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A unique hexaene hydrocarbon from a diatom (Skeletonema costatum)
SHORT COMMUNICATIONS
386 BBA 53258
A unique
hexaene
While observed
hydrocarbon
analyzing
that
from
the lipids of the marine
the hydrocarbon
fraction
(20)/b) and a trace
(Skeletonema
diatom,
constituted
in two cultures. Thin-layer chromatographic it was composed mainly of an unsaturated carotene
a diatom
Skeletonema
14.0-15.7%
costatum) costatum,
of the total
we lipids
analysis of the fraction indicated that hydrocarbon (So($,), a lesser amount of
of a homologous
series of C,,-C,,
n-alkanes.
Because
of the possible significance of this hydrocarbon to the marine food chain, to petroleogenesis, and to the ultrastructure of the organism, we undertook its identification. A previous investigation’ of hydrocarbons in S. costatum did not refer to such a compound. S. costatum was cultured at 16” for 8 days in 75 1 of a minimal media* composed of sterile
sea water to which nitrate,
phosphate,
silicate,
trace
metals,
vitamin
B,,
and biotin were added. The diatom was exposed to a 14-h light and 10-h dark cycle. The
cells were harvested
by centrifuging
at 7000
rev./min
in a Sharples
Super-
Centrifuge (Model T-I). The weight of dried cells from 75 1 of culture was 1.506 g. The lipids (8.6% of the dry cell weight) were obtained from the wet packed cells by 12-15
extractions
with lo-ml
portions
of chloroform-methanol
(2: I, v/v).
The combined extracts were evaporated under vacuum, and the hydrocarbon fraction was isolated by silicic acid column chromatography2, eluting with 3 column volumes of hexane. In thin-layer chromatographic analysis on silica gel-G coated plates developed with the indicated solvents, the following RF values were observed for the unknown hydrocarbon and the accompanying carotene, respectively: hexane, 0.23 and 0.07; isooctane, 0.29 and 0.11; hexane-benzene (I:I, v/v), 0.62 and 0.64. Preparative Instruments, employing off the
thin-layer plates coated with 0.4 mm of silica gel HR (Brinkmann Inc.) were used to separate the unknown hydrocarbon from the carotene, hexane for development.
silica
Recovery
gel in the appropriate
from the plate was effected by scraping
area and extracting
it repeatedly
with ethyl
ether. The unknown was further characterized by gas chromatography, using a Loenco Model 70 Hi-Flex apparatus and either an 8 cm x 0.125 inch outer diameter column of 10% diethylene glycol succinate polyester on 60/80 mesh Chromosorb W run at 156” and 30 lb/inch2 carrier gas pressure or a 6 cm x 0.125 inch column of 3% OV-I on 60/80 mesh Gas-Chrom P run at 170’ and 30 lb/inch2 (both columns were obtained from Applied Science Laboratories, ionization. The unknown had a carbon number**’
Inc.); detection was by flame of 20.6 on OV-I and 25.0 on
diethylene glycol succinate polyester. A number of control experiments were conducted to establish whether S. Costa&m was the actual source of the hydrocarbon. The sea water incubation medium, the extraction and chromatography solvents and procedures were eliminated as possible sources. The incubations were carried out in a manner to completely exclude bacteria and constant monitoring confirmed that the measures were effective. The compound was not detected in Dunaliella tertiolecta, a green alga, or from Calanus * Detailed composition available on request. ** Determined from a plot of total number alkanes run under identical condition9. Biochim.
Biophys.
Acta,
202
(1970)
386-388
of carbons
vs. lo,r~ retention
times of authentic
u-
SHORT COMMUNICATIONS
387
a copepod, when these organisms were cultured under conditions very similar to those used for S. costatum. It could not be detected in C. helgolandicus fed on S. costatum, which implies an active metabolism of the hydrocarbon by the copepod. A hydrocarbon exhibiting identical thin-layer and gas chromatographic behaviour was found in a second diatom, Chaetoceros septentrionale. Further evidence that S. costatum synthesized the hydrocarbon in question was obtained from the following experiment: 150 ml of a 6-day-old culture was incubated with 4.5 mC of %O, in a closed system for 60 h, after which the lipids were extracted as previously described. The lipid extract was separated with hexane on a thin-layer plate coated with silica gel G. The position of the radioactive components was determined by radioautography using Kodak Blue-sensitive Medical X-ray film. Two spots appeared in the hydrocarbon region, one which co-chromatographed with the carotene and the other with the unknown hydrocarbon. In order to establish the structure of the hydrocarbon, its mass spectrum (70 eV, Fig. I) was obtained from an LKB gas chromatograph-mass spectrometer (Model gooo), the hydrocarbon peak from gas chromatography being bled into the helgolandicus,
50
100
150
200
250
300
m/e
Fig. I. Mass
spectrum(70 eV) of the unsaturated hydrocarbon
isolated from S. costatum.
spectrometer. The unknown compound was also analyzed at 20 eV and a parent ion at 284 was observed, corresponding to an empircal formula of C,,H,,. Hydrogenation in hexane with an excess of platinum catalyst yielded a product whose mass spectrum showed the parent ion shifted to 296, indicating uptake of 12 hydrogens by 6 double bonds. It was established further that the mass spectrum and gas-liquid chromatographic behaviour of the hydrogenated compound were identical to those of authentic n-heneicosane (Chemical Samples Co., purity > 99% by both their and our assays). The unsaturated hydrocarbon at a concentration of 0.025 mg/ml in hexane showed essentially no radiant energy absorption between 200 and 400 m,u .The six double bonds are thus presumed to be non-conjugated. An all-&s configuration was confirmed for these double bonds by the lack of significant absorption in the infrared at 10.3 ,u, where bans double bonds absorb strongly. Mass fragments corresponding to CH,CH,CH=CH-(m/c 55) and to loss of CH&H,(m/c 255) in the mass spectrum of the unhydrogenated hydrocarbon suggest that the two ends of the molecule have the same structure. The mass spectra (not shown) of docosahexaenoic acid (22 : 6 o 3, Hormel Institute) and of the hydrocarbon derived by selectively reducing the carboxyl group of that acid by the method of SEIFERT et al.4, were very similar to that of the unknown hydrocarbon and included the major fragments m/c 67, 79, 93, Biochim.
Biqbhys.
Acta,
202
(1970) 386-388
3813
SHORT COMMUNICATIONS
108, rig and 133. Thus we tentatively
assign the symmetrical
structure
of all-&g,
G,g,rz,Ig,IS-heneicosahexaene to the unknown hydrocarbon. It could be metabolically by decarboxylation of the all-cis-4,7,10,13,16,Ig-docosahexaenoic which has been reported570 to constitute
0.7-1.7;(,
derived acid,
of the total fatty acids in the lipids
of S. costaturn cultured 6-14 days. However, we did not detect this acid in the lipids from our preparations, in agreement with CHUECAS AND RILEY~, perhaps because it was selectively decarboxylated to the hydrocarbon. We thank Elizabeth Baker, Laurel Loeblich, technical
assistance.
This research was supported
and Stanley
by Public
K. Freeman
for
Health Service Grants
HE 03632-11 and GM 12310. Scripps I&&&ion of Oceanography, P.O. Box 109 La Jolla, Calif 92037 (U.S.A.)
RICHARD F.LEE JUDD C. NEVENZEL G.-A. PAFFENH~FER A. A. BENSON
Lipids Laboratory, The Pennsylvania
STUART PATTON State University,
TERENCE
E. KAVANAGH
University Park, Pa. 16802 (U.S.A.) I R. C. CLARK, Tr. AND M. BLUMER, Limnol. Oceano~., 12 (1967)79. 2 D. L. FIUER& AND J, F. MEAD, Pvoc. Sac. ExptL Biol.‘Med:, ‘83 (1953) 574. 3 F. P. WOODFORD AND C. M. VAN GENT, J. Lipid Rcs., I (1960) 188. 4 W. Ii. SEIFERT, R. M. TEETER, W. G. HOWELLS AND 111. J.R. CANTOW, AnaZ.Chem.,
41 (1969)
1638. 5 R.G. ACKMAN,C.S.TOCHERAND J.MCLACHLAN. J.FisheriesRes.Roar.Canada.25(1968) 1603. 6 R. G. ACI(MAX,P. M. JANGAARD, R. J. HOYLE AND H. BROCKERHOFF, J, Fishevies Board Canada, 21 (1964)747. 7 I..CHUECAS ASD J. P. RILEY,J. Marine Biol. Assoc. U.K., 49 (1969) 97. Received
November
Ioth, 1969
Note added in pvooj’, We understand that M. BLUMER, Woods Hale Oceanographic Institute has recently detected in plankton a hydrocarbon such as we describe. (Received February Ioth, 1970). Biochim.
Biophys.
Acta,
202 (1970) 386-388
386 BBA 53258
A unique
hexaene
While observed
hydrocarbon
analyzing
that
from
the lipids of the marine
the hydrocarbon
fraction
(20)/b) and a trace
(Skeletonema
diatom,
constituted
in two cultures. Thin-layer chromatographic it was composed mainly of an unsaturated carotene
a diatom
Skeletonema
14.0-15.7%
costatum) costatum,
of the total
we lipids
analysis of the fraction indicated that hydrocarbon (So($,), a lesser amount of
of a homologous
series of C,,-C,,
n-alkanes.
Because
of the possible significance of this hydrocarbon to the marine food chain, to petroleogenesis, and to the ultrastructure of the organism, we undertook its identification. A previous investigation’ of hydrocarbons in S. costatum did not refer to such a compound. S. costatum was cultured at 16” for 8 days in 75 1 of a minimal media* composed of sterile
sea water to which nitrate,
phosphate,
silicate,
trace
metals,
vitamin
B,,
and biotin were added. The diatom was exposed to a 14-h light and 10-h dark cycle. The
cells were harvested
by centrifuging
at 7000
rev./min
in a Sharples
Super-
Centrifuge (Model T-I). The weight of dried cells from 75 1 of culture was 1.506 g. The lipids (8.6% of the dry cell weight) were obtained from the wet packed cells by 12-15
extractions
with lo-ml
portions
of chloroform-methanol
(2: I, v/v).
The combined extracts were evaporated under vacuum, and the hydrocarbon fraction was isolated by silicic acid column chromatography2, eluting with 3 column volumes of hexane. In thin-layer chromatographic analysis on silica gel-G coated plates developed with the indicated solvents, the following RF values were observed for the unknown hydrocarbon and the accompanying carotene, respectively: hexane, 0.23 and 0.07; isooctane, 0.29 and 0.11; hexane-benzene (I:I, v/v), 0.62 and 0.64. Preparative Instruments, employing off the
thin-layer plates coated with 0.4 mm of silica gel HR (Brinkmann Inc.) were used to separate the unknown hydrocarbon from the carotene, hexane for development.
silica
Recovery
gel in the appropriate
from the plate was effected by scraping
area and extracting
it repeatedly
with ethyl
ether. The unknown was further characterized by gas chromatography, using a Loenco Model 70 Hi-Flex apparatus and either an 8 cm x 0.125 inch outer diameter column of 10% diethylene glycol succinate polyester on 60/80 mesh Chromosorb W run at 156” and 30 lb/inch2 carrier gas pressure or a 6 cm x 0.125 inch column of 3% OV-I on 60/80 mesh Gas-Chrom P run at 170’ and 30 lb/inch2 (both columns were obtained from Applied Science Laboratories, ionization. The unknown had a carbon number**’
Inc.); detection was by flame of 20.6 on OV-I and 25.0 on
diethylene glycol succinate polyester. A number of control experiments were conducted to establish whether S. Costa&m was the actual source of the hydrocarbon. The sea water incubation medium, the extraction and chromatography solvents and procedures were eliminated as possible sources. The incubations were carried out in a manner to completely exclude bacteria and constant monitoring confirmed that the measures were effective. The compound was not detected in Dunaliella tertiolecta, a green alga, or from Calanus * Detailed composition available on request. ** Determined from a plot of total number alkanes run under identical condition9. Biochim.
Biophys.
Acta,
202
(1970)
386-388
of carbons
vs. lo,r~ retention
times of authentic
u-
SHORT COMMUNICATIONS
387
a copepod, when these organisms were cultured under conditions very similar to those used for S. costatum. It could not be detected in C. helgolandicus fed on S. costatum, which implies an active metabolism of the hydrocarbon by the copepod. A hydrocarbon exhibiting identical thin-layer and gas chromatographic behaviour was found in a second diatom, Chaetoceros septentrionale. Further evidence that S. costatum synthesized the hydrocarbon in question was obtained from the following experiment: 150 ml of a 6-day-old culture was incubated with 4.5 mC of %O, in a closed system for 60 h, after which the lipids were extracted as previously described. The lipid extract was separated with hexane on a thin-layer plate coated with silica gel G. The position of the radioactive components was determined by radioautography using Kodak Blue-sensitive Medical X-ray film. Two spots appeared in the hydrocarbon region, one which co-chromatographed with the carotene and the other with the unknown hydrocarbon. In order to establish the structure of the hydrocarbon, its mass spectrum (70 eV, Fig. I) was obtained from an LKB gas chromatograph-mass spectrometer (Model gooo), the hydrocarbon peak from gas chromatography being bled into the helgolandicus,
50
100
150
200
250
300
m/e
Fig. I. Mass
spectrum(70 eV) of the unsaturated hydrocarbon
isolated from S. costatum.
spectrometer. The unknown compound was also analyzed at 20 eV and a parent ion at 284 was observed, corresponding to an empircal formula of C,,H,,. Hydrogenation in hexane with an excess of platinum catalyst yielded a product whose mass spectrum showed the parent ion shifted to 296, indicating uptake of 12 hydrogens by 6 double bonds. It was established further that the mass spectrum and gas-liquid chromatographic behaviour of the hydrogenated compound were identical to those of authentic n-heneicosane (Chemical Samples Co., purity > 99% by both their and our assays). The unsaturated hydrocarbon at a concentration of 0.025 mg/ml in hexane showed essentially no radiant energy absorption between 200 and 400 m,u .The six double bonds are thus presumed to be non-conjugated. An all-&s configuration was confirmed for these double bonds by the lack of significant absorption in the infrared at 10.3 ,u, where bans double bonds absorb strongly. Mass fragments corresponding to CH,CH,CH=CH-(m/c 55) and to loss of CH&H,(m/c 255) in the mass spectrum of the unhydrogenated hydrocarbon suggest that the two ends of the molecule have the same structure. The mass spectra (not shown) of docosahexaenoic acid (22 : 6 o 3, Hormel Institute) and of the hydrocarbon derived by selectively reducing the carboxyl group of that acid by the method of SEIFERT et al.4, were very similar to that of the unknown hydrocarbon and included the major fragments m/c 67, 79, 93, Biochim.
Biqbhys.
Acta,
202
(1970) 386-388
3813
SHORT COMMUNICATIONS
108, rig and 133. Thus we tentatively
assign the symmetrical
structure
of all-&g,
G,g,rz,Ig,IS-heneicosahexaene to the unknown hydrocarbon. It could be metabolically by decarboxylation of the all-cis-4,7,10,13,16,Ig-docosahexaenoic which has been reported570 to constitute
0.7-1.7;(,
derived acid,
of the total fatty acids in the lipids
of S. costaturn cultured 6-14 days. However, we did not detect this acid in the lipids from our preparations, in agreement with CHUECAS AND RILEY~, perhaps because it was selectively decarboxylated to the hydrocarbon. We thank Elizabeth Baker, Laurel Loeblich, technical
assistance.
This research was supported
and Stanley
by Public
K. Freeman
for
Health Service Grants
HE 03632-11 and GM 12310. Scripps I&&&ion of Oceanography, P.O. Box 109 La Jolla, Calif 92037 (U.S.A.)
RICHARD F.LEE JUDD C. NEVENZEL G.-A. PAFFENH~FER A. A. BENSON
Lipids Laboratory, The Pennsylvania
STUART PATTON State University,
TERENCE
E. KAVANAGH
University Park, Pa. 16802 (U.S.A.) I R. C. CLARK, Tr. AND M. BLUMER, Limnol. Oceano~., 12 (1967)79. 2 D. L. FIUER& AND J, F. MEAD, Pvoc. Sac. ExptL Biol.‘Med:, ‘83 (1953) 574. 3 F. P. WOODFORD AND C. M. VAN GENT, J. Lipid Rcs., I (1960) 188. 4 W. Ii. SEIFERT, R. M. TEETER, W. G. HOWELLS AND 111. J.R. CANTOW, AnaZ.Chem.,
41 (1969)
1638. 5 R.G. ACKMAN,C.S.TOCHERAND J.MCLACHLAN. J.FisheriesRes.Roar.Canada.25(1968) 1603. 6 R. G. ACI(MAX,P. M. JANGAARD, R. J. HOYLE AND H. BROCKERHOFF, J, Fishevies Board Canada, 21 (1964)747. 7 I..CHUECAS ASD J. P. RILEY,J. Marine Biol. Assoc. U.K., 49 (1969) 97. Received
November
Ioth, 1969
Note added in pvooj’, We understand that M. BLUMER, Woods Hale Oceanographic Institute has recently detected in plankton a hydrocarbon such as we describe. (Received February Ioth, 1970). Biochim.
Biophys.
Acta,
202 (1970) 386-388