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Cholesterol in Physalia physalis
Comp. Biochem. Physiol., 1968, Vol. 24, pp. 507 to 510. Pergamon Press. Printed in Great Britain
C H O L E S T E R O L IN P H Y S A L I A P H Y S A L I S * ROBERT E. M I D D L E B R O O K t and CHARLES E. LANE Institute of Marine Sciences, University of Miami, Miami, Florida 33149
(Received 21 ffuly 1967) Abstract--l. Acetone extracts of whole floats of the siphonophore Physalia physalis included an abundant steroid. This was identified as cholesterol by thin-layer chromatography, by infrared absorption, by nuclear magnetic resonance and by mass spectroscopy. 2. Chemical confirmation was achieved by conversion to cholesteryl acetate, by hydrogenation to 3-fl-cholestanol and by the formation of the digitonide. 3. Most cholesterol occurred in the pneumatosaccus where it comprised approximately 9 per cent of the total dry weight. INTRODUCTION EhRLI~R studies from this laboratory described the preparation and physiological effects of the nematocyst toxin of Physalia physalis in a variety of animals (Lane, 1960; Lane & Larsen, 1965; Larsen & Lane, 1966; Lane, 1967). Clark & Lane (1961) confirmed Wittenberg's identification (1960) of carbon monoxide in the float gas. Lane et al. (1965) emphasized the high levels of protein and free amino acids in gastrovascular fluid of P. physalis compared with mesogleal fluid of Aurelia aurita. The present paper describes the isolation, identification and distribution of cholesterol in P. physalis. MATERIALS AND METHODS Surviving specimens of P. physalis were collected from the ocean beach, Key Biscayne, Florida, and quickly brought to the laboratory where they were immediately dissected and extracted. Physalia floats were exhaustively extracted with several changes of acetone at room temperature. After decantation of the acetone solution, the samples were transferred to a continuous percolator-extractor (Ciereszko, 1966) and refluxed with hot acetone for 24-48 hr. All acetone extracts were combined and concentrated on a rotary evaporator. The aqueous solution remaining was transferred to a separatory funnel and extracted with benzene. The combined benzene extracts were dried first by azeotropic distillation followed by several hours contact with anhydrous sodium sulfate. Removal of the solvent left * Contribution No. 857 from the Institute of Marine Sciences, University of Miami. Supported by USPHS Grant No. HE-5489. Present address: Monsanto Company, Agricultural Research Division, St. Louis, Missouri 63166.
508
ROBERT E. MIDDLEBROOK AND CHARLES E. LANE
a thick, greenish brown oil. This was dissolved in a small volume of benzene and chromatographed on a Florisil column (3 x 62 cm), using benzene as the eluant. A series of 1-1. fractions from the column was concentrated on a rotary evaporator. Products were analyzed by thin-layer chromatography on silica gel G (MerckDarmstadt) plates (5 x 20 cm). The plates were developed in 20% (v/v) ethyl acetate in benzene, except where noted, and the chromatogram rendered visible by exposure to iodine vapor. Infrared spectra were run on a Beckman IR-10 in chloroform solution. Nuclear magnetic resonance spectra were obtained on a Varian A-60 instrument. All samples for N M R were run in deuteriochloroform solution, using tetramethylsilane as internal standard. Mass spectra were determined on a Consolidated Electrodynamics Corporation Model 110B high-resolution mass spectrometer. C, H and O analyses were performed by Alfred Bernhardt Laboratories, Mulheim, Germany. RESULTS After concentration, the first fraction from the Florisil column was a clear yellow oil that was dissolved in hexane and rechromatographed on alumina. Addition of five equal increments of benzene (up to 10 per cent final concentration) separated this fraction into a hydrocarbon moiety and a mixture of fatty acid esters, all as yet unidentified. The infrared spectrum of the hydrocarbon fraction showed absorption at 2980, 2945 and 2990 cm -1 carbon-hydrogen stretching), 1468 cm -1 (methylene) and 1385 cm -1 (carbon-methyl). The ester mixture showed carbonyl absorption at 1740 cm -1. A small portion of this material could be crystallized, but melted over the range 65-70°C. The most prominent peak in its NMR spectrum occurred as a singlet at 1.25 ppm, the methylene region of the spectrum, indicative of a long chain, probably in both the acid and alcohol portions of the ester. Although this material displayed a single spot on thin-layer chromatography employing hexane (R! = 0.21), there was severe overlap of spots when the ester mixture was separated by thin-layer chromatography in other solvent systems. Successive fractions from the Florisil column (to 3 1. of benzene) provided a crystalline solid that was recrystallized several times from 95% ethanol, affording white plates melting at 147-148°C. The m.p. was unchanged on admixture of authentic cholesterol, m.p. 148°C. (Analysis: Calculated for C~7H460: C, 83.87; H, 11-99. Found: C, 84.08; H, 11.98.) The infrared spectrum was identical with that of a shelf sample of cholesterol. The N M R spectrum was a typical steroidal pattern and was very nearly superimposable on the spectrum of authentic cholesterol. The single discrepancy occurred at 1.26 ppm; the peak at this position was somewhat more prominent than in the standard itself. Enhancement of the methylene region of the spectrum may have been caused by slight hydrocarbon contamination. The mass spectrum showed a parent peak at role 386 and was identical to the cracking pattern of authentic cholesterol run immediately afterwards.
509
CHOLESTEROL IN P H Y S A L I A P H Y S A L I S
The compound was chemically confirmed as cholesterol by conversion to cholesteryl acetate on treatment with acetic anhydride in pyridine. Cholesteryl acetate showed a melting point of 114-116°C (lit. : 116°C). (Analysis: Calculated for C29H4sO2: C, 81.31; H, 11.22. Found: C, 81.47; H, 11.48.) The thin-layer chromatogram ( 1 : 1 benzene-hexane) of authentic cholesteryl acetate and our conversion product produced single spots with identical R 1 values of 0.58. The infrared spectra of these compounds were also identical. Hydrogenation of the compound dissolved in methanol with platinum oxide catalyst produced 3-fl-cholestanol, m.p. 142°C (lit. m.p. 142°C.) (Analysis: Calculated for C~TH4sO: C, 83-43; H, 12.45; O, 4-12. Found: C, 83-69; H, 12.13; O, 4.33.) Mass spectral analysis showed a parent peak at m/e 388 and a cracking pattern entirely consistent with that of cholestanol. Digitonide formation was observed when the Physalia sterol was treated with an equivalent of digitonin in 95% ethanol. The free sterol was recovered by Soxhlet extraction of the digitonide with toluene for 48 hr. Five newly collected Physalia, representing the normal range of sizes seen at the height of the season, were dissected into their principal components, tentacles, pneumatosaccus and pneumatocodon, to determine whether the distribution of cholesterol was uniform through the animal. By independent acetone extraction of these three components from five separate animals and comparison of the thinlayer patterns of the extracts, virtually all cholesterol was found to occur in the pneumatosaccus where it comprised approximately 9 per cent of the total dry weight (Table 1). The crystalline product could be isolated only from the acetone extracts of the pneumatosaccus. TABLE 1--STEROL CONTENT OF PNEUMATOSACCUS
Animal
D r y wt. of pneumatosaccus (rag)
Wt. of crude sterol (rag)
~o sterol
1 2 3 4 5
116"8 57"1 13"9 75"5 114.2
13"6 7-4 1-9 9"8 9"4
11"6 7"7 7"3 6"6 12"2
Pneumatosacci from several Physalia were divided so that one part contained the gas gland and the other embraced all the rest of the structure. These components were separately extracted with acetone. Thin-layer chromatography of the extracts revealed that cholesterol occurred in the gas gland as well as in the remainder of the pneumatosaccus. A few milligrams of crude crystalline cholesterol were isolated from the acetone extract of several gas glands. Examination of gastrovascular fluid by thin-layer chromatography revealed no cholesterol.
510
ROBERTE. MIDDLEBROOKAND CHARLESE. LANE
DISCUSSION T h e occurrence of a variety of sterols in animals of lower taxa was first described by Bergmann and his students (Bergmann & Domsky, 1960). Phylogenetic considerations alone would suggest that Cnidaria might contain an array of sterols. Aurelia aurita, however, when extracted by the procedure described above, contained minimal quantities of sterols. Millepora alcicornis and Pseudopterogorgia acerosa similarly appear to synthesize only small quantities of sterols (Middlebrook, unpublished). T h e occurrence of considerable quantities of cholesterol in P. physalis and its restricted distribution within the animal are noteworthy. T h e pneumatosaccus in intact Physalia is normally inflated to 5-6 m m water above atmospheric pressure by a carbon monoxide-rich gas mixture (Wittenberg, 1960; Clark & Lane, 1961; Latimer & Ashby, 1965). T h e epithelial surface not exposed to gas is bathed by gastrovascular fluid in which no cholesterol was detected by thin-layer chromatography. Conceivably gastrovascular fluid could transport hydrocarbon precursors of cholesterol derived from dietary sources to the pneumatosaccus. T h e biological significance of cholesterol in the quantities observed remains obscure. Acknowledgements--The authors gratefully acknowledge their indebtedness to Dr. Tom Karns and the Department of Chemistry, University of Oklahoma, for assistance with NMR spectra and to Dr. R. D. Grigsby, Continental Oil Company, Ponca City, Oklahoma, for supplying the mass spectral data. REFERENCES
BERGMANNW. & DOMSKYI. I. (1960) Sterols of some invertebrates. Ann. N. Y. Acad. Sci. 90, 906-910. CIERKSZKOL. S. (1966) A versatile continuous percolator-extractor. J. chem. Ed. 43, 252253. CLARK F. E. & LANE C. E. (1961) Composition of float gases in Physalia physalis. Proc. Soc. exp. Biol. Med. 107, 673-675. L~'B C. E. (1960) The toxin of Physalia nematocysts. Ann. N. Y. Acad. Sci. 90, 742-751. LANE C. E. (1967) Recent observations on the pharmacology of Physalia toxin. In Animal Toxins (Edited by RUSSELLF. E. & SAtr~E~S P. R.) Pergamon Press, New York. LANE C. E. & LARSENJ. B. (1965) Some effects of the toxin of Physalia physalis on the heart of the land crab, Cardisoma guanhumi (Latreille). Toxicon 3, 69-71. LANE C. E., PRINOLE E. & BERGm~ A. M. (1965) Amino acids in extracellular fluids of Physalia physalis and Aurelia aurita. Comp. Biochem. Physiol. 15, 259-262. LARIMERJ. L. & ASHBYE. A. (1962) Float gases, gas secretion and tissue respiration in the Portuguese man-of-war, Physalia. jT. cell. comp. Physiol. 60, 41-47. LARSEN J. B. & LANE C. E. (1966) Some effects of Physalia physalis toxin on the cardiovascular system of the rat. Toxicon 4, 199-203. WlTaXNB~O J. B. (1960) The source of carbon monoxide in the float of the Portuguese man-of-war, Physalia physalis L. 3t. exp. Biol. 37, 698-705.
C H O L E S T E R O L IN P H Y S A L I A P H Y S A L I S * ROBERT E. M I D D L E B R O O K t and CHARLES E. LANE Institute of Marine Sciences, University of Miami, Miami, Florida 33149
(Received 21 ffuly 1967) Abstract--l. Acetone extracts of whole floats of the siphonophore Physalia physalis included an abundant steroid. This was identified as cholesterol by thin-layer chromatography, by infrared absorption, by nuclear magnetic resonance and by mass spectroscopy. 2. Chemical confirmation was achieved by conversion to cholesteryl acetate, by hydrogenation to 3-fl-cholestanol and by the formation of the digitonide. 3. Most cholesterol occurred in the pneumatosaccus where it comprised approximately 9 per cent of the total dry weight. INTRODUCTION EhRLI~R studies from this laboratory described the preparation and physiological effects of the nematocyst toxin of Physalia physalis in a variety of animals (Lane, 1960; Lane & Larsen, 1965; Larsen & Lane, 1966; Lane, 1967). Clark & Lane (1961) confirmed Wittenberg's identification (1960) of carbon monoxide in the float gas. Lane et al. (1965) emphasized the high levels of protein and free amino acids in gastrovascular fluid of P. physalis compared with mesogleal fluid of Aurelia aurita. The present paper describes the isolation, identification and distribution of cholesterol in P. physalis. MATERIALS AND METHODS Surviving specimens of P. physalis were collected from the ocean beach, Key Biscayne, Florida, and quickly brought to the laboratory where they were immediately dissected and extracted. Physalia floats were exhaustively extracted with several changes of acetone at room temperature. After decantation of the acetone solution, the samples were transferred to a continuous percolator-extractor (Ciereszko, 1966) and refluxed with hot acetone for 24-48 hr. All acetone extracts were combined and concentrated on a rotary evaporator. The aqueous solution remaining was transferred to a separatory funnel and extracted with benzene. The combined benzene extracts were dried first by azeotropic distillation followed by several hours contact with anhydrous sodium sulfate. Removal of the solvent left * Contribution No. 857 from the Institute of Marine Sciences, University of Miami. Supported by USPHS Grant No. HE-5489. Present address: Monsanto Company, Agricultural Research Division, St. Louis, Missouri 63166.
508
ROBERT E. MIDDLEBROOK AND CHARLES E. LANE
a thick, greenish brown oil. This was dissolved in a small volume of benzene and chromatographed on a Florisil column (3 x 62 cm), using benzene as the eluant. A series of 1-1. fractions from the column was concentrated on a rotary evaporator. Products were analyzed by thin-layer chromatography on silica gel G (MerckDarmstadt) plates (5 x 20 cm). The plates were developed in 20% (v/v) ethyl acetate in benzene, except where noted, and the chromatogram rendered visible by exposure to iodine vapor. Infrared spectra were run on a Beckman IR-10 in chloroform solution. Nuclear magnetic resonance spectra were obtained on a Varian A-60 instrument. All samples for N M R were run in deuteriochloroform solution, using tetramethylsilane as internal standard. Mass spectra were determined on a Consolidated Electrodynamics Corporation Model 110B high-resolution mass spectrometer. C, H and O analyses were performed by Alfred Bernhardt Laboratories, Mulheim, Germany. RESULTS After concentration, the first fraction from the Florisil column was a clear yellow oil that was dissolved in hexane and rechromatographed on alumina. Addition of five equal increments of benzene (up to 10 per cent final concentration) separated this fraction into a hydrocarbon moiety and a mixture of fatty acid esters, all as yet unidentified. The infrared spectrum of the hydrocarbon fraction showed absorption at 2980, 2945 and 2990 cm -1 carbon-hydrogen stretching), 1468 cm -1 (methylene) and 1385 cm -1 (carbon-methyl). The ester mixture showed carbonyl absorption at 1740 cm -1. A small portion of this material could be crystallized, but melted over the range 65-70°C. The most prominent peak in its NMR spectrum occurred as a singlet at 1.25 ppm, the methylene region of the spectrum, indicative of a long chain, probably in both the acid and alcohol portions of the ester. Although this material displayed a single spot on thin-layer chromatography employing hexane (R! = 0.21), there was severe overlap of spots when the ester mixture was separated by thin-layer chromatography in other solvent systems. Successive fractions from the Florisil column (to 3 1. of benzene) provided a crystalline solid that was recrystallized several times from 95% ethanol, affording white plates melting at 147-148°C. The m.p. was unchanged on admixture of authentic cholesterol, m.p. 148°C. (Analysis: Calculated for C~7H460: C, 83.87; H, 11-99. Found: C, 84.08; H, 11.98.) The infrared spectrum was identical with that of a shelf sample of cholesterol. The N M R spectrum was a typical steroidal pattern and was very nearly superimposable on the spectrum of authentic cholesterol. The single discrepancy occurred at 1.26 ppm; the peak at this position was somewhat more prominent than in the standard itself. Enhancement of the methylene region of the spectrum may have been caused by slight hydrocarbon contamination. The mass spectrum showed a parent peak at role 386 and was identical to the cracking pattern of authentic cholesterol run immediately afterwards.
509
CHOLESTEROL IN P H Y S A L I A P H Y S A L I S
The compound was chemically confirmed as cholesterol by conversion to cholesteryl acetate on treatment with acetic anhydride in pyridine. Cholesteryl acetate showed a melting point of 114-116°C (lit. : 116°C). (Analysis: Calculated for C29H4sO2: C, 81.31; H, 11.22. Found: C, 81.47; H, 11.48.) The thin-layer chromatogram ( 1 : 1 benzene-hexane) of authentic cholesteryl acetate and our conversion product produced single spots with identical R 1 values of 0.58. The infrared spectra of these compounds were also identical. Hydrogenation of the compound dissolved in methanol with platinum oxide catalyst produced 3-fl-cholestanol, m.p. 142°C (lit. m.p. 142°C.) (Analysis: Calculated for C~TH4sO: C, 83-43; H, 12.45; O, 4-12. Found: C, 83-69; H, 12.13; O, 4.33.) Mass spectral analysis showed a parent peak at m/e 388 and a cracking pattern entirely consistent with that of cholestanol. Digitonide formation was observed when the Physalia sterol was treated with an equivalent of digitonin in 95% ethanol. The free sterol was recovered by Soxhlet extraction of the digitonide with toluene for 48 hr. Five newly collected Physalia, representing the normal range of sizes seen at the height of the season, were dissected into their principal components, tentacles, pneumatosaccus and pneumatocodon, to determine whether the distribution of cholesterol was uniform through the animal. By independent acetone extraction of these three components from five separate animals and comparison of the thinlayer patterns of the extracts, virtually all cholesterol was found to occur in the pneumatosaccus where it comprised approximately 9 per cent of the total dry weight (Table 1). The crystalline product could be isolated only from the acetone extracts of the pneumatosaccus. TABLE 1--STEROL CONTENT OF PNEUMATOSACCUS
Animal
D r y wt. of pneumatosaccus (rag)
Wt. of crude sterol (rag)
~o sterol
1 2 3 4 5
116"8 57"1 13"9 75"5 114.2
13"6 7-4 1-9 9"8 9"4
11"6 7"7 7"3 6"6 12"2
Pneumatosacci from several Physalia were divided so that one part contained the gas gland and the other embraced all the rest of the structure. These components were separately extracted with acetone. Thin-layer chromatography of the extracts revealed that cholesterol occurred in the gas gland as well as in the remainder of the pneumatosaccus. A few milligrams of crude crystalline cholesterol were isolated from the acetone extract of several gas glands. Examination of gastrovascular fluid by thin-layer chromatography revealed no cholesterol.
510
ROBERTE. MIDDLEBROOKAND CHARLESE. LANE
DISCUSSION T h e occurrence of a variety of sterols in animals of lower taxa was first described by Bergmann and his students (Bergmann & Domsky, 1960). Phylogenetic considerations alone would suggest that Cnidaria might contain an array of sterols. Aurelia aurita, however, when extracted by the procedure described above, contained minimal quantities of sterols. Millepora alcicornis and Pseudopterogorgia acerosa similarly appear to synthesize only small quantities of sterols (Middlebrook, unpublished). T h e occurrence of considerable quantities of cholesterol in P. physalis and its restricted distribution within the animal are noteworthy. T h e pneumatosaccus in intact Physalia is normally inflated to 5-6 m m water above atmospheric pressure by a carbon monoxide-rich gas mixture (Wittenberg, 1960; Clark & Lane, 1961; Latimer & Ashby, 1965). T h e epithelial surface not exposed to gas is bathed by gastrovascular fluid in which no cholesterol was detected by thin-layer chromatography. Conceivably gastrovascular fluid could transport hydrocarbon precursors of cholesterol derived from dietary sources to the pneumatosaccus. T h e biological significance of cholesterol in the quantities observed remains obscure. Acknowledgements--The authors gratefully acknowledge their indebtedness to Dr. Tom Karns and the Department of Chemistry, University of Oklahoma, for assistance with NMR spectra and to Dr. R. D. Grigsby, Continental Oil Company, Ponca City, Oklahoma, for supplying the mass spectral data. REFERENCES
BERGMANNW. & DOMSKYI. I. (1960) Sterols of some invertebrates. Ann. N. Y. Acad. Sci. 90, 906-910. CIERKSZKOL. S. (1966) A versatile continuous percolator-extractor. J. chem. Ed. 43, 252253. CLARK F. E. & LANE C. E. (1961) Composition of float gases in Physalia physalis. Proc. Soc. exp. Biol. Med. 107, 673-675. L~'B C. E. (1960) The toxin of Physalia nematocysts. Ann. N. Y. Acad. Sci. 90, 742-751. LANE C. E. (1967) Recent observations on the pharmacology of Physalia toxin. In Animal Toxins (Edited by RUSSELLF. E. & SAtr~E~S P. R.) Pergamon Press, New York. LANE C. E. & LARSENJ. B. (1965) Some effects of the toxin of Physalia physalis on the heart of the land crab, Cardisoma guanhumi (Latreille). Toxicon 3, 69-71. LANE C. E., PRINOLE E. & BERGm~ A. M. (1965) Amino acids in extracellular fluids of Physalia physalis and Aurelia aurita. Comp. Biochem. Physiol. 15, 259-262. LARIMERJ. L. & ASHBYE. A. (1962) Float gases, gas secretion and tissue respiration in the Portuguese man-of-war, Physalia. jT. cell. comp. Physiol. 60, 41-47. LARSEN J. B. & LANE C. E. (1966) Some effects of Physalia physalis toxin on the cardiovascular system of the rat. Toxicon 4, 199-203. WlTaXNB~O J. B. (1960) The source of carbon monoxide in the float of the Portuguese man-of-war, Physalia physalis L. 3t. exp. Biol. 37, 698-705.