New 2-methyl-13-icosenoic acid from the temperate calcisponge Leuconia johnstoni

Article abstract of DOI:10.1007/s11745-011-3631-4

The fatty acid composition of the temperate calcareous marine sponge Leuconia johnstoni Carter 1871 (Calcaronea, Calcarea) was characterized for the first time in specimens collected off the Brittany coast of France over four years from October 2005 to September 2008. Forty-one fatty acids (FA) with chain lengths ranging from C14 to C22 were identified as fatty methyl esters (FAME) and N-acyl pyrrolidide (NAP) derivatives by gas chromatography-mass spectrometry (GC-MS). Twenty-two saturated fatty acids (SFA) were identified accounting for 52.1-59.0% of the total FA and dimethylacetals (DMA). In addition, among the SFA, we noticed the presence of numerous methyl-branched iso and anteiso FA, suggesting a large number of associated bacteria within L. johnstoni. Thirteen monounsaturated fatty acids (MUFA, 28.0-36.0% of total FA? DMA) were also identified as well as six polyunsaturated fatty acids (PUFA, 4.0-8.2%). A noticeable DMA was detected at a high level, particularly in September 2008 (11.8%), indicating the presence of plasmalogens in this sponge species. This calcareous sponge lacked the non-methylene-interrupted FA (NMI FA) with a Δ5,9 system typical of siliceous Demosponges and Hexactinellids. The occurrence of the unusual 8,13-octadecadienoic acid was reported for the first time as a minor PUFA in a calcareous sponge. The major FA, representing 20-25% of this sponge FA, was identified as the new 2-methyl-13-icosenoic acid from mass spectra of its methyl ester and its corresponding N-acyl pyrrolidide derivatives as well as a dimethyl disulfide adduct. AOCS 2011.

Full text of DOI:10.1007/s11745-011-3631-4

Lipids (2012) 47:345–353
DOI 10.1007/s11745-011-3631-4
ORIGINAL ARTICLE
New 2-Methyl-13-Icosenoic Acid from the Temperate Calcisponge
Leuconia johnstoni
•
Elodie Quevrain Gilles Barnathan
•
´
Tarik Meziane Isabelle Domart-Coulon
•
•
•
Vony Rabesaotra Marie-Lise Bourguet-Kondracki
Received: 16 May 2011 / Accepted: 3 November 2011 / Published online: 27 November 2011
Ó AOCS 2011
Abstract The fatty acid composition of the temperate
calcareous marine sponge Leuconia johnstoni Carter 1871
(Calcaronea, Calcarea) was characterized for the first time in
specimens collected off the Brittany coast of France over
four years from October 2005 to September 2008. Forty-one
fatty acids (FA) with chain lengths ranging from C14 to C22
were identified as fatty methyl esters (FAME) and N-acyl
pyrrolidide (NAP) derivatives by gas chromatography–mass
spectrometry (GC–MS). Twenty-two saturated fatty acids
(SFA) were identified accounting for 52.1–59.0% of the total
FA and dimethylacetals (DMA). In addition, among the
SFA, we noticed the presence of numerous methyl-branched
iso and anteiso FA, suggesting a large number of associated
bacteria within L. johnstoni. Thirteen monounsaturated fatty
acids (MUFA, 28.0–36.0% of total FA ? DMA) were also
identified as well as six polyunsaturated fatty acids (PUFA,
4.0–8.2%). A noticeable DMA was detected at a high level,
particularly in September 2008 (11.8%), indicating the
presence of plasmalogens in this sponge species. This cal-
careous sponge lacked the non-methylene-interrupted FA
(NMI FA) with a D5,9 system typical of siliceous
Demosponges and Hexactinellids. The occurrence of the
unusual 8,13-octadecadienoic acid was reported for the first
time as a minor PUFA in a calcareous sponge. The major FA,
representing 20–25% of this sponge FA, was identified as the
new 2-methyl-13-icosenoic acid from mass spectra of its
methyl ester and its corresponding N-acyl pyrrolidide
derivatives as well as a dimethyl disulfide adduct.
Keywords Calcareous sponge ꢀ Leuconia johnstoni ꢀ
Fatty acids ꢀ 2-Methyl-13-icosenoic acid ꢀ
8,13-Octadecadienoic acid ꢀ Dimethylacetal
Abbreviations
amu
Atomic mass unit
DMA
DMDS
DUFA
ECL
Dimethylacetal(s)
Dimethyl disulfide
Diunsaturated fatty acid(s)
Equivalent chain length
Fatty acid(s)
FA
FAME
Fatty acid methyl ester(s)
GC–MS Gas chromatography–mass spectrometry
MS
Mass spectrum (spectra)
Monounsaturated fatty acid(s)
N-Acyl pyrrolidide(s)
´
MUFA
NAP
E. Quevrain ꢀ M.-L. Bourguet-Kondracki (&)
´
Molecules de Communication et Adaptation
des Micro-organismes UMR 7245 MNHN-CNRS,
NMI FA Non-methylene-interrupted fatty acid(s)
´
Museum National d’Histoire Naturelle, 57 rue Cuvier,
PUFA
SFA
Polyunsaturated fatty acid(s)
Saturated fatty acid(s)
C.P. 54, 75005 Paris, France
e-mail: bourguet@mnhn.fr
TLC
Thin layer chromatography
G. Barnathan ꢀ V. Rabesaotra
´
´
`
Groupe Mer Molecules Sante EA 2160, Equipe Lipides marins a
activite biologique, Faculte de Pharmacie, Universite de Nantes,
´
´
´
BP 53508, 44035 Nantes Cedex 1, France
Introduction
T. Meziane ꢀ I. Domart-Coulon
`
Biologie des Organismes et des Ecosystemes Aquatiques,
Sponges are very ancient metazoans, with fossils dating
back to the Early Cambrian period about 530 million years
´
UMR 7208 MNHN-CNRS-UPMC, Museum National d’Histoire
Naturelle, 57 rue Cuvier, C.P. 26, 75005 Paris, France
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Lipids (2012) 47:345–353
ago. Interestingly these sessile marine invertebrates have
the ability to biosynthesize a wide variety of structurally
unique secondary metabolites, with no equivalent in ter-
restrial natural products. Because many of these metabo-
lites display biological and pharmacological activities with
potential applications in human health and biotechnologi-
cal industry, they have become excellent candidates for the
discovery of new bioactive natural products [1, 2]. While
the siliceous Demospongia and Hexactinellida sponge
classes, which represent about 90% of the species in the
Porifera phylum, have been extensively studied, the
chemistry of the Calcarea sponges was barely investigated.
Chemical investigations on Calcispongiae have mostly
concerned sponges of the Calcinea subclass and very few
studies included representatives of the Calcaronea subclass
[3–5].
Materials and Methods
Sponge Material
Leuconia johnstoni Carter 1871 [16] is a small-sized cal-
careous sponge species with an encrusting growth form
(compressed lobes fused at the base, with a maximum size
of 50 mm in diameter and 15 mm thick) living on sub-
vertical rock surfaces in wave exposed sites with a distri-
bution ranging from the British Isles to the French Brittany
coasts [17]. Specimens of the intertidal calcareous sponge
L. johnstoni were collected off the Brittany coast (North–
`
East Atlantic, Finistere, France) near Concarneau (Plage
des Dames, 47°520N and 3°550W). Four successive col-
lections of specimens pooled from several rock-encrusting
colonies were realized on the same site on October 2005,
September 2007, March 2008 and September 2008
(through a permit granted to S. Derrien, MNHN-Concar-
neau). Samples were frozen immediately after collection and
stored at -80 °C until further investigations. Four voucher
Marine sponges have also proved to be a major source
of unusual fatty acids (FA) including very long-chain (up
to 34) non-methylene-interrupted fatty acids (NMI FA)
with the particular unsaturation pattern D5,9 [6]. Although
numerous usual and original FA have been identified from
marine Demosponges [7] and some Hexactinellids [5], to
the best of our knowledge, only five studies report on
the phospholipid FA composition of a few calcareous
sponge species [8–12], two of them revealing antimicro-
bial activity or protein kinase C inhibition activity
[11, 12].
´
specimens are available from the Museum National d’His-
toire Naturelle in Paris as collection number MNHN
n°C2009-3, n°C2009-10, n°C2009-15 and n°C2009-12,
respectively.
Extraction of Total Lipids and Preparation
of Fatty Acid Methyl Esters
The widespread association of marine sponges with a
great variety of microorganisms has generated much
recent interest, as in numerous chemical studies associ-
ated microorganisms proved to be the true producers of
sponge metabolites. Our investigations on the role of
sponge associated bacterial communities led us to detect
bacterial markers in their calcareous sponge hosts through
cellular localization, chemical and microbial approaches
[13, 14]. The temperate calcareous sponge Leuconia
johnstoni Carter 1871 (class Calcispongiae, subclass
Calcaronea, order Baerida, family Baeriidae) was selected
for investigation of its FA composition which can be
linked to the abundance of its resident bacteria. Indeed,
previous studies have reported that significant levels of
i/ai SFA can be assigned to bacterial sponge symbionts
[15]. However, due to the low available biomass of this
small encrusting calcareous species and the observation
by thin layer chromatography (TLC) that phospholipids
constituted the major class of this sponge lipids, the study
was focused on the characterization of its FA composi-
tion. These FA were identified through gas chromatog-
raphy coupled to mass spectrometry (GC–MS) on their
methyl esters (FAME) and N-acyl pyrrolidide (NAP)
derivatives to determine the chain length, degree and
position of unsaturations.
The extraction was based on the Bligh and Dyer method
[18] as amended by Meziane and Tsuchiya [19] and
included four successive steps: extraction of total lipids,
saponification, methylation and purification of FA by TLC.
Briefly, frozen sponge samples were lyophilized and
extracted twice with water/chloroform/methanol (1:1:2,
v/v/v) mixture. The combined chloroform extracts yielded
the crude total lipids.
The mixture was heated with 2 mL of methanol and
1 mL of sodium hydroxide (2 M). The saponification
reaction was stopped by adding an aliquot of hydro-
chloric acid (35%). After two successive washes with
chloroform, followed by centrifugation at 2,000 rpm for
5 min, the lipid fraction was evaporated under a nitrogen
stream.
Methylation of FA was obtained by adding 1 mL of a
solution of 10% of boron trifluoride (BF3) in methanol.
After 10 min at 90 °C, all FA of the total lipids were
converted into the fatty methyl esters (FAME). Samples
were then extracted twice by a water/chloroform (1:1, v/v)
mixture and centrifuged at 2,000 rpm during 5 min. The
chloroform was evaporated under nitrogen and FAME
were dissolved in a chloroform/methanol (2:1, v/v) mix-
ture, suitable for their preservation.
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Lipids (2012) 47:345–353
347
(25.8), 98 (18.1), 88 (100), 83 (22.6), 69 (34.6), 57 (16.5),
55 (46.0), 43 (20.5), 41 (26.5).
The lipids extracts were evaporated to dryness and
recovered with three washings of 500 lL of a chloroform/
methanol (98:2, v/v) mixture. Neutral and polar lipids were
separated by column chromatography on silica gel micro-
columns (30 9 5 mm I.D. Kieselgel 70-230 mesh, Merck)
using a chloroform/methanol (98:2, v/v) mixture to elute
the neutral lipids, followed by neat methanol to elute the
polar lipids. Each fraction was then treated in the same way
as the bulk sample.
2-Methyl-13-icosenoic acid pyrrolidide MS m/z (rel.
intensity %) [M?] 377 (8.4), 362 (0.8), 348 (1.5), 334 (1.9),
320 (4.6), 306 (3.1), 292 (1.1), 278 (0.8), 266 (0.8), 252
(2.3), 238 (1.5), 224 (1.5), 210 (2.3), 196 (4.4), 182 (4.6),
168 (2.3), 154 (3.8), 140 (26.7), 127 (100), 113 (15.3), 98
(22.1), 83 (9.2), 70 (19.1), 55 (40.5), 43 (22.1), 41 (18.3).
Dimethyl disulfide adduct of 2-methyl-13-icosenoic acid
methyl ester MS m/z (rel. intensity %) [M?] 418 (3.4),
403 (1.3), 386 (2.2), 369 (1.4), 355 (0.8), 339 (1.4), 322
(2.1), 307 (0.7), 291 (2.2), 273 (28.1), 241 (27.3), 255 (2.0),
208 (4.2), 193 (4.5), 177 (3.8), 165 (4.5), 145 (37.9), 125
(16.7), 111 (31.2), 109 (26.5), 97 (50.8), 95 (36.3), 85
(36.4), 83 (50.8), 82 (43.1), 69 (80.3), 55 (100), 43 (84.8),
41 (40.1).
Preparation of N-Acyl Pyrrolidides
N-acyl pyrrolidide derivatives (NAP) were prepared by
direct treatment of the FAME with a pyrrolidine/acetic acid
(10:1, v/v) mixture under reflux for 2 h [20].
Preparation of the Dimethyl Disulfide Adduct
8,13-Octadecadienoic acid methyl ester MS m/z (rel.
intensity %) [M?] 294 (5.3), 263 (4.5), 241 (1.4), 222 (2.3),
220 (2.1), 199 (2.3), 180 (2.4), 177 (4.2), 164 (5.1), 150
(10.5), 149 (6.8), 137 (8.9), 135 (10.5), 123 (15.8), 109
(29.3), 95 (60.2), 87 (28.6), 81 (85.7), 74 (44.3), 69 (54.2),
67 (100), 55 (87.2), 43 (54.1), 41 (76.7).
The dimethyl disulfide (DMDS) adduct was obtained from a
FAME mixture (2 mg) in dimethyl disulfide (0.48 mL) by
adding a solution (0.1 mL) of iodine in diethyl ether
(60 mg mL-1) under agitation for 24 h at room temperature
[21]. The reaction was stopped by adding hexane (6 mL),
washed with a dilute solution of sodium thiosulfate (1.2 mL
of a 7 mg/100 mL solution) and dried over anhydrous
sodium sulfate and evaporated to dryness. The product was
taken up in fresh hexane for injection onto the GC–MS.
8,13-Octadecadienoic acid pyrrolidide MS m/z (rel.
intensity %) [M?] 333 (6.8), 318 (0.4), 304 (0.6), 290 (1.6),
276 (1.3), 262 (1.4), 250 (1.6), 236 (3.0), 222 (6.8), 208
(3.8), 194 (4.6), 182 (7.6), 168 (6.1), 154 (4.5), 140 (7.6),
126 (64.4), 113 (100), 98 (26.5), 85 (16.0), 72 (27.4), 70
(41.7), 67 (20.4), 55 (42.4), 43 (25.8), 41 (25.1).
Gas Chromatography–Mass Spectrometry Analyses
Gas chromatography coupled with mass spectrometry
(GC–MS) experiments were performed on a Hewlett-
Packard HP-6890 chromatograph (Agilent Technologies,
Massy, France) linked to a HP-5989 spectrometer (70 eV)
equipped with a HP-9000-345 integrator and using a SLBTM
5-ms 60 mm 9 0.25 mm 9 0.25 lm fused silica capillary
column (splitless) (Supelco, Bellefonte, PA, USA). The
carrier gas was helium (flow rate 1 mL/min). Injector and
detector temperatures were used at 250 and 280 °C,
respectively. The oven temperature was maintained at
170 °C for 4 min and then programmed from 170 to
310 °C at a rate of 3 °C/min and at 200 °C for 4 min and
then programmed from 200 to 310 °C at a rate of 3 °C/min
for FAME and NAP derivatives, respectively.
Results
The fatty acid (FA) composition of L. johnstoni was
identified for four samples corresponding to four succes-
sive collections on the same geographical site, in the period
2005 to 2008 including autumn (3 replicates) and spring
(1 replicate) as reported in Table 1. FA were characterized
as their methyl esters (FAME) and N-acyl pyrrolidide
(NAP) derivatives using gas chromatography-mass spec-
trometry (GC–MS). Identification of FA was confirmed by
comparing their equivalent chain length values (ECL) and
their mass spectra (MS) with those of known standards.
Forty-one FA with chain lengths between C14 and C22
were identified in the different samples, as illustrated in
Table 1.
Mass spectral data for the two novel FA structures in the
calcareous sponge L. johnstoni, identified as their FAME and
NAP derivatives or DMDS adduct, are presented below.
Saturated Fatty Acids
2-Methyl-13-icosenoic acid methyl ester MS m/z (rel.
intensity %) [M?] 338 (1.3), 307 (4.0), 306 (6.1), 291 (0.7),
278 (1.2), 263 (0.5), 250 (1.2), 242 (0.4), 235 (0.4), 222
(0.3), 208 (1.4), 194 (0.7), 181 (0.5), 167 (0.8), 166 (1.0),
165 (1.1), 157 (2.2), 151 (1.2), 143 (2.3), 125 (3.8), 101
In all three samples collected in autumn from October 2005
to September 2008, similar SFA were determined whereas
in the sample collected in early spring, March 2008,
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Lipids (2012) 47:345–353
Table 1 Fatty acid composition of the sponge Leuconia johnstoni (4 samples collected off Concarneau, France, each sample pooled from several
colonies)
Fatty acids (symbol)
Abundance (wt%)
ECL
October 2005
September 2007
March 2008
September 2008
Saturated fatty acids
Tetradecanoic (14:0)
14.00
14.50
14.65
14.72
15.00
15.63
15.71
16.00
16.61
16.69
17.00
17.75
18.00
18.69
19.00
19.66
20.00
21.00
–
0.2
0.5
3.1
3.6
0.2
2.0
0.7
20.1
1.9
0.7
0.6
0.9
9.6
3.4
0.7
1.5
1.4
0.6
51.7
0.8
0.8
2.5
3.6
0.4
1.3
0.3
23.5
0.6
0.3
0.8
0.4
13.8
2.7
0.4
0.3
1.8
0.4
54.7
1.4
3.1
2.3
3.2
0.4
0.7
–
1.9
1.9
4.1
6.3
0.4
0.8
0.5
16.0
2.2
1.1
0.6
1.3
10.0
2.1
0.5
0.9
1.2
0.3
52.1
4,8,12-Trimethyltridecanoic (br-13:0)
13-Methyltetradecanoic (i-15:0)
12-Methyltetradecanoic (ai-15:0)
Pentadecanoic (15:0)
14-Methylpentadecanoic (i-16:0)
13-Methylpentadecanoic (ai-16:0)
Hexadecanoic (16:0)
22.8
0.9
1.4
0.9
1.6
14.9
2.9
–
15-Methylhexadecanoic (i-17:0)
14-Methylhexadecanoic (ai-17:0)
Heptadecanoic (17:0)
15-Methylheptadecanoic (ai-18:0)
Octadecanoic (18:0)
17-Methyloctadecanoic (i-19:0)
Nonadecanoic (19:0)
18-Methylnonadecanoic (i-20:0)
Icosanoic (20:0)
1.4
1.1
–
Heneicosanoic (21:0)
Total SFA
59.0
Monounsaturated fatty acids
5-Hexadecenoic (16:1)
15.80
15.87
17.80
17.90
19.75
20.18
20.86
–
0.5
1.8
1.0
1.8
0.3
0.9
1.4
0.7
0.8
0.7
–
9-Hexadecenoic (16:1)
9-Octadecenoic (18:1)
2.0
1.4
1.4
11-Octadecenoic (18:1)
13-Icosenoic (20:1)
2-Methyl-13-icosenoic (br-13-20:1)a
Heneicosenoic (21:1)b
4.1
3.2
2.0
1.9
2.5
1.5
24.3
1.4
22.4
1.5
20.8
1.1
25.8
–
Total MUFA
36.0
33.8
28.0
29.4
Di- and poly-unsaturated fatty acids
8,13-Octadecadienoic (18:2)c
9,12-Octadecadienoic (18:2)
11,15-Icosadienoic (20:2)
5,8,11,14-Icosatetraenoic (20:4)
5,8,11,14,17-Icosapentaenoic (20:5)
Total DUFA and PUFA
Dimethylacetal
17.82
17.85
20.30
18.96
19.20
–
0.8
0.8
3.1
1.7
1.8
8.2
0.6
0.8
1.9
1.9
1.5
6.7
0.9
1.1
1.4
1.4
0.9
5.7
0.7
1.0
0.8
0.8
0.7
4.0
DMA (21:2)b
20.75
2.9
3.2
5.9
11.8
FA identified as traces: SFA (ECL) 16-Methylheptadecanoic (17.64), 19-Methylicosanoic (20.66), 16-Methyloctadecanoic (18.79), Docosanoic
(22.00); MUFA 13-Methyl-4-tetradecenoic (14.32), 10-Hexadecenoic (15.90), 15-Methyl-5-hexadecenoic (16.40), 6-Octadecenoic (17.54),
14-Octadecenoic (17.94), 9-Icosenoic (19.30); DUFA 13, x-Docosadienoic (21.80)(not previously characterized in a calcareous sponge)
a
Not previously reported in nature
b
Undetermined unsaturations
c
Not previously characterized in a calcareous sponge
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349
ai-16:0, 19:0 and 21:0 were lacking. The linear SFA
hexadecanoic acid (16:0) and octadecanoic acid (18:0)
were always determined as dominant. They represented
between 16.0 and 23.5% and between 9.6 and 14.9% of the
total FA ? DMA, respectively. The minor linear SFA
(20:0) was observed at similar levels (between 1.1 and
1.8%) in the four samples. In contrast, the shortest length
SFA (14:0) was identified between 0.2 and 1.9% depending
on sample. Additional linear SFA were also observed at
noticeable levels of the total FA and DMA in all samples
(less than 0.5% for 15:0 and less than 1% for 17:0).
Furthermore, L. johnstoni also contained a high pro-
portion of branched SFA including iso (i) and anteiso (ai)
FA. The diversity of iso FA was similar to those of anteiso
FA (i/ai -15:0 to i/ai-19:0) excepted for i-18:0, ai-19:0 and
i-20:0, only identified as traces. The isoprenoid-type
4,8,12-tridecanoic acid (br-13:0) was also identified from
0.5 to 3.1% of the FA ? DMA (ECL value of the
FAME = 14.50).
compound was obviously a FAME. These key observations
allowed to identify this compound as a MUFA with a methyl
group at C-2, which was also supported by the MS of its NAP
derivative, showing the molecular ion at m/z 377 (Fig. 2).
The MS of its pyrrolidide derivative displayed the
expected McLafferty ion at m/z 113, while the base peak
was present at m/z 127, confirming the 2-methyl branching
nature of this MUFA. This observation was in accordance
with the occurrence of an elevated peak at m/z 140, as
depicted in the Lipid Library, for a 3-methyl branched
NAP. In addition, the gap of 12 amu between fragment ions
at m/z 278 and m/z 266 suggested localization of the double
bond between carbons C-13 and C-14 of the aliphatic
chain. In order to confirm this hypothesis, the dimethyl
disulfide (DMDS) adduct of this 2-methyl MUFA was
prepared as previously described by Imbs et al. [21].
Analysis of the MS of the corresponding DMDS adduct
showed the presence of characteristic fragments at m/z 145,
213, 241 and 273 (Fig. 3), confirming unambiguously the
localization of the unsaturation between C-13 and C-14.
However its configuration could not be determined.
This MUFA was unequivocally identified as 2-methyl-
13-icosenoic acid (2-Me-13-20:1). Its occurrence in all
samples of L. johnstoni collected at the same geographical
site at different seasons is noteworthy. Indeed, this unusual
2-methyl FA represented 22.4% and 24.3% in autumn
(September 2007 and October 2005 respectively) and
20.8% of the total FA ? DMA of specimens collected in
spring (March 2008). However in winter (February 2006
and 2007) the sponge lipid profile was dominated by SFA
and the abundance of this unusual 2-Me-13-20:1 dropped
to respectively 7.8 and 9.8% of FA ? DMA, although it
still dominated the sponge MUFA (data not shown, pub-
lication in preparation).
Monounsaturated Fatty Acids
Monounsaturated FA (MUFA) represented from 28.0 to
36.0% of the total FA ? DMA in the sponge. Surprisingly,
an unusual MUFA was present in all samples with an ali-
phatic chain of 20 carbons (ECL value of 20.18). The mass
spectrum (MS) of its methyl ester (FAME) revealed a
molecular ion [M]? at m/z 338 corresponding to a C20:1 FA
structure (Fig. 1).
In the MS of its FAME, the fragment ion at m/z 88 (C3
fragment ? H) as the base peak and the major ion at m/z 101
could remind a McLafferty rearrangement of a FA ethyl
ester. But, the presence in the MS of the relatively intense
fragment ion at m/z 306 ([M-31]?) indicated that the
Fig. 1 Mass spectrum of 2-methyl-13-icosenoic acid methyl ester from L. johnstoni
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Lipids (2012) 47:345–353
Fig. 2 Mass spectrum of 2-methyl-13-icosenoic acid pyrrolidide derivative from L. johnstoni
Fig. 3 Mass spectrum of 2-methyl-13-icosenoic acid dimethyl disulfide derivative from L. johnstoni
It appeared to us that the peak representing the new
2-methyl-13-icosenoic acid was highly represented in the
polar phospholipid fraction.
(15:1 FA structure), as well as fragment ions at m/z 139 and
152, and an intense fragment ion at m/z 166 (53%) indi-
cating a double bond between C-4 and C-5, as shown in the
Lipid Library [22]. Furthermore, a very weak ion at
m/z 264 indicated an iso-branched FA. Similarly, the MS of
the NAP of 15-methyl-5-hexadecenoic displayed the
molecular ion at m/z 321 (17:1 FA structure), and fragment
ions at m/z 140 and 153, indicating a double bond between
C-5 and C-6. In this case, the iso-branched was indicated
by a weak peak at m/z 292.
Two additional hexadecenoic-type MUFA, 5-hexadec-
enoic and 9-hexadecenoic, were also identified in low
amounts (0.3–1.4 and 0.7–1.8%, respectively) as well as
two octadecenoic-type MUFA: 9-octadecenoic (0.8–2.0%)
and 11-octadecenoic (0.7–4.1%). 13-icosenoic (1.5–2.5%)
and heneicosenoic acid (1.1–1.5%) were only identified in
three specimens out of four.
In addition, six MUFA were identified as traces:
13-methyl-4-tetradecenoic, 10-hexadecenoic, 15-methyl-5-
hexadecenoic, 6-octadecenoic, 14-octadecenoic, 9-icosenoic
(Table 1).
Polyunsaturated Fatty Acids
Four DUFA were observed, in low amounts (\2%).
The DUFA, eluted just before the 9,12-octadecadienoic
(0.8–1.1%), had an ECL value of 17.82 as methyl ester and
showed in its MS a molecular ion at m/z 294, indicating a
18:2 acid structure. The MS of its NAP showed a molecular
ion at m/z 333 (18:2). The two double bonds were located
by the 12 amu gaps between the peaks at m/z 182 (C-7) and
The two monomethyl branched MUFA, 13-methyl-4-
tetradecenoic and 15-methyl-5-hexadecenoic, were readily
identified from their GC mobilities (ECL of 14.32 and
16.40, respectively) and from their corresponding NAP
derivatives MS spectra. The MS of the NAP of 13-methyl-
4-tetradecenoic displayed the molecular ion at m/z 293
123

Lipids (2012) 47:345–353
351
Fig. 4 Mass spectrum of 8,13-octadecadienoic acid pyrrolidide derivative from L. johnstoni
m/z 194 (C-8) and between the peaks at m/z 250 (C-12) and
m/z 262 (C-13), indicating D8-D13 unsaturations. Major
diagnostic fragmentations of FAME and NAP of 8,13-oc-
tadecadienoic acid under electron impact are given in the
‘‘Materials and Methods’’ section. This DUFA 8,13-octa-
decadienoic acid occurred in low abundance in all samples
with values ranging from 0.6 to 0.9% of total FA ? DMA
(Fig. 4).
composition comparing four specimens collected at the same
site in a time series over 4 years.
Out of the forty-one identified FA, twenty-two were
saturated FA (SFA), including eighteen major SFA repre-
sented between 52.1 and 59.0% of the total FA ? DMA. In
addition to the known linear SFA, i/ai C14–C22, typical FA
of bacteria, were observed in all L. johnstoni specimens
at significant levels ranging from 9 to 16% of total
FA ? DMA. Previous studies had reported that i/ai FA
were found as minor compounds or traces in terrestrial
eukaryotes but in high amounts in some marine eukaryotes
such as sponges, nudibranch molluscs, asteroidea and tu-
nicates [9, 24, 25]. These mid-chain-branched i/ai FA,
which are considered as membrane constituent especially
in Gram-positive bacteria, have often been assigned to
bacterial sponge symbionts [15, 26, 27]. Recently, methyl-
branched fatty acids were proposed to be synthesized by
Poribacteria [28]. Nevertheless, 16-methyl-octadecanoic
acid (ai-19:0) which is reported as a major FA in several
Calcareous sponge species mostly of the Calcinea subclass
[9] was found in low amounts in L. johnstoni. Furthermore,
although 17-methyl-nonadecanoic acid (ai-20:0) was pre-
viously reported as a major compound of presumably
sponge cell origin in Calcispongiae [9], this FA was lack-
ing in our samples even as traces.
The two other detected DUFA are 11,15-icosadienoic
acid (0.8–3.1%) and 13,x-docosadienoic acid (traces).
Complete identification of the latter failed due to its very
small amount.
Two other PUFA 5,8,11,14-icosatetraenoic acid (0.8–
1.9%) and 5,8,11,14,17-icosapentaenoic acid (0.7–1.8%)
were also observed.
Dimethylacetals
Several DMA were detected at trace levels in the lipid
fraction of L. johnstoni. However, one of them was
detected at a significant amount in all samples, accounting
for 11.8% of total FA ? DMA in September 2008. MS of
this dominant DMA (ECL = 20.75), showed characteristic
fragment ions at m/z 75 ([(CH3O)2–CH]?) [23] and m/z 322
corresponding to [M-31]?, indicating a C21 saturated fatty
aldehyde with two unsaturations.
Other branched FA have also been considered as bac-
terial markers, especially some monounsaturated FA
(MUFA). In this study, thirteen MUFA were observed,
among which seven had major relative abundance
(28.0–36.0%). However, iso-15:1 and iso-17:1 MUFA with
various positions of the double bond, which were previ-
ously reported as characteristic of sulfate-reducing bacteria
of the genus Desulfovibrio [29, 30], were both lacking in
all samples of L. johnstoni.
Discussion
By contrast with siliceous Demosponges and Hexactinellids,
only a few reports have characterized the lipid composition
of Calcisponges [8–10]. To obtain further insight into the FA
content of Calcisponges, we selected Leuconia johnstoni as a
calcareous sponge model belonging to the chemically poorly
studied Calcaronea subclass. We investigated its FA
As a major result, our study led to the identification for
the first time in a calcareous sponge of a new natural
123

352
Lipids (2012) 47:345–353
MUFA identified as 2-methyl-13-icosenoic acid as the
dominant MUFA. Its high relative abundance, ranging
from 20.8 to 25.8% of the total FA ? DMA in all four
samples of L. johnstoni, suggests that it might play a sig-
nificant role in the biology of this sponge, potentially in the
function of its cellular membranes. Unfortunately, despite
of its high relative abundance, the purification of this
2-methyl FA remained difficult due to the low available
sponge biomass of L. johnstoni.
The finding of DMA among FAME in L. johnstoni
points to the presence of plasmalogens (1-O-10-enyl-2-
acyl-glycerophospholipids) in the sponge. The DMA are
produced from aldehydes, released from plasmalogens,
concomitantly with the FAME during the esterification step
[23]. These plasmalogens are widely distributed in the
animal kingdom and also occur in some anaerobic micro-
organisms and marine invertebrates [37]. In mammals, they
could have a role in physical properties of cell membranes,
storage of arachidonic acid, activation of phospholipase A2
and cellular signalization. They are also involved in pro-
tection against oxidative stress [38]. The presence of
plasmalogens was previously reported in the Demosponge
Polymastia penicillus but their structures could not be
determined due to both low amounts and co-elution with
other lipids during purification [7]. However, to date, the
biological role of plasmalogens in sponges is not clearly
determined. We speculate that they may have similar
functions as in more complex metazoan organisms
including protection against oxidative stress or against
attack by microorganisms.
Previously, some 2-methyl branched unsaturated FA
have already been isolated but only from siliceous marine
sponges. In 1990, the first 2-methyl branched FA, 2-meth-
yloctadecanoic acid was identified from the Demosponge
Plakortis halichondroides [31]. Later, two additional
2-methyl branched MUFA, 2-Me-24:1n-7 and 2-Me-26:
1n-9 were isolated in small amounts (7.1% of sponge total
FA) from the Demosponge Halichondria panicea [21].
Further chemotaxonomic studies would be necessary to
demonstrate the specificity of this new 2-methyl-13-icos-
enoic acid as potential biomarker for sponges of the genus
Leuconia.
Six polyunsaturated FA (PUFA) accounting between 4.0
and 8.2% of the total FA ? DMA, including the two
non-methylene-interrupted FA (NMA) 8,13-18:2 and
11,15-20:2 and one additional diunsaturated FA (DUFA)
9,12-18:2, were also detected. This is the first time that
the unusual 8,13-octadecadienoic acid was characterized
in a calcareous sponge. Numerous NMI FA, such as 5,11-
20:2, 5,13-20:2, 7,13-22:2 and 7,15-22:2 have already
been identified from marine sponges [6, 7, 32]. The
11,15-icosadienoic acid NMI observed in this study was
previously identified from the Caribbean Demosponge
Amphimedon complanata and the Axinellida sponge
Pseudaxinella cf. lunaecharta [7, 33, 34]. The origin of the
two PUFA 5,8,11,14-icosatetraenoic acid and 5,8,11,14,17-
icosapentaenoic acid, which were detected in all samples of
L. johnstoni, remains unknown [35]. They were observed at
a very low level, representing less than 2% of the total FA
and DMA content. No other PUFA with 3 or more double
bonds were identified.
Surprisingly, by contrast with all other FA, which were
in constant relative abundance in the four specimens col-
lected during two seasons over 4 years, detection of this
DMA was variable and fluctuated between 2.9 and 11.8%
without any seasonal corroboration.
In conclusion, this first investigation of the fatty acid
composition of the Calcaronea species Leuconia johnstoni
Carter 1871 identified the new 2-methyl-13-icosenoic FA
in a calcareous sponge. It also revealed the occurrence at
low levels of the unexpected PUFA 20:4 and 20:5 and of
the DMA 21:2 although in fluctuating ratios. The nearly
constant high relative abundance of the new 2-methyl-13-
icosenoic FA in L. johnstoni suggests that this MUFA
might be further explored as a potential biomarker. Further
investigations on lipids and FA composition of Leuconia
species remain to be carried out to substantiate this
hypothesis.
´
Acknowledgments This work is part of Elodie Quevrain’s Ph.D.
`
thesis, supported by a grant from the French Ministere de l’Ens-
eignement Superieur et de la Recherche. The authors also thank the
Furthermore, we noticed that demospongic acids,
defined by Christie for the database Lipid Library as long-
chain FA with a D5,9 unsaturation system and recently
revisited as NMI FA, were totally absent in L. johnstoni.
These NMI FA, observed in various marine sources with a
large range of chain-lengths (C16–C32) [36] were previ-
ously reported in specimens of calcareous sponges in the
genera Clathrina (Calcinea subclass) and Sycon (Calcaro-
nea subclass) [8].
´
´
director and staff of the Laboratoire de Biologie Marine du Museum
National d’Histoire Naturelle, Station Marine de Concarneau, France,
for providing access to facilities for regular sponge collections.
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