Camp. Biochem. Physiol.Vol. lOlC, No. 2, pp. 349-351, 1992
0306~4492/92 S5.00+ 0.00 0 1992Pergamon Press plc
Printed in Great Britain
L-CARNITINE PROTECTS FISH AGAINST ACUTE AMMONIA TOXICITY GEORGE C. TREMBLAY* and TERENCE M. BRADLEY? Departments of *Biochemistry and Biophysics and TFisheries, Animal and Veterinary Sciences, University of Rhode Island, Kingston, RI 02881, U.S.A. (Telephone: 401 792-2201) (Received 16 April 1991) Abstract-l. Juvenile chinook salmon (Oncorhynchus tshawytscha) were injected intraperitoneally (i.p.) with 0.25 M mannitol followed 1 hr later by an i.p. challenge of ammonium acetate. 2. At 10.75mmol ammonium acetate/kg body weight, 98% of the fish showed signs of ammonium
toxicity and 69% died. 3. Substitution of L-carnitine (lO-16mmol/kg) for mannitol afforded striking protection from the subsequent challenge with ammonium acetate; 67% showed no signs of ammonia toxicity and only 4% died. 4. Of other quaternary amines tested, trimethylamine oxide also afforded protection, but betaine and choline did not.
INTRODUCTION Ammonia
toxicityt
is associated
with a wide variety
of disorders in animals and humans. It is a major constraint in aquaculature of many species of fish (Piper et al., 1986), and a common clinical finding in humans afflicted with disorders that compromise liver function (Hoyumpa et al., 1975; Flannery et al., 1982). The report of O’Connor et al. (1984) claiming L-carnitine fully protected mice against what would otherwise have been a lethal injection of ammonium acetate, offered promise of less heroic clinical measures for treating ammonia toxicity. Since it is commonly held that ammonia interferes with energy metabolism, and since carnitine promotes fatty acid oxidation, O’Connor et al. (1984) suggested protection by carnitine might result from restored energy status and stimulation of the urea cycle. Evidence that ureagenesis is accelerated in mice treated with carnitine prior to an ammonia challenge was reported shortly thereafter (Costell et al., 1984). However, Kloiber et al. (1988) subsequently observed protection in mice with other quaternary amines, suggesting a different mechanism of action. Still more perplexing, Deshmukh and co-workers (1988, 1990), in exhaustive studies carefully designed to duplicate the conditions of O’Connor et al. (1984) concluded that the solvent, and not carnitine, afforded protection simply by dilution of the subsequent challenge of ammonia. Indeed, wide variation in the degree of protection afforded by carnitine has also been reported within a single study, using rats (Hearn et al., 1989). Our objectives were (i) to test for protection under conditions in which animals denied quaternary amine received an equal volume of isotonic fluid in its place to control for simple dilution of the subsequent challenge of ammonia and (ii) to $The term “ammonia” refers throughout to the sum of NH, and NH: under conditions specified.
narrow interpretations of the mechanism, should protection be observed, by conducting the test with an ammoniotelic species. MATERIALS AND
METHODS
Animals
Juvenile chinook salmon (Oncorhynchus rshawytscha) hatched and reared from the same parental stock at the University of Rhode Island aquaculture facility (East Farm) were transferred to 300-l holding tanks at least 4 days prior to use, and fasted the last 20-30 hr. Holding tanks were filled to capacity in a flow-through system operated at a rate of about 1 l/min. Groups of 5-7 fish were anesthetized in 6 1 aerated tricane methane sulfonate solution (75 mg/l, adjusted to pH 7 with NaOH). Anesthetized fish were individually blotted with a paper towel, weighed, and fin-clipped for later identification. Average weight was 19.6 k4.6g (Z-_+SD; N = 216). Each fish was then administered an intraperitoneal (i.p.) injection and transferred to an aerated 300-l holding tank with a flow rate of 1 l/min, in groups of 34 fish per tank, for observation. Elapsed time from transfer to the anesthetic solution to return to the holding tank for observation was generally less than 8 min. Fish routinely recovered from anesthesia within 5-10min after transfer from the anesthetic solution. Injection procedure
Salmonids possess small abdominal pores adjacent to the anal vent that drain the peritoneal cavity, an anatomical feature that limits retention of solutions injected into the peritoneal cavity (George er al., 1982). We routinely added food colorina (Cakolor Paste Food Color No. 3120. Maid of ScandinaGa Co., Minneapolis, MN) to solutions to be injected, to aid in the detection of leakage from these pores, and found no evidence of leakage if the sum of the volumes injected did not exceed 12 yl/g body weight; we adhered to that limit throughout. All solutions were injected at or near the ventral mid-line immediately anterior to the pelvic girdle, using a gas-tight 250~1 Hamilton syringe (Fisher Scientific, Medford, MA). Leakage from the injection site was avoided by using a 26 gauge 3/S” needle and drawing mucus from the ventral surface across the injection site with 349
G. C.
350
TREMBLAY
moistened tissue paper immediately upon withdrawing the needle. Owing to the limitation on volume to be injected, the solutions injected were quite concentrated: 2.4 M test solutions for protection and 2.58 M ammonium acetate for the ammonia challenge. The test solutions were injected 1 hr prior to the injection of ammonium acetate. Fish were anesthetized for each injection as described above. Control groups not receiving a test solution prior to the ammonia challenge were injected with an equal volume of isotonic (0.25 M) mannitol instead. The solution of mannitol provided a non-ionic iso-osmotic preparation of a metaboli~aliy inert solute to determine whether intraperitoneal dilution of the subsequent challenge of ammonium acetate might, in itself, confer protection.
and T. M.
BRADLEY
AMMONIA CHALLENGE MANNITOL c 100 ; 80t;
6Om
f
40.
$
20~
::
0
I
NOStCiNS
1
2
Chemicals
IO.S-1.7
Tricane methane sulfonate was purchased from Argent Chemicals (Redmond, WA). A refrigerated stock solution of 5 g/l, adjusted to pH 7 with NaOH, was used to prepare a fresh dilution at 75 mg/l for use as an anesthetic. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). L-carnitine was the inner salt (Sigma Cat. No. C-0158) of the same lot number (16F-0561). The concentration of ammonia in solutions of ammonium acetate was confirmed by Nesslerization.
4
DEATH
mmollkg
L-CARNITINE p
80
6
60
:
40
h
RESULTS
3
STAGE OF AMMONIA TOXICITY
E
20 n
” NOSIGNS Fish were closely monitored for 2.5-3.0 hr after the
challenge of ammonium acetate, and progressive signs of ammonia toxicity were recorded for each fish. Fish that did not recover from anesthesia within 10 min, which was rare, were excluded from the study. Progressive signs of ammonia toxicity were graded as follows: (1) ataxia, gulping at the water surface, (2) sinking to the bottom of the tank but struggling to swim, (3) immobility (settled to the bottom of the tank), muscle spasms (a shuddering action), forceful ventilation (exaggerated movement of the gill o~rculum), (4) convulsions (often with violent propulsion about the tank) and (5) death. Progression from ataxia to death generally occurred within 60-90 min after the challenge with 10.75 mmol ammonium acetate/kg body wt. Each test of a protective agent was accompanied by a companion test with 0.25 M mannitol, which accounts for the larger total pool of observations with mannitol-treated fish. Fish injected with 0.25 M mannitol 1 hr before being injected with ammonium acetate exhibited 69% mortality, with only 8% showing signs no more severe than grade 2 (Fig. 1, top panel). By sharp contrast, few fish treated with L-carnitine 1 hr before the ammonia challenge showed any signs of ammonia toxicity (Fig. I, center panel). At 16mmol L-carnitine/kg body wt there were no mortalities and 83% showed no signs of ammonia toxicity. At lower doses of 10-l 1.7 mmol L-carnitine/kg, only 6% died and 94% showed no signs more severe than grade 2. At 5 mmol L-carnitine/kg, mortalities rose to 27% but 64% still showed no signs more severe than grade 2. To test the possibility that protection against ammonia toxicity might be a non-specific response to quaternary amines, as suggested by Kloiber et nl. (1988), we substituted betaine and choline for Lcarnitine. We did not observe the protection in salmon that Kloiber et al. (1988) reported for similar doses of these amines in mice. On the contrary,
DEATH
ST&E
m
OF k4MONli
16 mmollkg m
10-11.7
TOXlCtTY
mmollkg
TRIMETHYLAMINE
“NO
SIGNS
0
OXIDE
1
STAGE OF h4MON1:
6 mm&kg
DEATH
TOXdTY
Fig. 1. Percent of population exhibiting designated stage as most severe sign of ammonia toxicity when challenged with 10.75 mmol ammonium acetate/kg body wt, administered i.p. Pretreatment 1 hr before ammonia challenge: top panel, 0.25 M mannitol (N = 52); center panel, L-carnitine at 16 (N = l2), 10-l 1.7 (N = 33) and 5 (N = 11) mmol/kg body wt; bottom panel, trimethylamine oxide at 16 (N = 16), 10 (N = 27) and 5 (N = 11) mmol/kg body wt. The volume of 0.25 M mannitol injected varied to equal that used for solutions of L-camitine or trimethyiamine oxide to which results were compared. Combined injections did not exceed 12$/g body wt.
betaine and choline at 7.5-12 mmol/kg body wt were quite toxic to juvenile chinook salmon; the fish did not survive the hour to be challenged with ammonia (data not shown). Fish administered choline at a lower dose of 2.3 mmole/kg body wt survived to be challenged with ammonia, but choline at this lower dose afforded no protection against ammonia toxicity (data not shown). Given these results, protection by t~methylamine oxide was quite unexpected. At doses
351
Carnitine and ammonia toxicity in fish
AMMONIA CHALLENGE MANNITOL, L-CARNITINE,
TMAO
60 % 0
70
F
60
P 0 P
50
U
40
!i
30
T
20
;
10
the suggestion of Kloiber et al. (1988) that protection against ammonia toxicity is an osmotic phenomenon. This could explain why trimethylamine oxide, thought to be a metabolically inert waste product of marine teleosts, compares favorably with carnitine in protecting against ammonia toxicity. Whether the two afford protection by the same mechanism remains to be resolved. Work directed at identifying a biochemical basis for the protective action of L-carnitine and trimethylamine oxide against ammonia toxicity in salmonids is currently in progress. Acknowledgements-This
Fig. 2. Comparison of effectiveness of mannitol, L-carnitine._ and trimethylamine oxide (TMAO) in protecting against a challenge of ammonium acetate. Data are pooled results with 1.0-1.7 mmol/kg body wt mannitol (N = 52), IO-16mmol/kg body wt L-carnitine (N= 45), or 1&16mmol/kg TMAO (N =43). Results shown are percentage of population exhibiting designated stage as most severe sign of ammonia toxicity resulting from subsequent challenge with ammonium acetate at 10.75 mmol/kg body wt.
of 16, 10, and 5 mmol/kg, 88, 79, and 45%, respectively, of fish injected with trimethylamine oxide showed signs no more severe than grade 2 when subsequently challenged with ammonia, though mortalities did occur in all groups (Fig. 1, bottom panel). Pooled results obtained at doses of quaternary amine ranging from 10 to 16 mmol/kg body wt allow a composite comparison of the effectiveness of L-carnitine and trimethylamine oxide in protecting against ammonia toxicity (Fig. 2). DISCUSSION Results reported above extend studies on the putative protective effect of L-carnitine on ammonia toxicity to include animals other than mammals, and support the controversial claim that L-carnitine affords protection against ammonia posioning. By demonstrating this effect in an ammoniotelic species,
acceleration of the urea cycle can no longer be entertained as the central mechanism, though it may still be an important aspect of protection in mammals. The observations of Kloiber et al. (1988) and our findings with trimethylamine oxide open to question whether protection by carnitine is at all related to its metabolic role in fatty acid oxidation. Recent work by Bellei et al. (1989) showing carnitine to prevent ammonia-induced mitochondrial swelling in the absence of fatty acid oxidation lends support to
work was partially supported by a arant (90-34123-5139) from the United States Denartment of Agriculture and by the Rhode Island Agricultural Experiment Station (contribution number 2654). REFERENCES
Bellei M., Battelli D., Guarriero D. M., Muscatello U., Di Lisa F., Siliprandi N. and Bobyleva-Guarriero V. (1989) Changes in mitochondrial activity caused by ammonium salts and the protective effect of carnitine. Biochem. biophys. Res. Commun. 158, 181-188.
Costell M., O’Connor J-E., Miguez M-P. and Grisolia S. (1984) Effects of L-carnitine on urea synthesis following acute ammonia intoxication in mice. Biochem. biophys. Res. Commun. 120, 726733.
Deshmukh D. R. and Rusk C. D. (1988) Failure of L-carnitine to protect mice against ammonia toxicity. Biochem. Med. Metab. Biol. 39, 126130. Deshmukh D. R., Singh K. R., Meert K. and Deshmukh G. D. (1990) Failure of L-carnitine to protect mice against hyperammonemia induced by ammonium acetate or urease injection. Pediatr. Res. 28, 256-260. Flannery D. B., Hsia Y. E. and Wolf B. (1982) Current status of hyperammonemic syndromes. Hepatology 2, 495-506.
George C. J., Ellis A. E. and Bruno D. W. (1982) On remembrance of the abdominal pores in rainbow trout, Salmo gairdneri Richardson, and some other salmonid spp. J. Fish Biol. 21, 643447. Heam T. J., Coleman A. E., Lai J. C. K., Griffith 0. W. and Cooper A. J. L. (1989) Effect of orally administered L-camitine on blood ammonia and L-carnitine concentrations in portacaval-shunted rats. Hepatology 10, 822-828.
Hoyumpa A. M., Greene H. L., Dunn G. D. and Schenker S. (1975) Fatty liver: biochemical and clinical considerations. Am. J. Dig. Dis. 20, 1142-1170. Kloiber O., Banjac B. and Drewes L. R. (1988) Protection against acute hyperammonemia: the role of quaternary amines. Toxicology 49, 83-90. O’Connor J-E., Costell M. and Grisolia S. (1984) Protective effect of L-carnitine on hyperammonemia. FEBS Lett. 166, 331-334.
Piper R. G., McElwain I. B., Orme L. E., McCraren J. P., Fowler L. G. and Leonard J. R. (1986) Fish Hatchery Management. 3rd Edn, pp. 3-59. American Fisheries Society, Washington, D.C.
0306~4492/92 S5.00+ 0.00 0 1992Pergamon Press plc
Printed in Great Britain
L-CARNITINE PROTECTS FISH AGAINST ACUTE AMMONIA TOXICITY GEORGE C. TREMBLAY* and TERENCE M. BRADLEY? Departments of *Biochemistry and Biophysics and TFisheries, Animal and Veterinary Sciences, University of Rhode Island, Kingston, RI 02881, U.S.A. (Telephone: 401 792-2201) (Received 16 April 1991) Abstract-l. Juvenile chinook salmon (Oncorhynchus tshawytscha) were injected intraperitoneally (i.p.) with 0.25 M mannitol followed 1 hr later by an i.p. challenge of ammonium acetate. 2. At 10.75mmol ammonium acetate/kg body weight, 98% of the fish showed signs of ammonium
toxicity and 69% died. 3. Substitution of L-carnitine (lO-16mmol/kg) for mannitol afforded striking protection from the subsequent challenge with ammonium acetate; 67% showed no signs of ammonia toxicity and only 4% died. 4. Of other quaternary amines tested, trimethylamine oxide also afforded protection, but betaine and choline did not.
INTRODUCTION Ammonia
toxicityt
is associated
with a wide variety
of disorders in animals and humans. It is a major constraint in aquaculature of many species of fish (Piper et al., 1986), and a common clinical finding in humans afflicted with disorders that compromise liver function (Hoyumpa et al., 1975; Flannery et al., 1982). The report of O’Connor et al. (1984) claiming L-carnitine fully protected mice against what would otherwise have been a lethal injection of ammonium acetate, offered promise of less heroic clinical measures for treating ammonia toxicity. Since it is commonly held that ammonia interferes with energy metabolism, and since carnitine promotes fatty acid oxidation, O’Connor et al. (1984) suggested protection by carnitine might result from restored energy status and stimulation of the urea cycle. Evidence that ureagenesis is accelerated in mice treated with carnitine prior to an ammonia challenge was reported shortly thereafter (Costell et al., 1984). However, Kloiber et al. (1988) subsequently observed protection in mice with other quaternary amines, suggesting a different mechanism of action. Still more perplexing, Deshmukh and co-workers (1988, 1990), in exhaustive studies carefully designed to duplicate the conditions of O’Connor et al. (1984) concluded that the solvent, and not carnitine, afforded protection simply by dilution of the subsequent challenge of ammonia. Indeed, wide variation in the degree of protection afforded by carnitine has also been reported within a single study, using rats (Hearn et al., 1989). Our objectives were (i) to test for protection under conditions in which animals denied quaternary amine received an equal volume of isotonic fluid in its place to control for simple dilution of the subsequent challenge of ammonia and (ii) to $The term “ammonia” refers throughout to the sum of NH, and NH: under conditions specified.
narrow interpretations of the mechanism, should protection be observed, by conducting the test with an ammoniotelic species. MATERIALS AND
METHODS
Animals
Juvenile chinook salmon (Oncorhynchus rshawytscha) hatched and reared from the same parental stock at the University of Rhode Island aquaculture facility (East Farm) were transferred to 300-l holding tanks at least 4 days prior to use, and fasted the last 20-30 hr. Holding tanks were filled to capacity in a flow-through system operated at a rate of about 1 l/min. Groups of 5-7 fish were anesthetized in 6 1 aerated tricane methane sulfonate solution (75 mg/l, adjusted to pH 7 with NaOH). Anesthetized fish were individually blotted with a paper towel, weighed, and fin-clipped for later identification. Average weight was 19.6 k4.6g (Z-_+SD; N = 216). Each fish was then administered an intraperitoneal (i.p.) injection and transferred to an aerated 300-l holding tank with a flow rate of 1 l/min, in groups of 34 fish per tank, for observation. Elapsed time from transfer to the anesthetic solution to return to the holding tank for observation was generally less than 8 min. Fish routinely recovered from anesthesia within 5-10min after transfer from the anesthetic solution. Injection procedure
Salmonids possess small abdominal pores adjacent to the anal vent that drain the peritoneal cavity, an anatomical feature that limits retention of solutions injected into the peritoneal cavity (George er al., 1982). We routinely added food colorina (Cakolor Paste Food Color No. 3120. Maid of ScandinaGa Co., Minneapolis, MN) to solutions to be injected, to aid in the detection of leakage from these pores, and found no evidence of leakage if the sum of the volumes injected did not exceed 12 yl/g body weight; we adhered to that limit throughout. All solutions were injected at or near the ventral mid-line immediately anterior to the pelvic girdle, using a gas-tight 250~1 Hamilton syringe (Fisher Scientific, Medford, MA). Leakage from the injection site was avoided by using a 26 gauge 3/S” needle and drawing mucus from the ventral surface across the injection site with 349
G. C.
350
TREMBLAY
moistened tissue paper immediately upon withdrawing the needle. Owing to the limitation on volume to be injected, the solutions injected were quite concentrated: 2.4 M test solutions for protection and 2.58 M ammonium acetate for the ammonia challenge. The test solutions were injected 1 hr prior to the injection of ammonium acetate. Fish were anesthetized for each injection as described above. Control groups not receiving a test solution prior to the ammonia challenge were injected with an equal volume of isotonic (0.25 M) mannitol instead. The solution of mannitol provided a non-ionic iso-osmotic preparation of a metaboli~aliy inert solute to determine whether intraperitoneal dilution of the subsequent challenge of ammonium acetate might, in itself, confer protection.
and T. M.
BRADLEY
AMMONIA CHALLENGE MANNITOL c 100 ; 80t;
6Om
f
40.
$
20~
::
0
I
NOStCiNS
1
2
Chemicals
IO.S-1.7
Tricane methane sulfonate was purchased from Argent Chemicals (Redmond, WA). A refrigerated stock solution of 5 g/l, adjusted to pH 7 with NaOH, was used to prepare a fresh dilution at 75 mg/l for use as an anesthetic. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). L-carnitine was the inner salt (Sigma Cat. No. C-0158) of the same lot number (16F-0561). The concentration of ammonia in solutions of ammonium acetate was confirmed by Nesslerization.
4
DEATH
mmollkg
L-CARNITINE p
80
6
60
:
40
h
RESULTS
3
STAGE OF AMMONIA TOXICITY
E
20 n
” NOSIGNS Fish were closely monitored for 2.5-3.0 hr after the
challenge of ammonium acetate, and progressive signs of ammonia toxicity were recorded for each fish. Fish that did not recover from anesthesia within 10 min, which was rare, were excluded from the study. Progressive signs of ammonia toxicity were graded as follows: (1) ataxia, gulping at the water surface, (2) sinking to the bottom of the tank but struggling to swim, (3) immobility (settled to the bottom of the tank), muscle spasms (a shuddering action), forceful ventilation (exaggerated movement of the gill o~rculum), (4) convulsions (often with violent propulsion about the tank) and (5) death. Progression from ataxia to death generally occurred within 60-90 min after the challenge with 10.75 mmol ammonium acetate/kg body wt. Each test of a protective agent was accompanied by a companion test with 0.25 M mannitol, which accounts for the larger total pool of observations with mannitol-treated fish. Fish injected with 0.25 M mannitol 1 hr before being injected with ammonium acetate exhibited 69% mortality, with only 8% showing signs no more severe than grade 2 (Fig. 1, top panel). By sharp contrast, few fish treated with L-carnitine 1 hr before the ammonia challenge showed any signs of ammonia toxicity (Fig. I, center panel). At 16mmol L-carnitine/kg body wt there were no mortalities and 83% showed no signs of ammonia toxicity. At lower doses of 10-l 1.7 mmol L-carnitine/kg, only 6% died and 94% showed no signs more severe than grade 2. At 5 mmol L-carnitine/kg, mortalities rose to 27% but 64% still showed no signs more severe than grade 2. To test the possibility that protection against ammonia toxicity might be a non-specific response to quaternary amines, as suggested by Kloiber et nl. (1988), we substituted betaine and choline for Lcarnitine. We did not observe the protection in salmon that Kloiber et al. (1988) reported for similar doses of these amines in mice. On the contrary,
DEATH
ST&E
m
OF k4MONli
16 mmollkg m
10-11.7
TOXlCtTY
mmollkg
TRIMETHYLAMINE
“NO
SIGNS
0
OXIDE
1
STAGE OF h4MON1:
6 mm&kg
DEATH
TOXdTY
Fig. 1. Percent of population exhibiting designated stage as most severe sign of ammonia toxicity when challenged with 10.75 mmol ammonium acetate/kg body wt, administered i.p. Pretreatment 1 hr before ammonia challenge: top panel, 0.25 M mannitol (N = 52); center panel, L-carnitine at 16 (N = l2), 10-l 1.7 (N = 33) and 5 (N = 11) mmol/kg body wt; bottom panel, trimethylamine oxide at 16 (N = 16), 10 (N = 27) and 5 (N = 11) mmol/kg body wt. The volume of 0.25 M mannitol injected varied to equal that used for solutions of L-camitine or trimethyiamine oxide to which results were compared. Combined injections did not exceed 12$/g body wt.
betaine and choline at 7.5-12 mmol/kg body wt were quite toxic to juvenile chinook salmon; the fish did not survive the hour to be challenged with ammonia (data not shown). Fish administered choline at a lower dose of 2.3 mmole/kg body wt survived to be challenged with ammonia, but choline at this lower dose afforded no protection against ammonia toxicity (data not shown). Given these results, protection by t~methylamine oxide was quite unexpected. At doses
351
Carnitine and ammonia toxicity in fish
AMMONIA CHALLENGE MANNITOL, L-CARNITINE,
TMAO
60 % 0
70
F
60
P 0 P
50
U
40
!i
30
T
20
;
10
the suggestion of Kloiber et al. (1988) that protection against ammonia toxicity is an osmotic phenomenon. This could explain why trimethylamine oxide, thought to be a metabolically inert waste product of marine teleosts, compares favorably with carnitine in protecting against ammonia toxicity. Whether the two afford protection by the same mechanism remains to be resolved. Work directed at identifying a biochemical basis for the protective action of L-carnitine and trimethylamine oxide against ammonia toxicity in salmonids is currently in progress. Acknowledgements-This
Fig. 2. Comparison of effectiveness of mannitol, L-carnitine._ and trimethylamine oxide (TMAO) in protecting against a challenge of ammonium acetate. Data are pooled results with 1.0-1.7 mmol/kg body wt mannitol (N = 52), IO-16mmol/kg body wt L-carnitine (N= 45), or 1&16mmol/kg TMAO (N =43). Results shown are percentage of population exhibiting designated stage as most severe sign of ammonia toxicity resulting from subsequent challenge with ammonium acetate at 10.75 mmol/kg body wt.
of 16, 10, and 5 mmol/kg, 88, 79, and 45%, respectively, of fish injected with trimethylamine oxide showed signs no more severe than grade 2 when subsequently challenged with ammonia, though mortalities did occur in all groups (Fig. 1, bottom panel). Pooled results obtained at doses of quaternary amine ranging from 10 to 16 mmol/kg body wt allow a composite comparison of the effectiveness of L-carnitine and trimethylamine oxide in protecting against ammonia toxicity (Fig. 2). DISCUSSION Results reported above extend studies on the putative protective effect of L-carnitine on ammonia toxicity to include animals other than mammals, and support the controversial claim that L-carnitine affords protection against ammonia posioning. By demonstrating this effect in an ammoniotelic species,
acceleration of the urea cycle can no longer be entertained as the central mechanism, though it may still be an important aspect of protection in mammals. The observations of Kloiber et al. (1988) and our findings with trimethylamine oxide open to question whether protection by carnitine is at all related to its metabolic role in fatty acid oxidation. Recent work by Bellei et al. (1989) showing carnitine to prevent ammonia-induced mitochondrial swelling in the absence of fatty acid oxidation lends support to
work was partially supported by a arant (90-34123-5139) from the United States Denartment of Agriculture and by the Rhode Island Agricultural Experiment Station (contribution number 2654). REFERENCES
Bellei M., Battelli D., Guarriero D. M., Muscatello U., Di Lisa F., Siliprandi N. and Bobyleva-Guarriero V. (1989) Changes in mitochondrial activity caused by ammonium salts and the protective effect of carnitine. Biochem. biophys. Res. Commun. 158, 181-188.
Costell M., O’Connor J-E., Miguez M-P. and Grisolia S. (1984) Effects of L-carnitine on urea synthesis following acute ammonia intoxication in mice. Biochem. biophys. Res. Commun. 120, 726733.
Deshmukh D. R. and Rusk C. D. (1988) Failure of L-carnitine to protect mice against ammonia toxicity. Biochem. Med. Metab. Biol. 39, 126130. Deshmukh D. R., Singh K. R., Meert K. and Deshmukh G. D. (1990) Failure of L-carnitine to protect mice against hyperammonemia induced by ammonium acetate or urease injection. Pediatr. Res. 28, 256-260. Flannery D. B., Hsia Y. E. and Wolf B. (1982) Current status of hyperammonemic syndromes. Hepatology 2, 495-506.
George C. J., Ellis A. E. and Bruno D. W. (1982) On remembrance of the abdominal pores in rainbow trout, Salmo gairdneri Richardson, and some other salmonid spp. J. Fish Biol. 21, 643447. Heam T. J., Coleman A. E., Lai J. C. K., Griffith 0. W. and Cooper A. J. L. (1989) Effect of orally administered L-camitine on blood ammonia and L-carnitine concentrations in portacaval-shunted rats. Hepatology 10, 822-828.
Hoyumpa A. M., Greene H. L., Dunn G. D. and Schenker S. (1975) Fatty liver: biochemical and clinical considerations. Am. J. Dig. Dis. 20, 1142-1170. Kloiber O., Banjac B. and Drewes L. R. (1988) Protection against acute hyperammonemia: the role of quaternary amines. Toxicology 49, 83-90. O’Connor J-E., Costell M. and Grisolia S. (1984) Protective effect of L-carnitine on hyperammonemia. FEBS Lett. 166, 331-334.
Piper R. G., McElwain I. B., Orme L. E., McCraren J. P., Fowler L. G. and Leonard J. R. (1986) Fish Hatchery Management. 3rd Edn, pp. 3-59. American Fisheries Society, Washington, D.C.
