p. 505 to 510. PergamonPress. Printed in t

OF THE CAPACITY OF IOLLUSCAmXIV. BIOSYI OF STEROLS IN SOME ULAMELLIBRANCHIA) P. A. VOOGT Physiology, The State University of Utr, The Netherlands

~SIZING ; AND eES

aan, Utrecht,

(Received 17 December 1973) Abstract--1. The incorporation of sodium acetate-l- or -2-14Cor DL-meva classes of the eulamellibranchianbivalves Anodonta cygnea, Cardium Cat edule arenaria was investigated. 2. It was demonstrated that the animals were able to synthesi rnthesize both sal able lipids. The presence of squalene could not be shown, however. howq 3. Sterols were non-radioactive after the administration of mevalonate i sodium acet radioactivity was present in the sterols after the injection of s, had lasted for 120 hr. 4. Sterols were radioactive after the injection of sodium acctate-2-14C. acel 5. Fifty per cent or more of the radioactivity in the sterols was presev twenty-seven carbon atoms. 6. It was concluded that the eulamellibranchian ulamellibranchianbivalves arq are able to s, alkylate these sterols to C~a and C29 ones. 7. The sterol composition of the animals was determined ant and was coml; myarian bivalves. A

INTRODUCTION revious paper (Voogt, 1975) we discussed the IN A prewous controver~ versy in the literature concerning the biosynthesis sis of sterols by bivalves. Idler and his coworkers ts (Fagerlund & Idler, 1960; Idler et aL, 1964; Tumura et al., 1964), in studying anisomyarian bivalve, ,~s, concluded that these animals are able to synthesize sterols. From their work it can be deduced that in addition these animals are able to terols by alkylation. transform Cs7 sterols into C28 sterols On the other hand, Salaque et! al. (1966) did not succeed in demonstrating any off these these capacities in the oyster Ostrea gryphea. We Ve also studied the capacity of synthesizing sterols in in anisomyarians by means of radioactive precursors but in all cases the isolated sterols were non-radioactive lctive (Voogt et al., 1974). th reference to the A similar situation exists with Eulamellibranchia. The findings of Idler and his co960, 1961b) for the workers (Fagerlund & Idler, 1960, ~xidomua eulamellibranchian bivalve Saxidomw mus giganteus were similar to those for the anisomyadans. I n addition, they showed that thiss animal is able to introduce ASa and A25 double bond.~ bonds into the side chain of sterols (Fagerhmd & Idler Idler, 1961a). However, Walton & Pennock (1972) could not show the biosynthesis of sterols in Cardium edule. This paper deals with our experiments eriments on the biosynthesis of sterols in the eulamellibr, amellibranchian bi-

Ire some lipid dica and Mya non-saponifiaC, but some n incubations are discussed. th more than gterols and to t of the aniso-

valves AJ Anodonta cygnea, Cardium edule, e& Cyprina the precursors islandica and Mya arenaria using the sodium acetate-l-uC, sodium acetate-2-x4C, acetatt and DL-mevalonate-2-uC (DBED sal0. Further, the composition of the sterols was determined by means of gas-liquid chromatograpy. MATERIALS AND METHODt gTHODS Cardium edule were Specimens of Anodonta cygnea and Cardil and at collected in the neighbourhood of Utrecht, Utn Kattendijke (Zeeland), respectively. Specimens of Cyprinaislandica and Mya arenaria were obtained c from the Netherlands Institute of Sea Research search aat Texel. The animals were each injected with an aqueous solution of either sodium acetate-l- or -2-x4C(Philips (Ph Duphar) or vL-mevalonic acid-2-uC (DBED salt, NE NEN Corp.). In the case of Anodonta and Cyprina, a small smaJ part of the shell was removed and injections were giver :iven directly into the hepatopancreas. Injections into Mya were placed between the ligament and the exhalant sipl,hon; those in Cardium were placed at random by forcing the needle of the syringe between the shells. The specimens of Anodonta were injected on the first, second secon and fourth days. The animals were incubated in well-aerated well, water for different times and then merificed. The~ywere stored at -20°C till they were taken into process ~rocess. Data about the animals and the radioactive precursors administered are shown in Table 1. Lipids were extracted from the animals t nimals using the procedure of Bligh & Dyer (1959) or its ~smodified modifi form after

5O5

P. A. VOOGT e saponified in a I under the usual ~odonta and Mya his solution. g and purifying he anisomyarian gt, 1 9 7 5 ) .

ranged from twent,. proportional coml: chromatograms an

r carbon atoms. The calculated from the Fable 5. r trimethylsilylethers and NPGS. Chro;embled those of the

Sterols were ana on the stationary 1 matograms obtaim

ne bivalves examined for their capacity o

Cardium edule cygnea

I

I

Cyprin III

10 14 July 1966

.ctive precursor ~pecific radioity

Sodium Sodium Sodium Sodium Sodium acetateacetateacetateacetateacetat 1-I4C 2-14C 2-14C 1-14C 1-14C 20 20 2 2 20 mCi/mM mCi/mM mCi/mM mCi/mM mCi/m

dministered to animal (t~Ci) tion time (hr)

3 × 12.5" 120

152 18 April 1967

103 26 June 1968

1

2

1

48

48

48

Mya arenaria

I

animals f collection

163 14 June 1968

sterols

I

9 17 Marc] 1966

II

10 21 17 March 19 Sept. 1966 1969 ~odium Sodium acetateacetate1-14C 1-14C 2O 2 mCi/mM mCi/mM

12.5 4.5

12'5 4'5

10 120

* Explanation in text. RESULTS The quantities of the isolated lipid fractions are listed in Table 2. radioactivity of each lipid fraction was deterThe radioactivit mined and the results are shown in Table 3. ols of Anodonta and Mya II were also purified Sterols arts of T LC as described previously (Voogt, by means et al. 1974). The results are given .,n in Table 4. ms were analysed by The crude hydrocarbon fractions ionary phase SE-52. gas chromatography on the stationar lot be demonstrated, The presence of squalene could not however. :lrogenated and after Samples of the sterols were hydrog, acetylation chromatographed on SE-30. Sterols

anisomya mrian bivalves. The calculated proportional ~osition of the sterols is given in T~ Table 6. composit Sterols of Anodonta, Cardium III and Mya II were hydrogenated and after acetylation subjected to )hy (PGLC) on preparati preparative gas-liquid chromatograph3 ca content SE-52; the th components with different carbon "adioactivity in each were trapped separately. The radioact fraction was measured. The results are summarized in Table 7. The total sterols of Cardium III were fractionated twice by means of PGLC. The total activity was 'sis of the almost equal to that in the analysis t hydrogenindividual ated sterols, viz. about 300 dis/min. The T from sterols, which were not completely separated sel •adioactivity in each each other, were trapped and radioacl

bivalves ;) and relative weights of the lipid fractions isolated from some bi Table 2. Quantities (in mg)

Cyprina islandica

Cardium edule Anodonta cygnea Fresh weight* Total lipids (% fresh wt.) Unsaponifiable lipids (% fresh wt.) Hydrocarbon fraction Crude sterol fraction 3/%Sterols (% fresh wt.)

800,000 358'0 0-04 88"0 82'5 0"01

I

196,000 1886"3 0"96 298"1 0"15 8"8 249"8 71"9 0"04

II 115,000 1175"7 1.02 195"5 0-17 12'7 134"4 40"7 0"04

* Fresh weight determined without the shells.

III

I

125,000 680,000 1 0 3 7 . 5 3271"1 0"48 0'83 536'4 860.0 0'43 0-13 140.5 119'4 0.10

228-8 183"9 0"03

II 160,000 1431"6 0"89 242-2 0.15 6"5 84"1 76"5 0"05

Mya arenaria I

II

262,800 1,278,000 5601-4 0.44 134"8 877.3 0"05 0.07 24-4 20"6 566.3 11-7 214.5 < 0.01 0.02

Sterols in some bivalves--II an of a radio-

d fractions isolated from some bivalves aJ ve precursor expressed in dis/rain per nag Cardium edule I

H

Mya arenaria

Cyprin~ III

I II ~.-1-14C)* (A.I-I'C)*

I

.l~,C)* (A-2-14C)t (A-2-1•C)t (A-I-I~C) 9x

6"749 x

2"286 x

2"498 x

)s

10a

l0 s

l0 s

3234

3740

1'69 ge administered) fiablelipids ~nifiablelipids ~arbon fraction sterolfraction 'ols 'olsafter three

2092.7 1984.6 2678.2 218-2 193.1

,stallizations •ols after four ,stallizations •ols after five ,stallizations

0"65

4943

810 45 336 9

75.8

26.8

9

5512"2

775 x l0 s

4.662 x l0 s 1690 0"11 2"03

566"1

2"50

0"8"

3121 2730 456 1183 330

9769"0 1374"7 40"5 907"5 113"2

865"0

396

122"8

65 "9

400

127"7

71 "5

424

128'9

66"7

1541"3 1781 '8 2711"1 352"6 8"1

1036"4 72"3 1"3

762"9 90"0 152"1 92"3

• Acetate.l.14C. ~-Acetate-2-x~C. :I:Mevalonic acid-2-14C (DBED salt).

,n was determined. The results are shown in 8. DISCUSSION id contents in eulamellibranchian bivalves are Lipid Values ranging from 0.8 to 1.0 per cent of the low. Values fresh weight were observed (Table 2), which is distinctly lower than the contents recorded for anisomayarians (Voogt, 1975). This holds also with reference to the sterol content, because values is/min per mg) of the Table 4. Specific radioactivity (dis/rain 3fl-sterols of Anodonta cygnea and Mya arenaria on purification via TLC A. cygnea M. arenaria

158 37

Crude sterols 193 Specific radioactivity of the 39 sterols after development in toluene-cthylacetate (4: 1, v/v) Specific radioactivity of the 31 hydrogenated sterylacetates after an additional development in hexane--diethylcther (90: 10, v/v)

27

yen less) of the value obtained o b t a i n e d here are o: for anisomyarians. Extremely low lipid lil contents ~resent in A. cygnea and M. arenaria. ar This were pre~ be due to a difference m i processing might partially pa~ since Ant nodonta and Mya I were sapon ~onified in toto, result in a reduced yiel :ield of lipids. which might m u., way and However,, Mya II is processed in the usual yields are only slightly higher. of radioactivity incorporated incol into The percentages pe~ rids show rather large variatior 'ariations (Table 3). total lipit only for 4.5 hr Cyprina I and Mya I were incubated or pe of and this is reflected in the lower percentages incorporation. Values of about 2 per ¢~nt are fully •ed in anisomyarians comparable Lblewith th, those encountered ar (Voogt, 1975). It is not quite clear whyy Cardium II, which possessed the highest lipid d content conte (Table 2), exhibited a reduced incorporation of radioactivity. r Of course, an obvious hypothesis would be that lipid biosynthesis is inhibited by the lipids already alr~ present, but this is rather speculative, because, lipid content is not at all high. Furthermore, Anodanta, Anoa with a very low lipid content, shows the ~e lowest lowe., incorporation of all the animals investigated ~d even after 120 hr. As could be expected, incorporatk ~oration of radioactivity into lipids from mevalonate is sstronger than from acetate.

~omposition, according to carbon content, of the sterols of some mebivalves bivalx Table 5. Proportional corn Species Anodonta eygnea Cardium edule Cyprina islandica Mya arenaria

C~6

C~

C2s

C2.

Cao

Trace 3.2 6.8 3-0

53.0 41-4 48.3 52.3

27.0 42.4 36-0 34-9

18.9 13.2 8.6 8.1

Trace Trace 1.7

P. A. Voo~r rtional composition of the sterols of som Anodonta cygnea

Trace 1 '9

4"2 47.4 --

Lyl-cholesta-5,22-dien-3fl-ol Lyl-cholest-5-en-3fl-ol tylenecholest-5-en-3fl-ol

18.5 8"5

L-cholesta-5,22-dien-3fl-ol I-cholest-5-en-3/3-ol lidenecholest-5-en-3fl-ol fucosterol) ol

8'5 10"2

--

--

Radioactivity (dis/min) in the various fractions :erols of Anodonta, Cardium III and Mya II after preparative gas-liquid chromatography action

A. cygnea

C. edule III

6 29 9 16

~'~27

C28 C29

M. arenaria II

26 50 64 189

5 19 3 13

Table 8. Radioactivity (dis/min) in the various fractions of the sterols of Cardium III after PGLC Sterols C~e sterol C2~A5,~ C27&5 C 2 8 A 5'22 + C27 A5'24

C~aA5+ C~sA5'~4c~a~+ C29A5'~2 C29As + C ~ 9 A 5 , 24~s~

First analysis 4 7 37 14 150 57

Second analysis 4 7 33 33 158 34

From Table 3 it can be concluded ded that the experimental animals are able to synthes hesize both saponifiable and non-saponifiable lipids. Generally, the crude sterol fraction :action possessed a higher specific radioactivity thann the hydrocarbon fraction. However, in Cyprina I and M y a I, which had been incubated only for 4.55 hr, hydrocarbons were labelled higher than the sterols. ~rols. This suggests that sterol biosynthesis proceeds ver very slowly and that possibly most activity is still incor ;orporated in intermediates of sterol biosynthesis. For this reason the hydrocarbon fractions were examined by gas chromatography for the presence of squalene, but it could not be detected. After the te addition of some carbon fraction of carrier squalene to the hydrocarbon :ochloride was preCyprina I the squalenehexahydrc

Cardium edule

4"2 (two compc nents) 0.7 6.9 24-9 5.0

a a

po-

Mya arenaria

3"4 (two components) 0"6 9"5 38'1 4"0

22.0 8.1 13"0

13"2 7.7 16.6

5"2 8.2 2"1

2"0 3-9 1"0

Trace

Trace

pared. This showe~ radioactivity of 2.9 dis/min per mg. TP concluded that the high radioactivity radio ot rbon fraction is not associated with squ~ re radioactive, but All cru,de sterol after isolation isola of the 3fl-sterols via their theil digitonides and eventual event~ purification via TLC (Tab "Tables 3 and 4), the pictun ~icture was less clear cut. Yet, some sore points are clear. Thus Th~ Cyprina did not synthesize sterols from mevalonal which is in accordance with mevalonate, wit] the results clue et al. (1966) for Ostrea gryph~ gryphea (an anisoof Salaque 'ennock (1972) for myarian!) and of Walton & Pennock Cardium edule. Furthermore, none of the animals rnthesize sterols from acetate-l-X4( Lte-l-x4C, unless the did synthe incubatior time was very long (120 hr, Anodonta incubation A and Mya II). However, sterols were indeed radioactive, when acetate-2-t4C had been adminis administered. This throws new light on the controversy in the tt literature: We were able to confirm, on the on e hand, the results of Salaque et aL (1966)and of o Walton & evalonate-2-14C or Pennock (1972) by using mevalona acetate-l-14C and on the other, the results of Fagerlund & Idler (1960).* This difference differel could be easily explained by assuming that radioa ctivity in the sterols was not due to a biosynthesis de novo of these sterols, but to a modification of pre-existing pre-exit sterols, for instance by alkylation at C24. The methyl n group of acetate might be preferentially utilized utili for this alkylation rather than the carboxyl group. gro~ To confirm this hypothesis, the sterols of Cardium Cart II were degraded according to Cox et al. (1958). (19 In this method the side-chain is cleaved from the tt nucleus of the sterol molecule. Indeed, specific :ific rad radioactivity of the nucleus part was lower than migh ;ht have been expected after de novo biosynthesis. To obtain more information sterols were hydrogenate enated and after acetylation analysed on the stationary 3' Iphase SE-30. * By using acetate-2-14C.

Sterols in some bivalves---H tp about half of old the second ing to carbon sble with those 'es. Sterol corndifferent from Css sterols are nt is lower and

28-isofucosterol a Goad et al. (1972) formation of play scheme stigmasterc ducts. If this schel accumulation of r understandable. Summarizing, w, neither biosynthesi~ demonstrated, but is a modest biosy~ capacity, however, by the c~ capacity of prompt two 1 new capacitie,~ )acities of synth, observed in anison mental conditions c~ ence ? Second: significan nificance of alky: functions which ca instance, stigmastei as yet, bu but an inves~ the physiological sterols.

y in stigmasterol. ~veral routes for the ,' chains. I n their ae possible end prolid for Cardium, the into stigmasterol is at in anisomyarians m of sterols could be lellibranchians there ~vo of sterols. This in a number of cases erols. These results ~'irst: why are the lkylating sterols not this due to experia systematic differthe physiological ? Are there essential :complished by, for ~¢ers cannot be given 3ceeding to establish of the individual

donta, Cardium reparative gascrtroma~ogrttpny. I SOle i snows ws the me distribuUlStrlouf radioactivity between the various sterols. I n nta and Mya (both have been incubated for ) almost equal amounts of radioactivity are tt in C~7 sterols and C~s+C~ sterols. We can tde from this that those animals are able to ~size C~7 sterols at a low rate and that they, lants, are able to alkylate these sterols. As y in C~ sterols is higher than in C~a ones (both solute and specific activity) it is probable that :thyl group used for alkylation was radioactive, 'dium III, in which acetate-2-x~C had been used, even more pronounced. The amount of radioy in C~s + C ~ sterols is five times more than in Acknow Acknowledgements wishes to thank Dr. .,7 sterols. Here, too, biosynthesis and alkyla- R . C . H . l v i . ~ ] u u u j d . l l ~ O . l l U . I J l . I . ] . J . v t u l M. Oudeja • J. van der Horst for :e performed by the animal, their contribution conU to the work on Cardium edule. sterol compositions were determined (Table O. The ~ey show a very close resemblance to those of REFERENCES the Anisom' nisomyaria (Voogt, 1974) and the comments given there there also hold here. Compared to the Aniso- BUGH BLIGHE. tO. 86DYERW. J. (1959) A rapid r~ dd method of total myaria the content of 24-methylenecholesterol lipid extraction ex and purification. Can. Can J. Biochem. Physiol. 37, 911-917. is somewhat ~ewhat higher in EulameUibranchia, whereas 22-cis-choles cholesta-5,22-dien-3fl-ol is somewhat lower. Cox J. S. G., HIGH L. B. • JONESE. R. ]H. (1958) The determination of steroid side chains. Proc. Pr, chem. Soc. )sterol could not be detected in Anodonta and determi~ Desmosterol 234-235. Cyprina. I n Anodonta, sterols containing the A ~*~s~ FAGERLUND U. H. M. & IDLER D. R. (1960) ( Marine double,"bond were absent. Previously it was supposed FAGER~tn~ sterols--VI. Sterol biosynthesis in molluscs and that this might be characteristic ;tic for non-marine echinoderms. Can. I. Bioehem. Physiol. : 997-1002. csioL 38, molluscs (Voogt, 1972). FAGERLUNDU. H. M. & IDLER, D. R. (1 (1961a) Marine After sterol composition had been determined, it sterols--VIII. In vivo transformations tions of the sterol side was interesting to determine whic lich of these sterols chain by a clam. Can.J. Biochem. Physiot ~siol. 39, 505-509. were radioactive. For this reasona, sterols of Cardium FAGERLtmOU. H. M. & IDLER D. R. (1! 1961b) Marine af PGLC. Table 8 sterols--IX. Biosynthesis of 24-methyk denecholesterol III were separated by means of in clams• Can. J. Biochem. Physiol. 39, 1347-1355. 1 shows that the results are easil[ly reproducible. It should be pointed out that I:~eaks are not fully GOADL. J., K~APP F. F., LE~rroN J. R. & GOODWIN T. W. (1972) Observations on the sterol stei side-chain separated, so that fractions are re certainly "crossalkylation mechanism in a Trcbouxia spo ~cies. contaminated". In the first anal nalysis 265 dis/min IDLERD. R., TAMta~ T. & WArNAIT. (I! 1964) Seasonal were trapped after C~6 had emerged from the variations in the sterol, fat and unsapo ~onifiable comcolumn. In the hydrogenated fraction, action (Table 7) 303 ponents of scallop muscle. J. Fish. Res. Bd Can. 21, dis/rain were trapped after Cse had sd emerl emerged. Thus it 1035-1042. can be calculated that in Table 8 we might expect SALAQUEA., BAmamRM. & LEDEP.~RE. (1966) (19( Sur la hioabout 45 dis/rain in the C~7 sterols, 'ols, about 58 in the synth~e des st6rols de l'hultre (Ostrea ggryphea) et de l'oursin (Paracentrotus lividus). Comp. Biochem. in in the C29 sterols. C2e sterols and about 170 dis/min Physiol. 19, 45-51. The separation between C~9A5+•Cs,A Cs, s , u t ~ and the e~ns that in the first TAMm~ T., TRUSCOTrB. & IDLER D. R. (1964) Sterol foregoing peak is good. This mca metabolism in the oyster. J. Fish. Res. Bd Can. 21, analysis about 110 dis/min are associated with the 1519-1522. C28A5,2z. Further calculation show.. ows that hardly any vAN DERHORSTD. J., VAs G E ~ A. H. & VOOaTP. A. radioactivity is present in desm ;mosterol and that (1969)A simplified method for extracting lipids from radioactivity is present in choleste terol, brassicasterol, large quantities of tissue abundant in wa water. Lipids 4, campesterol + 24 methylenecholesten sterol, fl-sitosterol + 300-301.

P. A. VOO~T phic analysis of nd Planorbarius 10, 697-704. the capacity of - X I I I . Biosyna some bivalves 91. 5013, 499-504. ',I-mENENJ. W . A . ~sition of sterols nes (Anthozoa).

WALTONM. J. & PEr the biosynthesis c squalene and sterol J. 127, 471--479.

972). Some studies on isoprenoid alcohols, tvertebrates. Biochem.

Key Word Index sterols of bivalves; cygnea; Cardium edu

ynthesis in bivalves; of sterols: Anodonta randica ; Mya arenaria.