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Biosynthesis and composition of 3β-sterols in the Ophiuroids Ophiura albida and Ophioderma longicauda
Comp. Biochon. Physiol., 1973, Vol. 45B, pp. 593 to 601. Pergamon Press. Printed in Great Britain
BIOSYNTHESIS AND COMPOSITION OF 3fl-STEROLS IN THE OPHIUROIDS OPHIURA ALBIDA AND OPHIODERMA LONGICA UDA P. A. V O O G T Laboratory of Chemical Animal Physiology, 40 Jan van Galenstraat, Utrecht, The Netherlands
(Received 13 November 1972) Abstract--1. The incorporation of sodium acetate-l-l*C into some classes of lipids in Ophiura albida and Ophioderma longicauda is investigated. 2. It is demonstrated that the animals utilize the acetate injected for the biosynthesis of various lipid classes, including sterols. 3. It is supposed that probably all ophiuroids and possibly all echinoderms are able to synthesize 3~-sterols. 4. The composition of the sterols is determined. In ophiuroids C27 sterols are not strongly predominant. The main sterol is brassicasterol, followed by cholesterol. These two features ophiuroids have in common with holothuroids. The sterol composition is rather simple and strongly resembles that of Echinoidea. INTRODUCTION IN A SEINESof papers on the composition and metabolism of sterols in echinoderms,
we reported the capacity of synthesizing 3fl-sterols for representatives of the classes Asteroidea, Echinoidea and Holothuroidea (Voogt, 1972b, 1973a; Voogt & Over, 1973) and it was supposed that this capacity should be a general property of all representatives of these classes. This paper deals with the Ophiuroidea. Recently Goad et al. (1972) and Walton & Pennock (1972) showed that Ophiocomina nigra did synthesize squalene, 4,4-dimethyl sterols, 4-monomethyl sterols and 4-demethyl sterols from mevalonate; label in 4-demethyl sterols being restricted to C2~ sterols. Only a few data about sterol composition in Ophiuroidea are available and most of them are from the time before gas-chromatography made its entrance in sterol analysis. Recently Gupta & Scheuer (1968) reported the sterol composition of Ophiocoma insularia, while Goad et al. (1972) reported on Ophiura albida. They agree that cholesterol is the main (or one of the main) sterol(s). Matsumoto & Tamura (1956), on the other hand, did not find any cholesterol in Ophioplocus japonicus. Several investigators indicated fl-sitosterol as one of the main sterols. This paper deals with the sterol composition and the biosynthesis of 3fl-sterols from the acetate of O. albida and Ophioderma longicauda. 593
594
P.A. VOOCT
MATERIALS AND METHODS Specimens of Ophiura albida were collected at the laboratory for marine biology "Laboratoire Arago", Banyuls-sur-mer, France; while specimens of Ophioderma longicauda were collected at "Stazione Zoologica", Naples, Italy. The animals were each injected with an aqueous solution of sodium acetate-l-'4C. The injections were given in the body-disc, close to the gonads. After the injection the animals were kept in running sea water and at the end of the incubation were fixed in ethanol. Data about the experimental animals and the acetate injected are summarized in Table 1. TABLE I ~ D A T A ABOUT THE TREATMENT OF O. albida AND O. longicauda EXAMINED FOR THEIR CAPACITY OF SYNTHESIZING 3ff-STEROLS
O. albida No. of animals Place and date of collection Sodium acetate-l-14C Dosage administered to each animal (in/~Ci) Incubation time (hr)
10 Banyuls-sur-mer 20 June 1967 Philips Duphar (20 mCi/mM)
O. longicauda
1"25
40 Naples 21 June 1971 New England Nuclear Corporation (1 mCi/l'35 mg) 12
72
72
Lipids were extracted from the animals using the procedure of Van der Horst et al. (1969) and then purified on Sephadex G-25 (Terner et al., 1970). The purified lipids of Ophiura were saponified with a solution of 1.5 N potassium hydroxide in 80% methanol, and the unsaponifiable and saponifiable lipids were obtained in the usual way. The unsaponifiable lipids were purified on basic cupricarbonate (Capella et al., 1960) and separated into a hydrocarbon fraction, a crude sterol fraction and the remaining fraction by means of column chromatography on alumina (Voogt, 1971a, b). Sterols were isolated via their digitonides. Phospholipids were removed from the purified lipids of Ophioderma by precipitating them in cold acetone (Lipsky et al., 1957) and by chromatography on silicic acid- Hyflo Super Cel (Hanahan et aI., 1957). The neutral lipids were separated into lipid classes according to Carroll (1961). The sterols from the sterolesters were, after hydrolysis, isolated by means of preparative thin-layer chromatography (TLC). Sterols were purified by chromatographing them thrice on thin-layer plates using as moving-phases consecutively: benzene-diethylether-ethanol-acetie acid (50 : 40 : 20 : 0"2 by volume), isopropylether-acetie acid ( 9 6 : 4 v/v) and benzene--ethylacetate ( 5 : 1 v/v). Radioactivities were measured with Packard Liquid Scintillation Counters, Models 3320 or 2420 in toluene containing 0"4% Omnifluor (NEN Chemicals). Gas-chromatography of the sterols, as their trimethylsilylethers, on the stationary phases SE-30 and NPGS, was performed as described elsewhere (Voogt, 1971c). Sterols were identified by means of their steroid numbers, which were calculated from the formulae given previously (Voogt, 1972d). Hydrogenation and acetylation of sterols was carried out as reported earlier (Voogt, 1971b). RESULTS T h e quantities of the isolated lipid fractions of Ophiura, expressed in m g and as a percentage of the total amount of lipids, are given in Table 2. This table
3 ~ - $ T E R O L S I N SOME OPHIUROIDS
595
TABLE 2---QUANTITIES ANn RADIOACTIVlTImOF THE ISOLATED LIPID FRACTIONS FROM O. albida, ExPItmS~ IN ms, FERCENTAOESOF THE TOTALAMOUNTOF LIPtDS, dis/min per mg AND PERCENTACESOF THE TOTAL RADIOACTIVITYINCORPORATEDINTO THE LIPIDS
Lipid fraction Fresh weight Purified lipids (% fresh weight) (% of total radioactivity administered) Saponifiable lipids Non-saponifiable lipids Hydrocarbon fraction Crude sterol fraction 3~-Sterols (% fresh weight)
mg 30,000 196 0"65
% of total lipids 100
dis/min per mg 1527
% of total radioactivity in lipids 100 1.09
134.4 44-5 22.7 15"0 11-5 0"038
67 25-4 11-6 9"4 7"2
1876"2 88.9 8 113-5 60
83.0 1.5 0"71 0"23
also shows the specific radioactivity of each fraction and the total activity present in these fractions expressed as percentages of the radioactivity incorporated into the total lipids. T h e corresponding values were also determined for Ophioderma. T h e y are s u m m a r i z e d in T a b l e 3. I n T L C the hydrocarbon fraction of Ophioderma appeared to contain some sterolesters, while the sterolester fraction also contained methylesters of fatty acids. F o r this reason the hydrocarbon and sterolester fractions were pooled and hydrolysed in the usual way, yielding 1187.4 m g of unsaponifiable matter. T h i s matter was separated on alumina in 150.8 m g of TABLE
3--QUANTITIES
AND RADIOACTIVITIES OF THE ISOLATED L I P I D FRACTIONS FROM IN r a g , PERCENTAGES OF THE TOTAL AMOUNT OF LIPIDS~ dis/rain AND PERCENTAGES OF THE TOTAL RADIOACTIVITY INCORPORATED INTO THE LIPIDS
O. longlcauda, EXPRESSED per mg
Lipid fraction Fresh weight Purified lipids (% fresh weight) (% total radioactivity administered) Phospholipids Neutral lipids Hydrocarbon fraction Sterolestera Triglycerides Sterol fraction Remaining fraction
mg 557,000 8137.8 1.46
% of total lipids 100
dis/min per mg 9871
~/o of total radioactivity in lipids 100 7"54
2170.0 5937.6 1503"6 1091 "4 1034"8 497-4 1734.6
26 "7 73"0 18"5 13"4 12"7 6-1 21"2
4434 11,861 20,067 2315 5508 498 I2,515
12.0 87"7 37"9 3"1 7"1 0"3 27"2
596
P.A. VOOGT
hydrocarbons, 44.8 mg of sterols and 896.4 mg of a remaining fraction. Specific radioactivities of these fractions, were 1863, 1771 and 9559 dis/min per mg, respectively. Some carrier squalene was added to the hydrocarbon fraction of Ophioderma (22.7 rag) and the squalenedodecabromide was prepared according to Mackenna et al. (1950). Total radioactivity in this material amounted to 9 dis/rain. Part of the hydrocarbon fraction of Ophioderma was chromatographed on a thin-layer plate, coated with silica gel-G and impregnated with silver nitrate, using hexane-diethylether ( 9 : 1 v/v) as the solvent. The zone co-chromatographing with reference squalene showed a specific radioactivity of 967 dis/min per rag. In gas chromatograms of this material performed on SE-52, several peaks were visible; squalene could not be detected, however. Sterols were purified by means of TLC. The results are shown in Table 4. TABLE 4---SPECIFIC RADIOACTIVITY OF THE STEROL8 OF O . albida AND O. PURIFYING THEM BY MEANS OF T L C
longicauda ON
O. longicauda O. albida Before purification After one purification After two purifications After three purifications
60 56
Free sterols Sterols from esters 498 95 50 48
1771 676 -51
A small amount of the sterols of both species was hydrogenated and after acetylation chromatographed on SE-52. The chromatograms showed four or five peaks, corresponding with sterols containing 26, 27, 28, 29 and 30 carbon atoms. The proportional compositions are given in Table 5. TABLE 5 - - P R O P O R T I O N A L COMPOSITION~ ACCORDING TO THE CARBON CONTEN% OF THE
STEROLSOF 0. alblda AND O. longicauda
Species O. albida O. longicauda
C~6
C2v
C2a
C29
C30
2.0 1.5
49.0 33.1
28.9 32.9
17"5 32"4
2.6 --
Sterols as their trimethylsilylethers were chromatographed on SE-30 and NPGS. Representative chromatograms of Ophioderma are depicted in Figs. 1 and 2; percentage composition and tentative identification are given at the foot of the figures. Chromatograms of Ophiura resembled those of Ophioderma and for this reason are not depicted; their data are summarized in Table 6. From the data in
8
7
6
5
4
4"62
3.95
3-52
2.97
2"64
30"20 (30.18) 30.59 (30-58) 31-15 (31" 19) 31 "53 (31"50) 32"04 (32-04)
(29"80)
28"50 29"01 29"80 (29-67)
SN
13"6
3.3
2"8
27-4
24"2
2"1 0"6 23"6
%
10
9
8
7
6
5
4
3
Isofueosterol ?
fl-Sitosterol
Stigmasterol
Campesterol
Brassieasterol
Cholesterol
22-Dehydroeholesterol (trans)
6-17
5"41
4"57
3.91
3-60
2"99
2"64
2"45
2"27
2
RRT 1-59 22-Dehydroeholesterol (eis)
NPGS
1
No.
The steroid numbers of the suitable sterols are given in parentheses.
fl-Sitosterol
Stigmasterol
Campesterol
Brassieasterol
Cholesterol
22-Dehydroeholesterol (trans)
1-58 1-84 2"34
1 2 3
22-Dehydroeholesterol (cis)
RRT
No.
SE-30
FOR T W O DIFFERENT STATIONARY PHASES
T A B L E 6 - - R E L A T I V E R E T E N T I O N TIMES~ STEROID NUMBERS AND P R O P O R T I O N A L C O M P O S I T I O N OF T H E STEROLS OF O .
33.22
30"32 (30"36) 30.75 (30"83) 31"39 (31.43 ) 31-67 (31"72) 32"20 (32-29) 32-77
30-07 (30"09)
29"80 (29.91)
2.4
4-0
10"0
3"6
3"1
27-1
24"9
15-3
7"1
2-5
%
DETERMINED
28-59
SN
albida
u0
O
O
598
P.A. VOOGT
S 3
\
6
FIG. 1. Chromatogram of the T M S derivates of the sterols of O. longicauda after GLC separation on SE-30. The percentage composition and the steroid number of each sterol is given. The calculated steroid numbers of the suitable sterols are given in parentheses. 1. 1% C~6 sterol, 28.51; 2. 5.3% 22-dehydrocholesterol (cis), 29"69 (29"67); 3. 5"6% 22-dehydrocholesterol (trans), 29"80 (29"80); 4. 22"6% cholesterol, 30"20 (30-18); 5.31.5 % brassicasterol, 30.60 (30"58); 6.5"8% campesterol, 31"17 (31-19); 7. 5"3% stigmasterol, 31"50 (31"50); 8.22"8% fl-sitosterol, 32"04 (32"04) or iso-fucosterol (32-02). Figs. 1 and 2 and T a b l e 6 the proportional compositions of the sterols could be drawn up. T h e s e are listed in T a b l e 7. TABLE 7--PROPORTIONAL COMPOSITION OF THE 3fl-STEROLS OF O. albida AND O.
Sterols C26 sterols
O. albida
2"6 (two components) 22-Cis-cholesta-5,22-dien-3fl-ol 7"3 22-Trans-cholesta-5,22-dien-3fl-ol 15.7 Cholesterol 25.2 Brassicasterol 27-0 Campesterol 2"8 Stigmasterol 3"3 ~-Sitosterol 9-9 Isofucosterol ? 3"9 Ca0 sterol 2"4
longicauda
O. longicauda 1"5 (two components) 5"5 6"6 23 "3 30 "9 3"6 5'3 18.5 4'3
3~-ST-~OLS IN SOME OPHIUROIDS
599
5
9
I
3
\ FIc. 2. Chromatogram of the TMS derivatives of the sterols of O. longicauda after GLC separation on NPGS. The percentage composition and the steroid number of each sterol is given. The calculated steroid numbers of the suitable sterols are given in parentheses. 1. 1"3% C~e sterol, 28.61; 2. 0"6% C26 sterol, 28.85; 3. 5.7% 22-dehydrocholesterol (cis), 29-81 (29.91); 4. 7"7% 22-dehydrocholesterol (trans), 30"08 (30"09); 5. 23"9% cholesterol 30"36, (30"36); 6. 30'3% brassicasterol, 30.79 (30.83); 7. 3"6% campesterol, 31.43 (31.43); 8.4"6% stigmasterol, 31.71 (31.72); 9. 17"5% fl-sitosterol, 32.25 (32.29); 10. 4"3% isofucosterol, 32-78. DISCUSSION Tables 2 and 3 show that the lipid contents of Ophiura and Ophiod~rma are strongly different, namely 0.65 and 1.46 per cent of the fresh weight, respectively. The first value and also that of the sterol content of Ophiura is rather low, being only slightly higher than the corresponding values found for Holothuroidea (Voogt & Over, 1973), the latter being well known for their low lipid and sterol content. In both experiments the acetate injected was utilized by the animals for the biosynthesis of lipids. Total incorporation of radioactivity into the lipids amounted to 1.09 and 7.54 per cent of the total dosage administered. All lipid fractions investigated turned out to be radioactive, pointing to an intensive lipid metabolism. The squalenedodecabromide prepared from the total hydrocarbon fraction of Ophiura (with 182 dis/min) possessed only 9 dis/rain, which possibly should be ascribed partly or fully to unsaturated components other than squalene. This
600
P.A. VOOGT
holds also for the radioactivity observed in the squalene fraction of Ophioderma. Here, with certainty, several components were present, but squalene could not be detected. Concluding, one should say that in both animals the presence of squalene is not shown unequivocally and that, for this reason, it is unknown whether squalene is synthesized by these animals or not. Table 4 shows that the sterols of both animals are radioactive, permitting the conclusion that these animals are capable of synthesizing sterols from acetate. Earlier, Goad et al. (1972) and Walton & Pennock (1972) reported that ophiuroids are able to synthesize sterols from mevalonate. From the data available at this moment it seems justified to suppose that all Ophiuroidea can synthesize sterols. The same has been found before for Asteroidea, Echinoidea and Holothuroidea. The fifth class of the Echinodermata, the Crinoidea, has not been studied as yet in this respect. It is likely, however, that all echinoderms possess the capacity of synthesizing sterols, although there may be great differences in the extent to which animals make use of this capacity. Contrary to Ophioderma, Ophiura also possessed sterols with thirty carbon atoms (Table 5). In their paper Goad et al. (1972) do not mention this type of sterols for Ophiura and they give the following composition: C2e, 5.9 per cent; C27, 49.8 per cent; C~s, 27.4 per cent; Cs9, 16.9 per cent. As can be seen agreement is excellent, with the exception of C~6. In Ophiura CaT sterols are the main ones, and in Ophioderma the amounts of sterols with 27, 28 and 29 carbon atoms are almost equal. It is obvious from Table 7 that not cholesterol, but brassicasterol is the main sterol; cholesterol in both cases holding the second place. In Holothuroidea, too, 24-methyl-cholesta-7,22-dien-3fl-ol is the main sterol (Voogt & Over, 1973). On the whole, the composition given here for Ophiura is in good agreement with the chromatogram depicted in the paper of Goad et al. (1972). Gupta & Scheuer (1968) observed six sterols in O. insularia. Summarizing we may say that all sterols reported until now to be present in ophiuroids have also been found in this study, but additionally some others have turned out to be present. The sterol composition of ophiuroids shows great similarity with that of echinoids (Voogt, 1973c); the differences being only quantitative. This similarity emphasizes the relationship that is supposed to exist between Ophiuroidea and Echinoidea.
Acknowledgement--The author is indebted to Mr. J. A. W. van Rheenen for his technical assistance. REFERENCES CAPELLA P., D E ZOTTX G., RXCCA G. S., VALENTINI A. F. S. & JACINI G. (1960) Chromatography on silicicacid of the unsaponifiable matter of fats..7. Am. Oil Chem. Soc. 37,
564-567. CARROLLK. K. (1961) Separation of lipid classes by chromatography on Florisil. J. Lipid Res. 2, 135-141. GOAD L. J., RUBINSTEIN1. & SMITHA. G. (1972) The sterols of echinoderms. Proc. R. Soc. Lond. B. 180, 223-246.
3ff-STEROLS IN SOME OPHIUROIDS
601
GUPTA K. C. & Scrmtnsa P. J. (1968) Echinoderm sterols. Tetrahedron 24, 5831-5837. HAN~a-IAN D. J., DITTMER, J. C. & WARASHINAE. (1957) A column chromatographic separation of classes of phospholipids, ft. biol. Chem. 228, 685-700. LIPSKY S. R., HAAVIKA., HOPPER C. L. & McDIVITT R. W. (1957) The biosynthesis of fatty acids of the plasma of m a n - - I . . 7 , clin. lnvest. 36, 233-244. MACKENNA R. M. B., WHEATLEYV. R. & WOaMALL A. (1950) The composition of the surface skin fat ("Sebum") from the human forearm, ft. invest. Derm. 15, 33--47. MATSUMOTO T. & TAMURA T. (1956) Unsaponifiable matter of echinoderms--III. The sterol components of Ophioplocus japonicus. Nippon Kagaku Zasshi 77, 376-378. Tsm~rsR CH., SZABOE. I. & SMITH N. A. (1970) Separation of gangliosides, corticosteroids and water-soluble non-lipids from lipid extracts by Sephadex columns, ft. Chromatog. 47, 15-19. VAN Dma HO~T D. J., VAN GF~NIP A. H. & VOOGT P. A. (1969) A simplified method for extracting lipids from large quantities of tissue abundant in water. Lipids 4, 300-301. VOOOT P. A. (1971a) Investigations of the capacity of synthesizing 3fl-sterols in Mollusca-V. The biosynthesis and composition of 3fl-sterols in the mesogastropods Crepidula fornicata and Natica cataena. Comp. Biochem. Physiol. 39B, 139-149. VOOGT P. A. (1971b) The biosynthesis of 3fl-sterols and identification of 3fl-sterols. In Experiments in Physiology and Biochemistry (Edited by KERKUT G.), Vol. IV, pp. 1-33. Academic Press, New York. VOOGT P. A. (1973a) On the biosynthesis and composition of 3fl-sterols in some representatives of the Asteroidea. lnt. ft. Biochem. (In press.) VOOOT P. A. (1972b) On the biosynthesis of 3fl-sterols in some representatives of the Echinoidea. Comp. Biochem. Physiol. 43B, 457-463. VOOGT P. A. (1973c) Sterols of some echinoids. Archs int. Physiol. Biochim. (In press.) VOOGT P. A. & OVER J. (1973) Biosynthesis and composition of 3fl-sterols in some holothurians. Comp. Biochem. Physiol. 45B, 71-80. WALTON M. J. & PSNNOCKJ. F. (1972) Some studies on the biosynthesis of ubiquinone, isoprenoid alcohols, squalene and sterols by marine invertebrates. Biochem. ft. 127, 471--479.
Key Word Index---Sterols ; Echinoderms; Ophiura albida ; Ophioderma longicauda.
BIOSYNTHESIS AND COMPOSITION OF 3fl-STEROLS IN THE OPHIUROIDS OPHIURA ALBIDA AND OPHIODERMA LONGICA UDA P. A. V O O G T Laboratory of Chemical Animal Physiology, 40 Jan van Galenstraat, Utrecht, The Netherlands
(Received 13 November 1972) Abstract--1. The incorporation of sodium acetate-l-l*C into some classes of lipids in Ophiura albida and Ophioderma longicauda is investigated. 2. It is demonstrated that the animals utilize the acetate injected for the biosynthesis of various lipid classes, including sterols. 3. It is supposed that probably all ophiuroids and possibly all echinoderms are able to synthesize 3~-sterols. 4. The composition of the sterols is determined. In ophiuroids C27 sterols are not strongly predominant. The main sterol is brassicasterol, followed by cholesterol. These two features ophiuroids have in common with holothuroids. The sterol composition is rather simple and strongly resembles that of Echinoidea. INTRODUCTION IN A SEINESof papers on the composition and metabolism of sterols in echinoderms,
we reported the capacity of synthesizing 3fl-sterols for representatives of the classes Asteroidea, Echinoidea and Holothuroidea (Voogt, 1972b, 1973a; Voogt & Over, 1973) and it was supposed that this capacity should be a general property of all representatives of these classes. This paper deals with the Ophiuroidea. Recently Goad et al. (1972) and Walton & Pennock (1972) showed that Ophiocomina nigra did synthesize squalene, 4,4-dimethyl sterols, 4-monomethyl sterols and 4-demethyl sterols from mevalonate; label in 4-demethyl sterols being restricted to C2~ sterols. Only a few data about sterol composition in Ophiuroidea are available and most of them are from the time before gas-chromatography made its entrance in sterol analysis. Recently Gupta & Scheuer (1968) reported the sterol composition of Ophiocoma insularia, while Goad et al. (1972) reported on Ophiura albida. They agree that cholesterol is the main (or one of the main) sterol(s). Matsumoto & Tamura (1956), on the other hand, did not find any cholesterol in Ophioplocus japonicus. Several investigators indicated fl-sitosterol as one of the main sterols. This paper deals with the sterol composition and the biosynthesis of 3fl-sterols from the acetate of O. albida and Ophioderma longicauda. 593
594
P.A. VOOCT
MATERIALS AND METHODS Specimens of Ophiura albida were collected at the laboratory for marine biology "Laboratoire Arago", Banyuls-sur-mer, France; while specimens of Ophioderma longicauda were collected at "Stazione Zoologica", Naples, Italy. The animals were each injected with an aqueous solution of sodium acetate-l-'4C. The injections were given in the body-disc, close to the gonads. After the injection the animals were kept in running sea water and at the end of the incubation were fixed in ethanol. Data about the experimental animals and the acetate injected are summarized in Table 1. TABLE I ~ D A T A ABOUT THE TREATMENT OF O. albida AND O. longicauda EXAMINED FOR THEIR CAPACITY OF SYNTHESIZING 3ff-STEROLS
O. albida No. of animals Place and date of collection Sodium acetate-l-14C Dosage administered to each animal (in/~Ci) Incubation time (hr)
10 Banyuls-sur-mer 20 June 1967 Philips Duphar (20 mCi/mM)
O. longicauda
1"25
40 Naples 21 June 1971 New England Nuclear Corporation (1 mCi/l'35 mg) 12
72
72
Lipids were extracted from the animals using the procedure of Van der Horst et al. (1969) and then purified on Sephadex G-25 (Terner et al., 1970). The purified lipids of Ophiura were saponified with a solution of 1.5 N potassium hydroxide in 80% methanol, and the unsaponifiable and saponifiable lipids were obtained in the usual way. The unsaponifiable lipids were purified on basic cupricarbonate (Capella et al., 1960) and separated into a hydrocarbon fraction, a crude sterol fraction and the remaining fraction by means of column chromatography on alumina (Voogt, 1971a, b). Sterols were isolated via their digitonides. Phospholipids were removed from the purified lipids of Ophioderma by precipitating them in cold acetone (Lipsky et al., 1957) and by chromatography on silicic acid- Hyflo Super Cel (Hanahan et aI., 1957). The neutral lipids were separated into lipid classes according to Carroll (1961). The sterols from the sterolesters were, after hydrolysis, isolated by means of preparative thin-layer chromatography (TLC). Sterols were purified by chromatographing them thrice on thin-layer plates using as moving-phases consecutively: benzene-diethylether-ethanol-acetie acid (50 : 40 : 20 : 0"2 by volume), isopropylether-acetie acid ( 9 6 : 4 v/v) and benzene--ethylacetate ( 5 : 1 v/v). Radioactivities were measured with Packard Liquid Scintillation Counters, Models 3320 or 2420 in toluene containing 0"4% Omnifluor (NEN Chemicals). Gas-chromatography of the sterols, as their trimethylsilylethers, on the stationary phases SE-30 and NPGS, was performed as described elsewhere (Voogt, 1971c). Sterols were identified by means of their steroid numbers, which were calculated from the formulae given previously (Voogt, 1972d). Hydrogenation and acetylation of sterols was carried out as reported earlier (Voogt, 1971b). RESULTS T h e quantities of the isolated lipid fractions of Ophiura, expressed in m g and as a percentage of the total amount of lipids, are given in Table 2. This table
3 ~ - $ T E R O L S I N SOME OPHIUROIDS
595
TABLE 2---QUANTITIES ANn RADIOACTIVlTImOF THE ISOLATED LIPID FRACTIONS FROM O. albida, ExPItmS~ IN ms, FERCENTAOESOF THE TOTALAMOUNTOF LIPtDS, dis/min per mg AND PERCENTACESOF THE TOTAL RADIOACTIVITYINCORPORATEDINTO THE LIPIDS
Lipid fraction Fresh weight Purified lipids (% fresh weight) (% of total radioactivity administered) Saponifiable lipids Non-saponifiable lipids Hydrocarbon fraction Crude sterol fraction 3~-Sterols (% fresh weight)
mg 30,000 196 0"65
% of total lipids 100
dis/min per mg 1527
% of total radioactivity in lipids 100 1.09
134.4 44-5 22.7 15"0 11-5 0"038
67 25-4 11-6 9"4 7"2
1876"2 88.9 8 113-5 60
83.0 1.5 0"71 0"23
also shows the specific radioactivity of each fraction and the total activity present in these fractions expressed as percentages of the radioactivity incorporated into the total lipids. T h e corresponding values were also determined for Ophioderma. T h e y are s u m m a r i z e d in T a b l e 3. I n T L C the hydrocarbon fraction of Ophioderma appeared to contain some sterolesters, while the sterolester fraction also contained methylesters of fatty acids. F o r this reason the hydrocarbon and sterolester fractions were pooled and hydrolysed in the usual way, yielding 1187.4 m g of unsaponifiable matter. T h i s matter was separated on alumina in 150.8 m g of TABLE
3--QUANTITIES
AND RADIOACTIVITIES OF THE ISOLATED L I P I D FRACTIONS FROM IN r a g , PERCENTAGES OF THE TOTAL AMOUNT OF LIPIDS~ dis/rain AND PERCENTAGES OF THE TOTAL RADIOACTIVITY INCORPORATED INTO THE LIPIDS
O. longlcauda, EXPRESSED per mg
Lipid fraction Fresh weight Purified lipids (% fresh weight) (% total radioactivity administered) Phospholipids Neutral lipids Hydrocarbon fraction Sterolestera Triglycerides Sterol fraction Remaining fraction
mg 557,000 8137.8 1.46
% of total lipids 100
dis/min per mg 9871
~/o of total radioactivity in lipids 100 7"54
2170.0 5937.6 1503"6 1091 "4 1034"8 497-4 1734.6
26 "7 73"0 18"5 13"4 12"7 6-1 21"2
4434 11,861 20,067 2315 5508 498 I2,515
12.0 87"7 37"9 3"1 7"1 0"3 27"2
596
P.A. VOOGT
hydrocarbons, 44.8 mg of sterols and 896.4 mg of a remaining fraction. Specific radioactivities of these fractions, were 1863, 1771 and 9559 dis/min per mg, respectively. Some carrier squalene was added to the hydrocarbon fraction of Ophioderma (22.7 rag) and the squalenedodecabromide was prepared according to Mackenna et al. (1950). Total radioactivity in this material amounted to 9 dis/rain. Part of the hydrocarbon fraction of Ophioderma was chromatographed on a thin-layer plate, coated with silica gel-G and impregnated with silver nitrate, using hexane-diethylether ( 9 : 1 v/v) as the solvent. The zone co-chromatographing with reference squalene showed a specific radioactivity of 967 dis/min per rag. In gas chromatograms of this material performed on SE-52, several peaks were visible; squalene could not be detected, however. Sterols were purified by means of TLC. The results are shown in Table 4. TABLE 4---SPECIFIC RADIOACTIVITY OF THE STEROL8 OF O . albida AND O. PURIFYING THEM BY MEANS OF T L C
longicauda ON
O. longicauda O. albida Before purification After one purification After two purifications After three purifications
60 56
Free sterols Sterols from esters 498 95 50 48
1771 676 -51
A small amount of the sterols of both species was hydrogenated and after acetylation chromatographed on SE-52. The chromatograms showed four or five peaks, corresponding with sterols containing 26, 27, 28, 29 and 30 carbon atoms. The proportional compositions are given in Table 5. TABLE 5 - - P R O P O R T I O N A L COMPOSITION~ ACCORDING TO THE CARBON CONTEN% OF THE
STEROLSOF 0. alblda AND O. longicauda
Species O. albida O. longicauda
C~6
C2v
C2a
C29
C30
2.0 1.5
49.0 33.1
28.9 32.9
17"5 32"4
2.6 --
Sterols as their trimethylsilylethers were chromatographed on SE-30 and NPGS. Representative chromatograms of Ophioderma are depicted in Figs. 1 and 2; percentage composition and tentative identification are given at the foot of the figures. Chromatograms of Ophiura resembled those of Ophioderma and for this reason are not depicted; their data are summarized in Table 6. From the data in
8
7
6
5
4
4"62
3.95
3-52
2.97
2"64
30"20 (30.18) 30.59 (30-58) 31-15 (31" 19) 31 "53 (31"50) 32"04 (32-04)
(29"80)
28"50 29"01 29"80 (29-67)
SN
13"6
3.3
2"8
27-4
24"2
2"1 0"6 23"6
%
10
9
8
7
6
5
4
3
Isofueosterol ?
fl-Sitosterol
Stigmasterol
Campesterol
Brassieasterol
Cholesterol
22-Dehydroeholesterol (trans)
6-17
5"41
4"57
3.91
3-60
2"99
2"64
2"45
2"27
2
RRT 1-59 22-Dehydroeholesterol (eis)
NPGS
1
No.
The steroid numbers of the suitable sterols are given in parentheses.
fl-Sitosterol
Stigmasterol
Campesterol
Brassieasterol
Cholesterol
22-Dehydroeholesterol (trans)
1-58 1-84 2"34
1 2 3
22-Dehydroeholesterol (cis)
RRT
No.
SE-30
FOR T W O DIFFERENT STATIONARY PHASES
T A B L E 6 - - R E L A T I V E R E T E N T I O N TIMES~ STEROID NUMBERS AND P R O P O R T I O N A L C O M P O S I T I O N OF T H E STEROLS OF O .
33.22
30"32 (30"36) 30.75 (30"83) 31"39 (31.43 ) 31-67 (31"72) 32"20 (32-29) 32-77
30-07 (30"09)
29"80 (29.91)
2.4
4-0
10"0
3"6
3"1
27-1
24"9
15-3
7"1
2-5
%
DETERMINED
28-59
SN
albida
u0
O
O
598
P.A. VOOGT
S 3
\
6
FIG. 1. Chromatogram of the T M S derivates of the sterols of O. longicauda after GLC separation on SE-30. The percentage composition and the steroid number of each sterol is given. The calculated steroid numbers of the suitable sterols are given in parentheses. 1. 1% C~6 sterol, 28.51; 2. 5.3% 22-dehydrocholesterol (cis), 29"69 (29"67); 3. 5"6% 22-dehydrocholesterol (trans), 29"80 (29"80); 4. 22"6% cholesterol, 30"20 (30-18); 5.31.5 % brassicasterol, 30.60 (30"58); 6.5"8% campesterol, 31"17 (31-19); 7. 5"3% stigmasterol, 31"50 (31"50); 8.22"8% fl-sitosterol, 32"04 (32"04) or iso-fucosterol (32-02). Figs. 1 and 2 and T a b l e 6 the proportional compositions of the sterols could be drawn up. T h e s e are listed in T a b l e 7. TABLE 7--PROPORTIONAL COMPOSITION OF THE 3fl-STEROLS OF O. albida AND O.
Sterols C26 sterols
O. albida
2"6 (two components) 22-Cis-cholesta-5,22-dien-3fl-ol 7"3 22-Trans-cholesta-5,22-dien-3fl-ol 15.7 Cholesterol 25.2 Brassicasterol 27-0 Campesterol 2"8 Stigmasterol 3"3 ~-Sitosterol 9-9 Isofucosterol ? 3"9 Ca0 sterol 2"4
longicauda
O. longicauda 1"5 (two components) 5"5 6"6 23 "3 30 "9 3"6 5'3 18.5 4'3
3~-ST-~OLS IN SOME OPHIUROIDS
599
5
9
I
3
\ FIc. 2. Chromatogram of the TMS derivatives of the sterols of O. longicauda after GLC separation on NPGS. The percentage composition and the steroid number of each sterol is given. The calculated steroid numbers of the suitable sterols are given in parentheses. 1. 1"3% C~e sterol, 28.61; 2. 0"6% C26 sterol, 28.85; 3. 5.7% 22-dehydrocholesterol (cis), 29-81 (29.91); 4. 7"7% 22-dehydrocholesterol (trans), 30"08 (30"09); 5. 23"9% cholesterol 30"36, (30"36); 6. 30'3% brassicasterol, 30.79 (30.83); 7. 3"6% campesterol, 31.43 (31.43); 8.4"6% stigmasterol, 31.71 (31.72); 9. 17"5% fl-sitosterol, 32.25 (32.29); 10. 4"3% isofucosterol, 32-78. DISCUSSION Tables 2 and 3 show that the lipid contents of Ophiura and Ophiod~rma are strongly different, namely 0.65 and 1.46 per cent of the fresh weight, respectively. The first value and also that of the sterol content of Ophiura is rather low, being only slightly higher than the corresponding values found for Holothuroidea (Voogt & Over, 1973), the latter being well known for their low lipid and sterol content. In both experiments the acetate injected was utilized by the animals for the biosynthesis of lipids. Total incorporation of radioactivity into the lipids amounted to 1.09 and 7.54 per cent of the total dosage administered. All lipid fractions investigated turned out to be radioactive, pointing to an intensive lipid metabolism. The squalenedodecabromide prepared from the total hydrocarbon fraction of Ophiura (with 182 dis/min) possessed only 9 dis/rain, which possibly should be ascribed partly or fully to unsaturated components other than squalene. This
600
P.A. VOOGT
holds also for the radioactivity observed in the squalene fraction of Ophioderma. Here, with certainty, several components were present, but squalene could not be detected. Concluding, one should say that in both animals the presence of squalene is not shown unequivocally and that, for this reason, it is unknown whether squalene is synthesized by these animals or not. Table 4 shows that the sterols of both animals are radioactive, permitting the conclusion that these animals are capable of synthesizing sterols from acetate. Earlier, Goad et al. (1972) and Walton & Pennock (1972) reported that ophiuroids are able to synthesize sterols from mevalonate. From the data available at this moment it seems justified to suppose that all Ophiuroidea can synthesize sterols. The same has been found before for Asteroidea, Echinoidea and Holothuroidea. The fifth class of the Echinodermata, the Crinoidea, has not been studied as yet in this respect. It is likely, however, that all echinoderms possess the capacity of synthesizing sterols, although there may be great differences in the extent to which animals make use of this capacity. Contrary to Ophioderma, Ophiura also possessed sterols with thirty carbon atoms (Table 5). In their paper Goad et al. (1972) do not mention this type of sterols for Ophiura and they give the following composition: C2e, 5.9 per cent; C27, 49.8 per cent; C~s, 27.4 per cent; Cs9, 16.9 per cent. As can be seen agreement is excellent, with the exception of C~6. In Ophiura CaT sterols are the main ones, and in Ophioderma the amounts of sterols with 27, 28 and 29 carbon atoms are almost equal. It is obvious from Table 7 that not cholesterol, but brassicasterol is the main sterol; cholesterol in both cases holding the second place. In Holothuroidea, too, 24-methyl-cholesta-7,22-dien-3fl-ol is the main sterol (Voogt & Over, 1973). On the whole, the composition given here for Ophiura is in good agreement with the chromatogram depicted in the paper of Goad et al. (1972). Gupta & Scheuer (1968) observed six sterols in O. insularia. Summarizing we may say that all sterols reported until now to be present in ophiuroids have also been found in this study, but additionally some others have turned out to be present. The sterol composition of ophiuroids shows great similarity with that of echinoids (Voogt, 1973c); the differences being only quantitative. This similarity emphasizes the relationship that is supposed to exist between Ophiuroidea and Echinoidea.
Acknowledgement--The author is indebted to Mr. J. A. W. van Rheenen for his technical assistance. REFERENCES CAPELLA P., D E ZOTTX G., RXCCA G. S., VALENTINI A. F. S. & JACINI G. (1960) Chromatography on silicicacid of the unsaponifiable matter of fats..7. Am. Oil Chem. Soc. 37,
564-567. CARROLLK. K. (1961) Separation of lipid classes by chromatography on Florisil. J. Lipid Res. 2, 135-141. GOAD L. J., RUBINSTEIN1. & SMITHA. G. (1972) The sterols of echinoderms. Proc. R. Soc. Lond. B. 180, 223-246.
3ff-STEROLS IN SOME OPHIUROIDS
601
GUPTA K. C. & Scrmtnsa P. J. (1968) Echinoderm sterols. Tetrahedron 24, 5831-5837. HAN~a-IAN D. J., DITTMER, J. C. & WARASHINAE. (1957) A column chromatographic separation of classes of phospholipids, ft. biol. Chem. 228, 685-700. LIPSKY S. R., HAAVIKA., HOPPER C. L. & McDIVITT R. W. (1957) The biosynthesis of fatty acids of the plasma of m a n - - I . . 7 , clin. lnvest. 36, 233-244. MACKENNA R. M. B., WHEATLEYV. R. & WOaMALL A. (1950) The composition of the surface skin fat ("Sebum") from the human forearm, ft. invest. Derm. 15, 33--47. MATSUMOTO T. & TAMURA T. (1956) Unsaponifiable matter of echinoderms--III. The sterol components of Ophioplocus japonicus. Nippon Kagaku Zasshi 77, 376-378. Tsm~rsR CH., SZABOE. I. & SMITH N. A. (1970) Separation of gangliosides, corticosteroids and water-soluble non-lipids from lipid extracts by Sephadex columns, ft. Chromatog. 47, 15-19. VAN Dma HO~T D. J., VAN GF~NIP A. H. & VOOGT P. A. (1969) A simplified method for extracting lipids from large quantities of tissue abundant in water. Lipids 4, 300-301. VOOOT P. A. (1971a) Investigations of the capacity of synthesizing 3fl-sterols in Mollusca-V. The biosynthesis and composition of 3fl-sterols in the mesogastropods Crepidula fornicata and Natica cataena. Comp. Biochem. Physiol. 39B, 139-149. VOOGT P. A. (1971b) The biosynthesis of 3fl-sterols and identification of 3fl-sterols. In Experiments in Physiology and Biochemistry (Edited by KERKUT G.), Vol. IV, pp. 1-33. Academic Press, New York. VOOGT P. A. (1973a) On the biosynthesis and composition of 3fl-sterols in some representatives of the Asteroidea. lnt. ft. Biochem. (In press.) VOOOT P. A. (1972b) On the biosynthesis of 3fl-sterols in some representatives of the Echinoidea. Comp. Biochem. Physiol. 43B, 457-463. VOOGT P. A. (1973c) Sterols of some echinoids. Archs int. Physiol. Biochim. (In press.) VOOGT P. A. & OVER J. (1973) Biosynthesis and composition of 3fl-sterols in some holothurians. Comp. Biochem. Physiol. 45B, 71-80. WALTON M. J. & PSNNOCKJ. F. (1972) Some studies on the biosynthesis of ubiquinone, isoprenoid alcohols, squalene and sterols by marine invertebrates. Biochem. ft. 127, 471--479.
Key Word Index---Sterols ; Echinoderms; Ophiura albida ; Ophioderma longicauda.