,p. 499 to 504. PergamonPress. Printed in

OF THE CAPACITY OF IOLLUSCA--XIII. BIOSY OF STEROLS IN SOME (ANISOMYARIA) P. A. VOOGT Physiology, The State University of Utr, The Netherlands

ESIZING AND tES

aan, Utrecht,

(Received 17 December 1973)

Abstract~l. The incorporation of sodium acetate-l- or -2-14( -2-14C into somq myarian bivalves Mytilus edulis, Atrina fragilis and Ostrea edulis edL was inv 2. It was demonstrated that the acetate was utilized for the biosynthes non-saponifiable lipids. aid chromatogra 3. Squalene could not be detected by means of gas-liquid cI 4. Sterols were not synthesized by the animals during the experiments. ex 5. The sterol composition of the animals was determined. Sterols witl~ main ones (45-50 per cent), followed by those with 28 carbon atoms at (35-3~ brassicasterol were quantitatively the most important sterols.

INTRODUCTION MANY investigators have studied the sterols of Bivalvia. The reasons for this interest are quite obvious. First, the animals possess economic value, and second, at the time their sterols were :red to be a rich source of provitamin D considered ,'tually a great deal of the studies on bivalve and actual] sterols was initiated with the aim to indicate new sources of these provitamins. Results have been trized in several reviews (Toyama, 1958; summarized an. 1962; Austin, 1970; Idler & Wiseman, Bergman, ately, most of these 1972; Voogt, 1972a). Unfortunatel le use because they data are out of date and of little ;eparation and suitwere obtained before powerful se re available. tilable. Recent able identification techniques were availal ~,1972a). In the past data have been given by Voogt (1972a). ~fbivalves has been few years interest in the sterols of bi :man, 1971a). This increasing again (Idler & Wiseman ;everal new sterols has led to the discovery of several (Idler et al., 1970, 1971; Idler & Wiseman, 1971b; Teshima et al., 1972). sterol composition Whereas our knowledge of the stere in Bivalvia increased very fast, our knowledge of sterol metabolism is still very fragment~ ~agmentary entary and the results are difficult to interprett because they are contradictory. Fagerlund & Idler (1960) rep ;ported that radioactivity was present in digitonin-] igitonin-precipitable material from Mytilus californianus nus and Saxidomus giganteus after the injection of sodium acetate-2x~C. Most activity was present in AS-monounsaturated sterols, with only traces traces in 24-methyl18

of the anisoonifiable and

ores were the tolesterol and

enecholesl ;holesterol (Fagerlund & Idler, 1961b). 19 They found that Saxidomus giganteus was , able to also foun nthesize 24-methylenecholesterol from fro~r cholesterol synthesize A 2~ (Fagcrlun, ~rlund & Idler, 1961b) and to introduce in It 1961a), and A u ddouble bonds (Fagerlund & Idler, varia of the F r o m their the: study on the seasonal variations 1 et aL sterols of ot Piacopecten magellanicus, Idler that in this animal metabolism (1964) concluded co and syntl ~athesis of cholesterol and 24-methylene24 ~terol were interrelated. T a m u r a , et al. (1964) cholestero obtained indications that Crassostrea is able to synthesize cholesterol and to convert this t into 24methylenecholesterol. Thus all the results resu obtained by Idler and coworkers suggest that in bivalves there is an active sterol metabolism an~ and that these animals are able to synthesize sterol(Cs). O n t h e that other hand, Salaque et aL (1966) reported re after incubation of Ostrea gryphea in i either Lmethionine-5-~4C or DL-mevalonic acid-2-1*C, aci no radioactivity was present in the sterols. Walton & Pennock (1972) concluded from their experiments ;dulis were not able that Cardium edule and Mytilus edulis w to synthesize sterols from DL-mevalon nevalonate-2-uC. In the scope of the long-term pro~ject of our laboratory on the biosynthesis off sterot. sterols in invertebrates, we started investigation of bivalves bival in 1966. Whereas, in the data mentioned above, there is no discrimination between reports on Anisomyaria or Eulamellibranchia (most reports are ar~ concerned with Anlsomyarla), we decided to study both orders separately. This paper deals with some some/~ Anisomyaria, viz. Ostrea edulis, Mytilus edulis and Atrina At fragilis.

499

P. A. VOOGT ments o n t h e i r ld f u r t h e r t h e m e a n s o f gas

)onifiable lipids were mixture in the usual cere separated into a terol fraction and a lumn chromatography

solution. Unsaponit isolated from the s~ way. The unsaponiJ hydrocarbon fractio remaining fraction b: on alumina (Voogt, 3 The hydrocarbon gas chromatograph, columns (180 × 0'38 c W (80-100 mesh, with 1% SE-52. In was hydrogenated in J (Voogt, 1971b) an carrier sq~ualene was and the squalene (Loud & Bucher, l 19( Sterols were isola via their ddigitonides from etha ethanol. Sterc by means of thin-la chromato~graphed tl developme ~ment consecu acetic acid (50 : 40 : 2 acetic acid ack (98 : 2,

)DS ollected on old he Netherlands), om the NetherI, while those of irujl/l..i Wi,l~ i,k/llr,,I.,I,i, ll u~' ll~ll~lllti, U l the tllF JL,O, UUIO," nl l . of Laborar Marine Biology Laboratoire Arago at Banyulsr, France. animals were each injected with an aqueous a of either sodium acetate-lA4C or sodium -2-~4C. Injections were given directly into the pancreas (in the case of Atrina) or into the foot other cases). In the experiment indicated Ostrea aaals were injected on the first, the third and the ty. The animals were incubated in running sea for varying times and then killed. They were at - 2 0 ° C or fixed in ethanol until they were Data about the animals and the radioactive or administered are given in Table 1. Is were extracted from the animals using the are of Bligh & Dyer (1959) with the lipids being ied in a solution of 1"5 N K O H in 80% methanol the usual conditions. In the experiment called I the animals were saponified in toto in this

analysed on a Becker or 2300. The glass lled with Chromosorb m d silanized) coated hydrocarbon fraction ; described previously matographed. Some hydrocarbon fraction oride was prepared crude sterol fraction ,y recrystallizing them [II were also purified graphy, sterols being icagel G using for -diethylet her-ethanolme) di-isopropyletherne--ethylacetate (4 : 1,

v/v). Gas ch chromatogra] ttification of the 3/3sterols were w, perfor n elsewhere (Voogt, 1972b). Radioac Radioactivity wa~' in a Packard TriCarb-Liquuid Scintillation Spectrometer, Model 3315. scinti] The scintillation medium consisted of tolu( toluene containing

Table 1. Data about some bivalves examined ned for their capacity of synthesizing sterols

Mytilus edulis I

Ostrea edulis Atrina fragilis

II

I

II

III

Number of animals 61 100 2 6 8 2 Date of collection 26 April 1967 14 June 1968 21 June 1967 2 Feb. 1966 16 March 1966 25 Nov. 1966 Radioactive preSodium Sodium Sodium Sodium Sodium Sodium cursor (specific acetate-l-14C acetate-2-x4C acetate-l-14C acetate-l-~4C acetate-l-x4C acetate-l-14C radioactivity) (21.6 mCi/mM) (2 mCi/mM) (20 mCi/mM) (20 mCi/mM) (20 mCi/mM) (20 mCi/mM) Dose administered 3.5 1 25 25 25 3 x 25* O,Ci) Incubation time (hr) 120 72 56 6.5 4"5 168 * Explanation in text. Table 2. Quantities and ative weights of the lipid fractions isolated from some bivalves nd relati~ relative bivalve

Mytilus edulis I Fresh weight* (mg) Total lipids (mg); % fresh wt. Unsaponifiable lipids (mg); % fresh wt. Hydrocarbon fraction (mg) Crude sterol fraction (mg) 3fl-Sterols (rag); % fresh wt.

II

354,000 392,000 5091 "7 6127.7 1"44 1.56 515.5 798.0 0.15 0"20 30-0 25.3 389-9 522"0 314.2 448-6 0"09 0"11

Ostrea edulis Atrina fragilis 92,000 533"2 0"58 99"7 0"11 11-2 51"7 16"6 0"02

I

II

III

295,700 242,000 210,000 2799"3t 3321"5 8191"0 3'90 0"95 1-37 964.7 477"1 448.0 0.33 0"20 0-21 18.4 19-3 307.2 231"2 180"0 223"9 179"1 153"6 0-07 0"08 0.07

* Fresh weight, determined led without the shells. "t"Determined by summation tion of ul unsaponifiable and saponifiable lipids.

Sterols in some bivalves---I d fractions isolated from some bivalves a e-l- or -2-"C expressed in dis rain-1 mg-

II

of sodium

~trea edulis

Mytilus edulis I

on

Atrina flag#is

1I

m

4.74×10 s 2.22×108 418"5 1"16

1.11x 10s 2581"1 1.24

¢.44 x 108 3.33 x l0 B 3450.6 1195.0 2.58 2.94

4396 439"0 40"8 106"3 7"6 7"2 6"8

3570"2 576"7 159"2 99"9 8"9 4"1

4397.3 2131.3

1430.9 840.8

695"0 886"7 374.0 212.7 289.0 325 "2

46-5 258"7 264"8 308"0 348 "3

mifiable lipids :arbon fraction ~terol fraction 3Is ~ls after three recrystallizations dis after four recrystallizations dis after five recrystallizations

-diphenyloxazole (PPO) and 50mg 1,4-di-2-(-53xazolyl)-benzene (POPOP) per litre (Hayes et ;6). RESULTS AND DISCUSSION quantities of the isolated lipid fractions were tined and are given in Table 2. This table determined shows that generally the lipid contents in bivalves are low. N, ranging from 1.0 to about 1.5 per cent of ,sh weight. Very different values were found the fresh for Atrina rrina and Ostrea HI. The relative amounts of unsaaponifiable lipids and 3/3-sterols obtained for A trina (Table 2) show that this animal is indeed poor In in lipid, whereas the corresponding values obtained ed for Ostrea III are in good agreement with those of the other animals and ~d suggest that the high lipid content in this case is due to the presence of saponifiable lipids, for examiple glycerides from the depot fat of the animals. The specific radioactivity of each lipid fraction was determined and the results are re shown in Table 3. This table shows that incorrporation of radioactivity into lipids is low. Evenn after the repeated injection of acetate in Ostrea HI--finally to a dose o f 75/zCi per animal and after an incubation time of 168hr--incorporationn of radioactivity into the lipids was no higher than 2.94 per cent. In all cases both saponifiable and aad non-saponifiable lipids were radioactive, allowin ing the conclusion that the animals are able to synthesize nthesize these lipids from acetate. The hydrocarbon fractions were 'ere analysed on the stationary phase SE-52. In thee chromatograrns a series of peaks--probably representing esenting a series of homologous hydrocarbons--was present, but squalene could not be detected :ted, neither could squalane in chrornatograms o f the hydrogenated hydrocarbons. Some carder s(qualene was added

325"0 109.9 51 "4 8"4 6"2 5"8

0

to the hyrdrocarbor and squalene hexahydrochl( ,drochloride was 1 ~is product appeared tO be no non-radioacl Leans that squalene was not synthes the experimental conditions. Table 3" shows that in contrary to the tl sterols of Mytilus, Atrina and Ostrea I, those tof Ostrea II and I I I aare rather radioactive. The sr ~ecific radioactivity' ol of these sterols increased on recrystalliTation rec~ and had not yet reached a constant value even after four fotn or five recrystallizations, At t the same time, the melting point of t h e sterols sterol decreased. The following follc melting points were observed c for I Ostrea II: after one recrystallizati( allization 136.3°C, after two 129.3°C, after three 128.7°~ 1.7°C and after five 121.8°C. This indicated that a radioactive component, which was not necessary a sterol, was enriched. F o r this reason the sterols of ¢ Ostrea HI were also purified by means of thin-layc eer chromatography. The initial specific radioactivilLy amounted to 289 diss/min per rag. After chromatography chrc in toluene--diethylether-ethanol-acetic acid specific radioactivity had decreased to 107 diss/~/min per mg. After an additional purification with tolueneethylacetate the specific radioactivit3 Ly amounted to 75 diss/min per nag and after a third purification in disopropylether-acetic acid this value was 63 diss/min per rag. This also confirm( :onfirmed that purification of the sterols of Ostrea was difficult. dt That is why the sterols were subjected to preparative gas-liquid chromatography (PGLC). Now radioactivity was eluated from the column before any of the sterols appeared. Further sterols were hydrogenated and their acetates purifie~ rifled via PGLC. This yielded the same results, viz. radioactivity was not concurrent with the sterols. F ] r o m this it can be concluded that in Ostrea III, too, sterols are not radioactive. Sterols of OstJ ~strea H were not available to subject them to PGLC, PGL but it is

P. A. VOOGT d have shown

111 I

~r experiments into sterols f r o m sodium vhere initially tctivity turned sterols. Thus those of Idler t biosynthesis 'fornianus and gtrea vwginica but are m Iull ull agreement ement ose o f Salaque et al. (1966). Yet we are not 1 to conclude that anisomyarian bivalves t capable of synthesizing sterols because fiosynthesis may proceed at an extremely low these animals. However, experiments of nd his coworkers lasted for a m u c h longer nd even then mostly only indications for fiosynthesis were obtained. nail a m o u n t of the sterols o f each species tdrogenated and after acetylation analysed stationary phase SE-30. In all c h r o m a t o five peaks were present, representing sterols • carbon content ranging from twenty-six :y carbon atoms. The proportional composiff the sterols were calculated and are given ill)l~ le 4.

Table 4.. Proportional composition, according to carbon content , of the hydrogenated steryl acetates of some bivalves C+,+

C27

C28

C~9

Cao

9.0 3"7 7"5

45.0 50"2 49"6

37-3 35.7 37'5

8.5 9.7 5.2

0.2 0.8 0.2

Mytilus edulis Atrinafragilis Ostrea edulis

6

rivates of the sterols Fig. 1. ChromatograJ Ch tion on the stationary tus edulis afteJ of Mytilus omposition and the phase SE-30. The steroid number nu ~, given. The steroid of e numbers of o the suital given in parentheses. •7 ~ 22-dehydrochol1. 5.9% C2G ( sterol lesterol 30.21 (30-18); esterol 29.85 29.', (29"80); 4. 31-5% brassicasterol 30"58 (30"58) or desmosterol (30"50); 5. 9"9~ campesterol 31"11 (31.19) or 24methylenecholesterol (31.07); 6. 1'5~ stigmasterol or 2831.50); 7. 2.3~ fl-sitosterol 32.06 (32.04) ( 31.52 (31.2 isofucosterol (32.02). stationary phases terols were analysed on the stati The ste SE-30 and N P G S as their trimethylsilylethers. ~ecles resembled C h r o m a t o g r a m s o f the three specie Chromat( each other very closely. F o r this reason only representative c h r o m a t o g r a m s of Mytilus are ~ortional comdepicted (Figs. 1 and 2). The propo

~roportional composition of the sterols of some bivalves Table 5. Pro Sterols C2e sterols

Mytilus edulis 5"0"

A trina fragilis 3"6"

Ostrea edulis 7"5*

Cholest-5-en-3/3-ol Cholesta-5,24-dien-3/3-ol

9.0 34"0 10'5

7"8 11.2 31.1 0'9

3-6 11.6 33'2 ?

24-Methyl-cholesta-5,22-dien-3/3-01 24-Methyl-cholest-5-en-3/3-01 24-Methylenecholest -5-en-3 fl-ol

21.5 3"0 9.0

23.2 1.8 7.3

24.7 2.6 9.5

24-Ethyl-cholesta-5,22-dien-3/3-ol 24-Ethyl-cholest-5-en-3/3-ol 24-Ethylidenecholest-5-en-3/3-01

3-0 3'5 0"3

2.7 5"1

1.5 3'4 1"7

Unidentified

0-3

5"4

0.4

22-cis-Cholesta-5,22-dien-3 /3-ol 22-trans-Cholesta-5,22-dien-3/3-ol

* Two components.

1 '0

Sterols in some bivalves--I

7

~

Chromatogram of TMS derivatives of the sterols ilus edulis after GLC separation on the stationary NPGS. The percentage composition and the number of each sterol are given. The steroid 7s of the suitable sterols are given in parentheses. /o C2~ sterol 28.60; 2. 1"07o 22-cis-dehydrochol29.87 (29.92); 3. 8.7y0 22-trans-dehydrocholesterol ~30.09); 4. 33.0% cholesterol 30.34 (30.36); 5. brassicasterol 30.77 (30.83); 6. 13.5% desmosterol (31.15) or campesterol (31-43); 7. 12.0% 24mecholesterol 31.72 (31.67) or stigmasterol x. . . . /,; 8. 4"0% /3-sitosterol 32.25 (32.29); 9. 0"3yo 28isofucosterol 32.78 (32.79).

position n of the sterols was calculated and is summarized in Table 5. Tables les 4 and 5 show the strong resemblance in the sterol composition of these bivalves, desmosterol being the sole component which is present in widely different amounts. The composition of vith that of Mytilus Mytilus is m good agreement with an (1971), although edulis given by Idler & Wiseman the latter authors do mention some lesser com)n atoms. The Css ponents with 28 and 29 carbon [3, are identical with sterols given in Table 5 probably 22-cis- and 22-trans-24-norcholesta-5 ~sta-5,22-dien-3fl-ol. Possibly trace a m o u n t s of desmosterol 3osterol are present in Ostrea edulis. I n each of the three aree sspecies (mostly) a minor component was presentt that could not be identified. It is striking that in bivalves C~7 sterols do not hold the predominant position that they have in most other molluscs. Especially has cholesterol, which in most other ter molluscs makes otal sterols, become up for over 50 per cent of the total less predominant. At the same time brassicasterol has become more important. This suggests that esterol in 31in brassicasterol might replace cholesterol h some of it functions. I n general, we can say that in comparison to other molluscs there is a strong shift from mono-unsaturated sterols towards lrds di-unsaturated ones. It would be interesting to, know whether this p h e n o m e n o n is related to the higgh content of polyunsaturated fatty acids in bivalves ~s (Voogt, 1972a).

AUSTINJ. 0970) The plants. In A d ~ Pharmacology (Ed pp. 73-96. AcadeE BERGMANN W. (19( distribution. In C( FLORKIN M. and Academic Press, N BLIGH E. G. & DYEJ total lipid extractiol Physiol. 37, 911-91 FAGERLUN U. H. FAGERLUND sterols--VI. Stere cchinod( 3derms. Can. FAGERLUND U. H. 1~, FAGERLUN sterols-sterols--VIII. In side cha chain by a ch

fine invertebrates and id Biochemistry and 6s M. H.), Vol. 1, York. their structure and )chemistry (Edited by Vol. 3, pp. 103-162. ) A rapid method of ion. Can. J. Biochem. 3. R. (1960) Marine s in molluscs and ~hysiol. 38, 997-1002. ). R. (1961a) Marine lations of the sterol 8iochem. Physiol. 39,

505-509.

FAGERLUNI U. H. I~ FAGERLUND

). R. (1961b) Marine sterols-sterols~IX. Biosy .methylenecholesterol in clams~. Can. J. . ioL 39, 1347-1355. HAYESF. N., ROGER aCHAM W. H. (1956) Countin:Lgsuspensio ntillators. Nucleonics 14, 48-51. IDLERD. R., ] SAFE L DONALD E. F. (1971) A new CZ-30 sterol (i enecholest-5-en-3fl-ol (29-mett (29-meth disofucos( t zu-memyllSOlUCOsterol). ~terolas 18, Its, 545-553. 34 IDLER D. R., ] TAMURAT. & WA~AX T. (1S (1964) Seasonal variatio~ ans in the sterol, fat and unsapo: ~onifiable components of scallop muscle. J. Fish Res. Bd Can. 21, 1035-1042. IDLER D. 1R. & WISEMANP. (1971a) Sterols Sterols of molluscs. Int. J. Biochem. 2, 516-528. IDLER D. R. & WISEMAN P. (1971b) Ide~ Identification of 22-cis-c[ 22-cis-cholesta-5,22-dien-3fl-ol and other s .~rscallop sterols chromatography and mass rr by gas-liquid gasspectreBiochem. Physiol. 38A, 581-590. 58 metry. Comp. ( Mollus~ sterols: a IDLERD. R. I & WISEMANP. (1972) Molluscan 385-398. review. J. Fish. Res. Bd Can. 29, 385-3~ IDLERD. R., WISEMANP. M. & SAFE L. M. (1970) A new marine sterol, 22-trans-24-norchole ~ta-5,22-dien3fl-oi. Steroids 16, 451--461. LOUDA. V. & BUCH~RN. L. R. (1958) The Th turnover of squalene in relation to the biosynthesis of o cholesterol. J. biol. Chem. 233, 37--41. SALAQUEA., BARBIER M. & LEDERER E. ((1966) Sur la biosynth6se des st6rols de l'huitre (Ostrea (Osl gryphea) et de l'oursin (Paracentrotus lividus). Co~ Comp. Biochem. Physiol. 19, 45-51. TAMURAT., TRUSCOTTB. & IDLER D. R. (1964) Sterol metabolism'in the oyster. J. Fish. Res. Bd Can. 21, 1519-1522. (1972) A C26-sterol TESnnaAS., KANAZAWAA. & ANDOT. (1972 Biochem. in the clam, Tapes phillippinarum. Comp. Cot Physiol. 41B 121-126. TOYAMAY. (1958) Die Sterine der fetten Ole von wirbellosen Wassertieren. Fette Seifen Anstrichmittel • 60,909-915. VOOGT P. A. (1971a) Investigations of the th capacity of synthesizing 3fl-sterols in Mollusca--V. The biosynthesis and composition of 3fl-sterols iin the mesogastropods Crepidula fornicata and Na Natica cataena. Comp. Biochem. Physiol. 39B, 139-149.

P. A. VOOGT )f 3~-sterols and riments in Phyy KERKUT G.), w York. :omponents and Zoology (Edited 7, pp. 245-300. sterols of the rrbustorum, and -496.

WALTONM. J. & P on the biosynthesis squalene and sterol J. 127, 471-479.

(1972) Some studies e, isoprenoid alcohols, tvertebrates. Biochem.

Key Word Indexsterols of bivalves; Ostrea edulis.

mthesis in molluscs; 'ulis; Atrina fragilis;