- Email: [email protected]
Studies on l -arginase of the small intestine
BIOCHEMICAL
14, 263-273
MEDICINE
Studies II.
(1975)
on L-Arginase
of the Small
Intestinal Arginase in Young its Role in Maintaining
L. Medical
KONARSKA
School
AND
of Warsan,.
02-097
Institute Warszalc*a,
Received
L.
and Adult Mammals, Urea Body Pool
and
TOMASZEWSKI
Department
of Clinical
of Biopharmacy ul.Banacha
August
Intestine
Biochemistry,
I (Poland)
27, 1975
INTRODUCTION
There are but scarce data in the literature concerning the arginase of intestinal mucosa. The enzyme has been found to occur in mucosa of the small intestine of dog (l), ox (l), mouse (2, 3), rat (4), some species of fish (5), and among the invertebrates, in the earthworm gut (6). However, the presence of arginase in mucosa of human small intestine has not so far been reported. In our previous paper, it has been demonstrated that many properties of the arginase from rat small intestine are identical with those of the enzyme from kidney and other rat tissues. From clinical observations it is known that in human blood the normal level of urea can be maintained even under conditions when the main pathway of its biosynthesis in liver is blocked due either to an inborn metabolic error in the omithine cycle, or to liver disease. Therefore, it seemed of interest to investigate whether under physiological conditions the arginase of small intestine can hydrolyze arginine and supply in this way the extracellular pool of urea. The occurrence of arginase in extrahepatic tissues of mammals is largely species-dependent; since in the rat the highest level of arginase was found,. apart from liver, in the small intestine and lactating mammary gland (4), this animal as well as the dog were chosen for our experiments. Moreover, data are presented on arginase activity in the small intestine of man and rat taking into account the age factor. MATERIALS
AND METHODS
Human rissrtes. Segments of the small intestine were taken from strictly determined regions ofduodenum, jejunum, and ileum, within 10-48 hr after death of persons who had not suffered from diseases of the alimentary 263 Copyright All rights
@ 1975 by Academic Press. Inc. of reproduction in any form reserved.
264
KONARSKA AND ?‘OMASZEWSKI
tract. Immediately after necropsy, the segments of the intestine were frozen and kept at - 10°C until taken for determinations. Samples of intestinal mucosa, weighing 20-30 mg, were taken on biopsis from the intestine in the neighborhood of the Treitz flexure. The position of the capsule of the catherer was controlled by X-rays. Erythrocytes were obtained from blood of healthy adults. Dogs. Healthy animals were killed by bleeding, and segments of the small intestine from the determined regions were immediately withdrawn. Before experiments in ~ivo, the animals were fasted for 24 hr. Ruts. White rats of the Wistar strain, adult (weighing 250-300 g) and young, were used. They were fed standard granulated laboratory diet containing vitamins and mineral salts. The animals were fasted for 24 hr before the experiment, killed by decapitation, bled, and segments of the small intestine from the determined regions were withdrawn. Homogenates. The isolated intestine was separated from mesentherium and washed with cold Tris-HCl buffer to remove intestinal contents. The mucosa was suspended in 5 vol ofcold 1 mM-MnCl,-5 mM-Tris-HCI buffer, pH 7.5, and homogenates prepared as described in the previous work (7). In the case of intestine from neonate rats. up to the 10th day of life, whole segments of the intestine were homogenized. Enzyme assay. The reaction mixture contained in 1 ml: 20 pmoles of L-arginine (adjusted to pH 7.5), 1 pmole of MnCl,, 50pmoles of Tris-HCl buffer, pH 9.5, and enzyme preparation. After 15 min incubation at 37°C. the reaction was stopped by immersing the samples for 5 min in a boilingwater bath. The activity of the enzyme was determined as described previously (7) by measuring the amount of ornithine formed. Protein determination. The method of Lowry et ul. (8) was used, with crystalline bovine serum albumin as standard. Experiments in lvitro. The experiments were performed on isolated rat intestine by the everted-sac technique of Wilson and Wiseman (9) in the modification of Crane and Wilson (10). i.e., by the so-called “test-tube” method (Fig. 1). The intestinal sac was placed in a tube containing 11.5 ml of Krebs-Ringer bicarbonate solution containing 0.2% glucose and the studied amount of arginine. A mixture of oxygen and carbon dioxide (95:5) was passed through the solution in the tube at a rate of 20-30 ml/min. The tubes were kept in water bath at 37°C for 60 min. After incubation, samples were taken both from within the intestinal sac (i.e., on the serosal side) and from the outer fluid (on the mucosal side). In the samples, urea was determined by the urease method (I I). Controls consisted of intestinal sacs incubated without arginine. Experiments in i!i\vo. These experiments were performed on dogs. The animals were anesthesized by intravenous injection of Eunarcon (0.4 ml/kg body wt) and kept throughout the experiment in a state of light sleep. A tube
L-ARGINASE
OF THE SMALL
INTESTINE
265
FIG. 1. Scheme of the apparatus for the experiments by the method of everted intestinal sacs, in the modification of Crane and Wilson (10). (A) injection needle serving as gas outlet from the tube; (B) rubber stopper; (C) tube of 15 ml volume; (D) fluid on the mucosal side: 11.5 ml of Krebs-Ringer solution containing 0.2% glucose and the studied amount of arginine; (E) fluid on the serosal side (0.9 ml of the same solution but containing no arginine); (F) as-cm long segment of thejejunum, turned inside out, tied at the bottom to form an “intestinal sack”; (G) injection needle with a fastened polyethylene tube, for introducing the mixture of Q and C@; (H) cannula.
was introduced into the alimentary tract. After opening of the abdominal cavity, the portal and femoral veins were cannulated with heparinized cannules. The position of the tube was checked and the studied volume of aqueous solution of arginine was introduced into the duodenum. Samples of blood from the portal and femoral veins were taken prior to administration of arginine, and then at intervals first of 5 min, and then of 15 and 30 min up to 2.5 hr. Urea was determined in parallel by the urease method (11) and according to Caraway (12) with diacetyl monoxime; control samples were checked for the absence of ammonia. RESULTS
The activity of arginase was determined in duodenum, jejunum, and ileum of three species of mammals: man, dog, and rat. The highest enzyme activity was observed in rat, and the lowest in man (Tabie 1). In all three species, the activity was the highest in duodenum, and the lowest in ileum.
266
KONARSKA
AND TOMASZEWSKI TABLE
ARGINASE
ACTIVITY SMALI.
I
IN THE MUCOSA OF SELECTED INTESTINE IN RAT. DOG, AND
SEGMENTS
OF I HE
MAY” Activity (ornithine formed)
Animal
Segment of intestine
h ___-___
~molesimg protein/l5 min
pmolesigm of mucosa/min
Rat
Duodenum Jejunum Ileum
20 20 20
8.lY 2 1.76 7.16 -c 2.16 2.63 k I.02
‘3.4 2 13.0 56.4 z 10.3 17.X r 5.6
Dotit
Duodenum Jejunum Ileum
5 5 5
0.51 2 0.11 0.40 + 0. IO 0 12 t 0.04
3.9 i 0.X 4.0 f I.2 I .o i- 0.4
Man material obtained at necropsy
Duodenum Jejunum Ileum
20 20 20
0.42 c 0.19 0.26 -c 0.13 0.20 t 0.09
1.7 2 0.X 1.7 2 0.6 0.6 ?- 0.2
material obtained at biopsy
Treitz flexure
15
0.20 + 0.13
1.3 + 0.7
C The results are mean values from the indicated number(N) of individual samples. i SD.
Activity of Intestinal Arginase during Development The activity of intestinal arginase in newborn rats was very low, about 1 pmole of omithine per gram of mucosa per minute, and remained at this level for about a fortnight (Fig. 2). At this time, the activity of the enzyme in duodenum, jejunum, and ileum showed no differences. Within the 3rd week of life, when the animals passed gradually from milk diet to normal laboratory diet, a distinct, statistically significant increase in arginase activity was observed up to values of 20 pmoles in duodenum, 17 pmoles in jejunum and 11 pmoles/g of mucosa per minute in ileum. These differences in the enzyme activity between the particular segments of the small intestine remained throughout the animal’s life. A further, statistically significant increase in arginase activity in all three segments of the small intestine occurred between the 3rd and 4th, and 4th and 5th weeks of life. In Sweek-old rats the arginase activity reached the level characteristic for adult animals; the differences in enzyme activity between those animals and the older ones (6-10 weeks), were not statistically significant. In man, age-dependent differences in .arginase activity were much less
L-ARGINASE
OF THE SMALL
INTESTINE
267
FIG. 2. Effect ofage on arginase activity in the mucosa ofrat small intestine. (A) Duodenum, (B) jejunum, (C) ileum. The activity is expressed as FmoIes of omithine/mg protein/IS min. The results for each age group are mean values from five to eight determinations.
pronounced (Table 2). In all three studied segments of the small intestine, the enzyme activity in children within the first year of life was usually lower than in adults, but the difference was statistically significant only for duodenum. TABLE ARGINASE ACTIVITY IN MUCOSA IN ADULTS AND CHILDREN
2 OF HUMAN SMALL INTESTINE, AGED UP TO 12 MONTHS”
Activity Segment of intestine
Age group
N
pmoles/mg protein/IS min
pmoles/g of mucosa/min
Duodenum
children adults
17 17
0.23 f 0.08 0.45 2 0.19
Jejunum
children adults
17 17
0.20 + 0.09 0.27 2 0.13
1.08 2 0.60 1.67 r 0.97 0.76 2 0.26 1.20 5 0.74
Ileum
children adults
17 17
0.16 k 0.05 0.20 5 0.09
0.56 c 0.33 0.59 f 0.26
@The results are mean values of the indicated number (N) of individual samples 2 SD.
268
KONARSKA
Hydrolysis of Exogenous Arginine by Isolated Rat Intestine
AND
TOMASZEWSKI
in Vitro
By the test-tube method of Crane and Wilson (10) it has been demonstrated that the greater was arginine concentration, the higher was the increase in the amount of urea formed (Fig. 3). It seems interesting that under similar conditions the arginase of human erythrocytes did not catalyze this reaction. Human erythrocytes were washed several times with physiological saline solution, centrifuged, and 1 ml introduced into a cellophane dialysing bag (0.8 x 4 cm). The bag was placed in a test tube containing0.2% glucose in Krebs-Ringer buffer and the studied amount of arginine, and the O,/CO, mixture (95:5) was passed through the solution. The determinations were performed in the same way as with the intestinal sacs, but no urea formation was observed. It should be noted that under the applied conditions erythrocytes did not undergo hemolysis. Experimrnts
in Vi\w
The experiments were aimed at determining whether arginase of the small intestine can catalyze in vivo hydrolysis of exogenous arginine to urea, and to what extent this process could contribute to the extracellular pool of urea in the organism. The results presented in Fig. 4 show that after intraduodenal administration ofarginine, ureaconcentration was increased in the blood, both from the portal and femoral vein.
TABLE 3 THE DEPENDENCE OF PRODUCTION OF UREA FROM THE ADDED ARGININE IN EXPERIMENTS in Virro ACCORDING TO CRANE AND WILSON” fimoles
Arginine @moles
External fluidmucosal side
of urea
Internal kidserosal side
Total
0.4 1.1 1.2 1.5 1.6
3.0 6.‘: I0.i IX.7 22.6
-.---__ 0 10 loo 500 1000 ” The
means
of three
2.6 5.6 9.5 17.2 21.0 to five experiments
are given.
--~
L-ARGINASE OF THE SMALL INTESTINE
269
FIG. 3. Effectof the amount of added arginineon formation of urea by isolated rat intestine. The experiments were performed according to Crane and Wilson (IO). Mean values from five determinations are given.
DISCUSSION
Arginase Activity in Mucosa of the Small Zntestine in Man, Dog, and Rat Among the mammals studied, the highest arginase activity in intestinal mucosa was found in the rat, and the lowest in man (Table 1). Specific activity of the enzyme from human intestine was about one-twentieth that in the rat, and when calculated per 1 g of mucosa, only about one-fortieth. In man, arginase activity in the mucosa obtained about 10-16 hr after death was, as a rule, somewhat higher than in mucosa obtained by biopsy. A similar difference was observed with arginase from human liver (13). Similarly as in rat (7), the highest activity of arginase in man and dog was found in duodenum, and the lowest in ileum. Activity of Zntestinal Arginase during Ontogenesis Several enzymes occurring in mucosa of rat small intestine, among others amylase, nonspecific esterase, lipase, as well as some proteolytic
270
KONARSKA
AND TOMASZEWSKI
25
I
0
30
60
90
120
,
I
150
time (min) FIG. 4. increase in urea concentration in the blood of portal and femoral veins of the dog after intraduodenal administration of arginine in GVO. Weight of the dog, 10 kg: amount of administered arginine, 420 mg; time of the experiment 2.5 hr. Urea was determined in samples of blood taken from the portal vein (-O-O-) and femoral vein (-O-O-).
enzymes, show much lower activity in suckling rats than in adult ones: the increase in activity occurring usually between the 14th and 21st day of life (14). The activity of intestinal arginase during the postembryonal development of the rat shows a similar course; it is very low throughout the first fortnight, with a distinct increase occurring the 3rd week of life. In the 5th week, the arginase activity in all the studied segments of the intestine raises to the values characteristic for adult animals (Fig. 2). As it appears from the studies OfGreengardetai. (4) and Porembska( IS), the development of arginase activity in liver shows two phases of intense increase: the first just before birth, and the second, which is the main one, during the 3rd week of life. We did not observe the first phase of increased activity of intestinal arginase. The rapid increase in the enzyme activity during the 3rd and 4th week of life appears in the rat at the time the young ones pass from the milk diet to the normal laboratory diet. The interpretation of this phenomenon presents some difficulties, as three factors should be considered: the adaptive character of the enzyme, effect of Mn2+ ion. and hormonal regulation.
L-ARGINASE
OF THE
SMALL
INTESTINE
271
The milk diet is low in Mn2+ and contains about 11.8% of protein, whereas the laboratory diet has 40 mg of M$+/kg and about 1% of protein. It could be supposed that the increase in arginase activity is due to the change in diet, which would point to the adaptive character of the enzyme or its activation by MnZ+ ions. These suppositions seem to be supported by the studies of Boyer et al. (16) who demonstrated that in adult rats kept on an Mn2+-low milk diet, the activity of liver arginase was consistently lower than in animals fed in a normal way. Enrichment of the diet in Mn2+ led to an increase in the enzyme activity in liver (17). High-protein diet is also known to cause a considerable increase in the activity of hepatic arginase (18, 19). One the other hand, the results of other authors point to a considerable role of steroid hormones in the enzyme activity. By administration of corticosteroids (20), the same increase in the activity of hepatic arginase could be obtained as by the high-protein diet. Moreover, in rats the increase in arginase activity in the 3rd week of life is coincident with the period of increased activity of pituitary gland and adrenal cortex (21). Greengard et al. (4) suggested that the activity of hepatic arginase is subject to hormonal regulation: in the first, embryonic phase it increases under the influence of thyroxine, and in the second, of glucocorticoids. At variance with the results obtained with the rat, human intestinal arginase did not show distinct age-dependence. In children aged up to 1 year, the enzyme activity was lower than in adults in all three intestinal segments studied, but the difference was statistically significant only for duodenum. Role of Intestinal Arginase in Maintaining the Extracellular Pool of Urea The results obtained in vitro and in viuo (Figs. 3 and 4) point to possible participation of the intestine in maintaining the extracellular pool of urea in the organism. This may be of importance in cases when the main pathway of urea formation in liver is blocked due to genetic impairment or as a result of liver disease. In all cases of inborn metabolic blocks (which may concern each of the five enzymes of the ornithine cycle), described so far, urea concentration in blood remained normal (22), despite high concomitant hyperammonemia. This leads to the conclusion that urea is formed in the organism even when one of the enzymes involved in its synthesis in liver is inactive. The pool of urea, measured as blood urea level, is decreased in patients kept on lowprotein diet (22). Several hypotheses have been advanced to explain these observations, Possible residual activity of the blocked enzyme (13, 23) has been considered, as well as an alternative pathway of urea synthesis (2426), activity in other tissues of the enzyme that was blocked in liver (23), and the activity
272
KONARSKAAND't'OMAS%EWSKI
of the mutant enzyme (27). However, none of these suggestions provides adequate explanation for the observed phenomena. Colombo (23). taking into account that arginase occurs in several tissues, assumed as a theoretical possibility, intra- or extrahepatic formation of urea through hydrolysis of arginine. The occurrence of urea in blood of patients with a metabolic block in the ornithine cycle is not a proof that urea is formed due to the action of the whole cycle, especially as this block is usually accompanied by hyperammonemia, which obviously points to disturbances in detoxication of ammonia. On this assumption it seems possible to accept that urea is formed during hydrolysis of arginine in every tissue which contains arginase. However, in most of the extrahepatic tissues. the activity of arginase is very low. The comparatively high activity of intestinal arginase permits to suggest that hydrolysis of exogenous arginase occurring in the intestinal wall may participate in maintaining the extracellular pool of urea under conditions when the hepatic pathway of its synthesis is blocked. This supposition is supported by the results obtained on isolated rat intestine: under the applied conditions, the amount of urea formed in vitro during 1 hr from the supplied arginine, when related to the whole length of the small intestine, corresponded to the average level of this compound in rat blood. Our experiments also demonstrated that the arginase of erythrocytes, which shows rather high activity in man, does not participate in supplying urea to the extracellular pool. The presented suggestion seems to be supported also by the clinically observed increase in urea concentration in the blood of patients with high bleeding into the gastrointestinal tract (28). Indirect support comes also from the studies on absorption of amino acids by the intestinal wall it7 vitro: Finch and Hird (29) observed that a decrease in the amount of arginine by about 90% was accompanied by the appearance of ornithine in an amount corresponding to about 56% of the arginine that had disappeared. Although the authors did not discuss this observation, it is also a direct proof for the metabolism of arginine in the intestinal wall. SUMMARY The activity of arginase was demonstrated in mucosa of human small intestine. The enzyme activity was lower in young children (up to 1 year old) than in adults. In newborn and suckling rats, the arginase activity was very low; a rapid increase in the activity occurred within the third week of life. with simultaneous quantitative differentiation of the activity in duodenum, jejunum, and ileum.
L-ARGINASE
OF THE SMALL
INTESTINE
273
Hydrolysis of exogenous arginine occurring in the intestinal wall can supply the extracellular pool of urea. In dogs, after intraduodenal administration of arginine in viva, concentration of urea increased, both in the blood from the portal vein and in peripheral blood. When nonhemolyzed human erythrocytes were incubated in the presence of arginine, no formation of urea was observed. REFERENCES I. 2. 3. 4. 5. 6. 7. 8.
Kossel, A., and Dakin, H. D., Z. Physiot. Chem. 41, 321 (1904). Greenstein, J. P., J. Nur. Cancer Inst. 4, 275 (1943). Kochakian, C. D., J. Bid/. Chem. 161, 115 (1945). Greengard, O., Sahib, M. K., and Knox, W. E.,Arch. Biochem. Bioph,ys. 137,477( 1970). Baret, R., Mourgue, M., and Pellegrin, J., Compt. Rend. Sot. Biol. 160, 1796 (1966). Reddy, S. R. R., and Campbell, J. W., Biochim. Biophys. Acra 159, 557 (1968). Konarska, L., and Tomaszewski, L., Biochem. Med. 14, 250-262 (1975). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.,J. Biol. Chem. 193,265 (1951). 9. Wilson. T. H., and Wiseman, G.. J. Physiol. 126, 116 (1954). 10. Crane, R. K., and Wilson, T. H., J. Appl. Physiol. 12, 145 (1958). II. Richterich, R., “Klinische Chemie.” Karger, Basel-New York, 1965. 12. Caraway, W. T., and Fanger, H., Amer. J. Clin. Pathol. 25, 317 (1!)55). 13. Levin, B., in “Advances in Clinical Chemistry” (0. Bodansky and A. L. Latner, Eds.), D. 66. Vol. 14. Academic Press. New York. London. 1971. 14. Koldovsky, O., “Development ofthe Functions of the Small Intestine in Mammals and Man,” p. 215. Karger, Basel-New York, 1969. 15. Porembska, Z., in “Enzyme,” (Y. Frei, W. E. Knox, 0. Greengard, Eds.), Vol. 15, p. 198. Karger, Basel, 1973. 16. Boyer, P. D., Shaw, J. H., and Phillips, P. H.. J. Biol. Chem. 143, 417 (1942). 17. Shils, M. E., and McCollum, E. V., J. Nlrtr. 26, 1 (1943). 18. Ashida, K., and Harper, A. E., Proc. Sot. Exp. Bio/. Med. 107, 151 (1961). 19. Schimke, R. T., J. Biol. Chem. 238, 1012 (1963). 20. McLean, P., and Gurney, M. W.. Biochem. J. 87, 96 (1963). 21. Levine, S., and Mullins, R. F.. Science 152, 1585 (1966). 22. McMurray, W. C., Rathbun, J. C., Mohyuddin, F., and Koegler, S. J , Pediatrics 32,347 (1963). 23. Colombo, J. P., in “Congenital Disorders of the Urea Cycle and Ammonia Detoxication.” (F. Falkner, N. Kretchmer, E. Rossi, Eds.), Monographs in Paediatrics, Vol. 1, p. 93. Karger, Basel, 1971. 24. Bach. S. J., and Smith, M., Biochem. J. 64, 417 (1956). 25. Cohen, B. D., and Stein, I. M., Amer. J. Med. 45, 63 (1968). 26. Stein, J. M., and Cohen, B. D., New Engl. J. Med. 280, 926 (1969). 27. Striver, C. R., &it. Med. Bull. 25, 35 (1969). 28. Cantarow, A., and Trumper, M., in “Clinical Biochemistry,” p. 167. W. B. Saunders Company, Philadelphia-London, 1962. 29. Finch, L. R., and Hird, F. J. R., Biochim. Biophys. Acta 43, 278 (1960).
14, 263-273
MEDICINE
Studies II.
(1975)
on L-Arginase
of the Small
Intestinal Arginase in Young its Role in Maintaining
L. Medical
KONARSKA
School
AND
of Warsan,.
02-097
Institute Warszalc*a,
Received
L.
and Adult Mammals, Urea Body Pool
and
TOMASZEWSKI
Department
of Clinical
of Biopharmacy ul.Banacha
August
Intestine
Biochemistry,
I (Poland)
27, 1975
INTRODUCTION
There are but scarce data in the literature concerning the arginase of intestinal mucosa. The enzyme has been found to occur in mucosa of the small intestine of dog (l), ox (l), mouse (2, 3), rat (4), some species of fish (5), and among the invertebrates, in the earthworm gut (6). However, the presence of arginase in mucosa of human small intestine has not so far been reported. In our previous paper, it has been demonstrated that many properties of the arginase from rat small intestine are identical with those of the enzyme from kidney and other rat tissues. From clinical observations it is known that in human blood the normal level of urea can be maintained even under conditions when the main pathway of its biosynthesis in liver is blocked due either to an inborn metabolic error in the omithine cycle, or to liver disease. Therefore, it seemed of interest to investigate whether under physiological conditions the arginase of small intestine can hydrolyze arginine and supply in this way the extracellular pool of urea. The occurrence of arginase in extrahepatic tissues of mammals is largely species-dependent; since in the rat the highest level of arginase was found,. apart from liver, in the small intestine and lactating mammary gland (4), this animal as well as the dog were chosen for our experiments. Moreover, data are presented on arginase activity in the small intestine of man and rat taking into account the age factor. MATERIALS
AND METHODS
Human rissrtes. Segments of the small intestine were taken from strictly determined regions ofduodenum, jejunum, and ileum, within 10-48 hr after death of persons who had not suffered from diseases of the alimentary 263 Copyright All rights
@ 1975 by Academic Press. Inc. of reproduction in any form reserved.
264
KONARSKA AND ?‘OMASZEWSKI
tract. Immediately after necropsy, the segments of the intestine were frozen and kept at - 10°C until taken for determinations. Samples of intestinal mucosa, weighing 20-30 mg, were taken on biopsis from the intestine in the neighborhood of the Treitz flexure. The position of the capsule of the catherer was controlled by X-rays. Erythrocytes were obtained from blood of healthy adults. Dogs. Healthy animals were killed by bleeding, and segments of the small intestine from the determined regions were immediately withdrawn. Before experiments in ~ivo, the animals were fasted for 24 hr. Ruts. White rats of the Wistar strain, adult (weighing 250-300 g) and young, were used. They were fed standard granulated laboratory diet containing vitamins and mineral salts. The animals were fasted for 24 hr before the experiment, killed by decapitation, bled, and segments of the small intestine from the determined regions were withdrawn. Homogenates. The isolated intestine was separated from mesentherium and washed with cold Tris-HCl buffer to remove intestinal contents. The mucosa was suspended in 5 vol ofcold 1 mM-MnCl,-5 mM-Tris-HCI buffer, pH 7.5, and homogenates prepared as described in the previous work (7). In the case of intestine from neonate rats. up to the 10th day of life, whole segments of the intestine were homogenized. Enzyme assay. The reaction mixture contained in 1 ml: 20 pmoles of L-arginine (adjusted to pH 7.5), 1 pmole of MnCl,, 50pmoles of Tris-HCl buffer, pH 9.5, and enzyme preparation. After 15 min incubation at 37°C. the reaction was stopped by immersing the samples for 5 min in a boilingwater bath. The activity of the enzyme was determined as described previously (7) by measuring the amount of ornithine formed. Protein determination. The method of Lowry et ul. (8) was used, with crystalline bovine serum albumin as standard. Experiments in lvitro. The experiments were performed on isolated rat intestine by the everted-sac technique of Wilson and Wiseman (9) in the modification of Crane and Wilson (10). i.e., by the so-called “test-tube” method (Fig. 1). The intestinal sac was placed in a tube containing 11.5 ml of Krebs-Ringer bicarbonate solution containing 0.2% glucose and the studied amount of arginine. A mixture of oxygen and carbon dioxide (95:5) was passed through the solution in the tube at a rate of 20-30 ml/min. The tubes were kept in water bath at 37°C for 60 min. After incubation, samples were taken both from within the intestinal sac (i.e., on the serosal side) and from the outer fluid (on the mucosal side). In the samples, urea was determined by the urease method (I I). Controls consisted of intestinal sacs incubated without arginine. Experiments in i!i\vo. These experiments were performed on dogs. The animals were anesthesized by intravenous injection of Eunarcon (0.4 ml/kg body wt) and kept throughout the experiment in a state of light sleep. A tube
L-ARGINASE
OF THE SMALL
INTESTINE
265
FIG. 1. Scheme of the apparatus for the experiments by the method of everted intestinal sacs, in the modification of Crane and Wilson (10). (A) injection needle serving as gas outlet from the tube; (B) rubber stopper; (C) tube of 15 ml volume; (D) fluid on the mucosal side: 11.5 ml of Krebs-Ringer solution containing 0.2% glucose and the studied amount of arginine; (E) fluid on the serosal side (0.9 ml of the same solution but containing no arginine); (F) as-cm long segment of thejejunum, turned inside out, tied at the bottom to form an “intestinal sack”; (G) injection needle with a fastened polyethylene tube, for introducing the mixture of Q and C@; (H) cannula.
was introduced into the alimentary tract. After opening of the abdominal cavity, the portal and femoral veins were cannulated with heparinized cannules. The position of the tube was checked and the studied volume of aqueous solution of arginine was introduced into the duodenum. Samples of blood from the portal and femoral veins were taken prior to administration of arginine, and then at intervals first of 5 min, and then of 15 and 30 min up to 2.5 hr. Urea was determined in parallel by the urease method (11) and according to Caraway (12) with diacetyl monoxime; control samples were checked for the absence of ammonia. RESULTS
The activity of arginase was determined in duodenum, jejunum, and ileum of three species of mammals: man, dog, and rat. The highest enzyme activity was observed in rat, and the lowest in man (Tabie 1). In all three species, the activity was the highest in duodenum, and the lowest in ileum.
266
KONARSKA
AND TOMASZEWSKI TABLE
ARGINASE
ACTIVITY SMALI.
I
IN THE MUCOSA OF SELECTED INTESTINE IN RAT. DOG, AND
SEGMENTS
OF I HE
MAY” Activity (ornithine formed)
Animal
Segment of intestine
h ___-___
~molesimg protein/l5 min
pmolesigm of mucosa/min
Rat
Duodenum Jejunum Ileum
20 20 20
8.lY 2 1.76 7.16 -c 2.16 2.63 k I.02
‘3.4 2 13.0 56.4 z 10.3 17.X r 5.6
Dotit
Duodenum Jejunum Ileum
5 5 5
0.51 2 0.11 0.40 + 0. IO 0 12 t 0.04
3.9 i 0.X 4.0 f I.2 I .o i- 0.4
Man material obtained at necropsy
Duodenum Jejunum Ileum
20 20 20
0.42 c 0.19 0.26 -c 0.13 0.20 t 0.09
1.7 2 0.X 1.7 2 0.6 0.6 ?- 0.2
material obtained at biopsy
Treitz flexure
15
0.20 + 0.13
1.3 + 0.7
C The results are mean values from the indicated number(N) of individual samples. i SD.
Activity of Intestinal Arginase during Development The activity of intestinal arginase in newborn rats was very low, about 1 pmole of omithine per gram of mucosa per minute, and remained at this level for about a fortnight (Fig. 2). At this time, the activity of the enzyme in duodenum, jejunum, and ileum showed no differences. Within the 3rd week of life, when the animals passed gradually from milk diet to normal laboratory diet, a distinct, statistically significant increase in arginase activity was observed up to values of 20 pmoles in duodenum, 17 pmoles in jejunum and 11 pmoles/g of mucosa per minute in ileum. These differences in the enzyme activity between the particular segments of the small intestine remained throughout the animal’s life. A further, statistically significant increase in arginase activity in all three segments of the small intestine occurred between the 3rd and 4th, and 4th and 5th weeks of life. In Sweek-old rats the arginase activity reached the level characteristic for adult animals; the differences in enzyme activity between those animals and the older ones (6-10 weeks), were not statistically significant. In man, age-dependent differences in .arginase activity were much less
L-ARGINASE
OF THE SMALL
INTESTINE
267
FIG. 2. Effect ofage on arginase activity in the mucosa ofrat small intestine. (A) Duodenum, (B) jejunum, (C) ileum. The activity is expressed as FmoIes of omithine/mg protein/IS min. The results for each age group are mean values from five to eight determinations.
pronounced (Table 2). In all three studied segments of the small intestine, the enzyme activity in children within the first year of life was usually lower than in adults, but the difference was statistically significant only for duodenum. TABLE ARGINASE ACTIVITY IN MUCOSA IN ADULTS AND CHILDREN
2 OF HUMAN SMALL INTESTINE, AGED UP TO 12 MONTHS”
Activity Segment of intestine
Age group
N
pmoles/mg protein/IS min
pmoles/g of mucosa/min
Duodenum
children adults
17 17
0.23 f 0.08 0.45 2 0.19
Jejunum
children adults
17 17
0.20 + 0.09 0.27 2 0.13
1.08 2 0.60 1.67 r 0.97 0.76 2 0.26 1.20 5 0.74
Ileum
children adults
17 17
0.16 k 0.05 0.20 5 0.09
0.56 c 0.33 0.59 f 0.26
@The results are mean values of the indicated number (N) of individual samples 2 SD.
268
KONARSKA
Hydrolysis of Exogenous Arginine by Isolated Rat Intestine
AND
TOMASZEWSKI
in Vitro
By the test-tube method of Crane and Wilson (10) it has been demonstrated that the greater was arginine concentration, the higher was the increase in the amount of urea formed (Fig. 3). It seems interesting that under similar conditions the arginase of human erythrocytes did not catalyze this reaction. Human erythrocytes were washed several times with physiological saline solution, centrifuged, and 1 ml introduced into a cellophane dialysing bag (0.8 x 4 cm). The bag was placed in a test tube containing0.2% glucose in Krebs-Ringer buffer and the studied amount of arginine, and the O,/CO, mixture (95:5) was passed through the solution. The determinations were performed in the same way as with the intestinal sacs, but no urea formation was observed. It should be noted that under the applied conditions erythrocytes did not undergo hemolysis. Experimrnts
in Vi\w
The experiments were aimed at determining whether arginase of the small intestine can catalyze in vivo hydrolysis of exogenous arginine to urea, and to what extent this process could contribute to the extracellular pool of urea in the organism. The results presented in Fig. 4 show that after intraduodenal administration ofarginine, ureaconcentration was increased in the blood, both from the portal and femoral vein.
TABLE 3 THE DEPENDENCE OF PRODUCTION OF UREA FROM THE ADDED ARGININE IN EXPERIMENTS in Virro ACCORDING TO CRANE AND WILSON” fimoles
Arginine @moles
External fluidmucosal side
of urea
Internal kidserosal side
Total
0.4 1.1 1.2 1.5 1.6
3.0 6.‘: I0.i IX.7 22.6
-.---__ 0 10 loo 500 1000 ” The
means
of three
2.6 5.6 9.5 17.2 21.0 to five experiments
are given.
--~
L-ARGINASE OF THE SMALL INTESTINE
269
FIG. 3. Effectof the amount of added arginineon formation of urea by isolated rat intestine. The experiments were performed according to Crane and Wilson (IO). Mean values from five determinations are given.
DISCUSSION
Arginase Activity in Mucosa of the Small Zntestine in Man, Dog, and Rat Among the mammals studied, the highest arginase activity in intestinal mucosa was found in the rat, and the lowest in man (Table 1). Specific activity of the enzyme from human intestine was about one-twentieth that in the rat, and when calculated per 1 g of mucosa, only about one-fortieth. In man, arginase activity in the mucosa obtained about 10-16 hr after death was, as a rule, somewhat higher than in mucosa obtained by biopsy. A similar difference was observed with arginase from human liver (13). Similarly as in rat (7), the highest activity of arginase in man and dog was found in duodenum, and the lowest in ileum. Activity of Zntestinal Arginase during Ontogenesis Several enzymes occurring in mucosa of rat small intestine, among others amylase, nonspecific esterase, lipase, as well as some proteolytic
270
KONARSKA
AND TOMASZEWSKI
25
I
0
30
60
90
120
,
I
150
time (min) FIG. 4. increase in urea concentration in the blood of portal and femoral veins of the dog after intraduodenal administration of arginine in GVO. Weight of the dog, 10 kg: amount of administered arginine, 420 mg; time of the experiment 2.5 hr. Urea was determined in samples of blood taken from the portal vein (-O-O-) and femoral vein (-O-O-).
enzymes, show much lower activity in suckling rats than in adult ones: the increase in activity occurring usually between the 14th and 21st day of life (14). The activity of intestinal arginase during the postembryonal development of the rat shows a similar course; it is very low throughout the first fortnight, with a distinct increase occurring the 3rd week of life. In the 5th week, the arginase activity in all the studied segments of the intestine raises to the values characteristic for adult animals (Fig. 2). As it appears from the studies OfGreengardetai. (4) and Porembska( IS), the development of arginase activity in liver shows two phases of intense increase: the first just before birth, and the second, which is the main one, during the 3rd week of life. We did not observe the first phase of increased activity of intestinal arginase. The rapid increase in the enzyme activity during the 3rd and 4th week of life appears in the rat at the time the young ones pass from the milk diet to the normal laboratory diet. The interpretation of this phenomenon presents some difficulties, as three factors should be considered: the adaptive character of the enzyme, effect of Mn2+ ion. and hormonal regulation.
L-ARGINASE
OF THE
SMALL
INTESTINE
271
The milk diet is low in Mn2+ and contains about 11.8% of protein, whereas the laboratory diet has 40 mg of M$+/kg and about 1% of protein. It could be supposed that the increase in arginase activity is due to the change in diet, which would point to the adaptive character of the enzyme or its activation by MnZ+ ions. These suppositions seem to be supported by the studies of Boyer et al. (16) who demonstrated that in adult rats kept on an Mn2+-low milk diet, the activity of liver arginase was consistently lower than in animals fed in a normal way. Enrichment of the diet in Mn2+ led to an increase in the enzyme activity in liver (17). High-protein diet is also known to cause a considerable increase in the activity of hepatic arginase (18, 19). One the other hand, the results of other authors point to a considerable role of steroid hormones in the enzyme activity. By administration of corticosteroids (20), the same increase in the activity of hepatic arginase could be obtained as by the high-protein diet. Moreover, in rats the increase in arginase activity in the 3rd week of life is coincident with the period of increased activity of pituitary gland and adrenal cortex (21). Greengard et al. (4) suggested that the activity of hepatic arginase is subject to hormonal regulation: in the first, embryonic phase it increases under the influence of thyroxine, and in the second, of glucocorticoids. At variance with the results obtained with the rat, human intestinal arginase did not show distinct age-dependence. In children aged up to 1 year, the enzyme activity was lower than in adults in all three intestinal segments studied, but the difference was statistically significant only for duodenum. Role of Intestinal Arginase in Maintaining the Extracellular Pool of Urea The results obtained in vitro and in viuo (Figs. 3 and 4) point to possible participation of the intestine in maintaining the extracellular pool of urea in the organism. This may be of importance in cases when the main pathway of urea formation in liver is blocked due to genetic impairment or as a result of liver disease. In all cases of inborn metabolic blocks (which may concern each of the five enzymes of the ornithine cycle), described so far, urea concentration in blood remained normal (22), despite high concomitant hyperammonemia. This leads to the conclusion that urea is formed in the organism even when one of the enzymes involved in its synthesis in liver is inactive. The pool of urea, measured as blood urea level, is decreased in patients kept on lowprotein diet (22). Several hypotheses have been advanced to explain these observations, Possible residual activity of the blocked enzyme (13, 23) has been considered, as well as an alternative pathway of urea synthesis (2426), activity in other tissues of the enzyme that was blocked in liver (23), and the activity
272
KONARSKAAND't'OMAS%EWSKI
of the mutant enzyme (27). However, none of these suggestions provides adequate explanation for the observed phenomena. Colombo (23). taking into account that arginase occurs in several tissues, assumed as a theoretical possibility, intra- or extrahepatic formation of urea through hydrolysis of arginine. The occurrence of urea in blood of patients with a metabolic block in the ornithine cycle is not a proof that urea is formed due to the action of the whole cycle, especially as this block is usually accompanied by hyperammonemia, which obviously points to disturbances in detoxication of ammonia. On this assumption it seems possible to accept that urea is formed during hydrolysis of arginine in every tissue which contains arginase. However, in most of the extrahepatic tissues. the activity of arginase is very low. The comparatively high activity of intestinal arginase permits to suggest that hydrolysis of exogenous arginase occurring in the intestinal wall may participate in maintaining the extracellular pool of urea under conditions when the hepatic pathway of its synthesis is blocked. This supposition is supported by the results obtained on isolated rat intestine: under the applied conditions, the amount of urea formed in vitro during 1 hr from the supplied arginine, when related to the whole length of the small intestine, corresponded to the average level of this compound in rat blood. Our experiments also demonstrated that the arginase of erythrocytes, which shows rather high activity in man, does not participate in supplying urea to the extracellular pool. The presented suggestion seems to be supported also by the clinically observed increase in urea concentration in the blood of patients with high bleeding into the gastrointestinal tract (28). Indirect support comes also from the studies on absorption of amino acids by the intestinal wall it7 vitro: Finch and Hird (29) observed that a decrease in the amount of arginine by about 90% was accompanied by the appearance of ornithine in an amount corresponding to about 56% of the arginine that had disappeared. Although the authors did not discuss this observation, it is also a direct proof for the metabolism of arginine in the intestinal wall. SUMMARY The activity of arginase was demonstrated in mucosa of human small intestine. The enzyme activity was lower in young children (up to 1 year old) than in adults. In newborn and suckling rats, the arginase activity was very low; a rapid increase in the activity occurred within the third week of life. with simultaneous quantitative differentiation of the activity in duodenum, jejunum, and ileum.
L-ARGINASE
OF THE SMALL
INTESTINE
273
Hydrolysis of exogenous arginine occurring in the intestinal wall can supply the extracellular pool of urea. In dogs, after intraduodenal administration of arginine in viva, concentration of urea increased, both in the blood from the portal vein and in peripheral blood. When nonhemolyzed human erythrocytes were incubated in the presence of arginine, no formation of urea was observed. REFERENCES I. 2. 3. 4. 5. 6. 7. 8.
Kossel, A., and Dakin, H. D., Z. Physiot. Chem. 41, 321 (1904). Greenstein, J. P., J. Nur. Cancer Inst. 4, 275 (1943). Kochakian, C. D., J. Bid/. Chem. 161, 115 (1945). Greengard, O., Sahib, M. K., and Knox, W. E.,Arch. Biochem. Bioph,ys. 137,477( 1970). Baret, R., Mourgue, M., and Pellegrin, J., Compt. Rend. Sot. Biol. 160, 1796 (1966). Reddy, S. R. R., and Campbell, J. W., Biochim. Biophys. Acra 159, 557 (1968). Konarska, L., and Tomaszewski, L., Biochem. Med. 14, 250-262 (1975). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.,J. Biol. Chem. 193,265 (1951). 9. Wilson. T. H., and Wiseman, G.. J. Physiol. 126, 116 (1954). 10. Crane, R. K., and Wilson, T. H., J. Appl. Physiol. 12, 145 (1958). II. Richterich, R., “Klinische Chemie.” Karger, Basel-New York, 1965. 12. Caraway, W. T., and Fanger, H., Amer. J. Clin. Pathol. 25, 317 (1!)55). 13. Levin, B., in “Advances in Clinical Chemistry” (0. Bodansky and A. L. Latner, Eds.), D. 66. Vol. 14. Academic Press. New York. London. 1971. 14. Koldovsky, O., “Development ofthe Functions of the Small Intestine in Mammals and Man,” p. 215. Karger, Basel-New York, 1969. 15. Porembska, Z., in “Enzyme,” (Y. Frei, W. E. Knox, 0. Greengard, Eds.), Vol. 15, p. 198. Karger, Basel, 1973. 16. Boyer, P. D., Shaw, J. H., and Phillips, P. H.. J. Biol. Chem. 143, 417 (1942). 17. Shils, M. E., and McCollum, E. V., J. Nlrtr. 26, 1 (1943). 18. Ashida, K., and Harper, A. E., Proc. Sot. Exp. Bio/. Med. 107, 151 (1961). 19. Schimke, R. T., J. Biol. Chem. 238, 1012 (1963). 20. McLean, P., and Gurney, M. W.. Biochem. J. 87, 96 (1963). 21. Levine, S., and Mullins, R. F.. Science 152, 1585 (1966). 22. McMurray, W. C., Rathbun, J. C., Mohyuddin, F., and Koegler, S. J , Pediatrics 32,347 (1963). 23. Colombo, J. P., in “Congenital Disorders of the Urea Cycle and Ammonia Detoxication.” (F. Falkner, N. Kretchmer, E. Rossi, Eds.), Monographs in Paediatrics, Vol. 1, p. 93. Karger, Basel, 1971. 24. Bach. S. J., and Smith, M., Biochem. J. 64, 417 (1956). 25. Cohen, B. D., and Stein, I. M., Amer. J. Med. 45, 63 (1968). 26. Stein, J. M., and Cohen, B. D., New Engl. J. Med. 280, 926 (1969). 27. Striver, C. R., &it. Med. Bull. 25, 35 (1969). 28. Cantarow, A., and Trumper, M., in “Clinical Biochemistry,” p. 167. W. B. Saunders Company, Philadelphia-London, 1962. 29. Finch, L. R., and Hird, F. J. R., Biochim. Biophys. Acta 43, 278 (1960).