The effect of different fatty acid sources on wound healing in rats assessed by matrix-assisted-laser-desorption-ionization mass-spectroscopy-imaging
Abstract
The objective of the present study was to compare the effects of dietary oils containing polyunsaturated fatty acids (PUFA) n-3 and n-6, respectively, on cutaneous wound healing in rats, and to demonstrate the usefulness of the matrix-assisted-laser-desorption-ionization mass spectroscopy-imaging (MALDI MSI) method in this type of experiment. Superiority of PUFA n-3 in this context was the tested hypothesis. Four groups of male Wistar rats by twelve animals each were fed a diet with added 5% of palm oil (P; control), fish oil (F), Schizochytrium microalga oil (Sch) and safflower oil (S), respectively, for eight weeks. Consequently, dorsal full-thickness cutaneous excisions were performed, and selected markers of wound healing were evaluated 18 days post excision. The median of signal intensity corresponding to an amount of collagen alpha-1 (III) fragment, quantified using MALDI MSI, decreased in a sequence P > F > Sch > S (P < 0.001). Using haematoxylin-eosin staining of the histological preparations, semiquantitatively assessed epithelium height tended to decrease in the order of P > S > Sch > F; the wound extent in the sequence of P > Sch > S > F; and the sequence of the progress of neoangiogenesis was assessed as S > P > F approximate to Sch. It was concluded that the tested hypothesis was confirmed only partially: PUFA n-3 showed better results regarding the wound extent, but were inferior in terms of epithelium height and progress of neo-angiogenesis. This was the first time MALDI MSI was successfully employed for evaluating skin wound healing in a rat model.
Full text
The e ec o di e en a y acid sou ces on wound healing in a s assessed
by ma ix-assis ed-lase -deso p ion-ioniza ion mass-spec oscopy-imaging
Tomáš Komp da1, Zbyšek Sládek2, Ve onika Š ehlo á1, Zuzana Lacko á3,4,
RomanGu áň3,4, Tomáš Do3, Jan Wijacki2, Ma cela Buch o á5, Jana Neuwi ho á6,
Bře isla Gál6,Voj ěchAdam3,4,OndřejZí ka3,4
1MendelUni e si yinB no,Depa men o FoodTechnology,B no,CzechRepublic
2MendelUni e si yinB no,Depa men o AnimalMo phology,PhysiologyandGene ics,B no,CzechRepublic
3MendelUni e si yinB no,Depa men o Chemis yandBiochemis y,B no,CzechRepublic
4B noUni e si yo Technology,Cen alEu opeanIns i u eo Technology,B no,CzechRepublic
5CzechAcademyo Sciences,Ins i u eo AnimalPhysiologyandGene ics, . .i.,B no,CzechRepublic
6Masa ykUni e si y,S .Anne’sHospi alandFacul yo Medicine,Depa men o O o hinola yngologyandHead
andNeckSu ge y,B no,CzechRepublic
Recei ed Ma ch 15, 2019
Accep ed Oc obe 29, 2019
Abs ac
The objec i e o he p esen s udy was o compa e he e ec s o die a y oils con aining
polyunsa u a ed a yacids(PUFA)n-3andn-6, espec i ely,oncu aneouswoundhealingin
a s,and odemons a e heuse ulnesso hema ix-assis ed-lase -deso p ion-ioniza ionmass
spec oscopy-imaging(MALDIMSI)me hodin his ypeo expe imen .Supe io i yo PUFA
n-3in hiscon ex was he es edhypo hesis.Fou g oupso maleWis a a sby wel eanimals
eachwe e edadie wi hadded5%o palmoil(P;con ol), ishoil(F),Schizochy iummic oalga
oil(Sch)andsa lowe oil(S), espec i ely, o eigh weeks.Consequen ly,do sal ull- hickness
cu aneous excisions we e pe o med, and selec ed ma ke s o wound healing we e e alua ed
18dayspos excision.Themediano signalin ensi yco esponding oanamoun o collagen
α-1 (III) agmen , quan i ied using MALDI MSI, dec eased in a sequence P > F > Sch > S
(P < 0.001). Using haema oxylin-eosin s aining o he his ological p epa a ions, semi-
quan i a i elyassessedepi heliumheigh ended odec easein heo de o P>S>Sch>F;
hewoundex en in hesequenceo P>Sch>S>F;and hesequenceo hep og esso neo-
angiogenesiswasassessedasS>P>F≈Sch.I wasconcluded ha he es edhypo hesiswas
con i medonlypa ially:PUFAn-3showedbe e esul s ega ding hewoundex en ,bu we e
in e io in e mso epi heliumheigh andp og esso neo-angiogenesis.Thiswas he i s ime
MALDIMSIwassuccess ullyemployed o e alua ingskinwoundhealingina a model.
Docosahexaenoic acid, eicosapen aenoic acid, Schizochy ium oil, eepi helializa ion, MALDI
MSI, collagen alpha-1(III)
Woundhealingisadynamicsequen ialp ocessconsis ingo h eeo e lappings ages:
in lamma ion,cellp oli e a ionand issue emodelling(Cae anoe al.2016).Thee ec s
o die a y a yacids,especiallylong-chainpolyunsa u a ed a yacids(LC-PUFAn-3)
eicosapen aenoicacid(EPA;20:5n-3)anddocosahexaenoicacid(DHA;22:6n-3),known
in lamma ion modula o s, on wound healing in animal models a e e y inconsis en
(Komp da2018):posi i ee ec s(Chene al.2012;Olsone al.2013)a econ adic ed
bynega i eones(D zymala-Czyze al.2012; dos San os Rosae al.2014),wi h
be e esul sbeingo enob ainedwi hoilscon ainingPUFAn-6(O an oe al.2010).
The main objec i e o he p esen s udy was o e alua e he e ec o ish oil (high
EPAcon en )andaSchizochy iumsp.mic oalgaex ac ( ichinDHA), espec i ely,in
compa isonwi hsa lowe oil(PUFAn-6linoleicacid)onselec edma ke so hehealing
o acu aneousexcisionin a s.Asmalle ex en o hewounda hegi en imein e al,
be e eepi helializa ion and neo- ascula iza ion sco e by die a y oils con aining LC-
PUFAn-3wasselec edas he es edhypo hesis.Oneo heobjec i eswasalso ouse
ACTA VET. BRNO 2019, 88: 443–449; h ps://doi.o g/10.2754/a b201988040443
Add ess o co espondence:
Tomáš Komp da
Depa men o FoodTechnology
MendelUni e si yinB no
Zemedelska1,61300B no,CzechRepublic
Phone:+420545133261
E-mail:[email p o ec ed]
h p://ac a e . u.cz/
o he i s ime hecu ing-edge me hod o ma ix-assis ed-lase -deso p ion-ioniza ion
mass-spec oscopyimaging(MALDIMSI;Angele al.2018) o moni o ing hespa ial
dis ibu iono aselec edcollagen agmen incu aneousexcisionsin a s.
Ma e ials and Me hods
Animalsanddie a yin e en ions
Fo y-eigh adul male a s o he labo a o ys ainWis a Albino(BioTes ,Koná o ice,Czech Republic)
a heageo 8weeks( hemeanli eweigh o 230±24.0g)we eused.The a swe ehousedinplas icboxes
(53.5×32.5×30.5cm)o ou animalseachina oommain aineda 23±1°C,humidi yo 60%and12/12h
o ligh /da kcycle(maximumin ensi yo 200lx).Theexpe imen waspe o medincompliancewi h heCzech
Na ionalCouncilAc No.246/1992Coll.on hep o ec iono animalsagains c uel yand heamendedAc No.
162/1993Coll.,andwasapp o edby heCommi ee o heP o ec iono Animalsagains C uel yo heMendel
Uni e si yinB no(S a emen No.16252-MZE-17214).
The a swe edi idedin o ou g oupsby12animalseachand edeigh weekswi hei he hecon oldie
consis ingo basic eedmix u e(BFM;pelle izedcomple echow o miceand a s;Biok on,Blučina,Czech
Republic)en ichedwi h5%o palmoil(P),o oneo he h eeexpe imen aldie s:BFMsupplemen edwi h5%
o ishoil(F),5%o oilex ac ed om heSchizochy iumsp.mic oalga(Sch),and5%o sa lowe oil(S;high
con en o linoleicacid), espec i ely.The eason o using hePdie asacon olins eado BFMalonewas he
equi emen okeep hedie sno onlyisocalo ic,bu alsoisolipidic.Fa yacidcomposi iono he ou die sis
showninTable1.
Thedie swe ep epa ed(a helabo a o y)as ollows:pelle izedchowwasg inded,homogenizedwi han
app op ia eamoun o hepa icula oilandcakesweighingapp oxima ely200gwe ep epa edbyhand.The
animalswe e eddailyad libi umandhad eeaccess od inkingwa e .Feedconsump ionwasmeasu eddaily
pe gi enbox;daily eedconsump ionpe a wascalcula edasaone- ou ho he o alin akepe cage(due o
he ad libi umaccess o he eed,majo di e encesinconsump ionbe weenanimalsinaboxwe eno p esumed).
Theanimalswe eweigheda weeklyin e als.
Excisionsandsamplecollec ion
A e 56 days o a ening, all animals we e anaes he ized by in amuscula adminis a ion o zolazepam
(Vi bac,Ca os,F ance),and wo ull- hickness(epide mis,de mis)cu aneousexcisionson hedo sumwe e
pe o medin hesagi alplaneusingaci cula punche wi hanin e naldiame e o 8mm(S ie elLabo a o ies,
B en o d,UK).Fo echnical easons, he o alnumbe o animalswas es ic ed o48;conside ing12animals
henecessa ynumbe o su icien cha ac e iza iono hedie a yg oupin hehealing imepe iod,wega eup
heideao assessingse e alsubsequen healingphasesand(basedonap elimina yexpe imen )op ed o one
18-dayin e al.A e heexcision, he a swe ehousedindi idually: woanimalswe eplacedin hesameplas ic
boxasmen ionedabo e,pa i ionedwi hac ossba in o wopa s.Thewoundswe echeckeddaily o in ec ions
andsince he ewe enone,noaddi ionalwound ea men waspe o med.A e 18dayso con inua ionon he
pa icula die (P,F,Sch,S), heanimalswe esac i icedbyiso lu aneo e dosingand he ollowing issueswe e
aken: wosampleso hewoundedskin o hep oduc iono his ologicalp epa a ions(includingMALDIMSI)
anda5galiquo o heli e o de e mina iono a yaciddeposi ion.
444
Table1.Fa yacidcon en in he a die ssupplemen edwi h5%o palmoil(P–con ol),5%o sa lowe oil(S),
5%o ishoil(F)and5%o Schizochy iumsp.mic oalgaex ac (Sch), espec i ely.
Die
P S F Sch
To allipids(%) 6.8 6.5 6.4 6.5
16:0 33.0 0.1 0.5 16.2
18:0 4.7 3.3 5.0 3.8
Fa yacidcon en (%o heƩ 18:1n-9 36.6 17.1 39.7 22.0
o allde e mined a yacids) 18:2n-6 21.1 73.3 24.3 21.8
18:3n-3 0.1 0.1 0.4 0.2
20:5n-3 0.2 0.5 6.3 1.0
22:6n-3 0.2 0.1 9.4 21.9
PUFAn-6/PUFAn-3 53.9 87.6 1.7 1.1
Fa yacidsde e mina ion
Thecon en o a yacidsin hedie sandin heli e wasde e minedaspe ou p e iouss udy(Komp dae
al.2017).
His ologicalp epa a ions
Skinwoundsamplesincludingca2mmo skinsu ounding hewoundwe e ixedin10%bu e ed o malin,
dehyd a edbyag adualalcoholse ies,clea edinxylene,embeddedinpa a inblocksandsec ionedin o he
sizeo 5–8µmusinga o a ymic o omeIn acoMic omodelMT5505(In acoMic oL d.,Tachlo ice,Czech
Republic).Sec ionswe esubsequen lys ainedwi hhaema oxylin-eosin(H&E).Thehis ologicalp epa a ions
we e hene alua edusingaligh mic oscopeOlympusBHTSPM1(OlympusAme icaInc.,USA).Mo phome y
ande alua iono hequali a i echangeswe eca iedou ineachsamplemanuallyusing heocula mic ome e
a ×5(woundex en )and×200( eepi helializa ionex en ),magni ica ions.A leas h eesec ionswe ep epa ed
omeach issuesample.Mo phome ywas hende e mineda e digi aliza iono hemic oscopicp epa a ions
bymeanso heso wa eLucia(Labo a o yImagingL d.,P ague,CzechRepublic).
MALDIMSIexpe imen
Fo malin- ixedandpa a in-embedded issuesec ionswi h8µm hickness,moun edon oindium- inoxide
(ITO)glassslides,we ep epa edacco ding oHege e al.(2016).
The mass spec ome y imaging was pe o med on a MALDI-TOF/TOF mass spec ome e B uke
ul a leX eme(B uke Dal onikGmbH,B emen,Ge many)usingap o ocolacco ding oHege e al.(2016).
A o al sample se spanned 8 ITO glass slides con aining 24 issue sec ions.As MALDI ma ix, 30 mg/ml
2,5-dihyd oxybenzoicacid(DHB)inme hanol/wa e (50:50, / )wi h1% i luo oace icacid(TFA)wasused.
The as e spo diame e was30μm.MALDIMSIo pep ideswaspe o medina e lec o posi i emodein he
m/z angeo 0–5kDa.A o alo 500spec awe esummed o eachspo using heRandomWalk as e pa e n,
wi hnoe alua ionc i e ia.Them/zimageswe egene a edand isualizedusing heSCiLSLab2014bso wa e
(SCiLS–B uke Dal onikGmbH,B emen,Ge many).FinalMALDIMSIimageswe ep epa edin heGIMP
2.8so wa e.
Fo on- issue diges ion collagenaseA om Clos idium his oly icum was used.App oxima ely 300 μl o
collagenaseAsolu ion(1mg/mlinwa e )pe oneslidewasappliedbyImageP ep(B uke Dal onikGmbH,
B emen,Ge many), heslidewaskep insideasmallboxwi hhumida mosphe ea 37°C o 24hand heDHB
ma ixsolu ionwasapplied.P io oMSIanalysis heslidewasd iedina acuumdesicca o o 15min.
F omdi e en ypeso collagen,collagenIII( oge he wi hcollagenI)a e ypical o heskin; he e o e,
collagenalpha-1(III)chain agmen GAPGFRGPAGPNGIPGEK(acco ding oOk aye al.2018)wasused
o analysis.
S a is icalanalysis
TheMSIda awe ee alua edbyAnde son-Da lingno mali y es andK uskal-Wallis es wi hSCiLSLab
so wa eacco ding oGu ane al.(2017).His ologicalp epa a ionswe eonlysemi-quan i a i elyassessed,and
he e o ewe eno s a is icallye alua ed.Rega dingsupplemen a ymeasu emen s(weigh cha ac e is ics, a y
aciddeposi ionin heli e ), hedi e encesbe weendie a yg oupswe ee alua edbyone-wayANOVAwi hpos
hocTukey’s es using heSTATISTICA12package(S a So ,Tulsa,OK,USA).
Resul s
Weigh cha ac e is icsand a yaciddeposi ionin heli e
Li eweigh o hecon ol(P-),S-,F-andSch- a sa e 56dayso a ening( inalli e
weigh be o eexcisions)was536.8±13.7,507.5±7.7,519.1±9.8and521.7±6.2g,
espec i ely, and did no di e be ween die a y g oups (P > 0.05). Daily weigh gains
(DWG;wi hin hepe iodun il heexcision,56days)o heP-,S-,F-andSch- a sa e
p esen ed in Fig. 1 which shows ha , in compa ison wi h con ol, only sa lowe oil
dec eased (P < 0.05) DWG o a s. Deposi ion o EPA+DHA in he li e is shown in
Fig.2.Thecon en o hebiologicallyac i esubs ances es edin hismainme abolically
ac i eo ganindica es hei a ailabili y o hep ocess o woundhealing, amongo he
hings.
His ologicalexamina ion
Semi-quan i a i elyassessed(H&Es aining)epi heliumheigh dec easedin heo de
o P>S>Sch>F(Pla eIII,Fig.3A).The o alex en o hewounddec easedin he
sequenceo P>Sch>S>F(Pla eIII,Fig.3B).Finally, hesequenceo heex en
o neo-angiogenesiswasassessedasS>P>F≈Sch.Collagen ib eswe eo ien ed
445
inpa allelin hebasalpa o heg anula ion issueo hecon olsamples(Pla eIII,
Fig.3Ba); heS-p epa a ions(Pla eIII,Fig.3Bb)didno di e in his ega d.Ho izon ally
a anged collagen ib es swi ched o an all-di ec ion o ien a ion in he g ea e dep h
o heF-p epa a ions(Fig.III3Bc).Thecollagen ib eso ien edinpa allel ha we e
accumula eda hebounda ybe ween heg anula ion issueand heco ium,swi ched
(simila as heF-samples) oanall-di ec iono ien a ionin heSch-p epa a ions(Pla e
III,Fig.3Bd).
446
Fig.1.Dailyweigh gaino a s ed56daysadie supplemen edwi h5%o palmoil(P;con ol),5%o sa lowe
oil(S),5%o ishoil(F)and5%o Schizochy ium sp. mic oalgaex ac (Sch), espec i ely;a,b–meanswi h
di e en le e sdi e a P<0.05,one-wayANOVAwi hpos hoc Tukey’s es
Fig.2.Deposi iono eicosapen aenoicacid(EPA)anddocosahexaenoicacid(DHA)in heli e o a s edadie
supplemen edwi h5%o palmoil(P;con ol),5%o sa lowe oil(S),5%o ishoil(F)and5%o Schizochy-
iummic oalgaex ac (Sch), espec i ely, o 74days(56daysbe o e+18daysa e excision);A,B–means
wi hdi e en le e sdi e a P<0.05(no indi idual a yacidsbu sumso EPA+DHAa ecompa ed);one-way
ANOVAwi hpos hocTukey’s es .
Die a y ishoilshowedmo ead anceda angemen o collagen ib esand ended o
dec ease hewoundex en 18daysa e excisionincompa isonwi hcon ol(P)which
wasaposi i e inding,bu simul aneously ended owo sen heou comein e mso he
epi heliumheigh andap og esso neo-angiogenesis.
MALDIMSIo acollagen agmen
The spa ial dis ibu ion o collagen alpha-1 (III) chain agmen
GAPGFRGPAGPNGIPGEK, m/z 1679.873 ± 0.100 Da, is shown in Fig. 4 (Pla e III,
Fig.4).The egionswi h hehighes in ensi ieso his agmen we e oundin heskin
issuesec iono con olg oupP; hein ensi ies ended odec easeina owP>F>S>Sch.
Mo eo e , heda ashowninFig.4(yellowlines)con i med ha hehealingp ocesss a ed
on hesideso heincisionandcon inued owa ds hecen e.Basedon hein ensi ybox
plo s(Pla eIV,Fig.5), hemediano in ensi ywasdec easing(P <0.001)inasequence
P>F>Sch>S.The e o e, heou pu o heMALDIMSImeasu emen sdi e edsomewha
om hehis ologyda a.
Discussion
Thee ec o die a yoilon heweigh cha ac e is icso a s
The epo ede ec so PUFAn-3onDWGo a sa econ adic o y:noe ec o die a y
ishoil(Campiolie al.2012);a endency oinc easeDWGin heF- a sincompa ison
wi h sa lowe oil ( his s udy; Fig. 1); a signi ican DWG dec ease in he Sch- ed a s
incompa isonwi hsa lowe oil(Komp dae al.2016).Thecon adic ionco obo a es
he con lic ing epidemiological e idence o a p esumed in e se associa ion be ween
consump iono LC-PUFAn-3andobesi y(Howee al.2014).
His ologicale alua ion
Ge ceke al.(2007) epo edsigni ican lysmalle meanwounddep hbu lowe mean
sco es o epide malandde mal egene a ion,g anula ion issue hicknessandangiogenesis
in a s ea ed wi h pa en e al ish oil emulsion 18 days a e he skin wounding in
compa isonwi h hecon ol,whichwas e ysimila oou esul s.
On heo he hand, hepe cen ageo woundclosu ese endaysa e heexcisionon
he back o a s was highe in con ol han in he ish oil- ed g oup in an expe imen
byO an oe al.(2010).Thepe cen ageo e-epi helializedwounda ea18dayspos
excisionwasno di e en be ween hesun lowe oiland ishoilg oup,bu waslowe in
he ishoilg oup hanin hecon ol.Theseda apa iallyco espondwi h he esul so he
p esen expe imen ,whe e heheigh o heepi heliumin heF- a s ended obelowe in
compa isonwi h hecon olg oup.
O an oe al. (2010)also epo ed delayedangiogenesis and impai ed o ma iono
he g anula ion issue in he ish oil g oup and in he sun lowe oil- ed a s; esul s o
he p esen expe imen a e pa ially in acco dance wi h he quo ed s udy: we can also
in e abe e sco eo die a ysa lowe oil han ishoilas a as e-epi helializa ionand
ascula iza ionisconce ned.
Mappingo collagendis ibu ionbyMALDIMSI
Themos ecen andac ually heonlys udyinwhichMALDIMSIwasusedinwound
healinge alua ed hedis ibu iono lipidsin ol edin heskinba ie epai /woundhealing
p ocessusinganin i oli ingskinequi alen model(Lewise al.2018).Howe e , he
animal( a )modelusedin hep esen s udyismo esui able hanin i omodels ega ding
he ele anceo gainedin o ma ion ona u albiochemicalp ocessesoccu inginali ing
o ganism.
447
Theuse o o malin- ixedandpa a in-embedded (FFPE) issuesec ions o MALDI
MSIhasi slimi a ions ega ding hep ocesseso depa a iniza ionandan igen e ie al,
du ingwhich heanaly eso in e es canbedilu edandmo edelocalizedin he issue.
Also, hee iciencyo hei ioniza ionisusuallywo se hanin hecaseo c yosec ions
(Pie owskae al.2016).
I canbeconcluded ha hehypo hesis es edin hep esen s udywasonlypa ially
con i med: he esul s show ambiguous e ec s o oils con aining PUFA n-3 on he
his ologicalma ke s,including hei inconclusi ee ec on heweigh cha ac e is icso
a s.On heo he hand, hep esen expe imen con i med hepowe o heMALDI-TOF
MSI echnique,whichwasapplied o he i s imein he a skinhealingexpe imen .In
o de oinc easee ec i i yo PUFAn-3inwoundhealing, heuse ulnesso hei opical
applica ion in a combina ion wi h nanopa icles would be wo hy o es ing in u u e
expe imen s.
Acknowledgemen s
Theexpe imen was inanciallysuppo edby heIn e nalG an Agencyo MendelUni e si yinB no(p ojec
no.TP3/2017)andbyCEITEC2020(LQ1601).
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Fig. 3. Reepi helializa ion (A) and wound ex end (B) 18 days pos excision in a s ed a die supplemen ed
wi h 5% o palm oil (P; a), 5% o sa lowe oil (S; b), 5% o ish oil (F; c) and 5% o Schizochy ium mi-
c oalga ex ac (Sch; d), espec i ely o 74 days (56 days be o e + 18 days a e excision). Haema oxylin&
eosin s aining. Magni ica ion was ×200 o (A) and ×5 o (B). The epi helium and i s wid h is highligh ed
by yellow dashed lines and a ows
Fig. 4. Haema oxylin-eosin images combined wi h ma ix-assis ed-lase -deso p ion-ioniza ion- ime-o -
ligh mass spec oscopy imaging o he m/z 1679.873 ± 0.100 Da collagen alpha-1(III) chain agmen
GAPGFRGPAGPNGIPGEK, in he de mis 18 days pos -excision in a s ed (56 days be o e + 18 days a e
excision) a die supplemen ed wi h 5% o palm oil (P – con ol; a), 5% o sa lowe oil (S; b), 5% o ish
oil (F; c) and 5% o Schizochy ium mic oalga ex ac (Sch; d), espec i ely. The egions wi h he highes
in ensi y o m/z 1679.873 ± 0.100 Da agmen a e highligh ed by ed o als
Pla e III
Komp da T. e al.: The e ec o di e en ... pp. 443-449
Fig. 5. In ensi y box plo o he m/z 1679.873 ± 0.100 Da collagen α-1(III) chain agmen
GAPGFRGPAGPNGIPGEK in he issue sec ions o he de mis 18 days pos excision in a s ed (56 days
be o e + 18 days a e excision) a die supplemen ed wi h 5% o palm oil (P – con ol), 5% o sa lowe
oil (S), 5% o ish oil (F) and 5% o Schizochy ium sp. mic oalga ex ac (Sch), espec i ely; each die a y
g oup is ep esen ed by six issue sec ions, se e al ens o housands o he mass spec a we e measu ed
wi hin each sec ion, and in ensi y o each mass spec um is depic ed by a spo wi hin he plo ; he in ensi y
dec eased in he o de o P > F > Sch > S (P< 0.001; K uskal-Wallis es )
Pla e IV