Fatty Acid Composition of Adult Schistosoma mansoni THOMAS M. SMITH, THOMAS J. BROOKS, JR. and HAROLD B. WHITE, JR.,
Departments of Biochemistry and Preventive Medicine, The University of Mississippi School of Medicine, Jackson, Mississippi 39216 ABSTRACT
ston Purina Company, St. Louis, Mo.) and water ad lib. Mice infected for 6 to 10 weeks with the Puerto Rican strain of S. mansoni were killed by skull fracture and the mature flukes dissected from the mesenteric venules. Prior to extraction with chloroform-methanol (2:1, v / v ) (7), these trematode parasites were washed in three changes of physiological saline and counted. Tissue lipid was separated into classes by ascending TLC on 20 x 20 cm glass plates coated with a 250 F silica gel G adsorbent layer (E. Merck AG, Darmstadt, Germany). Normal hexane-diethyl ether-acetic acid (70:30:1, v / v / v ) was used as the developing solvent (8). Reference mixtures for lipid class separations by TLC and methyl ester standards were obtained from the Hormel Institute, Austin, Minn. Prior to the application of lipid samples all chromatoplates except those coated with a 250/~ layer of silica gel H R (E. Merck AG, Darmstadt, Germany) were washed with methanol-diethyl ether (80:20, v / v ) to remove impurities in the silica gel layer (9). All solvents were routinely examined for contaminants by dissolving any residue remaining after evaporation of 100 ml in 10 /tl of nhexane and injecting this solution into a gas chromatograph. Operating conditions were always those used in subsequent analyses of fluke lipid. Contaminated solvents were purified by appropriate procedures detailed elsewhere(10). When necessary, 2,7-dichlorofluorescein and UV light were used to visualize standard class separations. Silica gel bands containing the separated components were scraped into individual 5 ml round bottom flasks. About 2 ml of 6% sulfuric acid in anhydrous methanol was added to lipid class or total lipid samples and each mixture refluxed for 6 hr at 80 ~ C. Methyl esters were routinely extracted into hexane and separated from other extractable reaction products by TLC using an n-hexanediethyl ether-acetic acid (90:10:1, v / v / v ) developing solvent (11). S. mansoni phospholipid components were further studied by rechromatographing the phospholipid fraction from above in chloroform-methanol-water (65:25:4, v / v / v ) and spraying the developed
The fatty acid composition of triglyceride and total phospholipid fractions of adult Schistosoma mansoni has been examined. Both triglyceride and phospholipid contained fatty acids varying in chain length from 12 through 24 carbons; trace amounts of shorter chain components were found in the triglyceride fraction. A docosahexaenoic acid in the triglyceride fraction represented the highest degree of unsaturation e n c o u n t e r e d . Branched chain fatty acids of 16 and 18 carbons were found in both phospholipid and triglyceride. Examination of fatty acids from fluke total lipid revealed the presence of small amounts of odd numbered carbon fatty acids varying in chain length from 13 through 23 carbons. INTRODUCTION
disease of Sportance. considerable medical and economic imThroughout the world nearly 200 C H I S T O S O M I A S I S IS
A
pARASITIC
million people are currently infected with one of the three species of blood flukes (1); the disease is particularly prevalent in the Orient (2). In the Western Hemisphere, schistosomiasis mansoni is widely distributed in Brazil, certain of the Caribbean Islands and in some areas of Puerto Rico (3). The accumulation of eggs from this blood fluke in the human liver and gut produces extensive fibrosis. Prognosis is poor in chronic cases with advanced cirrhosis of the liver (3). Carbohydrate and protein metabolism of Schistosoma mansoni have been investigated (4, 5) but the lipid composition of this important blood fluke has received little attention (6). The purpose of this paper is to identify the fatty acids found in the triglyceride and phospholipid fractions of S, rnansoni total lipid as an aid to the future understanding of lipid metabolism which may stimulate the development of new chemotherapeutic, prophylactic or suppressive agents. MATERIALS' AND METHODS
White Swiss mice or D B A / 2 mice were maintained on a diet of Purina rat chow (Ral31
32
TttOMAS M. SMITH, THOMAS J. BROOKS, JR. AND HAROLD B. WHITE, JR. cM TRIGLYCERCDE
PHOSPHOLtPID
o ~,
o
00
N
4
o -oo b,,
II 6
0
Oa
o
.. o .. ~_
214
_o OJ 4......
512
FIG. 1. GLC recording of fatty esters from adult S. mansoni phospholipid f r o m 470 pair. Glass column 4 ft • 4 mm i.d., packed with 5% D E G S on Diatoport S.
~-
-.
~.. O ~ X I ~
MIN.
-
0
8
o_ Io m ~. 0 .o9
m .0.9 f, co IB MIN.
"
J ~
I
o~ 6
~
r "~
~e~
Z4
FIG. 2, GLC recording of fatty esters from adult S. mansoni triglyceride from 470 pair. Column specifications detailed in Fig. 1 legend.
Fro. 3. Photograph of fractionation of fatty ester adducts by TLC according to number of double bonds. A, D, 1-6, standard mixture, in progressive order, from the origin: 1) docosahexaenoate and eicosapentaenoate, 2) arachidonate, 3) linolenate, 4) linoleate, 5) oleate, 6) stearate. B, C, adducts of fatty esters from adult S. mansoni total lipid, 1-6, in progressive order from the origin: 1) hexaenes and pentaenes, 2) tetraenes, 3) trienes, 4) dienes, 5) monoenes, 6) saturates. LIPIDS, VOL. 4, NO. 1
32
33
FATTY ACID COMPOSITION OF A BLOOD FLUKE
plates with either ammonium molybdate-perchloric acid reagent or ninhydrin and comparing calculated Rr values with recorded values for phosphatides (12). Fatty acid methyl esters were separated and qualitatively identified by two G L C procedures. In the first, methyl esters from fluke total lipid or lipid classes were chromatographed on either an F and M model 400 or 402 gas chromatograph equipped with hydrogen-flame ionization detectors. Results are shown in Figs. 1 and 2. The column used in both instruments was a 4 ft x 4 m m i.d. glass tube packed with 5% polydiethyleneglycolsuccinate ( D E G S ) coated on 80-100 mesh Diatoport S. Operating conditions were helium flow rate 75 ml/min, temperature programming from 100-210 C at 3 ~ and 400 attenuation of signal. Identification in this instance was made by comparison of unknown peak relative elution temperatures with those of standard methyl esters as previously described (8,13). In the two column technique used as the second G L C procedure, the problem of overlapping components so evident in Figs. I and 2 was resolved by collecting chain length fractions from the effluent port of either an F and M model 400 gas chromatograph as described elsewhere (14) or from the effluent port of an F and M model 500 gas chromatograph. In the latter chain length collection process, an 8 ft. x 88 in. i.d. stainless steel column packed with 20% S. E. 30 on 60-80 mesh Chromosorb W was used. Operating conditions were helium flow rate 120 m l / m i n , temperature programming from 250--375C at 4~ and a signal attenuation of 1. Block temperature was 390C and the effluent port was wrapped with asbestos string to a width of 2 in. to prevent condensation of higher molecular weight methyl esters in the effluent tube during the collection process. All collected chain length fractions were analyzed for double bond content by G L C on 5% D E G S with either an F and M model 400 or model 402 instrument as described above. To further aid in establishing degrees of unsaturation in individual fatty acids from fluke total lipid, phospholipid, or triglyceride, a complementary TLC, G L C procedure (15) modified by initially separating the methoxy, bromomercuri-adducts on longer (20 x 40 era) silica gel H R coated chromatoplates was used (Fig. 3). After adduct decomposition, the component chain lengths of the saturated, monoenoic and dienoic fatty acids were de-
HYDROGENATED TOTAL
METHYL
ESTERS
(D
0
Od
Od r
1 ~~
o
4
8
I
16
20
I
24
I
28
I
32
3[ 6
MIN
FIG. 4. GLC recording of hydrogenated fatty esters from adult S. mansoni total lipid. Column specifications detailed in Fig. 1 legend. termined by G L C of recovered methyl esters on 5% D E G S using operating conditions detailed above. Hydrogenated methyl esters from fluke total lipid, phospholipid and triglyceride (16) were analyzed by G L C on 5% D E G S to assist in resolving the complex general composition of each mixture, to verify chain length composition and to help in detecting the presence of branched chain fatty acids (Fig. 4). Peak areas were determined by multiplication of peak height by width at half-height. Percentages listed in Tables I and II represent the mean of the percentages of the total area under the curves contained in the peaks. RESULTS A N D D I S C U S S I O N
The first larval stages of all digenetic trematodes develop in molluscan intermediate hosts. This fact distinguishes this group of parasitic flatworms from all others. These original molluscan parasites apparently later developed an association with vertebrate hosts (17). Initial larval stages of S. mansoni develop in fresh water snails and this fresh water origin of these blood flukes is reflected in the large amounts of C16 and C1~ acids (18) found by us in adult S. mansoni triglyceride, phospholipid and total lipid (Table I). Indeed, the predominance of C1~ and Cis fatty acids in man, monkeys and wild rodents may be parLIPIDS, VOL. 4, No. 1
34
THOMAS M . S M I T H , T H O M A S J . BROOKS, JR. AND HAROLD B . W H I T E , JR. TABLE I F a t t y Acid C o m p o s i t i o n of A d u l t Schistosoma mansoni Tissue Lipids T o t a l L i p i da
Triglycerideb
Fatty Acid
Mean
S.D. Area
Min. %
Max.
Mean
12:0 13: 0e 14:0 15:0 16:0Br 16:0 i 16:1 17:0 18:0Br 18:0i 18:1 i 18:2 i 18:3 19:0 20:0 20: I i 20:2 20:3 20:Unsat. Unid. 20:4 20:5 21 : 0 22:0 22:1 22: U n s a t . Unid. 22: 2 22:3 22:4 22:5 22:6 23: 0 24:0J 24: lJ Unid.
0.1 Trace 0.7 0.3 Trace 17.6 1.1 0.5 0.3 11.8 22.2 9.4 Trace 0.6 0.6 10.7 4.3
0.1
0.1
0.2
0.5 0.1
0_5 0.2
1.2 0.4
0.2 Trace 0.8 Trace 0.2 14.5 2.2 0.4 0.2 5.3 32.4 14.7 Trace 0.4 0.4 8.8 3.1 f 1.1 r
9,5 h 0,4 Trace 0.9
0.8
1.6 Trace 5.0 0,2 1,4
1.4 0.0
0.3 1.5 0.3
16.3 19.5 1.0 1.1 0.5 0.5 Trace 0.3 10.7 13.2 21.0 24.8 9.1 9.8
0.1 0.8 0.4
0.4 0.4 9.9 3.7
0.8 0.7 11.9 4.7
0.4 0.1
9.4 0.3
10.0 0.6
0.1
0.8
1.0
0.3
1.2
0.4
0.9
0.7
2.5
0.5 0.1 1.2
4.3 0.2 0.1
5.5 0.3 2.5
5.7f OAf Trace 1.3f 0.9f
0.2e Trace 1.9f 0.3f 1.0f Trace 0.4 2.9 0.3
S.D. Min. Area % 0.1 0.4
6.0 0.9 0.2 1.4 1.0 1.1 0.1 0.1 3.0
0.1 1.i
0.2 1.1 0.1
0.1
Phospholipide Max. 0.2
0.4 Trace Trace 7.9 1.1 Trace 0.1 3.6 31.2 13.4
1.2 0.1 0.2 19.5 2.9 0.4 0.4 6.3 33.9 15.8
0.4 0.3 6.6 3.1 1.0
0.5 0.5 12.2 3.2 1H
5.6 0,3
5.9 0.5
0.9 0.8
1.8 1.1
0.2
0.2
1.7 0.3 0.9
2.2 0.4 1,2
0.3 2.0 0.3
0.5 4.3 0.4
Mean Traced Trace 0.7 0.4 1.2 28.6 0.6 0.4 0.6 21.8 17.2 2.7 Trace 0.4 0.8 11.6 2.1
S.D. Area
Min. %
Max.
0.3 0.1 0.3 5.1 0.1 0.1 0.1 2.8 6.1 1.5
0.4 0.2 0.7 22.6 0.5 0.3 0.5 19.0 12.8 1.4
1.2 0.5 1.6 36.1 0.9 0.4 0.8 ~.4 34.2 5.1
0.1 0.2 1.5 1.2
0.4 0.5 9.4 0.8
0~5 1.2 14.0 4.0
0.6g
0.5
0.6
Trace
Trace
0.4
0.I 3,0
0.3 1,3 2,l
0.5 8.4 2.2
0,2 3,2
0.4 0.6
0.8 7.6
Trace 0.4 4.0 2.1g
Trace 0.6 3.2
a Six determinations on two samples (145 pair and 205 pair + 2 male flukes). b F i v e determinations on two samples (243 pair and 200 p a i r ) . e N i n e determinations on four samples (243 pair, 1088 pair, 1087 pair + 180 males and 360 pair + 28 m a l e s ) . d Trace ~-~ less t h a n 0.1% of total area. e O d d chain length data f r o m hydrogenated samples. r Three determinations on one sample (243 p a i r ) . g F o u n d in three determinations on 360 p a i r + 28 males only. h 2 0 : 4 and 22:1 in t o t a l lipid. i M a j o r fatty acids composing 10% or more of t o t a l or either fraction. U n i d . = unidentified. Unsat. unsaturated fatty acid. B r - - b r a n c h e d chain fatty acid. J May represent a mixture with 22:3 or 22:4.
tially responsible for their selection as definitive hosts. An examination of S. mansoni cercarial total lipid fatty acid composition revealed a similar prevalence of C16 and Cls fatty acids in the larval stage which penetrates the skin to establish the parasite in the mesenteric venules and liver of primates and rodents (8). The fluke phospholipid fraction was found to contain materials corresponding in Rf to amino acids, lysolecithin, sphingomyelin and cardiolipin. Results of analyses of fatty acid moieties from S. mansoni total lipid, triglyceride and phospholipid are shown in Tables I and II. The triglyceride fraction contained 31 fatty acids varying in chain length from 12 through 24 carbons. Occasionally traces of LIPIDS, VOL. 4, No. l
shorter chain fatty acids were seen in GLC strip chart recordings. A similar chain length composition was found among the 27 fatty acids from S. mansoni total lipid and the 26 fatty acids of the phospholipid fraction (Tables I and II). A fatty acid composition nearly as complex has been reported for neutral lipid from female Moniliformis dubius, a thorny headed acanthocephalan parasite of rats which contained some 29 fatty acids differing in chain length from 10 through 22 carbons (19). A study of neutral lipid from the female swine acanthocephalan Macracanthorhyncus hirudenaceus revealed 16 fatty acids varying in chain length from 10 through 20 carbons (19). Analyses of major fatty
35
FATTY ACID COMPOSITION OF A BLOOD FLUKE TABLE II H y d r o g e n a t e d F a t t y A c i d Methyl Esters f r o m A d u l t S c h i s t o s o m a Total Lipid Swiss Mice Fatty Acid Less t h a n 14:0 14:0 15:0 16:0Br 16:0 h 17:0 18:0Br 18:0 h Unid. 19:0 20:0h 21:0 22:0 23:0 Unid. 24:0
(1)a
(2)b Area %
mansoni
Tissue Lipids
Triglycerided DBA Mice f (1)e
Traceg Trace 1.0 1.0 0.5 0.4 0.5 0.3 Trace 0.9 Trace 24.7 28.6 19.8 0.5 0.5 0.7 0.6 Trace 46.8 44.6 40.1 0.5 0.9 0.4 0.8 0.5 18.0 18.9 20.1 Trace Trace Trace 5.5 3.8 7.0 Trace Trace Trace 0.5 1.I 1.5 9.0
Phospholipid e
Swiss Mice Mean
Trace 0.8 Trace Trace 17.0 0.4 Trace 54.2
Swiss Mice
S.D. Min. Area %
Max.
0.2
1.0
0.7
0.2
16.8 Trace
17.2 0.4
0.6
53.4
54.7
0.4 21.6 Trace 4.5 Trace
0.l 0.8
0.4 20.8
0.5 21.7
0.1
4.1
5.0
1.1
0.1
1.1
1.3
a O n e d e t e r m i n a t i o n o n total lipid f r o m 330 pair. b O n e d e t e r m i n a t i o n on total lipid f r o m 863 pair. O n e d e t e r m i n a t i o n on total lipid f r o m 229 pair q- 26 males. a F o u r determinations on triglyceride f r o m 722 p a i r q- 13 males. 9 F o u r determinations on phospholipid f r o m 328 pair. t D i l u t e B r o w n A g o u t i mice. g T r a c e ~ less t h a n 0.1% o f total area. h M a j o r fatty acids c o m p o s i n g 10% or m o r e of the total o r either fraction. b r a n c h e d c h a i n fatty acid.
acids from Spirometra mansonoides sparganum larval lipid resulted in the detection of 15 acids varying in chain length from 14 through 22 carbons in the total lipid (20). The 14 fatty acids detected in the sparganum neutral lipid and the 15 fatty acids from its phospholipid exhibited a similar chain length constitution. In adult S. mansonoides, a tapeworm from domestic cats, 16 fatty acids varying in chain length from 14 through 22 carbons were identified in both neutral lipid and phospholipid. As in adult S. rnansoni, the C1~ and C18 acids were found to be generally predominant in lipid from each parasite mentioned above, however, the C~4 acids detected in adult S. mansoni represent the longest chain length detected in lipids from any of these parasites (19,20). A comparison of adult S. mansoni triglycer, ide and phospholipid fatty acid components with the same fractions from normal uninfected white Swiss mouse blood revealed a more complex fluke triglyceride fatty acid constitution but a very similar fatty acid composition for fluke and mouse blood phospholipid except that the predominant 20 carbon unsaturated fatty acid in fluke phospholipid was 20:1 in contrast to 20:4 in mouse blood
Mean
Trace 0.4 0.3 Trace 21.6 0.4 Trace 37.4 0.8 0.4 23.4 Trace 13.3 Trace 2.0
Unid. =
S.D. Min. Area %
Max.
0.1 0.1
0.4 0.2
0.5 0.4
0.6 0.1
21.1 0.3
21.4 0.4
0.9 0.1 0.1 0.2
36.6 0.7 0.4 23.2
38.3 0.9 0.5 23.7
0.2
12.6
13.9
0.1
1.9
"2.0
unidentified.
Br
phospholipid (21,22). S. mansoni thus appears to be capable of synthesizing its own triglyceride. It has been reported that S. mansonoides is able to synthesize triglyceride, sterol ester and phospholipid from exogenously supplied fatty acids and sterols (20). Branched chain fatty acids of 16 and 18 carbons were found in fluke total lipid, phospholipid and triglyceride along with small amounts of odd numbered carbon fatty acids differing in chain length from 13 through 23 carbons (Tables I and II). Trace amounts of 13 carbon branched chain fatty acids were identified in neutral lipid from M. hirudenaceus while M. dubius neutral ~ lipid contained traces of a 14 carbon branched chain acid. Small quantities of odd numbered carbon fatty acids differing in chain length from 11 through 17 carbons in M. dubius and 13 through 17 carbons in M. hirudenaceus have been reported (19). Neither branched chain nor odd numbered carbon chain acids were detected in lipid from the larvae or adult stage of S. mansonoides (20). The presence of highly unsaturated eicosatetraenoic acids in S. mansoni phospholipid and triglyceride and eicosapentaenoic and docosahexaenoic acids in the fluke triglyceride fracLIPIDS, VOL,
4, N O .
1
36
THOMAS i .
SMITH, THOMAS J . BROOKS, JR. AND HAROLD B. WHITE, JR.
tion at least suggests that pathways for fatty acid interconversions could exist in adult S. mansoni. ACKNOWLEDGMENTS This investigation was supported in p a r t by U S P H S G r a n t AI 07546, National Institute of Allergy and Infectious Diseases. H. B. White is a research career development awardee (6-K3-HE-18,345), United States Public Health Service. Technical assistance was given by Mrs. Katye R. Summerlin and Mrs. Shirley S. Powell. REFERENCES 1. Standen, O. D., Trans. Roy. Soc. Trop. IVied. Hyg. 61, 568 (1967). 2. Stoll, N. R., J. Parasitol. 33, 1-18 (1947). 3. Faust, E. C., and P. F. Russell, "Clinical Parasitology," Lea and Febiger, Philadelphia, 1964, pp. 545553. 4. Bueding, E., J. Gen. Physiol. 33, 475-495 (1950). 5. Senft, A. W., Ann. New Y o r k Acad. Sci. 113, 272288 (1963). 6. Kent, H. N., Ann. New York Acad. Sci. 113, 102 (1963). 7. Folch, J., M. Lees and G. H. Sloane Stanley, J. Biol. Chem. 226, 497-509 (1957). 8. Smith, T. M., T. J. Brooks, Jr. and H. B. White, Jr., Am. J. Trop. Med. Hyg. 15, 307-313 (1966).
LlPlOS, VOL. 4, N o .
I
9. Brown, T. L., and J. Benjamin, Anal. Chem. 30, 446-.447 (1964). 10. Fieser, L F., "Experiments in Organic Chemistry," D. C. Heath and Co., Boston, 1941, pp. 357-370. 11. Malins, D. C., and H. K. Mangold, JAOCS 37, 576578 (1960). 12. Wagner, H., L H o r h a m m e r and P. Wolff, Biochem. Z. 334, 175-184 (1961). 13. Schmit, J. A., and R. B. Wynne, J. Gas. Chromatog. 21, 325-328 (1966). 14. Smith, T. M., and H. B. White, Jr., J. Lipid Res. 7, 327-329 (1966). 15, White, H. B., Jr., J. Chromatog. 21, 213-222 (1966). 16. Poukka, R., L. Vasenius and O. Turpeinen, J. Lipid Res. 3, 128-129 (1962). 17. Smyth, J. D., "The Physiology of Trematodes," W. H. Freeman and Co., San Francisco, 1966, p. 3. 18. Wolfe, D. A., P. V. Rao and D. G. Cornwell, JAOCS 42, 633-637 (1965). 19. Beames, C. G., Jr., and F. M. Fisher, Jr., Comp. Biochem. Physiol. 13, 4 0 1 4 1 2 (1964). 20. Meyer, F., S. Kimura and J. F. Mueller, J. Biol. Chem. 241, 4224-4232 (1966). 21. Smith, T. M., unpublished observations. 22. Rehnborg, C. S., A. V. Nichols and J. K. Ashikawa, Proc. Soc. Exptl. Biol. Med. 106, 547-549 (1961).
[Received May 17, 1968]
