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The lipids and phospholipids of hydatid protoscolices of Echinococcus granulosus (cestoda)
0020-7519/80/0601-0213$02.00/0
International Journal for Parasitology, Vol. 10, pp. 213-216. Pergamon Press Ltd. 1980. Printed in Great Britain. 0 Australian Society for Parasitology.
THE LIPIDS AND PHOSPHOLIPIDS PROTOSCOLICES OF ECHIiVOCOCCUS (CESTODA)
OF HYDATID GRANULOSUS
GEORGE J. FRAYHA, GEORGE M. BAHR* and RIMA HADDAD Department
of Microbiology,
School of Medicine, American University of Beirut, Beirut, Lebanon (Received 22 October 1979)
Abstract-FRAYHA G. J., BAHRG. M. and HADDADR. 1980. The lipids and phospholipids of hydatid protoscolices of Echinococcusgranulosus (Cestoda). InternationalJournalfor Parasitology 10:213-216. The protoscolices of Echinococcus granulosus were isolated from hydatid cysts. The phospho- and neutral lipids of the protoscolices were determined. The seven major classes of lipids namely, phospholipids, fatty acids. mono-, di- and triglycerides, cholesterol and cholesterol esters were identified and quantitatively determined. The phospholipids isolated were cephalin, lecithin, lysolecithin, phosphatidyl inositol, phosphatidyl serine and sphingomyelin. Lecithin and cephalin were the most abundant. The significance of this finding is discussed. INDEX
KEY WORDS:
Lipids; phospholipids;
hydatid;
protoscolices;
Echinococcus
granulosus.
1952) and the protoscolices (Frayha, 1968, 1971). Acetic, propionic, valeric, succinic and higher fatty acids were identified in hydatid cyst fluid (Fliissner, 1924, 1925; Coutelen, 1931). Digenis, Thorson & Konyalian (1970) detected by gas-liquid chromatography seventeen fatty acids in the protoscolices of hydatid cysts with CIa:1, C,,:,, C&:,,, Czozabeing the most prevalent. The present study deals with the identification of seven classes of lipids and phospholipid components of the protoscolices of hydatid cysts. (Cmelik,
INTRODUCTION THE CHARACTERIZATIONof phospho- and neutral lipids of non-taeniid cestodes have been extensively studied in Moniezia expansa (Totterman & Kirk,
1939), Raillietina cesticiks (Reid, 1942; Botero & Reid, 1969), Hymenolepis diminuta and H. citelli (Warren M., unpublished Ph.D. thesis, The Rice Institute, Houston, Texas, 1957; Warren & Daugherty, 1957; Fairbairn, Wertein, Harpur & Schiller, 1961; Harrington, 1935; Ginger & Fairbairn, 1966a, b; Kilejian, Ginger & Fairbairn, 1968; Overturf & Dryer, 1968), Poecilancistrium caryophyIlum, Dasyrynchus giganteus, Thysanocephalum thysanocephalum, Grillotia simmonsi, Lacistorhynchus ten&, Orygmatobothrium musteli and Gariobothrium verticillatum (Buteau, Simmons & Fairbairn, 1969;
Buteau, Simmons, Beach, Holz & Sherman, 1971). The lipid composition of the taeniid tapeworms has been mainly confined to the larval stages. For example, Cysticercus fasciolaris displayed in its tissues phospholipids, cholesterol, cerebrosides, palmitic, stearic and arachidic fatty acids (Salisbury & Anderson, 1939). Kassis & Frayha (1973) demonstrated the presence of six phospholipids, 13 free as well as bound fatty acids, mono-, di-, and triglycerides, cholesterol and cholesterol esters in the cysticerci of Taenia hydatigena. Studies on the lipid composition of hydatid cysts of Echinococcus granulosus has been fragmentary. Cholesterol was isolated in the cyst membrane * Present Address: Middlesex Hospital, Medical School--School of Pathology, Riding House Street, London, W.l. 213
MATERTALS AND METHODS Parasite material. Viable protoscolices of Echinococcus grunulosus were collected according to the method of
Meymarian, Luttermoser, Frayha, Schwabe & Prescott (1963) from hydatid cysts in livers and lungs of cattle, obtained from the local abattoirs in Beirut a few hours after the slaughter of the hosts. The protoscolices were washed three times in physiological saline solution by suspension and sedimentation and their wet weight was recorded. Chemicals. The lipid standards monoolein, diolein, tripalmitin, cholesterol palmitate, lecithin (phosphatidyl choline), cephalin (phosphatidyl ethanolamine), sphingomyelin (phosphatidyl sphingoside), palmitic acid were purchased from Fluka AG, Buch SG, Switzerland. Cholesterol, lysolecithin (lysophosphatidyl choline), phosphatidyl inositol and phosphatidyl serine were obtained from Nutritional Biochemical Corporation, Cleveland, Ohio. Silica gel-G and Neatan for TLC were products of Fluka AG and Brinkmann Instruments Inc., Westbury, N.Y., respectively. All other chemicals used were reagent grades. Solvent mixtures were made on a v/v basis.
214
G. J. FRAYHA, G. M. BAHR and R. HADDAD
I.J.P. VOL. 10. 1980
TABLE 1. QUANTITATIVE DETERMINATION OF THE MAJOR LIPIDCLASSES IN THE PROTOSCOLICES OF E.granulosus
Range of four experiments Average
Phospholipids
Monoglycerides
55.8-59.40 56.80
1.68-2.08 1.87
Percentage (w/w) of total lipids DiglyTriglyFatty cerides cerides acids 2.33-5.30 3.67
2.26-3.60 3.16
3.86-5.87 5.32
Cholesterol
Cholesterol esters
20.30-30.90 26.00
2.90-3.73 3.54
TABLE 2. QUANTITATIVE DETERMINATIONOF PHOSPHOLIPIDSINTHE PROTOSCOLICESOF E.granulosus
Range of four experiments Average
Cephalin
Lecithin
18.50-19.89 19.29
25.71-29.94 28.03
Percentage (w/w) of total phospholipids Phosphatidyl Phosphatidyl Lysolecithin inositol serine 14.79-17.50 15.51
Isolation and identification of lipids. The total lipids of 5 g (wet weight) of the protoscolices were extracted according to the method of Folch, Lees & SloaneStanley (1957). Two to 5 mg of total lipids were dissolved in 0.5-l ml chloroform-methanol (2 : 1) and separated on TLC plates (Freeman & West, 1966) into the different classes of lipids, namely, phospholipids, monoglycerides, fatty acids, cholesterol, diglycerides, triglycerides and cholesterol esters. Each class was eluted from the TLC plate and quantitatively determined by the spectrophotometric method of Amenta (1964). Phospholipids were separated from total lipids on TLC and then fractionated into different components namely cephalin, lecithin, lysolecithin, phosphatidyl inositol, phosphatidyl serine and sphingomyelin by the chromatographic method of Parker & Paterson (1965). Each phospholipid component was then analyzed spectrophotometrically for quantitative estimation (Amenta, 1964). Placement and recovery studies with reference standards of phospholipids, monoglycerides, diglycerides, triglycerides, fatty acids, cholesterol and cholesterol esters were made, to assist in identifying and quantitatively analysing the lipids of hydatid protoscolices by chromatographic and spectrophotometric methods as well as to ascertain the efficiency of each step in the procedures.
RESULTS The average weight of the total lipids of the protoscolices of E. granulosus is 10.6 mg/g wet weight or 1.06% (w/w). The protoscolices showed the existence of all classes of lipids (Table 1); each class of lipid was quantitatively measured as mg/g wet weight of the protoscolices or percentage of total lipids and found to be: phospholipids 6.13 (56.80%), monoglycerides 0.19 (187 ‘A), diglycerides 0.39 (3.67 %), triglycerides 0.34 (3.16%) fatty acids 0.57 (5.32 %), cholesterol 2.81 (26.00%) and cholesterol esters 0.37 (3.54%).
10.67-14.50 11.65
13.36-15.35 13.67
Sphingomyelin 7.17-9.14 7.81
The fractionation of phospholipids of hydatid protoscolices revealed six components namely cephalin, lecithin, lysolecithin, phosphatidyl inositol, phosphatidyl serine and sphingomyelin (Table 2). The average mg weight of the phospholipid per g wet weight of protoscolices and the average percentages of each component per total phospholipids are: cephalin 1.13 (19.29x), lecithin 1.63 (28,03x), lysolecithin 0.90 (15.51 %), phosphatidyl inositol 0.67 (11.65 %), phosphatidyl serine 0.79 (13.67 %) and sphingomyelin 0.46 (781%).
DISCUSSION The value of the total lipids of E. granulosus protoscolices (10.6 mg/g wet weight) is equivalent to 13.52% of the dry weight (conversion method to dry weight by Frayha, 1971). This is almost identical to the 13.6% reported by Agosin, von Brand, Rivera & McMahon (1957) in the same parasite. The ratio of neutral to polar lipids in hydatid protoscolices is 0.75 : 1. This finding is different from those observed in Taenia hydatigenn cysticerci (Kassis & Frayha, 1973) and Hymenolepis diminuta (Ginger & Fairbairn, 1966a) in which the ratio of neutral to polar lipids were 2 : 1 and 3 : 1 respectively. The spectrophotometric determination of free cholesterol revealed an amount (2.81 mg/g wet weight) slightly different from that measured gravimetrically (3-4 mg/g wet weight) by Frayha (1971) in Echinococcus granulosus, E. multilocularis and T. hydatigena cysts. When the free cholesterol was calculated per 100 g of total lipids it amounted to 26% which is about 2: times that observed by Kassis & Frayha (1973) in the cysticerci of T. hydatigena (11.2%). On the other hand the value of
I.J.P. VOL. 10. 1980
Lipids of E. granulosus protoscolices
cholesterol esters in the protoscolices constituted 3.54 % of total lipids and was about half (7.1%) that observed in T. hydatigena cysticerci (Kassis & Frayha, 1973). This may be explained by the poor ability of E. grunulusus protoscolices to convert free cholesterol to its esterified form or by the ease with which the protoscolices hydrolyze cholesterol esters into free cholesterol as was suggested by Digenis et al. (1970). Contrary to previous findings in T. hydatigena cysticerci (Kassis & Frayha, 1973) in which the triglycerides were the major components of total lipids (2%20x), they were a minor fraction in E. granulosus protoscolices (3.38%) (Table 1). It can therefore be postulated that E. grunulosus protoscolices have a limited mechanism for triglycerides synthesis when compared to T. hydatigena cysticerci. The same argument probably applies to the mechanism of mono- and diglycerides synthesis since these 2 lipids were found to have lower values in E. granulosus protoscolices (0.8 % and 3.67 % of total lipids respectively) than in T. hydatigena cysticerci (4.4% and 6.3 ‘A respectively) (Kassis & Frayha, 1973). The free fatty acids in E. granulosus protoscolices are 5.32% of total lipids (Table 1). This value seems to be consistent in larval cestodes since it was reported by Kassis & Frayha (1973) to be 5.4% of total lipids of T. hydatigena cysticerci. The same phospholipids, namely cephalin, lecithin, lysolecithin phosphatidyl inositol, phosphatidyl serine and sphingomyelin, that are identified in E. granulosus protoscolices have also been reported to occur in H. diminuta (Fairbairn et al., 1961; Ginger & Fairbairn, 1966a) and T. hydatigena cysticerci (Kassis & Frayha, 1973). Among the phospholipids of E. grunulasus protoscolices, lecithin exhibited the highest percentage (28.03 %). This observation is not consistent with that reported by Kassis & Frayha (1973) concerning T. hydatigena cysticerci in which phosphatidyl inositol was the most concentrated among total phos?holipids (41.40%). However, the phosphatidyl inositol of E. granutosus protoscolices is quantitatively significant (11.65 ‘A of total phospholipids) thus indicating that this phospholipid is probably an important metabolic and structural component in certain larval cestodes. Cephalin which is 19.29% of total phospholipids makes a ratio of lecithin to cephalin in E. granufosus protoscolices of 1.45 : 1 which is similar to that observed in H. diminutn (1.6 : 1) by Ginger & Fairbairn (1966a). This ratio is however 3 times higher than that reported in T. hydatigena cysticerci by Kassis & Frayha (1973). No explanation can be given for this discrepancy. Acknowledgements-This study was supported in part by Grant No. 18-5216 from the Research Committee of the Faculties of Medical Sciences, American University of Beirut, and by Grant No. 38-5704 from the National Council for Scientific Research, Beirut, Lebanon.
215 REFERENCES
AGoslN M., VONBRANDT., RIVERAF. & MCMAHON P. 1957. Studies on the metabolism of Echinococcus wanulosus. 1. General chemical composition and respiratory reactions. Experimental Parasitology 6: 37-51. AMENTAJ. S. 1964. A rapid method for quantification of lipids separated by TLC. Journal ofLipid Research 5: 270-272. BOTERO H. & REID W. M. 1969. Raillietina cesticillus: fatty acid composition. Experimental Parasitology 25: 93-100. BUTEAUG. H., JR., SIMMONSJ. E. & FAIRBAIRND. 1969. Lipid metabolism in helminth parasites. IX. Fatty acid composition of shark tapeworms and of their hosts. Experimental Parasitology 26: 209-213. BUTEAUG. H , JR., SIMMONSJ. E., BEACHD. H., HOLZ G. G., JR., & SHERMANJ. W. 1971. The lipids of cestodes from Pacific and Atlantic coast triakid sharks. Journal qf Parasitology 51: 1272-l 278. CMELIK S. 1952. Zur kenntnis der lipoide aus den cystenmembranen von Taenia echinococcus. Zeitschrift ftir Physiologische Chemie 289: 78-79. COUTELENF. R. 1931. Histogenbse des cellules libres, I glycogene et B graisses, des Hydatides &chinococciques. Annales de Parasitologie Humaine et Comparte 9: 101-103. DIGENISG. A., THORSONR. E. & KONYALIANA. 1970. Cholesterol biosynthesis and lipid biochemistry in the scolex of Echinococcus granuloszs. Journal of Pharmaceutical Sciences 59: 676-679. FAIRBAIRND., WERTE~NG., HARPUR R. P. & SCHILLER E. L. 1961. Biochemistry of normal and irradiated strains of Hymeaolepis diminuta. Experimental Parasitology 11: 248-263. FL~~SSNER 0. N. 1924. Neue untersuchungen ueber die Echinokokkus-fluessigkeit. Zeitschrift fir Biologic 80: 255-260. FL~~SSNER 0. N. 1925. Neue untersuchungen ueber die Echinokokkus-fluessigkeit. Zeitschrift ftir Biologie 82: 297-30 1. FOLCH J., LEES M. & SLOANE-STANLEY G. H. 1957. A single method for the isolation and purification of total lipids from animal tissue. JournalofBiologkalChemistry 226: 497-509. FRAYHAG. J. 1968. A study on the synthesis and absorption of cholesterol in hydatid cysts (Echinococcus granulosus). 27: 875-878.
Comparative
Biochemistry
and Physiology
FRAYHAG. J. 1971. Comparative metabolism of acetate in the taeniid tapeworm Echinococcus granulosus, E. multilocularis, and Taenia hydatigena. Comparative Biochemistry and Physiology 39: 167-170. FREEMANC. P. & WESTD. 1966. Complete separation of lipid classes on a single thin-layer plate. Journal ofLipid Research 7: 324-327. GINGER C. D. & FAIRBAIRND. 1966a. Lipid metabolism of helminth parasites I. The lipids of Hymenolepis diminuta (Cestoda). Journal of Parasitology 52: 10861096. GINGERC. D. & FAIRBAIRND. 19666. Lipid metabolism in helminth parasites II. The major origins of the lipids of Hymenolepis diminuta (Cestoda). Journal of Parasitology 52: 1097-l 107.
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G. J. FRAYHA, G. M. BAHR and R. HADDAD
HARRINCTONG. W. 1965. The lipid content of Hymenolepis diminuta and Hymenolepis citelli. Experimental Parasitology 17: 287-295. KASSISA. I. & FRAYHA G. J. 1973. Lipids of the cysticerci of Taenia hydatigena (Cestoda). Comparative Biochemistry and Physiology 46B: 435443. KILEJIANA., GINGER C. D. & FAIRBAIRND. 1968. Lipid metabolism in helminth parasites. IV. Origins of the intestinal lipids available for absorption by Hymenolepis diminuta. Journal of Parasitology 54: 63-68 MEYMERIANE., LUTTERMOSERG. W., FRAYHA G. J., SCHWABE C. W. & PRESCOTTB. 1963. Hostparasite relationships in echinococcosis. X. Laboratory cvaluation of chemical scolicides as adjuncts to hydatid surgery. Annals of Surgery 158: 211-215. OVERTURFM. & DRYER R. L. 1968. Lipid metabolism in the adult cestode Hymenolepis diminuta. Comparative Biochemistry and Physiology 27: 145-175.
I.J.P. VOL. 10. 1980
PARKERF. &PATERSONN. F. 1965. Quantitative analysis of phospholipids and phospholipid fatty acids from silica gel thin-layer chromatograms. Journal of Lipid Research 6: 455-460. REID W. M. 1942. Certain nutritional requirements of the fowl cestode Raillietina cesticillus (Molin) as demonstrated by short periods of starvation of the host. Journal of Parasitology 28: 319-240. SALISBURYL. F. & ANDERSONR. J. 1939. Concerning the chemical co.mposition of Cysticerclts ,fasciol:rris. Journal of Biological Chemistry 129: 505-517. TOTTERMAN G. & KIRK E. 1939. Om innehallet av lipoideri Bothriocephalus latus. Nordisk Medicirl 3: 2715-2716. WARREN M. & DAUGHERTYJ. W. 1957. Host effects on the lipid fraction of Hymenolepis diminuta. Journal of’ Parasitology 43: 521-526.
International Journal for Parasitology, Vol. 10, pp. 213-216. Pergamon Press Ltd. 1980. Printed in Great Britain. 0 Australian Society for Parasitology.
THE LIPIDS AND PHOSPHOLIPIDS PROTOSCOLICES OF ECHIiVOCOCCUS (CESTODA)
OF HYDATID GRANULOSUS
GEORGE J. FRAYHA, GEORGE M. BAHR* and RIMA HADDAD Department
of Microbiology,
School of Medicine, American University of Beirut, Beirut, Lebanon (Received 22 October 1979)
Abstract-FRAYHA G. J., BAHRG. M. and HADDADR. 1980. The lipids and phospholipids of hydatid protoscolices of Echinococcusgranulosus (Cestoda). InternationalJournalfor Parasitology 10:213-216. The protoscolices of Echinococcus granulosus were isolated from hydatid cysts. The phospho- and neutral lipids of the protoscolices were determined. The seven major classes of lipids namely, phospholipids, fatty acids. mono-, di- and triglycerides, cholesterol and cholesterol esters were identified and quantitatively determined. The phospholipids isolated were cephalin, lecithin, lysolecithin, phosphatidyl inositol, phosphatidyl serine and sphingomyelin. Lecithin and cephalin were the most abundant. The significance of this finding is discussed. INDEX
KEY WORDS:
Lipids; phospholipids;
hydatid;
protoscolices;
Echinococcus
granulosus.
1952) and the protoscolices (Frayha, 1968, 1971). Acetic, propionic, valeric, succinic and higher fatty acids were identified in hydatid cyst fluid (Fliissner, 1924, 1925; Coutelen, 1931). Digenis, Thorson & Konyalian (1970) detected by gas-liquid chromatography seventeen fatty acids in the protoscolices of hydatid cysts with CIa:1, C,,:,, C&:,,, Czozabeing the most prevalent. The present study deals with the identification of seven classes of lipids and phospholipid components of the protoscolices of hydatid cysts. (Cmelik,
INTRODUCTION THE CHARACTERIZATIONof phospho- and neutral lipids of non-taeniid cestodes have been extensively studied in Moniezia expansa (Totterman & Kirk,
1939), Raillietina cesticiks (Reid, 1942; Botero & Reid, 1969), Hymenolepis diminuta and H. citelli (Warren M., unpublished Ph.D. thesis, The Rice Institute, Houston, Texas, 1957; Warren & Daugherty, 1957; Fairbairn, Wertein, Harpur & Schiller, 1961; Harrington, 1935; Ginger & Fairbairn, 1966a, b; Kilejian, Ginger & Fairbairn, 1968; Overturf & Dryer, 1968), Poecilancistrium caryophyIlum, Dasyrynchus giganteus, Thysanocephalum thysanocephalum, Grillotia simmonsi, Lacistorhynchus ten&, Orygmatobothrium musteli and Gariobothrium verticillatum (Buteau, Simmons & Fairbairn, 1969;
Buteau, Simmons, Beach, Holz & Sherman, 1971). The lipid composition of the taeniid tapeworms has been mainly confined to the larval stages. For example, Cysticercus fasciolaris displayed in its tissues phospholipids, cholesterol, cerebrosides, palmitic, stearic and arachidic fatty acids (Salisbury & Anderson, 1939). Kassis & Frayha (1973) demonstrated the presence of six phospholipids, 13 free as well as bound fatty acids, mono-, di-, and triglycerides, cholesterol and cholesterol esters in the cysticerci of Taenia hydatigena. Studies on the lipid composition of hydatid cysts of Echinococcus granulosus has been fragmentary. Cholesterol was isolated in the cyst membrane * Present Address: Middlesex Hospital, Medical School--School of Pathology, Riding House Street, London, W.l. 213
MATERTALS AND METHODS Parasite material. Viable protoscolices of Echinococcus grunulosus were collected according to the method of
Meymarian, Luttermoser, Frayha, Schwabe & Prescott (1963) from hydatid cysts in livers and lungs of cattle, obtained from the local abattoirs in Beirut a few hours after the slaughter of the hosts. The protoscolices were washed three times in physiological saline solution by suspension and sedimentation and their wet weight was recorded. Chemicals. The lipid standards monoolein, diolein, tripalmitin, cholesterol palmitate, lecithin (phosphatidyl choline), cephalin (phosphatidyl ethanolamine), sphingomyelin (phosphatidyl sphingoside), palmitic acid were purchased from Fluka AG, Buch SG, Switzerland. Cholesterol, lysolecithin (lysophosphatidyl choline), phosphatidyl inositol and phosphatidyl serine were obtained from Nutritional Biochemical Corporation, Cleveland, Ohio. Silica gel-G and Neatan for TLC were products of Fluka AG and Brinkmann Instruments Inc., Westbury, N.Y., respectively. All other chemicals used were reagent grades. Solvent mixtures were made on a v/v basis.
214
G. J. FRAYHA, G. M. BAHR and R. HADDAD
I.J.P. VOL. 10. 1980
TABLE 1. QUANTITATIVE DETERMINATION OF THE MAJOR LIPIDCLASSES IN THE PROTOSCOLICES OF E.granulosus
Range of four experiments Average
Phospholipids
Monoglycerides
55.8-59.40 56.80
1.68-2.08 1.87
Percentage (w/w) of total lipids DiglyTriglyFatty cerides cerides acids 2.33-5.30 3.67
2.26-3.60 3.16
3.86-5.87 5.32
Cholesterol
Cholesterol esters
20.30-30.90 26.00
2.90-3.73 3.54
TABLE 2. QUANTITATIVE DETERMINATIONOF PHOSPHOLIPIDSINTHE PROTOSCOLICESOF E.granulosus
Range of four experiments Average
Cephalin
Lecithin
18.50-19.89 19.29
25.71-29.94 28.03
Percentage (w/w) of total phospholipids Phosphatidyl Phosphatidyl Lysolecithin inositol serine 14.79-17.50 15.51
Isolation and identification of lipids. The total lipids of 5 g (wet weight) of the protoscolices were extracted according to the method of Folch, Lees & SloaneStanley (1957). Two to 5 mg of total lipids were dissolved in 0.5-l ml chloroform-methanol (2 : 1) and separated on TLC plates (Freeman & West, 1966) into the different classes of lipids, namely, phospholipids, monoglycerides, fatty acids, cholesterol, diglycerides, triglycerides and cholesterol esters. Each class was eluted from the TLC plate and quantitatively determined by the spectrophotometric method of Amenta (1964). Phospholipids were separated from total lipids on TLC and then fractionated into different components namely cephalin, lecithin, lysolecithin, phosphatidyl inositol, phosphatidyl serine and sphingomyelin by the chromatographic method of Parker & Paterson (1965). Each phospholipid component was then analyzed spectrophotometrically for quantitative estimation (Amenta, 1964). Placement and recovery studies with reference standards of phospholipids, monoglycerides, diglycerides, triglycerides, fatty acids, cholesterol and cholesterol esters were made, to assist in identifying and quantitatively analysing the lipids of hydatid protoscolices by chromatographic and spectrophotometric methods as well as to ascertain the efficiency of each step in the procedures.
RESULTS The average weight of the total lipids of the protoscolices of E. granulosus is 10.6 mg/g wet weight or 1.06% (w/w). The protoscolices showed the existence of all classes of lipids (Table 1); each class of lipid was quantitatively measured as mg/g wet weight of the protoscolices or percentage of total lipids and found to be: phospholipids 6.13 (56.80%), monoglycerides 0.19 (187 ‘A), diglycerides 0.39 (3.67 %), triglycerides 0.34 (3.16%) fatty acids 0.57 (5.32 %), cholesterol 2.81 (26.00%) and cholesterol esters 0.37 (3.54%).
10.67-14.50 11.65
13.36-15.35 13.67
Sphingomyelin 7.17-9.14 7.81
The fractionation of phospholipids of hydatid protoscolices revealed six components namely cephalin, lecithin, lysolecithin, phosphatidyl inositol, phosphatidyl serine and sphingomyelin (Table 2). The average mg weight of the phospholipid per g wet weight of protoscolices and the average percentages of each component per total phospholipids are: cephalin 1.13 (19.29x), lecithin 1.63 (28,03x), lysolecithin 0.90 (15.51 %), phosphatidyl inositol 0.67 (11.65 %), phosphatidyl serine 0.79 (13.67 %) and sphingomyelin 0.46 (781%).
DISCUSSION The value of the total lipids of E. granulosus protoscolices (10.6 mg/g wet weight) is equivalent to 13.52% of the dry weight (conversion method to dry weight by Frayha, 1971). This is almost identical to the 13.6% reported by Agosin, von Brand, Rivera & McMahon (1957) in the same parasite. The ratio of neutral to polar lipids in hydatid protoscolices is 0.75 : 1. This finding is different from those observed in Taenia hydatigenn cysticerci (Kassis & Frayha, 1973) and Hymenolepis diminuta (Ginger & Fairbairn, 1966a) in which the ratio of neutral to polar lipids were 2 : 1 and 3 : 1 respectively. The spectrophotometric determination of free cholesterol revealed an amount (2.81 mg/g wet weight) slightly different from that measured gravimetrically (3-4 mg/g wet weight) by Frayha (1971) in Echinococcus granulosus, E. multilocularis and T. hydatigena cysts. When the free cholesterol was calculated per 100 g of total lipids it amounted to 26% which is about 2: times that observed by Kassis & Frayha (1973) in the cysticerci of T. hydatigena (11.2%). On the other hand the value of
I.J.P. VOL. 10. 1980
Lipids of E. granulosus protoscolices
cholesterol esters in the protoscolices constituted 3.54 % of total lipids and was about half (7.1%) that observed in T. hydatigena cysticerci (Kassis & Frayha, 1973). This may be explained by the poor ability of E. grunulusus protoscolices to convert free cholesterol to its esterified form or by the ease with which the protoscolices hydrolyze cholesterol esters into free cholesterol as was suggested by Digenis et al. (1970). Contrary to previous findings in T. hydatigena cysticerci (Kassis & Frayha, 1973) in which the triglycerides were the major components of total lipids (2%20x), they were a minor fraction in E. granulosus protoscolices (3.38%) (Table 1). It can therefore be postulated that E. grunulosus protoscolices have a limited mechanism for triglycerides synthesis when compared to T. hydatigena cysticerci. The same argument probably applies to the mechanism of mono- and diglycerides synthesis since these 2 lipids were found to have lower values in E. granulosus protoscolices (0.8 % and 3.67 % of total lipids respectively) than in T. hydatigena cysticerci (4.4% and 6.3 ‘A respectively) (Kassis & Frayha, 1973). The free fatty acids in E. granulosus protoscolices are 5.32% of total lipids (Table 1). This value seems to be consistent in larval cestodes since it was reported by Kassis & Frayha (1973) to be 5.4% of total lipids of T. hydatigena cysticerci. The same phospholipids, namely cephalin, lecithin, lysolecithin phosphatidyl inositol, phosphatidyl serine and sphingomyelin, that are identified in E. granulosus protoscolices have also been reported to occur in H. diminuta (Fairbairn et al., 1961; Ginger & Fairbairn, 1966a) and T. hydatigena cysticerci (Kassis & Frayha, 1973). Among the phospholipids of E. grunulasus protoscolices, lecithin exhibited the highest percentage (28.03 %). This observation is not consistent with that reported by Kassis & Frayha (1973) concerning T. hydatigena cysticerci in which phosphatidyl inositol was the most concentrated among total phos?holipids (41.40%). However, the phosphatidyl inositol of E. granutosus protoscolices is quantitatively significant (11.65 ‘A of total phospholipids) thus indicating that this phospholipid is probably an important metabolic and structural component in certain larval cestodes. Cephalin which is 19.29% of total phospholipids makes a ratio of lecithin to cephalin in E. granufosus protoscolices of 1.45 : 1 which is similar to that observed in H. diminutn (1.6 : 1) by Ginger & Fairbairn (1966a). This ratio is however 3 times higher than that reported in T. hydatigena cysticerci by Kassis & Frayha (1973). No explanation can be given for this discrepancy. Acknowledgements-This study was supported in part by Grant No. 18-5216 from the Research Committee of the Faculties of Medical Sciences, American University of Beirut, and by Grant No. 38-5704 from the National Council for Scientific Research, Beirut, Lebanon.
215 REFERENCES
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