INFORMATION TO USERS
Thismanuscript has been reproducedfromthemicrofilmmaster. UMI films the text directlyfromtheoriginalor copy submitted. Thus,some thesis and dissertationcopies arein type'Nriter face,whileothers maybefrom any typeof com puter printer.
The quality of this reproduction is dependent upon the quality 01the copy submitted.Broken orindistinctprint,colored orpoorqualityillustrations andphotographs, printbleedthrough, substandard margins. and improper alignment can adverselyaffectreproduction.
In theunlikely eventthatthe authordid not send UMI a complete manuscript and therearemissing pages.thesewillbenoted. Also.ifunauthoriZecI copyrightmate rial hadtoberemoved,a note willindicate~e de~tion.
Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning theoriginal, beginningatthe upperleft-hand eeenerand continuing from lefttorightin equalsectionswithsmallovertaps.
Photographs ind uded in the originalmanuscript have been reproduced xercqrup hlcauy in this copy. Higher quality 6~ x 9~ black and ...mite photogra phicprintsare availablefor any photographs or illustrations appearing in thiscopyforanadditiona lctIarge.ContactUMIdirectlytoorder.
Sell&Howell Informationand Leaming 300 North ZeeO Road,Ann Arbor,MI48106-1346 USA
800-521.()6()()
UMl~
NOTE TO USERS
Page(s) not included in the original manuscript and are unavailable from the author or university. The manuscript
was microfilmed as received .
69
This reproduction is the best copy available.
UMl'
.",.
NationalofCanadalibrary Acquisitions and BibliographicServices 395We~ongIOtISUNt QnaWilONKIA 0N4 C;on;o4aBibliothequenationale duCanada Acquisitionset servicesbibliographiques J95.rueWelington OttawaONK1A0N4
e.-a
Theauthorhasgrantedanon- exclusivelicenceallowingthe NationalLibrary ofCanada to reproduce,loan, distributeorsell copies of thisthesisinmicroform, paperorelectronic formats.
The author retainsownershipof the copyrightinthisthesis.Neither the thesis norsubstantial extractsfrom it maybe printedorotherwise reproduced without theauthor's permission,
L•auteura accordeunelicencenon exclusivepennettaot
a
la BibliothequenationaleduCanada de reproduire ,preter,distribu erau vendre des copiesde cette these sous laforme demicrofiche/film,de reproduction sur papier ausurformat electronique.L'auteur conserve laproprietedu droitd'auteurqui protege cette these.
Nila theseni des extraitssubstaotiels de celle-ciDe doivent otre imprimes auautrernent reproduits sansson autorisation.
0-612·54891·0
Canada
LIPID COMPOSITIONOF SELECTED TISSUES AND MILK OF PHOCID SEALS OF EASTERN CANADA
By
©Edw ard A.D.Durnford
Athesis submittedto the Schoolof GraduateStudies inpartial fulfillmentof therequirementsfor
the degree of [he Masterof Science
Department of Biochemistry MemorialUniversity ofNewfoundland
April. 1998
St. John' s.Newfoundland.Canada
Abstract
Murineoilsandseafoodshave rece ivedmuchattentionsince the irconsumption has been associated with beneficial cardiovascular effects.Much of the research.
however.has focuse donfishoils andoily fishwith some recentreportson harp and grey ...ealblubberoils.This study investiga tedthelipidcompositionofseveraltissuesfromall six spec ies orEastern Canadian Phocid seals.The position of fatty acids ofthe trI:lcylglycero llT AG I back bonealsohasimportant dietaryand biochemicalimplicauons.
Therefore.the positionaldistributionoffattyacid softheTAGofharpsealmilk wasalso inve stigated.
Prox im atecomposition of thesamplesindicatedthatblubber. followedbymilk.
had thehighestlipidcontentandthelowest levelsof moi stureand protein.Muscle tissues hadthehighe stproteincontent and milk had thelowest amountofash.
Thin layer chromatography-flame Ionization detection fTlC·FIDl studies indic atedthatlipidclasscomposition was primarilydependentonthetis sue ascompared to speciesofseal.Furthermore.sometissueshad a veryuniqu ecompositio n.Blubberand milklipids were foundtobemainlycomposedof TAG whilebrainhad undetectable levels of TAG.Bra inlipids werehigh incholesterolandcerebrosi des .the latterbeinga polarlipidclassnot foundinanyother tissueexamined.
Thetanyacidsofall tissue lipids contained relative lyhigh proportionsof polyunsatu ratedfattyacids(PUF A) ofthero3type (12-23%)andthesewerecomposedof eicosapenraencic acid (EPA. 0.3-13%),docosapentaenoic acid (DPA. 0-5%), and
ii
dccosuhexaenoic acid(DHA. 3~16'7c). Multivariate analysis showedthat fattyacid composition varied morefromtissueto tissuethanspeciestospecies.Several unique featuresin thefatty acidcompositions ofvarious tissueswerealso identified.Blubber wasfoundtobe:highin monounsatu rated fan yacidsIMl:"FAI.butlowinarachidonic acid anddimethylacetals.Brainlipids.ontheotherhand.werehighindimethyl acerals.
Brainlipids"er ealso high in DHA.Lung tissuelipidswere veryhigh in saturatedfany J.~ld,.especiallypalmiticacid.and heartlipids had a higher contentof linoleic acid than lipids ofothertissuesexam ined.
vfulitvanareanalysisalsoindicated clear differences inthe neutraland polarlipid tracncn,of corresponding tissues.The polarlipidfractiontendedto be higherin dimethyl acctal v.vaturated fanyacidsandPUFAof theCJJ3configuration.The neutrallipidfraction of corte...ponding tissues.however.tended tobericher in~tL"FAand PUFAofthew3 configuration.
Thepos itionaldistributionofthefattyacidsof the triacylglycerclsofharpseal milkwasvery similartothatof harpseal blubber.Thew3 PUrAwere concentratedinthe J·ll- landSil-)positionswhilethe saturatedfattyacids were preferent iallyesterifiedtothe m·2position.
iii
Acknowledgements
[would liketoex.pressmy sincereappreciation toDr.F.Shahidi forprovidingme wnh theoppo rtunity tocomple temy degre eunder hissupervision.Hisguidance . support.
andencourage mentthro ugho ut thecourse of my studies and [hepreparationof this thesis are also g:reatly acknowledged. I would also like(0 express man ythanksto my -upe rvisorvcommitteemem ber s.Drs.E.Millerand R.Hoo verforthei r inputand guidancethroug hout this study.TheassistanceofDr.E.Millerinmultivariate analysisis also greatly apprecia ted.Iwould like10 thankall of my colleaguesin Dr.Shahidi' s resea rch groupfortheirsuppo rtandcreatingapleas antenviron me nt [0 studyand work . Finally.Iwouldliketothankmy lovingwifeSonja forher continuedsupport throughout my studies.
Table of Contents
Abstract Ackno wledge ments Tabl e ofContents List of Tables Listof Figures Listof Abbre..-ia tlons Cha pter 1.Int rod uct ion Cha pter2.LiteratureReview
S..:als
2.1.1 Phocidseals ofEasternCanada
2.1.1.1 The beardedse al.Erignathus barbatus 2.1.1.2 The grey seal.Halichoerus grypus 2.1.1.3 The harbourseal.Phoca vitulina 2.1.1A The harpseal.Phocagroenlandica 2.1.1.5 The hooded seal.Cvstophora cristata 2.1.1.6 The:ringedsea l.Phoca hispida
The Canadi an Sea lingIndu str y 2.1.1 Histc rv ofthe"SealHunt"
1.2.2 The Modem"Sea l Hunt'"
Marinelipidsandtheirsignificance
2.3.1 Che mistryandcompositionof marine lipids 2.3.1.1 Fat ty acids -Bac kgrou nd 2.3 .1.2 Fauvacidsofmarine mammals 1.3.1.3 Acylglycercls
2.3.IA Ste rols 1.3.1.5 Phospholipids D.1.6 Etherlipids 2.3.1.7 Sphingolipids
2.3.2 Marine lipidsin humannutritionand disease prevention
iv
viii
xii
5 5 5 7 8 10 11 11 14 14 16
17 17 18 19 23 16 16 31 31 35
2.-t Analysi s of lipidcomposition 43
.2.4.1 The analysis of fattyacids .2.·t.2 The analysis oflipid classes 2.4.3 Thestructural analysisoflipids
Chapter3•~!aterialsand~Iethods 51
).1
3.1
Materi als 3.1.1 Samples 3.1..2 Chem icals Me thods
3..2.1 Proximate compositi onandlipid extraction 3..2.1 .1 Moistureconte nt
3.1.1.2 Crude prote incontent 3.2. 1.3 Lipidextractionand quantifica tion 3..2.1A Ash con tent
3.2.1 Se paration of neutra l and polar lipidfractions by columnchromatography
3.2 .3 Lipidclassanalysisby thin la yer chro mato graphy- flu meionization detect ion (T LC-FIDIlatroscan 3.2..3.1 Inst rumenta tion
3.2..3.2 Preparation of chrom arods 3.2.3.3 Standardsandcalibratio n 3.2.3.4 latroscanIT LC- FID)analysis of seal
tissues 3.1..t Stereospecificanalysis
3.2A.1 Purificationof triacylglycer ols 3.2.·L ! Grignard reactiononpurified
triacylglycero ls
3.1A.3 Separatio nor ind ividua l lipidclassesfrom Grignardrea ct ion
3.2AA Preparationofsynthetic phospholipids fromdiac ylgly ce rolfraction 3.2.4.5 Stereospec ifichyd rol ysis ofsynthetic
phospholipidsbyphosp ho lipaseA, 3.2A.6 Hydro lysisbypacre atic lipase 3.2 .5 Analysisof fatty acid compositionof lipids
3.2.5.1 Preparatio n of fattyacid methylesters IFA.\ IEs)
3.2.5.2 Analysis of fattyacidmethylestersby gas chromatography
3.2.6 Statisticalanalysis
51 51 5\
52 52 52 52 53
5~
5~
5~
54 55 55 56 56 56 57 58 58 59 60 61 61 62 63 Cha pter '; • Resul tsand Discussion
.1.1 Proximatecomp ositi on
-1..1.1 Mo isture content
~.1.2 Proteincontent -1..1.3 Lipidcont ent -1..1.4 Ashconte nt Lipidclasses
-1..2.1 Neut ralandpolarlipids
·L~.2 Triacy lglycero ls
·L~.3 Freefattyacids -1..2.-1. Chole sterol -1..2.5 Cardiolipin
-1..2.6 Phosphatidylethanolam ineandphos phatidylseri ne
-1..2.7 Phosphatidylcholine
-1..2.8 Sphingom yelin
-1..2.9 Cerebroside s
Fattyacidcomposition
~.3.1 Blubber
-1..3.2 Muscle
-1..3.3 Brain -I.J.-4 Kidney
~.3.5 Heart
U.b Lung
-1..3.7 liver
'+.3.8 Com pari son oftissues andspecies .+.3.9 Com parison ofpolarandne utralfractions Posit ionaldistribution offattyacidsintriacylg lycero ls of harpsealmilk
Cha pter5•Co nclu sions Ref er en ces
64 64 67 70 70 70 73
7~
75 76 77 78 79 79
80 80
8~
88 91 93 98 100 I~
108
112 115 117
List of Tables
Table2.1 Commo n namesusedfur harpsealsofdifferent ages. 11 Table ....1 Moisture contentIgl lOOgtissue)of varioustissues fromsix species
o( ~al. 65
T able..l..~ Proteinconte nt(gllOOgtissueIofvarious tissues fromsixspec iesof
~JI. M
Table-\..3 Lipid content(git OOgtissue)of varioustissuesfromsixspec iesof
~~. 68
Table-+.4 Ash contentIgil OOgtissueIofvarious tis sues fromsix species of
"ca l. 71
Table....5 Lipidclass compositionfgllOOglipid)ofselected tissues of five
,pecles ofseal. 72
Table
-r.e
FattyacidcompositionIgl lOOg lipid}ofblubberof various speciesofseal. 81
Table....7 Fatty acid compositionIgllOOglipid)ofmuscleofvariousspecies
of ~~. 85
Tdole-1..8 Fatt yacidcomposi tionIgll OOglipid) ofbrain ofvariousspecies of
"eal. 89
Table...9 Fatt y acidcompositio n19/IOOglipid) of kidneyof variousspeci esof
seal. 92
Tabl<-I.IO Fatty acidcompos ition19/IOOglipid)ofheartofva riousspeciesof
seal. 95
Tablo-1.11 Fatly acidcompositionIgi lOOglipid)oflung of vario usspecies of
seal. 99
Tablc-U2 Fatt y acid composition19/IOOglipid ) of liverofvariousspeciesof
seal. 102
Table -1.13 Factoranalysissummary. 105
Tat·leJ.IJ Eigenvalues. 105
T-Ibl<-I.15 Factor analysis summary. 109
Table..U6 Eigenvalues. 109 Table-l.17 Fattyacidcomposition(g1I00 glipid! of polarand neutral lipid
fraction sofmuscle. kidney.heart.andlivertissuesof harpseal. 110 Table-l.18 Positional distributionoffattyacidsinrriacylglycerolsof harpseal
milk. 113
Listof Figures
Figure~.I Distributionofthe beardedsealIEngnu thusbarbatuss.
Figure 2.2 Disrribuuonof the greyIHalichoerusg0'puS).
Figure2.3 Distributionofthe harbourseal(Phocuvitulintu.
Figure2.4 Distribution oftheharpseal(Phocagroenlandlcav
Figure2.5 Distributionof the hoodedseal (Cvstophora cristata s. 13 Figure2.6 Distribution of the ringedseal(Phoca hispida v. 13
Figure2.7 Structuresofsomecommonfany acids. 20
Figure2.3 Four typesof etherlipids. 32
Figure:.9 Structuresofsomeco mmonsphingolipids. 34
Figure 2.
to
Bio syn thesis of eicosan oids from arachidonic t.20:4 <001 andcicosupentaenoic120:5 cu31acids. 38
Figure4.1 Contents of saturated tSAT). monounsa turated l~tL' FAI.
polyunsaturatedIPUFAl.omega 3.and omega 6fattyacidsin blubberlipidsof differentspeciesof EasternCanadianPhocid
seals. 83
Figure -1..2 Contents of saturated tSAT). monounsaturated IMUFA1. polyunsaturated(PUFA).omega3.andomega 6fattyacids in musclelipidsof different speciesofEasternCanadian Phocid
seals. 87
Figure-1..3 Contents of saturated (SAn. monounsaturated IMCFAI.
polyunsaturatedIPliFA).omega3.andomega 6fatty acids in brainlipids ofdifferentspeciesofEastern Canadian Phocid seals. 90 Figure-l...J Contents of saturated (SAT). monounsaturated IMUFA1.
polyunsaturated(PUFA).omega3.and omega6 fattyacidsin kidney lipids of differentspeciesofEasternCanadianPhocid seals. 94 Figure..J.5 Contents of saturated (SAT). monounsaturated a.tUFA).
polyunsaturated(PUFA).omega 3.andomega6 fattyacidsin heart lipidsofdifferentspeciesofEastern Canadian Phocidseals. 97
Figure-+.6 Contents of saturated ISAT I. mcncunsuturated i~tL'"FAI.
polyunsaturatedIPL:FAI.omega3.and omega 6fatty acids inlung lipids ofdifferentspeciesofEastern CanadianPhoc id seals. 101 Figure -+.7 Contents of saturated (SAT). monounsaturated 1~1L'FA).
polyunsaturatedIPUFAl.omega 3.and omega6 fattyacidsin liver lipids ofdifferentspeciesofEasternCanadian Phocid seals. 103 Figurcu.S A plot of a principle component analysis of the fatty acid
composition ofselectedtissuesofbearded. grey. har bour. and
harp.hooded . and ringed seals. 106
Figure-1..9 A plotof aprinciple component analysisof the fatty acid compositionofneutral andpolarlipid fract ionsofmuscle .kidney.
heart. andliverofharpsea l. III
.~A .~C S BHT
CE CHD CL
DAG DHA D\IA DP.~
DPPC EP.~
FA\ IE FF.~
FID FFSC GC HDL HPLC LA LDL
\lAG
List of Abbreviations
-Arachidonic acid - AmericanChemicalSociety - Butylatedhydroxytoluene - Cerebroside - Coronaryheartdisease - Cardiolipin -Diacylglycerol - Docosahexaenoicacid -Dimethylacetal -Docosapentaenoicacid - Dipalmitoylphosphatidylcholine .Eicosupentaenoicacid .Fattyacidmethylester -Free fattyacid -Harne ionizationdetector - Flexiblefused silicacolumn - Gas chromatography -High-densitylipoproteins
-High performanceliquid chromatography -Linoleic acid
-Low-densitylipoproteins -Monoacylglycerol
\I L"FA
:-;\ IR
P_~
PC PCA
PE PG PI PS PL-FA
S\I TAG
TLC TLC-FID
\-LDL
•Monounsaturatedfattyacids - Nuclearmagneticresonance - Phosphatidic acid
-Phosphatidylcholine - Princ iple componentanalysis
-Phosphatidylethanolamine
· Phosphatidylglycerol
-Phosphatidylinositcl -Phc sphand ylserine
· Polyunsaturated fatty acids
- Sphingomyelin
· Tnacylglycero l - Thin layerchromatography
·Thinlayer chromatography-flame ionizationdetection -Very-lo w-densitylipoproteins
CHAPTER1 INTRODUCTION
There has been continued interest in marine oils ..ince the 1970s when epidemio logica lstudies proposedthat low incide ncesofheart diseasein Greenland E..krrnosand someJapanese popula tionswere due[0thei runiquediets(halwere richin oilyfis h. whale blubber.andseal blubbertBangand Dyerberg.1972:Stan sb y.1990bLIt i,believed that humansoriginallyconsumeda diet with aratioofw610tl>3fany acids
o r
about I:I.whereastoda y the ratioisest imatedto rangefrom10:1to~O·~ 5:Iinthe we..tern diet.Therefore .westerndiets are deficientin003 (any acidscompared withthe diet on which humans evolved and from which their genetic profiles established .Stmopoutos.19911.Mere rece ntly.intere st inseal blubber.spec ificallyas a sourceof003 rallYacidshasinten sified.
EJ.r1 y res earc'ton003 polyunsaturatedfatty acidsIPCFAI focusedon fish.AsJ
result. much of the literature has provided evide nceon the beneficialeffects of eicosaperuaenoic acid (EPA l and docosahexaenoic acid tDHAI whic h are the predo minant003 Pl'FAsinfishoilsl~ewton.1996).However.seal blubber 011isrich in Jocosape mae noic acid(OPAl as well asEPA andDHAIShah idiet al..19961. Existing literaturecontains little informatio nontheeffectsofDPA.
MuchoftheresearchsuggestthatthereductionincoronaryheartdiseaseICHD) bymarineoils is throughloweringofseru mtriacylglycerols. reductionof the occurrence of arrhythmia.and their abilityto actas amiathercgeni c andantithro mboticagents
rAbbeyaal..1~90 ).Marine oils ha ve also bee nstudiedfor their effectson inflammato ry andautoim mune disorde rs.stroke.skin disorder s.variouscancers. and thedevelopment ofrenna andbrain ISimopoulos.1997 3).
Besidesfan y acidcomposition.thespeci ficclassesof lipidspresentalsohave
important biochem icaland nutri tional implications. Forexample.cho leste rolis an
e....enualcomponentof cellular membrane s (G ibbo nseral..1982).howe ver.highle vels 1)(choleste rolin the dietmay havenegative he althimplicationsIShah idiand Synow iecki.
l"N II.Besideschole sterolthelipid classescommo nly foun din animaltissues include Irl;'h:ylgly..:erols. free fuuy acids. rnonoacylgl ycerols. diacylglycerols. phospholipi ds cphos phatid ic acid. phosphat idy lglycerol. cardiolipin. phosphatidylcholine.
phosphundyle tha nola min e.phosphutidyls e rine.phosphat idylinos itc h.andsphingolipids I-phmgo myelin and ce re brosid e)IG unsto neand Norri s,19831.
Previ ou s-;tudieshave compar edthefattyac idcompositionof theblubber of -everal-pecresofsea lsand others have comparedthefattyacid co mpo sitionof several tissuesfromthe same speciesofseal.Howe ver.the re hasbeennoprevi ous study to compare the fattyacid compositionof differenttissues fromallSIXspeciesofEastern Canadi an Phoc id seals.Conseq uen tly.the firstobjectivewasto co mpa re the fattyacid ccrnpos incnor different organs of all six Eastern Can adi an Phocid seal species simultaneously.Thehypot hes isisthatwhencomp aring thefanyacid com positionacross both ..peciesand tissues.the tissueunderconsiderationwillhave a larg erimpactthan the
"pe":le s underconsideratio n.
Sincemost studies havefocused onthe blubber of seals.which is predominan tly macylglycercl s(T AGsl .there islittleinfo rmationin thelite ratureon thelipidclassesof various tissues ofdifferentspeciesof seals.The re is onestudy on the lipidclass composition of a Med ite rranean Monk seal using high performance liquid chromatographyIH PLCI IHenders onetal..19941.Therefore.theobje ct iveofthisaspect ofthe-tudywastodeterminethe lipid classcompositionofselectedtissue softhe EasternCanadianPhocidseals.Itishypothe si zed tha tthelipidclasses willdepe nd more onthetissueunder conside rationratherthanspeci e s and as a resultwill alsobesimilar to the gene ral trends obse rved in the analysis of the co rres ponding tissues in the Medit erraneanMo nkse al.
Be sidesthe differenttissues ofseal. milkof thesemammalsis oftenof interestto
~~lenustsbec auseitisaiso vervunique.The mostextraordinaryfeature ofseal milkisits
\er~htghfatcontentc30to6O~1IO ftedaletat..1988: Ackman andLamonthe.1989:
h cr...onetut.,19921..\tany studiesindica te thatthefatty acidcompositio nofseal milkis -tmtlartoblubber excep t that the levels ofpalmitic ac id(16:01arehighe r10milk.Since the fattyacid compositionof seal milk hasbeen wellestablishedandit iscomp o sed of prcdc mm .nelv TAGs. further stud ies in these areas would be unproduc tive or ururneresnng. Ho wever.there haveonly been alimi ted numberofstudieson the positionaldistributio noffattyacids in theT.-\Gsofseal milkelve rso net01..1991:
Pup pionet'Cul..1991)andneith er of theseprovidedthe positio nal distrib utio noffatty ac ids inallthree positionsof theTAG backbon e.Therefore. thefinal object iveof this
"itud~was[0det ermin e the positi onaldistribut ionof the fattyacidsin ailthree positions
viTAGbackboneofharpsealmilk.Thehypothesisisthat thedistributio nwillbesimilar
[0that of harpseal blubber.Thatis.the C1>3PUFA willbepreferentiall y esterified to the HI-I and.m -3pos itions while thesaturatedfatty acidswill be prefe rentiall y este rifiedto
the.)"1l-!position.
CHAPTER 2 LITERATURE REVIEW 2.1. Seals
Sealsbelongtothe OrderPinniped ia whichisdividedinto two main divisio ns:the Otuno idea.·...tii c hincludesthefurseals.sealionsandwalruses.andthePhocoidea.
whichmcludesthe"true" seals IKing.1983).Thetrueseals.also known as the"hair"
-cats. arecharacterizedbynegligibleexternalears.hindnippersthatdono ttumforward.
andthe absenceof an underfur.The Otanidae.however.haveall thesefeatureswiththe walru..es. Odclaenidae. beingan offshootofOtariidae(Sergean t.19911.
2.1.1.PhocidSeals of Easlern Canada
Sixspeciesofphocid seals arefound inEasternCanada.They incl ude the bearded.gre y.harbo ur.harp.hooded.andringed seals.Grey andhar bou r sealswhelp on land whilethe remainingfourspecies whelp on ice rMalouf.19861.
2.1.1.1.TheBeardedSeal,Erignathus barbatus
The beardedsealhas acircumpolardistributionIFigure 2.11and is essentiallyan Arcnc andsubarcticsealof relativelyshallowwatersIBonner.199Ol.Beardedsealstend
(0 be:sedentary. but will undertake regular longdistancemigrations inresponse
Figure2.1.Distribution of Ihebeardedseal(Erignarhus barbarus).
AdaptedfromKing(1983).
Figure2.2.Distributionof thegreyseal(Halichoerusgrypus).
Adapted from King(1983)
10 movementsofIcefieldsID~I.\"ISaal..19801.Since theygenerallydo no tmaintain
breathmghole sin theicethey are dependen t uponopenwater.Bearded sealsarelarge
with bothsexesreaching a weightof approxi mately~50kg.Theyare greyish incolour
\\ nhJ-cane ring of smalldarksPOtSand aredarker ontheback. Bearded sealshavean abundance oflongconspicuouswhiskersfromwhichit derivesItSnameIBonner.1990).
The world populationof beardedsealsis estimatedto bebetween500.000and 1.000.000
IBonner.1990: King. 19831 and the sizeofthe Canadianstock is unknown. butismuch lowerthan the:nnged sealstocktMulouf.19861.
The beardedsealishunted bytheInuit foritspeltwhichis prized forbeingtough andflexible .The meat is eaten by bothhumansanddogs rShahidi.19981.
2.1.1.2 The GreySeal.Halichoerusgrypus
There arethreedistinctpopulati ons of greyseals:thenortheas t Atlantic.theBaltic andthenorthwest Atlanticas showninFigure~.~IBonner. I990). Grey sealsare also knownusAtlanticseal.horsehead.Phoque gris. orCowmoresealtBeck.1983a l.Grey ..eals normally inhabitroc ky shoresandin thenortheas t Atlant icthe y areconcentratedin theGulfof St.LawrencerKing.1983 1.Grey sealsarefairlylargewiththemale being considerablylargerthan thefemale.Adult males reach approximat ely..50 kg andfemales 270kg.The male'scoat is dark. grey incolourwithsilver.grey spots.Thecoat ofthe femaleissilver greywith small scattereddarkspotst~1ans field.1988).
Priorto1990.the world populationofgreysea lswas estimatedto be betwee n 120.000and135.000 tBonner. 1990).However. in 1993.therewere anestimated 144.000
gr~y-eals10AtlanticCanadaalone.TheAtlantic Canadianpopulationis increasingat about9(013CCperyeartShahidi.1998).
lnCanada.grey sealsareof concernbecause of theirimpactonCanadian commercialfis heries.Forexample.theydamage fi shinggear. competewithfi shermen forfish.and arethe primaryhost for aparasitethat mustbe removedfromtheflesh 01 .:01.1.codworm:andother groundfi shtMalouf.19861.
2.1.1.3The Harbour Seal,Phoca vitulina
TheHabourseal.alsoknownas the commonorspottedseal(God win.1990).has
J.circumpolardistributionsimilarto theBeardedseal butatalowerlatitude.in cold- temperateand temperate watersIFigure .2.31.Thesearesmall tomediu msizedseals with male: ...reaching120kg andfemales90kg" The:colour andpatternofthe:coatvaries greatly.butisbasicallya mottleofdark.sPOtSon a lightbackgroundIBonner.19901,
The:Harbourseal isashore livinganimalfound principally.but notexclusivel y.
10e-ruane...and in areaswheresand banksare uncoveredatlow tidelKing. 1983J.
Several subspecies of theHarbourseal have beennamedincludingP.vitutina vitutina.P.
vituiinafOIlcosor.P.vitulina mellonae.P.vitulinarichardsi.P.vitulina geronimensis.
andP.vitulina stejnegeri.The totalworld populationofHarbour seals isbetween 300.000and-100.000IBonner.19901.
Harbour sealsare nothuntedcommercially inAtlantic Canada.excepton the Quebec XorrhShore.Theyare. however.regarded asa nuisancebyinshore fishermen
Figure2.3.Distrib utionof tileharbourseal(Phocavitulinas.
AdaptedfromKing(\983).
A11.ANTIC OCEAN
Figure2.~.Distributionof the harp seal(Phoca groenlandicat . Adapted fromKing(1983).
III
dueto damage tofixedfishing gear.competition fortish.and as a 000-preferredhost for the codworm
2.1.1.4The Harp Seal. Phocagroen/andica
The: ha rpse al. alsoknownasthe Greenlandorsaddle backseal. is found in[he open sea ofthe:ArcticAtlantic (King.1983),As shown inFigure 2.~therearethree populati on...of harp sealsbreeding intheWestern Atlanti c.off Jan Muyen.andinthe
\\'hu c:SeaIBon ner.1990 1.
Harpsealsaremediumsizedse alsran ging from 100to130 kg.dependingonthe limeoftheyearrSeargent. 199 1).TheCOalofthe harpchanges withmaturation.The adultmale is light silvery grey with an irre gul ar horse shoe shaped band of black ..rraddling (hebac k.The black bandor"harp' ispaler andlesswell definedinthe adult female.Harp.;(:J Isarebornye llowish.quick lytum white.and pas sthroughanumberof 'coats"Jurin g maturation.Dueto varyingcommerc ialvalueforthediffere ntcoats.each one hasacommon name as giveninTable :!.I .
Harpsea lsarethethirdmostabundan t speci esofsea linthe world andhave bee n hunted forthe irskin and oilsince historica ltime s tShahid i.1998 ).Harpseals are still the
focus of the Canadiancommercialseal humandmuch efforthas beenplacedinto
accuratel y determ ining theirpopulation.Variousapproacheshavebeen usedtoestimate theNort hwestAtlan tic harp seal populationbased principall y on pup production . catch- at-age. and pre gnanc y rate data IShelto n er al.. 1992: 199 5). Several models
Table2.1.Common naffi(Susedfurharp sealsofdiffe rentages(Conu ncau.19H9 ) Commo nNallk: Description
WhileCoil.; A harpseal pup from31010 daysold, nanwdfor usdistmcnvelong,whilehair.
Overgang Aweanedharpsealpup duringIhe prep aratorysldgeofitsfirstmouh:long,whilehairisslill firmly attache d,bUIsilver-Gre y beatercoalhairshavebegun10appearalappruximalely12daysof age.
Tanner A weaned harp seal pup during mid-stage of ils firstmoult.while hair isIoose andcane be easily pulledour OilapprOll.irnalely 16daysofage.
Raggedjackel
Bealer
Bedlamer
OldHasp
A weenedharpseal pup duringthelaslsiegesof ill;firstmoult,from 2.5104week s old.
Afullymoultedharpsealpup(fOOl3.5 week!;.10Iyearold.Bearershave short -haired .dar~spo iled,silver-greycoats
An immalure harpseal(romI 10 5year s old wuhiIspoiled coal.The Ji!ol incli vcsaddle or Ilarp-shaped JIldrkingsofthe allull harpsealdevel opgradually.
A mature sealofover5yearsold wilhdistincti ve~Jdleor harp- shaped markings.
U~ this JJ.tJ. toestim atethepopulat ion:using estima tedpopulationsandestim ated replacernen tyields.thecomme rcialse al harvest can be managed effectivel y.According 10Stensonl199 51.thepopulationofharpseals intheNorthwestAtlan tic rangesfrom3.6 to-L3million .
2.1.1.5TheHoodedSeal.Cystcph cf a crista/a
The:Hoodedseal has a distri butio nsimi lar tothat oftheharpseal.butisless nume ro us(IFigu re.!.5 J:IBonne r.1990)).Hoodedseals are alsomigra tor yandbreedon
h~a\'yice:flows.butare notas gregariousas theharpsea l. Theyte ndtobescatteredinto
111..11 \idualfam ilygroupsof a male.fema le.and pupIMalouf.19861.
;"1:.11.:hooded sealsare large.weighi ng300· -1.00 kg.while:femalesaresmalle r at about180kg.Bothse xesarepalegre ywith large black.blotchesanddar ker headsand hindflippers.The world populationofhoodedsealsis estimated at250.000to~OO.000
IBenner. 1990t
2.1.1.6TheRingedSeal.Ptioce hispida
The ringedsealisround aroun dthewhole Arcti cand subarctic regionsand inthe BelueSea.as sho wnin Figure2.6IBonne r. 1990}.The ringedsea lis themost abundan t ..ca l in theArctic andisfoundwhereverthereis open waterin thefast ice.evenas faras theNorth Pole.andinfj ords and bays.They arerare lyroundintheopensea oron floa tingpack ice(King.1983).
Ringed se alsaresmall withmalesrangingfrom65 to 95kg andfemales from ..5
Figure2.5.Distributionof thehoodedseal(Cystopho racristata).
AdaptedfromKing (1983).
Figure 2.6. Distributionoftheringed seal(Phocahispida).
Adapted fromKing (1983).
to80 kg.Thepeltislight greywithblack spots.Thespots areoftensurroundedwith lighter ringmarkings(he nce the nameI.andon theback thespots becomeconfluent.
gil,ingtheappea ranceofadarkstripedow ntheback.The bellyisalighter..liverygre y .Bonne r.IQ901.
Ringe d se als are the mostabundantspeciesofseals inthe worldwiththe total populationestimatedat7million(King.1983:Malouf.1986:Bonne r. 1990:Shahidi.
199:h The..izeof the Canadian stockisunknown.butprobab lynumber overamillion individuals rMalouf.19861.
.Althoughringedseals are an impo rtantsourceoffoodand clothin gfortheInuit l~blouf.19861.they are nothuntedcommerciallyinCanada.
2.2The CanadianSealing Industry 2.2.1His to ryofthe'SealHunl'
Seals havebeenhuntedby humansforapproxi matel y-1-00.000yearsin Europe and ..10.000yea rsin :-'-cnhAmericaIBonner. 1990).However.mostpre-industrialsealing In the'worldwasforsubsistence and not forcommercialexploucuoo.Early in the -axtecnthcenturyBasque fishermencametoNewfoun dland to catchcod and discove red that the ycouldcatch harpsealsbysettingnetsinthe Strai t ofBe lle Isle.By theearly 18th centuryatrad itio nal sea ling indus try.usingnets.was well establis hedinNew foundlan d .Bowe n.1985:Bo nner.1990>.
The'first step toward s thedevelo pme nt of the offshore seal hunt wasthe participat ion inl79~of the firstwooded sailingshipstohuntseals(Bowen.1985).By 1800the New foundlandschooner fleetwas underconstruction.Seal ingincreased rapidly
IS
tobecom eseco ndonly tocodfishinginthe Newfoundl andeconom y.Bythesecond Ljuan er oftheeighteenthce ntury up to 300sc hooners with 12.000 menweretaking more than halfJ.millionseals annually(Bonner.1990 1.Harvestsof680 .000.7"0.000.and 6S6.000-eal,werereponedin183 1.1832.and18-t4. respectivelyIColman. 1937t
ln1863 anot herimportantdevelopmen tinthesealingindustryoccurred when
';1~J.mpowered ships wereusedfor the first time.ln 1906the firststeel-hulledshipwas
usedinthesealhunt(Bowen.l 9851.
ln193 8thelargeNorv..egiansealingshipsbegantohuntthe NorthwestAtlantic ,c;lb .Bcwen. 19851resulting inthedemise oftheNewfoundland offshoresealing
vc we l... However.the Norwegians establishedcompan ies inCanada to operatethe
..calingenterprises(Bonner. 19901.
Sea llandings be tween 1863 and 189~averaged341.000 anddecl inedto an average
or
249.000 betwee n1895and1911.and159.000between1912and 1940.There was..liule scaling conducted during thewar.butfrom1949to 196 1 anaverageof310.000 -eals wereharvested.Aquota managementsystemwas intro ducedin1971andthe harp 'cal harvestfrom 1971to1981averaged172 .000IBowen .19851.Throughout the history ofseal ing small vesse lscontinued to hunt seals.
Fishe rmenwhohunted se als fromsmall boats1<65feeuwere know nas"landsmen'.
From 19.+9to 1982 the 'lands men' huntrepresented 15-..l5%of[he total landings (Bowe n.198 51.
Bythe1980s.[heefforts of anti-sea linggroups began[0have[heireffectsonthe sealingindustry.In1983 theimportationof pelts of harp and hood seal pupsinto
16
countries ofthe EuropeanEconom icCom munity(EEe)wasbanned.This destroyed markets forsealskinsresulting in animmediatereduc tioninthe sealhunt,[01987. the Canadiangovernmentofficially prohibited theharvesting ofsealpups.Jefinedasthose ..cabthaihadnotstartedthei rfirstmoultri.e.whitecoatsandbluebackst.rBonner.1990 1.
Theuseoflargevessels 165feetorlarger)forharvestingsealswasalso banned.
The basictechnologyofsealing wassimple.Adultseals werekilledbyshooting with high-powered riflesandpupswerekilledbyclubbing.Canadiansealers usedJ wood en club.shapedlike a baseballbat.but Norwegiansealers used aninstrument witha heavyironhook onalonghandle.called"hakapik",
Initiallytheproductsofthesealingindustry wereoilandsomeleather.However. inthe late 19"0sthe Norwegiansdevelopedpreservationand tanning methods that allowed peltstobeusedinthefurindustry.Pelts forfursoonbecame themainproductof the ..ealhunt.Carcass eswereleftonthe ice torthe most pan.Landsmendidtake..orne flipper..tor personal consumption and theywere marketedjustoutsidetheimmediate sealing.areas(Bonner.199 0 ).
2.2.2The Modern Seal Hunt
After[hecollapseofthemarketsforseal peltsin1982andsubsequent banningof large '..esselsand huntingsealpups.asmallsealindustrysurvived.It hasbecome and remainsan'inshore'fishery pursuedby'landsmen'orby thoseusing"mall«65 feet) fi shingvesselstShahidi.1998).
From1982 to1995the quotaortotalallowablecatch(TAC)remainedat186.000.
butonlyanaverageofabout 50.000 seals peryear were takenduring this period
17
{S tenson. 199.+1. as ares ultof limited markets.In1996.the quo ta wasraisedto 150.000 and forthefirsttimesince1982asubstantialportionofthe quota was harvested.A furtherincrease!O17; .000 occurredin1997which was nearlycompletely harvested IDFO.19971.
The technology of the industry is relative ly unchanged.howeve r.thefina l prod ucts arcmore varied.The policy ofthe modemsealind us tryisfullutiliza tio n.Pe lts In:culloneofthe mostimportantcomponentsand are primarilyused forthe furrier trade.The blubbe r isleft attachedto the peltanditused for ind ustrialoilproducts.but greaterpercentages are being con..-erted to pharmaceutical/n utraceuricalproductseach ye ar.The meatof thesealisalsoused.Rippersaresold as adel icac yin localmar kets ..1Od theremainderofthecarcass usedas humanfood orasanimal feed.Some organsarc currentlymarketedand effortsarebeingplacedintofindingopportunities for someof the:
internalorgans.
2.3Marinelipids and their significance 2.3.1 Chemistry and compositionof marine lipids
The term lipid isusedtodescribe a widerange ofnaturalproducts.However.a ..pecificdefinitionusedfrequentlyis onethatrestricts it to fatlyacidsandtheirnaturally- occurringderivativesandtocompoundscloselyrelated biosynthericall y10fatty acids lChristie.1981:Gunstc ne and Norris.1983).
IS
2.3.1.1Fatty acids- Background
Fattyacidsaregenerallyaliphaticrnonocarbox ylicacids.Trivial nameshave been green tomany ofthe morecommonfattyacids.butsystematicnames.based on the Inremaucnal Union of PureandApplied Chemists(lUPAC)systemofnomenclature.
ind icatethe chain lengthof theacid.the position.nature,and configuratio nof any unsaturated bonds.and the positionand nature ofsubstituents. The [l"PAC system numbersdouble bonds withrelation to their positionfrom the carboxyl end group.
However.anothercommonsystem.accountingfor biologicalactivity of molecules.
numbersthe doublebondsfromtheirmethylendgroup.Since mostnaturalfattyacids
contuming multiplebondsaremethylene.interrupted.itis onlynece ssary todesignate the fir~tdouble bondIGunstoneandSarris.1983 I.
One group ofcommon fatty acidsarefattyacids whichare straight-chained .even- numberedacidscontaining 12·22carbonatoms IGunstone.1994).Anothercommon group of fatty acidsarethemcnoencic acidsof whichmore thanone hundredhavebeen rdcnttfied.although most areveryrare.The9·cisisomer(palrrutcle ic I is themost widely occurrtnghexadecenoic acid. The9·cisisomer(oleicIoctadecenoic acidisthemost
\\"Idely distributedand amongthe mostextensively produced of all fattyacidstG unstone.
199.1.
Thethirdcommongroupoffatty acids are polyenoicacids havingtwo to sixcis double bonds arrangedina methylene-interruptedpattern.Theyareusually dividedinto families dependingon thedistanceofthe nearest doublebondtothemethylendgroup.
Thethree mainfamiliesareCJi).t:OO.and003.Someofthe morecommonimportant
19
methylene-interru pted polyenic acids arelinoleicIlg:~0061.a-linolenic (18:3 003,.
arachido nic(20:-.1-006), eicosapernacnoic(20:5003).docosapentaenotc (22:5003).and dccocahexae noic 122:6wJlacidsIGunstone andNorris,1983),
Ther e arc also someless commongroups of fatty acids.Branched fauyacids with J methylgroupon the penulti matelisaacids!orantepen ultimateumtesio acids)are -ornen me..found naturallyinsmallquantities.Acids containinga cyclopropaneor cyclopropcne groupandseveraloxygenatedacids suchas ricino leic acid alsoexist .Gun..tone and Norris.1983).
Structures of some of the common saturated. monoun satur ated. and polyun saturatedrauyacids areprovidedinFigure 2.7.
2.3.1.2FattyAcidsof MarineMammals
The compos itionofvirtually allmarineoils can be describedby8 totomajor i.m y acids.ignoringisomers.Numerousminor acidsare alsopresent.Awidevariationis obve rvedwithinidentical tissuesofthe samespecies.Thiscan be due[0bothseasonal variationsand geo graphicallocation.butthe mostimportantfactorisdietaryintake IPadleyettil.. 1994).
The (an y acidcompo sit ion ofmarinemammali anmuscletissue.blubber.and milkhasbeenthesubjectofmany investigat ions (AckmanandBurgher.1963:Jangaard and Ke. 1968:Ackmanetal..1911:1912:Ackman andHoope r.1974:Engelhart and Walker. 197..kWestetal.. 1919:AckmanandLamonthe.1989:Grompone eral..(990:
~OH o
Stearicacid Octadecanoicacid
~OH
Oleicacid
o
so-octadecen oicacid
Arachidonicacid 5e,8e,11e,l 4c -eieosatetraenoie aeid
OH
, 0
EPA
5c.8c.11c,14c.17c-eicosapentaen oicacid OH
DPA
7e,10e,13e,16e,1ge-doeosapentaenoicacid OH
o
DHA
4e,7e,1Oc,tac.tsc.tsc-coeosanexaenccacid Figure2.7.Structures ofsome commonfattyacids.
OH
Kakela and Hyvarinen.1993:Kakelaetal..1993:Shahidiet ul..199~:Hendersoncr <.II..
19q~:Grahl- NielsonandMjaavatten.1995:Fredheirnetill..1995:Wanasundaraand Shchid i.199i\
There aresevera l uniquefeaturesofmarineoils!.hatdifferentiate them frommost vegetable andlandbasedar umulIipids.Mostnotably.thelevel ofpolyunsaturatedfany acidslPL'FAlInmarineoilsishighandis mainlyfromthosewith5 or6doublebonds.
\10<; (ofthesefattyacidsarclong-chainIZOtoZ~carbonatoms}andof theomega-S famil y.
\tostofthePl'FAinmarineoilsare fann edinunicellularphytoplanktonand multicellul ar marinealgaethatget passed throughthefood chamandeventually become incorporatedintothe bodiesof fishandmarine mammalsiYongmannc haiand Ward.
IQS91.Thehighlevelsor omega-J fatty acidsin marine lipidsarebelieved tobea result
1.)1coldtemperature adaptation.becauseatlo w temperatures omega-J PCFA sremam hquid andrc-isr crystallizationIAckmanandLarnonthe.1989t
Thefatty acidcompositionofsea ls isdependentuponthespeciesandnssue analyzed.Inthe blubberof harpsealI~:O.16:0,16:1. 18:J.18:2.20:I.20:5. 2.2:l.22:5.
ami 22:6 are theprincipa l fattyacid stIangaardandKe.1968:Ackmanttlal.. 197 1:
ShahidietIll..199~:WanasundaraandShahidi.1997}.The predom inant fattyacidsof (he ringed and harbourscatblubbersare thesame as those forharp.except18:2.whilethe main fany acidsforgreysealblubberinclude l-t.t rAckmanand Hooper.197~:Westttl ,Ii..19791.The principalfan y acids of bearded sealblubberare alsosimilar to harp seal with the additionof 18:0.Inhoodedsealblubber 22:5isnot one of thepredominantfatty
acrdstIangaardandK~ .19681.The reasonsfer these imerspeciesdiffe rencesintau! acid compo sitionremainelusi ve.Fatty acidcompositi onof sea l blubberalsodepe ndsonthe age of rhe anima lrGrahl- NielsonandMjaavauen. 19951.
Lnlikethemilk of cows and goats .whic hha...'eISto23':'"of the irfattyacids with ..:hJIOle ngth ofshort e r thanI~carbon atoms . milk of marinemammals containsonly traceamounts.O.6C"'c) oftheshort er chain1< 1~Catorru fau yacids tAshwon het,d..
19661.The concentrationof falinthemilkofmarinemammalsishigh130 to60ccIand trI;lC~lgfycercls are always the mainclass oflipidsIOftedalaul..1988;Ac kman and Lamcruhc. 1989:ive rsonetat..199 2l.The fattyacidcompositionofsealmilk lipids rese mbles thatof the blub ber lipidsexceptthat milk lipidsha ve ahigher contentof 16:0 thanthe blub be r lipids rIangaard and Ke.1968l.
The main fatty acidsinharp sea l muscles are 14:0 . 16:0.18:0.16:1.18:1.~O:l.
~~:I. 1~:2_ ~O:5. ~1:5. end ~:!:6. Muscle lipi ds are riche rin both saturated and pol~unsaturatedfattyac ids than blubber lipids.butblubber isricher in moncuns taurated fJ uyacidsIShahidiand Synoweicki .1991:Shahidietat..19941.
Since marinemamma lse vol ved onlandand late r became purel y aquaticthefatty acid. compos itionof the internalorgans is similar to tharof terrestrialanimalsIAckman and Lamonthe.1989).The principlefauy acidsof sea l heart.liver.and lung tissues are I~:O.16:0.18:0.16:I.18:I.20 :I.18:2.20:~.20:5.22:5.and22:6 'Ackm an<Ial..197:':
Adman and Hooper.1974:Engelhart and Walker.197~).Severalinvestigation shave re porteddiffer encesin the fattyacid compo sitionbetween differentinte rnalorgansof the sa meseal. Ackmanand Hoopern97~)repone ddifferencesbetwee nheart.liver. and
blubbe r rauy acid composit ionsfrombothharbourand grey~akEnge lhartandWalker
IIqi~1demon strateddiffere ncesin the fatt y acidcom pos itionbetweenbrain.liver.aorta, muscle.and (estesofharpseal.
Braintissuehas averyuniquelipidcomposition.Brainlipids containless than1'(
mac vlgfvce rols and morethaniOC(polarlipids (Hendersonetal..1994 1.Themain fatly J~IJ"ofbra inlipidsare 16:0. 18:0. 14:1.16:1.18:1. 20:4.and 22:6-Engelhanand Walker.1074:Hendersonaal..19941.
2.3.1.3Acylglycerols
Themostcommon lipidsare acylesters or glycerolknownasmonoac ylglcero1s.
Jiacy l g l y,,~rols.andtricaylgly cerots.MonoacylglycerclsmaybeI-J,cyIIU -1or2-I~· I ac ylIsome r..IGunstc neandxoms.1983).
o
H,C-O-C- R -I HO-CH
H, C-OH 1-acylglycerol
o
H,C -OH" 1 R-C-O-CH
H,C-OH 2- acylgl ycerol
Dicey{glycerolsmaybea~11.2- and 2.3-diacylgfycerolstoran'11.3-diacylglycero l).
o "
o H '9 -
0 - C-R'W---C-O -CH H,C-OH 1.2-diacy lglycerol
o
HC-O-C- R'
'I
HO-CH 0H,C-O- C-R"
1,3-diacylglycerol
vlono-and diacyl gfycerolsare rare lypresentInmore thantraceamountsin treshanimal th~ue"I Christie.198~).
Tnacvlglycercls,themajorcomponentofnaturaloilsandfats.are composedof a glycerol molecule esterifiedwiththree fatty acid s:
o "
o H '9-
0 - C-R'R''-C-O-CH 0
H,c- O-C-R'"
1.2.3-Iriacylglyce rol 5".1 5".2 5".3
where R' .R". and R' " desi gnatethe hydrocarbonchains of fan y acids. Xatura l rn ac ylgtvce rotsseldomcontain threeide nticalacyl groups:mor e likelythe y containtwo crthree different acylgroupsselec tedfrom allthosepresent inthe ..ource material.The numberof possibletriac ylglyce rolsrisesquickly withthenumberof available acyl groups. Assuming that30 different acyl gro upsarepresentin marine oils. atota lof 27.000 isom erscould beformed. Evenifoptical andposition alisomerization were omitted. about 5.000differenttriacylglycerolmolecules couldbederivedtheoretically.
Suc h complexityalongwithsimilarityinphysicalandchemicalcharacteristics makes ..malvsesof mari ne oils fortheirrriac ylglycer olanalysis time-c onsum ing.extremely difficult andacomplete analysisisvirtuallyimpo ssible(Holme r.1989).
Hydr olysis of acylglycerolsbyacidoralkali givesglycero land a mixture offany acids.Enzy ma tic hydro lysis ismore specific:pancreatic lipase.forexample. hydrolyzes theacyl groupsattached to thetwoprimary hydrox ylgroups(sn - Iand.fn-3)andleaves the~-monoacylglycero lsin [heintact formtGunstone andNorris.1983).
Tnacylgf ycerols maybecharacterizedina numbe rof ways.The fattyacidsin
tri.rcylglyce rols may be identified and quantified. Tn ac ylglvcer cl s ma~ also be
characterizedonthebasis orthenumberofdouble bonds oracco rdingto carbon number.
Po..rticnul distributionoffatty acidson [he glyce rol backbone isanother meansof character izm gtriac ylglyc erol s.
Broc kerorf(1til.ll968 lreported thatin harbour sea l blubber thetwo a postuons.
~"p~l.:ially.m -3.wereoccupiedbythe polye nc icfa ttyacids20:5w3and22:6Ctl3.TheIn-
.:po-ano ni~esten fledwith highproport ion s of sat urated acids and C111-and Cw monocnoicratt~ acids.Thelonger mon oenesare foundin thesn- lposit ion.Similar find ing..han: beenreportediorharpsea l tBroc ker ho ff. 196 5:Wanasundara and Shahidi.
IlNi,.
The most comm onmethod ofstereos pecificana lys isof triacylglycerols uses a Gn gnard reaction. phospholi pase A:.and panc rea tic lipase [0elucidatethe positio nal JI..tn buno nofthe fattyacidsIBroc ke rho fft!ltil..1968:Wanasundaraand Shah idi.1997\.
However.seceralrecentpublicationshavereportedsimilarresultsusinghigh-re so lution
~:Cnuclearmagneticresonancespectrosco pyIe.g.Aursandet£II..19951.
Severalstudie son posit ionaldistributionof fatty acidsinse alrmlkindic atethat thePtFAarepreferentiallyesteri fied attheapositionof the triacyt glycero t molecul es.
-arrularto [hatofthedepo tfatsrlve rso net01.•1992:Puppionettl01.•1992),Existing literaturedoes not indicatefattyaciddist ributionoverallthreepositionsIi.e.. In-I.In-2.
and$11-31of[hemilktriacylglycerols.
:0
2.3.1.4Sterols
Mostlipidsamplescontainsome: freesterol. In plants.stigmasterol.~-sltosteroL campestercl. and ergosterolare themainsterols...hilein animals iti, predominantly cholesterol.A fractionof the sterol maybe esterifiedwitha fattyacidrGunsroneand 'corn s.198.31.ShahidiandSynowiecki 119911 reported that the cholesterol contentof harp seal meat wassimilar[0meatsfromotheranimals.However.thecholesterol content IShigherinorgan tissues ascomparedtocorresponding muscle tissues.Hendersonettil.
I 1')1.)-1-1reported the cholesterolcontentIq. totallipid} ofMediterranean monkleal blubber.muscle.liver.heart.end brain10be 1.2.9.i .12.3.12.8.and:!3.5.respectively.
In tact,choles terolwasreportedtobethe mastabundantneutrallipidinbraintissues.
2.3.1.5Phospholipids
The term phospholipid deno tesany lipidcontainingphosphe neacida...;J.mono- or diester and Includes (he glycerophcsp holipids and thesphingolipid. -pbingomyehn
IChristie.19821.They occurwidelythroughouttheanimalandplantkingdom s.being particularlyassociated with biologica l membrane s which provide a physicalburner -eparatmgcellsandsubcellular organelles fromtheirenvironmentand areintimately Involved inessential life processes. They facilitate and control the transport of metabolites betweentheenvironmentstheyseparate.and are Involved in many cell functionsIG unstone and Norri s. 1983).
Glycer ophosphcli pids are themost widespreadandabundan tgroup ofmembrane lipids.They aresimilarinthattheyare a diacylglycerol withaphosp hategroupesterified
tothe primary hydro,y!group of thesn-gl ycero!structu re.Withthe exceptionof phos phatidicac id.thephosphateforms a phosphcdiesrerbondwith the hydroxylgroupof a polar headgrcup substituent. The most commo n glycerophospho lipids are pho-phaudylchc fine(PCl. phosphatidylethanolami ne (PEl. phosphandylserine IPS).
pho-phandylinositcl IPlI. phosphatidylglycerol jPGI. diphcsphatidylg lycerol or cardioli pintC ll.and phosphatid icacidIPA l.tChris tie.1982:Mate.199031.
Within eachclassofphosphol ipid the reis a lar ge variation inthefanyacid cornpcsm on.In the majority of mammalianmembranelipidsunsaturated fatty acids are found inposition~ortheglycerolbackboneandsaturated fanyacidsinpositionl rMato.
1990aJ.
Phosphand ylchoiine is the mostabundant glyceroph ospho lipid inanimalsand higherplants,Prasad. 1996. Vasko\'sky.1989 l. Position m-l in PCof animals is
o
CHrO-C-R'
7! I
R''-C-O -CH 0 CH3
H,C-O-~-O-C-C-~=-CH3
O· H,H, CH 3 phosphal idylchol ine(PC)
almos t alwaysoccupiedlargely bysaturated fattyacids. whilepositionsn-2contains most or the Cili' C:o. and C:: polyu nsaturat ed fatty acids IChristie. 19821.
Lyvophc sphar idylcholineIl PC).where onlyone of the twoavailablehydroxylgroupsis esterifiedto afatty acid. isoftenfound whenPCis present(Pras ad.1996).Itis generally acce pted that in most instances position m-I is esterifiedto afattyacid{Christe. 19821.
Pho-phatidylethanolarrune •PEI is gene rally tile second most abundan t glyccrcph c spholipidin animalsand plants IPrasad.19961. Itscontentusuallyranges
o o
CH2- O-C-R'" I
R''---C-O-CH 0
, "
H2C-O- ~-O-C-C-N H,
O. H,H, phosphatidylethanolamine(PEl
(rom~oto~5C"'("IJfthetotalamountofphospholipidsinmanne animalsrYas kovsky.
19S91.Pho-p hundylethanclarninesin animalsusuallycontainmorePl'FAs thanthepes from the-umetissue.and thePCFAsare concen tratedat thesn·~position.Chnsue.
Phc ..phandyberine•PSiisa commoncomponentof marineanimalsrVuskovskv.
IYS91.ItISweaklyacidicand isusuallyisolatedasthepotassiumsalt.butma~alsobe
.l ..soctated withcalcium.sodium. ormagnesium ionsIChristie .1981 1.Phospbatidyl-
o
CH,O -C- R'
~
IR''---C-O- yH ~ ~H, H,C-O-~-O-~2-~-~-OH
O· 0
phosphatidylserine(PS)
-en ne has asimilarchromatographic behaviourtoPEandsomeauthors presenttogether thedata forbothof the lipidclasses.
Phospnatidylinositolcontains aheadgroupof six.carboncyclic sugaralcohol.
Inositol.and isacommonglycer opho spholipidofanimaland plantcells IPrasad.
o
CH2- 0 -C-R'
?,
I
R"- C-0 -~H ?, OH OH
H2C-0 -~ -0~H OH
O·
phosphatidyl-1-myo-lnosltot(PI)
19961.Phosphatid ylinosuolisstro ngly acidicandis usuallyisolate dinassociationwith magnesium orcalcium ions IChristie. \9821.Phosphand ylinosncl may be further pho..phorylated to gtve polyphosphoinositides such as diphospharidylinositol and tnphc vphandylinc sitoltvuskcvs ky.1989 1.
Phosp hutid ylgfycerol,PG l containsasecond moleculeofglyceroleste rifiedto phocphonc acid. Phosphand ylgl ycerolis normally present In sma ll quantitie s 10
CH~O -C-R' o
~ I
"R - C-O - CH 0
I "
H2C-0-~-0-yH2 OH CH-OH
H2CI-OH
3-sn-phosphatidyl- l'-sn-glycerol(PG)
ammaltissuesandis a minorglyceropho spho lipid in marinemammalsrvaskovsky. 1989:Prasad.1996 1.However.itis possiblethat PGismoreWidespread than hasbeen acceptedbecause most of the commonsolvent systemsforthinlaye rChro matography ITLCIorgtycerophospholipids donotresolvethemixtureofPGandPEtv askov skyand Tcrekhova.19i91.
}O
Dipho..pharidyl gfycerol.whichis commonly knownascardiolipin ICll.is .1 major glycerophospholip idof mitochondrialmembran elipids.especiallyof heartmuscle.
o "
CH2- O - C- R'
~ I
R" -C- O- CH 0
I "
H2C -0-~-0 -y H 2 O' CH-OHO
I "
H2C-0- ~- 0- yH2 ~ OH C~-0-C-R'"
R-<;i - 0 -CH 2
o
1'.3'-di-0 -(3-sn-phosphatidyl)-sn-glycerol,orcardiolipin(Ct.)
It10;,madeup ofaphosphatidyl gfycerolmolecule.whose 3' -hydroxylgroupatthesecond gfycerolmoietyis esterified to thephosphategroupof a secondphosphatidic acrd
rPraxad. 19961.The occurrence and properties of cardiolipin have been reviewed
rloannou and Golding .1979 ..
Phosphatidic acrd ,PAl is awidely distributed minor glycerophosphol ipid
IVuskovvky.l 9891.Althoughitisaminor gtyceropbos pholipidit is extremelyimport ant biovynthcucall ybeca useitis the precur sor toallother glycerophospholipids andof tnucylgfycerols tChristie. 1982 ).Phosphatidic acid is stronglyacidicand isoftenisolated
;ISmixedsalts.Similar to mostglycero phospholipidsin animal tissues.positionsn-l is occupied predominately by satura ted andsomemonoenoicfattyacids.whileposition .m-2usuallycontains pol yunsaturatedfatty acids.
o
CH,-O-C- R'
71
IR"-C-O-CH 0
, "
H,C-O -~-OH
O' 1.2-diacylglycerol-3-phosphate
phosphatidic acid (PAl
2.3.'.6Ether Lipids
Ether lipidsarevariations ofstruc tures previo usl ydiscussed.Inthese compounds the: acyl groupattac hedto the5n-lis replacedwithanalkyl or alkenylgrou p.There are tourtypes ofthesecompoundsdepending onwhether the etherlinkisintroducedintoa tna cylgf yccro l or aphosphatidy lesterIFigure.:!.81.Thealkyland alken- t-yl groups have the:..arne:chainlengt hsas thecommon fatty acidsIGunstoneand 'corn s.198.31.
The alken-J-yl ethe rphospholpids arc commonlycalled plasmaloge ns.They frequentl y have ethanolam ineorchol ine as their head groups I~lato. 1990bl.The plusmalo gens arecommon inmany tissuesbut are found at highle velsInvarioustissues ofmari ne mamma lsIHendersonetal.. !994 l.
Coder acidic transmethylationdimeth ylacetalsIDMA)areproduc edfromthe1- alk-l'-en-L' -yllinkedether chains.
2.3.1.7Sphingolipids
In contrast to all theesterlipidtype spreviouslydiscussed. sphingolip id,contain fau~ac idscombinedasamidesof long -chamcompoundscontai ningan amino and two
Ci/ yH,-O-~=~-R R''-C -O-CH 0
H,C-O-P-O-C-C-NH
cr
H,H, aAlk·l-enyl-ether phospholipidor Plasmal ogen
CH,- O- C- R'
Ci/
I
H,R''-C-0-9H Ci/
9
H3H,C-0-~-0-C-C-~=-CH3 0- H,H,CH3 Alkyl-etherphospholipid
o
H,C- 0-C=C-R'" I H H
R" -C-O-CH 0
I "
H,C -0-C-R'"
Alkenyldiacylglycerol (Neutral plasmalogens)
H2C -0-C-R'
~ I
H2R''--C-O-CH 0 H,G-O-C-R'"
Alkyldiac ylgl ycerol
Figure2.8.Fourtypes of ether lipids.
ormore hydroxylgroupsIGunstoneand~orris .1983).Sphingoli pidsinclude long- chain amines.ce rebrosides.gangtiosides.andsphingornyelins rFigure~.9J.
Cerumidesare amides of fatty acidswithlong-ch ain di- ortrihydro xyeases.
ccmairungtwelve[0twent y-two carbonatomsinthealipha tic chainrC hrisne. 198 ~1.In
"ornesphingolipidsthe ceramide islinke dwithJ.sugar moiety thro ughits primary hydro xyl group. The sugar may be simple rglucc seor galactoseI orcomple x.
Monogtycovylceramide s arekno wnascere brcsideswhile themorecomplex cerumides arc known as gunglic sides.The namecerebrosideisderivedfrombra inwhereitwas first isolated.However.theyaremmorcompo nentsofmostanimaltissuesand havealsobeen found m plants.Theassociatedfatty ac idsare mainlyalkanoic and 1-0 -hydro.'(yalka noic _'-'11.1:-.Gun-toneand Norris.19831.
In -pnm gcmvelin. position I of the ceramide unit b esterifi ed to pho- phc rylc hcline or phosphorylethanolamine IGunstone and 'corns. 19831.
Sphingomye linis foundas amajorcompo nentof thecomple x lipidsinnearlyall animal (ISsues,C hnstie.1981).Sphingo myelinsdiffe r inthe natureof thesphingosinebaseand tn theacylgroup.Themost commonsphingosine is the tx-carbon aminediol . 1.3·
dihvdroxy- j-umino-r-octadecene {~lato. 1990al. The most common funy acidsIn sphingomyelin are palm itic lI 6:0) and nervonyl Il ot: \. 1~:0. and ~ot:O ) acids
.Svennerholm er
zn..
1966).H H CH, (CH, ),CH =C-C- C-C- OH
H OHNHH,
I
COR ceramide
H H
9
p~CH,(CH,),CH =C- C- C- C-O-P - O-C- C- N:"' CH, H OHNHH, OH H,H, CH,
I
COR
ceramide phosphorylcholine(a sphingomyelin)
H, C- OH CH,(CH,),CH=C-9-9- C-O
,oo
OHH OHNH H, 0
I
COR OH
galactosylceramide(acerebroside)
Figure2.9.Structuresofsomecommonsphingolipids
35
2.3.2Marine lipi dsin human nutrit ionand diseasepreventio n
Recently.therehasbeenconsiderable publicityabo utthevalue ofmarineoilsin the diet;IS a meansofminimizingcertain diseases.especiallythose ofthe heart.
However. research inthisarea hasbeenongoin gforseveraldecades.Probabl y thefirst reported use ofmarine oilsinthetreatmen t ofdiseaserelates totheuse of cod-liveroil10 thetreatmentof arthritisatthe ManchesterInfirmaryinEngland.This studywas later re portedintheLondonMedicalJournal(Percival.17831. Inanother stud y.investig ators
InXorwayrep ortedthat the rateof heartdisease fell drasticallydunn g the SecondWorld War.whenthe availabilityofmeatdecreased andtheconsumptionof fishincrease d
IStram.1948:Stram andJensen .1951),Two long-termstudies werelaterconductedto mves ug aretheeffect s of marine oilconsumptiononheartdiseuse.A 19 ye arstudyby AvcrfyNelsontNelson.1972)inthe United States anda 20 yearstudyIn Hollandby Kromhout- ZutphenrKromhout etal..1985)showedthat therisk offatalheartattacks decreased withincreasedconsumptionof fish (Stansby.1990a ).
Interestin marineoils intensified inthe1970swhen epidemiologicalstudies proposedthatthelowincidenceofcoronaryheartdiseaseICHDlinEskimosmight be rel ated totheirunique diet.Greenland Eskimos wereconsuminglargeamount s ofoily fish as well as whaleandseal blubber intheirdiet(>lOOgi day).(Pique.19861.The purposeof theepidemiological studiesin the1970swas topinpoint thepossibleadverse effectsofahighfat.highprotein.lowfibre.and lowvitaminCdietonthe Eskimo's health. Surprisin gly. theresearchersfoundthatEskimosweregenerallyhealthyand remar kablyfree fromcardiovascular disease(Bangela!..1976 \.ln a prev ious stud y..
30
these rese arche rs found that the Greenland Eskimos had signif icantly lo wer concentrations of serum choles terol. rriac ylglycercls. very-low-densuy lipoproteins
tVLOllandlow-densitylipoproteins(LOllandahigherconcentra tion of high-densuy
lipoprotei nsIHDll thantheirDan ishcounterparts IBang andDyerberg, 197.21- Another situationsimilarto thatoftheGreenlandEskimo dietandits effect on hea rtdisease\V<lSnotedinJapan.Resear c hersfoundthatfamilies of fishermen living alongthe coast.whoatemuch morefishthanother Japanese families. demonstrateda decreased incidenceof CHD(Stansby. 1990bl.Areview of some of the Japanese investigationshasbeen compiledbyHirai t!ral.s1987l.
Theeffectof marine oils on CHDhas been extensively studiedtBurretal., 1989:
vtorns('IIll..1995:Delorgen letul.,199 6:Davidsonetul ..1997:Mizutamet al..1997:
Menetell.. 1997:Pieunen et al.1997 ).andmanyrevie...spublishedrSruckner.199:!:
Newton. 1996:Xcrd oy.1996:Simopou los.1997a.b l.However.thespecifi c mechanism oftheactionofmarinelipids in prevention ofCHDis stillunclear.Muchoftheresearch ..uggeststhatthe reduction inCHDbymarine oils is through lowering ofserum triacvlglycerols. reducin g the occurrence of arrhythmia and their abili ty to act as antiatherogenic andantithro mboticagentslAbbeyeral..1990).
Manyphysiolog icand pathoph ysiologicreactionssuchasvascularresistance.
thrombosis. wound healing. and inflammation are affected by eicosancids. The eicos anoi dsinclude prostaglandi ns.prostacyc lins.thrornboxanes. le ukotn enes.lipoxms.
andhydrox y fatty acids (We bereral.•1986),The activityof the eicosanoidsdependson
]7
minorstructural differences andoftentwodifferenttypesofprostaglandins canh3 \'C opposingeffectsonthe body(Pique. 1986).
Linoleic acidILAIisthe predominantPVFA in theWestern dietand isthe precursorofarachidonicacidIAAI.Arachidonicacidisintum theprecu rsorofthe prustanoids(prostaglandins andthrcmbcxaneslofthe .!seriesandofleukomenesofthe -l..ertes.Eicosapentaenoic acid and DHA.common PL'FAs of marineoils.are precu rsors oftheprcstanoidsof the 3 seriesand leukotriencs ofthe5 seriesr'ceecle rnent!la!..1979:
\\'t!ha etu!., !1.}86:Simopoulos.1990:Hwang.199::n .Adiagramof theformationof these differenteiccsunoidsis showninFigure~.I O.
TheeicosanoidsproducedfromEPA have aspectrum ofbiologicalactivitythatis moredesirablethan thatofthe eicosanoidsderivedfromAA.Thrombovane,-\:den..ed from ,-\,.\ISprcaggregatoryandvcsoconsm crorvwhereasthrcmboxaneA,derivedfrom EPAISnot prcaggregarcrvandisonly weakly vasconstrictcry.Prostaglandin I:derived from ,-\.-\ and prostaglandin b derived from EPA are both antiaggregatcry and vasodilarory.LeukotneneBJ fromAAisstronglychemotactic10contrasttoleukotrtene tromEPA which isonlyweaklychemotactic(WeberftCal..19861.
Byfarthe greatestamountofresearch has beenconducted ontherelationship hetweenmarine oils andheart disease.However.therehasbeen.andcontinuesto be.J great dealofeffortplacedondetermining t!"leeffectofmarineoilsonotherdiseases.
Manystudies indicate thatomega-JPL'F Asmayhavesometherapeuticbenefits byreducinginflammatoryandautoimmunedisorders(S imopoulos.1991:Boissonneault
Figure 2.10.Biosynthesisof eicosanoidsfrom arachidonic(20:~0:01and eicosapen tae noic120:5 (03) acids.
. j •
o I I
r-
0'
; - ' { ~o'
~ /
0_ 0t\
5
0=,
(
~.\
)
!I
>
el,
1 >
andHayek.199:!:Endreset al..19951including rheum atoidarthritistx remcrandJubiz.
1987:Fort in. 1995: Joeand Lokesh,1997).nephritis (T haisandStahl.1987:Rob inson er .1/..198,1.lupuserythernatos istAccinni andDixon.1979:Kelly et a/..1985),multiple
.,;..:lao~isrBateset a/..198 9).andulcerativecolitist~ewton.19961.Themec hanismsof
acno n forthebeneficialeffects of marine oilson intlarnrna toryand autoimm une diseases are not;l'iwellunderstood astheyarefor CHD.Howe ver.Higgs 11986 1studiedtherole of prosta gland insand leukotrienesininfl amm atory diseases.and Boissonneault and HayekI19921.haveconcluded thatboththe ratio ofillandw6fattyacids.aswell as their absol uteamounts. may have a profoundeffectsonimmune function.Theselatter investigators suggestedthatthe re we rethreepossiblemechan ismsinclud ingalterationsin eicosanoid vynthesis.changes inlipoprote in levels.and modification in membrane compo-auonthat affected transmembranesigna ltransduction.
Thereis ...ornedebat e abou t theeffects ofillpef Aconsum ptionand theriskof -rrokc.A.studyon GreenlandEskimos suggests that ofthepopulationsstudie d.theone Withthehigherintake ofw.3PL;FAs had a higherincidenceofstroketKromannand Green.19801.However.aJapane se studysuggeststhattheincidenceof strokedecreased 10populations withahigherrateofw.3PUFA consumption(Hiraieral..19871.Whethe r ornot consumptionofw.3fattyacidsaffec tstheincidenceofstroke.astudy byLands 11982).usingcatsshowedthatthere was reduced damage doneto cerebral tissue when thecon sum ptio n ofw.3fatty acids was high.
The reis alsoevidencethat consumpt ionofco3fattyacidslowersbloodpressure
ILands.1986:Knapp.1989:Gerst er.1993:Howe.1995:Adreassenetaf.•1997:Huanget
