. ft. biol. Chem. 54, 451--457. WARRENK. S. (1958) The differential toxicity of ammonium salts. J. din. Invest. 37, 497501. WARaF2q K. S. & S C H e m a S. (1960) Hypoxia and ammonia toxicity. Am.ft. Physiol. 199, 1105-1108. WARRENK. S. & SCrmNKERS. (1962) Differential effect of fixed acid and carbon dioxide on ammonia toxicity. Am..7. Physiol. 203, 903-906. WILSON R. P., DAvis L. E., MUHRER M. E. & BLOOMFIELDR. A. (1964) Toxicity of ammonium carbamate..7. Anim. Sci. 23, 1221. WILSON R. P., LETTERA. A., BLOOMFIELDR. A., DAVIS L. E. & MUHRER M. E. (1966) Comparative ammonia metabolism utilizing NlS-ammonia. J. Anim. Sci. 25, 1274. ZUIDEMAG. D., CULLEND., KOWALeZYKR. S. & WOLDMANE. F. (1963a) Blood ammonia reduction by potassium exchange resin. Archs Surg. 87, 296-300. ZUIDEMA G. D., GAIDFORDW. D., KOWALCZYK& WOLFMANE. F. (1963b) Whole-body hypothermia in ammonia intoxication. Archs Surg. 87, 578-582.
COMPARATIVE A M M O N I A T O X I C I T Y * R. P. W I L S O N , M. E. M U H R E R and R. A. B L O O M F I E L D Department of Agricultural Chemistry, University of Missouri, Columbia (Received 29 August 1967) A b s t r a c t - - 1 . Intravenous and intraperitoneal LDs0 and LD,,.. values for
ammonium acetate were determined in a uricotelic species (Gallus domesticus) and in a ureotelie species (Mus muscu/us). 2. The intravenous LVs0and LD,., values were 2"72 +0.101 and 4"87 _+0"101 m-mole/kg body weight for chicks and 5"64 _+0.083 and 7.67 _+0.083 m-mole/kg body weight for mice. 3. The intraperitoneal LDs0and I.Dga.gvalues were 10.44 _+0.405 and 26.2 _+ 0.405 m-mole/kg body weight for chicks and 10.84_+0.203 and 18.0_+0.203 m-mole/kg body weight for mice. 4. Intravenously, the ammonium acetate was twice as toxic in the chick as in the mouse, indicating in the chick a critical organ more sensitive to the ammonium ion. 5. Intraperitoneally, the ammonium acetate did not differ significantly in tolerance by mice and chicks, indicating that in the avian liver some other pathway may be as efficient for detoxifing exogenous ammonia as the urea cycle in the mouse liver. INTRODUCTION IN VERTEBRATESexisting in a water environment, the problem of detoxiiication and disposal of ammonia is handled by a simple diffusion of ammonia into the surrounding environment. T h e adaptation of the higher vertebrates to a terrestrial environment requires the organism to excrete excess ammonia in a non-toxic form, as either urea or uric acid. T h e various species may be divided into three classes according to the manner in which they excrete excess nitrogen or detoxify ammonia: the ammoniotelic, uricotelic and ureotelic species. Since the relationship between liver disease in man and ammonia toxicity has been established (McDermott, 1957; Brown et al., 1963 ; Dastur et al., 1963 ; Egense, 1963; Zuidema et al., 1963a, b), most of the studies on ammonia toxicity have been limited to the mammalian vertebrate or ureotelic species (Greenstein et al., 1956; Warren, 1958; Warren & Schenker, 1960, 1962; Navazio et al., 1961; Salvatore & Bocchini, 1961 ; Salvatore et al., 1964). In the mammalian vertebrate the collective actions of glutamic dehydrogenase, glutamine synthetase and carbamyl phosphate synthetase have been suggested as being responsible for the extremely low concentrations of ammonia in animal tissue (Brown et al., 1957; K a m i n & Handler, 1957; D u d a & Handler, 1958). However, as one attempts to differentiate the relative * Journal Series No. 5240, approved by the Director of the Missouri Agricultural Experimental Station. 295
296
R. P. WILSON,M. E. MUHRERANDR. A. BLOOMFIELD
importance of each of the various detoxification pathways for ammonia, it seems appropriate to study this toxicity in a uricotelic species which lacks one of the major pathways essential to urea formation, that of carbamyl phosphate synthetase (Bowers & Grisolia, 1962; Tamir &Ratner, 1963a, b; Mora et al., 1965). Snetsinger & Scott (1961) reported the intraperitoneal LDs0 and LDgg.9 values for ammonium sulfate in the chick of 8-3 and 14-2 m-mole/kg body weight, respectively. These values corresponded closely to those reported by Underhill & Kapsinow (1922) in the rat. This investigation was designed to compare the toxicity of exogenous ammonia in a uricotelic species (Gallus domesticus) to that in a ureotelic species (Mus
musculus). MATERIALS AND METHODS Young female albino mice (NLW) (Mus rnusculus) with an average weight of 20 g, and 3- to 4-week-old White Leghorn (Gallus domesticus) male chicks with an average weight of 260 and 346 g were used for the intravenous and intraperitoneal studies. A total of 200 chicks and 180 mice were used in this investigation. One-half to 1.5 ml of isotonic saline containing ammonium acetate (reagent grade) in varying concentrations was injected rapidly into the wing vein of the chicks and into a tail vein of the mice for the intravenous studies and into the peritoneal cavity for the intraperitoneal studies. The lethal effect was determined in ten animals for each of eight to eleven concentration levels. The animals were observed continually until all signs of acute toxicity disappeared and the survivors counted after 1 hr. The animals were maintained on a practical ration and fasted for 24 hr prior to the injection studies. The curves relating per cent mortality and administered dose were computed by the probit method (Cornfield, 1954) with the aid of a computer. The LDs0, LDso,LD,0 and LDg0 values along with the 95 per cent confidence intervals were obtained and plotted on semi-log paper (Figs. 1, 2). Thus the ammonium acetate LDs0value for chicks in Fig. 1 would be 2.72 m-mole/kg but the two dashed lines indicate that it might be as low as 2.53 m-mole or as high as 2.89 m-mole/kg. The standard deviations of tolerated doses were computed according to Gullino et al., 1956. RESULTS When the ammonium acetate was administered in a single rapid intravenous dose, a reaction began immediately in both species, characterized by hyperventilation and clonic convulsions. This was followed by a fatal tonic extensor convulsion among the non-survivors. However, in mice and chicks which survived the reaction differed. The mice which survived showed signs of hyperventilation and hyperirritability but did not go into a coma, whereas the chicks exhibited clonic convulsions followed by a gradual onset of coma. The chicks remained comatose for 20-45 min with signs of clonic convulsions but completely recovered in 50-60 min.
COMPARATIVE
AMMONIA
297
TOXICITY
The reaction to the intraperitoneal dose was quite similar in the two species. It began 10-15 min after injection followed by clonic convulsions accompanied by a gradual onset of coma. The animals then either exhibited a fatal tonic extensor convulsion or recovered in 50-60 rain. ,
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Dosage. m - m o l e s / k g b o ~ weight FIG. 1 . The dose response curves for ammonium acetate administered intravenously to mice (BB l l ) and chicks (@ @). The doses which gave 0 per cent and 100 per cent observed mortality are indicated as (~) and (~), respectively. The dashed lines indicate the 95 per cent confidence intervals.
There was a significant difference in the intravenous LDs0 values for the two species as can be seen in Fig. 1. The intravenous LDso values were 2"72 + 0-101 and 5.64_+0.083 m-mole/kg body weight for chicks and mice, respectively. The intravenous LDag.9 values were 4"87 _+0"101 and 7.67 _+0.083 m-mole/kg body weight for chicks and mice, respectively. However, the intraperitoneal LDs0 values were not significantly different (Fig. 2), being 10.44+0.405 and 10.84 _+0.203 m-mole/kg body weight for chicks and mice, respectively. The intraperitoneal L%9.a values were 26"2 + 0"405 and 18.0 _+0.203 m-mole/kg body weight for chicks and mice, respectively. These data indicate that ammonium acetate, w h e n a d m i n i s t e r e d i n t r a v e n o u s l y , w a s twice as t o x i c to the c h i c k s as to t h e mice.
However, the results show the intraperitoneal toxicity to be about the same in the t w o species.
298
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FIo. 2. The dose response curves for ammonium acetate administered intraperitoneally to mice ( • i ) and chicks (O • ) . The doses which gave 0 per cent and 100 per cent observed mortality are indicated as (~) and (~), respectively. The dashed lines indicate the 95 per cent confidence intervals. DISCUSSION In the mammalian vertebrate the collective actions of glutamic dehydrogenase, glutamine synthetase and carbarnyl phosphate synthetase have been suggested as being responsible for the extremely low concentrations of ammonia in animal tissue (Brown et a/.,' 1957; Kamin & Handler, 1957), indicating that these enzyme systems are utilized in the detoxification of exogenous ammonia. D u d a & Handler
COmPA~'~TXW~MOmA TOXmI~
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(1958), utilizing Nlb-NHs, reported that in the rat glutamine synthesis was the major fate of exogenous ammonia, followed in order of importance by carbamyl phosphate synthetase and glutamic dehydrogenase. Work in our laboratories has also shown glutamine synthesis to be the immediate fate of exogenous ammonia in rats, chicks and sheep (Wilson a al., 1966). Earlier studies by GuUino et al. (1956) and Greenstein et al. (1956) have demonstrated that under certain conditions arginine protects rats treated with a lethal intraperitoneal dose of various L-amino acids or ammonium acetate. An appropriate dose of an ornithine-aspartate mixture was shown to have a similar effect on rats injected intraperitoneally with a LD~0 dose of ammonium acetate (Salvatore & Bocchini, 1961). The protective treatments lowered the free blood ammonia level and increased the amount of plasma urea. Therefore it was assumed that both arginine and the ornithine-aspartate combination stimulated urea formation in vivo from the toxic ammonia. Snetsinger & Scott (1961) have reported that the chick's need for glycine and arginine is greatly accentuated when excess amino acids are incorporated in the ration. They postulated that glycine and arginine function in overcoming the amino acid toxicities by enhancing the excretion of excess nitrogen via the uric acid and urea cycles, respectively. However, recent investigators have been unable to detect carbamyl phosphate formation in the avian liver (Tamir & R a t n e r , 1963a, b; Bowers & Grisolia, 1962; Mora et al., 1965) and attribute the urea present to the action of arginase on dietary arginine (Brown, 1966). Salvatore et al. (1964) have reported evidence that arginine protects against ammonia toxicity through some mechanism other than the one involved in urea synthesis. Therefore, the protective effect indicated by Snetsinger & Scott (1961) may be attributed to a similar nonurea synthesis mechanism in the chick. Previous ammonia toxicity investigations conducted on dogs and sheep in our laboratories indicate that the animal dies from a cardiac death (Wilson et al., 1964). Cardiac effects were observed in the electrocardiograms immediately after a toxic dose of the ammonium salts was injected, and terminated in ventricular fibrillation. Based on these results it is not surprising that the intravenous administration is much more toxic than the intraperitoneal route because of the high blood levels of ammonia resulting immediately. However, it was surprising to note that ammonium acetate was twice as toxic to the chick as to the mouse. This possibly indicates that the heart of the chick may be more sensitive to the ammonium ion than the heart of the mouse. There was no significant difference between the intraperitoneal LDs0 values for the mouse and chick. In this case, one must consider the distribution of the ammonia after administration. Following intraperitoneal injection ofthe ammonium acetate, the ammonia is absorbed via the portal circulation system and passes through the liver before it enters the systemic circulatory system. Thus, this route of administration provides an index of the detoxification capabilities of the animal. As the capabilities of the detoxification enzyme systems are surpassed, systemic blood levels increase to a level which becomes toxic to a critical organ, perhaps the
300
R. P. WILSON, M. E. MUHaER AND R. A. BLOOMFIELD
heart. Therefore, the findings of this study indicate that the avian liver is able to detoxify exogenous ammonia as readily as the mouse liver, even in the absence of one of the major detoxification pathways functional in the mouse, namely carbamyl phosphate synthesis. Probably in the chick some other pathway, possibly the uric acid pathway, may be as efficient in detoxification as the urea cycle in the mouse. Investigations utilizing N15-NHs are being carried out in our laboratories to determine the relative kinetics of these ammonia detoxification enzyme systems in the rat and chick.
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