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Arginase and ornithine carbamoyltransferase activity in serum and liver of mice infected with Schistosoma mansoni
Comp. Biochem. Physiol., 1977, Vol. 57B, pp. 243 to 247. Per qamon Press. Printed in Great Britain
ARGINASE A N D O R N I T H I N E C A R B A M O Y L T R A N S F E R A S E ACTIVITY IN S E R U M A N D LIVER OF MICE I N F E C T E D WITH SCHISTOSOMA MANSONI C. R. PAGE III AND G. R. NEWPORT Laboratory of Parasitology, Department of Biology, Tulane University, New Orleans, Louisiana 70118, U.S.A. (Received 22 October 1976)
Abstract--1. In vitro activity of arginase (EC 3.5.3.1) and ornithine carbamoyltransferase (EC 2.1.3.3) was assayed in liver and serum of mice infected with Schistosoma mansoni for up to 19 weeks. 2. Prior to parasite egg deposition, the activity of both enzymes was comparable in the liver of infected and uninfected mice. 3. Nineteen weeks postinfection, total liver arginase increased by 61~o, while hepatic ornithine carbamoyltransferase activity decreased by 31~o. 4. Serum arginase was elevated in prepatent mice, and increased progressively into the patent stage of infection. 5. Serum ornithine carbamoyltransferase remained unaltered in infected mice. /
INTRODUCTION In vivo regulation of mammalian amino acid metabo-
lism through adaptive changes in enzyme levels is not completely understood. The high degree of complexity in this area of metabolism, as well as a lack of model systems amendable to genetic manipulation, has restricted investigation (Ratner, 1973). Considerable progress has nevertheless been realized during recent years, due in large measure to studies on the adaptive characteristics of the urea biosynthetic enzymes in mammalian liver. Activities of hepatic urea cycle enzymes in mammals appear to be positively correlated to levels of urea excretion (Schimke, 1962a). Changes in rates of urea excretion during starvation, corticosteroid treatment, or regimentation to a high protein diet are accompanied by a coordinated alteration in the activity of all five urea cycle enzymes (Schimke, 1962a, 1963; Nuzum & Snodgrass, 1971). Such data suggests integrated regulation of the entire pathway. The changes in arginase (E.C. 3.5.3.1) and ornithine carbamoyltransferase (E.C. 2.1.3.3) activity are due to changes in absolute enzyme levels, and not to a modification of their kinetic properties (Schimke, 1962a). Furthermore, adaptive changes in arginase levels appear to be attributable to shifts in the balance of synthetic versus degradative rates (Schimke, 1962a, 1964). There exist reports in the literature which indicate that under certain induced pathological conditions, hepatic arginase levels can be regulated independently from other urea cycle enzymes (Schimke, 1962b, 1963; Grillo, 1964). However, the dynamics of urea cycle enzymes in relation to diseases of the liver have seldom been investigated. In one such disease, experimental murine schistosomiasis mansoni, where increased activity of several hepatic amino acid catabolic enzymes has been detected (Daugherty et al., 1954; Brown & Smith, 1973), serum concentrations of certain urea cycle intermediates have been reported
to be present at abnormal levels (Senft, 1967). The present study was undertaken in order to characterize the hepatic and serum patterns of murine arginase and ornithine carbamoyltransferase to a Schistosoma mansoni infection. MATERIALS AND METHODS Animals
Female albino mice (Charles River CD-1), 2 months old at the beginning of the experiment, were housed 5 per cage in a photoperiod room with artificial illumination from 0600 to 1800 hr. The temperature was maintained at approximately 24°C, and the animals were fed on a diet of Purina Lab Chow and tap water ad libitum. Experimental mice received a single intraperitoneal injection of 100 cercariae of a Puerto Rican strain of Schistosoma
mansoni.
Hepatic enzyme assays
Mice were sacrificed by cervical dislocation between 1000 and 1400hr in order to eliminate possible diurnal variations. Livers were removed and homogenized in 0.l% cetyltrimethylammonium bromide according to method of Brown & Cohen (1959). Homogenates were centrifuged at 5000 0 for 15min at 4°C, after which a 1.0ml aliquot of the supernatant was combined with 9 vol of a saturated ammonium sulfate solution and centrifuged at 15,000 g for 30 min. The supernatant was discarded, the precipitate was resuspended to a volume of 10ml and the latter step repeated. This was found to considerably lower blank values during subsequent analysis of enzymatic activity. Ammonium sulfate precipitates were resuspended in 5.0 ml of cold distilled water, and arginase or ornithine carbamoyltransferase was assayed over a 10 rain incubation in the appropriate media described by Brown & Cohen (1959). Products of enzymatic catalysis were measured colorimetrically according to the method of Archibald (1944, 1945) as modified by Ratner (1955) using 1-propanedione-2-oxime for urea, and 2,3-butanedione-2oxime for citrulline. Serum enzyme assays
Blood collected from mice via cardiac puncture was allowed to clot for 15 min at room temperature in glass 243
244
C R. PAGk Ill ANt) G. R. NIiWPORT Table 1. Liver ornithine carbamoyltransferase and arginase in uninfected mice and mice infected with Schistosoma mansoni for 4 weeks
Experimental group
Liver weight (g) ± S.E.
Uninfected (8 mice) Infected (8 mice) P values
1.61 + 0.08 2.01 + 0.08 0.05
Ornithine carbamoyltransferase I~M citrultine/hr + S.E. Per g Total liver 16,780 + 1343 18,753 + 1422 0.40
centrifuge tubes and was then centrifuged at 60000 for 10 rain. Serum samples showing visible signs of hemolysis were discarded. Serum samples of 3001tl pooled from 2 animals (150/d/animal) were added to 2.7 ml of saturated ammonium sulfate solution, and centrifuged at 15,000 O for 15 rain at 4 C . The supernatants were discarded, and the precipitates were resuspended in 3.0ml of a saturated ammonium sulfate solution and recentrifuged. For ornithine carbamoyltransferase assays, the ammonium sulfate precipitates were suspended to a final volume of 2.0 ml in 0.1 M phosphate buffer (pH 7.0) containing 30 mg urease, and preincubated for 30 min at 37 C. Subsequently, 0.8 ml of the enzyme solution was added to 1.2 ml of freshly prepared phosphate buffer (pH 7.0) containing 0.1 M ornithine and 200/~moles of dilithium carbamyl phosphate and incubated for 1 hr at 37C. Reactions were terminated by the addition of 3.0 ml of 68!~; HCIO4. Blanks received HCIO~ prior to the addition of the enzyme. Citrulline was assayed as previously described. For arginase assays, the ammonium sulfate precipitates were resuspended to a final vol of 2.0ml with 0.05 M MnC12 (pH 9.4) and heated in a water bath set at 50 C for 15 rain. Denatured protein was removed by centrifugation. Aliquots of 0.5 ml were added to 1.5 ml of 0.2 M arginine (pH 9.4) and incubated for 30 min at 37C. Reactions were stopped with 3.0 ml of 68°,, HCIO4, and the precipitated protein was removed by centrifugation. Blanks received HC10,~ prior to enzyme addition and urea was measured as previously described. Statistical analysis Mean activity values of enzymes in infected and uninfected animals were compared by use of the Student's t test with P < 0.05 considered significant. RESU LTS Liver enzyme activity is expressed as the a m o u n t of product yielded per hour by an entire liver under
I(/,606 + 835 9538 + 1022 0.20
Arginase pM urea/hr + S.E. Total
g liver
56.798 + 6797 76,121 4-_ 6842 0.40
35,546 + 3261 38,233 + 3903 0.50
Per
the specified assay conditions. Since chronic murine schistosomiasis m a n s o n i is characterized by an intense hepatic inflammatory response, expression of enzyme activity as a function of tissue weight, or tissue protein content, seems inappropriate, owing to the presence of a heterogeneity of cellular and extracellular components during this stage of the infection. However, data on enzyme activity relative to liver wet weight is included as a source of reference in Tables 1 3. Four weeks postinfection, hepatic ornithine carbamoyltransferase and arginase activities in infected animals were comparable to those in uninfected animals (Table 1). It should be noted that this particular stage of the infection corresponds to a period .just prior to parasite egg deposition. Twelve weeks postinfection, approx 5-6 weeks after initial parasite egg deposition, total liver ornithine carbamoyltransferase was reduced by 27",~, in infected animals (Table 2). In contrast, the total hepatic arginase activity was elevated in these animals by approx 5300 (Table 2). The hepatic enzyme patterns in mice infected with Schistosoma mansoni for 19 weeks were more pronounced than those noted in animals infected for 12 weeks. Total liver ornithine carbamoyltransferase declined to 320;; below control levels (Table 3). A significant elevation in total liver arginase, represented by a 617,, increase, was also observed in these animals (Table 3). Serum enzyme activities are expressed in terms of product yield/ml serum/hr under the described assay conditions (Figs. 1-2). Each point represents a mean obtained from 5 separate determinations; each determ i n a t i o n was performed on serum pooled from 2 animals (i.e. total of 10 animals assayed per point/.
Table 2. Liver ornithine carbamoyltransferase and arginase in uninfected mice and mice infected with Schistosoma mansoni for 12 weeks
Experimental group Uninfected (6 mice) Infected (5 mice) P values
Liver weight (g) _+ S.E. 1.96 _+ 0.09 4.87 _+ 0.45 0.001
Ornithine carbamoyltransferase gM citrulline/hr _+ S.E. Per g Total liver 21,294 4-_ 1424 15.546 + 1801 0.025
10,916 + 1187 3281 + 40l 0.001
Arginase tiM urea/hr + S.E. Per g Total liver 77.961 _+ 4806 119,96l _+ 4386 0.025
39,894 + 2443 25.531 ± 2707 (I.005
Activity in serum and liver of mice
245
Table 3. Liver ornithine carbamoyltransferase and arginase in uninfected mice and mice infected with Schistosoma mansoni for 19 weeks
Experimental group
Liver weight (g) + S.E.
Uninfected (8 mice) Infected (8 mice) P values
2.01 ___ 0.13 5.17 + 0.37 0.001
Ornithine carbamoyltransferase #M citrulline/hr + S.E. Per g Total liver 21,518 + 1214 14,862 + 1122 0.005
Serum arginase activity was higher in infected mice than in uninfected mice (Fig. 1), except at 2 weeks where the enzyme activity of the two groups of animals were similar. Unlike the two liver enzymes examined in this study, serum arginase was elevated in infected mice at 4 weeks postinfection. The serum enzyme activity increased progressively after 4 weeks, and levels in mice infected for 12 weeks were approximately six-times higher than those in uninfected mice. Serum arginase increases in mice infected with Schistosoma mansoni for 4-12 weeks were all significant (P < 0.01). Unlike serum arginase, serum ornithine carbamoyltransferase showed none of the striking differences between infected and uninfected mice. Serum levels of this enzyme were unaltered in infected mice at all experimental time periods.
DISCUSSION Hepatic levels of arginase and ornithine carbamoyltransferase in mice are differentially affected by chronic Schistosoma mansoni infections. Specifically, whereas total arginase activity is increased in the liver of these animals, ornithine carbamoyltransferase is significantly reduced. As such, these data appear to extend the adrenalectomy studies of Schimke (1963) and hyperthyroidism studies of Grillo (1964) which suggest that, under certain conditions, hepatic arginase levels can be regulated separately from ornithine carbamoyltransferase. Ratner (1973) has proposed that such a distinction may allow for inde-
Arginase #M urea/hr _+ S.E. Per g Total liver
10,802 + 312 3002 + 232 0.001
78,128 + 7458 133,777 + 7418 0.001
38,989 ___ 3004 26,235 + 1785 0,005
pendent regulation of free arginine pools in mammals. However, the observation by Senft (1967) that free serum arginine is reduced to trace levels in mice during advanced schistosome infections obscures any adaptive significance for the increased arginase activity in these animals. Ultrastructural observations by Page & Knowles (1973) indicate that hepatocyte mitochondria may become swollen and disrupted during murine schistosome infections. Since ornithine carbamoyltransferase is compartamentalized within the mitochondria of mammalian liver cells, it is conceivable that the lack of coordination between this enzyme and cytoplasmic arginase represents a pathological condition rather than an adaptive response to schistosome infection. Arginase activity in eggs of Schistosoma mansoni has never been examined, hence the noted increase in level of this enzyme in liver of chronically infected mice is subject to qualification. Technical difficulties involved with quantitative recovery of viable schistosome eggs (i.e. free of host contaminants) from infected mouse liver restricts evaluation of this parameter. Arginase activity in parasitic helminths studied to date is many orders of magnitude lower than that found in mammalian liver (cf. Simmons, 1970), and would not be expected to contribute significantly to the assay system used in this study. Attempts to demonstrate arginase activity in eggs of a related trematode, Fasciola hepatica, have proven unsuccessful (Rijavec & Kurelec, 1966). Also, schistosome eggs in infected mouse liver exist in various stages of decomposition, with the increase in liver weight being attributable to an increased content of water, epithelioid
o Uninfected
o Uninfected
o. Infected
~
1.5
•
Infected
T
10
6 ~ ='-
2
o5
::k t
i
'
~
'
~
'
8
'
1'0
'
17
I
I
4
Weeks infected
Fig. 1. Serum arginase in uninfected mice and mice infected with Schistosoma mansoni. See text for details.
I
J
8
Weeks
I 12
I
I 16
infected
Fig. 2. Serum ornithine carbamoyltransferase in uninfected mice and mice infected with Schistosoma mansoni. See text for details.
246
C.R. PAGE Ill ANt) G. R. NEWPORT
cells, leukocytes, fibroblasts, and connective tissue (Meleney et al., 1953). Systemic h y p e r a m m o n e m i a is a well documented host response to schistosome infection. Daugherty et al. (1954) have noted that liver minces from chronically infected mice accumulate a m m o n i a in a buffered medium. Since certain a m m o n i a yielding processes are impaired in these minces, these authors hypothesized that the increase in a m m o n i a is not due to increased production of the compound, but rather to a decreased capacity for enzymatic detoxification. Similarly, Senti (1967) has noted that while argininc pools are depleted in serum of schistosome infected mice, ornithine and a m m o n i a levels are markedly increased, suggesting a defect in urea cycle activity. O t h e r investigators have proposed that the hyperammonemic condition in infected mice is due to a diversion of portal blood flow away from the liver (DeWitt & Warren, 1959). However, Warren & Reboucas (1966) have pointed out that portal-systemic collateral circulation, by itself, cannot account for increased blood a m m o n i a levels, since the presence of some form of hepatocellular damage is a necessary prerequisite. Furthermore. Page et al. (19721 have reported that increased serum a m m o n i a levels can exist in schistosome infected mice even in the absence of portal hypertension. While the present data are insufficient to account for elevated a m m o n i a concentrations in serum of infected mice, they do suggest that an alteration of host urea cycle activity may play a significant role in this phenomenon. The lack of coordination between arginase and ornithine carbamoyltransferase patterns in serum of schistosome infected mice is somewhat puzzling. Several investigators have proposed that elevated serum arginase values may be diagnostic for various forms of hepatocellular dysfunction, since this enzyme is found in significant a m o u n t s only in the liver (Manning & Grisolia, 1957: Pelikan eta/.. 1964). However. arginase is not present in the serum of certain mammals, and its physiological function is unknown, although it probably does not constitute a c o m p o n e n t of the n o r m a l route of degradation of the hepatic cnzyme (Schimke, 1964). A similar diagnostic use has been proposed for serum ornithinc carbamoyltransfcrasc (Brown & Grisolia, 1959). In the present investigation, serum arginase levels were generally elevated in schistosome infected mice, while ornithinc carbamoyltransferase was unaltered in activity, introducing a certain a m o u n t of ambiguity in interpretation of liver function. Indeed the metabolic and physiological status of the liver in schistosome infected mice is at present unclear, although various workers have indicated that no serious hepatic dysfunction is normally associated with the disease (Sadun & Williams, 1966: Stenger et al., 1967). It is thus evident that the rise of serum arginase and serum ornithine carbamoyltransferase for diagnostic purposes is subject to variability, and that a clearer understanding of their value awaits definitive ehlcidation of their relationship to the dynamics of urea cvcle activit\, in the liver.
Acknowled#ements This study was supported in part by a Biomedical Sciences Support Grant from Tulane University.
RI(FERENCES
ARCtHBALI) R. M. (1944) Demonstration of citrulline and allanloin and demonstration ,~f citrullinc m blood plasma. ,I. biol. Chem. 156, 121 142. ARCHmALD R. M. (1945) Colorimetric determination of urea. J. biol. Chem. 157, 507 518, BROWN J. N. & SMrIH T. M. (1973) Effects of Schi~tosonut mansotli infection on mductkm of tryptophan oxygenase in mouse livers. Comp. Biochem. Physiol. 45B, 487 489. Brown R. W, & GRlSOIbX S. 11959t Ornithine transcarbamylasc activity in scrunl. .I. Lah. C/in. Ah'd. 54. 617 620. BROWN W. (J. & COHIN P. P. (19591 Comparative biochemistry of urea synthesis I. Methods for the quantitative assay' of urea cycle enzymes in liver..I, biol. ('hem. 234, 1768 1774. DAt GHERIY J., GARSON S. & HIiSNEMAN D. (1954} The effect of Schistosoma t'aaIqsotli infections on liver function in mice I. Amino acid oxidase and ammonia. Am. d. Trop. Med. Hy.q. 3. 511 517. DFWH"I W. B. & WARRFN K. S. (1959) Hepaiosplenic schistosomiasis in mice. ,Ira..I. Trop. Med. H)'~t. 8, 440 446. GRiLLe M. A. [1964} l_flrea cycle in the liver of the hypcrthyroid rat. ('lit:. Chim. Acta 10, 259 261. MANNIN(; R. R. & GRISOt,I:, S. 11957) Serum arginase activity. Proc. Soc. exp. Biol. Med. 95. 225 226. M[-I,t';NIY H. E., SANI)GROUN1)J, H, MOOR! D. V. Mosl H. & ('ARN~?~"B. H. 119531 The histopathology of experimental schistosomiasis I1. Bisexual infections with S. manmmi, S. japonicttm, and S. Jtaematobizlm..tm, J. Trop. Med. tty,q. 2, 883 914. N / z t M 7". C. & SNOI)GRASS P..I. 119711 Urea cycle enzyme adaptation to dietary protcin in primates. Science. N.Y. 172, 1042 1043. P,',(~I ('. R. Ill. I=l(as E. J. & O~ai J. D. (1972! Experimental schistosomiasis mansoni: quantitali,,e analyses of proteins, enzyme activity, and free amino acids in mouse serum. E\p. Parasitol. 31, 341 349. P;,cn ('. R. I11 & KNowIl!S W. J. (19731 Experimental prepatcnt schistosomiasis mansoni: hepalic cylopathoIogy. Program and Abstratcs 48th Annual Meeting. Am. Soc. Parasitol.. p. 41. Pf!I.IKAN V., KALAB M. & Tn'llY J. 11964) Determination of arginasc activity in blood in epidemic hepatitis, ('Ira. Chim. 4eta 9, 259 261. RAINt!R S. (1955) Enzymatic synthesis of arginine (condensing and splitting enzymes). In Methods in Enzymolo,qy. (Edited by COLOWI('K S. P. & K.~,pl ,\Y N. O.). Vol. 2. pp. 2093 2096. R,'VINI!R S. 11973) Enzylne~, of argmme and nrea synthesis. 4dl, Enz.vmol. 39. 1 90. RIJ..\Vlt' K. & Kt:RILI(' B. (1966i Die actixitat der ornithinischen Transcarbamyhlsc und Arginasc im Gewebc verschiedener Entwicklungsstadien des grosscn l_eberagels (Fasciohl hepaticoi. Z. Parasit. Kde. 27, 99 105. SAI)I'N E. H. & WIllIAMS J. S. (1966) Biochemical aspects of schistosomiasis nlansoni ill mice m relation to worrn burdens and duration of infection. Evp. Parasitol. 18, 266 273. SCHIMKt R. T. (1962a1 Differential effects of fasting and protein-free diets on levels of urea cycle enzymes m rat liver..1, biol. Chem. 237. 1921 1924. SCHIMKt R T. (1962b) Adaptive characteristics of urea cycle cnzymes m lhc rat. J. hiol. Chem. 237, 459 46S. S('itlMKt R. T. 119631 Studies on factors affecting the levels of urea cycle enzymes in rat liver. ,l. hiol. ('hem. 238. 1012 1028. S('HIMKt R. T. (1964) The importance of both synthesis and degradation in the control of arginase levels in rat liver. J. biol. Chem. 239, 3808 3817.
Activity in serum and liver of mice SENrT A. W. (1967) Studies in arginine metabohsm by schistosomes--II. Arginine depletion in mammals and snails infected with S. mansoni or S. haematobium. Comp. Biochem. Physiol. 21, 299-306. STENGER R. J., WARREN K. S. & JOHNSON E. A. (1967) An electron microscopic study of the liver parenchyma and schistosome pigment in murine hepatosplenic schistosomiasis mansoni. Am. J. Trop. Med. Hyg. 16, 473~482.
247
SIMMONS J. E. (1970) Nitrogen metabolism in playthelminths In Comparative Biochemistry of Nitrogen Metabolism. (Edited by CAMPBELLJ. W.), pp. 91-102. Academic Press, New York. WARREN K. S. 8¢ REBOUCASG. (1966) Ammonia tolerance in compensated and decompensated schistosomiasis mansoni. Am. J. Trop. Med. Hyg. 15, 32-36.
ARGINASE A N D O R N I T H I N E C A R B A M O Y L T R A N S F E R A S E ACTIVITY IN S E R U M A N D LIVER OF MICE I N F E C T E D WITH SCHISTOSOMA MANSONI C. R. PAGE III AND G. R. NEWPORT Laboratory of Parasitology, Department of Biology, Tulane University, New Orleans, Louisiana 70118, U.S.A. (Received 22 October 1976)
Abstract--1. In vitro activity of arginase (EC 3.5.3.1) and ornithine carbamoyltransferase (EC 2.1.3.3) was assayed in liver and serum of mice infected with Schistosoma mansoni for up to 19 weeks. 2. Prior to parasite egg deposition, the activity of both enzymes was comparable in the liver of infected and uninfected mice. 3. Nineteen weeks postinfection, total liver arginase increased by 61~o, while hepatic ornithine carbamoyltransferase activity decreased by 31~o. 4. Serum arginase was elevated in prepatent mice, and increased progressively into the patent stage of infection. 5. Serum ornithine carbamoyltransferase remained unaltered in infected mice. /
INTRODUCTION In vivo regulation of mammalian amino acid metabo-
lism through adaptive changes in enzyme levels is not completely understood. The high degree of complexity in this area of metabolism, as well as a lack of model systems amendable to genetic manipulation, has restricted investigation (Ratner, 1973). Considerable progress has nevertheless been realized during recent years, due in large measure to studies on the adaptive characteristics of the urea biosynthetic enzymes in mammalian liver. Activities of hepatic urea cycle enzymes in mammals appear to be positively correlated to levels of urea excretion (Schimke, 1962a). Changes in rates of urea excretion during starvation, corticosteroid treatment, or regimentation to a high protein diet are accompanied by a coordinated alteration in the activity of all five urea cycle enzymes (Schimke, 1962a, 1963; Nuzum & Snodgrass, 1971). Such data suggests integrated regulation of the entire pathway. The changes in arginase (E.C. 3.5.3.1) and ornithine carbamoyltransferase (E.C. 2.1.3.3) activity are due to changes in absolute enzyme levels, and not to a modification of their kinetic properties (Schimke, 1962a). Furthermore, adaptive changes in arginase levels appear to be attributable to shifts in the balance of synthetic versus degradative rates (Schimke, 1962a, 1964). There exist reports in the literature which indicate that under certain induced pathological conditions, hepatic arginase levels can be regulated independently from other urea cycle enzymes (Schimke, 1962b, 1963; Grillo, 1964). However, the dynamics of urea cycle enzymes in relation to diseases of the liver have seldom been investigated. In one such disease, experimental murine schistosomiasis mansoni, where increased activity of several hepatic amino acid catabolic enzymes has been detected (Daugherty et al., 1954; Brown & Smith, 1973), serum concentrations of certain urea cycle intermediates have been reported
to be present at abnormal levels (Senft, 1967). The present study was undertaken in order to characterize the hepatic and serum patterns of murine arginase and ornithine carbamoyltransferase to a Schistosoma mansoni infection. MATERIALS AND METHODS Animals
Female albino mice (Charles River CD-1), 2 months old at the beginning of the experiment, were housed 5 per cage in a photoperiod room with artificial illumination from 0600 to 1800 hr. The temperature was maintained at approximately 24°C, and the animals were fed on a diet of Purina Lab Chow and tap water ad libitum. Experimental mice received a single intraperitoneal injection of 100 cercariae of a Puerto Rican strain of Schistosoma
mansoni.
Hepatic enzyme assays
Mice were sacrificed by cervical dislocation between 1000 and 1400hr in order to eliminate possible diurnal variations. Livers were removed and homogenized in 0.l% cetyltrimethylammonium bromide according to method of Brown & Cohen (1959). Homogenates were centrifuged at 5000 0 for 15min at 4°C, after which a 1.0ml aliquot of the supernatant was combined with 9 vol of a saturated ammonium sulfate solution and centrifuged at 15,000 g for 30 min. The supernatant was discarded, the precipitate was resuspended to a volume of 10ml and the latter step repeated. This was found to considerably lower blank values during subsequent analysis of enzymatic activity. Ammonium sulfate precipitates were resuspended in 5.0 ml of cold distilled water, and arginase or ornithine carbamoyltransferase was assayed over a 10 rain incubation in the appropriate media described by Brown & Cohen (1959). Products of enzymatic catalysis were measured colorimetrically according to the method of Archibald (1944, 1945) as modified by Ratner (1955) using 1-propanedione-2-oxime for urea, and 2,3-butanedione-2oxime for citrulline. Serum enzyme assays
Blood collected from mice via cardiac puncture was allowed to clot for 15 min at room temperature in glass 243
244
C R. PAGk Ill ANt) G. R. NIiWPORT Table 1. Liver ornithine carbamoyltransferase and arginase in uninfected mice and mice infected with Schistosoma mansoni for 4 weeks
Experimental group
Liver weight (g) ± S.E.
Uninfected (8 mice) Infected (8 mice) P values
1.61 + 0.08 2.01 + 0.08 0.05
Ornithine carbamoyltransferase I~M citrultine/hr + S.E. Per g Total liver 16,780 + 1343 18,753 + 1422 0.40
centrifuge tubes and was then centrifuged at 60000 for 10 rain. Serum samples showing visible signs of hemolysis were discarded. Serum samples of 3001tl pooled from 2 animals (150/d/animal) were added to 2.7 ml of saturated ammonium sulfate solution, and centrifuged at 15,000 O for 15 rain at 4 C . The supernatants were discarded, and the precipitates were resuspended in 3.0ml of a saturated ammonium sulfate solution and recentrifuged. For ornithine carbamoyltransferase assays, the ammonium sulfate precipitates were suspended to a final volume of 2.0 ml in 0.1 M phosphate buffer (pH 7.0) containing 30 mg urease, and preincubated for 30 min at 37 C. Subsequently, 0.8 ml of the enzyme solution was added to 1.2 ml of freshly prepared phosphate buffer (pH 7.0) containing 0.1 M ornithine and 200/~moles of dilithium carbamyl phosphate and incubated for 1 hr at 37C. Reactions were terminated by the addition of 3.0 ml of 68!~; HCIO4. Blanks received HCIO~ prior to the addition of the enzyme. Citrulline was assayed as previously described. For arginase assays, the ammonium sulfate precipitates were resuspended to a final vol of 2.0ml with 0.05 M MnC12 (pH 9.4) and heated in a water bath set at 50 C for 15 rain. Denatured protein was removed by centrifugation. Aliquots of 0.5 ml were added to 1.5 ml of 0.2 M arginine (pH 9.4) and incubated for 30 min at 37C. Reactions were stopped with 3.0 ml of 68°,, HCIO4, and the precipitated protein was removed by centrifugation. Blanks received HC10,~ prior to enzyme addition and urea was measured as previously described. Statistical analysis Mean activity values of enzymes in infected and uninfected animals were compared by use of the Student's t test with P < 0.05 considered significant. RESU LTS Liver enzyme activity is expressed as the a m o u n t of product yielded per hour by an entire liver under
I(/,606 + 835 9538 + 1022 0.20
Arginase pM urea/hr + S.E. Total
g liver
56.798 + 6797 76,121 4-_ 6842 0.40
35,546 + 3261 38,233 + 3903 0.50
Per
the specified assay conditions. Since chronic murine schistosomiasis m a n s o n i is characterized by an intense hepatic inflammatory response, expression of enzyme activity as a function of tissue weight, or tissue protein content, seems inappropriate, owing to the presence of a heterogeneity of cellular and extracellular components during this stage of the infection. However, data on enzyme activity relative to liver wet weight is included as a source of reference in Tables 1 3. Four weeks postinfection, hepatic ornithine carbamoyltransferase and arginase activities in infected animals were comparable to those in uninfected animals (Table 1). It should be noted that this particular stage of the infection corresponds to a period .just prior to parasite egg deposition. Twelve weeks postinfection, approx 5-6 weeks after initial parasite egg deposition, total liver ornithine carbamoyltransferase was reduced by 27",~, in infected animals (Table 2). In contrast, the total hepatic arginase activity was elevated in these animals by approx 5300 (Table 2). The hepatic enzyme patterns in mice infected with Schistosoma mansoni for 19 weeks were more pronounced than those noted in animals infected for 12 weeks. Total liver ornithine carbamoyltransferase declined to 320;; below control levels (Table 3). A significant elevation in total liver arginase, represented by a 617,, increase, was also observed in these animals (Table 3). Serum enzyme activities are expressed in terms of product yield/ml serum/hr under the described assay conditions (Figs. 1-2). Each point represents a mean obtained from 5 separate determinations; each determ i n a t i o n was performed on serum pooled from 2 animals (i.e. total of 10 animals assayed per point/.
Table 2. Liver ornithine carbamoyltransferase and arginase in uninfected mice and mice infected with Schistosoma mansoni for 12 weeks
Experimental group Uninfected (6 mice) Infected (5 mice) P values
Liver weight (g) _+ S.E. 1.96 _+ 0.09 4.87 _+ 0.45 0.001
Ornithine carbamoyltransferase gM citrulline/hr _+ S.E. Per g Total liver 21,294 4-_ 1424 15.546 + 1801 0.025
10,916 + 1187 3281 + 40l 0.001
Arginase tiM urea/hr + S.E. Per g Total liver 77.961 _+ 4806 119,96l _+ 4386 0.025
39,894 + 2443 25.531 ± 2707 (I.005
Activity in serum and liver of mice
245
Table 3. Liver ornithine carbamoyltransferase and arginase in uninfected mice and mice infected with Schistosoma mansoni for 19 weeks
Experimental group
Liver weight (g) + S.E.
Uninfected (8 mice) Infected (8 mice) P values
2.01 ___ 0.13 5.17 + 0.37 0.001
Ornithine carbamoyltransferase #M citrulline/hr + S.E. Per g Total liver 21,518 + 1214 14,862 + 1122 0.005
Serum arginase activity was higher in infected mice than in uninfected mice (Fig. 1), except at 2 weeks where the enzyme activity of the two groups of animals were similar. Unlike the two liver enzymes examined in this study, serum arginase was elevated in infected mice at 4 weeks postinfection. The serum enzyme activity increased progressively after 4 weeks, and levels in mice infected for 12 weeks were approximately six-times higher than those in uninfected mice. Serum arginase increases in mice infected with Schistosoma mansoni for 4-12 weeks were all significant (P < 0.01). Unlike serum arginase, serum ornithine carbamoyltransferase showed none of the striking differences between infected and uninfected mice. Serum levels of this enzyme were unaltered in infected mice at all experimental time periods.
DISCUSSION Hepatic levels of arginase and ornithine carbamoyltransferase in mice are differentially affected by chronic Schistosoma mansoni infections. Specifically, whereas total arginase activity is increased in the liver of these animals, ornithine carbamoyltransferase is significantly reduced. As such, these data appear to extend the adrenalectomy studies of Schimke (1963) and hyperthyroidism studies of Grillo (1964) which suggest that, under certain conditions, hepatic arginase levels can be regulated separately from ornithine carbamoyltransferase. Ratner (1973) has proposed that such a distinction may allow for inde-
Arginase #M urea/hr _+ S.E. Per g Total liver
10,802 + 312 3002 + 232 0.001
78,128 + 7458 133,777 + 7418 0.001
38,989 ___ 3004 26,235 + 1785 0,005
pendent regulation of free arginine pools in mammals. However, the observation by Senft (1967) that free serum arginine is reduced to trace levels in mice during advanced schistosome infections obscures any adaptive significance for the increased arginase activity in these animals. Ultrastructural observations by Page & Knowles (1973) indicate that hepatocyte mitochondria may become swollen and disrupted during murine schistosome infections. Since ornithine carbamoyltransferase is compartamentalized within the mitochondria of mammalian liver cells, it is conceivable that the lack of coordination between this enzyme and cytoplasmic arginase represents a pathological condition rather than an adaptive response to schistosome infection. Arginase activity in eggs of Schistosoma mansoni has never been examined, hence the noted increase in level of this enzyme in liver of chronically infected mice is subject to qualification. Technical difficulties involved with quantitative recovery of viable schistosome eggs (i.e. free of host contaminants) from infected mouse liver restricts evaluation of this parameter. Arginase activity in parasitic helminths studied to date is many orders of magnitude lower than that found in mammalian liver (cf. Simmons, 1970), and would not be expected to contribute significantly to the assay system used in this study. Attempts to demonstrate arginase activity in eggs of a related trematode, Fasciola hepatica, have proven unsuccessful (Rijavec & Kurelec, 1966). Also, schistosome eggs in infected mouse liver exist in various stages of decomposition, with the increase in liver weight being attributable to an increased content of water, epithelioid
o Uninfected
o Uninfected
o. Infected
~
1.5
•
Infected
T
10
6 ~ ='-
2
o5
::k t
i
'
~
'
~
'
8
'
1'0
'
17
I
I
4
Weeks infected
Fig. 1. Serum arginase in uninfected mice and mice infected with Schistosoma mansoni. See text for details.
I
J
8
Weeks
I 12
I
I 16
infected
Fig. 2. Serum ornithine carbamoyltransferase in uninfected mice and mice infected with Schistosoma mansoni. See text for details.
246
C.R. PAGE Ill ANt) G. R. NEWPORT
cells, leukocytes, fibroblasts, and connective tissue (Meleney et al., 1953). Systemic h y p e r a m m o n e m i a is a well documented host response to schistosome infection. Daugherty et al. (1954) have noted that liver minces from chronically infected mice accumulate a m m o n i a in a buffered medium. Since certain a m m o n i a yielding processes are impaired in these minces, these authors hypothesized that the increase in a m m o n i a is not due to increased production of the compound, but rather to a decreased capacity for enzymatic detoxification. Similarly, Senti (1967) has noted that while argininc pools are depleted in serum of schistosome infected mice, ornithine and a m m o n i a levels are markedly increased, suggesting a defect in urea cycle activity. O t h e r investigators have proposed that the hyperammonemic condition in infected mice is due to a diversion of portal blood flow away from the liver (DeWitt & Warren, 1959). However, Warren & Reboucas (1966) have pointed out that portal-systemic collateral circulation, by itself, cannot account for increased blood a m m o n i a levels, since the presence of some form of hepatocellular damage is a necessary prerequisite. Furthermore. Page et al. (19721 have reported that increased serum a m m o n i a levels can exist in schistosome infected mice even in the absence of portal hypertension. While the present data are insufficient to account for elevated a m m o n i a concentrations in serum of infected mice, they do suggest that an alteration of host urea cycle activity may play a significant role in this phenomenon. The lack of coordination between arginase and ornithine carbamoyltransferase patterns in serum of schistosome infected mice is somewhat puzzling. Several investigators have proposed that elevated serum arginase values may be diagnostic for various forms of hepatocellular dysfunction, since this enzyme is found in significant a m o u n t s only in the liver (Manning & Grisolia, 1957: Pelikan eta/.. 1964). However. arginase is not present in the serum of certain mammals, and its physiological function is unknown, although it probably does not constitute a c o m p o n e n t of the n o r m a l route of degradation of the hepatic cnzyme (Schimke, 1964). A similar diagnostic use has been proposed for serum ornithinc carbamoyltransfcrasc (Brown & Grisolia, 1959). In the present investigation, serum arginase levels were generally elevated in schistosome infected mice, while ornithinc carbamoyltransferase was unaltered in activity, introducing a certain a m o u n t of ambiguity in interpretation of liver function. Indeed the metabolic and physiological status of the liver in schistosome infected mice is at present unclear, although various workers have indicated that no serious hepatic dysfunction is normally associated with the disease (Sadun & Williams, 1966: Stenger et al., 1967). It is thus evident that the rise of serum arginase and serum ornithine carbamoyltransferase for diagnostic purposes is subject to variability, and that a clearer understanding of their value awaits definitive ehlcidation of their relationship to the dynamics of urea cvcle activit\, in the liver.
Acknowled#ements This study was supported in part by a Biomedical Sciences Support Grant from Tulane University.
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