Comp. Biochem. Physiol., 1974, VoL 48B, pp. 47 to 57. PergamonPress. Printed in Great Britain ON THE BIOSYNTHESIS AND COMPOSITION OF STEROLS AND STEROLESTERS IN SOME SEA ANEMONES (ANTHOZOA) P. A. VOOGT, J. M. VAN DE RUIT and J. W. A. VAN RHEENEN Laboratory of Chemical Animal Physiology, The State University of Utrecht, 40, Jan van Galenstraat, The Netherlands (Received 13 ffuly 1973) Abstract--1. The incorporation of radioactivity from either sodium acetate- 1-1'C or DL-mevalonate-2-14C into some classes of lipida in Metridiura senile, Aneraonia sulcata and Cerianthus membranaceus is investigated. 2. Lipid contents in these animals are relatively very high. Structural lipids make up a relatively small portion of these lipids, suggesting that part of the lipids is stored and functions as a reserve. This is also reflected in the composition of the neutral lipids. 3. It is demonstrated that these animals utilize the injected precursor for the biosynthesis of lipids. The high incorporation of radioactivity from mevalonate into phospholipids is rather enigmatic. 4. There was only a slight incorporation of radioactivity into the sterols and it is supposed that likely sea anemones can synthesize sterols. 5. Sterols with twenty-seven carbon atoms were predominant, cholesterol in all cases being the main sterol. 6. The composition of free and esterified sterols of each species, but also the sterol compositions of the different species closely resembled each other. INTRODUCTION OUR KNOWLEDGE about the sterols present in Coelenterata has been reviewed excellently by Bergman (1962). In this review data have been processed up to and inclusive of 1957. This means that it deals with studies carried out before suitable separation and identification techniques were available. Hammen (1968), rev;ewing the intermediate metabolism of coelenterates, was able to be very brief with reference to sterols because only one new report (Van Aarem et al., 1964) had appeared since that time. This clearly demonstrates that there was hardly any interest in the sterols of coelenterates in the past decade. This changed after 1968. Middlebrook & Lane (1968) demonstrated the presence of cholesterol in Physalia physalis, confirming the results of Hamilton & Van den Heuvel (1964). Thus far in Hydrozoa only cholesterol has been found. Ciereszko et al. (1968) studied several gorgonians and showed the presence of gorgosterol in four species belonging to different genera. Engelbrecht et al. (1972) studied an alcyonarian and they found cholesterol, 24o~-methyl-cholesterol, gorgosterol and a new sterol viz. 25- hydroxy-24-methyl-cholesterol. Gupta & Scheuer (1969) examined the sterols of 47
Transcript
Comp. Biochem. Physiol., 1974, VoL 48B, pp. 47 to 57. Pergamon Press. Printed in Great Britain
ON THE BIOSYNTHESIS AND COMPOSITION OF STEROLS AND STEROLESTERS IN SOME SEA ANEMONES
(ANTHOZOA)
P. A. VOOGT, J. M. VAN DE R U I T and J. W. A. VAN R H E E N E N
Laboratory of Chemical Animal Physiology, The State University of Utrecht, 40, Jan van Galenstraat, The Netherlands
(Received 13 ffuly 1973)
Abstract--1. The incorporation of radioactivity from either sodium acetate- 1-1'C or DL-mevalonate-2-14C into some classes of lipida in Metridiura senile, Aneraonia sulcata and Cerianthus membranaceus is investigated.
2. Lipid contents in these animals are relatively very high. Structural lipids make up a relatively small portion of these lipids, suggesting that part of the lipids is stored and functions as a reserve. This is also reflected in the composition of the neutral lipids.
3. It is demonstrated that these animals utilize the injected precursor for the biosynthesis of lipids. The high incorporation of radioactivity from mevalonate into phospholipids is rather enigmatic.
4. There was only a slight incorporation of radioactivity into the sterols and it is supposed that likely sea anemones can synthesize sterols.
5. Sterols with twenty-seven carbon atoms were predominant, cholesterol in all cases being the main sterol.
6. The composition of free and esterified sterols of each species, but also the sterol compositions of the different species closely resembled each other.
INTRODUCTION
OUR KNOWLEDGE about the sterols present in Coelenterata has been reviewed excellently by Bergman (1962). In this review data have been processed up to and inclusive of 1957. This means that it deals with studies carried out before suitable separation and identification techniques were available. Hammen (1968), rev;ewing the intermediate metabolism of coelenterates, was able to be very brief with reference to sterols because only one new report (Van Aarem et al., 1964) had appeared since that time. This clearly demonstrates that there was hardly any interest in the sterols of coelenterates in the past decade. This changed after 1968. Middlebrook & Lane (1968) demonstrated the presence of cholesterol in Physalia physalis, confirming the results of Hamilton & Van den Heuvel (1964). Thus far in Hydrozoa only cholesterol has been found. Ciereszko et al. (1968) studied several gorgonians and showed the presence of gorgosterol in four species belonging to different genera. Engelbrecht et al. (1972) studied an alcyonarian and they found cholesterol, 24o~-methyl-cholesterol, gorgosterol and a new sterol viz. 25- hydroxy-24-methyl-cholesterol. Gupta & Scheuer (1969) examined the sterols of
47
48 P. A. VOOGT, J. M. VAN DE RUIT AND J. W. A. VAN RHEENEN
three zoanthids. In Zoanthus confertus cholesterol, 24-methyl-cholesterol and 22,23-dihydrobrassicasterol were present besides 24-methylenecholesterol, which had been observed previously in Zoanthus proteus (Bergmann & Dusza, 1957). In Palythoa tuberculosa a sterol mixture was found which was apparently identical with the palysterol from Palythoa mammilosa (Bergmann et al., 1951). It con- sisted of cholesterol, 24fi-methyl-cholest-5-en-3fi-ol, 24fi-methyl-cholesta-5,22- dien-3fi-ol, 24-ethyl-cholest-5-en-3fl-ol and gorgosterol. In a Palythoa species from Tahiti only 24-methylenecholesterol was present.
Thus far gorgosterol has been observed only in Gorgoniaceae and Zoanthidea. Its structure was elucidated recently (Ling et al., 1970).
The sterol mixture of the actiniarian Calliactis parasitica (Ferezou et al., 1972) differed rather strongly from that of the coelenterates mentioned above. Chole- sterol was the main sterol, but cholestanol, 22-dehydrocholesterol, 24-methylene- cholesterol, brassicasterol and a C 26 sterol were present in addition. Remarkable is the presence of cholestanol, because this is the first time that a saturated sterol has been reported for coelenterates. Cholesterol was the sole sterol in Metridium senile (Mason, 1972).
Whereas our knowledge about the sterols present in coelenterates has increased rapidly in the past few years, our insight into the metabolism of sterols in these animals is still very sparse and is confined to a few reports.
Van Aarem et al. (1964) concluded that Rhizostoma sp. (a scyphozoan) was unable to synthesize sterols from acetate.
Ferezou et al. (1972) did not find radioactivity in the sterols of Calliactis parasitica after the injection of acetate-l-l~C into the animals, but mentioned that their experiment did not exclude the presence of a sterol biosynthesis which proceeds very slowly or is season-dependent or is strongly inhibited by the sterols from their diet. Walton & Pennock (1972) reported that Metridium senile was unable to synthesize sterols from mevalonate-2-14C. Apart from the unpublished work of Van Hedel (Vonk & Zandee, 1963), which suggested that acetate might be incorporated into sterols, all reports agree that no radioactivity was present in the sterols after the administration of a suitable radioactive precursor to the animals.
For the scope of the sterol project running on our laboratory we decided (1970) to study the composition of both the free and esterified sterols of three sea anemones and to investigate whether these animals are capable of synthesizing sterols either from acetate or mevalonate. Whilst this study was in progress the reports of Ferezou et al. (1972) and Walton & Pennock (1972) were published. In this paper the results of the investigation with Metridium senile, Anemonia sulcata and Cerianthus membranaceus are dealt with.
MATERIALS AND METHODS Specimens of Metridium senile were obtained from the Netherlands Institute for Sea
Research (N.I.S.R.) at Texel; those of Anemonia sulcata (like Metridium senile belonging to the order Actiniaria) and Cerianthus membranaeeus (belonging to the order Ceriantharia) were obtained from the Stazione Zoologica at Naples, Italy.
STEROLS IN SOMEANEMONES 49
The animals were each injected with either an aqueous solution of sodium acetate-l-x4c (NEN Corporation, Boston) or a saline solution of vL-mevalonic acid-2-~4C (DBED salt, N E N Corporation, Boston) into the body-wall and then maintained in well-aerated sea water. After incubation during which they were not fed, the animals were fixed in ethanol. Data about the animals and the precursors administered are given in Table 1.
TABLE 1 - - D A T A ABOUT THE TREATMENT OF SOME ANTHOZOANS EXAMINED FOR THEIR
Dosage administered 12"5 1.25 40 per animal (in/~Ci)
Total dosage Incubation time (hr)
125 50 200 24 48 72
Lipids were extracted from the animals using the procedure of Bligh & Dyer (1959) after the modification of Van der Horst et al. (1969). Some B H T (2,6-di-tert-butyl-4- methyl-phenol) was added to the lipids to prevent oxidation. This was repeated after each following step in the processing. Lipids were purified on columns with Sephadex G-25 according to Terner et al. (1970). Polar lipids were precipitated in cold acetone (Lipsky et al., 1957). Purity of both lipid fractions, polar and neutral lipids, was examined by thin-layer chromatography (TLC) according to Freeman & West (1966), in which the presence of phosphorus was detected by the spraying agent after Dit tmer & Lester (1964). When a fraction was still cross-contaminated it was purified by means of column chromato- graphy on silicic acid (Hanahan et al., 1957). The neutral lipids were separated into various lipid classes by column chromatography on Florisil as described by Carroll (1961).
The crude hydrocarbon fraction was chromatographed on thin-layer plates (silica gel G impregnated with 10% AgNOs) using hexane-diethylether (9 : 1, v/v) for development. The area co-chromatographing with reference to squalene was eluted and the eluted material was analysed with a Becket gas chromatograph, Model 2300, on the stationary phases SE-52 (4%) and PEGA (20% + 3% HsPO4).
Sterols were purified by means of T L C on silica gel G. First they were chromatographed in toluene-ethylacetate (4 : 1, v/v), then hydrogenated and after acetylation chromatographed in hexane--diethylether (9 : 1, v/v).
Sterol esters were hydrolysed with a solution of 1"5 N KOH in 80% methanol in the usual manner. Sterols were isolated from the saponification mixture by means of petroleum ether (b.p. 40-60°C) and then purified as described above.
Sterols were analysed as their trimethylsilylethers on the stationary phases SE-30 and N P G S under conditions only differing from those described before (Voogt, 1971a, b) in that the gas chromatograph used here was a Packard-Becker instrument, Model 420. The sterols were identified by means of their steroid numbers, which were calculated from the
50 P. A. VOOGT, J. M. VAN DE RUIT AND J. W. A. VAN RHEENEN
formulae given previously (Voogt, 1972). Hydrogenation and acetylation were performed as described earlier (Voogt, 1971a).
Preparative gas chromatography of 3]3-sterols was carried out on a Becker instrument, Model 1452, in the manner given previously (Voogt & Van Rheenen, 1973).
Infra-red spectra were measured in KBr with a Hitachi infra-red spectrometer, Model EPI-G3.
Radioactivity was determined in 10 ml toluene containing 0"4% Omnifluor (NEN Chemicals GmbH) with a Packard Liquid Scintillation Spectrometer, Model 2420.
RESULTS
Various lipid fractions were isolated from the animals. The i r quantities are summarized in Table 2. The specific radioactivity of each lipid fraction was determined and the results are shown in Table 3.
The poly-unsaturated hydrocarbons co-chromatographing with reference to squalene on silver-nitrate-impregnated silica gel G were analysed by gas chromato- graphy. Only the fraction from Anemonia (0.35 mg) showed a distinct squalene peak, whereas in the other fractions squalene could not be detected.
TABLE 2 - - W E I G H T S OF THE LIPID FRACTIONS ISOLATED FROM SOME ANTHOZOANS, EXPRESSED
IN m g AND AS PERCENTAGES OF THE TOTAL LIPIDS
M e tridium A nemonia C erian thus senile sulcata membranaceus
mg
% o f % of ~ of total total total lipids mg lipids mg lipids
Fresh weight of 396,000 522,000 268,700 the animals
Total lipids 4450"8 5199-5 3925"6 (% of the 1"12 1"00 1"46 fresh weight)
52 P. A. VOOGT, J. M. VAN DE RUIT AND J. W. A. VAN RHEENEN
Free sterols and sterols f rom the esters were purified by means of T L C and the puri ty was checked via preparative gas-l iquid chromatography (GLC) .
T h e results are given in Table 4. A small amount of the free sterols was hydrogenated and after acetylation
analysed by G L C on the stationary phase SE-30. Four peaks were visible in the chromatograms representing sterols containing 26 to 29 carbon atoms. The proport ional composit ion is given in Table 5. Trimethylsi lylethers of the sterols were prepared and analysed on the stationary phases SE-30 and NPGS. Repre- sentative chromatograms of the free sterols of Metridium are given in Figs. 1 and 2. Chromatograms of the ester sterols of Metridium and of the free and ester sterols of Cerianthus closely resembled those given in Figs. 1 and 2 and are not depicted
TABLE 5--PROPORTIONAL COMPOSITION, ACCORDING TO CARBON CONTENT, OF THE
HYDROGENATED STERYLACETATES OF SOME ANTHOZOANS
Metridium Anemonia C erianthus Sterols senile sulcata membranaceus
for this reason. Chromatograms of Anemonia sulcata were somewhat different as is shown in Fig. 3. T h e sterol compositions were calculated f rom the chromato- grams and are given in Table 6.
3
FIG. 1. Chromatogram of the trimethylsilyl derivatives of the sterols of Metridium senile after GLC separation on the stationary phase SE-30. The percentage com- position and the steroid number of each sterol are given. The steroid numbers of the suitable sterols are given in parentheses. 1 : 5"5%, C2e sterol, 28.53 ; 2: 10.8%, 22-dehydrocholesterol, 29"84 (29.80); 3: 70"1%, cholesterol, 30"22 (30"18); 4: 6"8%, brassicasterol, 30"61 (30"58) or desmosterol (30"50); 5: 5"4%, campesterol, 31"11 (31"19) or 24-methylenecholesterol (31"07); 6: 1"5%, /3-sitosterol, 32"13
(32"04) or iso-fucosterol (32"02) or stigmasterol (32.12).
4
5
FIG. 2. Chromatogram of the trimethylsilyl derivatives of the sterols of Metrid- ium senile after GLC separation on the stationary phase NPGS. The per- centage composition and the steroid number of each sterol are given. The steroid number of the suitable sterols are given in parentheses. 1: 5-8%, C~6 sterol, 28"60; 2: 1"0%, 22-cis dehydrocholesterol, 29"80 (29"92); 3: 10"3%, 22-trans dehydrocholesterol, 30-10 (30"09); 4: 68"0%, cholesterol, 30"34 (30"36); 5: 6"0%, brassieasterol, 30"77 (30"83); 6: 0"9%, desmosterol, 31.23 (31.15); 7: 1.79/o, campesterol, 31-38 (31-43); 8: 4.2~/o, 24-methylenecholesterol, 31"72 (31"67); 9: 1"1%, fl-sitosterol, 32-32 (32"29) or stigmasterol (32"24); 10: 1'1%,
isofucosterol, 32-78 (32"79).
54 P.A. VOOCT, J. M. VAN DE RUIT AND J. W. A. VAN RHEENEN
7
FIG. 3. Chromatogram of the trimethylsilyl derivatives of the sterols of Anemonia sulcata after GLC separation on the stationary phase SE-30. The percentage composition and the steroid number of each sterol are given. The steroid numbers of the suitable sterols are given in parentheses. 1: 0"3%, C2s sterol, 28"52; 2: 0"8%, 22-cis-dehydrocholesterol, 29.70 (29"67); 3: 8"7%, 22-trans-dehydro- cholesterol, 29"83 (29"80); 4: 62"3%, cholesterol, 30-23 (30'18); 5: 4.8%, desmo- sterol, 30"46 (30-50) and sterol X (see text). 6: 2"6%, brassicasterol, 30"63 (30"58); 7: 18"8%, campesterol, 31"10 (31"19) or 24-methylenecholesterol (31"01); 8: 0"5%, stigmasterol, 31 "50 (31.50) ; 9: 1 "2%, fl-sitosterol, 32"13 (32"04) or fucosterol (32-02).
DISCUSSION
Although this investigation was undertaken with the aim to study sterol com- position and sterol biosynthesis in the experimental animals, we also determined-- be it rather roughly-- the lipid composition in these animals (Table 2). Lipid contents expressed in percentages of the fresh weight are of the magnitude en- countered in molluscs. This means that the lipid contents in these animals are relatively very high, which would have become quite apparent if these contents had been expressed in percentages of the dry weight. This is in full agreement with the observations of Bergmann (1962) who mentions lipid contents of about 30 per cent of the organic matter. This high lipid content suggests that part of the lipids will be present in store. This is confirmed by the low content of phospho- lipids, whilst analysis of the neutral lipids shows that free fatty acids and glycerides are the main components. Sterols and sterolesters seem to be large fractions, but it should be borne in mind that they were collected as long as sterols or sterolesters were eluted from the column. After purification of these fractions, eventually after
STF-.ROLS IN SOME ANEMONES 55
hydrolysis, the values are considerably lower. Thus structural lipids, sterols and phospholipids make up only a small portion of the total lipids.
Incorporation of radioactivity into the total lipids amounted to 1-2 per cent from acetate and to 9 per cent from mevalonate (Table 3). The distribution of radioactivity between phospholipids and neutral lipids seems difficult to explain; Metridium and Cerianthus, both injected with acetate, show quite different pictures. Further, incorporation of a large part of the radioactivity from mevalonate into phospholipids is fully unexpected. The unexpected behaviour of the label from mevalonate is continued in the fractions of the neutral lipids: hydrocarbons, sterolesters and sterols are labelled poorly, whilst most activity is recovered in glycerides and fatty acids.
Hydrocarbons, generally, were labelled poorly, and only in Anemonia could the presence of squalene be shown. However, it could not be determined whether this squalene was radioactive.
Sterols, both free and esterified, were purified and their radioactivity was determined. The results given in Table 4 clearly show that no activity was found in the free sterols of Metridium and Cerianthus. A slight activity was present in the sterols from the esters in Metridium. In Anemonia (which had received mevalonate) low specific radioactivities in the sterols could be demonstrated. The results of the experiments with Metridium and Cerianthus are in full agreement with those obtained by Van Aarem et al. (1964), Ferezou et al. (1972) and Walton & Pennock (1972) and would lead to the same conclusion. However, radioactivity in the esterified sterols of Metridium is significant, whilst a slight activity is present in both the free and esterified sterols of Anemonia. In Anemonia too, the activity of the free sterols is surpassed by that of the esterified sterols. This phenomenon has been observed several times previously (Van der Horst & Voogt, 1972; Voogt, 1973a, b; Voogt & Van Rheenen, 1973). From the foregoing it can be concluded that likely sea anemones are able to synthesize sterols, but that, in accordance with the possibilities given by Ferezou et al. (1972), sterol biosynthesis proceeds very slowly or may be inhibited strongly by exogenous sterols. The higher specific radioactivity of the esterified sterols might be the result of the transport of newly synthesized sterols as sterolesters (Van der Horst & Voogt, 1972).
In sea anemones C27 sterols are the main sterols followed by those with 28 carbon atoms. In Anemonia the latter constitute up to about a quarter of the total sterols (Table 5).
The compositions of the free and esterified sterols of each species are very similar but also the sterol compositions of the different species show close resem- blances (Table 6). In all cases cholesterol is the main sterol, whilst 22-trans- cholesta-5,22-dien-33-ol and brassicasterol, respectively, follow. This order is changed in Anemonia due to the high content of 24-methylenecholesterol. The composition of Metridium and of Cerianthus differ only in detail. The CC26 content in Metridium is rather high. The complete absence of campesterol in the esterified sterols of Metridium is not certain but its presence could not be demonstrated. This holds also for stigmasterol in the free sterols of Metridium.
56 P. A. VOOGT, J. M. VAN DE RUIT AND J. W. A. VAN RHEENEN
In the esterified sterols of Cerianthus a component was present which on the ground of the steroid numbers determined on two different stationary phases, might be ergosterol. This identity has been not further confirmed.
T h e presence of desmosterol and 28-isofucosterol could not be demonstrated in the esterified sterols of Anemonia, but an unidentified sterol, indicated by sterol Y and containing probably 29 carbon atoms, was present.
In the free sterols of Anemonia a component was found, which on the ground of its steroid numbers might be 22-cis-24-methyl-cholesta-5,22-dien-3fl-ol. The 22-cis-bond has been identified recently in sterols with twenty-seven carbon atoms (Idler & Wiseman, 1971a) and supposed in C26 sterols (Idler & Wiseman, 1971b). Th is component was isolated by means of preparative G L C . T h e i.r. spec t rum showed a small absorbance at 690 cm -a, characteristic of the 22-cis-bond (Bellamy, 1964), but absorbance at about 970 cm -1, characteristic of the 22-trans bond, was much stronger. For this reason it is concluded that the isolated fraction is con- taminated with brassicasterol. Due to lack of material further trials to purify and to identify this sterol were impossible but will be undertaken in future.
Acknowledgements--The authors are indebted to Mr. A. F. Corsmit and Mr. F. van den Heuvel of the Solid State Department Physical Laboratory, Utrecht, for the i.r. spectroscopy.
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