Effects of Arginine Infusion in Infants: Increased Urea Synthesis Associated With Unchanged Ammonia Blood Levels Hans Kraus, Peter Stubbe, and Wolf von Berg Infusion of L-arginine hydrochloride in infants and children (ages ranging from 1 day to 12 yr) at a dosage of 0.5 g/kg body w e i g h t resulted in a dramatic increase in the arginine plasma concentration, w i t h highest values of approximately 7 m m o l e / liter immediately after the end of the infusion; 120 min later the mean plasma level of the amino acid had decreased to mean values of I mmole/liter. These fluctuations were paralleled by increased ornithine concentrations, although the mean plasma levels of this amino acid remained far below those of arginine, i.e., 0.73 and 0.22 mmole/liter after 30 and 90 min, respectively. When expressed on a
molar basis, arginine administration resuited in an almost stoichiometric rise in urinary urea excretion. These findings indicate that arginine is rapidly metabolized via urea and ornithine, the latter being transformed to glucose, as evidenced by a significant rise in the blood glucose concentration. Blood gas analyses and serum urea and blood ammonia concentrations determined after the load showed no significant deviations from preinfusion levels. Thus, in contrast to the effects to be expected from studies w i t h tissue culture homogenates, even when administered to newborn infants, arginine does not impair the turnover of the urea cycle.
R G I N I N E , like other amino acids, is widely used as a stimulus to evaluate glucagon, insulin, and growth h o r m o n e release in man. t,2 F o r this purpose it is administered to young children as a means for the diagnosis of endocrine and metabolic disorders. Arginine hydrochloride is also applied in infancy in the treatment of hypochloremic alkalosis. N o negative side effects of arginine have been noticed unless the solution was contaminated 3 or infused in uremic patients. 4 Using the dosage c o m m o n l y accepted, loading with arginine results in transitory high blood levels of the amino acid c o m p a r a b l e to or even higher than those reported in argininemia, a condition due to a congenital defect in the last step of the urea cycle. 5'6 Since the s y m p t o m s of this disease are vomiting, failure to thrive, ataxia, tremor, spastic paresis, convulsions, and, finally, mental retardation, the question arises whether arginine administration might possibly be harmful in any direct or indirect manner. With regard to the latter possibility, arginine, in concentrations in excess of the optimal, has been found to inhibit both argininosuccinate synthetase and argininosuccinate lyase in H e L a cell homogenates. 7 Whether this finding is applicable to the conditions of the urea cycle in liver is uncertain. The liver is the only organ containing all the enzymes of the urea cycle in amounts high enough to account for total urea synthesis. However, some of these enzymes, the activities of which are far below those found in
A
From the Department of Pediatrics, University of Goettingen, Goettingen, Germany. Received for publication January 15. 1976. Reprint requests should be addressed to Dr. Hans Kraus, Universiti~ts-Kinderklinik, Goettingen, Germany. 9 1976 by Grune & Stratton, lnc.
Metabolism,Vol. 25, No. 11 (November),1976
3400
1241
1242
KRAUS, STUBBE, AND van BERG
Table 1. Clinical Data and Number of Patients Investigated During Various Pracedures Determination of Blood gas
No. of Patients Investigated
Age at Investigation*
11
2-31 days
7
Weight at Investigation~ (g)
2310
• 8.2
• 95
• 112
2 - 6 wk
264 • 7
2452 • 148
2785 • 82
274 • 11.2
2721 • 174
2974 • 125
Serum urea
11
1-31 days
Urinary urea
11
12-31 days
excretion Blood ammonia
Birthweightt (g)
268
analysis
Arginine and ornithine
Gestational Age1" (days)
2715
268
3120
3312
• 1.2
• 712
-4- 597
3
1-2 days
261 • 17
2290 • 91
2216 • 153
3
7-8 days
2233 • 198 2080
2260 • 262 2346
• 319 --
• 70 32 kg
3
1 89 wk
263 • 4.5 244
5
9-12 yr
• 22 --
• 2.5 *Range. t M e a n • SD.
liver, are widely distributed in the b o d y tissues and, as mentioned above, have even been found in t u m o r cell lines. Whether these enzymes are under the same genetic control and have the same properties as those of the urea cycle in liver has been questioned, s Nevertheless, if similar mechanisms are operating both in H e L a cells and in the liver, inhibition of argininosuccinate synthetase which catalyses the rate-limiting step would slow down the rate of the urea cycle and thereby increase the blood a m m o n i a concentration. Beyond the possibility of specific interference it is not known whether or not the capacity of the urea cycle in newborn infants is high enough to cope with the additional nitrogen load caused by arginine infusion. It is well known that the capacity of some enzyme systems increases during fetal development, adult values not being reached at birth or soon after birth. In an attempt to elucidate some of the possible metabolic effects of arginine under conditions in viva, the amino acid was infused in infants and children. Arginine and ornithine plasma concentrations and blood glucose and blood a m m o n i a levels were measured before and after the load. Catabolism of arginine is expected to result in increased urea formation. Therefore, urinary urea excretion and the concentration of urea in the serum in response to arginine administration were observed and recorded. Since the load with arginine hydrochloride m a y also result in increased hydrogen ion concentration, blood gas analyses were simultaneously performed. MATERIALS AND METHODS The clinical data and number of patients investigated are shown in Table 1. Solutions of 6 ~ (w/v) L-arginine hydrochloride were infused 4 hr after the last feeding in infants or after an overnight fast in children with a dosage of 0.5 g/kg/30 min. Quantitative analyses of amino acids before and after the load were performed according to the method of Moore and Stein 9 by
ARGININE INFUSION
1243
m mot / l 10
~
I0
3
I
I
60
g0
I
170 rnin
Fig. 1. Disappearance rate of arginine in plasma determined in seven children (age range from 2 - 6 wk). The equation of the elimination curve is represented by log y = - 0 . 0 0 7 x - t 0.81; p < 0.001; r = - 0 . 9 2 . y, concentration of arginine in plasma (mmole/liter); x, time (min) after arginine infusion. The elimination constant K (K = [In 2 / ( t / 2 ) ] • 100) is 1 . 9 7 % / m i n .
means of an automatic amino acid autoanalyzer (Biocal BC 200). Deproteinization of the blood specimens was accomplished by adding 1 ml 5~ sulphosalicylic acid to each 0.5-ml sample of plasma. After refrigerated centrifugation, an aliquot of the supernatant was applied to the column. The disappearance rate of arginine was established with the aid of a semilogarithmic plot and expressed as K = [In 2/(t/2)] • 100. The elimination curve was calculated by the method of least squares. Ammonia was determined enzymatically by the method of Kun and Kearney. 1~ Heparinized blood (0.5 ml) was immediately deproteinized with 0.5 ml I N ice-cold perchloric acid. The precipitated protein was removed by centrifugation and the supernatant fluid was neutralized with 2 N KHCO 3. Blank readings were obtained according to the same method except that neutralized perchloric acid replaced the unknown blood sample. Recoveries were 96~ :~ 7.4~ using this system. Urea was determined with urease, using a test kit of Boehringer and Sons, Mannheim, Germany. Blood gas analysis was performed according to the Astrup-micromethod. Informed parental consent was obtained for study of all children. RESULTS
Arginine and Ornithine N o r m a l p l a s m a a r g i n i n e levels in seven i n f a n t s (age r a n g e 2 - 6 wk) were f o u n d to be in the r a n g e 0.04-0.12 m m o l e / l i t e r . L o a d i n g with a r g i n i n e resulted in a sharp increase in the c o n c e n t r a t i o n o f this a m i n o acid in p l a s m a , with highest values a b o u t 7 m m o l e / l i t e r . A t the e n d of the o b s e r v a t i o n period, i.e., 2 hr after the end of the i n f u s i o n , a r g i n i n e p l a s m a levels h a d drastically decreased to a m e a n v a l u e o f 1 m m o l e / l i t e r . T h e d i s a p p e a r a n c e rate o f a r g i n i n e (Fig. 1) expressed by the e l i m i n a t i o n c o n s t a n t K (K = [In 2 / ( t / 2 ) ] x 100) is 1.97~/min. A l o n g with the increase in arginine, a parallel b u t less m a r k e d increase in the o r n i t h i n e p l a s m a c o n c e n t r a t i o n was s h o w n in the same i n d i v i d u a l s . Peak values
1244
KRAUS, STUBBE, AND von BERG
Table 2. Levels of Ammonia in Blood ( # g / 1 0 0 ml) Before and After Intravenous Loading With Arginine (0.5 g / k g body weight) Minutes After Arginine Infusion Age (range) 1-2days 7-8 days 189 9-12 yr
0
15
30
45
60
69.7• 70.7• 88.2• 15.5 84.7+ 10.6 77.6• 10.2 (6) (3) (3) (3) (3) 57.9• 10.0 61.3• 11.1 4 5 . 5 • 62.3• 55.7• (7) (3) (3) (3) (3) 63.0• 72.4• 88.4• 60.7• 64.7• (5) (3) (3) (3) (3) 55.2 • 9.2 70.6 • 12.9 70.7 • 12.7 62.1 • 15.6 63.6 -k 15.1
(4)
(5)
(5)
(5)
(5)
90
120
94.1 • 16.0 (3) 66.0• (3) 73.9• (3) 77.8 • 16.4
88.2• (3) 62.3• (3) 77.4• (3) 73.6 • 15.9
(5)
(5)
The values are means • SEM. The figures in parentheses represent the number of observations.
(0.73 • 0.18 mmole/liter) were observed 30 rain after the end of the infusion; 60 min later the mean plasma level of this amino acid had decreased to values (0.22 4- 0.04 mmole/liter) in the range of the preinfusion concentration (0.15 • 0.06 mmole/liter). Plasma levels of other amino acids showed no significant changes in response to arginine administration. Ammonia
Blood levels of ammonia were determined in children aged 1 day-12 yr. Normal values observed in the postabsorptive state showed little variation, ranging from 50 to 70 #g/100 ml. To evaluate the effect of arginine, this amino acid was infused in some of the children. The mean blood ammonia concentrations determined during and after the load were not significantly different (p > 0.05) from preinfusion levels (Table 2). Urea
Urea formation was stimulated by arginine infusion. When expressed on a molar basis, the increase in urinary urea excretion almost equaled the amount of arginine applied (Table 3). Most of the surplus urea appeared during the first 12 hr after the arginine load (data not shown). The plasma level of urea showed no significant variation during arginine infusion (Table 4). Acid Base Balance
During a 2 hr period after the infusion pH and standard bicarbonate remained without significant changes (Table 4). DISCUSSION
At the dosage used in this study, arginine infusion leads to a short-term increase in the plasma concentration of this amino acid. This value is about seven times higher than the values observed in argininemia. 11 It seems unlikely that elevated blood levels of arginine per se are responsible for the pathologic symptoms seen in this disorder, since the clinical features of all congenital disorders of urea synthesis closely resemble each other. In all likelihood, raised blood levels of ammonia represent the c o m m o n pathogenetic principle of these disorders} As demonstrated in this paper, the blood ammonia content did not change significantly after arginine administration. This result excludes the pos-
ARGININE INFUSION
1245
Table 3. Relationship Between Arginine Administration (0.5 g/kg) and Urea Formation Urinary Urea Excretion Before Arginine Administration (mmole/day)
Arginine Applied (mrnole)
Increase in Urinary Urea Excretion After Arginine Administration (mmole/day)
19.8
10.0
23.4
9.2
8.4 7.0
24.8
9.2
10.5 6.4
10.8
7.8
28.0
8.1
8.5
22.2
9.2
7.8
21.2
10.1
9.5
12.8
10.3
8.1
13.5
8.4
6.5
16.8
6.9
5.4
16.7
7.2
6.0
19.1 •
1.6*
8.8 • 0.4*
7.7 • 0.5*
Urine urea excretion was determined 1 day before and during the d a y after arginine administration. Nutritional intake was held constant during the experimental period. The table shows the increase in daily urea excretion caused by arginlne administration. I n d i v i d u a l values of 11 children ~age r a n g e 1 - 6 wk) a r e listed, * M e a n • SEM.
sibility that argininosuccinate synthetase, the rate-limiting enzyme of urea synthesis in liver, is grossly inhibited by arginine, as was shown in H e L a cell cultures. 7 Urea synthesis in liver is controlled by substrate and allosteric effector concentrations rather than by enzyme capacityJ 2 In this context arginine plays a dominant role. It stimulates the synthesis of acetylglutamate, a cofactor controlling the activity o f carbamoyl phosphate synthetase. 13 Hydrolysis of arginine is mediated by arginase, an enzyme which has been demonstrated to be present in every metabolically active tissue and which has the highest activity among all enzymes of the urea cycle in liver. 8 Ornithine and urea are formed by this irreversible step. Urea is nontoxic and rapidly excreted by the kidney. This is evidenced by the almost stoichiometric relationship between the amount of arginine applied and the excess in urinary urea excretion observed after the load (Table 3). Normal ornithine concentration in liver is lower than the Km value of ornithine for ornithine transcarbamylase, 14 suggesting that ornithine may be rate limiting under conditions in vivo. Hence increasing the intracellular Table 4. pH, Standard Bicarbonate, and Serum Urea Concentration Before and After Intravenous Loading with Arginine (0.5 g/kg body weight) Minutes After Arginine Infusion Parameter
0
15
30
45
60
90
120
pH Standard bicarbonate (mmole/liter) Serum urea (rag/100 ml)
7.46 • 0.01 23.7 • 1,1
7.38 • 0.03 22.3 -k: 0,7
7.46 :a: 0.02 21.9 :: 0.9
7,39 d: 0.02 22,9 • 1.4
7.42 -k 0.03 23.2 ~. 0,6
7.45 • 0.01 23.7 • 1.1
7.46 • 0,03 24.1 • 0.5
28.3 • 1.2
30.1 • 2.4
28.4 • 1,3
29.7 • 0,9
30.2 • 3,9
29.1 • 1.0
28.7 • 1.4
The va}ues are means • SEM determined in 11 infants (age range 1-31 days).
1246
KRAUS, STUBBE, AND von BERG
ornithine concentration should increase the capacity of the liver to dispose of surplus ammonia. This prediction is supported by liver perfusion experiments of Krebs et al. 12 These authors have demonstrated that when alanine is added to the perfusion medium rat liver forms a great deal of ammonia; but when alanine and ornithine are added simultaneously increased urea and not a m m o n i a production is observed. Ornithine breakdown involves transamination to glutamate 3'semialdehyde, which may be converted to glutamate and a-oxoglutarate. 15 Apparently ornithine once formed is rapidly eliminated by this enzymatic machinery. Though large amounts of ornithine must have been liberated by the high activity of arginase, only moderate and short-term accumulation of this amino acid in plasma is observed. Urinary losses are probably low since in the case of arginine it was shown that these amounts are negligible. In conclusion, arginine administration is considered to cause an increase rather than a decrease in the rate of the urea cycle. Surplus a m m o n i a derived from breakdown of ornithine is thereby cleaved. Finally, the carbon skeleton of ornithine, a "glycogenic" amino acid, may be converted to glucose or glycogen in the reactions of gluconeogenesis) 5 Evidence for this reaction sequence arises from the observation that a small (10-15 mg/100 ml) but significant (p < 0.05) increase in the mean blood glucose level is seen in newborn infants (n = 7) during the first hour after arginine administration. Obviously, even in newborn infants the liver is capable of fulfilling these metabolic demands. This is demonstrated not only by the fact that the rise in urea formation almost equals the a m o u n t of arginine applied, but also by the finding that a m m o n i a blood levels showed no significant changes in response to arginine infusion. The results of this study are in agreement with the observation that toward the end of pregnancy human fetal liver already produces considerable amounts of urea. ~6 Argininosuccinate synthetase activity determined in necropsy specimens of h u m a n infant liver was found in the range of 28 39 ~zmole/hr/g liver) 7 As the mean weight of h u m a n infant liver is 125 g,t8 the maximal turnover of the enzyme approximates 100 mmole per total liver and per day. Thus the daily capacity of the rate-limiting enzyme is far in excess of the requirements for both endogenous urea production (up to 10 mmole) and the additional load (about 8 mmole) caused by arginine administration. REFERENCES
1. Unger RH, Aguilar-Parada E, MUller WA, Eisentraut AM: Studies on pancreatic alpha cell function in normal and diabetic subjects. J Clin Invest 49:837 852, 1970 2. Merimee TJ, Rabinowitz D, Riggs L, Burgess JA, Rimoin DL, McKusick VA: Plasma growth hormone after arginine infusion. N Engl J Med 276:434-439, 1967 3. Tiwary CM, Rosenbloom AL, Julius RL: Anaphylactic reaction to arginine infusion. N Engl J Med 288:218-221, 1973 4. Hertz P, Richardson JA: Arginine-induced hyperkalemia in renal failure patients. Arch Intern Med 130:778-783, 1972
5. Terheggen HG, Schwenk A, LiSwenthalA, van Sande M, Colombo JP: Hyperargininaemie mit Arginasedefekt. Eine neue familiare StoffwechselstiSrung. 1. Klinische Befunde. Z Kinderheilk 107:298-310, 1970 6. TerheggenHG, LOwenthal A, Lavinka F, Colombo JP: Familial hyperargininemia. Arch Dis Child 50:57-71, 1975 7. Schimke RT: Enzymes of arginine metabolism in mammalian cell culture. I. Repression of argininosuccinate synthetase and argininosuccinase. J Biol Chem 239:136 147, 1964 8. Levin B: Hereditary metabolic disorders of
ARGININE INFUSION
the urea cycle, in Bodansky O, Latner AL (eds): Advances in Clinical Chemistry, vol 14. New York, Academic Press, 1971, p 92 9. Moore S, Stein WH: Procedures for the chromatographic determination of amino acids on four per cent crosslinked sulfonated polystyrene resins. J Biol Chem 211:893 915, 1954 10. Kun E, Kearncy EB: Ammoniak, in Bergmeyer HU (ed): Methoden der enzymatischen Analyse. Verlag Chemic, Weinheim, Germany, 1970, p 1749 11. Terheggen HG, Schwenk A, LtSwenthal A, van Sande M, Colombo JP: Hyperargininaemie mit Arginasedefekt. Eine neue familiare Stoffwechselst6rung. 2. Biochemische Untersuchungen. Z Kinderheilk 107:313-328, 1970. 12. Krebs HA, Hems R, Lund P: Regulatory mechanism in the synthesis of urea, in Hommes FA, van den Berg CJ (eds): Inborn Errors of Metabolism. New York, Academic Press, 1973, p 201 13. Titibana M, Shigesasa K: Role of acetyl-
1247
glutamate in ureotelism. 1. Occurrence and biosynthesis of acetylglutamate in mouse and rat tissues. J Biol Chem 246:5588 5599, 1971 14. Reichard P: Ornithine carbamyl transferase from rat liver. Acta Chem Scand 11:523 536, 1957 15. Harper HA, Rodrell V: Protein and amino acid metabolism, in Harper HA (ed): Review of Physiological Chemistry, ed 14. Los Altos, Calif., Lange, 1973, p 311 16. Kennan AL, Cohen PP: Ammonia detoxication the liver. Proc Soc Exp Biol Med 106: 170 185, 1961 17. Short EM, Conn HO, Snodgrass PJ, Campell AGM, Rosenberg LE: Evidence for X-linked dominant inheritance of ornithine transcarbamylase deficiency. N Engl J Med 288: 7 16, 1973 18. Boyd E: Growth, including reproduction and morphological development, in Airman E, Dittmer F (eds): Biological Handbooks. Washington, D.C., Federation of American Societies for Experimental Biology, 1962, p 346
molar basis, arginine administration resuited in an almost stoichiometric rise in urinary urea excretion. These findings indicate that arginine is rapidly metabolized via urea and ornithine, the latter being transformed to glucose, as evidenced by a significant rise in the blood glucose concentration. Blood gas analyses and serum urea and blood ammonia concentrations determined after the load showed no significant deviations from preinfusion levels. Thus, in contrast to the effects to be expected from studies w i t h tissue culture homogenates, even when administered to newborn infants, arginine does not impair the turnover of the urea cycle.
R G I N I N E , like other amino acids, is widely used as a stimulus to evaluate glucagon, insulin, and growth h o r m o n e release in man. t,2 F o r this purpose it is administered to young children as a means for the diagnosis of endocrine and metabolic disorders. Arginine hydrochloride is also applied in infancy in the treatment of hypochloremic alkalosis. N o negative side effects of arginine have been noticed unless the solution was contaminated 3 or infused in uremic patients. 4 Using the dosage c o m m o n l y accepted, loading with arginine results in transitory high blood levels of the amino acid c o m p a r a b l e to or even higher than those reported in argininemia, a condition due to a congenital defect in the last step of the urea cycle. 5'6 Since the s y m p t o m s of this disease are vomiting, failure to thrive, ataxia, tremor, spastic paresis, convulsions, and, finally, mental retardation, the question arises whether arginine administration might possibly be harmful in any direct or indirect manner. With regard to the latter possibility, arginine, in concentrations in excess of the optimal, has been found to inhibit both argininosuccinate synthetase and argininosuccinate lyase in H e L a cell homogenates. 7 Whether this finding is applicable to the conditions of the urea cycle in liver is uncertain. The liver is the only organ containing all the enzymes of the urea cycle in amounts high enough to account for total urea synthesis. However, some of these enzymes, the activities of which are far below those found in
A
From the Department of Pediatrics, University of Goettingen, Goettingen, Germany. Received for publication January 15. 1976. Reprint requests should be addressed to Dr. Hans Kraus, Universiti~ts-Kinderklinik, Goettingen, Germany. 9 1976 by Grune & Stratton, lnc.
Metabolism,Vol. 25, No. 11 (November),1976
3400
1241
1242
KRAUS, STUBBE, AND van BERG
Table 1. Clinical Data and Number of Patients Investigated During Various Pracedures Determination of Blood gas
No. of Patients Investigated
Age at Investigation*
11
2-31 days
7
Weight at Investigation~ (g)
2310
• 8.2
• 95
• 112
2 - 6 wk
264 • 7
2452 • 148
2785 • 82
274 • 11.2
2721 • 174
2974 • 125
Serum urea
11
1-31 days
Urinary urea
11
12-31 days
excretion Blood ammonia
Birthweightt (g)
268
analysis
Arginine and ornithine
Gestational Age1" (days)
2715
268
3120
3312
• 1.2
• 712
-4- 597
3
1-2 days
261 • 17
2290 • 91
2216 • 153
3
7-8 days
2233 • 198 2080
2260 • 262 2346
• 319 --
• 70 32 kg
3
1 89 wk
263 • 4.5 244
5
9-12 yr
• 22 --
• 2.5 *Range. t M e a n • SD.
liver, are widely distributed in the b o d y tissues and, as mentioned above, have even been found in t u m o r cell lines. Whether these enzymes are under the same genetic control and have the same properties as those of the urea cycle in liver has been questioned, s Nevertheless, if similar mechanisms are operating both in H e L a cells and in the liver, inhibition of argininosuccinate synthetase which catalyses the rate-limiting step would slow down the rate of the urea cycle and thereby increase the blood a m m o n i a concentration. Beyond the possibility of specific interference it is not known whether or not the capacity of the urea cycle in newborn infants is high enough to cope with the additional nitrogen load caused by arginine infusion. It is well known that the capacity of some enzyme systems increases during fetal development, adult values not being reached at birth or soon after birth. In an attempt to elucidate some of the possible metabolic effects of arginine under conditions in viva, the amino acid was infused in infants and children. Arginine and ornithine plasma concentrations and blood glucose and blood a m m o n i a levels were measured before and after the load. Catabolism of arginine is expected to result in increased urea formation. Therefore, urinary urea excretion and the concentration of urea in the serum in response to arginine administration were observed and recorded. Since the load with arginine hydrochloride m a y also result in increased hydrogen ion concentration, blood gas analyses were simultaneously performed. MATERIALS AND METHODS The clinical data and number of patients investigated are shown in Table 1. Solutions of 6 ~ (w/v) L-arginine hydrochloride were infused 4 hr after the last feeding in infants or after an overnight fast in children with a dosage of 0.5 g/kg/30 min. Quantitative analyses of amino acids before and after the load were performed according to the method of Moore and Stein 9 by
ARGININE INFUSION
1243
m mot / l 10
~
I0
3
I
I
60
g0
I
170 rnin
Fig. 1. Disappearance rate of arginine in plasma determined in seven children (age range from 2 - 6 wk). The equation of the elimination curve is represented by log y = - 0 . 0 0 7 x - t 0.81; p < 0.001; r = - 0 . 9 2 . y, concentration of arginine in plasma (mmole/liter); x, time (min) after arginine infusion. The elimination constant K (K = [In 2 / ( t / 2 ) ] • 100) is 1 . 9 7 % / m i n .
means of an automatic amino acid autoanalyzer (Biocal BC 200). Deproteinization of the blood specimens was accomplished by adding 1 ml 5~ sulphosalicylic acid to each 0.5-ml sample of plasma. After refrigerated centrifugation, an aliquot of the supernatant was applied to the column. The disappearance rate of arginine was established with the aid of a semilogarithmic plot and expressed as K = [In 2/(t/2)] • 100. The elimination curve was calculated by the method of least squares. Ammonia was determined enzymatically by the method of Kun and Kearney. 1~ Heparinized blood (0.5 ml) was immediately deproteinized with 0.5 ml I N ice-cold perchloric acid. The precipitated protein was removed by centrifugation and the supernatant fluid was neutralized with 2 N KHCO 3. Blank readings were obtained according to the same method except that neutralized perchloric acid replaced the unknown blood sample. Recoveries were 96~ :~ 7.4~ using this system. Urea was determined with urease, using a test kit of Boehringer and Sons, Mannheim, Germany. Blood gas analysis was performed according to the Astrup-micromethod. Informed parental consent was obtained for study of all children. RESULTS
Arginine and Ornithine N o r m a l p l a s m a a r g i n i n e levels in seven i n f a n t s (age r a n g e 2 - 6 wk) were f o u n d to be in the r a n g e 0.04-0.12 m m o l e / l i t e r . L o a d i n g with a r g i n i n e resulted in a sharp increase in the c o n c e n t r a t i o n o f this a m i n o acid in p l a s m a , with highest values a b o u t 7 m m o l e / l i t e r . A t the e n d of the o b s e r v a t i o n period, i.e., 2 hr after the end of the i n f u s i o n , a r g i n i n e p l a s m a levels h a d drastically decreased to a m e a n v a l u e o f 1 m m o l e / l i t e r . T h e d i s a p p e a r a n c e rate o f a r g i n i n e (Fig. 1) expressed by the e l i m i n a t i o n c o n s t a n t K (K = [In 2 / ( t / 2 ) ] x 100) is 1.97~/min. A l o n g with the increase in arginine, a parallel b u t less m a r k e d increase in the o r n i t h i n e p l a s m a c o n c e n t r a t i o n was s h o w n in the same i n d i v i d u a l s . Peak values
1244
KRAUS, STUBBE, AND von BERG
Table 2. Levels of Ammonia in Blood ( # g / 1 0 0 ml) Before and After Intravenous Loading With Arginine (0.5 g / k g body weight) Minutes After Arginine Infusion Age (range) 1-2days 7-8 days 189 9-12 yr
0
15
30
45
60
69.7• 70.7• 88.2• 15.5 84.7+ 10.6 77.6• 10.2 (6) (3) (3) (3) (3) 57.9• 10.0 61.3• 11.1 4 5 . 5 • 62.3• 55.7• (7) (3) (3) (3) (3) 63.0• 72.4• 88.4• 60.7• 64.7• (5) (3) (3) (3) (3) 55.2 • 9.2 70.6 • 12.9 70.7 • 12.7 62.1 • 15.6 63.6 -k 15.1
(4)
(5)
(5)
(5)
(5)
90
120
94.1 • 16.0 (3) 66.0• (3) 73.9• (3) 77.8 • 16.4
88.2• (3) 62.3• (3) 77.4• (3) 73.6 • 15.9
(5)
(5)
The values are means • SEM. The figures in parentheses represent the number of observations.
(0.73 • 0.18 mmole/liter) were observed 30 rain after the end of the infusion; 60 min later the mean plasma level of this amino acid had decreased to values (0.22 4- 0.04 mmole/liter) in the range of the preinfusion concentration (0.15 • 0.06 mmole/liter). Plasma levels of other amino acids showed no significant changes in response to arginine administration. Ammonia
Blood levels of ammonia were determined in children aged 1 day-12 yr. Normal values observed in the postabsorptive state showed little variation, ranging from 50 to 70 #g/100 ml. To evaluate the effect of arginine, this amino acid was infused in some of the children. The mean blood ammonia concentrations determined during and after the load were not significantly different (p > 0.05) from preinfusion levels (Table 2). Urea
Urea formation was stimulated by arginine infusion. When expressed on a molar basis, the increase in urinary urea excretion almost equaled the amount of arginine applied (Table 3). Most of the surplus urea appeared during the first 12 hr after the arginine load (data not shown). The plasma level of urea showed no significant variation during arginine infusion (Table 4). Acid Base Balance
During a 2 hr period after the infusion pH and standard bicarbonate remained without significant changes (Table 4). DISCUSSION
At the dosage used in this study, arginine infusion leads to a short-term increase in the plasma concentration of this amino acid. This value is about seven times higher than the values observed in argininemia. 11 It seems unlikely that elevated blood levels of arginine per se are responsible for the pathologic symptoms seen in this disorder, since the clinical features of all congenital disorders of urea synthesis closely resemble each other. In all likelihood, raised blood levels of ammonia represent the c o m m o n pathogenetic principle of these disorders} As demonstrated in this paper, the blood ammonia content did not change significantly after arginine administration. This result excludes the pos-
ARGININE INFUSION
1245
Table 3. Relationship Between Arginine Administration (0.5 g/kg) and Urea Formation Urinary Urea Excretion Before Arginine Administration (mmole/day)
Arginine Applied (mrnole)
Increase in Urinary Urea Excretion After Arginine Administration (mmole/day)
19.8
10.0
23.4
9.2
8.4 7.0
24.8
9.2
10.5 6.4
10.8
7.8
28.0
8.1
8.5
22.2
9.2
7.8
21.2
10.1
9.5
12.8
10.3
8.1
13.5
8.4
6.5
16.8
6.9
5.4
16.7
7.2
6.0
19.1 •
1.6*
8.8 • 0.4*
7.7 • 0.5*
Urine urea excretion was determined 1 day before and during the d a y after arginine administration. Nutritional intake was held constant during the experimental period. The table shows the increase in daily urea excretion caused by arginlne administration. I n d i v i d u a l values of 11 children ~age r a n g e 1 - 6 wk) a r e listed, * M e a n • SEM.
sibility that argininosuccinate synthetase, the rate-limiting enzyme of urea synthesis in liver, is grossly inhibited by arginine, as was shown in H e L a cell cultures. 7 Urea synthesis in liver is controlled by substrate and allosteric effector concentrations rather than by enzyme capacityJ 2 In this context arginine plays a dominant role. It stimulates the synthesis of acetylglutamate, a cofactor controlling the activity o f carbamoyl phosphate synthetase. 13 Hydrolysis of arginine is mediated by arginase, an enzyme which has been demonstrated to be present in every metabolically active tissue and which has the highest activity among all enzymes of the urea cycle in liver. 8 Ornithine and urea are formed by this irreversible step. Urea is nontoxic and rapidly excreted by the kidney. This is evidenced by the almost stoichiometric relationship between the amount of arginine applied and the excess in urinary urea excretion observed after the load (Table 3). Normal ornithine concentration in liver is lower than the Km value of ornithine for ornithine transcarbamylase, 14 suggesting that ornithine may be rate limiting under conditions in vivo. Hence increasing the intracellular Table 4. pH, Standard Bicarbonate, and Serum Urea Concentration Before and After Intravenous Loading with Arginine (0.5 g/kg body weight) Minutes After Arginine Infusion Parameter
0
15
30
45
60
90
120
pH Standard bicarbonate (mmole/liter) Serum urea (rag/100 ml)
7.46 • 0.01 23.7 • 1,1
7.38 • 0.03 22.3 -k: 0,7
7.46 :a: 0.02 21.9 :: 0.9
7,39 d: 0.02 22,9 • 1.4
7.42 -k 0.03 23.2 ~. 0,6
7.45 • 0.01 23.7 • 1.1
7.46 • 0,03 24.1 • 0.5
28.3 • 1.2
30.1 • 2.4
28.4 • 1,3
29.7 • 0,9
30.2 • 3,9
29.1 • 1.0
28.7 • 1.4
The va}ues are means • SEM determined in 11 infants (age range 1-31 days).
1246
KRAUS, STUBBE, AND von BERG
ornithine concentration should increase the capacity of the liver to dispose of surplus ammonia. This prediction is supported by liver perfusion experiments of Krebs et al. 12 These authors have demonstrated that when alanine is added to the perfusion medium rat liver forms a great deal of ammonia; but when alanine and ornithine are added simultaneously increased urea and not a m m o n i a production is observed. Ornithine breakdown involves transamination to glutamate 3'semialdehyde, which may be converted to glutamate and a-oxoglutarate. 15 Apparently ornithine once formed is rapidly eliminated by this enzymatic machinery. Though large amounts of ornithine must have been liberated by the high activity of arginase, only moderate and short-term accumulation of this amino acid in plasma is observed. Urinary losses are probably low since in the case of arginine it was shown that these amounts are negligible. In conclusion, arginine administration is considered to cause an increase rather than a decrease in the rate of the urea cycle. Surplus a m m o n i a derived from breakdown of ornithine is thereby cleaved. Finally, the carbon skeleton of ornithine, a "glycogenic" amino acid, may be converted to glucose or glycogen in the reactions of gluconeogenesis) 5 Evidence for this reaction sequence arises from the observation that a small (10-15 mg/100 ml) but significant (p < 0.05) increase in the mean blood glucose level is seen in newborn infants (n = 7) during the first hour after arginine administration. Obviously, even in newborn infants the liver is capable of fulfilling these metabolic demands. This is demonstrated not only by the fact that the rise in urea formation almost equals the a m o u n t of arginine applied, but also by the finding that a m m o n i a blood levels showed no significant changes in response to arginine infusion. The results of this study are in agreement with the observation that toward the end of pregnancy human fetal liver already produces considerable amounts of urea. ~6 Argininosuccinate synthetase activity determined in necropsy specimens of h u m a n infant liver was found in the range of 28 39 ~zmole/hr/g liver) 7 As the mean weight of h u m a n infant liver is 125 g,t8 the maximal turnover of the enzyme approximates 100 mmole per total liver and per day. Thus the daily capacity of the rate-limiting enzyme is far in excess of the requirements for both endogenous urea production (up to 10 mmole) and the additional load (about 8 mmole) caused by arginine administration. REFERENCES
1. Unger RH, Aguilar-Parada E, MUller WA, Eisentraut AM: Studies on pancreatic alpha cell function in normal and diabetic subjects. J Clin Invest 49:837 852, 1970 2. Merimee TJ, Rabinowitz D, Riggs L, Burgess JA, Rimoin DL, McKusick VA: Plasma growth hormone after arginine infusion. N Engl J Med 276:434-439, 1967 3. Tiwary CM, Rosenbloom AL, Julius RL: Anaphylactic reaction to arginine infusion. N Engl J Med 288:218-221, 1973 4. Hertz P, Richardson JA: Arginine-induced hyperkalemia in renal failure patients. Arch Intern Med 130:778-783, 1972
5. Terheggen HG, Schwenk A, LiSwenthalA, van Sande M, Colombo JP: Hyperargininaemie mit Arginasedefekt. Eine neue familiare StoffwechselstiSrung. 1. Klinische Befunde. Z Kinderheilk 107:298-310, 1970 6. TerheggenHG, LOwenthal A, Lavinka F, Colombo JP: Familial hyperargininemia. Arch Dis Child 50:57-71, 1975 7. Schimke RT: Enzymes of arginine metabolism in mammalian cell culture. I. Repression of argininosuccinate synthetase and argininosuccinase. J Biol Chem 239:136 147, 1964 8. Levin B: Hereditary metabolic disorders of
ARGININE INFUSION
the urea cycle, in Bodansky O, Latner AL (eds): Advances in Clinical Chemistry, vol 14. New York, Academic Press, 1971, p 92 9. Moore S, Stein WH: Procedures for the chromatographic determination of amino acids on four per cent crosslinked sulfonated polystyrene resins. J Biol Chem 211:893 915, 1954 10. Kun E, Kearncy EB: Ammoniak, in Bergmeyer HU (ed): Methoden der enzymatischen Analyse. Verlag Chemic, Weinheim, Germany, 1970, p 1749 11. Terheggen HG, Schwenk A, LtSwenthal A, van Sande M, Colombo JP: Hyperargininaemie mit Arginasedefekt. Eine neue familiare Stoffwechselst6rung. 2. Biochemische Untersuchungen. Z Kinderheilk 107:313-328, 1970. 12. Krebs HA, Hems R, Lund P: Regulatory mechanism in the synthesis of urea, in Hommes FA, van den Berg CJ (eds): Inborn Errors of Metabolism. New York, Academic Press, 1973, p 201 13. Titibana M, Shigesasa K: Role of acetyl-
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glutamate in ureotelism. 1. Occurrence and biosynthesis of acetylglutamate in mouse and rat tissues. J Biol Chem 246:5588 5599, 1971 14. Reichard P: Ornithine carbamyl transferase from rat liver. Acta Chem Scand 11:523 536, 1957 15. Harper HA, Rodrell V: Protein and amino acid metabolism, in Harper HA (ed): Review of Physiological Chemistry, ed 14. Los Altos, Calif., Lange, 1973, p 311 16. Kennan AL, Cohen PP: Ammonia detoxication the liver. Proc Soc Exp Biol Med 106: 170 185, 1961 17. Short EM, Conn HO, Snodgrass PJ, Campell AGM, Rosenberg LE: Evidence for X-linked dominant inheritance of ornithine transcarbamylase deficiency. N Engl J Med 288: 7 16, 1973 18. Boyd E: Growth, including reproduction and morphological development, in Airman E, Dittmer F (eds): Biological Handbooks. Washington, D.C., Federation of American Societies for Experimental Biology, 1962, p 346
