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Schistosoma mansoni. I. Chemical composition of eggs
EXPENMENTAL
PARASITOLOGY 27, 265-272
Schistosoma
mansoni. Efrain
Department University
Toro-Goyco”
(1970)
I. Chemical and
Myrtha
Composition Rosas
del
of Eggs’
Valle
of Biochemistry of Puerto Rico
6 Nutrition and the Laboratory of Neurobiology, School of Medicine, and the San Juan Veterans Administration Hospital, San Juan, Puerto Rico ( Submitted
for publication,
5 June 1969)
Tono-GOYCO, EFRA~N AKD ROSAS DEL VALLE, MYRTHA. 1970. Schistosoma mansoni. I. Chemical Composition of Eggs. Experimental Parasitology 27, 265-272. Eggs of Schisto,soma mansoni have been isolated from tissues of infected mice by digestion with the proteolytic enzyme pingninain. The yield of eggs has been high enough to permit chemical characterization. These have been analyzed for carbohydrate, lipid, and protein contents after sonic disintegration. The water-soluble fraction of the sonicate contains a lipid-rich lipoprotein fraction which appears to be the only protein detectable in significant amounts. Glucose seems to be the most abundant hexose, while esterified cholesterol and neutral fats are the most abundant among the lipid entities. INDEX DESCRIPTORS: Schistosoma mansoni; Chemical composition; protein; Carbohydrates; Glucose; Cholesterol; Fats neutral.
Recent studies on Schistosoma mansoni, mostly of the adult form, place marked emphasis on biochemical and immunological aspects. It has been shown that the adult parasites in infected humans elicit the formation of various antibodies (Kagan and Pellegrino 1961). Antigenic preparations have been obtained from crude fractions of homogenized parasite and chemically analyzed in attempts to identify the chemical nature of the antigenic materials (Kagan and Goodchild 1963; Biguet et al. 1962). Although the antigenic nature of eggs has been established (Hunter and Crandall 1962; Toro-Goyco 1964; Cancio et al. 1965), no previous studies have been done to analyze or characterize the antigenic constituents present in eggs. Furthermore, no chemical study on the composition of the eggs of S. mansoni has been reported. This 1 Contribution No. 17, Laboratory of Neurobiology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico. 2 Lederle Medical Faculty Awardee, 1966-69. 265
Lipids; Lipo-
paper is a preliminary report on the chemical composition of eggs of S. munsoni resulting from studies undertaken as a preliminary step toward the identification of the chemical constituents of eggs responsible for their antigenicity. MATERIALS
AND METHODS
Mice were used as the source of eggs. These mice had been previously infected with S. mansoni according to the method developed by Ritchie and Berrios-Duran (1961). The mice were killed, and the intestines and livers removed. Livers were carefullv washed to remove all traces of blood. Intestines were washed to remove all residues of food prior to preparation of the homogenates. Since liver and intestinal tissues differ in their resistance to enzymic hydrolysis, homogenates were prepared from either one of the tissues but never from a mixture of tissues. The homogenates were incubated with the enzyme ping-
266
TORO-GOYCO
AND
uinain at pH 4.6 in 0.1 M acetate buffer. The details of the procedure for the preparation of the homogenates, incubation, and separation of the eggs have been described previously ( Toro-Goyco 1963). Isolated eggs were either kept refrigerated at 4” in 0.15 M sodium chloride to which 0.01% sodium azide had been added or lyophilized after repeated washing with water. The wet weight of eggs was calculated by packing aqueous suspensions of eggs in previously calibrated Wintrobe hematocrit tubes, centrifuging at 3000 rpm for 10 min in an International refrigerated centrifuge, removing the supernatant water, and weighing using as tare the previously weighed dry Wintrobe tube. This procedure yields the total wet weight. Since the tubes had been previously calibrated with a liquid of known density (water at ZOO), it was possible to calculate the weight per unit volume. For succeeding determinations of wet weight of eggs using these same tubes, it was only necessary to measure the packed volume after centrifugation. Dry weights were obtained by suspending the packed eggs in distilled water, placing them in weighing bottles of known weight, freezing the contents, and evaporating in the frozen state in a vacuum oven after which the bottles were dried to constant weight. Preparation of the soluble components of eggs was accomplished by suspending the wet eggs in phosphate saline buffer followed by sonic disintegration using a Raytheon sonic oscillator. The sonicate was centrifuged at 105,OOOgfor 1 hr in a Spinco Model L preparative ultracentrifuge, using a SW 25.1 swinging bucket rotor, to remove any remaining particulate material. The supernatant was removed for carbohydrate, lipid, protein, and amino acid analyses, gel filtration, and preparative ultracentrifugation. Gel filtration was performed using either Sephadex G-100 or G-200 columns with phosphate-buffered saline as the mobile
ROSA.5
DEL
VALLE
phase (0.15 M NaCI, 0.006 M monobasic sodium phosphate, 0.035 M dibasic sodium phosphate, pH 7.3). Filtration was carried out at room temperature. Carbohydrate analyses of the complete eggs and of the soluble and insoluble fractions of the sonicate were performed by the anthronc method using a modified anthrone reagent ( Fales 1951) . For total amino acid analysis, a 2-ml aliquot of the supernatant sonicate was removed, diluted to 4 ml with concentrated HCl, brought to 8 ml with constant boiling HCl, and sealed under reduced pressure. The sealed tubes were placed in the oven at 110” for 18 hr. For protein amino acid analysis, the supernatant was dialyzed for 24 hr against a 500-fold excess volume of water with change of water every 8 hr. The dialyzate was diluted as explained above and hydrolyzed under the same conditions. Amino acid analysis was performed by the method of Spackman et al. ( 1949)) using a Technicon amino acid analyzer. Detailed procedures for the analysis of the amino acids and calculation of the data has been described earlier ( Toro-Goyco et al. 1968). Total nitrogen determinations were performed by the micro-Kjeldahl technique using as blanks the corresponding solvent. Total protein was determined by the method of Lowry et al. (1951). For total lipid determinations, dry eggs were suspended in 2:l chloroform-methanol mixtures and ground by use of a glass, hand model tissue grinder. After centrifugation at 3000 rpm for 10 min to remove the insoluble material, the supernatant extract was removed, placed in a separatory funnel, and water was added (20% of the volume). The mixture was then shaken and separation of the two phases allowed to occur. The less dense water-methanol phase contained all the nonlipid constituents originally soluble in methanol while the chloroform phase contained the lipid material. The chloroform was evaporated to dryness
Schistosoma mansoni. I. under reduced pressure and stored until analysis. Analysis of the nonsoluble fraction of the sonicate was performed by using the same procedure. Thin-layer chromatography of lipid constituents was performed in 20 X 20-cm glass plates using silica as the stationary phase. The solvent used was ethyl ether, acetic acid, hexane ( 15:1:90), and the plates were stained with 2,7-dichlorofluorescein (Ruggieri 1962). Separation of the lipid constituents was also accompanied by silicic acid chromatography as suggested by Lis et al. ( 1961). Qualitative fatty acid analyses were performed using a Barber-Coleman gas chromatograph. Samples for gas chromatography were prepared by saponification of the lipid material followed by esterification with trifluoride in methanol and extraction with ethyl ether (Metcalfe and Schmitz 1961). Cholesterol was determined by the method of Zak ( 1957).
267 RESULTS
A. Yield of Eggs Consistently, the yield of eggs expressed in mg wet weight/g tissue was higher from intestinal tissue (4.2 -+ 0.4) than from liver tissue (2.2 t 0.2). B. Composition
of the Sonicate
The suspension obtained after sonication and centrifugation as explained above was of varying degrees of turbidity. The turbidity was found to be due to a lipid-rich lipoprotein fraction that was eluted with the void volume in Sephadex G-100 and G-200 chromatographic columns ( Fig. 1) . The lipoprotein nature of this component was ascertained by preparative ultracentrifugation as follows: The most concentrated fraction collected from the chromatographic columns (5 ml) was diluted to 12 ml with a solution of CsCl, bringing the density of the resultant solution to 1.21
20
IO FRACTION
30
NO
FIG. 1. Gel filtration pattern of a lipoprotein fraction isolated from soluble fraction of the sonicate (Sephadex G-100; phosphate saline buffer pH 7.3). Dimensions of column, 45 X 2.5 cm. Absorbancy at 280 rnp expressed in optical density units. Fractions 5 ml each. Protein concentration in peak 40 pg protein/ml.
268
TORO-GOYCO
AND
ROSAS
mg/ml (25” ), and centrifuged for 18 hr at 105,OOOgin a Spinco No. 40 rotor. At the end of this period the turbidity was concentrated at the top of the tube. The centrifuge tube was cut and four fractions analyzed for protein contents. No significant levels of protein nitrogen were found in any of the fractions, with the exception of the top one which also contained the turbidity. Thin-layer chromatography of the lipids extracted from the lipoprotein fraction indicated a pattern very similar to that shown by the lipids of the whole eggs (Fig. 2). C. Total Lipids A thin-layer chromatogram of the total lipid extract from whole eggs is shown in Fig. 2. Under the experimental conditions used, significant amounts of four main groups of lipids could easily be identified. The esterified cholesterol is shown as the fastest migrating component, followed by the triglycerides, the fatty acids and their
m
m,(j,,,+
. k
VALLE
esters (as a single component), and the slower migrating cholesterol. Only very slight traces of phospholipids staying at the origin could be detected. The marked contrast between the high levels of esterified cholesterol and other constituents, especially the practically nonexistent phospholipids, can be appreciated. Gravimetric analysis for the total lipids extracted from whole eggs isolated from intestinal tissue show that these components account for about one-fifth of the total dry weight (20.7 * 2.0). In eggs isolated from intestine, cholesterol and its esters account for one-fourth of the total lipids. The lipids present in eggs isolated from liver tissue account for 35 * 5% of the dry weight. Total lipid analysis was also performed in the soluble and nonsoluble portions of the sonicate. It was consistently found that the bulk of the lipid material (8CMO%) of the total lipid appeared in the soluble portion. Gas-chromatographic analyses have reSOLVENT -FRONT
111
Ir
. 8
DEL
.
.
.
.
.
5’
C
D
E
F
G
H
FIG. 2. Thin layer chromatogram of total lipid from whole eggs: A, cholesterol palmitate; B, tripalmitin; C, cholesterol; D, total lipid extract, eggs isolated from inatestine; E, total lipid extract, eggs isolated from liver; F, steak acid; G, methyl palmitate; H, egg lecithin. Solvent: ethyl ether, acetic acid, hexane, 15: 1:90.
Schistosoma mansoni. I. vealed that the most abundant fatty acids are palmitic, stearic, oleic, and linoleic acids. Collectively, these comprised some 90% of the total fatty acids (Fig. 3). The polyunsaturated linolenic and arachidonic acids account for 5% of the fatty acids in eggs isolated from both tissues. Arachidonic acid is more abundant in the triglvceride portion, where it accounts for 4%-of the total fatty acids. D. Carbohydrate
Content
Most of the carbohydrate was found in the insoluble fraction after sonication of the eggs. In eggs isolated from intestinal tissue, 16% of the total dry weight was accounted for as insoluble carbohydrate. The soluble fractions contain 5 to 6% of the total carbohydrate, as shown by the anthrone method. Sugar analysis performed by an enzymic method (Washko and Rice
269
1961) indicates that the hexose sugar present in this fraction is essentially glucose. E. Amino Acid Analyses and Protein Contents Preliminary analysis performed on the supernatant was revealed that there is a considerable amount of free amino acids present in the soluble fraction of the sonicate. The level of aromatic amino acids is significantly low (6.2%) while that of basic amino acids is considerably higher (34.4%).
DISCUSSION The higher unit weight of to liver tissue better source The difference tially explained
yield of eggs obtained per intestinal tissue as compared makes the former tissue a of this material for study. in yield may well be parby an easier digestibility of
cl6
I
TIME
IN MINUTES
FIG. 3. Gas chromatogram of ethyl esters of fatty acids present in triglyceride fraction of S. stand for the methyl esters of stearic, oleic, linoleic, and C&r”” mansonz eggs. 0, 8, C,,‘, C,,“, C,,“’ linolenic and arachidonic acids respectively. Peaks for C,,;, Crs~, C,,’ and C,,” taken with instrument of an attenuation of 300 K. All others taken with an attenuation of 30 K. Temperature of column 145-200°C.
270
TORO-GOYCO
AND ROSAS DEL VALLE
intestinal tissue as compared to liver tissue, which commonly revealed marked cirrhosis and therefore was less susceptible to enzyme digestion. We have found one disadvantage in eggs isolated from intestinal tissue. They are immunologically less reactive to sera of infected humans than those isolated from liver tissue using the same enzyme digestion procedure. It was surprising to find such low levels of protein in the soluble fraction of the sonicate. It may be argued that the process used for the isolation of eggs (enzymic digestion of tissues) may explain the low yield. Several factors, however, rule out this possibility. The permeability of the egg shell is small enough to restrict the inflow of proteins to the interior. In addition, the time of exposure to the enzyme is relatively short ( 1 to 2 hr ) so that taking into account diffusion factors, the possibility of inward flow because of damage to the egg shell is fairly small. Finally, we have not been able to detect any protease activity in the soluble fraction of sonicates prepared immediately after isolation of the ei%sThe high level of sterol esters among the lipid constituents is a significant finding. The role they may play in the further development of the parasite is unknown. Lipid analyses in cercarial and adult stages of S. mansoni (Smith et al. 1966, 1969) indicate that in these stages free sterol is a major component. They have also reported that phospholipids are the lowest among the lipid constituents. Studies now in progress using different solvent systems for thinlayer chromatographic analysis confirm the findings reported here. Among the fatty acids, the most abundant are Cl8 and Cl, acids. A similar finding has been reported by Smith and co-workers in their study of lipids from cercariae and adults of S. mansoni. The fact that only limited amounts of glucose were found in eggs can be ex-
plained by Bueding’s (1949) report that the parasite metabolizes glucose very fast. Although his studies were performed on adult parasites, it is not unreasonable to assume that because of low levels of glucose in the adult, low concentrations may be expected in its eggs. The role of much of the carbohydrate as structural units can be deduced from the fact that most of the carbohydrate is found in the insoluble fraction of the sonicate. This is in marked contrast with the lipid fraction which can be found mainly (N-90%) in the soluble fraction. It has been reported (Senft 1963) that adult parasites kept in vitro show marked utilization of basis amino acids but not of other amino acids. The results shown in Table I show that the most abundant amino acids in the dialyzable fraction of the sonicate are the basic amino acids, which comprise about 34% of the total amino acids. It is also stated by Senft that adult parasites produce significant amounts of urea. Whether urea production starts at
TABLE I Amino Add Composition of Dialyzable of a Sonicate of Schistosoma mansoni Amino
acid
Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine l/i Cystine Methionine Isoleucine Leucine Tyrosine @-Alanine NH, Lysine Histidine Arginine
% Total
Ff-action Eggs
N recovered 3.56 2.03 2.62 5.14 4.01 5.82 3.58 3.84 1.12 .15 4.14 6.49 2.78 3.48 15.43 11.14 7.65 15.64 98.62
Schistosoma mansoni. I. the early stage of the egg remains to be shown. Any urea present in eggs will be destroyed in our hydrolyzates, yielding ammonia. It appears appropriate to point out that ammonia accounts for about 15.4% of our hydrolyzates. The only protein in the soluble fraction of the sonicate detectable in any significant amount is the lipoprotein already described under Results. It is found in very small amounts ( 1% of the dry weight). Such low levels have been an impediment in our attempts to its physical-chemical characterization. This fact leads us to believe that this protein is structural, rather than metabolically functional. Probably, detailed amino acid and lipid analyses of this entity will supply the most relevant information as to its biological role. Identification of any other soluble protein will require more sensitive techniques than those used in this work. Preliminary work on the immunochemical constituents isolated from adult S. maasoni (Biguet et al. 1962) indicate that the adult parasite is immunochemically very complex, The preliminary data presented here tend to show that eggs are chemically less complex and characterization of these can be constituents immunochemical achieved with considerably less effort. ACKNOWLEDGMENTS
This work was supported by grant No. Al-07769 from the National Institutes of Health, U. S. Department of Health, Education and Welfare. REFERENCES J., CAPRON, A., AND TRAN VAN KY, P. 1962. Les antigens de Schistosoma mansoni. I. Etude electrophorktique et immunoelectrophoretique. Caracterization des antigens specifiques. Anales de Z’Institut Pasteur 103, 763-777. of parasite helBUEDING, E. 1949. Metabolism minths. Physiological Reviews 29, 195-218. BIGUET,
CANCIO, M., RIVERA LINA, R., AND
DE SALA,
A.,
ROD&GUEZ-MO-
TORO-GOYCO, E.
1965.
In
271
vitro quantitation of the circumoval precipitin test in infections with Schistosoma mansoni. Experimental Parasitology 16, 64-73. FALES, F. W. 1951. The assimilation and degradation of carbohydrates by yeast cells. JournaZ of Biological Chemistry 193, 113-124. HUNTER, G. W., AND CRANDALL, R. B. 1962. Studies on schistosomiasis. XIX. Results of preliminary experiments on the antigenic significance of the schistosome egg. Military Me&ine 127, 101-104. KAGAN, I. G., AND PELLEGRINO, J. 1961. A critical review of immunologic methods for the diagnosis of bilharziasis. Bulletin of the World Health Organization 25, 611-674. KAGAN, I. G., AND GOODCHILD, C. G. 1963. Polysaccharide content of schistosome skin test antigens and the reactivity of nitrogenous and carbohydrate components. American Journal of Tropical Medicine G Hygiene 12, 179183. LIZ., E. W., TINOCO, J., AND OKEY, R. 1961. A micromethod for fractionation of lipids by silicic acid chromatography. Analytical Biochemistry 2, 100-106. LOWKY, 0. H., ROSEBROUGH, N. J., FAIR, L., AND RANDALL, R. S. 1951. Protein measurements with the Folin-phenol reagent. Journal of Biological Chemistry 193, 256-261. METCALFE, L. D., AND SCHMITZ, A. A. 1961. The rapid preparation of fatty acid esters for gas chromatographic analysis. Analytical Chemistry 33, 363-364. RITCHIE, L. S., AND BERR~OS-DL&N, L. A. 1961. A simple procedure for recovering Schistosome eggs in mass from tissues. The Journal of Parasitology 47, 363-365. RUGGIERI, S. 1962. Separation of the methyl esters of fatty acids by thin layer chromatography. Nature 193, 1282-1283. SENFT, A. W. 1963. Observations on amino Xl ‘d metabolism of Schistosoma mansoni in a chemically defined medium. Annals of the New York Academy of Sciences 113, 272-288. SILIITH, T. M., BROOKS, T. J., AND WHITE, H. B. 1966. Thin layer and gas liquid chromatographic analysis of lipids from cercariae of Schistosoma mansoni. American Journal of Tropical Medicine G Hygiene 15, 307-313. SMITH,
T.
M.,
BROOKS,
T.
J., AND
WHITE,
H.
B.
1969. Fatty acid composition of adult Schistosomu munsoni. Lipids 4, 31-36. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S. 1958. Automatic recording apparatus for use in the chromatography of amino acids. Analytical Chemistry 30, 1190-1205.
272
TORO-GOYCO
AND
Togo-GOYCO, E. 1963. Schistosome ova: Rapid separation from mouse tissue by digestion with pinguinain. Science 142, 407-408. TORO-GOYCO, E. 1964. Circumoval precipitins: Localization by gel filtration in serum of humans infected with S. munsoni. Proceedings of the Society for Experimental Biology and Medicine 117, 921-923. TORO-GOYCO, E., MARETZKI, A., AND MATOS, M. 1968. Isolation, purification and partial
ROSAS
DEL
VALLE
characterization of pinguinain, the proteolytic enzyme from Bromelia pinguin L. Archioes of Biochemistry d7 Biophysics 126, 91-104. WASHKO, M. E., AND RICE, E. W. 1961. Determination of glucose by an improved enzymatic procedure. Clinical Chemistry 7, 542-545. for ZAK, B. 1957. Simple rapid microtechnique serum total cholesterol. American Journal of Clinical Pathology 27, 583-588.
PARASITOLOGY 27, 265-272
Schistosoma
mansoni. Efrain
Department University
Toro-Goyco”
(1970)
I. Chemical and
Myrtha
Composition Rosas
del
of Eggs’
Valle
of Biochemistry of Puerto Rico
6 Nutrition and the Laboratory of Neurobiology, School of Medicine, and the San Juan Veterans Administration Hospital, San Juan, Puerto Rico ( Submitted
for publication,
5 June 1969)
Tono-GOYCO, EFRA~N AKD ROSAS DEL VALLE, MYRTHA. 1970. Schistosoma mansoni. I. Chemical Composition of Eggs. Experimental Parasitology 27, 265-272. Eggs of Schisto,soma mansoni have been isolated from tissues of infected mice by digestion with the proteolytic enzyme pingninain. The yield of eggs has been high enough to permit chemical characterization. These have been analyzed for carbohydrate, lipid, and protein contents after sonic disintegration. The water-soluble fraction of the sonicate contains a lipid-rich lipoprotein fraction which appears to be the only protein detectable in significant amounts. Glucose seems to be the most abundant hexose, while esterified cholesterol and neutral fats are the most abundant among the lipid entities. INDEX DESCRIPTORS: Schistosoma mansoni; Chemical composition; protein; Carbohydrates; Glucose; Cholesterol; Fats neutral.
Recent studies on Schistosoma mansoni, mostly of the adult form, place marked emphasis on biochemical and immunological aspects. It has been shown that the adult parasites in infected humans elicit the formation of various antibodies (Kagan and Pellegrino 1961). Antigenic preparations have been obtained from crude fractions of homogenized parasite and chemically analyzed in attempts to identify the chemical nature of the antigenic materials (Kagan and Goodchild 1963; Biguet et al. 1962). Although the antigenic nature of eggs has been established (Hunter and Crandall 1962; Toro-Goyco 1964; Cancio et al. 1965), no previous studies have been done to analyze or characterize the antigenic constituents present in eggs. Furthermore, no chemical study on the composition of the eggs of S. mansoni has been reported. This 1 Contribution No. 17, Laboratory of Neurobiology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico. 2 Lederle Medical Faculty Awardee, 1966-69. 265
Lipids; Lipo-
paper is a preliminary report on the chemical composition of eggs of S. munsoni resulting from studies undertaken as a preliminary step toward the identification of the chemical constituents of eggs responsible for their antigenicity. MATERIALS
AND METHODS
Mice were used as the source of eggs. These mice had been previously infected with S. mansoni according to the method developed by Ritchie and Berrios-Duran (1961). The mice were killed, and the intestines and livers removed. Livers were carefullv washed to remove all traces of blood. Intestines were washed to remove all residues of food prior to preparation of the homogenates. Since liver and intestinal tissues differ in their resistance to enzymic hydrolysis, homogenates were prepared from either one of the tissues but never from a mixture of tissues. The homogenates were incubated with the enzyme ping-
266
TORO-GOYCO
AND
uinain at pH 4.6 in 0.1 M acetate buffer. The details of the procedure for the preparation of the homogenates, incubation, and separation of the eggs have been described previously ( Toro-Goyco 1963). Isolated eggs were either kept refrigerated at 4” in 0.15 M sodium chloride to which 0.01% sodium azide had been added or lyophilized after repeated washing with water. The wet weight of eggs was calculated by packing aqueous suspensions of eggs in previously calibrated Wintrobe hematocrit tubes, centrifuging at 3000 rpm for 10 min in an International refrigerated centrifuge, removing the supernatant water, and weighing using as tare the previously weighed dry Wintrobe tube. This procedure yields the total wet weight. Since the tubes had been previously calibrated with a liquid of known density (water at ZOO), it was possible to calculate the weight per unit volume. For succeeding determinations of wet weight of eggs using these same tubes, it was only necessary to measure the packed volume after centrifugation. Dry weights were obtained by suspending the packed eggs in distilled water, placing them in weighing bottles of known weight, freezing the contents, and evaporating in the frozen state in a vacuum oven after which the bottles were dried to constant weight. Preparation of the soluble components of eggs was accomplished by suspending the wet eggs in phosphate saline buffer followed by sonic disintegration using a Raytheon sonic oscillator. The sonicate was centrifuged at 105,OOOgfor 1 hr in a Spinco Model L preparative ultracentrifuge, using a SW 25.1 swinging bucket rotor, to remove any remaining particulate material. The supernatant was removed for carbohydrate, lipid, protein, and amino acid analyses, gel filtration, and preparative ultracentrifugation. Gel filtration was performed using either Sephadex G-100 or G-200 columns with phosphate-buffered saline as the mobile
ROSA.5
DEL
VALLE
phase (0.15 M NaCI, 0.006 M monobasic sodium phosphate, 0.035 M dibasic sodium phosphate, pH 7.3). Filtration was carried out at room temperature. Carbohydrate analyses of the complete eggs and of the soluble and insoluble fractions of the sonicate were performed by the anthronc method using a modified anthrone reagent ( Fales 1951) . For total amino acid analysis, a 2-ml aliquot of the supernatant sonicate was removed, diluted to 4 ml with concentrated HCl, brought to 8 ml with constant boiling HCl, and sealed under reduced pressure. The sealed tubes were placed in the oven at 110” for 18 hr. For protein amino acid analysis, the supernatant was dialyzed for 24 hr against a 500-fold excess volume of water with change of water every 8 hr. The dialyzate was diluted as explained above and hydrolyzed under the same conditions. Amino acid analysis was performed by the method of Spackman et al. ( 1949)) using a Technicon amino acid analyzer. Detailed procedures for the analysis of the amino acids and calculation of the data has been described earlier ( Toro-Goyco et al. 1968). Total nitrogen determinations were performed by the micro-Kjeldahl technique using as blanks the corresponding solvent. Total protein was determined by the method of Lowry et al. (1951). For total lipid determinations, dry eggs were suspended in 2:l chloroform-methanol mixtures and ground by use of a glass, hand model tissue grinder. After centrifugation at 3000 rpm for 10 min to remove the insoluble material, the supernatant extract was removed, placed in a separatory funnel, and water was added (20% of the volume). The mixture was then shaken and separation of the two phases allowed to occur. The less dense water-methanol phase contained all the nonlipid constituents originally soluble in methanol while the chloroform phase contained the lipid material. The chloroform was evaporated to dryness
Schistosoma mansoni. I. under reduced pressure and stored until analysis. Analysis of the nonsoluble fraction of the sonicate was performed by using the same procedure. Thin-layer chromatography of lipid constituents was performed in 20 X 20-cm glass plates using silica as the stationary phase. The solvent used was ethyl ether, acetic acid, hexane ( 15:1:90), and the plates were stained with 2,7-dichlorofluorescein (Ruggieri 1962). Separation of the lipid constituents was also accompanied by silicic acid chromatography as suggested by Lis et al. ( 1961). Qualitative fatty acid analyses were performed using a Barber-Coleman gas chromatograph. Samples for gas chromatography were prepared by saponification of the lipid material followed by esterification with trifluoride in methanol and extraction with ethyl ether (Metcalfe and Schmitz 1961). Cholesterol was determined by the method of Zak ( 1957).
267 RESULTS
A. Yield of Eggs Consistently, the yield of eggs expressed in mg wet weight/g tissue was higher from intestinal tissue (4.2 -+ 0.4) than from liver tissue (2.2 t 0.2). B. Composition
of the Sonicate
The suspension obtained after sonication and centrifugation as explained above was of varying degrees of turbidity. The turbidity was found to be due to a lipid-rich lipoprotein fraction that was eluted with the void volume in Sephadex G-100 and G-200 chromatographic columns ( Fig. 1) . The lipoprotein nature of this component was ascertained by preparative ultracentrifugation as follows: The most concentrated fraction collected from the chromatographic columns (5 ml) was diluted to 12 ml with a solution of CsCl, bringing the density of the resultant solution to 1.21
20
IO FRACTION
30
NO
FIG. 1. Gel filtration pattern of a lipoprotein fraction isolated from soluble fraction of the sonicate (Sephadex G-100; phosphate saline buffer pH 7.3). Dimensions of column, 45 X 2.5 cm. Absorbancy at 280 rnp expressed in optical density units. Fractions 5 ml each. Protein concentration in peak 40 pg protein/ml.
268
TORO-GOYCO
AND
ROSAS
mg/ml (25” ), and centrifuged for 18 hr at 105,OOOgin a Spinco No. 40 rotor. At the end of this period the turbidity was concentrated at the top of the tube. The centrifuge tube was cut and four fractions analyzed for protein contents. No significant levels of protein nitrogen were found in any of the fractions, with the exception of the top one which also contained the turbidity. Thin-layer chromatography of the lipids extracted from the lipoprotein fraction indicated a pattern very similar to that shown by the lipids of the whole eggs (Fig. 2). C. Total Lipids A thin-layer chromatogram of the total lipid extract from whole eggs is shown in Fig. 2. Under the experimental conditions used, significant amounts of four main groups of lipids could easily be identified. The esterified cholesterol is shown as the fastest migrating component, followed by the triglycerides, the fatty acids and their
m
m,(j,,,+
. k
VALLE
esters (as a single component), and the slower migrating cholesterol. Only very slight traces of phospholipids staying at the origin could be detected. The marked contrast between the high levels of esterified cholesterol and other constituents, especially the practically nonexistent phospholipids, can be appreciated. Gravimetric analysis for the total lipids extracted from whole eggs isolated from intestinal tissue show that these components account for about one-fifth of the total dry weight (20.7 * 2.0). In eggs isolated from intestine, cholesterol and its esters account for one-fourth of the total lipids. The lipids present in eggs isolated from liver tissue account for 35 * 5% of the dry weight. Total lipid analysis was also performed in the soluble and nonsoluble portions of the sonicate. It was consistently found that the bulk of the lipid material (8CMO%) of the total lipid appeared in the soluble portion. Gas-chromatographic analyses have reSOLVENT -FRONT
111
Ir
. 8
DEL
.
.
.
.
.
5’
C
D
E
F
G
H
FIG. 2. Thin layer chromatogram of total lipid from whole eggs: A, cholesterol palmitate; B, tripalmitin; C, cholesterol; D, total lipid extract, eggs isolated from inatestine; E, total lipid extract, eggs isolated from liver; F, steak acid; G, methyl palmitate; H, egg lecithin. Solvent: ethyl ether, acetic acid, hexane, 15: 1:90.
Schistosoma mansoni. I. vealed that the most abundant fatty acids are palmitic, stearic, oleic, and linoleic acids. Collectively, these comprised some 90% of the total fatty acids (Fig. 3). The polyunsaturated linolenic and arachidonic acids account for 5% of the fatty acids in eggs isolated from both tissues. Arachidonic acid is more abundant in the triglvceride portion, where it accounts for 4%-of the total fatty acids. D. Carbohydrate
Content
Most of the carbohydrate was found in the insoluble fraction after sonication of the eggs. In eggs isolated from intestinal tissue, 16% of the total dry weight was accounted for as insoluble carbohydrate. The soluble fractions contain 5 to 6% of the total carbohydrate, as shown by the anthrone method. Sugar analysis performed by an enzymic method (Washko and Rice
269
1961) indicates that the hexose sugar present in this fraction is essentially glucose. E. Amino Acid Analyses and Protein Contents Preliminary analysis performed on the supernatant was revealed that there is a considerable amount of free amino acids present in the soluble fraction of the sonicate. The level of aromatic amino acids is significantly low (6.2%) while that of basic amino acids is considerably higher (34.4%).
DISCUSSION The higher unit weight of to liver tissue better source The difference tially explained
yield of eggs obtained per intestinal tissue as compared makes the former tissue a of this material for study. in yield may well be parby an easier digestibility of
cl6
I
TIME
IN MINUTES
FIG. 3. Gas chromatogram of ethyl esters of fatty acids present in triglyceride fraction of S. stand for the methyl esters of stearic, oleic, linoleic, and C&r”” mansonz eggs. 0, 8, C,,‘, C,,“, C,,“’ linolenic and arachidonic acids respectively. Peaks for C,,;, Crs~, C,,’ and C,,” taken with instrument of an attenuation of 300 K. All others taken with an attenuation of 30 K. Temperature of column 145-200°C.
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TORO-GOYCO
AND ROSAS DEL VALLE
intestinal tissue as compared to liver tissue, which commonly revealed marked cirrhosis and therefore was less susceptible to enzyme digestion. We have found one disadvantage in eggs isolated from intestinal tissue. They are immunologically less reactive to sera of infected humans than those isolated from liver tissue using the same enzyme digestion procedure. It was surprising to find such low levels of protein in the soluble fraction of the sonicate. It may be argued that the process used for the isolation of eggs (enzymic digestion of tissues) may explain the low yield. Several factors, however, rule out this possibility. The permeability of the egg shell is small enough to restrict the inflow of proteins to the interior. In addition, the time of exposure to the enzyme is relatively short ( 1 to 2 hr ) so that taking into account diffusion factors, the possibility of inward flow because of damage to the egg shell is fairly small. Finally, we have not been able to detect any protease activity in the soluble fraction of sonicates prepared immediately after isolation of the ei%sThe high level of sterol esters among the lipid constituents is a significant finding. The role they may play in the further development of the parasite is unknown. Lipid analyses in cercarial and adult stages of S. mansoni (Smith et al. 1966, 1969) indicate that in these stages free sterol is a major component. They have also reported that phospholipids are the lowest among the lipid constituents. Studies now in progress using different solvent systems for thinlayer chromatographic analysis confirm the findings reported here. Among the fatty acids, the most abundant are Cl8 and Cl, acids. A similar finding has been reported by Smith and co-workers in their study of lipids from cercariae and adults of S. mansoni. The fact that only limited amounts of glucose were found in eggs can be ex-
plained by Bueding’s (1949) report that the parasite metabolizes glucose very fast. Although his studies were performed on adult parasites, it is not unreasonable to assume that because of low levels of glucose in the adult, low concentrations may be expected in its eggs. The role of much of the carbohydrate as structural units can be deduced from the fact that most of the carbohydrate is found in the insoluble fraction of the sonicate. This is in marked contrast with the lipid fraction which can be found mainly (N-90%) in the soluble fraction. It has been reported (Senft 1963) that adult parasites kept in vitro show marked utilization of basis amino acids but not of other amino acids. The results shown in Table I show that the most abundant amino acids in the dialyzable fraction of the sonicate are the basic amino acids, which comprise about 34% of the total amino acids. It is also stated by Senft that adult parasites produce significant amounts of urea. Whether urea production starts at
TABLE I Amino Add Composition of Dialyzable of a Sonicate of Schistosoma mansoni Amino
acid
Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine l/i Cystine Methionine Isoleucine Leucine Tyrosine @-Alanine NH, Lysine Histidine Arginine
% Total
Ff-action Eggs
N recovered 3.56 2.03 2.62 5.14 4.01 5.82 3.58 3.84 1.12 .15 4.14 6.49 2.78 3.48 15.43 11.14 7.65 15.64 98.62
Schistosoma mansoni. I. the early stage of the egg remains to be shown. Any urea present in eggs will be destroyed in our hydrolyzates, yielding ammonia. It appears appropriate to point out that ammonia accounts for about 15.4% of our hydrolyzates. The only protein in the soluble fraction of the sonicate detectable in any significant amount is the lipoprotein already described under Results. It is found in very small amounts ( 1% of the dry weight). Such low levels have been an impediment in our attempts to its physical-chemical characterization. This fact leads us to believe that this protein is structural, rather than metabolically functional. Probably, detailed amino acid and lipid analyses of this entity will supply the most relevant information as to its biological role. Identification of any other soluble protein will require more sensitive techniques than those used in this work. Preliminary work on the immunochemical constituents isolated from adult S. maasoni (Biguet et al. 1962) indicate that the adult parasite is immunochemically very complex, The preliminary data presented here tend to show that eggs are chemically less complex and characterization of these can be constituents immunochemical achieved with considerably less effort. ACKNOWLEDGMENTS
This work was supported by grant No. Al-07769 from the National Institutes of Health, U. S. Department of Health, Education and Welfare. REFERENCES J., CAPRON, A., AND TRAN VAN KY, P. 1962. Les antigens de Schistosoma mansoni. I. Etude electrophorktique et immunoelectrophoretique. Caracterization des antigens specifiques. Anales de Z’Institut Pasteur 103, 763-777. of parasite helBUEDING, E. 1949. Metabolism minths. Physiological Reviews 29, 195-218. BIGUET,
CANCIO, M., RIVERA LINA, R., AND
DE SALA,
A.,
ROD&GUEZ-MO-
TORO-GOYCO, E.
1965.
In
271
vitro quantitation of the circumoval precipitin test in infections with Schistosoma mansoni. Experimental Parasitology 16, 64-73. FALES, F. W. 1951. The assimilation and degradation of carbohydrates by yeast cells. JournaZ of Biological Chemistry 193, 113-124. HUNTER, G. W., AND CRANDALL, R. B. 1962. Studies on schistosomiasis. XIX. Results of preliminary experiments on the antigenic significance of the schistosome egg. Military Me&ine 127, 101-104. KAGAN, I. G., AND PELLEGRINO, J. 1961. A critical review of immunologic methods for the diagnosis of bilharziasis. Bulletin of the World Health Organization 25, 611-674. KAGAN, I. G., AND GOODCHILD, C. G. 1963. Polysaccharide content of schistosome skin test antigens and the reactivity of nitrogenous and carbohydrate components. American Journal of Tropical Medicine G Hygiene 12, 179183. LIZ., E. W., TINOCO, J., AND OKEY, R. 1961. A micromethod for fractionation of lipids by silicic acid chromatography. Analytical Biochemistry 2, 100-106. LOWKY, 0. H., ROSEBROUGH, N. J., FAIR, L., AND RANDALL, R. S. 1951. Protein measurements with the Folin-phenol reagent. Journal of Biological Chemistry 193, 256-261. METCALFE, L. D., AND SCHMITZ, A. A. 1961. The rapid preparation of fatty acid esters for gas chromatographic analysis. Analytical Chemistry 33, 363-364. RITCHIE, L. S., AND BERR~OS-DL&N, L. A. 1961. A simple procedure for recovering Schistosome eggs in mass from tissues. The Journal of Parasitology 47, 363-365. RUGGIERI, S. 1962. Separation of the methyl esters of fatty acids by thin layer chromatography. Nature 193, 1282-1283. SENFT, A. W. 1963. Observations on amino Xl ‘d metabolism of Schistosoma mansoni in a chemically defined medium. Annals of the New York Academy of Sciences 113, 272-288. SILIITH, T. M., BROOKS, T. J., AND WHITE, H. B. 1966. Thin layer and gas liquid chromatographic analysis of lipids from cercariae of Schistosoma mansoni. American Journal of Tropical Medicine G Hygiene 15, 307-313. SMITH,
T.
M.,
BROOKS,
T.
J., AND
WHITE,
H.
B.
1969. Fatty acid composition of adult Schistosomu munsoni. Lipids 4, 31-36. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S. 1958. Automatic recording apparatus for use in the chromatography of amino acids. Analytical Chemistry 30, 1190-1205.
272
TORO-GOYCO
AND
Togo-GOYCO, E. 1963. Schistosome ova: Rapid separation from mouse tissue by digestion with pinguinain. Science 142, 407-408. TORO-GOYCO, E. 1964. Circumoval precipitins: Localization by gel filtration in serum of humans infected with S. munsoni. Proceedings of the Society for Experimental Biology and Medicine 117, 921-923. TORO-GOYCO, E., MARETZKI, A., AND MATOS, M. 1968. Isolation, purification and partial
ROSAS
DEL
VALLE
characterization of pinguinain, the proteolytic enzyme from Bromelia pinguin L. Archioes of Biochemistry d7 Biophysics 126, 91-104. WASHKO, M. E., AND RICE, E. W. 1961. Determination of glucose by an improved enzymatic procedure. Clinical Chemistry 7, 542-545. for ZAK, B. 1957. Simple rapid microtechnique serum total cholesterol. American Journal of Clinical Pathology 27, 583-588.