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The biogeochemistry of sterols in Trin ity Bay. Newfound la nd.
and a new method (Thin Layer Chromatography-Pyrolys u- Gas Chromatography-Mass Spectrometry) for the ir An a lysis
Edward D.Hudson,B.Sc.
Athesissubmi n edtothe School ofGraduateStudies inpartialfulfilment oftberequimnmtsfor thedegreeof
Masterof Science MemorialUnivers ityofNewfOWMiland
St.John's, Newfoundl and
January 1999
Amlrlld
In the context of a muhidisciplinary study to determine currentand past ecosystem heahh, sterolswere analyzedinplankton,settling panicles and sedimentsfrom Trinity Bay, Newfoundlandby saponification. derivatization to their TMSethersandGCand GClMS.
Plankt o n net lowsamplesandsettlingpaniclescontainedCr,and~.sterolstypicalof marine plankton.However,higherplantC;.and~sterolswere prominentinsedimentsfromboth in-sho re and off-shore sites,indicatingan appreciable terre strial contnllutiontosedimentary organi ccarbon and either de gradationor effective recyclingof marine sterols.Nodecrease in totalorindividual sterols was observeddownthe cores, suggestinggood overall preservation .Tbe fecal sterol coprostanolwas not detected in offshore sediments, net tow materia] orsettlingparticles, and was present onlyal low levelsincertain in-shore sedime ntary horizons.This suggeststhat sewage dischargesin rural Newfoundlandarebeing efficientlydegraded ordispersed.
Total free sterolsin the samples weredetetmined bylatroscan TLC-FIDon Cbro marods, a widely-usedmethodwhicheffec t ive ly separatesandquantifieslipidclasses but provide snofurtber infonnationon thespeciesin each class. Thus, anewmethod (fLC . Pyrolysis-GClMS)was developedinwhich lip idbandsare desorbeddirectly fromthesilica Chro maro dsurface into a GCIMS foranalysis.Twelvelipidclasseswereeither desorbed without funhertreatment, converted to trimcthyIsilyI derivativesonthe Chromarod.or analyzed followinginsit uthermochemolysiswith tetramethylammoniwnhydro xid e.The
method' sutilitywasdemonstrated withlipidsfrom settling particles.especiallywhereTLe bandscontained morethanonelipidclass(waxl stery l esters,acetcoe-eoobue polar lipids).
Wax estersup to C.2were detected, with alkyl andacyl distnbutions cons istentwitha zooplankto nsource.Thewaxestet/steryl est erbBndcontained no more than 8%steryl esters, suggestingthat theircontr ibution to this bandisminimalbutthat theircontributio n tototal sterols maybe significant.Thefreesterolcarbon number distribution in settlingparticlesas detenninedby TLC-Pyrolysis-GCJMSmatcbed thatasdeterminedbyindividual molecular speciesanalysis(predominantly
Cn).
By extendingthescope o fl atro scanTLC-FrO,the new metho dshoWdhave app licatio ns in manyfieldsbesidesmarineenviro nme ntalstudies viz.food science,biomedicalscience andpetrolewn analysis.iii
AbsIract.••. TableofContents • LislofF igures...•••
LisiofT.bIes .
ListofAbbrevialio ns••.
Acknowlcdgmenls.... . Dedication
TableOrCOtlI~. ts
..
page. a
•• lv .vii
•••It xi ...xii xiii
I.Intr od uct ion .I
1.1Lipidsinthemarineenvironment.•. ..1
1.2LipKlOOIDeDcla hft• ..• . •. ••.•.• 1
1.2.1Sterols... . . . .... . 4
1.2.2 Sterolconjuaates. . . . ... . . . .... . 12 1.2.3 Fattyacids.fany alcohols andwaxesters. • .•• .. .. . 15 1.2.4Neutral K)'tglycerols.. ..•. • • . ... .. ..• • .. . 16
1.2.5Phospholipids ..16
1.3RelevanceoftrKing organic carbon in tbr marineenvironme'Dl•.••.•..•17 1.4 Lipkfsas biomadcninthe marineerMronmerJI.• .. .... . . 18 1.4.1Seero lsinthemarine environmmt.... .. . • . 19 1.4.1.1Slerols as biogeochemicalmarkers .20 1.4.1 .2Slerols asfecal biomar kers.. . ...22
l.5Methods for lipidanalysis•. . .•25
1.5.1Stero lanaiy5is.,. . .... .25
1.5.2LipidcLa.uanalysis. ...•. ..27
1.5.2.llatro scan TLC·FID.•... ....27
1.5.3Couplingof TLCwithGCorMS ... . . .30 1.6 0 bjecllves... •...•... • ... .... 31 1.6 .1StetolsinTrinityBay. NewfOWld1aDd .. .. . 31 1.6.2Lip", _ byTI.C-Pyro~.. .• ... . ... 33 Pan I:Biogeocbemistty ofstetobinplanlcton.scttlin&parlic~andsediments.,TmityBay.
NowfowodJand
2.Experimental•••••••••.•.••••• ••.•.•.•.• .• 2.1CbcmicaIs.glasswareand instrumentatioD 2.2 SlUtIpling .•.. .... .•.. . . ... ... 2.3SaqlIepreparation .
2.4Sapo nification. derivatizationanda." \lysis . iv
. ..34 J4 3S .36 . ..36
2.5 Analysis oftota! free sterols. 2.6 Floristic analysis and core dating
...40 . ..40
3.Resuhsanddiscussion. .43
3.1Methodvalidation 43
3.1.1Completencssofderivatization.. . 43
3.1.2 Linearity andequivalencyofdetector response. .43
3.1.3 Rccoveryoffrccstcrolsandstcrylcsters .45
3.1.4 Effectiveocssofthc Iiquidlliquidextraetion step .46
3.2 Plankton composition oftbc samples ... . . .46
3.3SterolsinTrinityBaysamples... .. . .. .. .. .. . ....•. .48 3.3. 1 Reported approaches to using sterol biomarkers .48
3.3.2 Plankton. ... 50
3.3.3Settlingparticlcs .57
3.3.4 Sediments . 60
3.3.4.1Spatial variation .. ... ...• . 64 3.3.4.2Historical(dowocore) variation. . 69 3.3.5Sterol-based sourceattnbution of organic matterinTrinity Bay..74
3.3.6Fecal biomarkers. 77
3.3.6.1St . .John"sHarbour scdimcm. ... .77 3.4 SterolsinNcwfouod1lmdmarinesamples: suggestions forfuture
approaches. ..79
Part II:DevelopmentofThiDlayercbromatography·PyroIysis-Gas cbromatographylMass -""0""",,
4.Experimental..• . .•...•. . .•. 4.1CbcmicaIsand glassware .
4.2Marine samples .
4.3Hydrogenation oftota!lipid extracts....
4.4 Cbromarod development and scanning
4.5Desorption.gaschromatography-massspectrometry••• 4.6TrimclhylsiJylation
4.7Thermochemolysis S.Resuhs and discussion.
5.1Hydrogenation . 5.2Neutral lipid classcs .
5.2.1Steryl andwaxesters . 5.2.2 Free sterols . 5.2.3Triacylglycerols .
.81 .81 ...81 ..82 .82 84 .87 88 ...89 ..89 91 .9 1 .96 99
5.2.4 Hydrocarbons...•... 100 5.2.5Freefattyacids... ... . . . ... • • .•... ... . . ...101 5.2.6 K.etones.,atiphatic akobols and methytesters.•. •.. . . ...101 53Polarlipid claMes.•• • • • •..••••.•.• • •• •103 5.3.1 Acetone-rnobiJepolarlipids .••.. . •••• •.. .•103
5.3.2 Stery lglucosides .•. ...105
5.3.3Pho spho lipids ... . .•. ... III S.4Pyroprobe interface:resolution and reproducibilityconsklerations 112 S.SAnticipateddirectionsinthedevelopment ofUC·pyrolysisGCJMS 113 6. Conclusions.
7.References..
...115 ..118 Appendix ACahbration curvesfOrcopro stano L cholesterolandstigmasterol. ... .131 Appendix8.Proportio nsofindividualsterols(%oftotal)insedimentcore horizons.133
vi
ListofFiprn
page 1.1 Clauesofmarine lipids and theirstnICt\Il'eS•••• •••
1.2Sterolstr\.ICtlJfeandnomencl&ture...
1.3 Sterolseceneesmentionedinthetext...
...2 . ..5
10 1.4 Repre sentativestructures of sterolspeciesoccurringinthe marine environment ..13 1.5,1s·Hydrogenati onof cbclesterolcan produce eitber cbolestanolOtcoprostanol...23 1.6 Studyareaand sampling locationsinwestern TrinityBay,Newfoundland. . .32
2.1Swnmaryofanalyticalproccd~usedforstero ls. ... . ..37 2.2Sterol derivatizati on ..• . . .•.. . ... . .• . . .. . 38
3.1 GC-FID chromatogramsoffWDconsecutive sets of extractionsofsedimen t
TLEsaponi fica tion mixture (St.7, 2-4 cm) ..47
3.2Seasonal variationin plankton sterolcontentduring 1995 ..52 3.3Spatial variation inplankton sterol contentduring 1995 ..53 3.4a.GC!MStotalionchrom atogram forthe H-9 horizontal planktontowsample
(April1995).b.sterol region... ... . .. . ... ...54
3.5 Meansterol compos itio nofTrinity Bayplanktontows. ..55
3.6Sterolcompositionofscttling particlesatSt-7. ..59 3.7a.GC/MS total ionchromalogmrlfortbc H-9oon::(24cmdcpth)Tl.E.b.sterol
region. .. ..,.62
3.8 Mass spectrumof thepeakat~"'20.45min in Figure 3.7
vii
...63
3.9Sterol composi tio nof Trinity Baysediments •. • . . .. . . .66 3.10Totalfree:sterol conlentsof sedimentsinindividualdepth horizons atin-sbore
stations••.••.••• .••.•••.••• • •.•.•.••••• •.••••..• •. • .71 3.11a Dcwecorecom bined percentagesof 24Et-29.6.',24Et-29.:1',24Et-2 9.6.' 'u,
24 Me-29.:1'and24Me-29.:1'atH-9.b.Downrorepercentagesof24Et-29.:1'
and 24Et-29.:1'atH-I .73
3.12Sources oforganic matterinTrinityBayplanktoo, settling particlesandsediments according to sterol biomarltercomposition.. ....76
4.1Typi cal latro scaJ1n.C·FIDchromatogram:hydroaena1edTLE ofsettlingparticles (St. 7,75m depth), showin g three devel opmen tsand scans ....83 4.2Pyroprobeand interfacewedtodesorblpyro lyzelipids off silicaChro marod
coating 85
5.1Effec tofhydrog enati onof the 11.E(senl ing particles,75m):thermocbemcly sis withmethanolicTMAHof:(a)Originaln.E(42.uatotallipidspottedonrod) ;
(b)Hydrogenatedn.E (l4.ug toca1lipid) . 90
5.2Three waxesters(16:0116:0,18:0116:0,and18:&18:0)andtwocholestetyl esters(cholesterylpalmitate and arachida1c) desorbcdfromaChram.ar04 sectionbya Pyroprobeset to450"C.. ... ... . . . ..92 5.3 Wax ester/stetylesterbandfromhydro a mated 11.Eof settling particles(SI- 7, 75 m
depth)dcsorbcdfromChromarod 93
5.4 Desorbtionoffreesterols;Pyroprobe setto300"C.(a ) 3.6.ug5a~holestallOl desorbed withoutfurthertreatment.(b)Mixedsterolstandard(9sterols pluscoprostanonc,1.3.ultotal)desorbcd\l,ithoutfurthertreatm era,(c)Mixed sterolstandard (0.78 j,o:gtow),desorbcdafteraddition of10.uLBSTFA...97 5.SFreesterolbandfromhydrogenated 11.E ofsettling particles(S t-7,7S m) dcsor bed
fromaChro marodasTMS ethers. . ...98
viii
5.6Monoacylglyceroldesorptio nafterOSTFA treatment;Pyroprobeset at500°C.
(a)Desorptionofa standard(I-monopalmitin).(b)AMPL band from 100 m
sertllng particles. ...104
5.7 Total ionchromatogramresultingfrom thedeso rbti on of eampesteryl,stigmasteryl
and sitosrerylglucoside(2.3~gtotal) froma Chromarod sectionafter BSTF A
application. 107
5.8 Isomeric sterad tenes proposed to resultfrom thermalcleavageof campesteryl
glucoside _ ..._. _..108
5.9Elutionof sterylglucosidestandardin thethirddevelopment.
ix
..Ito
1.1 Sterol names usedin the text 1.2Characteristicsterols ofvari o usalgaltaxa.
2.1Gaschroma tographi ccondi tions usedforsterol TMSethers. 2.2Elutio norder and characteristic GC· MS ions forstero ls detected
page
•..•.•.7 ...21
. 39
._.4 1
3.1Detector responsesfor equivalen t amounts of chole st erolinfreeandesterifie d
~. . ..45
3.2Free stero lcontribution(%)tototal lipids insettling particles at near-shore (51-9)
andoff-s hore [St-7) sites. .. 60
3.3Totalfreestero ls (mean±SO)insurf ace(0-2 em;n=2)andsubs wfacesediments
(n=7)sedimen ts. . ....67
3.4 Stanol/steno l ratiosin Trinity Bay sediments (calc ula tedfrom whole core mean
sterolcom pos itions ). ... 68
3.5Attributio nof stero lsinTrinityBaysamples to vari o ussourcesoforganic matter. 74
4.1Conditionsused totransfer lipids fromChromar odcoatingto GCcolumnbythermal
desorptio n/ pyrolysis.... ..86
5.1 Reproduc ibilityofdesotb tio olpyro lysisfromCbrom arodfragments. asillustratedfor
stcryl andwaxesters .113
AMPl ASG BSTFA OAG dw
E. r.
FAME FFA flO FS GC-FlD GC-MS HC HPl C 1.0.
IUPAC MAG OM Pl Pyr R, RSO SCE SO SE SEt SG TAG TlC TLC-FID TlE TMAlI TMCS TMS vtv WE w/v wlw
List
or
Abbrni.lioa.Acetooe-mobilepolarlipids Acylated steryl glycoside Bis-N; (J.(trimethylsilyl)trif]uoroacetamide DiacyJg.lyceroI
Dryweight Electron ionization Fatty acid methyl ester Free fatty acid Flameionization detector Free sterol
Gas chromatography withflameionization detection Gas chromatography/massspectrometry Hydrocarl>on
High performance liquid chromatography Internal diameter
International Union forPurrand AppliedChe mistry Moooacylglycerol
Organicmatter Phospholipid Pyrolysis Retention factor
Relativestandarddeviation(=coefficient of variation) Steryl chlorinester
Standard deviation Sterylester Sterol ether Sterol glycoside Triacylglycerol Thin layer chromatography
Thin layer chromatographywithflame ionization detection Totallipid extract
TetramethyJammonium hydroxide Trimethylchlorosilane Trimethylsilyl Volumelvolwne Wax ester Weight/volume Weight/weight
Ac:kDowledemeatl
Firstly.twish10 thank mysupervi sors,Dr.RobertJ.Helle urand ChristopherC.
Parrish. fortheir guidance, suppo rt, encouragement and patie nceduring thisproject.
Tomyfellow graduate studentsinmarine andanalytical chemistry (YvetteFavaro.
Jerry Pulcban.ShuTang.HannahDayan,Tanya MacGillivray.NevenaPopovi c.JimFarrell andespeciallySue Budge):thankyouforyOU!'s~rtandfriendship.Tomyfamily(Joke, Jim.Helen.Dave):thankyo u for allyour lo ve aadencouragement.
Thanksarealso duetoDr.Graham Bodwellforhis constructiveroleon my superviso ry committee.toStewart"DiscoStu"ParsonsandJeane tte Wellsfortechnical support,10 Linda Wind sor forloans ofsolventsand equipme nt,toDr. D.J.Burnell,Dr.P.
D. Golding andDr.T.A. Abrajano.Jr.forhelpful discussions .and toDr.M.D.MackeyfOI access 10the Varian3400 GC. [am.also gratefultoMike Rhieland the erew ofthe R.V.
Karland JackieIJfOI teac hingmetheinsandouts ofmarine sampling durin g thesumm er of 19%.
Lastl y, theroleof the NationalSciencesandEngineeringResearchCounc il and the Departmentof Chemistry inpreventing mystarvation and evicti on is gratefully acknowledged.
xii
Dedicated tothe memoryof VirginiaCatherine Augemari (1972 - 1994).
xiii
1.Introduction
1.1Lip klsIn lhe marineenvi ronmenl
Lip idsarc abroadcia»ofbiornolec ule swhichare~nera1Jyoperatio nally defi ned asbeingpoorlysolubleinwat er.butsolublein non-po larorganic solvents suchas ch lorofonn.althoug hsomeauthorspreferthenarro werdefinitionof"fatty acids and structurallyorbiosymhencah yrelatedcompo unds"(Christie.1989).adefinitio nwhich would.forexample.excl udepolycyclic(polyn uclear) aromatichydrocarbonsandsterols.
Awidevarietyoflipid clas sesare found ineverycompart mentofthemarine en viron ment:
inorgan is ms.inthesea-surfacemicrolaycr.adsorbedontoorincorporatedintosettling particulates.inancient(Wrecemsediments.andinwaterindissolvedor collo idal form.They include (inorderof elutiononsilica:Parrish.1988)alip hatichyd rocar bons.polycyclic arom at ichydrocarbo ns(P AHs).wax esters. shortchain(e.g.•methyl) este rs.acylate d glycerolethers. triacylg lyce rols.free fattyacids.aliphaticalco hols.ste rols.diacylglycero ls . monoacy lglyce rols.glyc oglyc erolipids.pigments.and phospholipids.Fig.1.1givestypical examplesofsomeoftheselipidclasses.Thefunction s of Jipidsinorgan isms(Zubay.1993) range from energycapture and storage.tobuo yanc ycontrol. insu lation.structural com ponents(especial lyof ce llmembranes).andvitam in andhormon alroles.
1.2Lip id nonwc:laCuR'
Given thevarie ty of lipidsfoundinmari ne material.anunde rs tandi ng oftheir
Aliphatic hydrocarbon(pnsune)
PAH(anthracene}
o
Waxester(heudecyl teU'adecanoat e)
~OM' o
Fatty acidmethyl ester (methyl palmitate)
~ o
Ketone(heudeean~3-<1ne)
Triacylglycercl(tripalmitin)
~ o
Freefattyacid(palmitic acid)
Aliphati c alcohol(fr-hexadecanol) Filur~1.1Classesof marinelipids andtheir structures.After Parrish (1988)
[)jacyl.~erol (1,2-di~mitin)
~ .
~ - ,
Pigmen t (ch lorophylla)
~a'"Y'"'OH
OHMonoacylglycerol(I-monolinolein)
G1ycoglycerolipid(1,2-dipalmitoyl_3-(131'-6-gaIaC1osyl~gaJactosyI8lycerol )
~~o-~cr-...-N(CH,)'
~O'
Phosph olipid(l,2-dipal m itoy lphos phati dylc holine)
Figure 1.1(continued )
nomenclature isan essential prerequisitetoany discussion oftheiranalysis.,occurrenceand biogeochemistry.Thesystems used fornamingthelipid classesstudied herewillbe briefly introduced.
1.2.tSterols
Sterolsarcformedinnature bythe cyclization of(3S)- 2.3 -oxid osqualeDe(squalene epoxide)(CatellandCeruti, 1991)and modificationoftheresuhingtetraey clicmolecule co giveawidevariety ofstructures,havingincommontheperhydrocyclopentanophenanchrene
"steroid nucleus"withringsA·DgenerallyjoinedalJ4ra ns(Fig.I.la}(Zubay,1993).In the conventio nal representation of1hestero idnucleus(Fig. 1.2b ),thisabsolute stereochemistry (thatof cholesterol)isasswnedunlessanotheris shown. Athorough discussionofstero l norrenclatureispresentedbyJanssen~tal.(199 1).A5 illustrated herebydebydrodinosteroI.
sterolsare formallynamedas derivativesof cholesterol, withcarbons numberedasin 1.2c.
Thedesignatio nsaandIIindicatesubstituents below andabo ve the plaoeofthesteroid nucle us, asdrawn.Confusingly, a andIIare alsoused to indicate stereochemistryatC-2 4 onthe sidechain-withaC·24 methylsubstituent.24aisequivalent to24R sterochemistry (Goad,1991 ). Theterms stenol and stanoldescribeunsaturatedand saturatedspecies.
respec tive ly,althoughina biogeochemical contexttheymay referto analogouspainof4-5 unsaturatedand saturated structures,in which the sidechain is still unsatwated. Chole stero l itselfistherefore cholest - 5-en- 311-o1 BOO debydrodinost erolis 4.23 .24-trimethylcholesta- 5,22(E)-d ien-3 jl-oL Ina saturated nucleus, designation oftheC-Sbridgehead asaor
p
H
b.
rigu~1.2.Sterol snuctueeandnomenclature.a.Cholesterol.showing stereochemisny.
b.Theconventionalresprescntationofcholesterol.CoDehydrodinost efol(44,23,24- trimethylcholest-S.22(£)-.dien-lll-oI). d.The latest IUPACconventionsfor numbering substituenn onthesterolnucleus
indicatesa subs titue nt{HIbelo wor abovetheplane of the ste roid nucleus . respectively.
Furthermethylsubsti tuerus(no tsho w n) are designat ed C·29 (onC-28), C·3 1(onC-4.13)and C·32(on C-14.0.).While1989 IUPACnomenc latu rerevisions(Goad. 1991 ) resultedina newnum berin g system(1.2d ).theolde rconventio ns are still widelyencounte red in the literature andarethereforeuse dhere.Many sterols alsoha vetrivialnames,ofte nindica tive of thesource fromwhich the ywere firstisolat ed .
Abbreviationofoflenunwield y sterol namesishamperedbythediffi cu h y in readily describing allaspec tofaste ro l'sstructure.The form 296S.2OU 18l£maybeused .this example indica tinga stero l with atotalof 29 carbo ns, with doublebondsat the 5 posit ion and between carbons 24 and 28,thelatt e rwithEconfiguration .Stereoc he m icalconsideratio ns(a or 13) can bede si gnat edthus:313,5 13-276° for 5p-eho les tan-313-01(coprostano l)(6° indicatinga saturatedspecies).Howe ver .thedifficulty ofident ifyin gthepositi on ofsubs t ituemson.or deletio ns from (ind icated bytheprefix "r-noe", where carbonato m numberx is deleted),the cho lesterolparentsti llremains. Many abbrevia tionsystemsare either insu ffici e ntl yprecise tobeuniversal(e.g..Lietai.199 5 ) or arcpreci sebut arbitr ary,giving littleintrinsic indicati o n of the struct ure(e.g.,Patte rson ,1991 ). Some au thors(e .g.,Ma yzaud etat., 1989) usethe trivialnam e of oneparticularstero lC-24 epime r to represe nteithe rep ime r.since the two are often not separated in bio geoch emi call y-orien te d works.Thus."sitos te rol"would designate both sitos terol(24a-ethylc ho lest-5-e n-313-oI)andclionosrerol(2413-ethylcho ]est.S.
en.313-ol),anapproac h whi chis val idaslongasit is exp licit lystated.Thenaming system used here (Table 1.1)designate stheposition ofsubst itue n ts(or deleti o n s ) with respect to
Tab le1.1.Sterolnames usedinthetext.Letter/rwmberdesignatioos(bold text) following the abbreviation refer to Fig.I.J
TriviaJ_ Sysl~MOIjc _
(A bbf'rVioriOfl)
cis-22-ddJydrocbo n ero l cbo",",-S.22(Z)-d;m-JIl-<>I (27b.,·nl)A7
cbolestanol 5a:-<:boIeslao-Jjkll
(27b.~C l
cbolestero l cbolest-5~J~1
(27b.')Al
copro stanol Sp,-<:bolestan-Jp.ot
(SP-27"'>81
dehydrodinosterol 4a:,23,24~trimethylcholest·5,22(E)-d.ien-Jp,-01 (4a,23,24triMe~30A'.w)AI4
desmosterol cholesta-5,24·d~J p-01
(27Au· )M
dimethyldebydrocbo'estanol 4a.,24-dimethyI.Sa-cbo lest· 22( E)-e ool (41l,24diM e-29Ant)D19
dinosterol 4a..23 ,2 4-trimethyt- 5Il-cbo lest- 22( E}eo-J p-01 (4a.2 J .2 4triMe- J 04nt)DI4
brassicastanol· 24- methy l-5a -cho Iest.22(£)-en-J J!-oI (24Me _2842#:) C9
bnssica:sterol· 24-m<tbykbol=o-S.22(E)-<ticn-JIl-<>I (24Me _284' .nE)A9
epico prostanol 5lkbokstao-Jo.-ol
(3a,Sp,-274~FI
ethylchoiesta.5,22E-dieno1 24-<thy k bo",",-S.22( E)-d ;e"..JIl-<>I (24Et-29b.'.nE)Al I
ethylcholest. 22E-eno1 24-ethyI-5a-cbo lest-22(E)-en- Jp -01 (24Ec-29A2lf:)CII
Table1.1.(Continued) elhy lcho leslanol 24 -c thyl-Sa-choleslan-JjH>1
(24Et-29b.~CJ
ethylchcle srerol 24-et hylchole sl-S-en.JP-oI
(24Et-29 b.')AJ
ethylco prost anol 24-ethyl~,Sjkholestan·J13-o1
(24EI·5l3-2911~83
fucosran ol 24-c lh ylc ho les l·24(28 )(E)-e n-Jj3-01 (24EI_2911:",:aoL)CIl
fucosrero! 24-clhylcholesla -S.24(28 )(E.:')-dien.Jll-ol (24 Et· 2911''':''':l oE)Al l
isofuco stanol 24-clnylcholesl -24(28 )(Z)-cn-JI'-ol (24Et~29 6:"'!I12)CI1
iso fuco srero l 24-clhylc holesla·5.24(28)(Z)-dien-3I'-o1 (24Et_296,..:",:a,z)AI2
lanostero l 4 .4.14·trime th yl·5a-c ho les la- 8.24 -d ien.3ll-ol (4.4.14 tri Me-30l1w )E5
24- me lhylcholestano l 24-mc:lhyl-Sa-cholestan. JjH>1
(24Me-286~C2
4-mc:lhylcholeslallo l 4a.melhyl·Sa-choleslan-J Ji-oI
{4aMe·28A~D1
24-melhylcho leslerol 24-methyk ho leSl.'s-en-Jj3-01
(24 Me -2 M')A2
24-methylenechole Sianoi 24-mc:thylchoIeSl-24( 28 )-cn·3j3-01 (24 Me · 28 A!",:a,)CI O
24-methylenecholes lerol 24-me:thylcholesta·S.24(28)-dien-J j3-01 (24 Me.28A'.1-Io!a')AIO
24- no rde hydrocho leslanol 24-nor-Sa-chole sla·22(£)-cn -3 j}-o1 (24no r-26Al l l)C4
TaMe1.1.(Continued) 24-no rdehydrocbo lcstcro l 24-DOrcbo 'esta -5.22( £)-dien- J j3.a1 (24nor-26&5..nE)Af
cccejastero l 24-methyl-27-DOrcboIcsta-S.22(E)-dien-Jp..o l (24Me-27DOr.2745.n£ )AI
lrans-22-de hydrocbo kstano l Sa:<bolesta-22(E)-dien-lj3.a1
(274nE)C6
lTans -22-de bydrocholcsterol cbolcsta-5.22(E)-dieo-JJ)-oI (2745.:1£)A6
Inthis work. brassicasterol and brass icas lanolwerenot separatedfromtheir24P epimers (epibrassacasteroland epibnls.sic.astanol.. respectively). Hereafter.
''brass icasterol" thusreferstobothbrassicasterolandepibrassicasterol.likewise.
"brassicastanoj"designatesbothbrassicastanolandepibrassicastanoL
$
A#
B# $
c
0H$ ~ ..q¢
E
H#
GFigure1.3.Sterolstru ctur es mentioned in the text.R0 ;side chain(structu res 1-14).
Nee=steroid nucleus (structures A-G) 10
~ 0 rt
~ ~ ~
'Cly ~ 0
' 0 r t 0
1. 11 12
0 m
13 U
Figure1.3.(Continued)
\I
choles terol.followed by configurat ionat importantchiraJcentres, andfinal ly the carbon number andthepositionsof anyunsarurancn s.Thehydroxy groupis assu medto be 3(3and the stereochemistryof a satura tednucleusSa,unless otherwisestate d,andC-24 stereoche mistryisnot designated.
1.2.2Sterol oonj ugat6
Estersof sterolswithfany acids(commo nlycalledsrerytesters.SE)arenamedby combiningthename ofthe steroland the fattyacid(thenomenc latureofwhichis discussed below).Thus the sterylesterin Fig.1.4isnam ed choles t-5-en-3(3-yl he xadeca noate . or cholesterylpalmitate.
A sterylglycoside(SG)isasterolof whichthe Clhydroxylgroup isre placed by an ether(glycosidic ) linkage tothe C,positio nofa mon o- or oligosaccharide co ntainingupto five glycosylresidu es inlinear seq ue nce(Heinz.199 6 ).Inhigherplants. a singleglucose residueisthe mostcommonsaccharidemoiety(Wo jciec ho wski,1991).Anacylatedsteryl glycoside(ASO) additionallycontains afattyacidesteri fiedto ahydro xylon the saccharide, mostoftenat C6(Heinz,1996 ). Giventhe varietyof monosaccharideswhich theymay contain(e .g..galactose.ramnose, mannose, xylose,ribose . glucuronic acid),the positio ns throughwhich these maybelinked ( 131-6,1.l1-3. or (.\1- 4),thenatureandpositionof any acylsubstituent.and thevarioussterol speciesthemsel ves,thenamingofspecificSGs and ASGs canbecom plex;a detailed discus sionispresentedbyHe inz (1996). The sterol is ind icated eitherbefore (Wojciec hows ki,199 1:Heinz,1996) or after(Fuj inoandOhnishi,
12
Non-poIar species
Freesterol (FS)
Sterylester(SE)
Steryl ether (SEt)
Figun1.4.Representativestructu res of sterolspecies occurring in the marine environment.
13
Polar species
Sterylglycoside (SG)
Acylatedsterylglycoside(ASG)
Ste ryl chlorin ester(SeE)
FigureI....(Cont inued )
14
1979 )afull desc ripnonofthe glycosylponion.Fig.1.4shows 24-ethykholeste ryl~[)..
glucopyranos yl(lM-4HJ-o.gluco p)'Tal1os ide(SO),ofwhich the glycosyl ponionm.Jybe abbrevia ted to lJ-o.Glu(I.4>-tl-[)..G lu. and 24-ethylc holes teryl (6·..()..steroyl>-{l-[)..
glucopyranoside(ASG).
Steryl chlorinesters (SC E.'i)(Fig. 1.4)arerecenny discoveredsterolconjugat esin which sterols are esterified tophytopheophorbidc:Q,a porphy rin-likedegrada tionproduct of chlorophyll(Eckhardt et al.,1991).No particularnomenclatureiscurrentlyspecified(ot
sees .
1.2.3Fatty acids.fauyalcoho lsandwax esters
Individ ualfaltyacids are oftenabbreviatedto the fonn x:y(l)Z,where x indicatesthe numberofcarbons,ythenumberofdouble bondsand z thenumber ofcarbonsfromthe terminal
« (I)
methyl group(inclusive) to the firstdouble bond.Unless otherwiseindicated . all bondsare assumed10becis(Z)in configurationand methyleneimerrupeed,Forexample.octadeca-9. 12-dienoicacid(linoleic acid) (Fig.1.1)wouldbe abbreviatedto18:26J6.More unusual structuressuch asiso-oranteiso- bnnching(indicating amethyl grouponthew2 or(1)3carbo ns.respectively).ortrans(£)doublebondconfiguration.areindicated bythe appropriate prefixe s.Trivi alna mesformanyacids are still Wide lyused.
Longchainaliphatic(fauy)alcohols,tnough notasabundanta consntuentoflipid materialasfauyacids,may be similarlyabbreviated, aslon g as itisclearwhichclass of compound ismeant.Thus,waxesters,theestersoffeuyacidswith Iauyalcohols,arehere
IS
abbreviated as x:y!x':y'.indicatingthe nwnbcr of carbons (x)anddoublebonds(y)inthe alkyland acyl
0
portions, respectively.1.1.4 Neutral.cyl&!Ynrvll
Several important lipid classes consist of glycerol (1.2.3-propanetriol)esterifiedto oneor more fatty acids;thosehavingone. two andthree fatty acids aretermed moooacylglycerois (MAGs). diacylglycerols (DAGs)andtriacylglycerols (fAGs).
respectively.For acylglycerols containing the same fatty acid at each position, the acid's trivialnameisreadily used tonametheacylglycero1. Using the example of palmitic (hexadecanoic)acid,I~monopalmitindesignatestheMAGl~moDOhexadecanoylglycerol.
1.3-dipalmitinwould be the DAG 1.3-dihcxadccanoylglycerol.and tripalmitin istheTAG tribcxadccanoylglycerol.The namingsystemusedhere does noedesignatestereochemical configura tio n around the glycerolC-2.since this isunimportantinthis work; this issue is discus sed byPerkins(199 1).WhereDAGs or TAGs contain CWoorthree different fatty acids.lengthier systematic names are needed, althoughthe uivial names ofCbcfattyacids maybeincorporatedintothese.
u.s
Phospholipid.Pbosphoglyccridcs, the phospbolipidsstudiedhere.arc derivativesofg1ycerol-3~
phospha tewhich maybe esterifiedat the phosphoric acid portion as well as at both remaininghydtoxyls on glycerol. Theyare namedKCOtdinBto thesubstituenton phosphoric
'6
acid (Zubay,1993)aDdthe£anyacidson the glycerol bydroxyb areindicatedas rnfixes.
Thus,the pbospbolipid lD Fie.1.1is disIc:aro)'lpbospbatidykboline.Pbospbatidykbolines area150knownas kcitbins. OthercornmODsubstitueDtIesterifiedtothe pbospbale moeity include serine,M)'O-iDositol,aDd.g.Iycerol. Hthis substituent is. hydrogenatom.thelipid istermed •pbospbatid.ic:acid.
1.3 Reln. .«or tneillllorpaiearbollliatile • •rille nYirolUll eaL
Marineprimaryproductionby phytoplanktoninsurfacewateTSunderpins the entire marine food web, includingcommncialand IUbsistencefisheries.Whilemost primary production is also consumed orremineraliud in surface walen (Wakebamand Lee,1991), an importantfractionsinksthroughtheWlItetcolumnandnourishes mid-wateraDd.benthic organisms.Furthermore,thissinlring organic matter transports atmosphericearboointo sediments and deepoceanwaJtn.,wbereitmayremainfor centuries(Libes. 1992).thereby influencing globalclimate(sannieruoaDd.Sundquist,1992).Therefore,an undcn:tanding ofthe biogeochemistry(sourceorganisms,processesand forms.rates of production and degradation,and ultimate fate)of organic carbon in the marifte environmentisofvital importance.
Organic compounds &omtaKstrialsources which enter themarineenvironmentin.
nm-off orthrouBbatmospheric:depositionprovidean additional energy input which can be utilized and degndcd b)' marine orJanisms (Libes,1992).Human activities onlandwhich affect this input (e.I .,agriculture,deforestation)caDthereforeaffectmarine ecosystems.
17
Furthermore, the magnitude and dispersal patterns oftcrrestrial input may indicate which partsof the marine environmentmaybe impactM by anthropogcnicaUy introduced pollutants (e.g.,petroleum products).
1.4 Lipid. . . biomarkeniD.the mariDe eariroDacat
Lipids canprovidea wealth of information about organisms and processesinthe marine environment, reflecting sources of cqanic matter and its transformational historyin the water column or sediments (Cranwell, 1982). Certain fatty acids can betraced unmetabolized through food webs. Bnmchedfattyacids {e.g.iso 14:0 andiso 16:0, and 15:0 and 17:0iso and anuiso acids) in sediments and estuarine particles arc markers of bacterial carbon input (e.g.,Scribe~t
m.,
1991). Toxic dinoflagellatesanddiatoms produce characteristic fatty acids (parrishetaI.,1992), such as 16:4<01inthe case of Nitzsc#lio pungens (parrish etaI.,1991).Moreover,lipidsateoften the slowest ofall biomolcculcs to degradeinthe carly stages of diagenesis (Huvcy etm.,
1995; Ishiwatari etm .,
1995), particularly inaooxic sediments. The use of lipids as biomarkersinthis fashion is reviewed bySaliotet al. (1991).Inaddition to using individual molecular species as biomarkers,lipid classdatacan yieldcnvirOnDl.CIltal information. Goutx etaI.(1990) obtained cbatac:teristic lipid class profiles for common marine microalgac. Furthermore, lipid class composition can indicate the physiological state of organisms. Applications have included using total triac:ylglyceroUsterolratios to gauge the condition of fish,crustaceanand bivalve larvae
'8
(Fraser. 1989)andgauging the quality ofbalibut eggs by determining storage lipid content (Danielel 01., 1993).
1.4.1SterolsiathemariIleearil'olUlle.t
Sterolsare foundinalleukaryotes,aswellasincyanobacteria (patterson, 1991).
Their principal function is as a membrane component,inwhichtheymodulatethe fluidity of cell membranes; furtbcrmore.theyarc precursors to certain vitaminsandregulatory substances (Goad, 1991;Zubay,1993).While some organisms (mammals. higher plants) canS)1lthesizesterolst:krrovo,others(e.g.,crustaceans) (SerTazanettietaI.,1989)can only modify dietary sterols andstillothers (e.g.,manymolluscs: NapolitanoetaI.•1993) inco rporate unmodified dietary sterols.
A wide varietyof sterolstructura occurintheoceans.either originatingthen:orfrom input from terrestrial systems (see Volkman, 1986).BrassellandEglinton (1981) isolated 69 different sterolsfrom sediments from the Japan trench,andthe identification of 30 or moresterolsinsettlingparticlesinvariousmarineenvironments (SmitheloJ.,1983;Bayona etal.•1989;Harvey and Johnston, 1995) is not unusual. These figuresateDot su:pri.sing considering the variety of organisms producing these sterols.withup to74 sterolsbaving beenisolatedfroma single organism(ltohetal.•1983),andthe variety of processes which may subsequently transform these (reduction, dealkylation.etc.)(Cranwell,1982).
Most organisms whichsyn~izeC-24 alkylated sterolsproduceonly oneC-24 epimer,althougbafewtettestriaJ.plants. including certain CucurbitaeeaeandCras sutaceae such asKmlJMhoepinnata (air plant) (Akihisa etoJ.,1991b)synthesize both C·24epimen
19
of aparticular structure.GeDcra1ly,higher plantswillcontaiD the24a cpimer(e.g.,240·
ethylcbo lesta.S,12(E)-dic:n-31S-oI,stigmasterol).whilealgae contain the24~epimer (e.g., 24p-ethylcholesta-S,22(E)-dien-3~I,poriferasterol) (Maxwell etai.,1980).
SterolsmayOCCUI"inaquatic environments asfreesterols,orinstayl esters
(Wakehammd Frew,1982).sterylethers (BooaaDdDeleeuw,1919).steryl glycosides or acyla1ed sterylgIycosides(Gt.du~tol.,1991;Veron~tIll,1996), orsaerylddorinesters (Eckardtrt al,1991;HmwiiDertal..1996),the last formed duringzoopIanktoobcrtriwry.
Insomeenvironmellts the sterol composition intheseforms diffcn&om thatofthe free sterols.Sedimentary steryl estersinLoch Clair,UK.hadalowerSlanoUA'-stcnolratio than freesterolsinthe samesediment(Cran we ll, 1982).Conversely, in settling particlesinthe equalOriaiAtlan tic,free sterolaDd stetyl estermoleculardistributionswere welleom:lated (Wakeham~I aJ..1980).andsedimc1:lwysterylchloriDestersmaybe abetter indicator of thesterol composition of sourceorpDismsthan free sterolstbcmselva(pearceetaI.,1998).
1.4.1.1Stero....bio&eocb~lIIical lll.rken
Even among lipid classes,sterols arc some of thebestbiogeochemicalmarker
compounds.duetotheirresistmce 10degradatioa(Saliot:elaI.,1991;Qucmmeur and Marty, 1992 )and theitwidevarietyof muctures.Intactsterols haveC'VCIlbeenfoundinlignite coals(DelRio~t aJ..1992)
Thesterolprofileofdialoms can be cbarxteristicof . patticularcLass,family,acnus or even species(8am1tetal.,1995);23,244imethylcholest-5,22Eodien-31l-01maybe •
20
·uniquemarkerforthe widespreadFragillmWpitWlla.Table 1.2iDdieatescenai.n 5tcT01 strue:turesbroadlyassociucdwithvariousalpl. taxa, although then: are many exceptions.
T~includethepredominaDa:offueostcrolinmostbrown algae (Pbaeopbyceac)aDdof cholesterolin manyredalgae (R.hodophyccae),withdesmostcTOlalso frequentlypresentin the latter.4a-Me:thy1sterolsliemostwidespread lImOD&the dinoflagellates(Dinopbyceae), and 24-methylenedtolester'Olaod (epl)brassic.asterolare frequentlyfoundindiatoms (Bacillariopbyceae ).
T.ble1.1. Characteristicsterolsof various allal tax.a(afterPatterson,1991) Algal clau PrindpaJ01'char acrerirticsle,ols Dinopb)Ulle(dino fla&eUatn) 4-Methyl sterols,
e.a-.
dinosterol;cholestero l ,."".,..-opbyceoe 4'sttrols;(epi)bnssjcasterol Phaeo phyccae(brownalgae) F _IBaa:ilariophyccae (di ato ms) Varies;many 41andC:z.sterols Rhodophyceee (red algae) Cholesterol,desmosterol
Chlo rophyceae(greenalgae) Varies;4'sterols,some 41or AUsterols;~sterols
Xanthopbyccae Ethylcbo lesterol
Choroph ... Ethykbolesterol
Theuse of sterol biomarkersis complicatedbythe variabili tyofthesterol compositionwhic:h may occur even withincenaiD.algal &eoer&(Patterson,1991),andthe possibility thata1pJ.8fO\\'1h cooditions may altertheirsterolcomposition (VeronetaI., 1996).24-Ethylcbolesterol.until recently thought tobean exclusivelyterrestrial sterol,is
21
alsoproduced by some algae(Volkman, 1986;Pattc:non.,I991;Li,.,al.1995).TbestCTOI Wnostero lwasonce thought tobe solely produced by dinoOagcllates.but bas also been
ro und
incertainmarine diatoms (Volkman,. , 01.,1993).Furthermore,wtWesome invertebratn accumulat eWUDDdi6ed dietarysterols.otbensuch ascopepods dealkylate dietary phytosterols(Scrnnmcttit'l01.,1989).tbetefore modifying thestero lmputintosinking particles. No netbc ltss. certain pettems in the sterol compositionof marine samples can provideuseful biogeochc1nical information. A predominanceof4-metbylstero ls Iilc.ely indicates a significantdinoflagellate contnbution.\\/bile cenainalgae produce predominantly C;;.and
c;..
sterols, proponionatelybighabundancesof tbcscan:inmany cases still indicative of terrestrialinput(e.g.,Laureillard and Saliot, 1993).Additionally.examining the ratios of certain sterols to othen (Liet aJ.,1995),orthe co-variation ofeenain sterols with eachotber (Pockingtonet01.,1981) and withothe r biomarkers(YWlker etal.,1995)yieLdsfurther information.1.4.1.1Sterols as fecalb~ .,.bn
Sewage dischatgnfromcoastal communities may hive variousimpaasOD
me
marine environme nt.TheymaydisruP'lIrarioc life by loweringdissolved oxygen levels(Libcs, 1992 ).foul the gillsoffishand filtcr fcediogorganisms(Franke l 1995).and aherfish abundances (Mearns.1982).HumanbeaIthmayalso beimpactedthroughpathoien5(e.g., skin and eyeinfections, hepalitis A)andhearymetaisinsewage being introduced into the diet throughcontaminatedscafood (Frankel,t995).Thereforeitis imponant 10determinethe22
extent and distribution ofhuman sewage contamination,espec iallyin areas such as Newfo und land, which has ttaditionallydependedon marine fisheries and where thereis great inte restinthefurther developmentof aquaculture.
Cholesterol(choIest·5-en-3lJ..o1)
"~
HCholestanol(Sa -<:holesta n-3l3-ol)
Coprosta none (5P-choIestan-3-one)
Coprostanol (5t,kholestan-3j}.o1)
Epiooprostanol(5ll-dlolestan-3a..ol) Figure1.5.4.'-Hydrogenat ioD of cholesterolcan produce eitber cbo5estano1orcopro stanol, Cop rostaoo IlCandepicoprostanoi are important in differentiating sewage inputsfrom other possible coprostanol sources.
23
The sterol coprostanol (5tl-cbolestan-3JkJI,)has long been used as a marker of sewage contamination (e.g .• HatcherandMacGillivary.1979; Pierce and Brown, 19&4;
Writeret01.,1995), providing an alternative 10 fecal coliform counts, which may be unreliable due to die variability in coliform survivalindifferent environments (Nichols and Espey,1991).BacterialAs· hydrogenati on of dietary cholesterolindie intestines ofmammals produces this sterol ralhertban itsisomer.dxllestanol (Sa-cbolestan-3JkJI)(Fig. 1.5)aDdits distributioninriver. barbour andcoastalsediments (e.g., Writeret01.,1995;Venkasetan and Kaplan, 1990)inbeachsands and greases, andinthe sea surface microlayer (Nichols etoJ..
1996)has therefore been widelystudied.
Pocldingtonet01.(1987) have suggestedthat coprostanol may not be an unambiguous fecal biomarker.sinceits concentrationinBedford Basin, Nova Scotia.was clo sel ycorrelated with severaldistinctivealp! biomarkers. Furthermore,anoxic sedimentary environmentsappearto favor the formation of 5j}-stanols over 5a-stanolsfrom tt,s_stenol s(Meyers and lshiwatari,1993). Writeret01.(1995) used a coprostanol concentrati on of 0.010~i/idwscdimeDtas a eut-offto indicate sewage contamination, but coprostanolconcentrationsfarhigher thanthismay occurinsediments from remote marine locations(e.g.,Grimaltetai.,1991;Colomboeral.,1997), suggesting sources other than sewage.Moreover. itisnecessaryto differentiate fecal input due to livestock fromthatdue todom esticsewage(Nichols etaI.,1996).Nonetheless, toascenai.nwhether human. fecal contaminationispresentinwastewaters orsediments.S~lsand ketone intermediates may be determined, and theirabundancescompamllothatof other sterols. Mudge and
24
Bebianno (1997)usedthe coprostanolfc:bolesterol ratio to ascertain the extent of sewage contaminationinsediments.QuemeneuraDdMarty (1992) c:alc:u1atedthis ratio and the 24- eth ylc:o prostanoUsitosterol ratio asindie:atorsoffreshdomestic: wastewater. Other biomarkers,suc:has the 18:1(011118:1009fatty acid ratio(Quemencw" and Marty,I992)can corroborateevidence of sewage input Theratio of coprostanol to coprostanol plus cho lestanol, Spt(Sa+SP), bas alsobeenused (GrimahetaI.,1990;LietoJ.,I99S;Mudge and BebianDo,1997), as hasthe ratioof c:oprostanol to dinos=ol (Venkantesao andKaplan, 1990).The epic:oprostaDOlto coprostanol ratio can distinguishthefeces ofcertainmarine mammals from thai ofbumans(Venkantesanand Santiago,19 89), although ep;c:oprostanol isalsoprevalentinsewagctreatedinsludge digeston (Grimaltetal.,1990).Lastly,the44•
st eno ne and stanooe intermediates in stanolformationmaybe examined.
1.5 Methods (or lipid _&lysis 1.5.1Sterolaaalyits
While individualsterolsmaybe separated and analysedbyHPLC (AkihisaetaI., 1991 a), GCisthe most commonly used c:hromatographic: method forCheiranalysis.Thefree alcoholsmaybecbromatograpbed (e.g.,Mayzaudet
m .,
1989;Quemeneur&.Marty,1992), but mo st methodsprecedeGC with some form of dcrivatiDtion.Conversion to less polar derivativesallowsconvenientanalysis on commonoon-polar$fati0Dat)'phases, onwbic:hthe free alcoholsteDdtotailexcessively.Sterolaceta1csmaybe prepared (e.g.,WakehametaI., 1980;Itchetal.,1982;ConwayandMcDowcU Capizzo,1991) but trimethylsilylcthcn arc2S
byfar the mostcommonlyusedderivativesformarinesamples.aDd·manyminorvariatioos intheirpreparation havebeenrqlOrted.alongwithreviewsofrel..ativeretention timesaDd ma5Sspeetra(Jooesel al..1994).Wherethefreealcobo lsareanalyz.ed,theirpriorisolation bysilica columnduomatograpby,~vcn.C(e.g.,Mayzaudet01.•1989)or HPLC (e. g., Qucmeneur&.Marty,1992)maybenecessary,although manystudiesemploy this approacbanyway,sinceitinaeases thecertaintythatmainlysterolswillappearmtheGC cbromatopam(e.g. Venbtesanetal.•1987;Colombo et01.,1996;Laurril1ardandSaliot, 1993).SIICb.JR-fractioaatioD.maya150be usedtolsolalcspcc:ificsterolsubclasses,such as SjktaDols(Buller01.•1998),or tosepame4,4-dimethyl.4-mooometbyland 4-desmethyl sterols(Volkman. 1986).
A notablesboncominaofmost GCmethodsforsterolsis their inability to separate C-24 cpimers.SuchseparationhasbeenacbiendODlongcapillary columns(100 mor more)(MaxweUet01.,1980;Thompso netal.,198 1),butanalysis timesan:toolong (3-10 brs)forroutine usc. Reverse- phaseHPLCis useful for separating specificpairsofC-24 epimers(A.lcihisaet01.•199 1b),and IHnuclear magDC'licresonance(NMR) (e.g..Goad, 1991)and X-raycrystallography(Ling etai.,1970)areabletodistinguishthem.although theserequireisolalionofsignificantquantitiesof the iDdividualsterol. Due to sample unavailability andcomplexity,C-24 cpimcn are rarely distinguisbcd
m
marine biogeochemicalstUdies.despite thepocemiaIlOdiffcrmlialc algalBDdhigherplantinputsby C-245teTeOCbemisrry.Sterylesters ateDOldifferentiated from&tiesleI'Olsbycommonmethodswhichbegin
2 .
with saponification (base bydrolysis) of thetotallipKl extract. However,they may be analyzcdintaetbyGC orGC-MS(Wakeham4c Frew,1982; Bulletal.,1998),althoughbigb temperatures arerequiredandanalysistimes are l)'pic.a!.Iylong. Sterylctbmcanbe:
similulyanalyzed(BooDandDeLeeuw,197 9).Polar sterol species present the greatest analyticalchalJenaes. Basehydrolysisdoesnot release sterols&om steryI gJywsides or acyla1cd stef)'lgtycosidcs.andtheyan: thereforeDOCde1ecledby conventional methods for marine sterol analysis which ameralJy involve a1Winc saponification. Fully trimethylsilylatcd ster)'1 glucoside!have been analyz.ed by GC on a short packed column (Lain eandElbein, 1971). Reverse-phaseHPLC maybe:used.althoughdetection is problematic(Heinz, 1996).The acidhydrolysisusedbysome workers (e.g.,Veron etoJ.•
199 6)torelease sterols fromthesepolarconjugatesmay alter thestrue:~of those sterols (Heinz,1996);pcriodate cleavageof tbeetherlin.bceispreferable.
1.5.1 Upiddau_aJyI;iI
AspreviouslyDOted,a pttpamive lipid class sepantionmaybeauseful prccedeut tomolecular spccic:sanalysisofanindividualclass..Often.quantitation by lipid classisthe desiredrcsulL\\Ihile HPLC has beenusedfor this(Gonzal cz -Castroet aL,1996),most wodenhave used some form ofTI..C.
1.5.:1.1latrol UII TLC·FID
Inthe pastfifteenyears,Iauoscanthinlayer chromatography-flameionization 27
detection(JLC~FID)bas become a widelyKCCJKCdtechniquefor lipid class analysis.The analyticalsystemCOIlSistsoflXmquctzrods (Ouvmarods).0.9mmind..iamc1.cr.coated witha75-J411thicklayerofpxoussilicI.gelparticles(5-.umdiameter)sinten::dinto&glass flit(Ac:kman n al..I990).1hesampleis~&1oaeend:oftherodanddeveloped &Sin pla1cTLC.Itisthenpassedthrough•flamcioDiDlioade1a:rorintheIatroscan insaumenl, where each sep.med baldisquantifiedbycombustion.Up to1mrodscanbescanned sim ultaneously. providina:hipsample throughput.. Partialscanning of the rods,andthe further developmentof uncombusted.material, allows multidimensional separationand detection impossibleinconventiooal plate
nc.
Applied tomarine lipid classes,Iauo5C&D TLC-FIDcandistinguishenergystorage classes, pollutants, mcmbrulc lipids, orindicatorsof orpnic matter degradation(c.s.•Goutx
~Iai.,1990).Thetedmiqueisnpec:iaUyuseful foranalyzinj:suchdasses as phospholipids (Hazel,19&5;GeriDandGouct,1993)andpigments whKh are100involatile orpolarfor GC (VolkmanandNKbols.1991);it hasalso been applied totherapiddetenninatioo of tot.al lipidcarboo(Harvey andPanoo,198 1;Oauraet01..1987)and inother methodsasasupport for quantitation rather thIID•separationmedium1""Ie(Kramer~IaI.,1991). Like conven tio Dal TLCplates,theIilic:aCbromarodcoatinaCIabeimprqnalcd withvarious modifiers,forexample.copper(II)sulfalc to increase FIDrnponseand improW' detection uniformity(Kaima1andShantha,19&4),oxalic acid10dIicientJyseparatephospholipidtypes (Banerjee~IaI.1985).boric acid to separa1e isomeric acylalycerols (Tanakaer01.,1980;
TataraetaI.,1983),or silvCT(I)nitrate to separate lipids bydegreeandIorgeometry of
2.
uosaruratioo (Scbedio andAckman.1981;ScbedioetoJ.,1985).Ouuideoflipidanalysis, app licati oDllof n..c-FID have iJxludedqUlll1tiwion of the aliphatic,aromaric.resinand asbphaltene &actionsofcrudeoils(KarlsenandLaner,199 1) and analysisofpolystym::w:and polyisoprene polymen(TakenakaetoJ.,1995).
Theprincipaldrawb8ckofCbromarod
n.c.
indeed 11.Cgeoaally,isiuiDabilityto resolve individualdosdy-rdatedmolecular speciesincomplexsamples. AsDOledabove, molecular speciesanaJysiJofapatticularlipidclassisusuallydoneby GC(Kubisand Myher,1989) orHPLC (Kubis et oJ.•1991). often requiringpre-6actionation, sapo nification, derivatizationor other treatment(Wood.1991). A method allowing individualcompoundswithina band on aChromarod to be analyzedfurtherwouldincrease theamountof information available &omalipidclass separation.Sterols,inpartic:u1ar,illu:stralcthe difficultiesin TI.C-FIDascurrcutly pncticcd.As DOt edabove,theyoccurinthemarine environmentinseveraldifferentformsof varying polariti es.In.typical Chromarodlipid class scpuation usedinourlabontory(Parrish, t981) ,sterolspeciesappearinseveral different bands. Free sterols (FS) are generally resolved intoa pure bandafterdevelopmentinthesecoed solventsystem,although some contaminationof thisbaDdby1,.3-diacy tglycerolsispossible.Sterylesters(SE)co-elute withwax:esters.Lastly,aD)'steryI&Jycosides(SO)oracyIau:dsteryIaIYeosides(ASO)could beexpectedtoeluteinthethird development,although their~valueshave never been determined.Irisexpected thai s&erylchlorin esters wouldalsoelutein this region.
29
1.5.3 CoupliDlofTLCwitbGC orMS
Variousmethodshave beenreported10 coupletherapid,inexpensive, low-resolution n.Cseparation dire<:t1y to further separation oralXuratequantitarion (GC), or superior specificityand identification(mass spectrometry,MS).Direatransfer from
nc
to massspec trometrybasbeen accomplished byXe"bombardment to remove phospholipidsfrom TLCplatesas secondary ions (Xushi andHmda, 1985).andbylaserdesorptionofTI.C- separatedsimple mixtures ofpoJypeptidesanddyes (Gusevetal.,1995),various quincnes, alcoh ols,acidsandaromatic esters (Ramaley et
m.,
198 5), andthe dzug Napmxen (Fanibanda etaL,1994).Laserdesotptionbas alsobeenused to transfer pesticidesfrom nConto aOCcolumn (ZhuandYeung. 1989).Lyle and Tehrani (198130b) reportedthe intr oductio nof polymersandwaler-solublevitaminsfromcut TLCplates intoa gas chromat ographby pyrolysis,althoughthe GC stepwas used primarily for accurate quantitation (already possible in n.c+FID)ratherthanfor further separation, especially since the pyrolytic introductionledto multipleproductsfroma single substanceonthen.c
plate.Ho wever ,allof these methods havebeendeveloped for plate TLC ratherthanto complemen t the specific advantagesof Chromarods, particularlyinlipid analysis.Furthermore.theyrely on 1LCasthe principalsepandionstep for individual molecular species.rather thantaking advantageof thebroadprofiling capabilities of
n.c
for complex,naturally occurring mixtures.30
1.6Objectives
1.6.1 Sterull i.b TriDity Bay. Newtoaadlaad
The collapse ofNewfoUDdlandgroundfishstocks.with its devastating eccecmieand socialimpacts.,providedanimpetusforthe multi-disciplinary Eco-Researchprogram.This three-year study,fundedunder EnvironmentCanada's"GreenPlan".aimed to examine factors contributing tosustainabilityinNewf'mmdlandas a reprcscntative Nonh. Atlantic cold-oceanecosystemaDdsociety.Within this framework,theMarine Team. a~unitof the Eco-researcbprogramcomprising 7workersinMemoria]'s Chemistryand EarthScicnc.es departments and theOceanSciences Centre, aimed10gaugeecosystembc:aIthbyexamining specific sets ofbiomarken (various lipids, phenolic compounds) todetmnincSOlRCS,fates and transformations of organic matterinthemarine enviromnent, and the relative contributions ofmarineV.I'.terrestrialandDatura1V.I'.anthropogenic input (Section1.3).Tbis aspectof the study focused on thewesternTrinityBay region (Fig.1.6).
Inrural Newfoundland,sewageis typicallydisposed ofinbaysand harbourswith littleor no pre-treatmcnt.Thepotential use of SP-stanols as afecal.markers for this input initiallymotivated the study ofsterolsinmarine samples from the region. Inconjunction with other lipid biomarker studies ofCCOS)'stem.health (Budge and Parrish. 1998; Favaro, 1998; Parrish.199 8b), thestudy alsoaimed.to use sterols 10 identify sources and transformations oforganic:matterinmarine environment ofwestem Trinity Bay, as well as ascertain wbetbersewageinputswererecognizableinthecurrent ecosystem orinthe region 'ssedimenwy record.
31
Figurr1.6.Study areaandsampling locauc nsinwesternTrinityBay.Newfoundland.
32
U.2 Lip idaaalylil by11.C.Pynlyli:I-GCIMS
Thesc:coodputof thiswork:aimedtoextmd the~ofChromarod
n.c
by tbennally desorbingthelipidclass baDdsdirectly0I'd0.GC columnforfWtbcranalysis of lhcirc:onstituc:otsbyGC-MS.M wellasprovidiDa;furtber informalioaon molc:cularspecies withineachsepamcdclass.,this would e:larifythepurityand composition ofdiebaDdsbeiDa c:ooco mitaDtly quaDti6ed by FID scanning.espccial1y for tbosc bands whichgroupscveral classes of lipids,such asthesterylesterlwaxesterb&ndaDdtheso-c:alJedacetone-mobile polarlipids (AMPL),a bandinch.tinggIycog1ycerolipids,pigmentsandIDODOlIC)'lglycero ls.Furthermore,the workaimedtodevelopthe tec:hniquesuch thaiit wouldbeofgcner.alutility forawid erange oflipid c1uses,using widely available: columnsandavoidingextremely high elutiontemperaturesandlonge:luti on times.
The effortwastargetedatstcrol-<Ol1tainingbands inCbromatod separations,in conjunctionwiththefirstputof this work.However,otherbands,particularlyaqllipid bands,arealsoof in1eresl:due10their role asenergyIk:Jn&ec:ompouodsiDmarineorganisms.
lbc:DeWtechnique would be applicableDOtoaly tothelipidbiogeocbemM:alstudies CODdudcdin the6'mDeworkofdleEc:o-Resc:archpropam,butalso10other suchstudies and thoseutilizingIatroscan 1l.C.FIDinfood science, biomedical scienceand1qWK:u1nue nutritionstudies.
33
Part I.Biogeochembtryor sterolsinplanktoD, settling particles and sediments in Trinity Bay,Newfoundland
2. Experimental
2.1 Che mie8ls, glassware . .d ialll'1lmeatalloD
AUglasswareused was heated at 450°Covernight or,wherethiswas not possible, rinsed atleastthreetimeswiththe solventtobe usedbefore use.Initially,aUsolventsused forrinsingand for dilutionwere traceanalysisgrade(Omnisolv(BOil) orOptima (Fisher».
Ho wever,blankH~Lhexane plugsinjected into a GC-FIDdid not give appreciablydifferent chro matograms regardJessofwhetherOptimahexane or ACS-gradehexane(BDH,delivered inglass bottles)wasusedfor rinsingorforinjection. Subsequently.ACS·grade hexanewas usedforrinses,andOptima fordilutions.Newly-receivedbottles ofACS-gradehexane were checked forpossible impuritiesbyGC before use.Waterused forrinsingwasdistilled,de- ionized andfiltered(0.2,um) (Nanopure Usystem.Barnstead~Thermo!yDe,Dubuque.lA).
Additionally,water usedto prepare solutionswasextracted 3 timeswithOmnisolv.grade chloroformto ensureaminimumoflip klcontaminants. This extraction is consistent withthe treatmentofwaterused to preparelipidextracts frommarine material (see Parrish. 1998a).
Nine individual neat sterolstandards,plus a coprostanone(5p.ehoIestan-3-one) standard,wereusedforsterolidentification.Thefollowingstandardmixtures wereprepared inhexaneorI:1hexane/chloroform:MixtureI(epico prostaDO!. coprostanone,cbc jesterc l,
34
cholestanol,stigmasterol),85.ugl mLIOta!sterols;Mixnuc2(coprostanol,campesterol, lanosterol,silostero l, fucosterol).57,.,gImltotal sterols.lanostm>I(-65%),fucosterol and coprostanolwereobtained from Sten.&oids (Wahon, NH),whileaDother sterols, coprostanone and cholestane(intcmal standard)were purchasedfrom Sigma(St.Louis,MO ).
Sampleswereanalyzed using • Varian]400 SaJchromatograph with•flame ionization de1ector.GC-MS analysis was done0110.HcwIm·Pw;:kard 5890 UGCwith.
5971AMassSelective Detectorusing70 eV electronionizatto n.
2.2Sa m pliDI
Sediment coreswereobtained from an off-shoresiteinTrinityBay(St.7)(Fig.1.6) andIWOin-sboreshesinthe Northwest Arm ofTrirUtyBay(H-I,H·9)witha 30-<:mboll corerduring tbespringandSUJnmeTofl994.Mooredsediment trap arrays (Parrish, 1998b) weredeployed atSI-9and$t-7during 1994and1995 atdcplmof 50,75and 100m.with fourcoUectonateach depth. FinaUy.vertical(100mdqxhtosurface)and horizontal planktonnet tows(20«m mesh)(thelatterat thedepthofmaximumcbJorophyll fluo rescence. asdetermined withaSeabird
em
equippedv.itbaSeaTech fluorimeter)were takenat sitesSt-7,51-9,St-IIduringMarch,April,MayandJW)Cof 1996,atH-9inMarch andApril1996,and alongatransect(T-I,2,3,4)leading intothebarbourofthe10wn of Trinity during April1995.SamplecoOection and$lorllge aredescribedindetailinParrish (199 8b).35
2.3 S. m pleprep....tio.
Anoverviewof the extractionandanalysismethods usedis showninFig.2.1.
Sediments were processed asdcscribcdbyFavaro (l998).Cores werescctioocd into2-em portions (depth) after removal oftheoutc r Iem (diameter) cfeachccre.Sections wereair- dried overnightinlow light conditionsand visibledebrisremoved.Sedimentswereground witha mortarandpestl cand -10g of eachsection.,plus- Igprc-e1caDcdanhydrous NazS O.wasSoxhletcxtractedfor 24 hours with 9:1dichlo ro metbanc:mctbanol in a pre- extracted ccUuJoscthimble. One quarter oftileextract. previously evaporated todryncssand storedfrozen.,was re-dissolvedinImLof2:1[v/v}hcxaDe:chloroform by sonication(icc bath.I minute) foranalysis.
Sediment trapandnet tow samples were ccljected on pre-eombusted GF!Cglassfibre filters,extracted with 2:Ichloro form:rnethanol by amodifiedFolchprocedure(Folchet ai., 1959;Parrish,1998a),and thelipidsrecoveredinchloroformto giveatotal lipidextract (UE).Sincetherewasoften a considerablelengthoftime(u pto severalmonths)between extra ctio n and analysis.,allTLEswerestoredin the darkWIder N2at·20°Cuntil analysis.
2.4Sapo ni flal tio a , deriv.tiDlioa.ad •••lysis
For sterol analysis,0.1or0.5mLof sample extract (depending on availabilityand anticipatedsterol concentration),wasevaporated to drynessunder a ge ntleflow of nitrogen (UHPor HP+grade),thensaponifiedwith0.1mLsaturated(l2%w/v )KOHinmethanol (75
°C,I b).Afterthe addition of water(0.4mL),thesolutionwasshakenandextracted with 36
FiIU ~1.1.Summaryof analytical proceduresused forsterols 37
6aliquots(0.1-0.2 mLeach)of2:1bexane:cblorofonn.To assistphasescparalaoD,adrop of saturatedNaC IsoJutaoo wasaddedorthesample wascentrifu ged.Tbepooledextracts were evaporated to dryness and derivatizcd with S0-200 .uL of his-N,D- (trime tbylsilyl)tritl uoroacetamide(BSTFA) containing 1'1,trimetbyk:h1orosilane(ThlCS) (Supeko,Bellefonle.PAl (60"C.1h)toproducetheIrimethylsilyJ(TMS)etbers(Fig.2.2 ).
Whereeecessary, 6dropsof silylatioo-gradep)Tidine(Supeko.Bellefont e.PAlwereadded todissolveallthematerialbeforederivatization. Anysamplesoot chromato@l'&Phed inuned"'telyafterderivatizatioowerestoredina desM:catorat-20
"c
withthe derivatizing agent stillinplace.Ho v.rever.inllCOOrdancelNitb theobservatio nsof Joneseta/.(1994 ) coJ¥:.erDingS10rageof sterolTMS ethers,sampb ~geoc:raUyanaIyzcd....ithi:n24 hours ofderMttizatioll.Immediate ly priortoanalysis,thederivatizi:n&agC'Dlwas n'&POra ted underNJ•The TMSethersweredissolved in10-2S.uLbexanecontainingcbolcstancasanintema.lstandard (48.uglmLor later.90 .u&!rnl.) andanalyzedbycapiUaryGC·F1D00 a 30mx0.2S mm 1.0.
(S%phenyJ)polydimethylslloxanecolumn (DB-S.0.12~film;J&WSciemific.PaloAlto, 38
CA).Cho lestane isa widely-usedinternalstandardinsterol analysis (e.g.•Smithet af.1983;
Nichols and Espey.1991;QuemeDeurandMarty,1992).Steranes had been previously confirmed as being absentfrom the TIEs (Favaro.1998).
Table1.1. GasChro matographic conditionsusedfor sterol TMS ethers.
GC- Fl D GC·MS
Injec torporttemperature 290"C 290"c
Detectorltransfer port 315
-c
220"Ctemperature
Oven temperature 80"CforImin., 80"Cfor1 min., pro... 50"Clmin to 235"C. 50"Clmin to 200"C,
5"Clmin to 30 5"C, 5"Clmin to 300"C,
305"C for15min. 305"C for 5 min.
Carrie r gas pressurelflow 20 psi constantpressure 1mUminconstant flow
Eacb samplewasalso analyzed byGC-MS on a25mx0.25mm1.0.CP-Sil
sca
column (100%p.llydimethylsiJoxane;0.12 «m film; OB-I equivalent;Chrompac k.the Nethe rlands). Table 2.1inclicates GC conditions for GC-FlD and GC-MS analysis;helium wasthecarriergas in both cases.ForGC·MS ,the mass selective detectorwasscanned from 35-530 am uat 1.5 scaes/second.SterolTMSetberswere identified by their mass spectra and retention times comparedwithstandardsandpublished spectral data (Goad, 1991;Joneset 01.,1994 )(Table 2.2).Peak areas from GC-ADcbromatograms (Varian Star4.Sintegration software )wereusedfor quantitationexceptwhereotherwise stated.
39
1.5~aIy.iI;oftotal free .teroll
TotalfreesterolsinallTLEs'Weredc1erminedby TI.C-FID on Chromarods
s-m
(parri sh.,1981)usinaan Ia!roscanMk V(IatroD Laboratories.Tolcyo,Japan). Tbc devel opmentprocedureisdiKUSSCd in moredetailin Section 4.4. Each extnet was simultaneouslyanalyzedinttipli uteonadjKCDt Cbromarods.Cholesterolwu wedas an exteTDa1quantitatiorl.standaniandbcxadccm-3~as aninttmalstaDdani.
2.6 Floristic:.aaly•• ud eon dadal
Subs am plesof theplankton tow and sedimenttrapsamp lesweretakenpriorto filtrationandpreservedwith Lugol'siodiDeand10%bufferedaqueous formaldehyde(ImL each).A 200-;.:L aliquotwas observedat200 x magnificationunder aninverted Zeiss compound microscope.Anocularmicrometerand appropriate p>meuicshapes wereused to calculate biovolumes(BudacandParrish, 1991).
Subsamples&omthreelIC:dime:Dtcom;(St.7.H·l,H-9)~datedusiDgJ~byDr.
Jack CornettatMycore Inc.•DeepRiver , ON.Jlopb(...22yean)isadecay product of atmospbcricU1RawtDcbisdeposited.fromtheatI:D05pberconto theocean surfaceAIa stable rateandincorporatedintosettlingparticlesandthus intosediments (Schell. 1982).
40
T.ble2.2.Elution orderand characleristicGC~MSions forstero ls detected.Nosingle samplecontainedallthesterols described.
Trivialname Abbreviatio" Characteristic ions 24-nordchydrocbolesterol 24nor-26~'.22E 442,255,313,129,97 24-nordchydrocholcstanol 24DOr_26~:t2E 444,374, 345,257
coprostanol 5P_27~o 445,370,316
ep icopro stanol 31l,5ll-274° 445,370,316
occclasterol 24Mc_24nor_27..:1'.22E 456,366,255,III rrons-22-dchydrocholcsterol 274'.JJE 456,366,255,III
rrans-22-dcbydrocbolesunol 274m: 374,257
cholesterol 274' 458,443,368,353,129
cholestanol 274° 460,445,370
dcsmosterol 274'.2· 456,372,343,129
brassicasterol 24Me-28~'.22E 470,455.380,341,129
brassicastaool 24Me_28~Z2E 472.374.34S
24-methylenecbolesterol 24Me_28~'.2ol(11} 470.386,374,129 24-metbylenecbolestanol 24Me--284!....211 472.255,2IS 24-mcthylcholesterol 24Me--284' 472,382,129
4-methylcbolestanol 4uMe-284° 474,459,384,229
24-methylcbolestanol 24Mc-284° 474,459,384.215
ethylcoprostanol 24Et-5P-294° 473,398,257,215
dimethyldehydrocbolcstanol 4u,24diMe--294nE 486,388, 357 ethylchoJcsta-S,22E-dicnol 24Et-294',m- 484,394, 255, 129 ethylcholest-22E-eno1 24Et-29422£ 486,374, 345,257
ethylcholcsterol 24Et-294' 486,396.129
ethylcholestanol 24Et_29~o 488. 398,215
fucosterol 24Et_29~u'(2IlC 484,386,296,129
41
T.ble%.2.(Co ntinued)
"""""""I 24Et-2942~ 486.388,373,34 5
debydrodinostcrol 4a..23,24triMe-3M'.zu 318,359 , 297,129.69
isofucosteroJ 24Et-294'.2oOl2N 484. 386,296,129
isofucostmol 24Et-294 N(1t),Z 486, 388,373,345
dinomrol 4a..23,24triMe-30422E SOOt388,359
unidentified~'Renlrienol 304'... 496,313,129
42
3.Resul ts aDddiscussioD
3.1Method vaUdatioa 3.1.1Completeaeuo(derlntizatio_
Conversion of sterolstandards totheirTMSethers (Section 2.4)wasgenera.lly~ry efficient,with residualfreesterols detected in only one instance in theexamina t ionof a GC!MS extracted ion chromatogram. These free coprostanol, choksteroland stigmasterol peaks had areas of 3.85%, 3.66%and 10.6"... respectively,oflbe peaks fortbeirTMS ether derivatives(quantitation byGCIMS peak areas, assuming equal detector responses),although the firstis an underestimate due to the contnbution ofcoprostanone to the coprostanol TMS etherpeak.Thetemperatureand reactiontimeused are similar to those found satisfactory bymanyworkers(e.g.,Bayonaet al.,1989;Grimahetal.,1991;Laureillardand SaIiot,1993;
Jones et al. ,1994;Nicholset ai.,1996 ), ahboughBulletal.(1998) allowed 12 boursfor
5p·
stanols from soils to react completely with OSTIA. No rearrangements oflbe standard stero ls occurred duringthe saponification step. wlUch each one being detectedbothbefore and afterthe procedure. Lastly,itwasassumed that derivati2ation efficiency differed negligiblybetweendifferentstero ls, since the unavailability of a widerangeof standards would in any case DOtallowthisto be tested foraUthe sterols detected in thesamples.
3.1.2 Liourlty . .deq. iTale_eyofdeteetor rnpo_se
SinceJoneselai.(J 994) cautionthatf1Dresponse fordifferent sterol structures may
43
differ up totwo-fol d,the FlOresponsesof difftm1tsterolswerestudied todetamioe if corTtttionfac1Of'SwoukIbe required.. Tbm:-com.ponentstandards(choleskrol, coprostanol andstigmasterol)werepttparedinhexaneat3differal1c:onc:entntions;scriaIdilutionsfrom these lave afurther four concentra tions.Tbesethreesterols werechosentoreprnenta varietyof structural features(~Sp...stanol,c;,-A'-s1efOI,~steradic:nol).A 1OO-~Laliquot of eachstan dard was evaporatedtodryness,derivarizedwith50~LBSTFA (Section 2.4), andmadeup102S~Lwithhexanecontaining 0.0899mglrnLcholestane.Eachwasanalyzed intriplicatebyGC~FID. Calibration curves,with95%confidence inlervaiSaroundthe regressionlines,are shownAppendixI.Tbefollowinglinearfluwereobcaincd..withxand yrepresenting sterol conc:cntration(mglmL)and(SIeTOIFlOrespooseIintcmalstandard F1D response ) (unitless), respectively:
Coprostano l.
Cholestero l:
Stigmast ero l:
yo.lOS(±6)x-O.O(±O.3 ), y-I 06(± 4 ) x-0.8(±O.3) , y-107(±I3) x -0.9 (±I.l),
r-0.946(0-21) r-0.978(0=>2 1)
r
-0. 767(n=2 1)Whilediecorrelati on coefficien t for thestigmasterolcalibntionc:urvewaspoorer (r
=0.767),allthree correlationsweresignificant(p<:t>.01) (Sokaland Ro hlf.1931).
Furthennore,thestandarddeviationsforthe line slopesalloverlap,suiiesting thatdetector response isnotsigni fican tlydiffere n t forthese threesterols, andIhestandard deviations of
44
mey·in tercepts ailover lapped zero.Given this.andgiven thatthepauciEyofstandards available wouldprecludethe calculationof FIDresponsecorrectionfactors for all the sterols detected.aUsubseq uen tdatawasprocessedassumingequalddec10r responsefOf'allsterols.
3.1.3Recovery of free .tero haad.teryl nfen
Standard solutio ns of free cholesterolandof cbole sterylhexadecaaoate(paImitale ) were preparedinhexane.and a1iquo ts of eachdispensedin ordertodeliver 3.8.uSof cholesteroloritsequivalenLTwofreecholesterolandtwocholesterylpalmita tesampks were saponified,extractedandderivatized asperSection2.4.
Table 3.1Detectorresponses: forequi valen tamountsof cholesterolinfreeandesterified
'ann
Formofch o lestero l Chole sterolFlOresponse/ln temalstandan:IFlO response(meaneSO.•n-2)
Freecholesterol 1.94±O.OJ
Cholesterylpalmitate 1.9 ±O.2
Theamount of choleste roldetectedafter saponificationandderivatiza tiondid not differsigni ficantly(two- tailedStudent'st-test,P<O.OS)between cholcsteJyl palmitateand freecholesterol(Tabl eJ.t).Thisresult suggests lhatsterylestersarereadilydetectedbythe method.althoughonlyonecompoundfrom eachclasswasused.
45
