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Unsaponifiable lipids of Taenia taeniaeformis and Moniezia sp.
EXPERIMENTAL
PARASITOLOGY
Unsaponifiable Malcolm
9,
127-130 (1960)
Lipids
of Tuenia
J. Thompson,’
Erich
taeniaeformis Mosettig’
and
and Theodor
Monieziu von
Brand2
U. S, Department of Health, Education and Welfare, Public Health Service, National Institutes National Institute of Arthritis and Metabolic Diseases1 and National Institute of Allergy and Infectious Diseases,= Bethesda, Maryland (Submitted
for publication,
sp.
of Health
25 June 1959)
The experiments presented above show that in Taenia taeniaejormis and in Moniezia sp. cholesterol is by far the most prevalent unsaponifiable substance. In the former tapeworm it amounted to at least 98% of total unsaponifiable material, in the latter to approximately 85%. The fact that a search for friedelin in Taenia taeniaejormis was negative strengthens the assumption by Cmelik and Bartl (1956) that the friedelin found in Taenia saginata did originate from cork stoppers. Our finding of 7-ketocholesterol in the Moniezia sample requires a comment. It is known that hot alkaline saponification of 7-ketocholesterol ester produces cholesta-3,5-diene-7one.3 (Milburn et al., 1956; Mauthner and Suida, 1896). Since the latter compound was not present in our sample, it may be concluded that the 7-ketocholesterol was not originally present, but was formed during isolation and storage of the unsaponifiable material’ from the Moniezia sp. No trace of 7-ketocholesterol could be isolated from the unsaponifiable material of the Taenia taeniaejormis.
The unsaponifiable lipid fraction of parasitic worms has been studied rarely in detail, leaving the question in doubt whether it is as diversified as in some invertebrate phyla, such as Mollusca or Porifera. The nematodes Ascaris and Parascaris have been studied most extensively. Their unsaponifiable fraction contains a large amount of t’he glycosides ascaroside A, B, and C (Fouquey et al., 1957, 1958 a, b) and a smaller amount of a mixture consisting of 25 % saturated and 75 % unsaturated sterols (Fairbairn, 1955). The latter has been identified unequivocally as cholesterol (Fairbairn and Jones, 1956). As far as cestodes are concerned, cholesi Laboratory of Chemistry. 2 Laboratory of Parasitic Diseases. 3 We refluxed a mixture of 7-ketocholesterol and 2% methanolic potassium hydroxide solution for milder condione hour, i.e., under considerably tions than employed by us in the saponification of the acetone soluble lipids and isolated predominantly cholesta-3,5-diene-7-one. 4 Fieser et al. (1957) have isolated 7-ketocholesterol from aged and air-oxidized cholesterol. 127
terol has been found in Diphyllobothrium Zatum by Faust and Tallqvist (1907) and in Cysticercus fasciolaris by Salisbury and Anderson (1939). Van Brand (1933) isolated from the unsaponifiable material of Moniexia expansa a sterol which he did not’ identify. More recently Cmelik (1952) found 75 % of the unsaponifiable mat’erial isolated from the cyst membranes of Echinococcus granulosus to be cholesterol. In all these instances the characterization of the isolated cholesterol was based primarily on melting point and mixture melting point. In some cases color react’ions were also performed. The isolation of friedelin along with cholesterol from Taenia saginata by Cmelik and Bartl (1956) appears of some interest. The authors themselves suspected cork stoppers used in their apparatus might be the origin of this pentacyclic triterpene, but left the question of the origin of the compound open. It appeared desirable to reinvestigate the nonsaponifiable fract’ions of tapeworms, to search carefully for products accompanying cholesterol and finally to characterize all
128
THOMPSON,
MOSETTIG
the purified compounds that were isolated. Two species were investigated, Taenia taeniaejormis from cats, a carnivorous host, and Moniezia sp. from sheep, an herbivorous host. Taenia taeniaejormis The tapeworms were collected from the intestines of narcotized cats. They were washed with Tyrode’s solution, wiped dry on filter paper, dried at lOO”C, and pulverized in a mortar. Twenty grams of the pulverized material was extracted for 12 hours with ether in a Soxhlet apparatus, yielding after evaporation of the ether 991 mg of ether extract. This material was dissolved in about 2 ml of ether and the phospholipids were separated by addition of 12 ml of acetone. The acetone-soluble lipids, obtained after centrifuging out the phospholipids and evaporating the decanted acetone solution, were saponified by refluxing for 3 hours with 20 ml of 5 % ethanolic NaOH. After cooling, 50 ml of distilled water were added. During storage in the cold room overnight a white precipitate developed which was collected by centrifugation. It was dissolved in ether and washed with water. Evaporation of the ether yielded white crystals. The aqueous fluid from which these crystals had been isolated was extracted with ether in a separatory funnel. The ether was washed with water and after evaporation of the ether a small amount, of white crystalline material was recovered which was combined with the first crystalline fraction. This material weighed 300 mg and represents the total unsaponifiable material which was studied further as follows. The unsaponifiable material dissolved in 15 ml of (9: 1) benzene-petroleum ether (6070°C) was placed on 8 g of benzene-petro6 All melting points were determined on Kofler block. Rotations were determined in approximately 1% solutions in chloroform and ultraviolet spectra in absolute ethanol with a Cary Model 11 recording spectrophotometer. Infrared spectra were obtained with a Perkin-Elmer Model 21 double beam spectrophotometer with sodium chloride prism and cells. Infrared spectra were determined by Mr. H. K. Miller and ultraviolet spectra by Mrs. A. Wright.
AND
VON BRAND
leum ether (9: 1) washed alumina, aluminum oxide “Woelm,” non-alkaline (almost neutral), and eluted with 25 ml portions of solvent. The fractions 6 to 12 eluted with benzene-chloroform (9: l), recrystallized from dilute ethanol yielded 280 mg of plates, m.p. 148-148.5”, [oL]~’ - 42”. This material is designated as compound A. The mother liquor afforded an additional 15 mg of A in the form of needles, m.p. 146-148’. Compound A was compared with an authentic sample of cholesterol by mixture melting point and infrared spectra; both were found to be identical in every respect. The acetate of A (acetic anhydride-pyridine, 18 hours, 25’) was obtained as colorless plates, m.p. 113-114”, [ol]E” - 45”. It was identical with cholesterol acetate (mixture melting point, infrared spectra). No other identifiable material was isolated from the unsaponifiable fraction of the Taenia taeniaeformis. Moniexia sp . We are indebted for the sheep tapeworms to Dr. N. F. Baker, University of California, Davis, California. He collected the worms from lambs and states that over 95 % of the Moniezia found in this region are M. expansa. Small numbers of Thysanosoma actinoides, representing certainly much less than 1% of the weight of the sample, may have also been present. The fresh worms weighing about 600 g were washed repeatedly in sterile saline and dried for approximately 48 hours at 25 inches of vacuum and 55°C. The dried worms were sent to Bethesda and treated as follows. The powdered material (45 g) was extracted with ether for 24 hours in a Soxhlet apparatus. Evaporation of the ethereal extract yielded 11 g lipids which were dissolved in 30 ml of ether and precipitated with 150 ml of acetone. The acetone-soluble lipids, after removal of the acetone, were saponified by refluxing for 3 hours with 50 ml of 5% ethanolic NaOH. The solution was cooled, diluted with 150 ml distilled water and placed in a cold room overnight. The slightly yellowish material which precipitated from solution was collected by centrifugation and washed with distilled water. The precipitate was taken up in ether and again washed with
UNSAPONIFIABLE
LIPIDS OF TAPEWORMS
water. Removal of the ether gave 0.85 g of unsaponifiable crystalline material which was analyzed as follows. Approximately 750 mg of this material (m.p. 135-144”), dissolved in 30 ml of benzene, was placed on 24 g of benzene-washed alumina, aluminum oxide ” Woelm, ” nonalkaline (almost neutral) and eluted with 100 ml portions of solvent. The first four elutions with benzene yielded no material. Fractions 5 to 10 eluted with benzene-chloroform (9: 1) yielded 650 mg of material melting unsharply from 139 to 144’. Fractions 11 and 12 were rechromatographed and will be discussed below. Crystallization of fractions 5 to 10 from dilute ethanol yielded 590 mg of plates, m.p. 145-146.5”, [oL];’ - 41”. This material is designated as compound B. It was directly compared with an authentic sample of cholesterol by mixture melting point and infrared spectra and found to be identical in every respect. The acetate of B (acet’ic anhydride-pyridine, 18 hours, 25”) was obtained as colorless plates, m.p. 114-115”, [LY]~~- 46”. It was identical with cholesterol acetate (mixture melting point, infrared spectra). The mother liquor from compound B, when concentrated to dryness in vacua yielded approximateIy 50 mg of semicrystalline material which exhibited weak absorption in the carbonyl region of the infrared. Fractions 11 and 12, m.p. 125-145” also exhibited weak absorption in the carbonyl region and gave an ultraviolet absorption maximum at 236 rnp. Fractions 11, 12 and the semi-crystalline residue were combined and rechromatographed over 4 g of benzenewashed alumina and collected in 10 ml portions. The first four fractions (4, 5, 6, and 7) eluted with benzene-chloroform (9: I>, yielded an additional 50 mg of cholesterol (m.p. 146-147” after recrystallization from dilute ethanol). Fractions 8 and 9 exhibited strong absorption bands in the infrared at 1680 and 1631 cm-l. These strong absorption bands suggested LX,/%unsaturated carbony]. Two recrystallizations of fractions 8 and 9 from dilute ethanol afforded 3 mg of needles. This compound, designated as compound C, had the following characteristics:
129
m.p. 166-168”, X max 238 rnp, 6 12,890; ycs2 3590 cm-’ (hydroxyl), 1680 cm-’ strong (7ketone), and 1631 cm-’ strong (double bond in conjugation with carbonyl). The mother liquors of fractions 8 and 9 were concentrated to dryness and rechromatographed. The fractions collected were monitored by ultraviolet analysis. Two recrystalhzations of the two fractions giving high E values afforded an additional 4 mg of C as needles, m.p. 167-168”, X max 238, E 13,100. The total yield of C was hence 7 mg. An authentic sample of 7-ketocholesterol melting at 165-166”, [ol]E” - 107”, X max 238, E 13,000 gave no melting point depression when mixed with C and was identical in respect to infrared spectra. The acetat’e of C (acetic anhydride-pyridine, 18 hours, 25”) was obtained as colorless plates from dilute methanol, m.p. l53155”, X max 235, e 11,830, ycs2 1735 cm+ strong (acetate), 1680 cm-l (kebone) and 1636 cm-’ (double bond in conjugation with ketone). An authentic sample of 7-ketocholesterol acetate melting at 1555157’, [oL]~’- loo”, X max 235, E 12,000 was directly compared with the acetate of C by mixture melting point and infrared spectra and found identical in every respect.
REFERENCES
T. 1933. Untersuchungen iiber den Stoffbestand einiger Cestoden und den Stoff-
VON BRAND,
wechsel von Moniezia expansa. Z. vergleich. Physiol. 18.562-596. CMELIR, S. 1952. Zur Kenntnis der Lipoide aus den Cystenmembranen von Taenia echinococcus. Z. physiol. Chem. 289, 78-79. CMELIK, S., AND BARTL, A. 1956. Zusammensetzung der Lipidevon Taeninsaginata. Z. physiol. Chem. 305,170-176. FAIRBAIRN, D. 1955. Lipids of the female reproductive organs in Ascaris lumbricoides. Can. J. Riochem. Physiol. 33, 31-37. FAIRBAIRN, D. AND JONES, R. N. 1956. Cholesterol from Ascaris lumbricoides (Nematoda). Can. J. Chem. 34, 182-184. FAUST, E. S., AND TALLQVIST, T. W. 1907. Vber die Ursachen der Bothriocephalus Anamie.Arch. exptl. Pathol. Pharmakol. 67, 370-385. FIESER, L. F., HUANG, W. Y., AND BHATTACHARYYA, B. K. 1957. Cholesterol and companions. X. The diol fraction. J. Org. Chem. 22, 13801384.
130
THOMPSON,
MOSETTIG
FOUQUEY, C., POLONSKY, J., AND LEDERER, E. 1957. Sur la structure-chimique de l’alcool ascarylique isole de Parascaris equorum. Bull. sot. chim. biol. 39, 101-132. FOUQUEY, C., LEDERER, E., L~~DERITZ, O., PoLONSKY, J., STAUB, A., STIRN, S., TINELLI, R., AND WESTPHAL, 0. 1958a. Syntheses de 3,6didesoxyhexoses: determination de la structure des sucres naturels: abequose, colitose, tyvelose et ascarylose. Compt. rend. 246,24172420. FOUQUEY, C., POLONSKY, J., AND LEDERER, E.
AND
VON
BRAND
195813. Structure chimique de l’ascarylose. Bull sot. chim. biol. 40.315-325. MAUTHNER, J., AND SUIDA, W. 1896. Beitriige zur Kenntnis des Cholesterins. Monatsh. Chem. 17, 570. MILBURN, A. H., TRUTER, E. V., AND WOODFORD, F. P. 1956. The Components of Wool Wax. Part IV. The identification of steroidal derivatives. J. Chem. Sot. 174&1743. SALISBURY, L. F., AND ANDERSON, R. J. 1939. Concerning the chemical composition of Cysticercus fasciolaris. J. Biol. Chem. 129, 505-517.
PARASITOLOGY
Unsaponifiable Malcolm
9,
127-130 (1960)
Lipids
of Tuenia
J. Thompson,’
Erich
taeniaeformis Mosettig’
and
and Theodor
Monieziu von
Brand2
U. S, Department of Health, Education and Welfare, Public Health Service, National Institutes National Institute of Arthritis and Metabolic Diseases1 and National Institute of Allergy and Infectious Diseases,= Bethesda, Maryland (Submitted
for publication,
sp.
of Health
25 June 1959)
The experiments presented above show that in Taenia taeniaejormis and in Moniezia sp. cholesterol is by far the most prevalent unsaponifiable substance. In the former tapeworm it amounted to at least 98% of total unsaponifiable material, in the latter to approximately 85%. The fact that a search for friedelin in Taenia taeniaejormis was negative strengthens the assumption by Cmelik and Bartl (1956) that the friedelin found in Taenia saginata did originate from cork stoppers. Our finding of 7-ketocholesterol in the Moniezia sample requires a comment. It is known that hot alkaline saponification of 7-ketocholesterol ester produces cholesta-3,5-diene-7one.3 (Milburn et al., 1956; Mauthner and Suida, 1896). Since the latter compound was not present in our sample, it may be concluded that the 7-ketocholesterol was not originally present, but was formed during isolation and storage of the unsaponifiable material’ from the Moniezia sp. No trace of 7-ketocholesterol could be isolated from the unsaponifiable material of the Taenia taeniaejormis.
The unsaponifiable lipid fraction of parasitic worms has been studied rarely in detail, leaving the question in doubt whether it is as diversified as in some invertebrate phyla, such as Mollusca or Porifera. The nematodes Ascaris and Parascaris have been studied most extensively. Their unsaponifiable fraction contains a large amount of t’he glycosides ascaroside A, B, and C (Fouquey et al., 1957, 1958 a, b) and a smaller amount of a mixture consisting of 25 % saturated and 75 % unsaturated sterols (Fairbairn, 1955). The latter has been identified unequivocally as cholesterol (Fairbairn and Jones, 1956). As far as cestodes are concerned, cholesi Laboratory of Chemistry. 2 Laboratory of Parasitic Diseases. 3 We refluxed a mixture of 7-ketocholesterol and 2% methanolic potassium hydroxide solution for milder condione hour, i.e., under considerably tions than employed by us in the saponification of the acetone soluble lipids and isolated predominantly cholesta-3,5-diene-7-one. 4 Fieser et al. (1957) have isolated 7-ketocholesterol from aged and air-oxidized cholesterol. 127
terol has been found in Diphyllobothrium Zatum by Faust and Tallqvist (1907) and in Cysticercus fasciolaris by Salisbury and Anderson (1939). Van Brand (1933) isolated from the unsaponifiable material of Moniexia expansa a sterol which he did not’ identify. More recently Cmelik (1952) found 75 % of the unsaponifiable mat’erial isolated from the cyst membranes of Echinococcus granulosus to be cholesterol. In all these instances the characterization of the isolated cholesterol was based primarily on melting point and mixture melting point. In some cases color react’ions were also performed. The isolation of friedelin along with cholesterol from Taenia saginata by Cmelik and Bartl (1956) appears of some interest. The authors themselves suspected cork stoppers used in their apparatus might be the origin of this pentacyclic triterpene, but left the question of the origin of the compound open. It appeared desirable to reinvestigate the nonsaponifiable fract’ions of tapeworms, to search carefully for products accompanying cholesterol and finally to characterize all
128
THOMPSON,
MOSETTIG
the purified compounds that were isolated. Two species were investigated, Taenia taeniaejormis from cats, a carnivorous host, and Moniezia sp. from sheep, an herbivorous host. Taenia taeniaejormis The tapeworms were collected from the intestines of narcotized cats. They were washed with Tyrode’s solution, wiped dry on filter paper, dried at lOO”C, and pulverized in a mortar. Twenty grams of the pulverized material was extracted for 12 hours with ether in a Soxhlet apparatus, yielding after evaporation of the ether 991 mg of ether extract. This material was dissolved in about 2 ml of ether and the phospholipids were separated by addition of 12 ml of acetone. The acetone-soluble lipids, obtained after centrifuging out the phospholipids and evaporating the decanted acetone solution, were saponified by refluxing for 3 hours with 20 ml of 5 % ethanolic NaOH. After cooling, 50 ml of distilled water were added. During storage in the cold room overnight a white precipitate developed which was collected by centrifugation. It was dissolved in ether and washed with water. Evaporation of the ether yielded white crystals. The aqueous fluid from which these crystals had been isolated was extracted with ether in a separatory funnel. The ether was washed with water and after evaporation of the ether a small amount, of white crystalline material was recovered which was combined with the first crystalline fraction. This material weighed 300 mg and represents the total unsaponifiable material which was studied further as follows. The unsaponifiable material dissolved in 15 ml of (9: 1) benzene-petroleum ether (6070°C) was placed on 8 g of benzene-petro6 All melting points were determined on Kofler block. Rotations were determined in approximately 1% solutions in chloroform and ultraviolet spectra in absolute ethanol with a Cary Model 11 recording spectrophotometer. Infrared spectra were obtained with a Perkin-Elmer Model 21 double beam spectrophotometer with sodium chloride prism and cells. Infrared spectra were determined by Mr. H. K. Miller and ultraviolet spectra by Mrs. A. Wright.
AND
VON BRAND
leum ether (9: 1) washed alumina, aluminum oxide “Woelm,” non-alkaline (almost neutral), and eluted with 25 ml portions of solvent. The fractions 6 to 12 eluted with benzene-chloroform (9: l), recrystallized from dilute ethanol yielded 280 mg of plates, m.p. 148-148.5”, [oL]~’ - 42”. This material is designated as compound A. The mother liquor afforded an additional 15 mg of A in the form of needles, m.p. 146-148’. Compound A was compared with an authentic sample of cholesterol by mixture melting point and infrared spectra; both were found to be identical in every respect. The acetate of A (acetic anhydride-pyridine, 18 hours, 25’) was obtained as colorless plates, m.p. 113-114”, [ol]E” - 45”. It was identical with cholesterol acetate (mixture melting point, infrared spectra). No other identifiable material was isolated from the unsaponifiable fraction of the Taenia taeniaeformis. Moniexia sp . We are indebted for the sheep tapeworms to Dr. N. F. Baker, University of California, Davis, California. He collected the worms from lambs and states that over 95 % of the Moniezia found in this region are M. expansa. Small numbers of Thysanosoma actinoides, representing certainly much less than 1% of the weight of the sample, may have also been present. The fresh worms weighing about 600 g were washed repeatedly in sterile saline and dried for approximately 48 hours at 25 inches of vacuum and 55°C. The dried worms were sent to Bethesda and treated as follows. The powdered material (45 g) was extracted with ether for 24 hours in a Soxhlet apparatus. Evaporation of the ethereal extract yielded 11 g lipids which were dissolved in 30 ml of ether and precipitated with 150 ml of acetone. The acetone-soluble lipids, after removal of the acetone, were saponified by refluxing for 3 hours with 50 ml of 5% ethanolic NaOH. The solution was cooled, diluted with 150 ml distilled water and placed in a cold room overnight. The slightly yellowish material which precipitated from solution was collected by centrifugation and washed with distilled water. The precipitate was taken up in ether and again washed with
UNSAPONIFIABLE
LIPIDS OF TAPEWORMS
water. Removal of the ether gave 0.85 g of unsaponifiable crystalline material which was analyzed as follows. Approximately 750 mg of this material (m.p. 135-144”), dissolved in 30 ml of benzene, was placed on 24 g of benzene-washed alumina, aluminum oxide ” Woelm, ” nonalkaline (almost neutral) and eluted with 100 ml portions of solvent. The first four elutions with benzene yielded no material. Fractions 5 to 10 eluted with benzene-chloroform (9: 1) yielded 650 mg of material melting unsharply from 139 to 144’. Fractions 11 and 12 were rechromatographed and will be discussed below. Crystallization of fractions 5 to 10 from dilute ethanol yielded 590 mg of plates, m.p. 145-146.5”, [oL];’ - 41”. This material is designated as compound B. It was directly compared with an authentic sample of cholesterol by mixture melting point and infrared spectra and found to be identical in every respect. The acetate of B (acet’ic anhydride-pyridine, 18 hours, 25”) was obtained as colorless plates, m.p. 114-115”, [LY]~~- 46”. It was identical with cholesterol acetate (mixture melting point, infrared spectra). The mother liquor from compound B, when concentrated to dryness in vacua yielded approximateIy 50 mg of semicrystalline material which exhibited weak absorption in the carbonyl region of the infrared. Fractions 11 and 12, m.p. 125-145” also exhibited weak absorption in the carbonyl region and gave an ultraviolet absorption maximum at 236 rnp. Fractions 11, 12 and the semi-crystalline residue were combined and rechromatographed over 4 g of benzenewashed alumina and collected in 10 ml portions. The first four fractions (4, 5, 6, and 7) eluted with benzene-chloroform (9: I>, yielded an additional 50 mg of cholesterol (m.p. 146-147” after recrystallization from dilute ethanol). Fractions 8 and 9 exhibited strong absorption bands in the infrared at 1680 and 1631 cm-l. These strong absorption bands suggested LX,/%unsaturated carbony]. Two recrystallizations of fractions 8 and 9 from dilute ethanol afforded 3 mg of needles. This compound, designated as compound C, had the following characteristics:
129
m.p. 166-168”, X max 238 rnp, 6 12,890; ycs2 3590 cm-’ (hydroxyl), 1680 cm-’ strong (7ketone), and 1631 cm-’ strong (double bond in conjugation with carbonyl). The mother liquors of fractions 8 and 9 were concentrated to dryness and rechromatographed. The fractions collected were monitored by ultraviolet analysis. Two recrystalhzations of the two fractions giving high E values afforded an additional 4 mg of C as needles, m.p. 167-168”, X max 238, E 13,100. The total yield of C was hence 7 mg. An authentic sample of 7-ketocholesterol melting at 165-166”, [ol]E” - 107”, X max 238, E 13,000 gave no melting point depression when mixed with C and was identical in respect to infrared spectra. The acetat’e of C (acetic anhydride-pyridine, 18 hours, 25”) was obtained as colorless plates from dilute methanol, m.p. l53155”, X max 235, e 11,830, ycs2 1735 cm+ strong (acetate), 1680 cm-l (kebone) and 1636 cm-’ (double bond in conjugation with ketone). An authentic sample of 7-ketocholesterol acetate melting at 1555157’, [oL]~’- loo”, X max 235, E 12,000 was directly compared with the acetate of C by mixture melting point and infrared spectra and found identical in every respect.
REFERENCES
T. 1933. Untersuchungen iiber den Stoffbestand einiger Cestoden und den Stoff-
VON BRAND,
wechsel von Moniezia expansa. Z. vergleich. Physiol. 18.562-596. CMELIR, S. 1952. Zur Kenntnis der Lipoide aus den Cystenmembranen von Taenia echinococcus. Z. physiol. Chem. 289, 78-79. CMELIK, S., AND BARTL, A. 1956. Zusammensetzung der Lipidevon Taeninsaginata. Z. physiol. Chem. 305,170-176. FAIRBAIRN, D. 1955. Lipids of the female reproductive organs in Ascaris lumbricoides. Can. J. Riochem. Physiol. 33, 31-37. FAIRBAIRN, D. AND JONES, R. N. 1956. Cholesterol from Ascaris lumbricoides (Nematoda). Can. J. Chem. 34, 182-184. FAUST, E. S., AND TALLQVIST, T. W. 1907. Vber die Ursachen der Bothriocephalus Anamie.Arch. exptl. Pathol. Pharmakol. 67, 370-385. FIESER, L. F., HUANG, W. Y., AND BHATTACHARYYA, B. K. 1957. Cholesterol and companions. X. The diol fraction. J. Org. Chem. 22, 13801384.
130
THOMPSON,
MOSETTIG
FOUQUEY, C., POLONSKY, J., AND LEDERER, E. 1957. Sur la structure-chimique de l’alcool ascarylique isole de Parascaris equorum. Bull. sot. chim. biol. 39, 101-132. FOUQUEY, C., LEDERER, E., L~~DERITZ, O., PoLONSKY, J., STAUB, A., STIRN, S., TINELLI, R., AND WESTPHAL, 0. 1958a. Syntheses de 3,6didesoxyhexoses: determination de la structure des sucres naturels: abequose, colitose, tyvelose et ascarylose. Compt. rend. 246,24172420. FOUQUEY, C., POLONSKY, J., AND LEDERER, E.
AND
VON
BRAND
195813. Structure chimique de l’ascarylose. Bull sot. chim. biol. 40.315-325. MAUTHNER, J., AND SUIDA, W. 1896. Beitriige zur Kenntnis des Cholesterins. Monatsh. Chem. 17, 570. MILBURN, A. H., TRUTER, E. V., AND WOODFORD, F. P. 1956. The Components of Wool Wax. Part IV. The identification of steroidal derivatives. J. Chem. Sot. 174&1743. SALISBURY, L. F., AND ANDERSON, R. J. 1939. Concerning the chemical composition of Cysticercus fasciolaris. J. Biol. Chem. 129, 505-517.