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Hyperammonemia in anorectic tumor-bearing rats
Pergamon Press
Life Sciences, Vol. 43, pp. 67-74 Printed in the U.S.A.
HYPERAMMONEMIA IN ANORECTIC TUMOR-BEARING RATS William T. Chance, Lequn Cao, Jeffrey L. Nelson, Teri Foley-Nelson, and Josef E. Fischer Department of Surgery University of Cincinnati Medical Center Cincinnati, OH 45267 (Received in final form May 9, 1988) Summary Plasma ammonia concentrations were significantly elevated by 150% in anorectic rats bearing methylcholanthrene sarcomas. Assessment of ammonia levels in blood draining these sarcomas indicated nearly a 20-fold increase as compared with venous blood in control rats, suggesting the tumor mass as the source of this increase in ammonia. Infusing increasing concentrations of annnoniumsalts produced anorexia and alterations in brain amino acids in normal rats that were similar to those observed in anorectic tumor-bearing rats. Therefore, these results suggest that ammonia released by tumor tissue may be an important factor in the etiology of cancer anorexia. Anorexia and the associated loss of body tissue (cachexia) are well recognized aspects of neoplastic disease that contribute to morbidity and mortality of cancer patients (1,2). Although animal models of cancer anorexia have been available for several years, studies in this area have not revealed the etiologic mechanisms of this paradoxic response in neoplastic disease (2-5). However, the recent demonstration of the transfer of anorexia to nontumorbearing parabiotic rats that shared a portion of their circulating blood with anorectic tumor-bearing (TB) rats suggests that an anorexigenic factor may be elaborated humorally (6). Although this circulating anorexigenic factor was not identified, reports of a rapid increase in food intake following the resection of tumors in anorectic rats suggest that the source of this factor in vivo experiments suggesting may be the tumor mass (6,7). From in vitro and _that several lines of tumors utiliz~g~utamie as a metabolic substrate (8-ll), we hypothesized that ammonia released from glutamine metabolism may contribute to the anorexia and the neurochemical aberrations observed in TB rats (7). In the present series of experiments, we investigated this hypothesis by measuring plasma ammonia, plasma amino acid and brain amino acid concentrations in anorectic TB rats. In addition, we also assessed the effects of intravenous infusion of ammonia on these parameters and on food intake in normal non-TB rats. Materials and Methods Experiment 1 Twelve adult (250-300 g) male, Fischer 344 rats tories, Wilmington, MA) were anesthetized with ether with approximately 50 mg of fresh methylcholanthrene a 4 mm diameter trocar. This tumor tissue was taken
(Charles River Laboraand were inoculated (SC) (MCA) sarcoma tissue using from a donor rat and has
0024-3205/88 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
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Hyperammonemia and Cancer
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been maintained by serial transplantation for several years. Following transplantation of this sarcoma, food intake is typically reduced to a significant degree within three weeks, with the TB rats usually surviving an additional two to three weeks depending on tumor growth rate (12,13). An additional 6 rats served as controls, being anesthetized and subjected to sham inoculations. All rats were housed individually and were maintained -ad lib. on rat chow and water. Bodyweight, food intake, and water intake were determined for these animals for the next 37 days. Thirty days after tumor inoculation, each rat was anesthetized with ether and 1 ml of blood was taken by cardiac puncture for the determination of ammonia concentrations. All rats were sacrificed by decapitation on day 37. Blood and brain tissue (cerebral cortex and diencephalon) were retained for the assay of plasma and brain amino acid concentrations and plasma ammonia levels. Tumors were removed and weighed. Concentrations of ammonia were determined enzymatically (14) in 100 ~1 of plasma employing the glutamate dehydrogenase reaction. Levels of free amino acids were assayed in plasma and brain following deproteinization with 5% sulfosalicylic acid on a Beckman 121-MB automated amino acid analyzer according to our previously-published procedures (10). Experiment 2 In order to determine whether the tumor mass was the source of elevated ammonia concentration in the blood, MCA sarcomas were transplanted into 6 Fischer 344 rats. When the tumor weight was estimated from planar measurement (13) to be greater than 50 g, the 6 TB and 6 control rats were anesthetized with ether and the surface of the sarcomas was surgically exposed. Using a 25 ga heparinized needle and a 1 ml syringe, blood was taken from the superficial veins draining the tumor mass for the determination of ammonia concentrations in the plasma. Similar samples were obtained from the descending aorta and the vena cava of these TB and control rats. Experiment 3 To assess the effects of elevated blood ammonia levels on food intake and amino acid profiles, silastic catheters (#602-155, Dow Corning, Midland, MI) were implanted into the external jugular vein of 12 Fischer 344 rats (225-250 9). These surgeries were conducted under aseptic conditions according to our previously-published procedures (10). An additional 5 rats served as operated control subjects in this experiment. Immediately following surgery, the catheter of each rat was connected to a feed-through swivel and a peristaltic pump was employed to infuse normal saline at a rate of 2 ml/hr. Three days later, ammonia infusions were begun in one group of 6 rats at the same rate and an initial concentration of 0.14 M (equal parts of ammonium acetate and amnonium bicarbonate; pH = 7.9). On each subsequent day the concentration of ammonia was increased by approximately 0.1 M, until a final concentration of 0.4 M ammonium salts was reached on the fourth day of infusion. The other group of catheterized rats continued to be infused with normal saline across these four days, while no treatment was administered to the operated control rats. All rats had rat chow and water available -7 ad lib. during this experiment. Food intake and bodyweight were determined dally and all rats were sacrificed 24 hrs after the initiation of the 0.4 M ammonia infusion. Plasma and brain tissue were retained at sacrifice for the analysis of amino acid concentrations, Statistical evaluation of the results was accomplished using analysis of variance techniques. -Post hoc comparisons of individual pairs of means were accomplished by J tests.
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Results wstrated in Fig 1, consistently-reduced intake of rat chow was first observed 21 days aft& the transplantation of fresh tumor tissue. Mean estimated tumor weight at this time point was 26 + 2 g and constituted approximately 8% of the mean total body weight (315 + 6 g). The mean body weight of control rats at this time (day 21) was 312 * 3 g. Food intake by the T8 rats continued to decrease for the duration of the experiment. An additional drop in feeding was observed in both groups of rats following the cardiac punctures on day 30. However, the TB rats exhibited a greater decrease in food intake following this removal of 1 ml of blood. Although water intake (data not shown) roughly followed food intake, water consumption was not reduced significantly in TB rats until day 30 (20.4 +1.3 g vs. 25.4 + 1.0 g). Similar differences in water intake were maintained until sacrifice.
0 TUMOR h= 12) 6
CONTROL (n- 6)
0”“““““““““““““““““” 1 3 6 6 11 13
16 I7
13 21 23 26 27 26 31 33 31 37
DAYS POST TUMOR IMPLANTATION
FIG. 1 Mean (* SEM) daily intake of rat chow by tumor-bearing and control rats. Significantly (~(0.01 by t statistic) reduced food intake was consistently observed beginning on day 21. On day 30 all rats were anesthetized and 1 ml of blood was drawn by cardiac puncture for the analysis of ammonia and amino acid concentrations. The enzymatic assay of amnia concentration revealed significant (~(0.01) increases in ammonia of TB rats in blood taken by cardiac puncture on day 30 (138 + 56 vs. 56 ? 10 nmol/ml) as well as following decapitation on day 37 (243 f 18 vs. 60 + 4 nmol/ml). Furthermore, ammonia levels increased significantly (p
Hyperammonemia and Cancer
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Vol. 43, No. 1, 1988
BCAA being unaltered and concentrations of glutamine being nearly doubled. The other LNAA were also greatly elevated by factors of 2 to 3 in the brains of these TB rats. TABLE 1 Mean (2 SEM) concentrations of glutamine and neutral amino acids in the plasma (nmol/ml) and the brain (nmol/g) of control and tumor-bearing (TB) rats. Amino Acid -Glutamine Valine Isoleucine Leucine Methionine Tyrosine Phenylalanine Tryptophan Histidine
Brain
Plasma Control 799 f 55 277 + 20 123 + 9 191 f 9 49 + 2 113 + 7 70 f 3 123 f 6 83 ?r 3
515 139 60 86 73 142 100 105 96
TB 23* f 5* f 2* + 4* f 2” + 5* * 3* * 3* + 5
Control 5016 * 119 79 f 2 32 2 1 68 f 1 26 + 1 64 f 1 37 f 1 13 f 1 56 k 2
9104 78 34 63 82 204 120 23 135
TB 298* lr 2 * 1 + 2 f 4* f 9* f 5* t l* 1 5*
*p
Hyperammonemiaand Cancer
vol. 43, No. 1, 1988
71
0.0 -
7.0 -
0 g 5 2
s.o-
LO -
iiij
4.0-
2
5.0 -
Y 2.0 -
LO-
I
I
I
I
I
8
9
0.14
0.20
O.SS
NH3
CWCEllTRATKm
0.40 0
FIG. 2 Mean (k SEM) daily intake of rat chow by operated control, saline-
infused and amnonia-infused rats. Normal saline was infused initially at a rate of 2 ml/hr. Three days later, ammonia infusions were begun in one roup of 6 rats at the same rate and an initial concentration of 0.14 M 9equal parts of ammonium acetate and ammonium bicarbonate; pH = 7.9). The concentration of ammonium salts was increased on each subsequent infusion day to a maximum of 0.4 M on the fourth day of atmnonia infusion.
Mean plasma and brain amino acid profiles for this experiment are presented in Table 2. Plasma levels of glutamine, phenylalanine and tryptophan were increased significantly, while concentrations of tyrosine, methionine and the BCAA were decreased in ansnonia-infused rats. As in the previous experiment, a different amino acid profile was observed in the brain with all of the LNAA and glutamine exhibiting large increases in ammonia-infused rats.
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vol. 43, No. 1, 1988
Hyperammonemia and Cancer
TABLE 2 Mean (k SEM) concentrations of glutamine and neutral amino acids in the plasma (nmol/ml) and the brain (nmol/g) of saline-infused (n = 6), ammonia-infused (n = 6) and operated control (n = 5) rats. Amino Acid -Glutamine Valine Is01 eucine Leucine Methionine Tyrosine Phenylalanine Tryptophan Histidine
Control Plasma --
591+11
Brain
4231544
Saline-Infused -Plasma
617526
Brain --
4608+75
272k13
94*2
264+10
88*5
20727 57*2 lOOk3 65?2 1151t2 71*2
82*1 34+1 65k2 33*1 15*1 46~1
21529 6121 106kl 6922 11723 7822
721t4 28+2 60+_2 30+1 15*1 47+1
119k3
42+1
119k6
36k3
Ammonia-Infused -Plasma
Brain
942?55*t 24074+1364*t 142+5*t
70+3*t 123+4*t 48+1*t
71r3*t 93+.1*t 128+4*t 71+2
135*11*4
70+4*t
132+10*?
70+3*t
167+8*t
187+14*t 44*5*t 154+8*t
*p
Vol. 43, No. 1, 1988
Hyperammonemia and Cancer
73
secondary to the accumulation of ammonia in the brain or secondary to the biochemical alterations associated with cerebral detoxification of ammonia. Significant elevation in blood urea nitrogen (BUN) has been reported for MCA sarcoma-bearing rats (6). A decrease in gluconeogenesis from lactate has also been observed in hepatocytes taken from rats bearing MCA sarcomas that were at least 14% of total body weight (19). The contribution of ammonia to ureagenesis and the reduction of gluconeogenesis by amnonia (20) suggest that both of these observations may be secondary to hyperannnonemia in anorectic TB rats. The detoxification of ammonia by cerebral tissue results in greatly elevated concentrations of glutamine in the brain. Additional alterations in brain chemistry associated with elevated ammonia concentrations involve the increased influx of LNAA (21). The influx of LNAA appears to be coupled with the efflux of glutamine from the brain through the sharing of a corrmOncarrier system for transport across the blood-brain barrier (21). Therefore, as glutamine is transported out of the brain, tyrosine, tryptophan, phenylalanine, methionine, histidine and the BCAA are allowed increased entry. Since the LNAA share this transport mechanism, they compete with each other for transport across the blood-brain barrier (22). The blood concentrations also influence the transport so that those LNAA that are in higher concentration will compete for carrier sites more effectively. Thus, the BCAA are not elevated in the brains of fB rats because of their reduction in plasma. The elevation in BCAA observed in the ammonia-infused rat brains appears to be related to their higher content of glutamine, which would tend to increase transport of all of the LNAA. Although tryptophan does not appear to follow the above rule, having low plasma and high brain concentrations, previous research.in our laboratory has demonstrated that the percentage of tryptophan that is not bound to albumin is actually increased in TB rats (12). Thus, the free portion of tryptophan, which competes more effectively for transport into the brain, is increased as compared to control rats. Therefore, the end result of cerebral detoxification of ammonia in TB rats appears to be greatly increased brain concentrations of glutamine, tyrosine, tryptophan, phenylalanine, methionine and histidine. Although these alterations in ammonia and amino acids may be sufficient to cause anorexia (23), tyrosine and tryptophan are precursors for the synthesis of the amine neurotransmitters, dopamine (DA), norepinephrine (NE) and serotonin (5-HT). Under appropriate conditions, elevations in these precursors in the brain result in increased synthesis and release of DA and 5-HT (24,25). Previous studies sug est that both DA and 5-HT metabolism are elevated in anorectic TB rats (7B. Although the elevation in 5-HT metabolism may be secondary to the anorexia (26,27), the increase in DA metabolism may have more direct effects on food intake (27). Therefore, several alterations are present in the brains of anorectic TB rats that appear sufficient to cause anorexia. However, this cascade of biochemical aberrations appears to be set in motion by the metabolism of specific amino acids by tumor tissue and the subsequent release of arrmonia. Thus, the most logical treatment regimen to correct this anorexia may be to: 1) reduce amino acid metabolism by the tumor, 2) facilitate detoxification of ammonia by the host, or 3) reduce the transport of LNAA across the blood-brain barrier. Acknowledgement Supported in part by grant 886834 from the American Institute for Cancer Research.
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References 1. S. WARREN,Am. J. Med. Sci. 184 610-615 (1932). 2. S.D. MORRISON, Physiol. Behav.17 705-714 (1976). 3. S. GARATTINI, A. BIZZI, M.G. DOELLI, A. GUAITANI, R. SAMANINand F. SPREAFICO, Cancer Treat. Rep. 7 115-140 (1980). 4. W.D. DeWYS, Cancer 43 2013-2019 (1979). 5, A. THEOLOGIDES,AnnrN.Y. Acad. Sci. 230 14-22 (1974). 6. J.A. NORTON, J.F. MOLEY, M.V. GREEN, m. CARSON and S.D. MORRISON, Cancer Res. 45 5547-5552 (1985). 7. W.T. mANCE, L. CAO, J.L. NELSON, T. FOLEY-NELSON and J.E. FISCHER, Am. J. Surg. 155 124-130 (1988). 8. L.A. SmR, J.W. STAYMAN and R.T. DAUCHY, III, Cancer Res, 42 4090-4097 (1982). 9. G. WEBER, M.S. LUI, J. SEBOLDT and M.A. FADERAN, Glutamine Metabolism in Mammalian Tissues, D. Haussinger and H. Sies (eds),m8-291, Sprin?j%Verlag, Bem984). 10. W.T. CHANCE, L. CAO, J.L. NELSON, T. FOLEY-NELSON and J.E. FISCHER, Surgery 102 386-394 (1987). 11. m. CARRASCOSA, P. MARTINEZ and I.N. DE CASTRO, Cancer Res. -44 3831-3835 (1984). 12. W.T. CHANCE, M.F. VON MEYENFELDT and J.E. FISCHER, Neurosci. Biobehav. Rev. 7 471-479 (1983). 13. W.T. CHANCE, F.M. VAN LAMMEREN, M.H. CHEN, R.F. MURPHY, S.N. JOFFE and J.E, FISCHER, J. Surg. Res. 36 490-498 (1984). 14, J. FOLBERGROVA, J.V. POEONNEAU, O.H. LOWRY and D.W. SCHULZ, J. Neurochem. 16 191-203 (1969). 15, KT. CHANCE, L. CAO, M.W. KIM, J.L. NELSON and J.E. FISCHER, Life Sci. -42 87-94 (1988). 16. K. NODA, J. Nutr, 105 508-516 (1975). 17. M.H. KYU and J.B. i%iANAGH, Br. J. Exp. Path. 51 217-227 (1970). 18, M.D. TRICKLEBANK,J.L. SMART, D.L. BLOXAM and c CURZON, Pharmacol. Biochem. Behav,'9 181-189 (1978). 19. R.I. INi%LET, J.L. PEACOCK, C.M. GORSCHBOTH and J.A. NORTON, J. Natl. Cancer Inst. 79 1039-1046 (1987). 20. H.A. KEBS, P. LUND and M. STUBBS, Gluconeogenesis: Its Regulation in Mammalian Species, R.W. Hanson and M.A. Hehlman (eds),pp. 269-291,Wiley & Sons, New York (1976). 21. J.H. JAMES, B. JEPPSSON, V. ZIPARO and J.E. FISCHER, Lancet 1. 772-775 (1979). 22. W.M. PARDRIDGE, J. Neurochem. 28 103-108 (1977). 23, Q.U. ROGERS and P.M.B. LEUNG, Ed. Proc. 32 1709-1719 (1973). 24. R.J. WURTMAN and J.D. FERNSTROM, Biochem.Pharmacol. 25 1691-1696 (1976). 25. A. CARLSSON and M. LINDQVIST, Naunyn-Schmiedeberg's AEh. Pharmacol. -303 157-164 (1978). 26. W.T. CHANCE,M.F. VON MEYENFELDT and J.E. FISCHER, Pharmacol. Biochem. Behav. 18 115-121 (1983). 27. W.T. CHmCE, T. FOLEY-NELSON, J.L. NELSON and J.E. FISCHER, Brain Res. 416 228-234 (1987).
Life Sciences, Vol. 43, pp. 67-74 Printed in the U.S.A.
HYPERAMMONEMIA IN ANORECTIC TUMOR-BEARING RATS William T. Chance, Lequn Cao, Jeffrey L. Nelson, Teri Foley-Nelson, and Josef E. Fischer Department of Surgery University of Cincinnati Medical Center Cincinnati, OH 45267 (Received in final form May 9, 1988) Summary Plasma ammonia concentrations were significantly elevated by 150% in anorectic rats bearing methylcholanthrene sarcomas. Assessment of ammonia levels in blood draining these sarcomas indicated nearly a 20-fold increase as compared with venous blood in control rats, suggesting the tumor mass as the source of this increase in ammonia. Infusing increasing concentrations of annnoniumsalts produced anorexia and alterations in brain amino acids in normal rats that were similar to those observed in anorectic tumor-bearing rats. Therefore, these results suggest that ammonia released by tumor tissue may be an important factor in the etiology of cancer anorexia. Anorexia and the associated loss of body tissue (cachexia) are well recognized aspects of neoplastic disease that contribute to morbidity and mortality of cancer patients (1,2). Although animal models of cancer anorexia have been available for several years, studies in this area have not revealed the etiologic mechanisms of this paradoxic response in neoplastic disease (2-5). However, the recent demonstration of the transfer of anorexia to nontumorbearing parabiotic rats that shared a portion of their circulating blood with anorectic tumor-bearing (TB) rats suggests that an anorexigenic factor may be elaborated humorally (6). Although this circulating anorexigenic factor was not identified, reports of a rapid increase in food intake following the resection of tumors in anorectic rats suggest that the source of this factor in vivo experiments suggesting may be the tumor mass (6,7). From in vitro and _that several lines of tumors utiliz~g~utamie as a metabolic substrate (8-ll), we hypothesized that ammonia released from glutamine metabolism may contribute to the anorexia and the neurochemical aberrations observed in TB rats (7). In the present series of experiments, we investigated this hypothesis by measuring plasma ammonia, plasma amino acid and brain amino acid concentrations in anorectic TB rats. In addition, we also assessed the effects of intravenous infusion of ammonia on these parameters and on food intake in normal non-TB rats. Materials and Methods Experiment 1 Twelve adult (250-300 g) male, Fischer 344 rats tories, Wilmington, MA) were anesthetized with ether with approximately 50 mg of fresh methylcholanthrene a 4 mm diameter trocar. This tumor tissue was taken
(Charles River Laboraand were inoculated (SC) (MCA) sarcoma tissue using from a donor rat and has
0024-3205/88 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
68
Hyperammonemia and Cancer
Vol. 43, No. 1, 1988
been maintained by serial transplantation for several years. Following transplantation of this sarcoma, food intake is typically reduced to a significant degree within three weeks, with the TB rats usually surviving an additional two to three weeks depending on tumor growth rate (12,13). An additional 6 rats served as controls, being anesthetized and subjected to sham inoculations. All rats were housed individually and were maintained -ad lib. on rat chow and water. Bodyweight, food intake, and water intake were determined for these animals for the next 37 days. Thirty days after tumor inoculation, each rat was anesthetized with ether and 1 ml of blood was taken by cardiac puncture for the determination of ammonia concentrations. All rats were sacrificed by decapitation on day 37. Blood and brain tissue (cerebral cortex and diencephalon) were retained for the assay of plasma and brain amino acid concentrations and plasma ammonia levels. Tumors were removed and weighed. Concentrations of ammonia were determined enzymatically (14) in 100 ~1 of plasma employing the glutamate dehydrogenase reaction. Levels of free amino acids were assayed in plasma and brain following deproteinization with 5% sulfosalicylic acid on a Beckman 121-MB automated amino acid analyzer according to our previously-published procedures (10). Experiment 2 In order to determine whether the tumor mass was the source of elevated ammonia concentration in the blood, MCA sarcomas were transplanted into 6 Fischer 344 rats. When the tumor weight was estimated from planar measurement (13) to be greater than 50 g, the 6 TB and 6 control rats were anesthetized with ether and the surface of the sarcomas was surgically exposed. Using a 25 ga heparinized needle and a 1 ml syringe, blood was taken from the superficial veins draining the tumor mass for the determination of ammonia concentrations in the plasma. Similar samples were obtained from the descending aorta and the vena cava of these TB and control rats. Experiment 3 To assess the effects of elevated blood ammonia levels on food intake and amino acid profiles, silastic catheters (#602-155, Dow Corning, Midland, MI) were implanted into the external jugular vein of 12 Fischer 344 rats (225-250 9). These surgeries were conducted under aseptic conditions according to our previously-published procedures (10). An additional 5 rats served as operated control subjects in this experiment. Immediately following surgery, the catheter of each rat was connected to a feed-through swivel and a peristaltic pump was employed to infuse normal saline at a rate of 2 ml/hr. Three days later, ammonia infusions were begun in one group of 6 rats at the same rate and an initial concentration of 0.14 M (equal parts of ammonium acetate and amnonium bicarbonate; pH = 7.9). On each subsequent day the concentration of ammonia was increased by approximately 0.1 M, until a final concentration of 0.4 M ammonium salts was reached on the fourth day of infusion. The other group of catheterized rats continued to be infused with normal saline across these four days, while no treatment was administered to the operated control rats. All rats had rat chow and water available -7 ad lib. during this experiment. Food intake and bodyweight were determined dally and all rats were sacrificed 24 hrs after the initiation of the 0.4 M ammonia infusion. Plasma and brain tissue were retained at sacrifice for the analysis of amino acid concentrations, Statistical evaluation of the results was accomplished using analysis of variance techniques. -Post hoc comparisons of individual pairs of means were accomplished by J tests.
Hyperammonemia and Cancer
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69
Results wstrated in Fig 1, consistently-reduced intake of rat chow was first observed 21 days aft& the transplantation of fresh tumor tissue. Mean estimated tumor weight at this time point was 26 + 2 g and constituted approximately 8% of the mean total body weight (315 + 6 g). The mean body weight of control rats at this time (day 21) was 312 * 3 g. Food intake by the T8 rats continued to decrease for the duration of the experiment. An additional drop in feeding was observed in both groups of rats following the cardiac punctures on day 30. However, the TB rats exhibited a greater decrease in food intake following this removal of 1 ml of blood. Although water intake (data not shown) roughly followed food intake, water consumption was not reduced significantly in TB rats until day 30 (20.4 +1.3 g vs. 25.4 + 1.0 g). Similar differences in water intake were maintained until sacrifice.
0 TUMOR h= 12) 6
CONTROL (n- 6)
0”“““““““““““““““““” 1 3 6 6 11 13
16 I7
13 21 23 26 27 26 31 33 31 37
DAYS POST TUMOR IMPLANTATION
FIG. 1 Mean (* SEM) daily intake of rat chow by tumor-bearing and control rats. Significantly (~(0.01 by t statistic) reduced food intake was consistently observed beginning on day 21. On day 30 all rats were anesthetized and 1 ml of blood was drawn by cardiac puncture for the analysis of ammonia and amino acid concentrations. The enzymatic assay of amnia concentration revealed significant (~(0.01) increases in ammonia of TB rats in blood taken by cardiac puncture on day 30 (138 + 56 vs. 56 ? 10 nmol/ml) as well as following decapitation on day 37 (243 f 18 vs. 60 + 4 nmol/ml). Furthermore, ammonia levels increased significantly (p
Hyperammonemia and Cancer
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Vol. 43, No. 1, 1988
BCAA being unaltered and concentrations of glutamine being nearly doubled. The other LNAA were also greatly elevated by factors of 2 to 3 in the brains of these TB rats. TABLE 1 Mean (2 SEM) concentrations of glutamine and neutral amino acids in the plasma (nmol/ml) and the brain (nmol/g) of control and tumor-bearing (TB) rats. Amino Acid -Glutamine Valine Isoleucine Leucine Methionine Tyrosine Phenylalanine Tryptophan Histidine
Brain
Plasma Control 799 f 55 277 + 20 123 + 9 191 f 9 49 + 2 113 + 7 70 f 3 123 f 6 83 ?r 3
515 139 60 86 73 142 100 105 96
TB 23* f 5* f 2* + 4* f 2” + 5* * 3* * 3* + 5
Control 5016 * 119 79 f 2 32 2 1 68 f 1 26 + 1 64 f 1 37 f 1 13 f 1 56 k 2
9104 78 34 63 82 204 120 23 135
TB 298* lr 2 * 1 + 2 f 4* f 9* f 5* t l* 1 5*
*p
Hyperammonemiaand Cancer
vol. 43, No. 1, 1988
71
0.0 -
7.0 -
0 g 5 2
s.o-
LO -
iiij
4.0-
2
5.0 -
Y 2.0 -
LO-
I
I
I
I
I
8
9
0.14
0.20
O.SS
NH3
CWCEllTRATKm
0.40 0
FIG. 2 Mean (k SEM) daily intake of rat chow by operated control, saline-
infused and amnonia-infused rats. Normal saline was infused initially at a rate of 2 ml/hr. Three days later, ammonia infusions were begun in one roup of 6 rats at the same rate and an initial concentration of 0.14 M 9equal parts of ammonium acetate and ammonium bicarbonate; pH = 7.9). The concentration of ammonium salts was increased on each subsequent infusion day to a maximum of 0.4 M on the fourth day of atmnonia infusion.
Mean plasma and brain amino acid profiles for this experiment are presented in Table 2. Plasma levels of glutamine, phenylalanine and tryptophan were increased significantly, while concentrations of tyrosine, methionine and the BCAA were decreased in ansnonia-infused rats. As in the previous experiment, a different amino acid profile was observed in the brain with all of the LNAA and glutamine exhibiting large increases in ammonia-infused rats.
72
vol. 43, No. 1, 1988
Hyperammonemia and Cancer
TABLE 2 Mean (k SEM) concentrations of glutamine and neutral amino acids in the plasma (nmol/ml) and the brain (nmol/g) of saline-infused (n = 6), ammonia-infused (n = 6) and operated control (n = 5) rats. Amino Acid -Glutamine Valine Is01 eucine Leucine Methionine Tyrosine Phenylalanine Tryptophan Histidine
Control Plasma --
591+11
Brain
4231544
Saline-Infused -Plasma
617526
Brain --
4608+75
272k13
94*2
264+10
88*5
20727 57*2 lOOk3 65?2 1151t2 71*2
82*1 34+1 65k2 33*1 15*1 46~1
21529 6121 106kl 6922 11723 7822
721t4 28+2 60+_2 30+1 15*1 47+1
119k3
42+1
119k6
36k3
Ammonia-Infused -Plasma
Brain
942?55*t 24074+1364*t 142+5*t
70+3*t 123+4*t 48+1*t
71r3*t 93+.1*t 128+4*t 71+2
135*11*4
70+4*t
132+10*?
70+3*t
167+8*t
187+14*t 44*5*t 154+8*t
*p
Vol. 43, No. 1, 1988
Hyperammonemia and Cancer
73
secondary to the accumulation of ammonia in the brain or secondary to the biochemical alterations associated with cerebral detoxification of ammonia. Significant elevation in blood urea nitrogen (BUN) has been reported for MCA sarcoma-bearing rats (6). A decrease in gluconeogenesis from lactate has also been observed in hepatocytes taken from rats bearing MCA sarcomas that were at least 14% of total body weight (19). The contribution of ammonia to ureagenesis and the reduction of gluconeogenesis by amnonia (20) suggest that both of these observations may be secondary to hyperannnonemia in anorectic TB rats. The detoxification of ammonia by cerebral tissue results in greatly elevated concentrations of glutamine in the brain. Additional alterations in brain chemistry associated with elevated ammonia concentrations involve the increased influx of LNAA (21). The influx of LNAA appears to be coupled with the efflux of glutamine from the brain through the sharing of a corrmOncarrier system for transport across the blood-brain barrier (21). Therefore, as glutamine is transported out of the brain, tyrosine, tryptophan, phenylalanine, methionine, histidine and the BCAA are allowed increased entry. Since the LNAA share this transport mechanism, they compete with each other for transport across the blood-brain barrier (22). The blood concentrations also influence the transport so that those LNAA that are in higher concentration will compete for carrier sites more effectively. Thus, the BCAA are not elevated in the brains of fB rats because of their reduction in plasma. The elevation in BCAA observed in the ammonia-infused rat brains appears to be related to their higher content of glutamine, which would tend to increase transport of all of the LNAA. Although tryptophan does not appear to follow the above rule, having low plasma and high brain concentrations, previous research.in our laboratory has demonstrated that the percentage of tryptophan that is not bound to albumin is actually increased in TB rats (12). Thus, the free portion of tryptophan, which competes more effectively for transport into the brain, is increased as compared to control rats. Therefore, the end result of cerebral detoxification of ammonia in TB rats appears to be greatly increased brain concentrations of glutamine, tyrosine, tryptophan, phenylalanine, methionine and histidine. Although these alterations in ammonia and amino acids may be sufficient to cause anorexia (23), tyrosine and tryptophan are precursors for the synthesis of the amine neurotransmitters, dopamine (DA), norepinephrine (NE) and serotonin (5-HT). Under appropriate conditions, elevations in these precursors in the brain result in increased synthesis and release of DA and 5-HT (24,25). Previous studies sug est that both DA and 5-HT metabolism are elevated in anorectic TB rats (7B. Although the elevation in 5-HT metabolism may be secondary to the anorexia (26,27), the increase in DA metabolism may have more direct effects on food intake (27). Therefore, several alterations are present in the brains of anorectic TB rats that appear sufficient to cause anorexia. However, this cascade of biochemical aberrations appears to be set in motion by the metabolism of specific amino acids by tumor tissue and the subsequent release of arrmonia. Thus, the most logical treatment regimen to correct this anorexia may be to: 1) reduce amino acid metabolism by the tumor, 2) facilitate detoxification of ammonia by the host, or 3) reduce the transport of LNAA across the blood-brain barrier. Acknowledgement Supported in part by grant 886834 from the American Institute for Cancer Research.
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Vol. 43, No. 1, 1988
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