Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue12.Dec2000:
Organic Geochemistry 31 (2000) 1703±1712
www.elsevier.nl/locate/orggeochem
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, Switzerland
Abstract
Investigation of chlorins in the oxic surface sediment of a small eutrophic alpine lake (Motte lake) revealed the presence of a new series of steryl chlorin esters containing the pheophorbide a nucleus, together with their pyropheophorbide a steryl ester counterparts previously observed in the anoxic surface sediment of the same lake.
Identi®cation of the pheophorbide a steryl esters was based on comparison of spectroscopic, chromatographic and
mass spectrometric characteristics of the compounds with those of a synthetic standard and of pyropheophorbide a
steryl esters. Combined liquid chromatography-mass spectrometry analysis con®rmed the absence of pheophorbide a
steryl esters in the anoxic sediment but allowed their detection in traces in the water column, indicating that pheophorbide a steryl esters are, like their pyropheophorbide a analogs, formed in the water column. The distribution of
sterols released by hydrolysis of the pheophorbide a steryl esters shows close similarities to that of the free sterols in the
water column and of the sterols of the pyropheophorbide a steryl esters. It appears that, like their pyropheophorbide a
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 zooplankton grazing on phytoplankton. 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 substituent in the anoxic environment. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Steryl chlorin esters; Sterols; Pheophorbide a; Pyropheophorbide a; Sediments; LC±MS
1. Introduction
Steryl chlorin esters, known to encompass a variety of
sterols esteri®ed to a common pyropheophorbide a tetrapyrrolic 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 occurrence and relative abundance [up to 40% of the total
HPLC-measured, extractable chlorins in a surface sediment 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
* Corresponding author. Fax: +41-22-321-5606.
E-mail address: [email protected] (F.O. GuÈlacËar).
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 sapropel 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 chlorophyll a.
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 phytoplankton blooms. The pyropheophorbide a steryl 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;
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00072-3
1704
C. RieÂ-Chalard et al. / Organic Geochemistry 31 (2000) 1703±1712
conclusion was therefore consistent with previous
reports, i.e. pyropheophorbide a steryl 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 pyropheophorbide 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 chlorins.
2. Experimental
2.1. General
All the solvents used were doubly-distilled or of
HPLC-grade. After each extraction and isolation, solvents were evaporated to dryness and compounds were
stored in the dark at ÿ18 C.
2.2. Sediment samples and extraction
Fig. 1. Structures of chlorins cited in the text.
King and Wakeham, 1996). Recently, the production of
pyropheophorbide b steryl esters during grazing has also
been demonstrated during a laboratory feeding experiment involving the copepod Calanus helgolandicus grazing 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 pyropheophorbide a steryl 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 pyropheophorbide a esters were the same. The
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, approximately 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
Na2SO4 and 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 40 mm; ®ltration time 1 h, 280 l/
min). This procedure produced less than 500 mg (dry
C. RieÂ-Chalard et al. / Organic Geochemistry 31 (2000) 1703±1712
weight) of particulate matter for each water column
sample. Organic compounds were ultrasonically extracted 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 120 C for 2 h, eluent: acetone/
hexane 25/75 v/v) of the total organic extract was carried out using pyropheophorbide a methyl ester (Sigma)
as a reference compound (Rf=0.30). The green-brown
bands containing neutral chlorins (0.2
www.elsevier.nl/locate/orggeochem
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, Switzerland
Abstract
Investigation of chlorins in the oxic surface sediment of a small eutrophic alpine lake (Motte lake) revealed the presence of a new series of steryl chlorin esters containing the pheophorbide a nucleus, together with their pyropheophorbide a steryl ester counterparts previously observed in the anoxic surface sediment of the same lake.
Identi®cation of the pheophorbide a steryl esters was based on comparison of spectroscopic, chromatographic and
mass spectrometric characteristics of the compounds with those of a synthetic standard and of pyropheophorbide a
steryl esters. Combined liquid chromatography-mass spectrometry analysis con®rmed the absence of pheophorbide a
steryl esters in the anoxic sediment but allowed their detection in traces in the water column, indicating that pheophorbide a steryl esters are, like their pyropheophorbide a analogs, formed in the water column. The distribution of
sterols released by hydrolysis of the pheophorbide a steryl esters shows close similarities to that of the free sterols in the
water column and of the sterols of the pyropheophorbide a steryl esters. It appears that, like their pyropheophorbide a
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 zooplankton grazing on phytoplankton. 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 substituent in the anoxic environment. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Steryl chlorin esters; Sterols; Pheophorbide a; Pyropheophorbide a; Sediments; LC±MS
1. Introduction
Steryl chlorin esters, known to encompass a variety of
sterols esteri®ed to a common pyropheophorbide a tetrapyrrolic 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 occurrence and relative abundance [up to 40% of the total
HPLC-measured, extractable chlorins in a surface sediment 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
* Corresponding author. Fax: +41-22-321-5606.
E-mail address: [email protected] (F.O. GuÈlacËar).
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 sapropel 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 chlorophyll a.
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 phytoplankton blooms. The pyropheophorbide a steryl 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;
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00072-3
1704
C. RieÂ-Chalard et al. / Organic Geochemistry 31 (2000) 1703±1712
conclusion was therefore consistent with previous
reports, i.e. pyropheophorbide a steryl 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 pyropheophorbide 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 chlorins.
2. Experimental
2.1. General
All the solvents used were doubly-distilled or of
HPLC-grade. After each extraction and isolation, solvents were evaporated to dryness and compounds were
stored in the dark at ÿ18 C.
2.2. Sediment samples and extraction
Fig. 1. Structures of chlorins cited in the text.
King and Wakeham, 1996). Recently, the production of
pyropheophorbide b steryl esters during grazing has also
been demonstrated during a laboratory feeding experiment involving the copepod Calanus helgolandicus grazing 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 pyropheophorbide a steryl 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 pyropheophorbide a esters were the same. The
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, approximately 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
Na2SO4 and 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 40 mm; ®ltration time 1 h, 280 l/
min). This procedure produced less than 500 mg (dry
C. RieÂ-Chalard et al. / Organic Geochemistry 31 (2000) 1703±1712
weight) of particulate matter for each water column
sample. Organic compounds were ultrasonically extracted 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 120 C for 2 h, eluent: acetone/
hexane 25/75 v/v) of the total organic extract was carried out using pyropheophorbide a methyl ester (Sigma)
as a reference compound (Rf=0.30). The green-brown
bands containing neutral chlorins (0.2