A new series of steryl chlorin esters: pheophorbide
a
steryl
esters in an oxic surface sediment
Catherine RieÂ-Chalard, Ludovica Verzegnassi, Fazil O. GuÈlacËar *
Laboratoire de SpectromeÂtrie de Masse, Universite de GeneÁve, 16 bd d'Yvoy, 1211 Geneva 4, SwitzerlandAbstract
Investigation of chlorins in the oxic surface sediment of a small eutrophic alpine lake (Motte lake) revealed the pre-sence of a new series of steryl chlorin esters containing the pheophorbide a nucleus, together with their pyr-opheophorbide a steryl ester counterparts previously observed in the anoxic surface sediment of the same lake. Identi®cation of the pheophorbide asteryl esters was based on comparison of spectroscopic, chromatographic and mass spectrometric characteristics of the compounds with those of a synthetic standard and of pyropheophorbidea
steryl esters. Combined liquid chromatography-mass spectrometry analysis con®rmed the absence of pheophorbidea
steryl esters in the anoxic sediment but allowed their detection in traces in the water column, indicating that pheo-phorbideasteryl esters are, like their pyropheophorbideaanalogs, formed in the water column. The distribution of sterols released by hydrolysis of the pheophorbideasteryl esters shows close similarities to that of the free sterols in the water column and of the sterols of the pyropheophorbideasteryl esters. It appears that, like their pyropheophorbidea
counterparts, pheophorbide a steryl esters incorporate mainly sterols of phytoplanktonic origin. Their formation probably involves the same mechanism as for pyropheophorbide a steryl ester formation, i.e. metabolism by zoo-plankton grazing on phytozoo-plankton. The presence of pheophorbide a steryl esters in the oxic sediment and their absence from the anoxic sediment is probably due to a lower stability of compounds containing a carbomethoxy sub-stituent in the anoxic environment.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Steryl chlorin esters; Sterols; Pheophorbidea; Pyropheophorbidea; Sediments; LC±MS
1. Introduction
Steryl chlorin esters, known to encompass a variety of sterols esteri®ed to a common pyropheophorbidea tet-rapyrrolic macrocycle (Fig. 1,1S), have been reported to occur widely in marine and lacustrine environments (Eckardt et al., 1991, 1992; King and Repeta, 1991; Prowse and Maxwell, 1991; Pearce et al., 1993, 1998; Chillier and GuÈlacËar, 1995). Their widespread occur-rence and relative abundance [up to 40% of the total HPLC-measured, extractable chlorins in a surface sedi-ment reported by King and Repeta (1991)] indicate that they form an important transformation pool of sterols and chlorophyll a (2) in sediments. To date, the only other chlorophyll a derived tetrapyrrolic macrocycle
found as a steryl chlorin ester is mesopyropheophorbide
a(3) in an immature lacustrine sediment of Miocene age (Prowse and Maxwell, 1991). Pyropheophorbide b (4) steryl esters have also been reported in recent lacustrine sediments (Pearce et al., 1993), in Mediterranean sapro-pel samples (Cariou-Le Gall et al., 1998) and in a Baltic Sea sediment (Kowalewska et al., 1999), suggesting that steryl chlorin esters can originate from chlorophylls other than chlorophylla.
Two processes for the formation of steryl chlorin esters were originally proposed. While King and Repeta (1991, 1994) considered that the esteri®cation of the pigment occurs by zooplankton herbivory, Eckardt et al. (1991, 1992) and Kowalewska (1994) were in favor of an esteri®cation during senescence following phyto-plankton blooms. The pyropheophorbideasteryl esters, detected in zooplankton guts and fecal material, were ®nally shown, however, to be formed by zooplankton herbivory (Harris et al., 1995; Harradine et al., 1996;
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* Corresponding author. Fax: +41-22-321-5606.
King and Wakeham, 1996). Recently, the production of pyropheophorbidebsteryl esters during grazing has also been demonstrated during a laboratory feeding experi-ment involving the copepodCalanus helgolandicus graz-ing on the prasinophyte Tetraselmis suicica (Talbot et al., 1999a).
It is now clear that steryl chlorin esters present great potential as sedimentary biomarkers since they re¯ect the original distribution of the autochthonous sterols at the time of deposition more accurately than do the free sterols (Eckardt et al., 1992; King and Repeta, 1994; Pearce et al., 1998). Recent laboratory studies showed, however, that the sterol distribution in steryl chlorin esters isolated from fecal pellets of copepods may dier to some extent from the distribution of the sterols in the phytoplankton on which the copepod grazes (Talbot, 1999; Talbot et al., 1999a±c).
In an earlier study carried out in our laboratory, we identi®ed pyropheophorbideasteryl esters in the anoxic surface sediment of a small eutrophic lake (Motte lake; Chillier and GuÈlacËar, 1995). We also showed that the distributions of free sterols from a sediment trap placed in the anoxic zone of the water column and of sterols from the pyropheophorbideaesters were the same. The
conclusion was therefore consistent with previous reports, i.e. pyropheophorbideasteryl esters are formed in the water column and they only incorporate sterols of autochthonous biomass.
As part of a study of chlorophyll transformation in the water column and in the sediments of Motte lake, we have investigated the distribution of steryl chlorin esters in oxic and anoxic surface sediments as well as in particulate matter from the oxic and anoxic zones of the water column. We report here the identi®cation of pheophorbide a steryl esters (5S) in the oxic surface sediment of Motte lake together with their pyr-opheophorbide a steryl ester counterparts. The origin and geochemical implications of these new steryl chlorin esters are discussed on the basis of a comparison between the distributions of free sedimentary sterols, water column free sterols and sterols esteri®ed to chlor-ins.
2. Experimental
2.1. General
All the solvents used were doubly-distilled or of HPLC-grade. After each extraction and isolation, sol-vents were evaporated to dryness and compounds were stored in the dark atÿ18C.
2.2. Sediment samples and extraction
Surface sediments were collected from Motte lake, a small freshwater eutrophic alpine lake near Thonon (France). The ®rst sampling site, under 9 m water depth, remains permanently anoxic. The second sampling site, under 4 m water depth, is in the oxic zone of the lake. Extraction and separation of pigments were done according to published procedures (King and Repeta, 1991; Chillier and GuÈlacËar, 1995). Brie¯y, approxi-mately 500 g of wet surface sediment were extracted ultrasonically (5 min) with four portions of acetone (250 ml) followed by three portions of methylene chloride (250 ml). The combined extracts were concentrated to 200 ml and the same volume of a solution of hexane/ diethyl ether (30/70 v/v) was added. After removal of the aqueous phase, the organic extract was dried over Na2SO4and evaporated to dryness, yielding about 100 mg of a dark brown residue.
2.3. Water column samples and extraction
Suspended particulate matter was collected at 7 m depth in the anoxic zone of the water column, and at 3.50 m depth in the oxic zone by pumping and ®ltration of the water (porosity 40mm; ®ltration time 1 h, 280 l/ min). This procedure produced less than 500 mg (dry
weight) of particulate matter for each water column sample. Organic compounds were ultrasonically extrac-ted as described above.
2.4. Separation of chlorins from total organic extract
Preparative thin layer chromatography (TLC) separation (Merck, Kieselgel 60, 0.5 mm, washed with acetone and activated at 120C for 2 h, eluent: acetone/ hexane 25/75 v/v) of the total organic extract was car-ried out using pyropheophorbideamethyl ester (Sigma) as a reference compound (Rf=0.30). The green-brown bands containing neutral chlorins (0.2<Rf<0.50) were collected and combined in acetone.
2.5. Isolation of steryl chlorin esters
In order to allow their distinct analysis, pheophorbide
a steryl esters (Fig. 2b, fraction X) and pyr-opheophorbide a steryl esters were isolated from the total organic extract by TLC using the same conditions as above. The two uppermost green-brown bands were removed and eluted with CH2Cl2and acetone. The ®rst band (Rf=0.47) contained mainly pyropheophytina(6) and pyropheophorbideasteryl esters while the second band (Rf=0.42) contained pheophytina(7) and pheo-phorbideasteryl esters. The contents of each band were then submitted to preparative HPLC (Merck LiChro-spher 100 RP-18, 10mm, 25010 mm, methanol/acet-one 50/50 v/v, ¯ow 3 ml/min) for further puri®cation of the two classes of esters.
2.6. Synthesis of a pheophorbide a steryl ester standard (8)
The starting product pheophorbidea(5) was obtained from the spray-dried blue alga Spirulina platensis
(Fischer et al., 1994; Fischer, 1995). Brie¯y, chlorophyll
awas extracted with methanol/oxalic acid (0.01% w/w) and precipitated by addition of dioxane and water. After demetallation and hydrolysis of the phytyl esters with aqueous HCl (30%), carotenoids were removed with diethyl ether. The aqueous phase was diluted with the same volume of water and extracted with chloroform. The organic phase was then dried over Na2SO4and eva-porated, yielding the dark-brown pheophorbidea.
Pheophorbideastigmasteryl ester (8) was synthesized from pheophorbide a and stigmasterol (recrystallized) according to King and Repeta (1994). The compound was separated from unreacted products by preparative HPLC (Merck LiChrospher 100 RP-18, 10mm, 250 10 mm) with methanol/acetone 50/50 v/v (¯ow 3 ml/ min) and puri®ed with methanol/acetone 60/40 v/v. Pheophorbideastigmasteryl ester was analyzed by LC± MS (see conditions below) and aorded typical ions at
m/z 987 (MH+), m/z 955 ([MH-32 Da]+), m/z 929 ([MH-58 Da]+), andm/z593 ([MH-sterene]+).
2.7. Isolation of free sterols
Free sterols were isolated from total organic extracts by preparative TLC (Merck, Kieselgel 60, 0.5 mm, washed with acetone and activated at 120C for 2 h, eluent CH2Cl2) using stigmasterol (Rf=0.16) as the reference compound. The band containing the free ster-ols (0.1<Rf<0.46) was collected and eluted with CH2Cl2.
2.8. Isolation of esterifying sterols from steryl chlorin esters
Alkaline hydrolysis of steryl chlorin esters was per-formed according to Chillier and GuÈlacËar (1995). The fraction containing the steryl chlorin esters (2 mg) was re¯uxed with a mixture of THF/NaOH 4M (1/1 v/ v) for 1 h. After adding diethyl ether in order to obtain a phase separation, the organic layer was separated and washed with three portions of acetic acid (5%), fol-lowed by ®ve portions of distilled water. The organic extract was dried over Na2SO4 and evaporated under N2.
2.9. High performance liquid chromatography (HPLC) and combined liquid chromatography±mass spectrometry (LC±MS) analysis
HPLC analyses were performed with a Merck Hitachi HPLC instrument equipped with a Rheodyne 7125 injector ®tted with a 20 ml loop and a Merck LiChro-spher 100 RP-18 column (5mm, 250 x 4 mm). Table 1 shows the solvent (Merck, HPLC-grade) system used. Quanti®cation of chlorins was performed using pyr-opheophorbide amethyl ester as an external standard and a Merck Hitachi L-4520 UV±visible detector (366 nm). An on-line diode-array detector (Merck Hitachi L-4500) was also used to record UV±vis spectra (350±800 nm).
LC±MS was performed using the same HPLC system coupled to a SSQ 7000 Finnigan MAT via a Finnigan MAT atmospheric pressure chemical ionization (APCI) interface. The APCI interface conditions were: sheath gas (nitrogen) pressure 40 psi (no auxiliary gas), vapor-izer temperature 550C, ion transfer capillary 230C, corona electrode 5mA. The capillary voltage was set at +8.8 V, and CID oset was o. Spectra were obtained in the positive ionization mode, scanning fromm/z400 tom/z1300 in 2 s.
2.10. Derivatization and gas chromatography±mass spectrometry (GC±MS) analysis
performed with a HP 5890 series II chromatograph coupled to a VG Mass Lab Trio-2 quadrupole mass spectrometer. The chromatograph was equipped with a split/splitless injector (carrier gas helium 50 kPa), and a capillary column (Alltech EC-5; 30 m0.25 mm, 0.25 mm). The temperature program was 1 min at 40C, 10C/min to 200C, 3C/min to 280C and ®nally 30 min at 280C. The mass spectrometer operated in the electron ionization mode (70 eV) with a source tem-perature of 220C. Spectra were recorded in full scan mode from 50 to 650 Da (1 scan/s).
Compounds were identi®ed by their mass spectra and relative retention times and the relative amounts of sterols were determined using peak areas in the total ion chromatogram.
Table 1
Solvent system for HPLC separation of tetrapyrroles on a Merck RP-18 column (Lichrospher 100, 2504 mm) with a 1 ml/min ¯owa
Time (min) A (%) B (%) C (%)
0 0 90 10
2 5 85 10
10 50 40 10
18 50 45 5
22 50 40 10
50 65 35 0
110 90 10 0
a A, acetone; B, methanol; C, water (containing 2.5%
methanol).
3. Results and discussion
3.1. Steryl chlorin esters in anoxic sediments
Fig. 2a shows the HPLC chromatogram (366 nm) of the chlorin fraction from the anoxic surface sediment of Motte lake. The major chlorins were identi®ed from their on-line UV±vis spectra (lmax: 400, 503, 635 nm) and their retention times as pheophytin a(7) (with its epimer70) and pyropheophytina(6). These well known chlorophylladegradation products were followed by a discrete group of components eluting between 54 and 60 min and having electronic spectra consistent with a pheophytin aor pyropheophytinatype of chlorin and presumably corresponding to the pyropheophorbide a
steryl esters.
LC±MS analysis aorded spectra for the individual peaks with a typical pattern (Harris et al., 1995; Ver-zegnassi et al., 1999). Thus, pheophytina(7) gave MH+ as base peak atm/z871, [MH-58 Da]+atm/z813 cor-responding to the loss of the C-132carbomethoxy sub-stituent with hydrogen transfer, and [MH-phytadiene]+ atm/z593. Similarly, pyropheophytina(6) gave MH+ as base peak atm/z813 and [MH-phytadiene]+atm/z 535. The compounds eluting between 54 and 60 min were con®rmed as pyropheophorbide a steryl esters: MH+was the base peak and a common fragment ion at
m/z 535 corresponded to the loss from the C-172 side chain of the corresponding alkene. Them/zvalues of the protonated molecules of individual peaks showed that the major esterifying sterols were C27:1, C28:2, C29:1and C29:2(Table 2).
3.2. Steryl chlorin esters in oxic sediments
The HPLC chromatogram (366 nm) of the chlorins from the oxic surface sediment (Fig. 2b) resembles that of the anoxic sediment. However, one can observe the presence of another group of compounds eluting between 48 and 54 min (fractionX). This group has the same distribution pattern as the pyropheophorbide a
steryl esters. Based on their retention times and their on-line electronic absorption spectra (lmax: 400, 503, 635 nm), these compounds were suspected to be another series of steryl chlorin esters.
On LC±MS analysis, the fraction X compounds (tR=48±54 min) exhibited protonated molecules (MH+) ranging fromm/z961 tom/z991, indicating molecular weights greater than those of pyropheophorbideasteryl esters. Furthermore, the spectra of all components had a fragment ion atm/z593 instead ofm/z535, which sug-gested for these compounds a pheophorbide achlorin nucleus. As the mass spectra included other fragments in the region of MH+ and some overlapping with the pyropheophorbideasteryl esters occurred, we isolated fractionXfrom the total chlorins by preparative TLC and preparative HPLC (see Experimental). Pyro-pheophorbide a steryl esters were separated from the TLC band containing pyropheophytin a, while the fractionXcompounds eluted with pheophytina, giving further evidence of their pheophorbideanucleus. Fig. 3 shows the HPLC chromatogram of the isolated fraction
Xand the LC±MS spectrum of peaka. The synthesized pheophorbide a stigmasteryl ester coeluted perfectly with peak a and exhibited the same UV±visible (400,
Table 2
LC±MS analysis of steryl chlorin esters Ð principal MS peaks (base peaks are underlined and trace components are given in brackets) and corresponding sterols
Steryl chlorin esters Anoxic sedimentm/z(uma) Oxic sedimentm/z(uma) Corresponding sterol
Pheophorbidea 593, 915, 941, 973 C28:2
steryl esters Not detected 593, 903, 929, 961 C27:1
(FractionX) 593, 929, 955, 987 C29:2
Pyropheophorbidea (535, 901) (535, 901) C27:2
steryl esters 535, 915 535, 915 C28:2
503, 635 nm) and mass spectral characteristics: a proto-nated molecule atm/z987 (MH+) as base peak,m/z955 ([MH-32 Da]+) corresponding to the loss of CH
3OH from the C-132 carbomethoxy substituent, m/z 929 ([MH-58 Da]+) corresponding to the loss of the whole C-132carbomethoxy substituent with hydrogen transfer, andm/z593 corresponding to the loss of the sterol as the corresponding sterene. The other peaks eluting between 47 and 54 min provided LC±MS spectra (Table 2) with the same fragmentation pattern and could all be identi®ed as pheophorbide a steryl esters. Thus, the major esterifying alcohols of the pheophorbideasteryl esters could be recognized as C27:1, C28:2, C28:1, C29:2, C29:1sterols, and a C29stanol. Peakbhad a retention time and a mass spectrum (MH+atm/z843 and [MH-phytadiene]+ atm/z 565) consistent with purpurin-18-phytyl ester (9), an oxidative chlorophyll degradation product recently identi®ed in two contemporary lacus-trine sediments (Naylor and Keely, 1998).
The isolated pyropheophorbide a steryl esters were also analyzed by HPLC and LC±MS (Table 2) and appeared to contain mainly C27:1, C28:2, C28:1, C29:2, and C29:1sterols. Traces of other pyropheophorbideaesters could be detected and seemed to include a C27:2 ester-ifying sterol and several esterester-ifying stanols (C28:0, C29:0 and C30:0).
The quanti®cation results for the pheophorbide a
steryl esters, pyropheophorbide a steryl esters, pheo-phytinaand pyropheophytinaare given in Table 3.
3.3. Steryl chlorin esters in the suspended particulate matter of the oxic and anoxic water column
Extraction of suspended particulate matter from the oxic and anoxic parts of the water column yielded 25 and 45 mg of total organic matter, respectively. Because of the sample limitation, we decided to use all of the oxic water column extract to investigate the steryl
chlorin esters, while the anoxic water column extract was separated in two aliquots: 15% for the investigation of free sterols, and 85% for the study of chlorins. HPLC and LC±MS analyses were performed separately on the two uppermost TLC bands (Rf0.47 and 0.42) but sam-ple limitation did not allow isolation and quanti®cation of steryl chlorin esters.
In spite of their low concentration, we could detect pyropheophorbideaand pheophorbideasteryl esters in oxic and anoxic water column samples thanks to their retention times and their absorption maxima observed by HPLC-DAD (350±800 nm). In particular, LC±MS analysis of the oxic water column sample exhibited mass fragmentograms form/z 593 and m/z 961 which con-®rmed the presence of pheophorbide a esteri®ed to a C27:1sterol.
The presence of pheophorbideasteryl esters in susp-ended particulate matter suggests that they are, like the pyropheophorbideasteryl esters, produced in the water column. However, are they produced by the same proc-ess and do they incorporate the same sterols? In order to answer these questions, further investigation of the esterifying alcohols was achieved by hydrolysis of steryl chlorin esters isolated from the oxic sediment and comparison of their distribution with the free sterols distribution.
Fig. 3. Partial HPLC chromatogram (366 nm) of the isolated pheophorbideasteryl esters with coinjection of pheophorbidea stig-masteryl ester and LC±MS spectrum of the peaka.
Table 3
Quanti®cation of chlorins in oxic and anoxic surface sediments (mg/g dry sediment)
Compound Oxic surface sediment
Anoxic surface sediment
3.4. Free sterols and sterols bound to chlorins
The distributions of cyclic alcohols in the anoxic sur-face sediment, those associated with the suspended par-ticulate matter from the water column and the sterols released by hydrolysis of the pheophorbideaand pyr-opheophorbide aesters from the oxic surface sediment are shown in Fig. 5. Compound names and structures are given in Table 4 and Fig. 4, respectively. The water column particulate matter yields a free sterol distribu-tion characteristic of a mainly phytoplanktonic origin, as previously observed by WuÈnsche (1987) in sediment trap and plankton samples from the same lake. The major constituents are cholesterol (C) and b-sitosterol (N) and C27sterols are approximately 75% of C29 ster-ols. In contrast, C29 stenols/stanols and 4a-methyl-stenols/stanols predominate in the sedimentary distribution where C27sterols are less than 10% of C29 sterols. A higher contribution of the more refractory higher plant sterols (N,O,Q, R, T), originating from plant debris, is therefore obvious.
The distributions of sterols released from pheo-phorbide a esters and pyropheophorbide a esters are qualitatively and quantitatively similar to that of the water column. As evidence, statistical analyses of the
distributions showed that the relative amounts of free sterols in the water column strongly correlate with those of the pyropheophorbidea and pheophorbide aesters (R=0.94 and 0.91, respectively).
As previously reported for the pyropheophorbide a
steryl esters from the anoxic surface sediment of the
Fig. 4. Structures of sterol nuclei, cyclic alcohols and sterol side chains cited in the text.
same lake (Chillier and GuÈlacËar, 1995), the steryl chlorin esters from the oxic sediment incorporate mainly the autochthonous sterols from microplankton. It may, however, appear surprising that the 4a-methyl sterols with a 23,24-dimethyl substitution on the side chain (i.e. componentsP andS), speci®c to the unicellular dino-¯agellate algae, are abundant in the surface sediment while they are minor in or absent from the water column and in the chlorin esters. The same observation has been made by King and Repeta (1991) and may be attributed to the fact that the dino¯agellates appear as blooms which may disappear quickly and for long periods. Another possible explanation is the higher stability of sedimentary 4-methylsterols relative to desmethylsterols (Dreier et al., 1988; King and Repeta, 1994). Regarding the other 4-methyl sterols with a 24-ethyl substituent (componentsQ andT), we have shown that, in Motte lake, they originate from an aquatic plant Utricularia neglecta(Klink et al., 1992); they are probably incorpo-rated into sediments by very rapidly sinking plant debris so that they are quasi absent from suspended particles. Finally, the triterpenoid alcohols isoarborinol (W) and diplopterol (X) which originate mainly from land plants (Graminae and ferns, respectively) are not detected as chlorin esters.
In spite of the close resemblance between chlorin ester sterols and particulate matter sterols, some small but signi®cant dierences can be observed. The relative amount of campesterol (I) is lower in chlorin esters when compared to the water column sterols. On the other hand, gorgosterol (U) and gorgostanol (V) are detected in chlorin esters as well as in the surface sedi-ment although they are absent from the suspended par-ticles. The latter compounds, considered to be speci®c to marine sponges (Gorgonians), have already been repor-ted in the surface sediment of Motte lake and the possi-bility of a freshwater dino¯agellate origin was envisaged (WuÈnsche et al., 1987). The above dierences are prob-ably due to dissimilarities in the modes of transport of biogenic sterols to the sediment which, in turn, depends on the nature of the source organisms.
Because of the sample size limitation we could not analyze the sterols of the chlorin esters in the suspended particulate matter by GC±MS after hydrolysis. How-ever, the results we report show clearly that the pheo-phorbideaesters are formed in the water column, as are the pyropheophorbide a esters, and they are all pro-duced by the same process, i.e. zooplankton grazing. It would be tempting to postulate that the pheophorbidea
esters are intermediate products in the formation of the
Table 4
Cyclic alcohols in Motte lake samplesa
Polycyclic alcohols No. Structure RRTb
27-Nor-24-methyl-cholesta-5,22-dien-3b-ol A Ig 0.965
Cholesta-5,22-dien-3b-ol B If 0.974
Cholest-5-en-3b-ol C Ia 1.000
5a-Cholestan-3b-ol D IIa 1.006
24-Methyl-cholesta-5,22-dien-3b-ol E Ib 1.026
24-Methyl-5a-cholest-22-en-3b-ol F IIb 1.032
5a-Cholest-7-en-3b-ol G IIIa 1.033
24-Ethyl-5b-cholestan-3b-ol H IVe 1.064
24-Methyl-cholest-5-en-3b-ol I Ic 1.068
24-Methyl-5a-cholestan-3b-ol J IIc 1.077
24-Ethyl-cholesta-5,22-dien-3b-ol K Id 1.094
24-Ethyl-5a-cholest-22-en-3b-ol L IId 1.102
24-Methyl-5a-cholest-7-en-3b-ol M IIIc 1.113
24-Ethyl-cholest-5-en-3b-ol N Ie 1.144
24-Ethyl-5a-cholestan-3b-ol O IIe 1.153
4a,23,24-Trimethyl-5a-cholest-22-en-3b-ol P Vh 1.184 4a-Methyl-24-ethyl-5a-cholest-22-en-3b-ol Q Vd 1.189
24-Ethyl-5a-cholest-7-en-3b-ol R IIIe 1.192
4a,23,24-Trimethyl-5a-cholest-17(20)-en-3b-ol S Vj 1.214 4a-Methyl-24-ethyl-5a-cholestan-3b-ol T Ve 1.236 22,23-Methylene-23,24-dimethyl-cholest-5-en-3b-ol U Ii 1.268 22,23-Methylene-23,24-dimethyl-5a-cholestan-3b-ol V IIi 1.280
Isoarborinol W VI 1.303
Diplopterol X VII 1.361
a For structures refer to Fig. 4.
pyropheophorbide a esters. However, if this was the case, Harradine et al. (1996), Talbot (1999) and Talbot et al. (1999a,b) should have observed them together with the pyropheophorbide aesters found in the fecal pellets of the copepodCalanus helgolandicuswhich was fed in laboratory experiments with various phytoplank-tonic species (a diatom, a prasinophyte, several chlor-ophytes, dino¯agellates and haptophytes). Likewise, King and Wakeham (1996) detected only pyr-opheophorbide aesters in the guts of salps sampled in the Sargasso Sea and not their pheophorbide a coun-terparts. However, as pointed out by King and Wake-ham (1996), the identi®cation of this new series of steryl chlorin esters with a pheophorbideanucleus may pro-vide support for a biochemical pathway for steryl chlorin ester formation starting with the transesteri®ca-tion of the phytol side-chain of chlorophyll a, sequen-tially followed by demetallation and then by the loss of the carbomethoxy group.
During our laboratory experiments, we observed that the pheophorbide a steryl esters are more sensitive to alkaline hydrolysis than are the pyropheophorbide a
esters. This may suggest a greater lability of pheo-phorbideaesters in the water column or in the sediment (pH of the water in Motte lake varies from 8 at the surface to 6.5 at the bottom). Investigation of steryl chlorin esters in a sediment core sampled in the oxic zone showed that the pheophorbide aesters disappear within a few cm below the surface, in contrast to the pyropheophorbideaesters (unpublished results). Com-parison of the data for the oxic and anoxic sediments (Table 3) reveals the relative preservation of pyr-opheophytinaand pyropheophorbideasteryl esters vs. pheophytin aand pheophorbide asteryl esters. There-fore, the presence of the pheophorbideasteryl esters in the oxic surface sediment and their absence from the anoxic sediment can be explained by lower stability of pheophorbide macrocycle in the anoxic environment.
4. Conclusions
A novel series of pheophorbideasteryl esters isolated from an oxic surface sediment has a sterol distribution quite similar to that of the co-occurring pyr-opheophorbideasteryl esters and these components are probably produced by the same mechanism, i.e. meta-bolism during zooplankton grazing on phytoplankton. The presence of pheophorbideasteryl esters in the oxic sediment and their absence from the anoxic sediment is most likely due to a lower stability of the carbo-methoxy-containing compounds in anoxia. The ques-tion remains whether pheophorbide a steryl esters are widespread by-products of zooplankton herbivory which have not been detected till now because of inap-propriate sampling or analysis, or if they are produced
by speci®c zooplanktonic populations present at Motte lake.
Acknowledgements
We thank Dr W. Behr (D-Bonn) for kindly furnishing the spray-dried blue algaeSpirulina Platensis. This work was supported by the Fonds National Suisse de la Recherche Scienti®que (Grant No. 2000-52429.97).
References
Cariou-Le Gall, V., Rosell-Mele, A., Maxwell, J.R., 1998. Data report: characterization of distributions of photosynthetic pigments in sapropels from holes 966D and 969C1.
Proceed-ings of the Ocean Drilling Program, Scienti®c Results 160, 297±302.
Chillier, X.Fr.D., GuÈlacËar, F.O., 1995. Characterisation of chlorin steryl esters in sediments by desorption/mass spec-trometry and geochemical signi®cation. Archives des Sci-ences GeneÁve 48, 29±40.
Dreier, F., Buchs, A., GuÈlacËar, F.O., 1988. The degradation rates of some sterols in a heated sediment. Geochimica et Cosmochimica Acta 52, 1663±1666.
Eckardt, C.B., Keely, B.J., Maxwell, J.R., 1991. Identi®cation of chlorophyll transformation products in a lake sediment by combined liquid chromatography±mass spectrometry. Jour-nal of Chromatography 557, 271±288.
Eckardt, C.B., Pearce, G.E.S., Keely, B.J., Kowalewska, G., JaeÂ, R., Maxwell, J.R., 1992. A widespread chlorophyll transformation pathway in the aquatic environment. Organic Geochemistry 19, 217±227.
Fischer, R., 1995. Chlorophyllabbau in Seidenraupen (Bombyx moriL.): BeitraÈge zur StrukturaufklaÈrung der prosthetischen Gruppe eines rot ¯uoreszierenden chromoproteins. Inaugu-ral dissertation, University of Freiburg, Switzerland. Fischer, R., Engel, N., Henseler, A., Gossauer, A., 1994.
Synthesis of chlorophyll a labeled at C(32) from
pheo-phorbideamethyl ester. Helvetica Chimica Acta 77, 1046± 1050.
Harradine, P.J., Harris, P.G., Head, R.N., Harris, R.P., Max-well, J.R., 1996. Steryl chlorin esters are formed by zoo-plankton herbivory. Geochimica et Cosmochimica Acta 60, 2265±2270.
Harris, P.G., Carter, J.F., Head, R.N., Harris, R.P., Eglinton, G., Maxwell, J.R., 1995. Identi®cation of chlorophyll trans-formation products in zooplankton faecal pellets and marine sediment extracts by liquid chromatography/mass spectro-metry atmospheric pressure chemical ionization. Rapid Communications in Mass Spectrometry 9, 1177±1183. King, L.L., Repeta, D.J., 1991. Novel pyropheophorbide steryl
esters in Black Sea sediments. Geochimica et Cosmochimica Acta 55, 2067±2074.
King, L.L., Wakeham, S.G., 1996. Phorbin steryl ester forma-tion by macrozooplankton in the Sargasso Sea. Organic Geochemistry 24, 581±585.
Klink, G., Dreier, F., Buchs, A., GuÈlacËar, F.O., 1992. A new source for 4-methyl sterols in freshwater sediments: Utricu-laria neglectaL. (Lentibulariaceae). Organic Geochemistry 18, 757±763.
Kowalewska, G., 1994. Steryl chlorin esters in sediments of the Southern Baltic Sea. Netherlands Journal of Aquatic Ecol-ogy 28, 149±156.
Kowalewska, G., Winterhalter, B., Talbot, H.M., Maxwell, J.R., Konat, J., 1999. Chlorins in sediments of the Gotland Deep (Baltic Sea). Oceanologia 41, 81±97.
Naylor, C.C., Keely, B.J., 1998. Sedimentary purpurins: oxi-dative transformation products of chlorophylls. Organic Geochemistry 28, 417±422.
Pearce, G.E.S., Keely, B.J., Harradine, P.J., Eckardt, C.B., Maxwell, J.R., 1993. Characterisation of naturally occurring steryl esters derived from chlorophyll-a. Tetrahedron Letters 34, 2989±2992.
Pearce, G.E.S., Harradine, P.J., Talbot, H.M., Maxwell, J.R., 1998. Sedimentary sterols and steryl chlorin esters: distribution dierences and signi®cance. Organic Geochemistry 28, 3±10. Prowse, W.G., Maxwell, J.R., 1991. High molecular weight
chlor-ins in a lacustrine shale. Organic Geochemistry 17, 877±886. Talbot, H.M., 1999. Steryl chlorin esters: Origin, Signi®cance
and Potential as Indicators of Phytoplankton Community Structure. PhD thesis, University of Bristol, Bristol, UK. Talbot, H.M., Head, R.N., Harris, R.P., Maxwell, J.R., 1999a.
Steryl esters of pyrophaeophorbide b: a sedimentary sink for chlorophyll b. Organic Geochemistry 30, 1403±1410. Talbot, H.M., Head, R.N., Harris, R.P., Maxwell, J.R., 1999b.
Distribution and stability of steryl chlorin esters in copepod faecal pellets from diatom grazing. Organic Geochemistry 30, 1163±1174.
Talbot, H.M., Head, R.N., Course, P.A., Harris, R.P., Max-well, J.R., 1999c. Biogeochemical implications of phyto-planktonic photosynthetic pigment changes during herbivory. In: 19th International Meeting on Organic Geo-chemistry, Istanbul, Turkey. Abstracts Part I, pp. 153±154. Verzegnassi, L., RieÂ-Chalard, C., Kloeti, W., GuÈlacËar, F.O.,
1999. Analysis of tetrapyrrolic pigments and derivatives in sediments by high performance liquid chromatography/ atmospheric pressure chemical ionization mass spectrometry. Fresenius' Journal of Analytical Chemistry 364, 249±253. WuÈnsche, L., 1987. GeÂochimie des lipides neutres et diageneÁse
preÂcoce des steÂrols dans des seÂdiments du bassin leÂmanique. PhD thesis, no.2260, University of Geneva, Geneva, Swit-zerland.
