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Blood ammonia levels and hepatic encephalopathy induced by CCl4 in rats
TOXlCOLOGYANDAPPLIEDPHARMACOLOGY
91,46l-468(1987)
Blood Ammonia Levels and Hepatic Encephalopathy Induced by Ccl4 in Rats HIRO-AKI
YAMAMOTO’
AND NARUMI
SUGIHARA
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima, 729-02 Japan
Received January 23, 1987; accepted July 21. 1987 Blood Ammonia Levels and Hepatic Encephalopathy Induced by CCL in Rats. YAMAMOTO, AND SUGIHARA, N. (1987). Toxicol. Appl. Pharmacol. 91, 461-468. An investigation of the mechanism of development of hepatic encephalopathy induced by CC& was performed in rats. CC& (1 .O ml/kg three times per week for over 10 weeks) caused hepatic encephalopathy in 80% of the treated rats. Accompanying the hepatic encephalopathy were hematemesis, abdominal dropsy, and hyperammonemia, conditions observed in hepatic coma patients. The blood ammonia levels were tremendously increased in only those rats with hepatic encephalopathy. Hepatic activities of carbamylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), important enzymes of the urea cycle, were significantly inhibited by CC&. However, the causality between the inhibition of CPS or ASS activity and the increase in blood ammonia Levelswas not observed. On the other hand, the content of ATP, which is a substrate of CPS and ASS, was decreased by 60% in liver of rats with hepatic encephalopathy. The activity of Mg’+-ATPase which can decompose hepatic ATP was increased by 60 and 300% in mitochondria and microsomes, respectively, of livers of rats with CC&-induced encephalopathy. There was a good correlation between the decreased hepatic ATP content and the increased mitochondrial Mg*+-ATPase activity. Furthermore, there was also a good correlation between the increase in blood ammonia levels and the increase in Mg*+-ATPase activity in microsomes. These findings suggest that hyperammonemia, which was produced by the decrease in hepatic content and by the inhibition of CPS and ASS, may play an important role in induction of hepatic encephalopathy. o 19x7 H.-A.,
Academic
Press. Inc.
Hepatic coma is one of the most frequent terminal episodes of primary hepatic disease. It is important to elucidate the mechanism of development of hepatic encephalopathy, since the death rate in these patients is high. Knowledge of the mechanism may result in appropriate therapy. Many kinds of animal models have been developed to mimic hepatic coma. Such ’ To whom reprint requests should be addressed at Department of the First Hygienic Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima, 729-02 Japan.
461
methods include administration of toxic substances (Zieve et al., 1974) or portacaval anastomosis (Smith et al., 1978; James et al.. 1979). However, these animal models have not completely reproduced the clinical signs of hepatic encephalopathy. To develop hepatic coma in rats, with signs which are similar to those in patients with hepatic encephalopathy, we administered CC& for long periods of time to rats. These rats were used to attempt to elucidate the mechanism responsible for hepatic encephalopathy. A number of reports have suggested that a failure of damaged or bypassed liver to metabolize cerebro-
462
YAMAMOTO
AND SUGIHARA
toxic substances such as ammonia (McDermott, 1958; Walker et al., 1970), amines (Fischer et al., 197 1; Smith et al., 1978), amino acids (Fischer et al., 1975; Ishikawa et al., 1985), short chain fatty acids (Zieve et al., 1974), or mercaptans (Zieve et al., 1974) may cause hepatic coma. Therefore, we investigated the relationship between an increase of cerebrotoxic substances, especially ammonia and the development of hepatic coma in C&treated rats. We measured the activities of the hepatic urea cycle enzymes (carbamyl phosphate synthetase, CPS; argininosuccinate synthetase, ASS) and their substrate (ATP) content in rats with hepatic encephalopathy.
METHODS Male Wistar rats ( 150- 180 g) were purchased from lshikawa Pref. Animal Laboratories (Ishikawa, Japan). The animals were housed in a temperature- and light ( 12hr dark/light)-controlled room. They were given a diet of standard laboratory chow (oriental kobo) and water ad libitum. Hepatic encephalopathy was produced by the intraperitoneal injection ofCC& in olive oil, 1.Oml/kg per day for 3 days per week (every other day) for over 10 weeks. This treatment caused hepatic encephalopathy in 80% of the rats. Accompanying the hepatic encephalopathy were hematemesis, abdominal dropsy, and hyperammonemia, conditions observed in hepatic coma patients. Blood was taken from the heart and the ammonia concentration was immediately determined by the method of Okuda and Fujii (1966). Determination ofATP. A sample of liver tissue was immediately frozen in liquid nitrogen and homogenized in ice-cold 0.6 N HC104. After centrifugation, ATP in the supematant was determined by a modification of the method of Biicher (1947). Preparation of liver mitochondria, microsomes, and cyrosol. Rats were killed by decapitation and the liver was homogenized in ice-cold 250 mM sucrose buffer, pH 6.8, containing 5 mM Hepes, 1 mM dithiothreitol, and 1 mM EGTA. This 10% homogenate was centrifuged at 1OOOg for 5 min. The supernatant was centrifuged at 7800g for 10 min to give a mitochondrial pellet. The supematant was further centrifuged at 10,500Og for 60 min to yield a cytosol fraction and a microsomal pellet. Pellets were washed with 20 mM Hepes buffer, pH 6.8, by centrifugation at 7800 or 10,500Og. These pellets were homoge-
FIG. 1. Effects of CC& on blood ammonia. Ammonia concentration of blood was determined as described under Methods. The abscissa represents the time (hours or weeks) after the single or consecutive injection (three times per week) of CCL,, 1.Oml/kg, ip; the ordinate represents the increase (times) in blood ammonia levels as compared to those of control. Values represent the mean ? SE; n = 6. Asterisks indicate significantly different from control (p < 0.01).
nized with 20 mM Hepes buffer, pH 6.8, for use in the experiments described below. Determination of Mg”-A TPase activity. Mg’+-ATPase activity was measured by the calorimetric determination of phosphate hydrolyzed from ATP (Dawson et a/., 1983). To measure mitochondrial Mg*+-ATPase activity, aliquots containing 280-620 pg of protein were incubated at 37’C for 10 min in 1 ml of 20 mM Hepes buffer, pH 6.8, containing 5 mM MgCl,, 1 mM EGTA, and 1 mrvt ATP. After incubation the reaction was stopped by the addition of 1 ml of 10% (w/v) trichloroacetic acid (TCA). To measure microsomal Mg*+-ATPase activity, aliquots containing 360-490 pg of protein were incubated as described for the determination of mitochondrial ATPase activity, except 1 PLgof oligomycin was added. Determination of CPS activity. Mitochondria (75- 160 pg protein) were incubated at 37°C for 10 min in 1 ml of 50 mivr Hepes buffer, pH 8.0, containing 5 mM N-acetylglutamate, 15 mM MgC&, 10 mM NH,Cl, 50 mM NaHCOl ,5 mM omithine, and 10 mM ATP. After incubation, the reaction was stopped by the addition of 1 ml of 10% TCA. After centrifugation, the amount of citrulline produced in the supematant was determined by diacetylmonooxime method (Mori et a/. 1978). Determination ofASS activity. Cytosol (about 600 pg ofprotein) was incubated at 37’C for 10 min in 1 ml of 50
CCI,-INDUCED
HEPATIC
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FIG. 2. Effects of CCI, on carbamylphosphate synthetase (CPS) or argininosuccinate synthetase (ASS) activity in rats. CPS and ASS were assayed as described under Methods. The abscissa represents the time (hours or weeks) after the single or consecutive injection (three times per week) of Ccl,, 1.O ml/kg, ip: the ordinate represents the percentage of inhibition of CPS or ASS activity as compared to that of control. Values represent the mean ? SE; n = 5. Asterisks indicate significantly different from control (*I, < 0.05: **p < 0.01).
mM Tris-HCl buffer, pH 7.5, containing 5 mM MgC12, 5 mM aspartate, 0.5 mM citrulline, and 1 mM ATP. After incubation the reaction was stopped by the addition of I ml of 10% TCA. After centrifugation, the amount of citrulline used by the reaction was determined by diacetylmonooxime method (Takada et al., 1979). Determination of GPT activity. Glutamic pyruvic transaminase activity was assayedin plasma with a shino test reagent kit according to the calorimetric method of Reitman and Frankel(1957). Other methods. Protein was determined by the phenol method (Lowry et al., 195 1). All data were compared by an analysis of variance. When the analysis indicated that a significant difference existed, the means of selected groups were compared by Student’s t test.
RESULTS CC&-Induced Hepatic Coma and Blood Ammonia Levels The dosing regimen with CCL, (three times per week for 10 weeks) caused hepatic encephalopathy in 80% of rats. Accompaning hepatic encephalopathy were loss of righting reflex, hematemesis, abdominal dropsy, and hyperammonemia. Blood ammonia levels of
the hepatic encephalopathy rats (574.7 f 97.2 pg N/dl) were increased eightfold over those of the corresponding control group (75.4 + 11.1 pg N/dl). However, the blood ammonia levels in rats administered CCL, for 6 (90.9 f 9.0 pgN/dl) or9 weeks(141 + 15.7 pg N/dl) were only slightly increased as compared to those of the corresponding controls (79.3 AI 17.7 or 77.1 f 10.5 pg N/dl) (Fig. 1). The livers from hepatic encephalopathy rats were small and hard. Furthermore, at the time of hepatic encephalopathy, GPT activity was increased eightfold as compared to that of controls. These results suggest that a tremendous increase in blood ammonia levels may play an important role in the development of CC&-induced hepatic encephalopathy in rats. Efects of Ccl4 on CPS and ASS Activities CPS and ASS activities, which can control blood ammonia levels, were measured in rats after CCL treatment. The CPS activity (0.65
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AND SUGIHARA
+- 0.05 pmol citrulline/mg protein) at 24 hr after a single injection of CCL ( 1.O ml/kg) was inhibited by 38% from that of corresponding controls (1.03 f 0.05 pm01 citrulline/mg protein). Furthermore, similar inhibition was observed by injections of Ccl,, 1.O ml/kg per day three times per week (every other day) for 1 or 9 weeks. On the other hand, the mitochondrial CPS activity (0.18 f 0.04 pmol citrulline/mg protein) from liver of rats with hepatic encephalopathy was inhibited by 84% when compared to that of controls (1.14 f 0.15 pmol citrulline/mg protein). These data indicate that the marked inhibition of CPS and ASS may cause an increase in blood ammonia levels. ASS activity, which is a rate-limiting enzyme of the urea cycle, was not significantly changed by treatment with CC4, 1.O ml/kg per day three times per week for 1 week (0.278 + 0.1 pmol citrulline/mg protein) as compared to vehicle-treated controls (0.308 f 0.1 pmol citrulline/mg protein). Furthermore, the ASS activity (0.173 ? 0.01 pmol citrulline/mg protein) in cytosol of liver from hepatic encephalopathy rats demonstrating a tremendous increase in blood ammonia levels was inhibited to the same extent as that of rats demonstrating only a small increase in blood ammonia levels (0.208 + 0.0 1 pmol citrulline/mg protein) (Figs. 1 and 2). These results indicate that the marked increase in blood ammonia levels produced by the development of hepatic encephalopathy may not be due solely to inhibition of urea cycle enzymes such as CPS and ASS.
Hepatic Encephalopathy and Liver A TP Content The content of ATP, which is one of the substrates of CPS and ASS, was determined in the liver of rats after CC4 treatment. The ATP content (1.2 k 0.09 pmol/g liver) 24 hr after CCI, administration was significantly decreased (20%) as compared to that of con-
RG. 3. Effects of CC& on hepatic ATP contents in rats. Hepatic ATP contents were determined as described under Methods. The abscissa represents the time (hours or weeks) after the single or consecutive injection (three times per week) of CCb, 1.O ml/kg, ip; the ordinate represents the percentage of decrease of ATP content liver as compared to that of control. Values represent the mean + SE; n = 4. Asterisks indicate significantly different from control (*p c: 0.05, **p < 0.01).
trols (1.53 f 0.06 pmol/g liver). The liver ATP content was not significantly different between rats administered CC4, 1.O ml/kg per day (three times per week), for 1 (1.30 f 0.05 pmol/g liver) or 9 weeks ( 1.49 t- 0.14 pmol/g liver). These values were not different from corresponding control values (1.44 _+0.06 rmol/g liver, 1.46 + 0.15 pmol/g liver). However, administration of CC4 for over 10 weeks, which resulted in hepatic encephalopathy, decreased the liver ATP content by 60% (Fig. 3).
Eflect ofA TP Concentration on CPS and ASS Activities in Vitro As shown in Fig. 4, CPS or ASS activity was dependent on ATP concentration. CPS or ASS had little activity with low ATP con-
CClJNDUCED 3 {
HEPATIC 1
CPS
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ENCEPHALOPATHY ASS 0.2.
0.8. bl -! 4
FIG. 4. Relationship between CPS or ASS activity and ATP concentration. CPS and ASS activity were assayed as described under Methods. The ordinate represents the concentration (mM) of ATP added; the abscissa represents the enzyme activity (citrulline pmol/mg protein) produced by each addition. Values represent the mean rfr.SE; n = 3.
centration and was enhanced by the addition of ATP. The data indicate that CPS or ASS activity may be decreased by the deple-
tion of hepatic ATP content, which occurred during the development of hepatic encephalopathy.
Mitochondria
Microsomes 10 .
3 .
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24h.
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9 w.
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24 h.
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FIG. 5. Effects of CC& on M$‘-ATPase activity of hepatic mitochondria or microsomes. Mg*+-ATPase activity of mitochondria or microsomes in liver was determined as described under Methods. Microsomes and mitochondria were prepared From whole liver homogenate from rats. Values represent the mean f SE, n = 6. Asterisks indicate significantly different from control (*p < 0.05, **p < 0.01).
466
YAMAMOTO
AND SUGIHARA
Eflect of CC& on Mg”-A TPaseActivity Mg2’-ATPase activity in mitochondria from rats injected with a single dose of CC& (1.0 ml/kg) was significantly increased by 25% over that ofcontrols (0.9 -t 0.04 prnol ip/ mg protein). Furthermore, the mitochondrial Mg2+-ATPase activity in rats administered CCIJ, 1.O ml/kg per day three times per week for 1 or 9 weeks, was not different when compared to that of controls (Fig. 5). In addition, Mg2+-ATPase activity in liver mitochondria from rats with hepatic encephalopathy was significantly increased by 60% from that of corresponding controls (0.88 + 0.03 pmol ip/ mg protein). Microsomal Mg2+-ATPase activity of rats injected with a single dose of CCL was increased by 20% from that of controls (0.82 t 0.05 wmol ip/protein). However, the Mg2+ATPase activity of rats administered CCL, 1.O ml/kg per day three times per week for 1 week, was not different from that of the corresponding controls. On the other hand, the Mg2+-ATPase activity of rats administered Ccl4 for 9 weeks (2.41 -+ 0.09 pmol ip/mg protein) or over 10 weeks (3.30 f 0.05 pmol ip/mg protein) was significantly increased by 60 and 300%, respectively. DISCUSSION Hepatic coma is one of the most frequent consequences of primary hepatic disease and the death rate is high. However, the mechanism of development of hepatic coma is poorly understood at present. In these experiments, we used rats having signs similar to those of hepatic patients. This model was produced by the administration of CC& for over 10 weeks. All rats demonstrating hepatic encephalopathy displayed a high ammonia level in blood. This result suggests that the increase in blood ammonia levels may play an important role in development of hepatic encephalopathy induced by Ccl,.
8 of
Decrease
( ATP Content
)
FIG. 6. Correlation between the increase of mitochondrial Me-ATPase activity and the decrease of hepatic ATP content. The abscissa represents the percentage increase of Mg’+-ATPase activity from mitochondria after CCL treatment in rats; the ordinate represents the percentage decrease of hepatic ATP content. The correlation coefficient was 0.989. Values represent the mean tSE;n=6.
Since Tamai ( 1970) have reported that various enzyme activities in the urea cycle, which can control ammonia levels in blood, were inhibited by CC&, it was suggested that the increase in blood ammonia might be due to the inhibition of enzymes of the urea cycle. Therefore, we measured CPS and ASS activities in liver from rats injected with CCL,. Both CPS and ASS activities were significantly decreased by Ccl, treatment. However, a link between the inhibition of CPS or ASS activity and the increase in blood ammonia levels was not observed (Figs. 1 and 2). In addition, Sherlock et al. (1967) have reported that the resection of 80% of the liver did not change the capacity for urea synthesis in rats. These results suggest that the increase in ammonia levels in blood produced by the development of hepatic encephalopathy may be not due solely to inhibition of urea cycle enzymes such as CPS and ASS. Hyams et al. ( 1964) have reported that hepatic ATP content decreased by 50% in rats
Ccl,-INDUCED
a OK.
200
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400 ( NH3
HEPATIC
604 Concentration
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FIG. 7. Correlation between the increased microsomal Mg2+-ATPase activity and increased blood ammonialevels. The abscissa represents the percentage increase of Mg2+-ATPase activity from microsomes after CC1, treatment; the ordinate represents the percentage increase of blood ammonia levels. The correlation coefficient was 0.997. Values represent the mean + SE; n = 6.
administered Ccl, (5 ml/kg, PO). Therefore, we determined hepatic ATP content in rats administered CCL. The hepatic ATP content in rats with hepatic encephalopathy decreased by 60% as compared to that of controls. In addition, CPS and ASS activities were dependent on ATP concentration in vifro. These findings suggest that CPS or ASS activity partially inhibited by direct effects of CCL may be further inhibited by the indirect action of CC4 on hepatic ATP content. Mg*+-ATPase activity of mitochondria or microsomes was measured in rats administered CCL,. It was observed that Mg*+-ATPase activity in rats with hepatic encephalopathy increased by 60% in mitochondria and by 300% in microsomes. Furthermore, there was a good correlation between the increased Mg2+-ATPase activity in mitochondria and the decreased hepatic ATP content. This suggests that the decreased hepatic ATP content may be due to increased Mg*+-ATPase activity (Fig. 6). On the other hand there was also a good correlation between the increase in
ENCEPHALOPATHY
467
Mg*+-ATPase activity of microsomes and the increase in blood ammonia levels (Fig. 7). Since it has been found that ASS is a ratelimiting enzyme in the urea cycle, it is suggested that the decreased ATP content of the cytosol (perhaps produced by the increase of Mg*+-ATPase activity of microsomes) may further inhibit ASS activity and thereby increase blood ammonia levels. James et al. (1979) have found that hyperammonemia contributes to encephalopathy indirectly, by raising the brain concentration of neutral amino acids which alter neurotransmitter metabolism, rather than directly, by toxic effects on neuronal metabolism. Therefore, our present results suggest that hyperammonemia produced by CC& administration may contribute to encephalopathy by raising the brain concentration of neuronal amino acids. However, since it has been reported that organic amines (Fischer et al.. 197 1; Smith et al., 1978) short chain fatty acids (Zieve et al., 1974) or mercaptans (Zieve et al., 1974) may also have a role in hepatic encephalopathy, it could be important to further investigate the relationship between blood ammonia levels and the other factors involved in hepatic encephalopathy. REFERENCES B~CHER, T. (1947). Uber ein phosphatubertragendes garungsferment. Biochim. Biophys. Ada 1,292-3 14. DAWSON, A. P., AND FULTON, D. V. (1983). Some properties of the Ca++-stimulated ATPase of a rat liver microsomal fraction. &o&em. J. 210,405-410. FISCHER, J. E., AND BALDESSARINI, R. J. ( 197 1). False neurotransmitters and hepatic failure. Lancer 2, 7% 80. FISCHER, J. E., FUNOVIS, J. M.. AGUIRRE, A., JAMES, J. H., KEANE, J. M., WESDORP, R. I. C., YOSHIMURA. N., AND WESTMAN, T. (1975). The role of plasma amino acids in hepatic encephalopathy. Surgery 78, 276-288. HYAMS, D. E., AND ISSELBACHER,K. J. (1964). Prevention of fatty liver by administration of adenosine triphosphate. Nature(London) 204,1196-l 197. ISHIKAWA, A., ISHIYAMA, H., ENOMOTO, T., OZAKI, A., F~KAO, K.. OKAMURA, T., IWASAKI, Y., AND YAMA-
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MOTO, H.-A. (1985). Hepatic coma and amino acids in the nerve endings ofthe central nervous system. Lz> Sci. 37,2 129-2 134. JAMES, J. H., ZIPARO, V., JEPPSSON,B., AND FISCHER, J. E. (1979). Hyperammonemia, plasma amino acid in balance and blood-brain amino acid transport: A unified theory of portal systemic encephalophathy. Lancet 13,772-775. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951). Protein measurement with Folin phenol reagent. .I. Biol. Chem. 193, 262275.
MCDERMOTT, W. V. (1958). The role of ammonia intoxication in hepatic coma. Bull. N. Y. Acad. Med. 34, 352-357.
MORI, M., AND COHEN, P. P. (1978). Preparation of crystalline carbamyl-phosphate synthetase I from frog liver. J. Biol. Chem. 25,8337-8339. OKUDA, H., AND FUJII, S. ( 1966). Direct calorimetric determination of blood ammonia. Saishin Zgaku, 21, 622-627.
REITMAN, S., AND FRANKEL, S. A. (1957). A colorimetric method pyruvic transaminases. Amer. J. C/in. Pathol. 28,53-56.
SHERLOCK, S. (1967). The Liver (A. E. Read, Ed.), p. 24 1. Butterworths, London. SMITH, A., ROSSI-FANELLI, ZIPARO, V., JAMES, J. H., PERELLE, B., AND FISCHER, J. E. (1978). Alterations in plasma and CSF amino acids, amines and metabolites in hepatic coma. Ann Surg. 187,343-350. TAKADA, S., SAHEKI, T., IGARASHI, Y., AND KATSUMUNA, T. (1979). Studies on rat liver argininosuccinate synthetase inhibition by various amino acids. J. Biochem. 85,1309- 13 14. TAMAI, Y. (1970). Enzymological studies on ammonium detoxication in liver. I. Measurement of the activities of urea cycle enzymes and the enzymes concerning glutamic acid metabolism in liver after ammonium chloride administration. Nippon Shokakibyo Gakkai Zasshi 67,856-865. WALKER, C. O., AND SCHENKER, S. (1970). Pathogenesis ofhepatic encephalophathy with special reference to the role of ammonia. Amer. J. Clin. Nutr. 23,6 19-632. ZIEVE, L., DOIZAKI, W. M., AND ZIEVE, F. J. (1974). Synergism between mercaptans and ammonia or fatty acids in the production of coma: A possible role for mercaptans in the pathogenesis of hepatic coma. J. Lab. Clin. Med. 83, 16-28.
91,46l-468(1987)
Blood Ammonia Levels and Hepatic Encephalopathy Induced by Ccl4 in Rats HIRO-AKI
YAMAMOTO’
AND NARUMI
SUGIHARA
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima, 729-02 Japan
Received January 23, 1987; accepted July 21. 1987 Blood Ammonia Levels and Hepatic Encephalopathy Induced by CCL in Rats. YAMAMOTO, AND SUGIHARA, N. (1987). Toxicol. Appl. Pharmacol. 91, 461-468. An investigation of the mechanism of development of hepatic encephalopathy induced by CC& was performed in rats. CC& (1 .O ml/kg three times per week for over 10 weeks) caused hepatic encephalopathy in 80% of the treated rats. Accompanying the hepatic encephalopathy were hematemesis, abdominal dropsy, and hyperammonemia, conditions observed in hepatic coma patients. The blood ammonia levels were tremendously increased in only those rats with hepatic encephalopathy. Hepatic activities of carbamylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), important enzymes of the urea cycle, were significantly inhibited by CC&. However, the causality between the inhibition of CPS or ASS activity and the increase in blood ammonia Levelswas not observed. On the other hand, the content of ATP, which is a substrate of CPS and ASS, was decreased by 60% in liver of rats with hepatic encephalopathy. The activity of Mg’+-ATPase which can decompose hepatic ATP was increased by 60 and 300% in mitochondria and microsomes, respectively, of livers of rats with CC&-induced encephalopathy. There was a good correlation between the decreased hepatic ATP content and the increased mitochondrial Mg*+-ATPase activity. Furthermore, there was also a good correlation between the increase in blood ammonia levels and the increase in Mg*+-ATPase activity in microsomes. These findings suggest that hyperammonemia, which was produced by the decrease in hepatic content and by the inhibition of CPS and ASS, may play an important role in induction of hepatic encephalopathy. o 19x7 H.-A.,
Academic
Press. Inc.
Hepatic coma is one of the most frequent terminal episodes of primary hepatic disease. It is important to elucidate the mechanism of development of hepatic encephalopathy, since the death rate in these patients is high. Knowledge of the mechanism may result in appropriate therapy. Many kinds of animal models have been developed to mimic hepatic coma. Such ’ To whom reprint requests should be addressed at Department of the First Hygienic Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima, 729-02 Japan.
461
methods include administration of toxic substances (Zieve et al., 1974) or portacaval anastomosis (Smith et al., 1978; James et al.. 1979). However, these animal models have not completely reproduced the clinical signs of hepatic encephalopathy. To develop hepatic coma in rats, with signs which are similar to those in patients with hepatic encephalopathy, we administered CC& for long periods of time to rats. These rats were used to attempt to elucidate the mechanism responsible for hepatic encephalopathy. A number of reports have suggested that a failure of damaged or bypassed liver to metabolize cerebro-
462
YAMAMOTO
AND SUGIHARA
toxic substances such as ammonia (McDermott, 1958; Walker et al., 1970), amines (Fischer et al., 197 1; Smith et al., 1978), amino acids (Fischer et al., 1975; Ishikawa et al., 1985), short chain fatty acids (Zieve et al., 1974), or mercaptans (Zieve et al., 1974) may cause hepatic coma. Therefore, we investigated the relationship between an increase of cerebrotoxic substances, especially ammonia and the development of hepatic coma in C&treated rats. We measured the activities of the hepatic urea cycle enzymes (carbamyl phosphate synthetase, CPS; argininosuccinate synthetase, ASS) and their substrate (ATP) content in rats with hepatic encephalopathy.
METHODS Male Wistar rats ( 150- 180 g) were purchased from lshikawa Pref. Animal Laboratories (Ishikawa, Japan). The animals were housed in a temperature- and light ( 12hr dark/light)-controlled room. They were given a diet of standard laboratory chow (oriental kobo) and water ad libitum. Hepatic encephalopathy was produced by the intraperitoneal injection ofCC& in olive oil, 1.Oml/kg per day for 3 days per week (every other day) for over 10 weeks. This treatment caused hepatic encephalopathy in 80% of the rats. Accompanying the hepatic encephalopathy were hematemesis, abdominal dropsy, and hyperammonemia, conditions observed in hepatic coma patients. Blood was taken from the heart and the ammonia concentration was immediately determined by the method of Okuda and Fujii (1966). Determination ofATP. A sample of liver tissue was immediately frozen in liquid nitrogen and homogenized in ice-cold 0.6 N HC104. After centrifugation, ATP in the supematant was determined by a modification of the method of Biicher (1947). Preparation of liver mitochondria, microsomes, and cyrosol. Rats were killed by decapitation and the liver was homogenized in ice-cold 250 mM sucrose buffer, pH 6.8, containing 5 mM Hepes, 1 mM dithiothreitol, and 1 mM EGTA. This 10% homogenate was centrifuged at 1OOOg for 5 min. The supernatant was centrifuged at 7800g for 10 min to give a mitochondrial pellet. The supematant was further centrifuged at 10,500Og for 60 min to yield a cytosol fraction and a microsomal pellet. Pellets were washed with 20 mM Hepes buffer, pH 6.8, by centrifugation at 7800 or 10,500Og. These pellets were homoge-
FIG. 1. Effects of CC& on blood ammonia. Ammonia concentration of blood was determined as described under Methods. The abscissa represents the time (hours or weeks) after the single or consecutive injection (three times per week) of CCL,, 1.Oml/kg, ip; the ordinate represents the increase (times) in blood ammonia levels as compared to those of control. Values represent the mean ? SE; n = 6. Asterisks indicate significantly different from control (p < 0.01).
nized with 20 mM Hepes buffer, pH 6.8, for use in the experiments described below. Determination of Mg”-A TPase activity. Mg’+-ATPase activity was measured by the calorimetric determination of phosphate hydrolyzed from ATP (Dawson et a/., 1983). To measure mitochondrial Mg*+-ATPase activity, aliquots containing 280-620 pg of protein were incubated at 37’C for 10 min in 1 ml of 20 mM Hepes buffer, pH 6.8, containing 5 mM MgCl,, 1 mM EGTA, and 1 mrvt ATP. After incubation the reaction was stopped by the addition of 1 ml of 10% (w/v) trichloroacetic acid (TCA). To measure microsomal Mg*+-ATPase activity, aliquots containing 360-490 pg of protein were incubated as described for the determination of mitochondrial ATPase activity, except 1 PLgof oligomycin was added. Determination of CPS activity. Mitochondria (75- 160 pg protein) were incubated at 37°C for 10 min in 1 ml of 50 mivr Hepes buffer, pH 8.0, containing 5 mM N-acetylglutamate, 15 mM MgC&, 10 mM NH,Cl, 50 mM NaHCOl ,5 mM omithine, and 10 mM ATP. After incubation, the reaction was stopped by the addition of 1 ml of 10% TCA. After centrifugation, the amount of citrulline produced in the supematant was determined by diacetylmonooxime method (Mori et a/. 1978). Determination ofASS activity. Cytosol (about 600 pg ofprotein) was incubated at 37’C for 10 min in 1 ml of 50
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ENCEPHALOPATHY
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80
0
24 h. 1 W. 9
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10 W. Card)
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24 h. lw.
9 w.10 w. wei=tic Gmd
FIG. 2. Effects of CCI, on carbamylphosphate synthetase (CPS) or argininosuccinate synthetase (ASS) activity in rats. CPS and ASS were assayed as described under Methods. The abscissa represents the time (hours or weeks) after the single or consecutive injection (three times per week) of Ccl,, 1.O ml/kg, ip: the ordinate represents the percentage of inhibition of CPS or ASS activity as compared to that of control. Values represent the mean ? SE; n = 5. Asterisks indicate significantly different from control (*I, < 0.05: **p < 0.01).
mM Tris-HCl buffer, pH 7.5, containing 5 mM MgC12, 5 mM aspartate, 0.5 mM citrulline, and 1 mM ATP. After incubation the reaction was stopped by the addition of I ml of 10% TCA. After centrifugation, the amount of citrulline used by the reaction was determined by diacetylmonooxime method (Takada et al., 1979). Determination of GPT activity. Glutamic pyruvic transaminase activity was assayedin plasma with a shino test reagent kit according to the calorimetric method of Reitman and Frankel(1957). Other methods. Protein was determined by the phenol method (Lowry et al., 195 1). All data were compared by an analysis of variance. When the analysis indicated that a significant difference existed, the means of selected groups were compared by Student’s t test.
RESULTS CC&-Induced Hepatic Coma and Blood Ammonia Levels The dosing regimen with CCL, (three times per week for 10 weeks) caused hepatic encephalopathy in 80% of rats. Accompaning hepatic encephalopathy were loss of righting reflex, hematemesis, abdominal dropsy, and hyperammonemia. Blood ammonia levels of
the hepatic encephalopathy rats (574.7 f 97.2 pg N/dl) were increased eightfold over those of the corresponding control group (75.4 + 11.1 pg N/dl). However, the blood ammonia levels in rats administered CCL, for 6 (90.9 f 9.0 pgN/dl) or9 weeks(141 + 15.7 pg N/dl) were only slightly increased as compared to those of the corresponding controls (79.3 AI 17.7 or 77.1 f 10.5 pg N/dl) (Fig. 1). The livers from hepatic encephalopathy rats were small and hard. Furthermore, at the time of hepatic encephalopathy, GPT activity was increased eightfold as compared to that of controls. These results suggest that a tremendous increase in blood ammonia levels may play an important role in the development of CC&-induced hepatic encephalopathy in rats. Efects of Ccl4 on CPS and ASS Activities CPS and ASS activities, which can control blood ammonia levels, were measured in rats after CCL treatment. The CPS activity (0.65
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+- 0.05 pmol citrulline/mg protein) at 24 hr after a single injection of CCL ( 1.O ml/kg) was inhibited by 38% from that of corresponding controls (1.03 f 0.05 pm01 citrulline/mg protein). Furthermore, similar inhibition was observed by injections of Ccl,, 1.O ml/kg per day three times per week (every other day) for 1 or 9 weeks. On the other hand, the mitochondrial CPS activity (0.18 f 0.04 pmol citrulline/mg protein) from liver of rats with hepatic encephalopathy was inhibited by 84% when compared to that of controls (1.14 f 0.15 pmol citrulline/mg protein). These data indicate that the marked inhibition of CPS and ASS may cause an increase in blood ammonia levels. ASS activity, which is a rate-limiting enzyme of the urea cycle, was not significantly changed by treatment with CC4, 1.O ml/kg per day three times per week for 1 week (0.278 + 0.1 pmol citrulline/mg protein) as compared to vehicle-treated controls (0.308 f 0.1 pmol citrulline/mg protein). Furthermore, the ASS activity (0.173 ? 0.01 pmol citrulline/mg protein) in cytosol of liver from hepatic encephalopathy rats demonstrating a tremendous increase in blood ammonia levels was inhibited to the same extent as that of rats demonstrating only a small increase in blood ammonia levels (0.208 + 0.0 1 pmol citrulline/mg protein) (Figs. 1 and 2). These results indicate that the marked increase in blood ammonia levels produced by the development of hepatic encephalopathy may not be due solely to inhibition of urea cycle enzymes such as CPS and ASS.
Hepatic Encephalopathy and Liver A TP Content The content of ATP, which is one of the substrates of CPS and ASS, was determined in the liver of rats after CC4 treatment. The ATP content (1.2 k 0.09 pmol/g liver) 24 hr after CCI, administration was significantly decreased (20%) as compared to that of con-
RG. 3. Effects of CC& on hepatic ATP contents in rats. Hepatic ATP contents were determined as described under Methods. The abscissa represents the time (hours or weeks) after the single or consecutive injection (three times per week) of CCb, 1.O ml/kg, ip; the ordinate represents the percentage of decrease of ATP content liver as compared to that of control. Values represent the mean + SE; n = 4. Asterisks indicate significantly different from control (*p c: 0.05, **p < 0.01).
trols (1.53 f 0.06 pmol/g liver). The liver ATP content was not significantly different between rats administered CC4, 1.O ml/kg per day (three times per week), for 1 (1.30 f 0.05 pmol/g liver) or 9 weeks ( 1.49 t- 0.14 pmol/g liver). These values were not different from corresponding control values (1.44 _+0.06 rmol/g liver, 1.46 + 0.15 pmol/g liver). However, administration of CC4 for over 10 weeks, which resulted in hepatic encephalopathy, decreased the liver ATP content by 60% (Fig. 3).
Eflect ofA TP Concentration on CPS and ASS Activities in Vitro As shown in Fig. 4, CPS or ASS activity was dependent on ATP concentration. CPS or ASS had little activity with low ATP con-
CClJNDUCED 3 {
HEPATIC 1
CPS
465
ENCEPHALOPATHY ASS 0.2.
0.8. bl -! 4
FIG. 4. Relationship between CPS or ASS activity and ATP concentration. CPS and ASS activity were assayed as described under Methods. The ordinate represents the concentration (mM) of ATP added; the abscissa represents the enzyme activity (citrulline pmol/mg protein) produced by each addition. Values represent the mean rfr.SE; n = 3.
centration and was enhanced by the addition of ATP. The data indicate that CPS or ASS activity may be decreased by the deple-
tion of hepatic ATP content, which occurred during the development of hepatic encephalopathy.
Mitochondria
Microsomes 10 .
3 .
I -
IL
24h.
lw.
9 w.
orw mm*
10 w. ccma)
24 h.
lw.
9 W. aver meatic
10 w. CaM)
FIG. 5. Effects of CC& on M$‘-ATPase activity of hepatic mitochondria or microsomes. Mg*+-ATPase activity of mitochondria or microsomes in liver was determined as described under Methods. Microsomes and mitochondria were prepared From whole liver homogenate from rats. Values represent the mean f SE, n = 6. Asterisks indicate significantly different from control (*p < 0.05, **p < 0.01).
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Eflect of CC& on Mg”-A TPaseActivity Mg2’-ATPase activity in mitochondria from rats injected with a single dose of CC& (1.0 ml/kg) was significantly increased by 25% over that ofcontrols (0.9 -t 0.04 prnol ip/ mg protein). Furthermore, the mitochondrial Mg2+-ATPase activity in rats administered CCIJ, 1.O ml/kg per day three times per week for 1 or 9 weeks, was not different when compared to that of controls (Fig. 5). In addition, Mg2+-ATPase activity in liver mitochondria from rats with hepatic encephalopathy was significantly increased by 60% from that of corresponding controls (0.88 + 0.03 pmol ip/ mg protein). Microsomal Mg2+-ATPase activity of rats injected with a single dose of CCL was increased by 20% from that of controls (0.82 t 0.05 wmol ip/protein). However, the Mg2+ATPase activity of rats administered CCL, 1.O ml/kg per day three times per week for 1 week, was not different from that of the corresponding controls. On the other hand, the Mg2+-ATPase activity of rats administered Ccl4 for 9 weeks (2.41 -+ 0.09 pmol ip/mg protein) or over 10 weeks (3.30 f 0.05 pmol ip/mg protein) was significantly increased by 60 and 300%, respectively. DISCUSSION Hepatic coma is one of the most frequent consequences of primary hepatic disease and the death rate is high. However, the mechanism of development of hepatic coma is poorly understood at present. In these experiments, we used rats having signs similar to those of hepatic patients. This model was produced by the administration of CC& for over 10 weeks. All rats demonstrating hepatic encephalopathy displayed a high ammonia level in blood. This result suggests that the increase in blood ammonia levels may play an important role in development of hepatic encephalopathy induced by Ccl,.
8 of
Decrease
( ATP Content
)
FIG. 6. Correlation between the increase of mitochondrial Me-ATPase activity and the decrease of hepatic ATP content. The abscissa represents the percentage increase of Mg’+-ATPase activity from mitochondria after CCL treatment in rats; the ordinate represents the percentage decrease of hepatic ATP content. The correlation coefficient was 0.989. Values represent the mean tSE;n=6.
Since Tamai ( 1970) have reported that various enzyme activities in the urea cycle, which can control ammonia levels in blood, were inhibited by CC&, it was suggested that the increase in blood ammonia might be due to the inhibition of enzymes of the urea cycle. Therefore, we measured CPS and ASS activities in liver from rats injected with CCL,. Both CPS and ASS activities were significantly decreased by Ccl, treatment. However, a link between the inhibition of CPS or ASS activity and the increase in blood ammonia levels was not observed (Figs. 1 and 2). In addition, Sherlock et al. (1967) have reported that the resection of 80% of the liver did not change the capacity for urea synthesis in rats. These results suggest that the increase in ammonia levels in blood produced by the development of hepatic encephalopathy may be not due solely to inhibition of urea cycle enzymes such as CPS and ASS. Hyams et al. ( 1964) have reported that hepatic ATP content decreased by 50% in rats
Ccl,-INDUCED
a OK.
200
8 of
lnctease
400 ( NH3
HEPATIC
604 Concentration
)
FIG. 7. Correlation between the increased microsomal Mg2+-ATPase activity and increased blood ammonialevels. The abscissa represents the percentage increase of Mg2+-ATPase activity from microsomes after CC1, treatment; the ordinate represents the percentage increase of blood ammonia levels. The correlation coefficient was 0.997. Values represent the mean + SE; n = 6.
administered Ccl, (5 ml/kg, PO). Therefore, we determined hepatic ATP content in rats administered CCL. The hepatic ATP content in rats with hepatic encephalopathy decreased by 60% as compared to that of controls. In addition, CPS and ASS activities were dependent on ATP concentration in vifro. These findings suggest that CPS or ASS activity partially inhibited by direct effects of CCL may be further inhibited by the indirect action of CC4 on hepatic ATP content. Mg*+-ATPase activity of mitochondria or microsomes was measured in rats administered CCL,. It was observed that Mg*+-ATPase activity in rats with hepatic encephalopathy increased by 60% in mitochondria and by 300% in microsomes. Furthermore, there was a good correlation between the increased Mg2+-ATPase activity in mitochondria and the decreased hepatic ATP content. This suggests that the decreased hepatic ATP content may be due to increased Mg*+-ATPase activity (Fig. 6). On the other hand there was also a good correlation between the increase in
ENCEPHALOPATHY
467
Mg*+-ATPase activity of microsomes and the increase in blood ammonia levels (Fig. 7). Since it has been found that ASS is a ratelimiting enzyme in the urea cycle, it is suggested that the decreased ATP content of the cytosol (perhaps produced by the increase of Mg*+-ATPase activity of microsomes) may further inhibit ASS activity and thereby increase blood ammonia levels. James et al. (1979) have found that hyperammonemia contributes to encephalopathy indirectly, by raising the brain concentration of neutral amino acids which alter neurotransmitter metabolism, rather than directly, by toxic effects on neuronal metabolism. Therefore, our present results suggest that hyperammonemia produced by CC& administration may contribute to encephalopathy by raising the brain concentration of neuronal amino acids. However, since it has been reported that organic amines (Fischer et al.. 197 1; Smith et al., 1978) short chain fatty acids (Zieve et al., 1974) or mercaptans (Zieve et al., 1974) may also have a role in hepatic encephalopathy, it could be important to further investigate the relationship between blood ammonia levels and the other factors involved in hepatic encephalopathy. REFERENCES B~CHER, T. (1947). Uber ein phosphatubertragendes garungsferment. Biochim. Biophys. Ada 1,292-3 14. DAWSON, A. P., AND FULTON, D. V. (1983). Some properties of the Ca++-stimulated ATPase of a rat liver microsomal fraction. &o&em. J. 210,405-410. FISCHER, J. E., AND BALDESSARINI, R. J. ( 197 1). False neurotransmitters and hepatic failure. Lancer 2, 7% 80. FISCHER, J. E., FUNOVIS, J. M.. AGUIRRE, A., JAMES, J. H., KEANE, J. M., WESDORP, R. I. C., YOSHIMURA. N., AND WESTMAN, T. (1975). The role of plasma amino acids in hepatic encephalopathy. Surgery 78, 276-288. HYAMS, D. E., AND ISSELBACHER,K. J. (1964). Prevention of fatty liver by administration of adenosine triphosphate. Nature(London) 204,1196-l 197. ISHIKAWA, A., ISHIYAMA, H., ENOMOTO, T., OZAKI, A., F~KAO, K.. OKAMURA, T., IWASAKI, Y., AND YAMA-
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MOTO, H.-A. (1985). Hepatic coma and amino acids in the nerve endings ofthe central nervous system. Lz> Sci. 37,2 129-2 134. JAMES, J. H., ZIPARO, V., JEPPSSON,B., AND FISCHER, J. E. (1979). Hyperammonemia, plasma amino acid in balance and blood-brain amino acid transport: A unified theory of portal systemic encephalophathy. Lancet 13,772-775. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951). Protein measurement with Folin phenol reagent. .I. Biol. Chem. 193, 262275.
MCDERMOTT, W. V. (1958). The role of ammonia intoxication in hepatic coma. Bull. N. Y. Acad. Med. 34, 352-357.
MORI, M., AND COHEN, P. P. (1978). Preparation of crystalline carbamyl-phosphate synthetase I from frog liver. J. Biol. Chem. 25,8337-8339. OKUDA, H., AND FUJII, S. ( 1966). Direct calorimetric determination of blood ammonia. Saishin Zgaku, 21, 622-627.
REITMAN, S., AND FRANKEL, S. A. (1957). A colorimetric method pyruvic transaminases. Amer. J. C/in. Pathol. 28,53-56.
SHERLOCK, S. (1967). The Liver (A. E. Read, Ed.), p. 24 1. Butterworths, London. SMITH, A., ROSSI-FANELLI, ZIPARO, V., JAMES, J. H., PERELLE, B., AND FISCHER, J. E. (1978). Alterations in plasma and CSF amino acids, amines and metabolites in hepatic coma. Ann Surg. 187,343-350. TAKADA, S., SAHEKI, T., IGARASHI, Y., AND KATSUMUNA, T. (1979). Studies on rat liver argininosuccinate synthetase inhibition by various amino acids. J. Biochem. 85,1309- 13 14. TAMAI, Y. (1970). Enzymological studies on ammonium detoxication in liver. I. Measurement of the activities of urea cycle enzymes and the enzymes concerning glutamic acid metabolism in liver after ammonium chloride administration. Nippon Shokakibyo Gakkai Zasshi 67,856-865. WALKER, C. O., AND SCHENKER, S. (1970). Pathogenesis ofhepatic encephalophathy with special reference to the role of ammonia. Amer. J. Clin. Nutr. 23,6 19-632. ZIEVE, L., DOIZAKI, W. M., AND ZIEVE, F. J. (1974). Synergism between mercaptans and ammonia or fatty acids in the production of coma: A possible role for mercaptans in the pathogenesis of hepatic coma. J. Lab. Clin. Med. 83, 16-28.