`© Elsevier/North-Holland Biomedical Press
`
`MARINE STEROLS. XII. THE STEROLS OF SOME
`PELAGIC MARINE CRUSTACEANS
`
`JAMES A. BALLANTINE and JOHN c. ROBERTS
`Department of Chemistry. Institute.for Marine Studies, University College <~{Swansea. Wales
`
`and
`
`ROBERT J. MORRIS
`lnstitllle <~/'Oceanographic Sciences, Wormley, Surrey, EnglC111cl
`
`Abstract: The sterol composition of some pelagic crustaceans (three species of pelagic euphausiids. one
`species of a bathypelagic marine mysid and one species of an estuarine mysid. two species of pelagic
`marine copepod~. and one species of a bathypelagic marine copepod) have been determined by gas chro(cid:173)
`matographic--mass spectrometric te\:hniques. The cuphausiids appear to have developed an almost uni·
`specific requirement for cholesterol. irrespective of trophic level. whilst the mysids show a l'onsidcrablc
`variation in sterol composition. The two near-surface copepod species contain a significantly greater
`variety of stcrols than their bathypelagic counterpart. The results are compared with the known sterol
`compositions of other crustaceans in the marine food web and with phytoplankton. The extent of
`modification of dietary sterols after ingestion is discussed.
`
`INTRODUCTION
`
`Whilst the sterol chemistry of some of the marine and freshwater crustaceans has
`received ccnsiderable attention (see reviews by Goad, 1976, 1978; Morris & Culkin.
`1977) comparatively few data are available on the sterol composition of pelagic
`marine copepods, euphausiids, and mysids. One species of euphausiid (NyclliJ/umes
`norvegica) has been analysed by Idler & Wiseman ( 1971 ). one species of mysid (Neo(cid:173)
`mysis intermedia) by Teshima & Kanazawa ( 1971 ), and one species of copepod
`(Euchaetajaponica) by Lee et al. (1974).
`These crustacean groups are major components of the marine ecosystem. The
`copepods especially dominate the marine zooplankton in numbers and together with
`the other small crustaceans provide the link between the primary producers in the
`marine food web, the phytoplankton, and the larger carnivorous plankton. Know(cid:173)
`ledge of their sterol chemistry and hence their contribution to the pool of sterols in
`the marine food web is necessary for a proper understanding of the relative require(cid:173)
`ments of sterols by marine organisms and the biochemical potential for sterol syn(cid:173)
`thesis-modification within the various trophic levels. Similarly such knowledge is
`necessary if the geochemical pathways of sterol diagenesis during and after sedimenta(cid:173)
`tion of dead organisms are to be confidently identified.
`This paper presents the sterol composition of three species of pelagic to meso-
`25
`
`RIMFROST EXHIBIT 1167 Page 0001
`
`
`
`26
`
`JAMES A. BALLANTINE ET AL.
`
`pelagic euphausiids, one species of a bathypelagic marine mysid and one species of
`an estuarine mysid, two species of pelagic marine copepods and one species of a
`bathypelagic marine copepod. Th~ paper compares the sterol composition with
`those of other crustaceans in the marine food web and with phytoplankton and at(cid:173)
`tempts to draw some conclusions as to whether the composition of the crustacean
`sterols is controlled metabolically by the animal as a result of its own specific physio(cid:173)
`logical or biochemical requirements, or merely represents the type of dietary input,
`the animal having little control over its sterol make-up.
`
`MATERIAL, METHODS AND RESULTS
`
`\he animals were caught by net during biological cruises of R.R.S. Discovery to
`the northeastern Atlantic. They were sorted immediately by hand and where pos(cid:173)
`sible were kept alive for 3 h in clean, filtered sea water at 8 °C under low light condi(cid:173)
`tions so that they might clear their guts. They were then frozen under nitrogen at
`- 25 °C in specially cleaned glass jars. They were kept frozen at - 25 °C until ana(cid:173)
`lysis.
`Total lipids were extracted from the animals and the free sterol and sterol ester
`fractions crudely separated by methods previously described (Ballantine et al., 1975,
`1976). These fractions were then saponified with 10~,;~ potassium hydroxide in ethyl
`alcohol for 24 h under nitrogen in the dark and the non-saponified fraction purified
`by preparative TLC. The separated sterols which originated from the free and
`bound fractions were then determined using an SE30 0.3~>;, textured glass bead
`column (Table I).
`The sterols were identified and their relative amounts determined by the G LC of
`their trimethylsilyether (TMSE) derivatives on l l,~~ Dexsil 300 GC and I~~ Sitar
`SCP (2.5 m x 4 mm i.d. at 260 and 220 °C, respectively). These columns were coupled
`
`TABLE I
`
`Details of samples.
`
`Depth of net
`Sterol
`Total lipid
`Species
`(m)
`Position caught
`0 total lipid)
`(0
`0 wet wt)
`-·-- -- ----- -- ------------ - - - - - - - - - - - - - - - - -- -- --~----- ------------------ - -
`M eganyct iphanes nonegica M. Sa rs
`.l4
`25°N; 17°W
`250-0
`1.9
`4.4
`Thysa11opoda suhaequalis Boden
`1.2
`20°N ~ 23 °W
`1000-0
`Styloclwiron ahhn•\•iatum G.O. Sa rs
`3.3
`I. 7
`25 °N: 17 °W
`250-0
`37"N; 25 cw
`Eucopia sculpticauda Faxon
`1000--950
`9.7
`0.6
`Neomysis integer (leach)
`4.3
`Totton Marsh
`surface
`1.9
`(Southampton)
`20°N: 22°W
`60°N~ 20°w
`38 °N; 28 °W
`
`Lahidocera acm{fiw1s (Dana)
`Calmws flnmarchicus (Gunnerus)
`Bathycalam1s sp.
`
`surface
`surface
`1258-1264
`
`1.3
`2.7
`7.7
`
`0
`
`(
`
`Sterol ester
`0 total lipid)
`
`(0
`
`-------- ---~---·--
`0.007
`0.036
`
`0.002
`0.012
`
`0.026
`
`3.6
`0.7
`0.2
`
`RIMFROST EXHIBIT 1167 Page 0002
`
`
`
`STEROLS OF SOME PELAGIC MARINE CRUSTACEANS
`
`27
`
`through a silicone membrane separator to a fast scanning MS9 mass spectrometer.
`as previously described (Ballantine et al .• 1975). The results of the GLC-MS analysis
`upon the TMSE are tabulated in Tables II and III.
`
`5
`
`3
`
`a
`
`7, 10
`
`2
`
`10
`
`20
`
`30mins
`
`5.6
`
`3
`
`b
`
`4
`
`7, 8
`
`2
`
`10
`
`20
`
`30mins
`
`Fig. 1. Sterol profile of Lahidocaa m·111ifrons on Dcxsil 300 GC (a) and Sitar SCP (b).
`
`Fig. I represents the sterol profile of Labidocera acut(frons', which has been
`selected as an example because it contain~d almost all of the marine sterols found in
`the pelagic marine crustaceans. The TMSE derivatives of L. acut(fi·ons were observed
`
`1 For taxonomic authorities see Table I.
`
`RIMFROST EXHIBIT 1167 Page 0003
`
`
`
`TABLE II
`
`Sterol composition of sample .. : tr. trace.
`
`Sterolsa
`
`Species
`
`2
`
`3
`
`4
`
`5
`
`6
`
`...,
`I
`
`8
`
`9
`
`10
`
`Mega ll) ·ct iphanes norreg i c ·a
`Thysanopoda suhaequalis
`S tylocheiron ahhrei·ia tum
`Eucopia scu/pticauda
`Neomysis imeger
`Lahidocera acut(li·om;
`Ca/anus fin march icus
`Bathycalanus sp.
`Thalassionema nit=ochioides
`(Ballantine et al .. 1979b)
`
`a Key list for .Herols
`Sterol
`I 25C:' 22E
`'J 27C5. 22::*
`3 27C5.22E
`4 27C.22E
`5 27C5
`6 27C
`7 28C5.22E
`8 28C.22E
`9 27C5.7
`I 0 27C5.24(25)
`11
`28C5
`12 28C5.24(28)
`13 29C5.22E
`14 29C5
`15 29C5.24(28)Z
`16 Unknown
`
`0.6
`LI
`1.7
`0.5
`3.2
`14.3
`14.1
`16.9
`22.7
`
`98.2
`96.6
`96.0
`96.l
`88.0
`73.2
`45.4
`80.5
`39.6
`
`tr
`tr
`
`tr
`2.1
`
`tr
`
`1.0
`1.2
`27.7
`
`5.0
`
`0.6
`
`tr
`
`0.8
`4.2
`3.0
`
`3.6
`
`0.7
`2.7
`
`l.5
`
`1.4
`1.5
`
`tr
`
`22(E)-24-norcholesta-5.22-dien-3fJ-ol
`occelasterol
`22(E)-cholesta-5.22-dien-3/J-ol
`22(E)-5~-cholest-22-en-3fJ-ol
`cholesterol
`5:x-cholestan-3fJ-ol
`22(E).24(E)-24-methylcholesta-5.22-dien-3/J-ol
`22(E).24(E)-24-methyl cholest-22-en-3{::,.01
`cholesta-5, 7-dien-3fJ-ol
`desmosterol
`24(E)-24-methylcholest-5-en-3fJ-ol
`24-methylene cholesterol
`22( E).24(£ )-24-ethylchclesta-5.22-dien-3fJ-ol
`24(r. )-24-ethylcholest-5-en-3fJ-ol
`24(28)-24-ethylcholesta-5.24(28)-dien-3fJ-ol
`
`11
`
`0.2
`0.2
`tr
`0.5
`LO
`0.3
`1.5
`0.2
`2.9
`
`12
`
`0.2
`0.5
`tr
`0.8
`2.9
`1.6
`
`0.5
`2.2
`
`13
`
`0.3
`0.5
`tr
`l.l
`0.6
`0.9
`
`0.3
`-
`
`14
`
`0.6
`I.I
`tr
`1.1
`2.4
`1.6
`2.0
`1.5
`2.0
`
`15
`
`16
`
`tr
`tr
`
`0.8
`
`2.2
`
`N
`oc
`
`'-> :::
`tT1
`r.n
`>
`= > r-
`r-
`> z
`~ z
`tT1
`...,
`h'l
`~
`r""
`
`RIMFROST EXHIBIT 1167 Page 0004
`
`
`
`STEROLS OF SOME PELAGIC MARINE CRUSTACEANS
`
`29
`
`as eight partly resolved peaks in both the Dexsil and Sitar chromatograms. By
`multiple mass spectrometric :iCanning of each of these peaks it was established that
`14 different marine sterols were present in the extracts and their retention times were
`determined.
`
`GL-MS characteristics of the sterol TMSE derivatives of labidocera acutifrons.
`
`TABLE III
`
`Sterols
`
`Identity
`
`Dexsil
`RRT
`
`Silar
`RRT
`
`I
`2
`3
`4
`5
`6
`7
`8
`10
`11
`12
`13
`14
`IS
`
`26C5. 22E
`27C5. 22Ec
`27C5, 22E
`27C 22E
`27C5
`27C
`28C5, 22E
`28C 22E
`27C5 24(25)
`28C5
`28C5, 24(28)
`29CS. 22E
`29C5
`29C5, 24(28)Z
`
`0.63
`0.84
`0.89
`0.93
`1.00
`1.04
`1.10
`1.14
`1.10
`1.29
`1.29
`1.36
`1.61
`1.69
`
`0.65
`0.88
`0.93
`0.90
`1.00
`0.97
`1.22
`1.09
`1.34
`1.32
`1.43
`1.49
`1.61
`1.92
`
`M+
`
`442
`456
`456
`458
`458
`460
`470
`472
`456
`472
`470
`484
`486
`484
`
`Characteristic
`fragment ionsb
`
`427,372.352,337,313.281,255. 129
`441, 366. 351, 327. 255. 215, 213, 129
`441, 366. 351, 327. 255, 215, 213, 129
`443.374,346,345,257,217,215
`443, 368.353,329,275,255.247. 129
`445.370,355,306,305,217,216,2/5
`455, 380,365.341, 337. 255. 129. 125
`457, 374,346.345,257.217,215
`441.366.351.343,327,255,253. 129
`457. 382,367.343,289.261.255, 129
`455.386,380,365,343.341,257. 129
`469,394,379.~55,351.255. 139, 129
`471.396,381,357,275,255.213. 129
`386,371.296.281.257,255.213. 129
`
`a For key see Table II.
`b Only ions above m/z 125 are quoted in this table and the base peak is italicized.
`c Occelasterol.
`
`The sterols were identified (Table III) by a combination of their GLC retention
`times on the two different 'liquid phases and their characteristic mass spectrometric
`fragmentation patterns, which were compared with the GC-MS data for authentic
`sterol TMSE derivatives and for TM SE derivatives of known marine sterols obtained
`from other marine organisms as previously described (Ballantine et al., 1975, 1976,
`1977, 1978, 1979a-b). It must be noted that neither the GLC nor MS analysis is able
`to determine the stereochemistry at C24 in the sterol side chain and there was in(cid:173)
`sufficient sample available to determine the stereochemistry by other means.
`In this way sterols I, 3, 5, 6, 7, 10, 11, 12. 13, 14 and 15 were identified (Tables II
`and Ill) as common sterols which were present in many marine organisms. Sterol 2
`was identified as occelasterol obtained from an annelid by Kobayashi & Mitsuhaski
`( 1974) and from the jellyfish Pyrosoma sp. by Ballantine et al. ( 1977). Sterol 4 was
`identified as 22-dehydrocholestanol and Sterol 8 was neospongesterol (or its C24 epi(cid:173)
`mer) both of which were isolated by Erdman & Thomson (1972) from the sponge
`Hymeniacidon per/eve and which were shown to be present in jellyfish (Ballantine
`et al., 1975) and tunicates (Ballantine et al., 1977). Sterol 9 was a minor sterol in the
`extracts of Stylocheiron abbrevicatum and was easily identified as cholesta-5,7-dien-
`3{J-ol by comparison with authentic sterol.
`
`RIMFROST EXHIBIT 1167 Page 0005
`
`
`
`30
`
`JAMES A. BALLANTINE ET AL.
`
`The sterols were determined by G LC using the two liquid phases. The flame
`ionization detector response factors for a series of standard marine sterols on these
`phases were determined and found, within experimental error, to be identical. Each
`sterol in a mixture was located by mass spectrometry within the peaks of the chroma(cid:173)
`tograms, and the peak areas of the various regions of the chromatograms were
`determined by cutting out and weighing on an analytical balance. By comparing the
`areas containing each individual sterol and by using subtractive techniques from
`the results of the two different columns, the relative amounts of each sterol could be
`determined. Cross-checks using different column systems were employed and the
`results averaged. In general, there was good agreement in the cross-correlations
`and the results have an estimated error range of the order of ± 2% for abundant
`sterols and ± 0.3°~ for minor sterols.
`
`DISCUSSION AND CONCLUSIONS
`
`Before any discussion of the results it should be pointed .. 'ut that although a period
`was allowed for the animals used to clear their guts prior to sacrifice, Htrace" sterols
`detected in the animals could be coming from residual, non-absorbed gut contents.
`The predominance of cholesterol in the euphausiids is in agreement with the fin(cid:173)
`dings of Idler & Wiseman ( 1971) for Nyctiphanes norvegica. The minor sterols they
`found in the mixture were, however, 22(E)-cholesta-5,22-dien-3{J-ol, 22(E),24(r. )-24-
`methylcholesta-5,22-dien-3{J-ol (0.1 °1~), desmosterol (0.4 !:», and three unknown ste(cid:173)
`rols. This result is at variance with the present work. It is possible, however, that sin~e
`Idler and Wiseman used a single column to determine the sterol composition of
`the animal, the desmosterol they found was in fact cholesta-5,7-dien-3{J-ol as the
`column (3~;, XE. 60-3?~ NGS) does not resolve these two sterols (desmosterol
`RRT = 1.29 and 7-dehydrocholesterol RRT = 1.30).
`The predominance of cholesterol in the mysids is in agreement with the findings
`of Teshima & Kanazawa (1971) but the large quantities of 22(E),24(e)-methyl(cid:173)
`cholesta-5,22-dien-3{J-ol and methylenecholesterol reported by these authors to be
`present in Neomysis intermedia were not found in the species examined in the present
`work. The bathypelagic species Eucopia sculpticauda was found to have a relatively
`low percentage of sterol in its lipids but this was due to the large amount of wax
`esters in its total lipid (e.g. Morris, 1972).
`In agreement with the results of Lee et al. ( 1974) with the species Euchaetajaponica,
`the copepod Ca/anus finmarchicus analysed in the present work also contained a
`large proportion of desmosterol (27. 7~,~»· This sterol was, however, either absent
`·or present as a minor component in the other two copepod species analysed.
`The majority of the other copepod sterols, 1, 2, 3, 4, 6, 7, (8,9), 11, 12, 13(14), and
`15 reported in this work are new to the copepods and the stanols, 4, 8, and sterol 2
`are new to the crustaceans as a group.
`
`RIMFROST EXHIBIT 1167 Page 0006
`
`
`
`STEROLS OF SOME PELAGIC MARINE CRUSTACEANS
`
`31
`
`The thr.ee~ euphausiid species examined in this work are generally found in dif(cid:173)
`ferent geographical areas and at different environmental depths and hence are
`thought to occupy rather different trophic levels. Meganyctiphanes norvegica and
`Thysanopoda subaequalis almost certainly feed for part of their time on mixed phyto(cid:173)
`plankton and smaller zooplankton in the euphotic zone whilst Stylocheiron ahhre(cid:173)
`viatum does not normally enter the upper euphotic zone. From our results it would
`appear that irrespective of trophic level these euphausiids have developed an almost
`uni-specific requirement for cholesterol as their body sterol. Only the macrurid
`decapods (unpubl. data), to which euphausiids are fairly closely related, and possibly
`the bathypelagic mysids (this work, see later) amongst the crustac~an.i have a similar
`sterol composition, completely dominated by cholesterol.
`In contrast, the results for the mysids together with the data of Teshima &
`Kanazawa (1971) show a considerable variation in sterol composition. The
`carnivorous bathypelagic Eucopia species (see Table II) has the same cholesterol(cid:173)
`dominated composition as the euphausiids but the two surface omnivorous Neomysis
`species (see Table II and Teshima & Kanazawa, 1971) have a far more diverse compo(cid:173)
`sition with 22·dehydrocholesterol, brassicasterol (or crinosterol), methylene choles(cid:173)
`terol, and P-sitosterol being important sterol components.
`The results of the copepod analyses indicate that the two near-surface species con(cid:173)
`tain a significantly greater variety of sterols than their bathypelagic counterpart
`Bathycalanus sp. For example sterols L 2, 4, 6. 7, 8, and 10 are present in the
`omnivorous surface species Lahidocera acut(fi·ons but not in the carnivorous bathy(cid:173)
`pelagic species. It is now accepted that members of the Arthropoda cannot bio(cid:173)
`synthesize sterols in vivo but can convert ingested phytosterols to cholesterol via a
`desmosterol intermediate (e.g. Thompson et al., 1972). Thus. the sterols observed in
`the surface species are probably of either exogenous origin or are modifications
`of sterols of exogenous origin. Unfortunately little is known of the inter-relation(cid:173)
`ships between sterols of natural field populations of phytoplankton and zooplankton
`due to the difficulties involved in obtaining field samples containing only one species
`(see discussion in Ballantine et al., 1979b). It seems probable however, that the large
`quantity of Hphytosterol" produced by the phytoplankton in the upper reaches of
`the sea is converted by the crustacean members of the plankton into cholesterol.
`Because the phytoplankton are found in the upper 50 m or so of the oceans then one
`would expect the surface crustacean species to contain the greatest number of sterols.
`Certainly most pelagic copepods will be omnivorous (Marshall, 1973), and Ca/anus
`finmarchicus and Lahidocera aclll(frons are thought to be predominantly herbi-
`vorous.
`In comparing the published sterol analyses of phytoplankton cultures with the
`present data. it is essential to consider the possible effects of culture condition and
`stage of growth in the composition of the phytoplankton (see Ballantine et al .•
`1979b). Therefore. we have mainly compared the copepod data with the published
`sterol composition of a single species of marine diatom taken from a single natural
`
`RIMFROST EXHIBIT 1167 Page 0007
`
`
`
`32
`
`JAMES A. BALLANTINE ET AL.
`
`field population in the northwestern Atlantic, i.e. the same general area from which
`the copepods were taken (Ballantine et al., 1979bt In detail, the sterol compo(cid:173)
`sition of Labidocera acut~frons is very similar to that found in the free-living diatom
`Tha/assionema nitzschioides (see Table II) although cholesterol is a more major
`component of the copepod sterols and stanols were not found in the diatom. The
`surface calanoid species (Ca/anus finmarchicus) contains many of the sterols found
`in phytoplankton and its major sterols, 22-dehydrocholesterol, cholesterol, and
`desmcsterol are also the major sterols of the free-living diatom, although they are
`present in different relative amounts - desmosterol in particular appearing to be
`of importance to this copepod. The deep-water calanoid species (Bathycalanus sp.)
`has a much simpler sterol composition than is normally found in phytoplankton
`with 22-dehydrocholesterol and cholesterol comprising over 97% of the component
`sterols. The complete absence of desmosterol in this species compared with its
`presence as a major sterol in the surface calanoid species may indicate a particular
`requirement for desmosterol in Ca/anus finmarchicus.
`The results suggest that many of the sterols found in the surface copepods may be
`of a dietary origin although the animals have clearly modified them to a varying
`degree, presumably to suit their particular needs. The presence of stanols in these
`species but not in marine diatoms (Ballantine et al., I 979b) which are likely to form
`part of their diet, may indicate that either the stanols are intermediates involved in
`the conversion of phytosterols to sterols required by the copepods in vivo or that
`they are being synthesized by the copepods for specific physiological-biochemical
`purposes.
`
`ACKNOWLEDGEMENTS
`
`Tlt!-fi authors gratefully acknowledge support from the Institute of Oceanographic
`Sciences in the form of a research contract (NERC F60/B 1 /6), the Science Research
`Council for a studentship to one of us (J.C. R.), and the help of colleagues in the
`Biology Department of the 1.0.S. in the collection and identification of the animals
`used in this study.
`
`REFERENCES
`
`BALLANTINE, J. A .. J.C. ROBERTS & R. J. MORRIS, 1975. Sterols of the cockle Cerastoderma edule.
`Evaluation of thermostable liquid phases for the gas chromatography-mass spectrometric analysis
`of the trimethylsilyl ethers of marine sterols. J. Chromatogr .. Vol. 103. pp. 289-304.
`BALLANTINE, J. A .• J. c. ROBERTS & R. J. MORRIS, 1976. Marine sterols. Ill. The sterol compositions
`of oceanic jellyfir,h. The use of gas chromatography-mass spectrometry techniques to identify un(cid:173)
`resolved components. Biomecl. Mass Spectrom., Vol. 3, pp. 14-20.
`BALLANTINE, J. A .• A. LAVIS. J.C. RoBFRTS & R. J. MORRIS, 1977. Marine sterols. V. Sterols of some
`Tunicata. The occurrence of saturated ring sterols in these filter-feeding organisms. J. exp. mar.
`Biol. Ecol., Vol. 30, pp. 29-44.
`·
`BALLANTINE. J. A., A. LAVIS, J.C. ROBERTS, R. J. MORRIS, J. F. ELSWORTH & G. M. L. CRAGG, 1978.
`
`RIMFROST EXHIBIT 1167 Page 0008
`
`
`
`STEROLS OF SOME PELAGIC MARINE CRUSTACEANS
`
`33
`
`Marine sterols. VII. The sterol compositions of oceanic and coastal marine Annelida species. Comp.
`Biochem. Physio/., Vol. 61 B. pp. 43-47.
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