- Email: [email protected]
A new source for 4-methyl sterols in freshwater sediments: Utricularia neglecta L. (Lentibulariaceae)
Org. Geochem.Vol. 18, No. 6, pp. 757-763, 1992 Printed in Great Britain. All rights reserved
0146-6380/92$5.00+ 0.00 Copyright© 1992PergamonPress Ltd
A new source for 4-methyl sterols in freshwater sediments: Utricularia neglecta L. (Lentibulariaceae) G. KLINK, F. DREmR, A. BucHs and F. O. GOLA(;AR Laboratoire de Spectromttrie de Masse, Universit6 de Gentve, 16 boulevard d'Yvoy, 1211 Gendve 4, Switzerland (Received 13 April 1992; accepted in revised form 2 June 1992)
Abstract--Lipid analyses of several aquatic plants growing in a freshwater lake allowed us to show that Utricularia neglecta L. (Lentibulariaceae), a world-wide distributed submerged aquatic plant, is an important potential source for sedimentary 4-methyl sterols in lacustrine environments. This plant contains, besides the 4-desmethyl sterols, high proportions of 4-methyl sterols, 4*t-methyl-24-ethyl-5~tcholestan-3/Lol (citrostanol) being predominant. Other 4-methyl sterols include 4a-methyl-5a-cholestan3fl-ol, 4a,24-dimethyl-5a-cholestan-3fl-ol, 4a,23,24-trimethyl-5a-cholest-22-en-3/~-ol (dinosterol) and 4a, 23, 24-trimethylcholesta-5,22-dien-3fl-ol. Steroidal ketones comprising both 4-desmethyl and 4-methyl components are also present in significant amounts. The similarity of the distribution of the 4-methyl sterols in the surface sediment with those in U. neglecta L. suggests that this plant is an important source for sedimentary 4-methyl sterols. Key words--sterols, 4-methyl sterols, citrostanol, Utricularia neglecta L., Utricularia australis, Lentibulariaceae, lacustrine sediments
4-Methyl steroids occur widely in sediments and, up to recent years, they have been considered as specific indicators for a dinoflagellate contribution both to freshwater and marine sediments at the time of deposition (see for example Robinson et ai., 1984a, b; Boudou et aL, 1986; Brassell and Eglinton, 1986; de Leeuw and Baas, 1986; Macquaker et aL, 1986; Cranwell, 1988). Virtually all marine and freshwater dinoflagellate species biosynthesizc 4-methyl sterols with different unsaturation patterns for the nucleus (As, A7, As(14), A 14, A 1~('°)) and for the side chain (A22, A24(24"))and different alkyl substitution patterns for the side-chain (no substituent, 23-methyl-, 24methyl-, 23,24-dimethyl-, 24-ethyl, 22,23-methylene23,24-dimethyl). Regarding the C30 sterols, most of the dinoflagellate species contain 4a,23,24trimethyl-5a-cholest-22-en-3fl-ol (dinosterol) as the major component and although some species contain significant proportions of 24-ethyl substituted components (Bohlin et al., 1981; Kokke et al., 1981), there is no report of a dinoflagellate in which 4-methyl-24-ethyl sterols are predominant. Ten years ago we had shown that in the recent sediments of a small lake, Voua de la Motte, rich in 4.methylsterols, 4ct-methyl-24-ethyl-5a-cholestan3fl-oi (citrostanol) predominated over all other sterols including the 4-desmethyl sterols (Mermoud et al., 1982). Dinosteroi or dinostanol were absent or present only in trace amounts. Similar distributions had been reported in two other freshwater sediments (de Leeuw et ai., 1976; Mattern et al., 1970). At that time we could not find any algal or terrestrial source for these 4-methyl sterols and, as
some bacteria were known to biosynthesize sterols and more particularly 4.methyl sterols (Bird et al., 1971; Bouvier et al., 1976; Nes and McKean, 1977), we had postulated for them a bacterial origin. However, numerous in situ incubation experiments which we have performed since, with deuterium labelled or radiolabelled (3H or 14C) steroids (Mermoud et al., 1982; Mermoud et al., 1984; Wiinsche, 1987), [U-~4C]acetate and DL-[214C]mevalonate (unpublished results) have not allowed us to confirm any bacterial transformation of 4-desmethylinto 4-methyl sterols or a de novo biosynthesis of 4.methyl sterols in the sediment. Therefore, the problem of the origin of 4-methyl sterols in this lacustrine sediment remained unresolved and we went on searching for other possible sources, sampling and analysing periodically the algal population of the lake and the aquatic plants in the water and on the shores of the lake. In this paper we report the sterol composition of one of the aquatic macrophytes we have studied, Utricularia neglecta (Lentibulariaceae), a submerged plant very abundant in the shallow waters of Voua de la Motte from May to October. This plant, reported to be widely distributed throughout the world (Taylor, 1989), contains high proportions of 4-methyl sterols with a distribution strikingly similar to that of the underlying sediments, citrostanol being the predominant sterol. The data we will present suggest a significant contribution of that plant to the sedimentary organic matter of the lake. We shall discuss also the possible contribution of this plant to the organic matter in older sediments.
757
758
G. KLINK et al. EXPERIMENTAL
Sample The Utricularia sample was collected during August 1989 and identified at the Botanical Department of the University of Geneva as U. neglecta L. It should be pointed out that the identification of species of Utricularia seems to be a delicate matter and species like U. vulgaris and U. neglecta are often confused. Utricularia australis and U. neglecta are synonyms (Taylor, 1989). The plant sample was lyophilized to constant weight and stored at - 2 5 ° C until analysis.
Lipid extraction and separation The lyophilized plant (4.6g) was extracted by ultrasonication twice with 15 ml of acetone and thrice with 15 ml of methylene chloride to give the total organic extract which was separated into acidic and neutral fractions by column chromatography on KOH impregnated SiO~. The neutral lipids were further separated by flash chromatography (Wiinsche et al., 1988) using a SiO2 column (1.5g SiO2, 30 crn x 0.6 cm) into aliphatic hydrocarbons (15 ml of n-hexane), polyunsaturated and aromatic hydrocarbons (15 ml of 37.5% toluene in n-hexane), esters (15 ml of 50% methylene chloride in n-hexane) and ketones + alcohols (20 ml of 50% ethyl acetate in n-hexane). Part of the last fraction containing the sterols and the steroidal ketones was further separated by a second flash chromatography (1 g SiO2, 30cm x 0.4cm, 50ml of 12% ethyl acetate in nhexane) into two subfractions containing unsaturated steroidal ketones + 4-methyl sterols and 4-desmethyl sterols.
Reference compounds 4-Methyl sterols 2e, 2d and ld (see Appendix for structures) were prepared from 24-ethylcholesta-4,22-dien-3-one by the following sequence of reactions described by Knapp and Sehroepfer (1974, 1975): (i) methylation with methyl iodide in t-butanol containing potassium t-butoxide gave 4-methyl-24-ethyl-cholesta-4,22-dien-3-one, (ii) bromination of this 4-methyl ketone with Nbromosuccinimide lead to 6fl-hromo-4-methyl-24ethyl-cholesta-4,22-dien-3-one, (iii) reduction of this compound with a large excess of LiA1H 4 gave 4~methyl-24-ethyl-cholesta-5,22-dien-3fl-ol (2e). Catalytic hydrogenation of 2e (PtO2/H~, ~ atmospheric pressure, room temperature) in hexane/acetic acid (4:1) for 4 h gave a mixture of 2d and ld. Compounds 2a and la were prepared from cholest-4-en-3-one by the same sequence of reactions.
(2 3R,2 4 R )-4`',23,2 4-trimethyl- 5`'-cholestan- 3fl-ol (lf, dinostanol) was obtained from hydrogenation (PtO2/H2, ~ atmospheric pressure, room temperature) of natural dinosterol (lg) in hexane/acetic acid (4:1) for 12h (Zielinski et al., 1983).
(5~ H, 14`"H, 17,, H)-4~-methyl steranes corresponding to citrostanol and dinostanol were prepared by conversion of ld and If to the tosylates with tosyl chloride in pyridine at room temperature for 24 h (Steele and Mosettig, 1963). Reduction of the tosylates with an excess of lithium aluminium hydride in anhydrous ether for 4 days at reflux (Corey et al., 1956) gave the desired steranes.
Analysis by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) Quantitative analysis of the steroidal lipids were performed on a Carlo Erba 5300 Mega Series gas chromatograph equipped with an FID using a cold on-column injector. Before analysis the sterols were converted to their trimethylsilyl ethers using BSTFA. Separations were done on an Altech SE-54 fused silica capillary column (30 m x 0.32 mm, 0.25 #m film thickness, 50 cm retention gap) using helium as carrier gas (32 cm/s at 40°C). Samples were injected at 40°C and the temperature program was: 1 min at 40°C, 10°C/min to 200°C, 3°C/min to 280°C and finally 30 min at 280°C. Peak areas were quantified with a Hewlett Packard 3388A integrator using 5`'androstane as internal standard. Compound identifications were based mainly on GC-MS data obtained on a VG Masslah Trio-2 mass spectrometer coupled to Hewlett Packard 5890 series II GC. The gas chromatographic conditions were the same as for the GC analysis described above. Typical operating conditions of the mass spectrometer were: electron impact mode with a nominal electron energy of 70cV; source temperature of 220°C; mass range of 45-600 daltons and a scan rate of 1 scan/s. GC-MS data were obtained for underivatised sterols as well as for TMS derivatives. Co-injections of standards and samples were made as free sterols and as TMS ethers on different capillary columns coated with OV-1, SE-54 and OV-1701. RESULTS AND DISCUSSION
Figure l(a) and l(b) show the partial gas chromatograms obtained of the total and the 4-methyl sterol fractions respectively from U. neglecta. Components are listed in Table 1. The 4-desmethyl sterols are mainly composed of common higher plant sterols (24-ethylcholesta-5,22-dien-3fl-ol, 24-ethylcholest-5en-3fl-ol, 24-methylcholesta-5,22-dien-3fl-ol, 24methylcholest-5-en-3fl-ol) and cholest-5-en-3fl-ol. These sterols are also found as major or significant components in the underlying sediments (Mermoud et al., 1982; Wiinsche et al., 1988), but since they are widely distributed in living organisms they cannot be used as source indicators. In U. neglecta these sterols are accompanied in high proportions by the corresponding 5`'-stanols [Fig. l(a)] and ster-4-en-3-ones [Fig. l(b)] while 5a-stan-3-ones are present (Table 1) in lesser or trace amounts. Because of co-elutions with other sterols most of the ketones could not be
Utricularia neglecta L.I4-methyl sterol source
/
759
1 19+4
/
5
/
6
a)
22+n-oi
23+12+13
.Asl l ' , ' l JA ]
I
[
I
I
I
I
19\
b) 23+12+13 /16
[
22+n-oi
n-ol ", 17
I
I
I
t
I
t
I
12+13
n-ol
t 46
)
16
c)
9
~
I
I
t
I
J
48
50
52
5/*
56
58
> C£me ( m i t t )
Fig. 1. Partial gas chromatograms showing the distributions of the steroidal compounds in (a) total sterol fraction from U. neglecta, (b) 4-methyl sterol and unsaturated ketone fraction from U. neglecta, and (c) 4-methyl sterol and unsaturated ketone fraction from the surface sediment of Voua de la Motte. Sterols are derivatised to TMS ethers. The capillary column is a 30 m fused silica SE-54 column and the oven temperature program is described in the experimental part. Peak numbers refer to compounds in Table 1 and 'n-or represents a linear alcohol as TMS ether. rigorously quantified by GC, but by comparisons of their mass spectral data and retention times with those of synthetic or commercial reference compounds they were unambiguously identified and semiquantitatively determined by mass fragmentograms of appropriate ions. Low concentrations of nuclear saturated- and A4-steroidal ketones are encountered ubiquitously in recent sediments where they are considered to be the intermediate products of the microbial transformation of A~-sterols to 5~t(H)- and 5fl(H)-stanols (Mermoud et aL, 1984; de Leeuw and Baas, 1986 and refs therein). However partial input from the water column via direct biosynthesis by algae or higher organisms is also possible since minor amounts of steroidal ketones have also been detected
in diverse organisms (Popov et al., 1976; Mermoud et al., 1981; Robinson et al., 1948b). In U. neglecta the distribution of 4-desmethyl-ster-4-en-3-ones parallels that of the corresponding AS-sterols. To our knowledge no organism having such high proportions of A4-sterones has previously been reported. This may, at least in the particular case of the Motte lake sediments, support the hypothesis of a direct input. However, in the surface sediments, the ratio of the Ad-sterones to the corresponding AS-sterols is considerably lower than in U. neglecta (Table 1) presumably due to two different factors: (i) dilution of the A4-sterones by input of AS-sterols from terrestrial or planktonic sources with less or no sterones, (ii) rapid diagenesis of A4-sterones in surface
G. KLINK et al.
760
Table 1. Steroidal compounds in Utricularia neglecta Peak Not
M W Systematic name (structure)~/
RRT~
RRTI~
Weight (/tg/g)~
1.000 1.005 1.026 1.050 1.067 1.091 1.134 1.143
1.000 1.014 1.078 1.144 1.203 1.278 1.417 1.438
102 7.7 23 tr* 56 81 45 5*
4-desmethyl sterols 1 2 3 4 5 6 7 8
458 460 470 468 472 484 486 488
Cholest-5-en-3fl -oI-TMS 5~,-Cholestan-3fl -ol-TMS 24-Methylcholesta-5,22-dien-3fl-oI-TMS 24-Methylcholesta-5,7,22-dien-3~-oI-TMS 24-Methyl-cholest-5-en-3/~ -oI-TMS 24-Ethylcholesta-5,22-dien-3fl-oI-TMS 24-Ethylcholest-5-en-3fl-oI-TMS 24-Ethyl-5~-cholestan-3fl-oI-TMS
9 l0 I1 12 13 14 15 16
474 472 488 498 500 486 484 502
4~t-Methyl-5a-cholestan-3fl-oI-TMS (la) 4~t-Methyl-5,,-cholest-7-en-3fl-oI-TMS (3a) 4ct,24-Dimethyl-Se,-cholestan-3fl-ol-TMS (le) 4~t,23,24-Trimethylcholesta-5,22-dien-3~8-ol-TMS (2g) 4¢t,23,24-trimethyl-5~t-cholest-22-en-3fl-ol-TMS (lg) 4~,24-Dimethyleholest-7-en-3fl-ol-TMS (3e) 4~,24-Dimethyl-5~t-cholesta-7,24(24')-dien-3fl-ol-TMS (3b) 4~'t-Methyl-24--ethyl-5~t-cholestan-3~-oI-TMS (hi)
4-Methyl sterols 1.065 1.082 1.141 1.162 1.165 1.186 1.187 1.226
1.504 1.510
1.720
5.9 tr* 2.7 3* 2.8* < 4.0" 45
Steroidal ketones -17 18 19 -20 21 -22 -23 24 25 ----
386 382 400 384 382 396 414 426 398 414 410 428 412 410 412 424
5~t-Cholestan-3-one Cholesta-4,22-dien-3-one 4a-Methyl-Sa-cholestan-3-one (4a) Cholest-4-en-3-one Cholesta-4,6-dien-3-one 24-Methyl-cholesta-4,22-dien-3-one 4~,24-Dimethyl-5~t-cholestan-3-one (4e) 4~t,23,24-trimethyl-5a-chnlest-22-en-3-one (411) 24-Methyl-cholest-4-en-3-one 24-Ethyl-5,,-cholestan-3-one 24-Ethyl-¢holesta-4,22-dien-3-one 4a-methyl-24-ethyl-5~t-cholestan-3-one (4d) 24-Ethyl-cholest-4-en-3-one 24-Ethyl-cholesta-4,6-dien-3-one 24-Methyl-cholest-4- zn-3,6-dione 24-Ethyl-cholesta-4,22-dien-3,6-dione
1.003 1.025 1.033 1.050 1.070 1.082 1.109 l.l I 1 1.129 1.143 1.158 1.185 1.210 1.228 1.255 1.282
1.098 1.144
1.387 1.479 1.570 1.647
tr 2.2 1.5 86 tr 13" 6.4 tr 37* tr 66* 45 29 tr tr tr
t P e a k numbers refer to G C peaks in Fig. 1. ~Structures are given in the Appendix §Values with an * are obtained from C-C/MS data and are only approximate because of coelutions, tr = trace. RRTpro = Retention time relative to cholesterol-TMS ether on SE-54 capillary column operated with temperature program defined in the experimental part. RRTi~ o = as above but on a SE-54 capillary column operated isothermally at 260°C.
sediments. The latter was demonstrated by in situ incubation experiments of deuterium labelled cholest4-en-3-one in the surface sediment of Motte lake which have shown that after 291 days of incubation approximately 60% of cholest-4-ene-3-one was transformed to 5~t-cholestan-3-one (15.8%), 5~-cholestan3fl-ol (38.4%), 5fl-cholestan-3-one (4.7%) and 5fl-cholestan-3fl-oi (traces) (Wfinsche, 1987). The identification and abundances of the 4-methyl steroidal compounds present in U. neglecta are given in Table 1. Structures are shown in the Appendix. The major component is 4ct-methyl-24-ethyl-5~cholestan-3fl-ol (citrostanol, ld) which is also the most abundant sterol in the underlying sediment [Fig. l(c)]. Other 4-methyl sterols are 4~t-methyl-5~cholestan-3fl-ol (la), 4~,24-dimethyl-5~-cholestan3fl-ol (le), 4~,23,24-trimethyl-5~-cholest-22-en-3fl-ol (lg) and 4ct,23,24-trimethylcholesta-5,22-dien-3fl-ol (2g) which are also found in the surface sediment with a similar distribution, suggesting that, if not the only one, U. neglecta is a major source for the sedimentary 4-methyl sterols. These components were identified by comparison of mass spectra, GC retention times and co-injections with authentic standards on columns of different polarities, except for 2g. The
identify of sedimentary citrostanol has also been confirmed by conversion to 4~-methyl sterane and coinjection with authentic 4~-methyl-24-ethyl5~(H),14~(H),17~(H)-cholestane (see experimental
part). The occurrence of 24-ethyl- (ld) and 23,24dimethyl-substituted 4-methyl steroids (lg and 2g) in U. neglecta is surprising. Two hypothesis may be invoked: (i) the plant has two different competing side-chain alkylation pathways to biosynthesize C30 4-methyl sterols, or (ii) the minor amounts of lg and 2g are due to the ingestion of microalgae (including dinoflagellates) by the plant since Utricularia are known to capture tiny animals and microscopic organisms such as protozoa, rotifers and diatoms (Grave, 1957). Further research is needed to clarify this point. It should be pointed out that the mass spectra of citrostanol (ld) and dinostanol (If) present only a few distinguishing characters. Mass spectra of If in free form (Zielinski et al., 1983), as TMS ether (Prowse and Maxwell, 1991) and as acetate (Volkman et al., 1984) all show a significant ion at m/z 98 (CTH +) arising from the cleavage of the C-22-C-23 bond with rearrangement of a hydrogen atom. In the
Utricularia neglecta L.---4-methyl sterol source mass spectra of ld (Mermoud et al., 1982) and its TMS and acetate derivatives, this fragment is almost absent. Another significant difference is a more abundant m/z 261 ion in the mass spectrum of the TMS derivative of I f relative to ld (Prowse and Maxwell, 1991). The corresponding steranes are also distinguished by the m/z 98 fragment (Wolff et al., 1986, Summons et al., 1987). Indeed we observe easily these differences when analysing synthetic standards. However with complex sediment samples where coelutions can occur and especially with samples containing low proportions of ld and/or If, the identifications based on these differences are often more difficult, especially in the case of samples conraining both compounds. Moreover the GC separation of steroidal pairs differing only by their side chain substitution pattern as 23,24-dimethyl- versus 24-ethyl- is difficult. For example, on G C - M S analyses with 30m OV-101 or SE-54 coated capillary columns, ld and If are not resolved although the mass fragmentogram of m/z 98 shows that I f elutes in the second half of the TIC peak. With the corresponding 4ct-methyl-5~t(H)-steranes the situation is comparable, i.e. ((23S,24S)-4,,,23,24trimethyl-5a(H),14a(H),17~t(H)-cholestane and ~ methyl-24~-ethyl-5ct(H), 14~t(H), 17ct(H)-cholestane are not resolved but the m/z 98 mass fragmentogram again shows that the former elutes in the second part of the peak. A splitting of the peaks was observed using 50 m capillary columns but, with amounts of the two compounds higher than 200 ng in the column, we could never obtain a separation down to the baseline. A reasonable resolution was obtained only on a semi-polar OV-1701 stationary phase. These considerations indicate that the published occurrences of dinostanol and/or 4-methyl-24-ethyl-cholesterol in sediments should be considered with some suspicion when they are based only on the GC-MS retention times and mass spectra of minor amounts without co-injection experiments with authentic standards and/or m/z 98 mass fragmentographic measures. Some recent work with lacustrine sediments indicate in fact that ld might be mistaken for If (Prowse and Maxwell, 1991) and the former might be much frequently present than reported in the geochemical literature. Other minor or trace 4-methyl sterols detected in U. neglecta are quoted in Table I. For these compounds with a AT-unsaturation, standards were not available and the identifications are based on GC retention characteristics and mass spectral patterns. Like the 4-desmethyl sterols, which are accompanied by abundant corresponding ketones, significant amounts of C2s-C30 4-methyl-steran-3-ones, with a distribution similar to that of the fully saturated 4-methylsterols, are also present. Utricularia neglecta may therefore be the direct source of the 4-methyl ketones present in trace amounts in the underlying sediment. The finding of a source for 4-methyl sterols in recent lacustrine sediments has also a geochemical
761
implication for palaeoenvironments. Recent studies of C30 steranes (in which side-chain structures were determined) in diverse sediments and crude oils showed that samples of marine origin contain compounds with both side-chain structures d and f, while lacustrine samples are characterized by a dominance of steranes having ethyl-substituted side-chains, dinosteranes being absent or in very low abundance (Summons et al., 1987; Goodwin et al., 1988). The lacustrine Messel oil shale (Eocene) is also characterized by high abundance of citrostanol (ld) relative to dinosterol (lg) (Robinson et al., 1989). Citrostanol has been recently reported in four species of marine prymnesiophytes of the genus Pavlova (Volkman et al., 1990) along with its A22-unsaturated counterpart (le). In all cases the unsaturated compound was 13-35 times more abundant. This finding provided a plausible source for the 4-methyl sterols having a 24-ethyl substituent (ld and le) in recent marine sediments and the corresponding steranes in ancient sediments and crude oils of marine origin. Our resuits, on the other hand, provide a possible source for 4-methyl sterols present in recent lacustrine sediments. But, contrarily to prymnesiophytes whose importance in past geological periods is well documented by coceolith deposits dating back at least to the Jurassic, we have no knowledge on the geological time extension of Utricularia. If this latter can also be reasonably taken as the source for the 4-methyl-24ethyl sterols and/or corresponding 4-methyl steranes reported in ancient lacustrine crude oils or oil shales, then one can deduce that the plant has existed at least since the Cretaceous period. CONCLUSION The freshwater plant Utricularia neglecta contains large amounts of 4~-methyl sterols with 4~-methyl24-ethyl-5u-cholestan-3fl-ol as the major component. The similarity of the distribution of these compounds in the plant and in the underlying sediment suggests that this world-wide spread plant is an important source of the fully saturated 4-methyl sterols in recent lacustrine sediments. It may also be the source of the 24-ethyl substituted 4-methyl steranes in older sediments and crude oils of lacustrine origin.
Acknowledgements--Tiffs work was supported by the Fonds National Suisse de la Recherche Scientifique (Grant No. 20-26301.89) We thank Professor C. Djerassi (Standford University, U.S.A.) for kindly providing the natural dinosterol sample and D. Schefer and W. Kloeti for valuable technical assistance. REFERENCES
Bird C. W., Lynch J. M., Pirt F. J., Reid W. W., Brooks C. J. W. and Middleditch B. S. (1971) Steroids and squalene in Methylococcus capsulatus grown on methane. Nature 230, 473-474. Bohlin L., Kokke W. C. M. C., Fenical W. and Djerassi C. (1981) 4~-Methyl-24S-ethyl-5u-cholestan-3~ol and 4~-methyl-24S-ethyl-5~-cholest-8(14)-en-3/~-ol,
762
G. KLINK et al.
two new sterols from a cultured marine dinoflagellate. Phytochemistry 20, 2397-2401. Boudou J. P., Trichet J., Robinson N. and Brassell S. C. (1986) Lipid composition of a Recent Polynesian microbial mat sequence. Org. Geochem. lfl, 705-709. Bouvier P., Rohmer M., Benveniste P. and Ourisson G. (1976) Amo-Steroids in the bacterium Methyloccus capsulatus. Biochem. J. 159, 267-271. Brassell S. C. and Egiinton G. (1986) Molecular geochemical indicators in sediments. In Organic Marine Geochemistry (Edited by Sohn M. L.), pp. 10-32. ACS Symposium Series, No 305. Corey E. J., Howell M. G., Boston A., Young R. L. and Sneen R. A. (1956) Spectral and stereochemical studies with deuterated cyclohexanes. J. Am. Chem. Soc. 78, 5036-5040. Cranwell P. A. (1988) Lipid geochemistry of late Pleistocene lacustrine sediments from Burland, Cheshire, U.K. Chem. Geol. 68, 181-197. Goodwin N. S., Mann A. L. and Patience R. L. (1988) Structure and significance of C30 4-methyl steranes in lacustrine shales and oils. Org. Geochem. 12, 495-506. Grave E. V. (1957) A plant that captures animals under water. Nat. Hist., 74-97. Knapp F. F. Jr. and Schroepfer G. J. Jr. (1974) A novel synthesis of 4~- and 4fl-methylcholest-5-ene-3//-ol from 6//-bromo-4-methylcholest-4-en-3-one. J. Org. Chem. 39, 3247-3250. Knapp F. F. Jr. and Schroepfer G. J. Jr (1975) Chemical synthesis, spectral properties, and chromatography of 4~-methyl and 4//-methyl isomers of (24R)-24-ethyl-5~cholestan-3//-ol and (24S)-24-ethyl-cholesta-5,22-dien3//-ol. Steroids 26, 339-356. Kokke W. C. M. C., Fenical W. and Djerassi C. (1981) Sterols with unusual nuclear unsaturation from three cultured marine dinoflagellates. Phytochemistry 20, 127-134. de Leeuw J. W., Rijpstra W. I. C., Boon J. J., de Lange F. and Schenk P. A. (1976) The relationship between lipids from Fontinalis antipyretica, its detritus and the underlying sediment: the fate of waxesters and sterolesters. In Interaction Between Sediments and Fresh Water (Edited by Golterman H. L.), pp. 141-147. Proc. Int. Symposium, Amsterdam. de Leeuw J. W. and Baas M. (1986) Early stage diagenesis of steroids. In Biological Markers in the Sedimentary Record (Edited by Johns R. B.), pp. 101-123. Elsevier, Amsterdam. Macquaker J. H. S., Farrimond P. and Brassell S. C. (1986) Biological markers in the Rhaetian black shales of South West Britain. Org. Geochem. 10, 93-100. Mattern G., Albrecht P. and Ourisson G. (1970) 4-Methylsterols and sterols in Messel shale (Eocene). Chem. Cornman. 1570-1571. Mermoud F., Clerc O., Giila~;ar F. O. and Buchs A. (1981) Analyse des acides gras et des sterols dam le plancton du lac L6man. Arch. Sci. (Gendve) 34, 367-382.
Mermoud F., Gfilagar F. O., Siles S., Chassaing B. and Buchs A. (1982) 4-Methylsterols in recent lacustrine sediments: terrestrial, planktonic or some other origin7 Chemosphere l l , 557-567. Mermoud F., Wiinsche L., Clerc O., G~laqar F. O. and Buchs A. 0984) Steroidal ketones in the early transformations of AS-sterols in different types of sediments. Org. Geochem. 6, 25-29. Nes W. R. and McKean M. L. (1977) In Biochemistry of Steroids and Other lsopentenoids, pp. 416- 418. University Press, Baltimore, MD. Popov S., Carlson R. M. K., Wegmann A. and Djerassi C. (1976) Minor and trace sterols in marine invertebrates I. General methods of analysis. Steroids 28, 699-732. Prowse W. G. and Maxwell J. R. (1991) High molecular weight chlorins in a lacustrine shale. Org. Geochem. 17, 877-886. Robinson N., Cranwell P. A., Finlay B. J. and Egiinton G. (1984a) Lipids of aquatic organisms as potential contributors to lacustrine sediments. Org. Geochem. 6, 143-152. Robinson N., Egiinton G., Brassell S. C. and Cranwell P. A. (1984b) Dinoflagellate origin for sedimentary 4a-methylsteroids and 5#(H)-stanols. Nature 308, 439-442. Robinson N., Eglinton G., Cranwell P. A. and Zeng Y. B. (1989) Messel oil shale (western Germany): assessment of depositional paleoenvironment from the content of biological marker compounds. Chem. Geol. 76, 153-173. Steele J. A. and Mosettig E. (1963) The solvolysis of stigmasteryl tosylate J. Org. Chem. 28, 571-572. Summons R. E., Volkman J. K. and Boreham C. J. (1987) Dinosterane and other steroidal hydrocarbons of dinoflagellate origin in sediments and petroleum. Geochim. Cosmochim. Acta 51, 3075-3082. Taylor P. (1989) In The Genus Utricularia--A Taxonomic Monograph, pp. 598-605. HMSO Books, London. Volkman J. K., Gagosian R. B. and Wakeham S. (1984) free and esterified sterols of the marine dinottagellate Gonyaulax polygramma. Lipids 19, 457-465. Volkman J. K., Kearney P. and Jeffrey S. W. (1990) A new source of 4-methyl sterois and 5a(H)-stanols in sediments: prymnesiophyte microalgae of the genus Pavlova. Org. Geochem. 15, 489-497. Wolff G. A., Lamb N. A. and Maxwell J. R. (1986) The origin and fate of 4-methyl steroids-II. Dehydration of stanols and occurrence of C30 4-methyl steranes. Org. Geochem. lfl, 965-974. Wiinsche L. (1987) G6ochimie des lipides neutres et diagen6se pr6coce des st6rols dans des s6diments du bassin l~manique. Ph.D. thesis, University of Geneva. Wiinsche L., Mendoza Y. and Gfilaqar F. (1988) Lipid geochemistry of a post-glacial lacustrine sediment. Org. Geochem. 13, 1131-1143. Zielinski J., Kokke W. C. M. C., Tam Ha T. B., Shu A. Y. L., Duax W. L. and Djerassi C. (1983) Isolation, partial synthesis and structure determination of sterols with the four possible 23,24-dimethyl substituted side chains. J. Org. Chem. 48, 3471-3477.
Appendix opposite
Utricularia neglecta
L . - - 4 - m e t h y l sterol s o u r c e
763
APPENDIX
SC:
'"
a
IIII1,,
(~
., n
%
-
•
.
b
%.
H
1
R I , , H , R2=OH
2
R I " H, R 2 " O H ,
5 A
3
RI = H ,
"7 A
4
R2=OH ,
R1,R2sO
"'"~'..._~~ g H
f
0146-6380/92$5.00+ 0.00 Copyright© 1992PergamonPress Ltd
A new source for 4-methyl sterols in freshwater sediments: Utricularia neglecta L. (Lentibulariaceae) G. KLINK, F. DREmR, A. BucHs and F. O. GOLA(;AR Laboratoire de Spectromttrie de Masse, Universit6 de Gentve, 16 boulevard d'Yvoy, 1211 Gendve 4, Switzerland (Received 13 April 1992; accepted in revised form 2 June 1992)
Abstract--Lipid analyses of several aquatic plants growing in a freshwater lake allowed us to show that Utricularia neglecta L. (Lentibulariaceae), a world-wide distributed submerged aquatic plant, is an important potential source for sedimentary 4-methyl sterols in lacustrine environments. This plant contains, besides the 4-desmethyl sterols, high proportions of 4-methyl sterols, 4*t-methyl-24-ethyl-5~tcholestan-3/Lol (citrostanol) being predominant. Other 4-methyl sterols include 4a-methyl-5a-cholestan3fl-ol, 4a,24-dimethyl-5a-cholestan-3fl-ol, 4a,23,24-trimethyl-5a-cholest-22-en-3/~-ol (dinosterol) and 4a, 23, 24-trimethylcholesta-5,22-dien-3fl-ol. Steroidal ketones comprising both 4-desmethyl and 4-methyl components are also present in significant amounts. The similarity of the distribution of the 4-methyl sterols in the surface sediment with those in U. neglecta L. suggests that this plant is an important source for sedimentary 4-methyl sterols. Key words--sterols, 4-methyl sterols, citrostanol, Utricularia neglecta L., Utricularia australis, Lentibulariaceae, lacustrine sediments
4-Methyl steroids occur widely in sediments and, up to recent years, they have been considered as specific indicators for a dinoflagellate contribution both to freshwater and marine sediments at the time of deposition (see for example Robinson et ai., 1984a, b; Boudou et aL, 1986; Brassell and Eglinton, 1986; de Leeuw and Baas, 1986; Macquaker et aL, 1986; Cranwell, 1988). Virtually all marine and freshwater dinoflagellate species biosynthesizc 4-methyl sterols with different unsaturation patterns for the nucleus (As, A7, As(14), A 14, A 1~('°)) and for the side chain (A22, A24(24"))and different alkyl substitution patterns for the side-chain (no substituent, 23-methyl-, 24methyl-, 23,24-dimethyl-, 24-ethyl, 22,23-methylene23,24-dimethyl). Regarding the C30 sterols, most of the dinoflagellate species contain 4a,23,24trimethyl-5a-cholest-22-en-3fl-ol (dinosterol) as the major component and although some species contain significant proportions of 24-ethyl substituted components (Bohlin et al., 1981; Kokke et al., 1981), there is no report of a dinoflagellate in which 4-methyl-24-ethyl sterols are predominant. Ten years ago we had shown that in the recent sediments of a small lake, Voua de la Motte, rich in 4.methylsterols, 4ct-methyl-24-ethyl-5a-cholestan3fl-oi (citrostanol) predominated over all other sterols including the 4-desmethyl sterols (Mermoud et al., 1982). Dinosteroi or dinostanol were absent or present only in trace amounts. Similar distributions had been reported in two other freshwater sediments (de Leeuw et ai., 1976; Mattern et al., 1970). At that time we could not find any algal or terrestrial source for these 4-methyl sterols and, as
some bacteria were known to biosynthesize sterols and more particularly 4.methyl sterols (Bird et al., 1971; Bouvier et al., 1976; Nes and McKean, 1977), we had postulated for them a bacterial origin. However, numerous in situ incubation experiments which we have performed since, with deuterium labelled or radiolabelled (3H or 14C) steroids (Mermoud et al., 1982; Mermoud et al., 1984; Wiinsche, 1987), [U-~4C]acetate and DL-[214C]mevalonate (unpublished results) have not allowed us to confirm any bacterial transformation of 4-desmethylinto 4-methyl sterols or a de novo biosynthesis of 4.methyl sterols in the sediment. Therefore, the problem of the origin of 4-methyl sterols in this lacustrine sediment remained unresolved and we went on searching for other possible sources, sampling and analysing periodically the algal population of the lake and the aquatic plants in the water and on the shores of the lake. In this paper we report the sterol composition of one of the aquatic macrophytes we have studied, Utricularia neglecta (Lentibulariaceae), a submerged plant very abundant in the shallow waters of Voua de la Motte from May to October. This plant, reported to be widely distributed throughout the world (Taylor, 1989), contains high proportions of 4-methyl sterols with a distribution strikingly similar to that of the underlying sediments, citrostanol being the predominant sterol. The data we will present suggest a significant contribution of that plant to the sedimentary organic matter of the lake. We shall discuss also the possible contribution of this plant to the organic matter in older sediments.
757
758
G. KLINK et al. EXPERIMENTAL
Sample The Utricularia sample was collected during August 1989 and identified at the Botanical Department of the University of Geneva as U. neglecta L. It should be pointed out that the identification of species of Utricularia seems to be a delicate matter and species like U. vulgaris and U. neglecta are often confused. Utricularia australis and U. neglecta are synonyms (Taylor, 1989). The plant sample was lyophilized to constant weight and stored at - 2 5 ° C until analysis.
Lipid extraction and separation The lyophilized plant (4.6g) was extracted by ultrasonication twice with 15 ml of acetone and thrice with 15 ml of methylene chloride to give the total organic extract which was separated into acidic and neutral fractions by column chromatography on KOH impregnated SiO~. The neutral lipids were further separated by flash chromatography (Wiinsche et al., 1988) using a SiO2 column (1.5g SiO2, 30 crn x 0.6 cm) into aliphatic hydrocarbons (15 ml of n-hexane), polyunsaturated and aromatic hydrocarbons (15 ml of 37.5% toluene in n-hexane), esters (15 ml of 50% methylene chloride in n-hexane) and ketones + alcohols (20 ml of 50% ethyl acetate in n-hexane). Part of the last fraction containing the sterols and the steroidal ketones was further separated by a second flash chromatography (1 g SiO2, 30cm x 0.4cm, 50ml of 12% ethyl acetate in nhexane) into two subfractions containing unsaturated steroidal ketones + 4-methyl sterols and 4-desmethyl sterols.
Reference compounds 4-Methyl sterols 2e, 2d and ld (see Appendix for structures) were prepared from 24-ethylcholesta-4,22-dien-3-one by the following sequence of reactions described by Knapp and Sehroepfer (1974, 1975): (i) methylation with methyl iodide in t-butanol containing potassium t-butoxide gave 4-methyl-24-ethyl-cholesta-4,22-dien-3-one, (ii) bromination of this 4-methyl ketone with Nbromosuccinimide lead to 6fl-hromo-4-methyl-24ethyl-cholesta-4,22-dien-3-one, (iii) reduction of this compound with a large excess of LiA1H 4 gave 4~methyl-24-ethyl-cholesta-5,22-dien-3fl-ol (2e). Catalytic hydrogenation of 2e (PtO2/H~, ~ atmospheric pressure, room temperature) in hexane/acetic acid (4:1) for 4 h gave a mixture of 2d and ld. Compounds 2a and la were prepared from cholest-4-en-3-one by the same sequence of reactions.
(2 3R,2 4 R )-4`',23,2 4-trimethyl- 5`'-cholestan- 3fl-ol (lf, dinostanol) was obtained from hydrogenation (PtO2/H2, ~ atmospheric pressure, room temperature) of natural dinosterol (lg) in hexane/acetic acid (4:1) for 12h (Zielinski et al., 1983).
(5~ H, 14`"H, 17,, H)-4~-methyl steranes corresponding to citrostanol and dinostanol were prepared by conversion of ld and If to the tosylates with tosyl chloride in pyridine at room temperature for 24 h (Steele and Mosettig, 1963). Reduction of the tosylates with an excess of lithium aluminium hydride in anhydrous ether for 4 days at reflux (Corey et al., 1956) gave the desired steranes.
Analysis by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) Quantitative analysis of the steroidal lipids were performed on a Carlo Erba 5300 Mega Series gas chromatograph equipped with an FID using a cold on-column injector. Before analysis the sterols were converted to their trimethylsilyl ethers using BSTFA. Separations were done on an Altech SE-54 fused silica capillary column (30 m x 0.32 mm, 0.25 #m film thickness, 50 cm retention gap) using helium as carrier gas (32 cm/s at 40°C). Samples were injected at 40°C and the temperature program was: 1 min at 40°C, 10°C/min to 200°C, 3°C/min to 280°C and finally 30 min at 280°C. Peak areas were quantified with a Hewlett Packard 3388A integrator using 5`'androstane as internal standard. Compound identifications were based mainly on GC-MS data obtained on a VG Masslah Trio-2 mass spectrometer coupled to Hewlett Packard 5890 series II GC. The gas chromatographic conditions were the same as for the GC analysis described above. Typical operating conditions of the mass spectrometer were: electron impact mode with a nominal electron energy of 70cV; source temperature of 220°C; mass range of 45-600 daltons and a scan rate of 1 scan/s. GC-MS data were obtained for underivatised sterols as well as for TMS derivatives. Co-injections of standards and samples were made as free sterols and as TMS ethers on different capillary columns coated with OV-1, SE-54 and OV-1701. RESULTS AND DISCUSSION
Figure l(a) and l(b) show the partial gas chromatograms obtained of the total and the 4-methyl sterol fractions respectively from U. neglecta. Components are listed in Table 1. The 4-desmethyl sterols are mainly composed of common higher plant sterols (24-ethylcholesta-5,22-dien-3fl-ol, 24-ethylcholest-5en-3fl-ol, 24-methylcholesta-5,22-dien-3fl-ol, 24methylcholest-5-en-3fl-ol) and cholest-5-en-3fl-ol. These sterols are also found as major or significant components in the underlying sediments (Mermoud et al., 1982; Wiinsche et al., 1988), but since they are widely distributed in living organisms they cannot be used as source indicators. In U. neglecta these sterols are accompanied in high proportions by the corresponding 5`'-stanols [Fig. l(a)] and ster-4-en-3-ones [Fig. l(b)] while 5a-stan-3-ones are present (Table 1) in lesser or trace amounts. Because of co-elutions with other sterols most of the ketones could not be
Utricularia neglecta L.I4-methyl sterol source
/
759
1 19+4
/
5
/
6
a)
22+n-oi
23+12+13
.Asl l ' , ' l JA ]
I
[
I
I
I
I
19\
b) 23+12+13 /16
[
22+n-oi
n-ol ", 17
I
I
I
t
I
t
I
12+13
n-ol
t 46
)
16
c)
9
~
I
I
t
I
J
48
50
52
5/*
56
58
> C£me ( m i t t )
Fig. 1. Partial gas chromatograms showing the distributions of the steroidal compounds in (a) total sterol fraction from U. neglecta, (b) 4-methyl sterol and unsaturated ketone fraction from U. neglecta, and (c) 4-methyl sterol and unsaturated ketone fraction from the surface sediment of Voua de la Motte. Sterols are derivatised to TMS ethers. The capillary column is a 30 m fused silica SE-54 column and the oven temperature program is described in the experimental part. Peak numbers refer to compounds in Table 1 and 'n-or represents a linear alcohol as TMS ether. rigorously quantified by GC, but by comparisons of their mass spectral data and retention times with those of synthetic or commercial reference compounds they were unambiguously identified and semiquantitatively determined by mass fragmentograms of appropriate ions. Low concentrations of nuclear saturated- and A4-steroidal ketones are encountered ubiquitously in recent sediments where they are considered to be the intermediate products of the microbial transformation of A~-sterols to 5~t(H)- and 5fl(H)-stanols (Mermoud et aL, 1984; de Leeuw and Baas, 1986 and refs therein). However partial input from the water column via direct biosynthesis by algae or higher organisms is also possible since minor amounts of steroidal ketones have also been detected
in diverse organisms (Popov et al., 1976; Mermoud et al., 1981; Robinson et al., 1948b). In U. neglecta the distribution of 4-desmethyl-ster-4-en-3-ones parallels that of the corresponding AS-sterols. To our knowledge no organism having such high proportions of A4-sterones has previously been reported. This may, at least in the particular case of the Motte lake sediments, support the hypothesis of a direct input. However, in the surface sediments, the ratio of the Ad-sterones to the corresponding AS-sterols is considerably lower than in U. neglecta (Table 1) presumably due to two different factors: (i) dilution of the A4-sterones by input of AS-sterols from terrestrial or planktonic sources with less or no sterones, (ii) rapid diagenesis of A4-sterones in surface
G. KLINK et al.
760
Table 1. Steroidal compounds in Utricularia neglecta Peak Not
M W Systematic name (structure)~/
RRT~
RRTI~
Weight (/tg/g)~
1.000 1.005 1.026 1.050 1.067 1.091 1.134 1.143
1.000 1.014 1.078 1.144 1.203 1.278 1.417 1.438
102 7.7 23 tr* 56 81 45 5*
4-desmethyl sterols 1 2 3 4 5 6 7 8
458 460 470 468 472 484 486 488
Cholest-5-en-3fl -oI-TMS 5~,-Cholestan-3fl -ol-TMS 24-Methylcholesta-5,22-dien-3fl-oI-TMS 24-Methylcholesta-5,7,22-dien-3~-oI-TMS 24-Methyl-cholest-5-en-3/~ -oI-TMS 24-Ethylcholesta-5,22-dien-3fl-oI-TMS 24-Ethylcholest-5-en-3fl-oI-TMS 24-Ethyl-5~-cholestan-3fl-oI-TMS
9 l0 I1 12 13 14 15 16
474 472 488 498 500 486 484 502
4~t-Methyl-5a-cholestan-3fl-oI-TMS (la) 4~t-Methyl-5,,-cholest-7-en-3fl-oI-TMS (3a) 4ct,24-Dimethyl-Se,-cholestan-3fl-ol-TMS (le) 4~t,23,24-Trimethylcholesta-5,22-dien-3~8-ol-TMS (2g) 4¢t,23,24-trimethyl-5~t-cholest-22-en-3fl-ol-TMS (lg) 4~,24-Dimethyleholest-7-en-3fl-ol-TMS (3e) 4~,24-Dimethyl-5~t-cholesta-7,24(24')-dien-3fl-ol-TMS (3b) 4~'t-Methyl-24--ethyl-5~t-cholestan-3~-oI-TMS (hi)
4-Methyl sterols 1.065 1.082 1.141 1.162 1.165 1.186 1.187 1.226
1.504 1.510
1.720
5.9 tr* 2.7 3* 2.8* < 4.0" 45
Steroidal ketones -17 18 19 -20 21 -22 -23 24 25 ----
386 382 400 384 382 396 414 426 398 414 410 428 412 410 412 424
5~t-Cholestan-3-one Cholesta-4,22-dien-3-one 4a-Methyl-Sa-cholestan-3-one (4a) Cholest-4-en-3-one Cholesta-4,6-dien-3-one 24-Methyl-cholesta-4,22-dien-3-one 4~,24-Dimethyl-5~t-cholestan-3-one (4e) 4~t,23,24-trimethyl-5a-chnlest-22-en-3-one (411) 24-Methyl-cholest-4-en-3-one 24-Ethyl-5,,-cholestan-3-one 24-Ethyl-¢holesta-4,22-dien-3-one 4a-methyl-24-ethyl-5~t-cholestan-3-one (4d) 24-Ethyl-cholest-4-en-3-one 24-Ethyl-cholesta-4,6-dien-3-one 24-Methyl-cholest-4- zn-3,6-dione 24-Ethyl-cholesta-4,22-dien-3,6-dione
1.003 1.025 1.033 1.050 1.070 1.082 1.109 l.l I 1 1.129 1.143 1.158 1.185 1.210 1.228 1.255 1.282
1.098 1.144
1.387 1.479 1.570 1.647
tr 2.2 1.5 86 tr 13" 6.4 tr 37* tr 66* 45 29 tr tr tr
t P e a k numbers refer to G C peaks in Fig. 1. ~Structures are given in the Appendix §Values with an * are obtained from C-C/MS data and are only approximate because of coelutions, tr = trace. RRTpro = Retention time relative to cholesterol-TMS ether on SE-54 capillary column operated with temperature program defined in the experimental part. RRTi~ o = as above but on a SE-54 capillary column operated isothermally at 260°C.
sediments. The latter was demonstrated by in situ incubation experiments of deuterium labelled cholest4-en-3-one in the surface sediment of Motte lake which have shown that after 291 days of incubation approximately 60% of cholest-4-ene-3-one was transformed to 5~t-cholestan-3-one (15.8%), 5~-cholestan3fl-ol (38.4%), 5fl-cholestan-3-one (4.7%) and 5fl-cholestan-3fl-oi (traces) (Wfinsche, 1987). The identification and abundances of the 4-methyl steroidal compounds present in U. neglecta are given in Table 1. Structures are shown in the Appendix. The major component is 4ct-methyl-24-ethyl-5~cholestan-3fl-ol (citrostanol, ld) which is also the most abundant sterol in the underlying sediment [Fig. l(c)]. Other 4-methyl sterols are 4~t-methyl-5~cholestan-3fl-ol (la), 4~,24-dimethyl-5~-cholestan3fl-ol (le), 4~,23,24-trimethyl-5~-cholest-22-en-3fl-ol (lg) and 4ct,23,24-trimethylcholesta-5,22-dien-3fl-ol (2g) which are also found in the surface sediment with a similar distribution, suggesting that, if not the only one, U. neglecta is a major source for the sedimentary 4-methyl sterols. These components were identified by comparison of mass spectra, GC retention times and co-injections with authentic standards on columns of different polarities, except for 2g. The
identify of sedimentary citrostanol has also been confirmed by conversion to 4~-methyl sterane and coinjection with authentic 4~-methyl-24-ethyl5~(H),14~(H),17~(H)-cholestane (see experimental
part). The occurrence of 24-ethyl- (ld) and 23,24dimethyl-substituted 4-methyl steroids (lg and 2g) in U. neglecta is surprising. Two hypothesis may be invoked: (i) the plant has two different competing side-chain alkylation pathways to biosynthesize C30 4-methyl sterols, or (ii) the minor amounts of lg and 2g are due to the ingestion of microalgae (including dinoflagellates) by the plant since Utricularia are known to capture tiny animals and microscopic organisms such as protozoa, rotifers and diatoms (Grave, 1957). Further research is needed to clarify this point. It should be pointed out that the mass spectra of citrostanol (ld) and dinostanol (If) present only a few distinguishing characters. Mass spectra of If in free form (Zielinski et al., 1983), as TMS ether (Prowse and Maxwell, 1991) and as acetate (Volkman et al., 1984) all show a significant ion at m/z 98 (CTH +) arising from the cleavage of the C-22-C-23 bond with rearrangement of a hydrogen atom. In the
Utricularia neglecta L.---4-methyl sterol source mass spectra of ld (Mermoud et al., 1982) and its TMS and acetate derivatives, this fragment is almost absent. Another significant difference is a more abundant m/z 261 ion in the mass spectrum of the TMS derivative of I f relative to ld (Prowse and Maxwell, 1991). The corresponding steranes are also distinguished by the m/z 98 fragment (Wolff et al., 1986, Summons et al., 1987). Indeed we observe easily these differences when analysing synthetic standards. However with complex sediment samples where coelutions can occur and especially with samples containing low proportions of ld and/or If, the identifications based on these differences are often more difficult, especially in the case of samples conraining both compounds. Moreover the GC separation of steroidal pairs differing only by their side chain substitution pattern as 23,24-dimethyl- versus 24-ethyl- is difficult. For example, on G C - M S analyses with 30m OV-101 or SE-54 coated capillary columns, ld and If are not resolved although the mass fragmentogram of m/z 98 shows that I f elutes in the second half of the TIC peak. With the corresponding 4ct-methyl-5~t(H)-steranes the situation is comparable, i.e. ((23S,24S)-4,,,23,24trimethyl-5a(H),14a(H),17~t(H)-cholestane and ~ methyl-24~-ethyl-5ct(H), 14~t(H), 17ct(H)-cholestane are not resolved but the m/z 98 mass fragmentogram again shows that the former elutes in the second part of the peak. A splitting of the peaks was observed using 50 m capillary columns but, with amounts of the two compounds higher than 200 ng in the column, we could never obtain a separation down to the baseline. A reasonable resolution was obtained only on a semi-polar OV-1701 stationary phase. These considerations indicate that the published occurrences of dinostanol and/or 4-methyl-24-ethyl-cholesterol in sediments should be considered with some suspicion when they are based only on the GC-MS retention times and mass spectra of minor amounts without co-injection experiments with authentic standards and/or m/z 98 mass fragmentographic measures. Some recent work with lacustrine sediments indicate in fact that ld might be mistaken for If (Prowse and Maxwell, 1991) and the former might be much frequently present than reported in the geochemical literature. Other minor or trace 4-methyl sterols detected in U. neglecta are quoted in Table I. For these compounds with a AT-unsaturation, standards were not available and the identifications are based on GC retention characteristics and mass spectral patterns. Like the 4-desmethyl sterols, which are accompanied by abundant corresponding ketones, significant amounts of C2s-C30 4-methyl-steran-3-ones, with a distribution similar to that of the fully saturated 4-methylsterols, are also present. Utricularia neglecta may therefore be the direct source of the 4-methyl ketones present in trace amounts in the underlying sediment. The finding of a source for 4-methyl sterols in recent lacustrine sediments has also a geochemical
761
implication for palaeoenvironments. Recent studies of C30 steranes (in which side-chain structures were determined) in diverse sediments and crude oils showed that samples of marine origin contain compounds with both side-chain structures d and f, while lacustrine samples are characterized by a dominance of steranes having ethyl-substituted side-chains, dinosteranes being absent or in very low abundance (Summons et al., 1987; Goodwin et al., 1988). The lacustrine Messel oil shale (Eocene) is also characterized by high abundance of citrostanol (ld) relative to dinosterol (lg) (Robinson et al., 1989). Citrostanol has been recently reported in four species of marine prymnesiophytes of the genus Pavlova (Volkman et al., 1990) along with its A22-unsaturated counterpart (le). In all cases the unsaturated compound was 13-35 times more abundant. This finding provided a plausible source for the 4-methyl sterols having a 24-ethyl substituent (ld and le) in recent marine sediments and the corresponding steranes in ancient sediments and crude oils of marine origin. Our resuits, on the other hand, provide a possible source for 4-methyl sterols present in recent lacustrine sediments. But, contrarily to prymnesiophytes whose importance in past geological periods is well documented by coceolith deposits dating back at least to the Jurassic, we have no knowledge on the geological time extension of Utricularia. If this latter can also be reasonably taken as the source for the 4-methyl-24ethyl sterols and/or corresponding 4-methyl steranes reported in ancient lacustrine crude oils or oil shales, then one can deduce that the plant has existed at least since the Cretaceous period. CONCLUSION The freshwater plant Utricularia neglecta contains large amounts of 4~-methyl sterols with 4~-methyl24-ethyl-5u-cholestan-3fl-ol as the major component. The similarity of the distribution of these compounds in the plant and in the underlying sediment suggests that this world-wide spread plant is an important source of the fully saturated 4-methyl sterols in recent lacustrine sediments. It may also be the source of the 24-ethyl substituted 4-methyl steranes in older sediments and crude oils of lacustrine origin.
Acknowledgements--Tiffs work was supported by the Fonds National Suisse de la Recherche Scientifique (Grant No. 20-26301.89) We thank Professor C. Djerassi (Standford University, U.S.A.) for kindly providing the natural dinosterol sample and D. Schefer and W. Kloeti for valuable technical assistance. REFERENCES
Bird C. W., Lynch J. M., Pirt F. J., Reid W. W., Brooks C. J. W. and Middleditch B. S. (1971) Steroids and squalene in Methylococcus capsulatus grown on methane. Nature 230, 473-474. Bohlin L., Kokke W. C. M. C., Fenical W. and Djerassi C. (1981) 4~-Methyl-24S-ethyl-5u-cholestan-3~ol and 4~-methyl-24S-ethyl-5~-cholest-8(14)-en-3/~-ol,
762
G. KLINK et al.
two new sterols from a cultured marine dinoflagellate. Phytochemistry 20, 2397-2401. Boudou J. P., Trichet J., Robinson N. and Brassell S. C. (1986) Lipid composition of a Recent Polynesian microbial mat sequence. Org. Geochem. lfl, 705-709. Bouvier P., Rohmer M., Benveniste P. and Ourisson G. (1976) Amo-Steroids in the bacterium Methyloccus capsulatus. Biochem. J. 159, 267-271. Brassell S. C. and Egiinton G. (1986) Molecular geochemical indicators in sediments. In Organic Marine Geochemistry (Edited by Sohn M. L.), pp. 10-32. ACS Symposium Series, No 305. Corey E. J., Howell M. G., Boston A., Young R. L. and Sneen R. A. (1956) Spectral and stereochemical studies with deuterated cyclohexanes. J. Am. Chem. Soc. 78, 5036-5040. Cranwell P. A. (1988) Lipid geochemistry of late Pleistocene lacustrine sediments from Burland, Cheshire, U.K. Chem. Geol. 68, 181-197. Goodwin N. S., Mann A. L. and Patience R. L. (1988) Structure and significance of C30 4-methyl steranes in lacustrine shales and oils. Org. Geochem. 12, 495-506. Grave E. V. (1957) A plant that captures animals under water. Nat. Hist., 74-97. Knapp F. F. Jr. and Schroepfer G. J. Jr. (1974) A novel synthesis of 4~- and 4fl-methylcholest-5-ene-3//-ol from 6//-bromo-4-methylcholest-4-en-3-one. J. Org. Chem. 39, 3247-3250. Knapp F. F. Jr. and Schroepfer G. J. Jr (1975) Chemical synthesis, spectral properties, and chromatography of 4~-methyl and 4//-methyl isomers of (24R)-24-ethyl-5~cholestan-3//-ol and (24S)-24-ethyl-cholesta-5,22-dien3//-ol. Steroids 26, 339-356. Kokke W. C. M. C., Fenical W. and Djerassi C. (1981) Sterols with unusual nuclear unsaturation from three cultured marine dinoflagellates. Phytochemistry 20, 127-134. de Leeuw J. W., Rijpstra W. I. C., Boon J. J., de Lange F. and Schenk P. A. (1976) The relationship between lipids from Fontinalis antipyretica, its detritus and the underlying sediment: the fate of waxesters and sterolesters. In Interaction Between Sediments and Fresh Water (Edited by Golterman H. L.), pp. 141-147. Proc. Int. Symposium, Amsterdam. de Leeuw J. W. and Baas M. (1986) Early stage diagenesis of steroids. In Biological Markers in the Sedimentary Record (Edited by Johns R. B.), pp. 101-123. Elsevier, Amsterdam. Macquaker J. H. S., Farrimond P. and Brassell S. C. (1986) Biological markers in the Rhaetian black shales of South West Britain. Org. Geochem. 10, 93-100. Mattern G., Albrecht P. and Ourisson G. (1970) 4-Methylsterols and sterols in Messel shale (Eocene). Chem. Cornman. 1570-1571. Mermoud F., Clerc O., Giila~;ar F. O. and Buchs A. (1981) Analyse des acides gras et des sterols dam le plancton du lac L6man. Arch. Sci. (Gendve) 34, 367-382.
Mermoud F., Gfilagar F. O., Siles S., Chassaing B. and Buchs A. (1982) 4-Methylsterols in recent lacustrine sediments: terrestrial, planktonic or some other origin7 Chemosphere l l , 557-567. Mermoud F., Wiinsche L., Clerc O., G~laqar F. O. and Buchs A. 0984) Steroidal ketones in the early transformations of AS-sterols in different types of sediments. Org. Geochem. 6, 25-29. Nes W. R. and McKean M. L. (1977) In Biochemistry of Steroids and Other lsopentenoids, pp. 416- 418. University Press, Baltimore, MD. Popov S., Carlson R. M. K., Wegmann A. and Djerassi C. (1976) Minor and trace sterols in marine invertebrates I. General methods of analysis. Steroids 28, 699-732. Prowse W. G. and Maxwell J. R. (1991) High molecular weight chlorins in a lacustrine shale. Org. Geochem. 17, 877-886. Robinson N., Cranwell P. A., Finlay B. J. and Egiinton G. (1984a) Lipids of aquatic organisms as potential contributors to lacustrine sediments. Org. Geochem. 6, 143-152. Robinson N., Egiinton G., Brassell S. C. and Cranwell P. A. (1984b) Dinoflagellate origin for sedimentary 4a-methylsteroids and 5#(H)-stanols. Nature 308, 439-442. Robinson N., Eglinton G., Cranwell P. A. and Zeng Y. B. (1989) Messel oil shale (western Germany): assessment of depositional paleoenvironment from the content of biological marker compounds. Chem. Geol. 76, 153-173. Steele J. A. and Mosettig E. (1963) The solvolysis of stigmasteryl tosylate J. Org. Chem. 28, 571-572. Summons R. E., Volkman J. K. and Boreham C. J. (1987) Dinosterane and other steroidal hydrocarbons of dinoflagellate origin in sediments and petroleum. Geochim. Cosmochim. Acta 51, 3075-3082. Taylor P. (1989) In The Genus Utricularia--A Taxonomic Monograph, pp. 598-605. HMSO Books, London. Volkman J. K., Gagosian R. B. and Wakeham S. (1984) free and esterified sterols of the marine dinottagellate Gonyaulax polygramma. Lipids 19, 457-465. Volkman J. K., Kearney P. and Jeffrey S. W. (1990) A new source of 4-methyl sterois and 5a(H)-stanols in sediments: prymnesiophyte microalgae of the genus Pavlova. Org. Geochem. 15, 489-497. Wolff G. A., Lamb N. A. and Maxwell J. R. (1986) The origin and fate of 4-methyl steroids-II. Dehydration of stanols and occurrence of C30 4-methyl steranes. Org. Geochem. lfl, 965-974. Wiinsche L. (1987) G6ochimie des lipides neutres et diagen6se pr6coce des st6rols dans des s6diments du bassin l~manique. Ph.D. thesis, University of Geneva. Wiinsche L., Mendoza Y. and Gfilaqar F. (1988) Lipid geochemistry of a post-glacial lacustrine sediment. Org. Geochem. 13, 1131-1143. Zielinski J., Kokke W. C. M. C., Tam Ha T. B., Shu A. Y. L., Duax W. L. and Djerassi C. (1983) Isolation, partial synthesis and structure determination of sterols with the four possible 23,24-dimethyl substituted side chains. J. Org. Chem. 48, 3471-3477.
Appendix opposite
Utricularia neglecta
L . - - 4 - m e t h y l sterol s o u r c e
763
APPENDIX
SC:
'"
a
IIII1,,
(~
., n
%
-
•
.
b
%.
H
1
R I , , H , R2=OH
2
R I " H, R 2 " O H ,
5 A
3
RI = H ,
"7 A
4
R2=OH ,
R1,R2sO
"'"~'..._~~ g H
f