Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 437 to 44l. Pergamon Press. Printed in Great Britain

BIOSYNTHESIS OF STEROLS IN A STARFISH, LAIASTER LEACHII SHIN-ICHI TESHIMAAND AKIO KANAZAWA Laboratory of Fisheries Chemistry, Faculty of Fisheries, University of Kagoshima, Japan (Received 8 July 1974)

Abstract--1. This study deals with the biosynthesis of sterols from mevalonate in a starfish, Laiaster leachii. 2. After injection of mevalonate-2-14C, the incorporation of mevalonate into the unsaponifiable matters and sterols was investigated by using thin-layer, column, and gas-liquid chromatographic techniques. 3. The results indicated that the starfish is capable of synthesizing squalene, lanosterol, and desmethylsterols from mevalonate, and also that AT-cholestenol is a main desmethylsterol formed under the present experimental conditions. 4. On the other hand, it was suggested that this animal possesses no ability for both alkylation at C-24 and introduction of double bond at C-22 of AT-cholestenol.

INTRODUCTION ECHINODERMS generally contain complex sterol mixtures in their tissues. Advances in analytical methods have clarified the detailed composition of sterols in them. Austin (1970) and Goad et al. (1972) have reviewed the investigations about the sterols of echinoderms. It is generally recognized that asteroids and holothurians usually contain AT-sterols, while ophiuraids and echinoids contain AS-sterols. Plants are capable of synthesizing sterols from lower units, and it is conceived that they differ from animals in the respect that they form C2s- and C29-sterols from C27-sterols by successive transmethylations (Lederer, 1969). However, the occurrence of large amounts of C28- and C29-

sterols in the tissues of asteroids and holothurians conjectures the possibility that a part of C28- and C29sterols may be synthesized by themselves. Several workers have demonstrated that the asteroids, Asterias rubens (Goad et al., 1970; Smith & Goad, 1971a; Walton & Pennock, 1972; Voogt, 1973), Henricia saouinolenta (Goad et al., 1970; Smith & Goad, 1971a), Marthasterias glacialis (Voogt, 1973), Astropecten aurantianus (Voogt, 1973) and Echinaster sepositus (Voogt, 1973), and the holothurians, Cucumaria planci (Voogt & Over, 1973), Holothuria tubulosa (Voogt & Over, 1973), Stichopus regalis (Voogt & Over, 1973) and Cucumaria elonoata (Voogt & Over, 1973) are capable of synthesizing sterols from acetate or mevalonate.

Unsaponifiable matters

I

I Digitonin method J TLC on Silicogel G-Silicagel GF2~4 3~6- Hydroxysterols Rodiooutogrophy' Alumina column chromatography 20-50% Ether in hexone Methylsterols

] 50-90*/. Ether in hexone I Desmethylsterols

I Acetylotion Desmethylsteryl Ac

I Preparative GLC Each peak

I

Scintillation counting

J Silicic ocid.AgNO 3 column chromatography Saturdted steryl Ac

I

Monoene steryl Ac Preporotive ,

Diene stery Ac l

,

Triene stelryl Ac

[

(T) (TT) ( m l

(]~r) (~r}

{X) (~'TI

Fig. 1. The outline of procedures for separation of metabolites and for detection of radioactivity.Abbreviations are as follows: Ac, acetates; A7'22, AV,22-steryl acetates; A7,24, A7.24-steryl acetates; A7'24~2a) A7,24(2a~.stery1acetates; (I), cholestan-3fl-yl Ac; (II), A7-stigmastenyl Ac; (III), A7-ergostenyl Ac; (IV), A7 cholestenyl Ac; (V), cholest-5-en-3fl-yl Ac; (VI), AT'2Z-stigmastadienylAc; (VII), AT'Z2-ergostadienylAc; (VIII), AT'22-cholestadienyl Ac; (IX), AT'22-24-norcholestadienyl Ac; (X) and (XI), unknown steryl Ac; (XII), AT'24t28LstigmastadienylAc; (XIII), AT'24(zS)-ergostadienylAc; (XIV), 24-methylcholesta-7,22,25trien-3fl-yl Ac. 437

438

SHIN-ICH1 TESHIMA AND AKIO KANAZAWA

1.0

Although the sterols of the echinoderms are composed of a number of sterol components, it is obscure whether all sterols are formed by themselves or some sterols originate from exogenous sources. In a previous study (Kanazawa et al., 1973), the authors have elucidated in detail the sterol composition of a starfish, Laiaster leachii (phylum Echinodermata, class Asteroidea). This paper deals with the biosynthesis of sterols from mevalonate in this animal.

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MATERIALS AND METHODS The starfish, L. leachii, 60 g body weight, was collected near Sakurajima in Kagoshima in October, 1973. The animal was injected with 20/~Ci of mevalonate-2-14C (specific activity, 39mCi/mg of DL-mevalonic acid DBED salt; Radiochemical Centre, Amersham, England) into the body cavity from the root of legs and then kept in an aquarium at 18-20°C for l0 days. From the animal injected with mevalonate-2-t4C, the unsaponifiable matters were obtained in the usual manner, and the metabolites were investigated. 3fl-Hydroxysterols were isolated by the digitonin method (Idler & Baumann, 1952). Acetylation was made with acetic anhydride-dry pyridine (1:1, v/v) at room temperature. Thin-layer chromatography (TLC) was carried out by using Silicagel G-Silicagel GF25 + (4:1, w/w) developed with chloroform-ethyl acetate (20:1, v/v)and a mixture of Silicagel HF25 + + 366-silver nitrate (5: 1, w/w) developed with hexane-benzene (5:2, v/v) twice. Analytical gas-liquid chromatography (GLC) was conducted as described previously (Teshima et al., 1971). Preparative GLC was performed by using a column (6ram i.d. × 3 m long) packed with 3~o OV-17 on 80-100 mesh Shimalite W. The column and detector temperatures were 265°C and 285°C, respectively. Trapped samples were subjected to the measurement of radioactivity and to analytical GLC for check of homogenity. Column chromatography on alumina (50g, Merck, Grade II-III) was carried out according to the essentially same method as reported by Dickson et al. (1972). In this chromatography, elution was done with 200 ml each of solvents containing 0, 10, 20, 30, 40, 50, 70 and 90~o of ether in hexane. Column chromatography on a mixture (10 g) of silicic acid and silver nitrate (4: 1, w/w) was conducted by the similar method to that reported previously (Teshima et al., 1973),and elution was done with 100 ml each of solvents containing 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50~ of benzene in hexane; the sterol composition of fractions was monitored by analytical GLC. The detection of radioactivity was made with two methods, scintillation counting and radioautography. Radioactivity was measured with a Beckman Liquid Scintillation Counter, Model LS-230, using a toluene solution of PPO (0"6Yo).The efficiency of counting of radioactive materials was about 90~o. Radioautographs were prepared on a Sakura X-ray film (Konishiroku Photo Ind. Co., Japan) after 3-week exposure. The outline of procedures for the separation of metabolites from mevalonate-2-~4C and for the detection of radioactivity is given in Fig. 1. RESULTS

The unsaponifiable matters (300 mg) isolated from the starfish injected with mevalonate-2-t4C gave high radioactivity (5,000,000 counts/min). An aliquot of the unsaponifiable matters was chromatographed by TLC on Silicagel G-Silicagel GF254 and radioautographed. The radioautogram (Fig. 2) indicated the presence of radioactive metabolites corresponding to squalene (Ry 0.72), AT-cholestenol (cholest-7-en-3fl-ol) (RI 0'28), and three unknown metabolites (Rs; 0.80, 0.41, and

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o o

o d

0.5 0 f

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Fig. 2. Radioautography of the unsaponifiable matters and 3fl-hydroxysterols isolated from the starfish, L. leachii, injected with mevalonate-2-14C. The radioactive samples were subjected to TLC on Silicagel G-Silicagel GF and radioautographed. U and S indicate the radioactive unsaponifiable matters and 3fl-hydroxysterols, respectively, a, the sterol mixture isolated from another starfish injected with no radioactive precursor; b, cholestanol; c, AV-cholestenol; d, cholest-4-en-3-one; e, 5~-cholestan-3-one; f, lanosterol; g, squalene.

0'36). The highest radioactivity was detected in the zone corresponding to AV-cholestenol. However, the zones corresponding to 5:t-cholestane-3-one and cholest-4-en-3-one gave no radioactive spot. From the radioautogram of 3fl-hydroxysterols, one (Ry 0.41) of the unknown metabolites was identified as lanosterol. Furthermore, 3fl-hydroxysterols were chromatographed on alumina with hexane-ether. As shown in Table 1, the radioactivity of 3fl-hydroxysterols was recovered mainly in the desmethylsterol fraction, and small amounts of radioactivity were present in the methylsterols fraction. Smith and Goad (1971a) have demonstrated that mevalonic acid-2-14C injected into the starfish, A. rubens, was incorporated highly into squalene and methylsterols fractions, but relatively poorly into desmethylsterols fraction at 17 and 41 hr after injection. The difference in the incorporation of mevalonate between L. leachii and A. rubens was attributed to the discrepancy in the keeping periods after injection of mevalonate-2-14C. In any case, it seems clearly that the starfish, L. leachii, is capable of synthesizing some desmethylsterols from mevalonate probably via squalene and lanosterol. The analytical GLC showed that the isolated desmethylsterols contained a variety of types of sterols as shown in Table 2. AT-Cholestenol, AV'22-ergostadienol (24-methylcholesta-7,22-dien-3fl-ol), AT-ergostenol (24methylcholest-7-en-3fl-ol), AT'24~28)-ergostadienol (24methylenecholest-7-en-3fl-ol), AV-stigmastenol (24-ethylcholest-7-en-3fl-ol), and AT'24128Lstigmastadienol (24-ethylidenecholest-7-en-3fl-ol) were detected as prominent sterols. The sterol composition of desmethylsterols was almost similar to that of another sample of the sterols from L. leaehii reported previously (Kanazawa et al., 1973). Hence, in order to clarify the approximate distribution of radioactivity in the individual sterols, desmethylsterols were first subjected to preparative GLC, and then the radioactivity of trapped materials was measured with a scintillation

439

Biosynthesis of sterols in a starfish Table 1. Alumina column chromatography of the unsaponifiable matters isolated from the starfish, L. leachii, injected with mevalonate-2J4C Fractions

Solvents

1 2 3 4 5 6 7 8 9

Hexane 10~o Ether in hexane 20~o Ether in hexane 30~ Ether in hexane 40~ Ether in hexane 50~o Ether in hexane 70~ Ether in hexane 90~ Ether in hexane Ether

counter. As shown in Fig. 3, most of the radioactivity was associated with the portion C corresponding to AT-cholestenol, only small radioactivity being found in the other portions. Next, the radioactive desmethylsterols were acetylated with acetic anhydride-dry pyridine, and the acetates (Ac) of desmethylsterols were chromatographed on a silver nitrate-impregnated silicic acid with hexane-benzene. As shown in Fig. 4, the fractions 18-27 and 37-48 gave high radioactivity. The radioactivity and steryl acetate composition of the notable fractions are as follows: fraction 18, 2090 counts/min cholestanyl Ac (100°/0); fraction 21, 864,000 counts/rain a mixture of AT-cholestenyl Ac (63~), AT-ergostenyl Ac (14~o) and AT-stigrnastenyl Ac (23~); fraction 24, 23,600 counts/min a mixture of AT-cholestenyt Ac (33~) and AV'22-stigrnastadienyl Ac (67~); fraction 27, 2000 counts/min AT'22-ergostadienyl Ac (1130%); fraction 40, 20,500 counts/min unknown steryl Ac; fraction 44, 29,000 counts/min unknown steryl Ac. However, the fractions containing A7'22-cholesta dienyl Ac (fractions 30--33), AT'22-24-norcholestadienyl Ac (24-norcholesta-7,22-dien-3fl-yl Ac) (fractions 3436), A7'24(2S)-stigmastadienyl Ac (fractions 50-53), A7.24(2S)-ergostadienyl Ac (fractions 52-55) and 24methylcholesta-7,22,25-trien-3fl-yl Ac (fractions 58-63) gave no significant radioactivity. Moreover, the radioactive substances of the fractions 18, 21, 27, 40, and 44 were chromatographed by

Radioactivity (c.p.m.)

Sterols

0 300 1300 800 5000 2,200,000 31000 3000 611

--Methylsterols Methylsterols -Desmethylsterols Desmethylsterols ---

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0

D~E

I

40

20

RETENTION TIME (MIN)

Fig. 3. Distribution of the radioactivity in the radioactive desmethylsterols isolated from the starfish, L. leachii, injected with mevalonate-2-14C. The radioactive desmethylsterols were subjected to preparative GLC on 3'0~ OV-17, and the portions A, B, C, D, E, F and G were trapped. The radioactivity of each fraction was measured by a liquid scintillation counter.

Table 2. The composition of desmethylsterols isolated from the starfish, L. leachii, determined by GLC Peaks 1 2 3 4 5 6 7 8 9 10 11

Sterols A7'22-24-Norcholestadienol Cholestanol Cholesterol AT'22-Cholestadienol AV-Cholestenol AV'22-Ergostadienol 24-Methyleholesta-7,22,25-trien-3fl-ol AV-Ergostenol A7,24(2S).Ergostadienol A7'22-Stigmastadienol Unknown sterols A and B A7-Stigmastenol A7,24(28)_Stigmastadienol Unknown sterol C Unknown sterol D

Present study

Composition (~) Kanazawa et al. (1973)

1-5 4-4

1.0 4.9

5.0 36"2 15.8 1.1 20.3

3'3 30.2 20.1 2-3 18-6

2.2

2.2

12'1

14-1

2'0 1.3

2.8 0.5

The unknown sterols gave the following relative retention times to cholesterol in GLC on 1-5~ OV-17; unknown A, 1.83; unknown B, 1.93; unknown C, 2.02; unknown D, 2-21. CBPIB)52/3~

440

SHIN-ICHI TESHIMA AND AK[O KANAZAWA

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Fig 4. Column chromatography of the radioactive desmethylsteryl acetate. The radioactive desmethylsteryl acetates were subjected to column chromatography on a mixture of silicic acid and silver nitrate, and the radioactivity and steryl acetate composition of each fraction were monitored. TLC on a silver nitrate-impregnated Silicagel HF254+ 366 followed by radioautography. The radioautograms (Fig. 5) indicated that the substance of fraction 21 gave the same mobility as authentic A7cholestenyl Ac, whereas those of fractions 40 and 44 were more polar than AT'Z2-stadienyl Ac usually occurring in echinoderms and less polar than A 7'24(2 S)_ stadienyl Ac. Also, as to the fractions 18 and 27 a definite spot was not detected in the zones corresponding to authentic cholestanyl Ac and AV'22-ergostadienyl Ac. The fraction 21 was further subjected to preparative GLC in order to separate A%steryl Ac differing only in the length of side-chain. As given in Fig. 6,

TIME

C W'~l 40 (MIN)

Fig. 6. Distribution of the radioactivity in the fraction 21 obtained by column chromatography on a mixture of silicic acid and silver nitrate. The fraction 21 obtained by column chromatography on a mixture of silicic acid and silver nitrate was subjected to preparative GLC on 3.0% OV-17. The portions corresponding to AV-cholestenyl Ac (portion A), AV-ergostenyl Ac (portion B) and AT-stigmastenyl Ac (portion C) were trapped and the radioactivity of them was measured. most of the radioactivity was associated with the portion of AV-cholestenyl Ac. This result shows that the starfish, L. leachii, is capable of AT-cholestenol, the predominant sterol in this animal, but hardly AV-ergos tenol and AV-stigmastenol. From the above data, it was concluded that the starfish, L. leachii, possesses the enzyme systems for biosynthesis of AV-cholestenol from mevalonate. In this animal, however, AT-cholestenol appears not to be undergone the alkylation at C-24 and the unsaturation at C-22. In addition, the present study showed the formation of unkown desmethylsterols from mevalonate. The authors assume that these unknown sterols are probably AT'24-sterol and AT'25-sterol. However, further elucidation of these metabolites was not performed in this study.

DISCUSSION

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3

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Fig. 5. Radioautography of the fractions obtained by column chromatography on a mixture of silicic acid and silver nitrate. The fractions (F-21, F-40 and F-44) obtained by column chromatography were subjected to TLC on a silver nitrate-impregnated Silicagel HF254+366 and radioautographed. 1, cholesteryl Ac; 2, AV-cholestenyl Ac; 3, AT,22-cholestadienyl Ac; 4, Av'24(2a)-stigmastadienyl Ac; 5, AT'24(2a)-ergostadienylAc.

Recently, the sterol biosynthesis has been investigated about a number of marine invertebrates including echinoderms. All echinoderms examined so far, except for a sea-urchin, Paracentrotus lividus (Salaque et al., 1966), and sea-cucumber, Stichopus japonicus (Nomura et al., 1969), have been shown to be capable of synthesizing sterols from acetate and mevalonate. However, since the sterols of echinoderms, especially asteroids and holothurians, are generally complex mixtures, the question is raised whether all or some sterols are de novo synthesized by themselves. As to this question, the informations available are little. Goad et al. (1972) have demonstrated that the starfish, A. rubens, is capable of at least A%cholestenol from mevalonate but not A 7 ' 2 2 - e r g o s t a d i e n o l . On the other hand, Fagerlund & Idler (1960) have shown the bioconversion of cholesterol to AV-choles tenol in a starfish, Pisaster ochraceus. Recently, Smith & Goad (1971b) have reconfirmed the bioconversion of cholesterol to AV-cholestenol in A. rubens and Solaster papposus. Moreover, it has been proved by using A.

Biosynthesis of sterols in a starfish

rubens a n d Porania pulvillus that the above bioconversion proceeds possibly via cholest-5-en-3-one, cholest4-en-3-one, 5~t-cholestan-3-one, a n d 5a-cholestan-3flol ( G o a d et al., 1972; Smith et al., 1972). These reports have shown that some A7-sterols occurring in the tissues of asteroids originate partly from exogenous sources of AS-sterols. In the present study, it was clarified that A7-choles tenol was synthesized from mevalonate in the starfish, L. leachii. However, this animal seems not to possess the ability for b o t h alkylation at C-24 a n d the introduction of double b o n d at C-22 of AT-cholestenol: Therefore, it may be assumed that in the starfish, L. leachii, C27-sterols such as AT-cholestenol are derived by b o t h biosynthesis a n d exogenous sources but C26 , C28 , C29 , a n d C3o-sterols depend o n only diets. Acknowledgements--This work was supported in part by a grant from the Ministry of Education (Japan). The authors thank Miss T. Ozeki for her technical assistance during this study. REFERENCES AUSTIN J. (1970) The sterols of marine invertebrates and plants. In Advances in Steroid Biochemistry and Pharmacology (Edited by BRIGGS M. H.), Vol. 1, pp. 73-96, Academic Press, London. DICKSON L. G., PATTERSON G. W., COHEN C. F. & DUTKY S. R. (1972) Two novel sterols from inhibited Chlorella ellipsoidea. Phytochem. 11, 3473-3477. FAGERLUND U. H. M. • IDLER D. R. (1960) Marine sterols --VI. Sterol biosynthesis in Molluscs and Echinoderms. Can. J. Biochem. Physiol. 38, 997-1002. GOAD L. J., SMITH A. G. & GOODWIN T. W. (1970) Triterpene biosynthesis in echinoderms. J. Am. Oil Chem. Soc. 47, 90A. GOAD L. J., RUaINSTEIN I. & SMITHA. G. (1972) The sterols of echinoderms. Proc. R. Soc. Lond.,B. 180, 223-246. IDLER D. R. & BAUMAr~r~C. A. (1952) Skin sterols--II. Isolation of 7-cholestenol. J. Biol. Chem. 195, 623-628.

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KANAZAWAA., TESHIMAS. & ANDO T. (1973) (E)-24-Ethylidene-cholest-7-en-3fl-ol and other sterols in asteroids. Mem. Fac. Fish., Kagoshima Univ. 22, 21-31. LEDERER E. (1969) Some problems concerning biological Calkylation reactions and phytosterol biosynthesis. Quart. Rev. Chem. Soc. 2,3, 453-481. NOMURA T., TSUCHIYAY., ANDRE D. & BARBIERM. (1969) Sur la biosynth6se des st6rols de rHolothurie Stichopus japonicus. Bull. Jap. Soc. Sci. Fish. 35, 299-302. SALAQUEA., BARBIER M. t~; LEDERER E. (1966) Sur la biosynth6se des st6rols de rhultre (Ostrea #ryphea) et de l'oursin (Paracentrotus lividus). Comp. Biochem. Physiol. 19, 45-51. SMITH A. G. & GOAD L. J. (1971a) Sterol biosynthesis in the starfish Asterias rubens and Henricia saouinolenta. BiDchem. J. 123, 671-673. SMITH A. G. & GOAD L. J. (1971b) The metabolism of cholesterol by the echinoderms Asterias rubens and Solaster papposus. FEBS Lett. 12, 233-235. SMITH A. G., GOODFELLOW R. & GOAD L. J. (1972) The intermediacy of 3-oxo steroids in the conversion of cholest5-en-3fl-ol into 5~-cholestan-3fl-ol by the starfish Asterias rubens and Porania pulvillus. Biochem. J. 128, 1371-1372. TESHIMA S., KANAZAWAA. & ANDO T. (1971) Occurrence of desmosterol and other sterols in the clam, Tapes philippinarum. Mem. Fac. Fish., Kaooshima Univ. 20, 131-139. TESHIMAS., KANAZAWAA. & ANDO T. (1973) Column chromatography of steryl acetates on a silver nitrate-impregnated silicic acid. Mem. Fac. Fish., Kagoshima Univ. 22, 7-13. VOOGTP. A. (1973) On the biosynthesis and composition of 3fl-sterols in some representatives of the asteroidea. Int. J. Biochem. 4, 42-50. VOOGTP. A. & OVER J. (1973) Biosynthesis and composition of 3fl-sterols in some holothurians. Comp. Biochem. Physiol. 45B, 71-80. WALTON M. J. & PENNOCK J. F. (1972) Some studies on the biosynthesis ofubiquinone, isoprenoid alcohols, squalene and sterol by marine invertebrates. Biochem. J. 127, 471479.

Key Word Index--Biosynthesis; sterols; starfish; Laiaster leachii; invertebrates; asteroids; 7-cholestenol.