Comp. Blochem. Physiol. Vol. 85B, No. I, pp. 131-134, 1986
`Printed in Great Britain
`
`0305-049 l /86 $3.00 + 0.00
`Pergamon Journals Ltd
`
`H. FRICKE and G. GHRCKEN
`Institute for Biochemistry and Food Chemistry, University of Hamburg, Martin-Luther-King-Platz 6,
`D-2000 Hamburg 13, FRG
`J. 0EHLBNSCHLAGER
`Institute for Biochemistry and Technology in the Federal Research Centre for Fisheries, Palmaille 9,
`D-2000 Hamburg 50, FRG
`
`(Received 7 NO!lember 1985)
`
`Abm11ct-l. Antarctic Krill contains 1-0-alkyllipids as minor components ranging from 0.3 to 0.6% of
`total lipid content.
`2. 1-0-Alkyllipids were present in whole Krill samples and in isolated abdominal muscle as well.
`3. In both phospholipids and neutral lipids the presence of 1-0-alkyllipids could be found.
`4. A number of 16 individual species of 1-0-alkylglycerols could be separated by GLC and identified
`using GLC/MS after enzymatic and chemical degradation of total lipids.
`5. The predominant alkyl chains found were: 16:0 (40.S-52.7%), 14:0 (10.9-13.0%) and 16: 1
`(8.3-l l.8%, one of two isomers).
`6. 1-0-Alk-1'-enyllipids (plasmalogens) and 2-0-alkyllipids were not detected.
`
`INTRODUCTION
`
`Small amounts of alkoxylipids, commonly referred
`to as glyceryl ethers or ether lipids, are present in
`the lipids of many marine animals (Mangold, 1979;
`Horrocks and Sharma, 1982). Though the alkoxy(cid:173)
`lipids of fishes are investigated to some extent there
`is only limited information available on inverte·
`brates and especially on crustaceans. The occurrence
`of alkenyl moieties (a metabolic succursor of alkyl
`groups) was shown by Dembitskii (1976 and 1979) in
`the phospholipids of marine invertebrates and by
`Isay et al. (l 976) in crustaceans from tropical waters
`and other invertebrates (1984). The amounts found
`by lsay (1976) varied from 0.5 to 5.9% of total
`lipids. Clarke (1977) demonstrated that the Antarctic
`crustacean Chorismus antarcticus Pfeffer contained
`I ·0-alk • l' -enyl-glycerolipids in its polar lipids and
`also small amounts of 1-0-a1kyl-2,3,-0-diacyl(cid:173)
`glycerols. The presence of alkyldiacylglycerols in the
`Antarctic crustaceans Serolis cornuta and Serolis
`pagenstecheri (lsopoda) from 0.32% (males)
`to
`1.48% (females) could be shown by the same author
`(1984).
`Little information is present on the physiological
`function of 1-0-alkyllipids. Recently Demonopoulos
`et al.
`(1979)
`reported
`l-O-a!kyl-2-0-acetyl-sn(cid:173)
`glycero·3-phosphocholine
`to be
`the platelet(cid:173)
`activating factor (PAF) and Berdel et al. (1981)
`reviewed the anti-tumor action of alkyl-lysophos(cid:173)
`pholipids.
`lipid composition
`While investigating the
`of Antarctic Krill (Euphausia superba Dana) (Fricke
`et al., 1984) we failed in finding plasmalogens but
`there was some evidence for
`the
`of
`Jglifcerol:iPi<1s in trace amounts.
`on board a '"'"''"''<.; ..
`m'l'est1i<;atea were
`and 1981 and could
`be mv•estigatea
`
`some months of frozen storage. The amounts of free
`fatty acids found in these samples indicated that some
`degradation processes had taken place during stor(cid:173)
`age. Therefore it was .necessary to confirm and verify
`our findings. This was done during the 1985 expedi(cid:173)
`tion of the Federal Republic of Germany with FRV
`"Walther Herwig". Lipid extracts of freshly caught
`Krill were prepared on board.
`
`MATEIUALS AND METHODS
`
`Krill samples were collected from the Scotia Sea in
`December 1977 (A) and from the Gerlache Strait in March
`1981 (B) and in March/April 1985 (C-F) during the second
`(1977/78), the third (1980/81) and the fourth (1984/85)
`Antarctic expedition of the Federal Republic of Germany
`with FRY "Walther Herwig" using a 1219 mesh pelagic
`Krill net. In 1977 and 1981 Krill samples of 5 kg each were
`quick frozen on board and stored at - 35°C until analyzed.
`Lipid extraction was performed according to Folch et al.
`(1957) after some months of frozen storage. Samples from
`1985 were homogenized in a 20-fold excess of dichloro·
`methane:methanol (2:1, v/v) immediately after catching
`(almost all specimens were alive until this procedure). In
`addition to the lipid extracts of whole Krill a sample of Krill
`muscle was prepared (F) on board during the 1985 expedi(cid:173)
`tion. This sample was prepared from the Krill muscles of
`approx. 80 specimens which were carefully removed from
`the exoskeleton by hand and then treated like the whole
`Krill samples. These crude extracts were stored at - 30°C
`until further treatment.
`
`Lipid separation and derivatisation
`The alkylglycero!ipids from the 1977 and 1981 samples
`were isolated after
`hydrolysis of the total lipids
`according to Pugh et
`(1977). Phospholipids and neutral
`lipids were separated
`thin layer chromatography (TLC)
`on silica gel (E.
`Darmstadt, FRG) with diethyl·
`ether:water (100:1, v/v).
`incubation ofphospholipids
`with phospholipase C (from Bacillus cereus, Boehringer,
`
`131
`
`RIMFROST EXHIBIT 1160 Page 0001
`
`

`

`132
`
`H. FRICKE ec al.
`
`Mannheim, FRG) using the procedure of Blank et al. ( 1975)
`the alkylglycerols were prepared from the phospholipids
`and the neutral lipids by concentrated methanolic hydro(cid:173)
`chloric acid (2 hr, 80°C). The alkylglycerols were isolated by
`TLC on silica gel using the above mentioned system. The
`2,3-0-isopropylidene derivatives of alkylglycerols were pro(cid:173)
`duced after Hanahan et al. (1963) using acetone/perchloric
`acid.
`The lipid extracts of the 1985 samples were treated
`according to Snyder et al. (1971) with Vitride (sodium-di(cid:173)
`hydro-bis-(2-methoxyethoxy)-aluminate) to form the free
`alkylglycerols.
`
`Gas chromatography/mass spectrometry (GLC/MS)
`The separation and identificatoin of alkylglycerols was
`achieved on a 25 m WCOT Silar l0°C (Packard Instrn(cid:173)
`ments) column, temperature programmed from 110 to
`210°C, 3°C/min, and on a 50m WCOT SIL 5 CB column
`(Chrompack), temperature programmed from 220 to 320°C,
`3°C/min, on a Packard 428 gas chromatograph equipped
`with a FID and a HP 3371 integrator. Helium was used as
`a carrier gas with a flow rate of I ml/min, split ratio was
`100: !.
`GLC/MS of 1-0-alkyl-2,3-0·isopropylideneglycerols was
`performed with a HP 5985A quadrupole mass spectrometer,
`ionization energy 70 eV, ion source temperature 200°C,
`GLC column: 25 m WCOT CP Sil 5 (Chrompack). The
`identification of individual alkylglycerols was achieved by
`cochromatography of underivatised alkylglycerols and
`their isopropylidene derivatives as well using standard sub(cid:173)
`stances on a polar and a non-polar column and by the
`characteristic mass spectra of l-0-alkyl-2,3-0·isopropyl(cid:173)
`ideneglycerols (m/e 101 and M+ - 15). Quantification was
`attained using 1-0-heptadecylglycerol as internal standard
`added to the crude lipid extract.
`
`RESULTS AND DISCUSSION
`
`In all Krill samples investigated
`lipids were found as minor lipid components. The
`amounts ranged from 0.3 to 0.6% of total lipid
`content of Antarctic Krill (Table 1). No differences
`were found between the lipid samples caught in
`different years and seasons. Also the differing treat·
`ments of the samples-frozen storage before lipid
`extraction vs lipid extraction of freshly caught
`material-showed no influence on the alkylglycerol
`content From the qualitative investigation in the
`phospholipids and neutral lipids in the 1977 and 1981
`Krill samples we found that 1-0-alkylglycerolipids
`were present in both phospholipids and neutral lipids.
`2-0-Alkyllipids could not be detected in any sample.
`The amount of 1-0-alkylglycerolipids is lower than
`reported by Isay (1976) for crustaceans from tropical
`regions and is similar to the content in the Antarctic
`benthic prawn Chorismus antarcticus and the Antarc·
`tic isopods Sero/is in which Clarke (1977 and 1984)
`reported
`the presence of small amounts of
`1-0-alkylglycerols. In Chorismus a. Clarke found also
`high amounts of I-0-alk-l'-enyllipids. This was in
`agreement with the findings of Dembitskii (l 976 and
`1979) who showed the presence of plasmalogens in a
`number of marine invertebrates including crus(cid:173)
`taceans. During our analyses of the 1977 and 1981
`Krill samples (Fricke et al., 1984) and during the
`analyses of the 1985 samples no plasmalogens were
`found. This discrepancy might be explained by the
`use of hydrochloric acid fume for reaction thin layer
`
`*Krill caught in December 1977.
`tKrill caught in March 1981.
`:f:Krill caught in March/ April 1985. Krill samples extracted immediately after catching.
`§Krill muscle analyzed.
`I/On a wet weight basis.
`•tKrill samples stored at -30°C.
`
`Table 2. 1·0-Alkylglycerol composition in total lipids of Antarctic Krill (E11phausia superba Dana) samples. Data are
`expressed as wt % of total 1 ·0-alkylglycerols and represent means and standard deviation of at least three separate
`experiments. Numbering corresponds to peak-numbering in Fig. I. Samples A-F, see explanation in Table !
`Samples
`
`No. M+ -15*
`I
`311
`313
`2
`3
`325
`325
`4
`327
`5
`327
`6
`327
`7
`339
`8
`9
`339
`341
`JO
`ll
`353
`355
`12
`365
`13
`14
`367
`15
`367
`16
`369
`
`14:1
`14:0
`15:1
`15:1
`15:0 brt
`l5:0br
`15:0
`16:1
`16:1
`16:0
`17:1
`17:0
`18:2
`18:1
`18:1
`18:0
`
`A
`1.5 ± 0.1
`12.3 ± 1.8
`0.7±0.I
`0.9 ±0.2
`0.9 ±0.2
`0.7±0.2
`2.6 ± 0.2
`8.6 0.l
`4.0
`44.1 ± l.8
`0.9 ±0.2
`I.I ± 0.1
`4.6± l.7
`6.3 ±0.6
`3.1 ±0.3
`4.9 ± 0.5
`
`B
`0.6±0.l
`13.0 ± 1.3
`0.2 ±0.I
`1.0±0.7
`3.0 ±0.3
`1.6 ± 1.0
`1.3 ± 0.7
`11.8 ± l.8
`2.3 ± l.4
`52.7 ± 6.0
`0.6 ± 0.4
`0.7 ± 0.4
`l.5 ±0.9
`3.0 ± 0.5
`4.4 ± 0.6
`1.1
`l.7
`
`c
`l.l ± 0.1
`11.4 ± 0.5
`3.1 ±0.2
`2.1 ±0.5
`4.4 ± 0.2
`1.5 ± 0.4
`1.5 ± 0.2
`8.3 ±0.l
`3.2 ± 0.2
`40.5 ± 0.9
`0.9 ±0.l
`I.l ± 0.2
`3.3 ± 0.2
`4.8 ±0.3
`5.4 ± 0.4
`4.6 0.2
`
`D
`l.6 ± 0.2
`12.4 ± 1.0
`3.2 ± 0.2
`1.0±0.l
`1.7±0.2
`0.7±0.l
`2.0 ±0.3
`9,7 ±0.5
`3.5±0.1
`43.6 ± 2.0
`0.8 ± 0.2
`0.9 ±0.2
`3.2 ± 0.2
`5.2±0.3
`5.4 ± 0.3
`4.7
`0.1
`
`E
`LO± OJ
`10.9 ± 0.4
`3.2±0.2
`l.O ± 0.1
`2.2 ±0.2
`0.8 ± 0.1
`l.7±0.l
`9.1 ±0.2
`3.1 ±0.l
`42.l ±0.7
`0.6±0.1
`0.8 ±0.2
`4.0±0.4
`5.5 ±0.6
`5.8 ±0.7
`4.8 O.l
`
`F
`l.2±0.l
`10.9± LO
`2.6±0.l
`J.2 ±0.l
`3.4±0.2
`l.9 ± O.l
`1.4±0.2
`8.5 ± 0.2
`3.2±0.l
`44.1 ± J.4
`1.1 ±0.2
`0.8 ±0.2
`3.3 ±0.2
`S.I ± 0.2
`.5.9±0.2
`4.8
`0.1
`
`RIMFROST EXHIBIT 1160 Page 0002
`
`

`

`1-0-Alkylglycerolipids in Antarctic Krill (Euphausia superba Dana)
`
`133
`
`15
`
`14
`
`16
`
`10
`
`8
`
`2
`
`5
`
`9
`
`7
`
`~
`~
`~
`
`....
`B
`
`0
`
`min
`Fig. 1. GLC-separation of 1-0-alkylglycerols in Antarctic Krill (Euphausia superba Dana) (50 m WCOT
`column coated with SIL 5 CB, temperature programmed 220-320°C, 3°C/min). Peaks numbered 1-16
`show individual 1-0-alkylglycerols as listed in Table 2. X is contamination by phthalic acid ester.
`
`chromatography by Dembitskii and Clarke in esti(cid:173)
`mating the amounts of plasmalogens. According to
`Touchstone (1984) this method can produce mis(cid:173)
`leading results.
`In Fig. l a separation of l-0-alkylglycerols into
`individual species is shown. The quantification and
`mass spectrometrical identification of the com(cid:173)
`ponents in Fig. 1 are given in Table 2.
`In total a number of 16 different 1-0-alkylglycerols
`were found and identified by GLC/MS. The alkyl
`moieties 16:0 and 14:0 (with 44.09 and 12.33% of
`total alkyl chains) were dominant. The alkyl chains
`16: 1, 18: 1and15:1 were each presentin two isomeric
`forms. The positions of the double bonds of these
`chains could not be determined because of the very
`small amount of these alkyl chains which allowed no
`further analysis. The same problem arose for the
`two branched alkyl chainitof 15:0. 15:0 and 17:0 are
`the only saturated and 15:1 and 17:1
`the only
`unsaturated odd numbered alkyl chains. Only one
`double unsaturated alkyl chain (18:2) was found.
`Alkyl groups with more than 18 C-atoms or with
`more than two double bonds were absent.
`All alkyl side chains found correspond to the acyl
`chains present in glycerolipids of Krill (Fricke et al.,
`1984).
`The
`different sannp!1es
`
`the
`within
`1977 to 1985 did not differ
`the data in Table 2 it is evident
`
`that the frozen storage of samples A and B for several
`months had no influence on the 1-0-alkylglycerol
`composition. Furthermore this composition is not
`influenced by the total lipid content of the Krill
`samples ranging from 2.7 to 10.3% on a wet wt basis
`(Table 1). The 1-0-alkylglycerol composition and the
`l-0-alkylglycerol content were very similar in the
`samples from whole Krill (A-E) and in the Krill
`muscle sample (F). These findings suggest that the
`l-0-alkylglycerolipids form an integral component of
`the Krill and are not only located in the digestive
`tract where they possibly may be present from un(cid:173)
`digested diet.
`
`REFERENCES
`
`Berdel W. E., Bausert W. R. E., Fink U., Rastetter J. and
`Munder P. G. (1981) Anti-tumor action of alkyl(cid:173)
`lysophospholipids. Anticancer Res. 1, 345---352.
`Blank M. L., Cress E. A., P:iantadosi C. and Snyder F.
`(1975) A method for the quantitative determination of
`glycerolipids containing 0-alkyl and O-alk-1-enyl moi(cid:173)
`eties. Biochim. Biophys. Acta 380, 208-218.
`Clarke A. (1977) Lipid class and fatty acid composition of
`Chorismus antarcticus (Pfeffer) (Crustacea: Decapoda) at
`South Georgia. J. exp. mar. Biol. Ecol. 28, 297-314.
`Clarke A. ( 1984) Lipid composition of two
`of Serolis
`(Crustacea, Isopoda) from Antarctica.
`Suro.
`Bull. 64, 37-53.
`Dembitskii V. M. and Vaskovskii V. E. (1976) Distribution
`
`RIMFROST EXHIBIT 1160 Page 0003
`
`

`

`134
`
`H. FRICKE et al.
`
`of plasmalogens in various classes of phospholipids of
`marine invertebrates. Sov. J. mar. Biol. 2, 329-332.
`Dembitskii V. M. (1979) Plasmalogens in phospholipids of
`marine invertebrates. Biol. Morya (Vladivost.) S, 86-90.
`Demopoulos C. A., Pinckard R. N. and Hanahan D. J.
`(1979)
`Platelet-activating
`factor.
`Evidence
`for
`1-0-alkyl-2-acetyl-sn -glyceryl-3·phosphorylcholine as the
`active component (a new class of lipid chemical media(cid:173)
`tors). J. biol. Chem. 254, 9355-9358.
`Folch J., Lees M. and Sloane Stanley G. H. (1957) A simple
`method for the isolation and purification of total lipids
`from animal tissues. J. biol. Chem. 226, 497-509.
`Fricke H., Gercken G., Schreiber W. and Oehlenschlager J.
`(1984) Lipid, sterol and fatty acid composition of Antarc(cid:173)
`tic Krill (Eupltausia St1perba Dana). Lipids 19, 821-827.
`Hanahan J. D., Eckholm J. and Jackson C. M. (1963)
`Studies on the structure of glyceryl ethers and the glyceryl
`ether phospholipids of bovine erythrocytes. Biochem. 2,
`630--641.
`Horrocks L.A. and Sharma M. (1982) Plasmalogens and
`In: New Comprehensive
`0-alkylglycerophospholipids.
`Biochemistry, Vol. 4. Phospholipids (Edited by Haw-
`
`thorne J. N. and Ansell G. B.), pp. 5!-93. Elsevier
`Biomedical
`Amsterdam.
`Isay S. V.,
`M.A. and Vaskovskii V. E. (1976)
`A study of glyceryl ethers-I. Content of a-glyceryl ethers
`in marine invertebrates from the Sea of Japan and
`tropical regions of the Pacific ocean. Comp. Biochem.
`Physiol. SSS, 301-305.
`Isay S. V., Osheva 0. N. and Makarchenko M. A. (1984)
`Study on glyceryl ethers-IL a-Glyceryl ether content in
`tissues of Octopus dojleini. Comp. Biochem. Physiol. 77B,
`799-801.
`Mangold H. K. (1979) Synthesis and biosynthesis of
`alkoxylipids. Angew. Chemie Intern. Ed. 18, 493-503.
`Pugh E. L., Kates M. and Hanahan D. J. (1977) Character·
`ization of the alkyl ether species of phosphatidylcholine
`in bovine heart. J. Lipid Res. 18, 710-716.
`Snyder F., Blank M. L. and Wykle R. L. (1971) The enzymic
`synthesis of ethanolamine plasmalogens. J. biol. Chem.
`246, 3639-3645.
`Touchstone J. C., Snyder K. A. and Levin S. S. (!984)
`Analysis ofplasmalogens by in situ reaction on thin layer
`chromatograms. J. Liquid Chromatogr. 7, 2725-2733.
`
`RIMFROST EXHIBIT 1160 Page 0004
`
`