Dietary Conjugated Linoleic Acid-Enriched Cheeses Influence the Levels of Circulating n-3 Highly

16 Pages·2017·1.2 MB·English

International Journal o f
Molecular Sciences
Article
Dietary Conjugated Linoleic Acid-Enriched Cheeses
Influence the Levels of Circulating n-3 Highly
Unsaturated Fatty Acids in Humans
ElisabettaMurru1,†,GianfrancaCarta1,† ID,LinaCordeddu1,MariaPaolaMelis1,
ErikaDesogus1,HastimansoorehAnsar1,‡ ID,YvesChilliard2,AnneFerlay2,
CatherineStanton3,MairéadCoakley3,R.PaulRoss4,GiovanniPiredda5,MargheritaAddis5,
MariaCristinaMele6 ID,GiorgioCannelli6,SebastianoBanni1,*andClaudiaManca1
1 DipartimentoScienzeBiomediche,UniversitàdegliStudidiCagliari,09042Monserrato,Italy;
[email protected](E.M.);[email protected](G.C.);[email protected](L.C.);
[email protected](M.P.M.);[email protected](E.D.);[email protected](H.A.);
[email protected](C.M.)
2 UniversitéClermontAuvergne,INRA,VetAgroSup,UMRHerbivores,F-63122Saint-Genès-Champanelle,
France;[email protected](Y.C.);[email protected](A.F.)
3 APCMicrobiomeIreland,TeagascFoodResearchCentre,Moorepark,Fermoy,P61C996Co.Cork,Ireland;
[email protected](C.S.);[email protected](M.C.)
4 APCMicrobiomeIreland,UniversityCollegeCork,T12YT20Cork,Ireland;[email protected]
5 ServizioperlaRicercaneiProdottidiOrigineAnimale,AGRISSardegna,Loc.Bonassai,07100Sassari,Italy;
[email protected](G.P.);[email protected](M.A.)
6 FondazionePoliclinicoUniversitarioA.GemelliIRCCS,00168Roma,Italy;
[email protected](M.C.M.);[email protected](G.C.)
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
† Theseauthorshavecontributedequallytothiswork.
‡ Currentaddress:MinimallyInvasiveSurgeryResearchCenter,IranUniversityofMedicalSciences,
1449614535Teheran,Iran.
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:17April2018;Accepted:5June2018;Published:11June2018
Abstract: n-3 highly unsaturated fatty acids (n-3 HUFA) directly and indirectly regulate lipid
metabolism, energy balance and the inflammatory response. We investigated changes to the n-3
HUFAscoreofhealthyadults,inducedbydifferenttypesandamountsofconjugatedlinoleicacid
(CLA)-enriched (ENCH) cheeses consumed for different periods of time, compared to dietary
fish oil (FO) pills (500 mg, each containing 100 mg of eicosapentaenoic and docosahexaenoic
acids—EPA+DHA)orα-linolenicacid(ALA)-richlinseedoil(4g,containing2gofALA).Asignificant
increaseinthen-3HUFAscorewasobserved, inadose-dependentmanner, afteradministration
oftheFOsupplement. Intermsoftheimpactonthen-3HUFAscore,theintakeofENCHcheese
(90 g/day) for two or four weeks was equivalent to the administration of one or two FO pills,
respectively. Conversely, the linseed oil intake did not significantly impact the n-3 HUFA score.
FeedingENCHcheesesfromdifferentsources(bovine,ovineandcaprine)fortwomonthsimproved
then-3HUFAscorebyincreasingplasmaDHA,andtheeffectwasproportionaltotheCLAcontent
inthecheese. Wesuggestthattheimprovedn-3HUFAscoreresultingfromENCHcheeseintake
maybeattributedtoincreasedperoxisomeproliferator-activatedreceptoralpha(PPAR-α)activity.
ThisstudydemonstratesthatnaturalENCHcheeseisanalternativenutritionalsourceofn-3HUFA
inhumans.
Keywords:conjugatedlinoleicacids(CLA);cheese;ruminant;highlyunsaturatedfattyacids(HUFA);
fattyacids(FA);plasma;clinicaltrial
Int.J.Mol.Sci.2018,19,1730;doi:10.3390/ijms19061730 www.mdpi.com/journal/ijms
Int.J.Mol.Sci.2018,19,1730 2of16
1. Introduction
Theeffectsofn-3fattyacids(n-3FA)onhumanhealthhavebeenextensivelystudied,andthe
useofhealthsupplementscontainingthesefattyacidsisincreasing[1]. Infact,n-3andn-6FAare
essentiallipidsthatcannotbesynthesizeddenovoandconsequentlyneedtobeingestedaspartofthe
diet. Inhumans,theprecursorofthen-6FA,linoleicacid(LA,18:2n-6),andthatofthen-3FAfamilies,
α-linolenicacid(ALA,18:3n-3),areelongatedanddesaturatedtotheirmorehighlyunsaturatedforms
(HUFA),whicharen-3andn-6FAwith≥20atomsofcarbonand≥3doublebonds,i.e.,principally,
arachidonicacid(ARA,20:4n-6)anddocosahexaenoicacid(DHA,22:6n-3)[2]. Thereiscompetitive
inhibitionbetweenfamiliesforenzymaticmechanisms,with,forexample,delta-6-desaturasefavoring
theconversionofn-3FAoverthatofn-6FA[3,4]. However,ahighLAintakemayshiftthebalance
towardstheconversionofn-6FAandcaninhibittheconversionofALAtoeicosapentaenoicacid(EPA,
20:5n-3)orDHA[5].
Inhumans,thissynthesisisacontroversialissue,asanALA-richdietdoesnotnecessarilyleadto
increasedtissueDHA.Healthysubjectsreceiving30mL/dayofALA-richlinseedoilforfourweeks
demonstrateda70%increaseinDHAintheserumphospholipids[6]. Inalong-termstudyofelderly
subjects,theintakeof3g/dayofALAincreasedtheplasmaDHAlevelsby20%by10months,butnot
byfourmonths[7]. Incontrast,thesupplementationofALA,upto15g/dayforfourweeks,ledto
increasedALAandEPAlevelsinplasmatriacylglycerolsandphospholipids,withverylittleincrease
intheDHAlevelsinplasma,platelets,andwhiteandredbloodcells[8–10]. Alsoinotherstudies,
ahighdoseofALAforaprolongedperiod(upto40g/dayforupto42weeks)significantlyincreased
plasmaEPA,butnotDHA[11,12]. Pawloskyandcolleaguesstudiedtheconversionof1goraldose
ofanisotopetracerofALA,namely,penta-deuteratedethylester[13]. Theyindicatedthatonly0.2%
of plasma ALA was destined for the synthesis of EPA, around 63% of plasma EPA was accessible
for the production of docosapentaenoic acid (DPA, 22:5n-3), and 37% of this was available for the
synthesisofDHA.Inaddition,thecorrelationbetweenDHAanditsdirectprecursor,24:6n-3,was
studied. Inculturedskinfibroblasts,thepreferredmetabolicfateof24:6n-3-CoA,afteritssynthesisin
theendoplasmicreticulum,isitstransfertoperoxisomes,whereitisbeta-oxidizedtoDHA-CoA[14].
The n-3 and n-6 HUFA are involved in various physiological processes and influence human
health in many different ways [15]. DHA, EPA, and ARA are important components of neuronal
membranes[16,17].DHAisthemajorHUFAintheadultmammalianbrainandretina,anditsdeficiency
can lead to memory loss, learning disabilities, and impaired visual acuity [18]. EPA contributes to
cardiovascularhealth[1,19].ARAandEPAareprecursorsofeicosanoids,pro-inflammatoryeicosanoids,
suchasthe2-seriesprostaglandins,2-seriesthromboxanes,4-seriesleukotrienes,andhydroxyfatty
acids, and anti-inflammatory eicosanoids, such as 3-series prostaglandins, 3-series thromboxanes,
and 5-series leukotrienes [3,20–22]. A number of studies have provided strong evidence that the
resolutionofinflammationisabiosyntheticallyactiveprocess,regulatedbybiochemicalmediators
andreceptor-dependentsignalingpathways,drivenbyspecializedpro-resolvingmediators[23]. These
specialized pro-resolving mediators lead to the resolution of inflammatory processes and restore
homeostasis.[24].
With current dietary practices, our dietary n-6/n-3 HUFA ratio is elevated for the overall
population, with the optimal ratio being 4:1 [25,26]. However, this imbalance can be corrected by
dietary supplementation with n-3 HUFA or by eating fish rich in EPA and DHA, such as salmon,
herring,mackerel,andtuna[27]. Recommendationsareintherangeof0.5–0.8g/dayofn-3HUFA,
ortheconsumptionofatleasttwoservingsoffishperweek[28]. Aphysiologicalmarkerofn-3FAin
tissuesisthen-3HUFAscoreobtainedastheratiobetweenthen-3HUFA(FA≥20carbonsand≥3
doublebonds)andthetotalFA;HUFAaremainlyincorporatedintoplasmalipids(phospholipids)
andarepotentialprecursorsofthebiologicallyactiveeicosanoidsanddocosahexaenoids[29].
Functionalfoods,nutraceuticalsorfoodproductswithenhancedn-3HUFAlevelsarebecoming
more popular, as a result of innovative development strategies. Another option to enrich for n-3
HUFAinhumansmaybetheincorporationofflaxseedproductsinlivestockfeed,resultinginthe
Int.J.Mol.Sci.2018,19,1730 3of16
accumulationofHUFAintissuesandotheranimalproducts(milk,cheesesormeat). Pastureand/or
lipid supplementation of ruminants’ diet was used to increase by up to four-fold milk fat ALA,
aprecursorofn-3HUFA,thec9t11isomerofconjugatedlinoleicacid(CLA,18:2c9t11),andvaccenic
acid(VA,18:1t11)andtodecreaseby23%thesaturatedfattyacids(SFA)[30–33].
CLA,anunusualfattyacidcharacterizedbyconjugateddoublebonds[34],issynthetizedwithin
therumenasaresultoftheactivityofanaerobicbacteria[35,36],andthemajorityofthec9t11isomer
ofCLAisendogenouslysynthesizedinthemammaryglandofruminants[33]andalsoinhumans
fromVA[37,38].
The c9t11 isomer of CLA is metabolized in a similar manner to LA and ALA, competes with
themforthesameenzymaticsystems,andplaysaroleinlipidmetabolism[39]. Inapreviousstudy
onhypercholesterolemicsubjects,wedemonstratedthattheintakeof90g/dayofafunctionalfood
(a CLA-enriched (ENCH) cheese), was an efficient approach to enhance the n-3 HUFA score [40].
Interestingly, improved lipid metabolism was achieved with a 90 g/day and not with a 45 g/day
intake of ENCH cheese, suggesting a dose-dependent effect, at least after three weeks of dietary
intervention[40].
Inthisstudy,weinvestigatedtheplasmafattyacidprofilesofhealthyhumanvolunteersand
evaluatedtheeffectofnaturallyCLA-enrichedcheeseonn-3FAmetabolismtooptimizethen-3HUFA
scores. We compared the modifications in the plasma fatty acid profiles following different daily
intakesofENCHcheese(90g/day)fortwoandfourweeks,or50g/dayofcheesefortwomonths.
Inalongertreatment,wecomparedtheeffectsofdifferenttypesofENCHcheeses,producedfromthe
milkofthreedifferentruminantspecies. Theeffectsofafishoil(FO)supplementorofALA-containing
linseedoilwerecomparedtothoseofENCHcheese.
2. Results
The pilot study consisted of 15 healthy human subjects fed 90 g per day of an ENCH cheese
(sheep-1-ENCH) for an initial two-week period; plasma fatty acids were then measured (Table 1),
andthefeedingcontinuedforafurthertwoweeks. Theplasmaconcentrationsofpalmiticacid(PA,
16:0)weremarkedlyreducedinthesubjectsoverthefourweeksofthedietaryinterventioncompared
totimezero(baseline). Adailyintakeof90g/dayofsheep-1-ENCHcheeseresultedinasignificant
increase in EPA and DHA plasma levels corresponding to 80% and 20%, respectively, by week 4
(Table1). Inaddition,therewasagreaterincorporationofCLAasaresultofsheep-1-ENCHcheese
intake,leadingtoanincreaseof220%aftertwoweeksandof240%afterfourweekscomparedtothe
baseline(Table1).
Table1.Mainfattyacids(FA)intheplasmaofsubjectsconsuming90g/dayofaconjugatedlinoleic
acid(CLA)-enriched(ENCH)sheepcheese(sheep-1-ENCH).PA,palmiticacid;OA,oleicacid;ALA,
α-linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; LA linoleic acid, ARA,
arachidonicacid.Thedataareexpressedasmol%andrepresentthemean±SEMfor15individuals
pergroup;*p<0.05;**p<0.01;***p<0.001versusbaseline(time0).
FAmol%in Baseline(Time0) Sheep-1-ENCH(2Weeks) Sheep-1-ENCH(4Weeks)
Plasma
Mean SEM Mean SEM Mean SEM
14:0 1.25 0.24 1.16 0.16 1.36 0.26
16:0(PA) 30.28 0.54 29.52 0.34 24.72 0.63***
17:0 7.27 0.22 7.49 0.12 6.02 0.21***
18:0 1.03 0.06 1.16 0.06 1.03 0.08
16:1 2.54 0.15 2.53 0.22 2.32 0.30
18:1n-9(OA) 18.20 0.60 18.62 1.53 19.95 1.03
18:3n-3(ALA) 0.25 0.03 0.42 0.04** 0.52 0.05***
Int.J.Mol.Sci.2018,19,1730 4of16
Table1.Cont.
FAmol%in Baseline(Time0) Sheep-1-ENCH(2Weeks) Sheep-1-ENCH(4Weeks)
Plasma
Mean SEM Mean SEM Mean SEM
18:4n-3 0.03 0.01 0.03 0.00 0.03 0.00
20:5n-3(EPA) 0.51 0.05 0.71 0.05* 0.93 0.13***
22:6n-3(DHA) 1.81 0.15 1.75 0.11 2.51 0.15*
18:2n-6(LA) 27.05 0.85 29.68 1.59 31.63 0.62
Int. J. Mo1l8. :S3cni.- 26018, 19, x FO0R.5 P4EER REVI0E.0W5 0.49 0.03 0.49 0.04 4 of 15
20:3n-6 1.52 0.12 1.53 0.19 1.64 0.13
20:4n-260:(4AnR-6A ()ARA) 6.79 6.79 0.37 0.37 6.466.46 0.04.343 77..1111 0.03.83 8
22:4n-6 0.14 0.02 0.11 0.01 0.11 0.00
22:4n-6 0.14 0.02 0.11 0.01 0.11 0.00
18:2c9t11(CLA) 0.13 0.10 0.42 0.23*** 0.45 0.07***
18:2c9t11 (CLA) 0.13 0.10 0.42 0.23 *** 0.45 0.07 ***
FFuurrththeerrmmoorere,,t htheei nintatkakeeo fof9 090g /gd/daayyo offs shheeeepp-1-1-E-ENNCCHHf oforrt wtwooa nadndfo fuorurw weeekeskism imprporvoevdedth tehne -n3-
H3U HFUAFsAc osrceor(fer o(fmrom11 %11%to t2o8 2%8,%re, srpesepceticvteivlye)lyw) whehnenco cmompapraerdedto toth teheb absaesleinliene( F(iFgiguurere1 )1.).I nIno ordrdeerrt oto
ccoommppaarreet thheei immppaacctt oonn tthhee ppllaassmmaa nn--33 HHUUFFAA ssccoorree ooff 9900 gg//ddaayy oof fshsheeepep-1--1E-ENNCCHH chceheesees ewwithit hthtahta otf
oifnicnrceraesainsign gFOFO suspupplpelmemenetnattaiotino n(0(.05.,5 1, 1aanndd 11.5.5 gg ppeerr ddaayy)),, tthhee ssuubbjjeeccttss wweerree ttrreeaatteedd ffoorr ttwwoow weeeekkss
wwitihtho onnee ooff tthhee iinnddiiccaatteedd FFOO ddoosseess, ,wwhhicihch rerseusulteltde dini na lainlienaer aprlapslmasam inacionrcpoorrpaotiroanti oonf no-f3 nH-3UHFAU iFnA a
indoased-odseep-ednedpeenntd menatnmnearn (nFiegru(rFei g1u). rTeh1e) .n-T3h HeUn-F3AH sUcoFrAes saccohrieesveadch wieitvhe da dwaiitlhy iantdaakiely oifn 9t0a kge oof sfh9e0egp-
o1f-EshNeCepH- 1c-hEeNesCeH focrh teweos eanfodr ftowuor wanedekfso ucorrwreesepkosndcoerdr etosp tohnodsee dobttoaitnheods ewoitbht aai ndeadilyw FitOh iandgeasiltyionF Oof
in0.g5e gst iFoOn (oofn0e.5 sugpFpOle(moneents)u apnpdle 1m.0e gn tF)Oan (dtw1o.0 sgupFOple(tmweonstsu)p ppelre mdaeyn,t sre)sppeercdtiavye,lrye (sFpiegcutirvee 1ly). (Figure1).
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aanndd1 1.5.5g g//ddaayy ooff fifisshh ooiill ((FFOO)) ((◆b lbuleued idaimamonodn)do) ro9r 090g /gd/daayyo offs hsheeeepp-1-1-E-ENNCCHHc chheeeesseef oforrt wtwoow weeeekkss
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**p p< <0 0.0.055;;* *****p p< <0 0.0.00011v veerrssuussb baasseelilninee( (ttimimee0 0))..
Since n-3 HUFA, particularly DHA and CLA, are PPAR-α ligands [41], we also evaluated the
relative expression levels of the PPAR-α gene. PPAR-α gene expression analysis indicated a
significant two-fold increase following dietary intervention with 90 g/day of sheep-1-ENCH cheese
for four weeks (p < 0.01) when compared to the baseline or to the intake for two weeks of sheep-1-
ENCH cheese (p < 0.01). However, there were no significant differences following the intake of the
FO supplement at 1.5 g/day (Figure 2).
We evaluated whether the results obtained in the pilot study with a sheep-1-ENCH cheese were
reproducible during a longer period (two months) and with different ruminant ENCH cheeses of
bovine, ovine and caprine origin. In this experiment, 50 g per day of cheese was ingested for two
months, as the amount consumed in the pilot study (90 g/day) may be considered high for the longer
feeding period. This lower intake was calculated on the basis of the regular intake of cheese in the
Italian diet [42].
Int.J.Mol.Sci.2018,19,1730 5of16
Sincen-3HUFA,particularlyDHAandCLA,arePPAR-αligands[41], wealsoevaluatedthe
relativeexpressionlevelsofthePPAR-αgene. PPAR-αgeneexpressionanalysisindicatedasignificant
two-fold increase following dietary intervention with 90 g/day of sheep-1-ENCH cheese for four
weeks(p<0.01)whencomparedtothebaselineortotheintakefortwoweeksofsheep-1-ENCHcheese
(p<0.01). However,therewerenosignificantdifferencesfollowingtheintakeoftheFOsupplementat
1.5g/day(Figure2).
Weevaluatedwhethertheresultsobtainedinthepilotstudywithasheep-1-ENCHcheesewere
reproducible during a longer period (two months) and with different ruminant ENCH cheeses of
bovine, ovineandcaprineorigin. Inthisexperiment, 50gperdayofcheesewasingestedfortwo
months,astheamountconsumedinthepilotstudy(90g/day)maybeconsideredhighforthelonger
feedingperiod. Thislowerintakewascalculatedonthebasisoftheregularintakeofcheeseinthe
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 15
Italiandiet[42].
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Figure2. RelativePPAR-αgeneexpressioninthebuffycoatofsubjectsconsuming1.5g/dayofFO
Figure 2. Relative PPAR-α gene expression in the buffy coat of subjects consuming 1.5 g/day of FO
and90g/dayofsheep-1-ENCHcheesefortwoandfourweeks. Thedataareexpressedasmean±
and 90 g/day of sheep-1-ENCH cheese for two and four weeks. The data are expressed as mean ±
SEM,with15individualspergroup;**p<0.01;versusbaseline(time0).
SEM, with 15 individuals per group; ** p < 0.01; versus baseline (time 0).
TableTa2bloeu 2t loinuetlsintehse thpel apslmasmaaF AFAle lveveelslsi nin ssuubbjjeeccttss iinnggeestsitnign gENENCHC Hchecehseees s(e5s0 (g5 p0egr dpaeyr) dfraoym) from
different sources.
differentsources.
Table 2. Main FA in the plasma of subjects consuming 50 g/day of different types of ENCH cheese for
Table2.MainFAintheplasmaofsubjectsconsuming50g/dayofdifferenttypesofENCHcheesefor
two months. The cheeses were produced from the milk of cows (cow-ENCH), sheep (sheep-2-ENCH)
twomonths.Thecheeseswereproducedfromthemilkofcows(cow-ENCH),sheep(sheep-2-ENCH)
and goats (goat-ENCH). PA, palmitic acid; OA, oleic acid; ALA, α-linolenic acid; EPA,
andgoats(goat-ENCH).PA,palmiticacid;OA,oleicacid;ALA,α-linolenicacid;EPA,eicosapentaenoic
eicosapentaenoic acid; DHA, docosahexaenoic acid; LA linoleic acid, ARA, arachidonic acid; VA,
acid;DHA,docosahexaenoicacid;LAlinoleicacid,ARA,arachidonicacid;VA,vaccenicacidandCLA,
vaccenic acid and CLA, conjugated linoleic acid. The data are expressed as mol % and represent mean
conjugatedlinoleicacid.Thedataareexpressedasmol%andrepresentmean±SEMof36individuals
± SEM of 36 individuals per group; * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (CRTL) group.
pergroup;*p<0.05;**p<0.01;***p<0.001versuscontrol(CRTL)group.
CRTL Cow-ENCH Sheep-2-ENCH Goat-ENCH
FA mol % in Plasma
Mean SEM Mean SEM Mean SEM Mean SEM
FAmol%in CRTL Cow-ENCH Sheep-2-ENCH Goat-ENCH
14:0 0.64 0.02 1.31 0.12 *** 0.66 0.06 0.58 0.07
Plasma
15:0 Mean 0S.2E2M 0.00M ea0n.42 0S.0E3M *** 0M.17e an 0.01 S* EM0.22 Me0a.n02 SEM
16:0 (PA) 11.53 0.25 11.26 2.27 11.13 0.30 11.93 0.85
14:0 0.64 0.02 1.31 0.12*** 0.66 0.06 0.58 0.07
17:0 0.50 0.02 0.38 0.04 0.34 0.02 ** 0.50 0.05
15:0 0.22 0.00 0.42 0.03*** 0.17 0.01* 0.22 0.02
18:0 9.03 0.29 6.90 0.42 6.95 0.21 * 7.89 0.78
14:1 0.09 0.01 0.07 0.01 0.05 0.00 0.07 0.01
16:1 1.82 0.12 2.07 0.24 1.77 0.06 1.57 0.11
18:1n-9 (OA) 26.31 1.02 29.96 1.90 27.48 1.30 26.70 1.19
18:3n-3 (ALA) 0.58 0.03 0.69 0.04 0.54 0.05 0.67 0.08
18:4n-3 0.02 0.00 0.02 0.01 0.02 0.00 0.02 0.00
20:5n-3 (EPA) 0.54 0.03 0.67 0.07 0.73 0.11 0.76 0.14
22:6n-3 (DHA) 1.99 0.09 2.73 0.33 *** 2.47 0.08 ** 2.74 0.28 ***
18:2n-6 (LA) 35.58 0.76 40.73 2.47 * 38.67 1.56 34.32 1.24
18:3n-6 0.41 0.03 0.50 0.06 0.39 0.03 0.37 0.05
20:3n-6 1.89 0.08 2.32 0.11 * 1.58 0.09 1.96 0.16
20:4n-6 (ARA) 8.06 0.35 7.79 0.85 7.20 0.31 7.52 0.33
Int.J.Mol.Sci.2018,19,1730 6of16
Table2.Cont.
FAmol%in CRTL Cow-ENCH Sheep-2-ENCH Goat-ENCH
Plasma
Mean SEM Mean SEM Mean SEM Mean SEM
16:0(PA) 11.53 0.25 11.26 2.27 11.13 0.30 11.93 0.85
17:0 0.50 0.02 0.38 0.04 0.34 0.02** 0.50 0.05
18:0 9.03 0.29 6.90 0.42 6.95 0.21* 7.89 0.78
14:1 0.09 0.01 0.07 0.01 0.05 0.00 0.07 0.01
16:1 1.82 0.12 2.07 0.24 1.77 0.06 1.57 0.11
18:1n-9(OA) 26.31 1.02 29.96 1.90 27.48 1.30 26.70 1.19
18:3n-3(ALA) 0.58 0.03 0.69 0.04 0.54 0.05 0.67 0.08
18:4n-3 0.02 0.00 0.02 0.01 0.02 0.00 0.02 0.00
20:5n-3(EPA) 0.54 0.03 0.67 0.07 0.73 0.11 0.76 0.14
22:6n-3(DHA) 1.99 0.09 2.73 0.33*** 2.47 0.08** 2.74 0.28***
18:2n-6(LA) 35.58 0.76 40.73 2.47* 38.67 1.56 34.32 1.24
18:3n-6 0.41 0.03 0.50 0.06 0.39 0.03 0.37 0.05
20:3n-6 1.89 0.08 2.32 0.11* 1.58 0.09 1.96 0.16
20:4n-6(ARA) 8.06 0.35 7.79 0.85 7.20 0.31 7.52 0.33
22:4n-6 0.27 0.01 0.31 0.01 0.23 0.02 0.27 0.03
18:1t11(VA) 0.36 0.02 0.69 0.07** 0.57 0.06* 1.03 0.15***
18:2c9t11(CLA) 0.22 0.01 0.53 0.11** 0.41 0.04 0.85 0.11***
The plasma levels of ALA and CLA increased in all subjects after ENCH cheese intake when
comparedtothecontrolvalues. TherewasasignificantincreaseintheincorporationofCLA(>280%)
intotheplasmaofsubjectsingestinggoat-ENCHcheesecomparedtosubjectsconsumingthecontrol
(CRTL)cheese. Asimilarresultwasobservedforthecow-ENCHcheese(>140%increaseinCLA)
whencomparedtotheCRTLvalues.
ThesehighplasmaCLAlevelsdetectedinthesubjectsconsuminggoat-ENCHcheesereflected
the1.5-foldgreaterCLAconcentrationingoat-ENCHcheesewithrespecttootherENCHcheeses(see
Table2). NosignificantvariationswerefoundintheplasmalevelsofALAandEPAwhenthedifferent
ENCHcheeseswereconsumed;onlytheDHAlevelsincreasedby~24–37%followingtheintakeofall
typesofENCHcheeses(seeTable2).
Wealsoevaluatedwhetherasubstantialintake(2g)ofthen-3HUFAprecursor(ALA)significantly
modifiedtheplasmaDHAlevelsand,hence,impactedthen-3HUFAscores. Subjectsadministeredfor
twomonths4g/dayoflinseedoil,containing2gofALA,werecomparedwithsubjectsadministered
4g/dayofoliveoil(controlgroup). WeobservedasignificantriseinALAandEPA(78%and50%,
respectively),butnotofDHA(13%increase,Figure3A)inthelinseedoilgroup. Figure3comparesthe
plasmalevelsofALA,EPA,DHA,andCLAfollowingthedifferenttreatmentsofthisstudy. Itwas
observedthathighcirculatingCLAlevelsinducedDHAbiosynthesismorestronglythantheDHA
precursorALA.
Figure4representstheplasman-3HUFAscoresofsubjectsonthedifferenttreatments. Significant
increases were observed with the intake of ENCH cheese for longer periods or with the higher
concentrationsofFO.
Int.J.Mol.Sci.2018,19,1730 7of16
Figure3.Levelsofthemainn-3FA(ALA,EPA,andDHA)andCLAmeasuredintheplasmaofsubjects
consumingdifferentdiets:(A)CRTLdiet(4g/dayoliveoil)or4g/daylinseedoil(~2gofALA)for
twomonths,n=12/group;(B)0.5g/day,1.0g/day,or1.5g/dayofFOfortwoweeks,n=15/group;
(C)90g/dayofsheep-1-ENCHcheesefortwoandfourweeks,n=15/group;(D)50g/dayofdifferent
ENCHorCRTLcheesesfortwomonths,n=36/group.Thedataareexpressedasmol%andrepresent
mean±SEMpergroup;*p<0.05;**p<0.01;***p<0.001versusbaseline(time0)orcontrolgroup.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 15
30 A B *** C * D *** ***
*
*
25
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As
F15
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H
3
n-
10
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0 CRTL ti2m102A.e2.gL2/A 0d ay baseline (FO)1p020i.15l.(l9.gF 4F/OdO)a y 1.0(gF/Od)a y 12.5(pgF2/iO1ld4l).sa 0. y8F O baseline (ENCH) 3sheep-1-ENCHp2 (2wks)i1l7l.s2.4 FO sheep-1-ENCH (4wks)time12 .100 CRTL.80 9 0cow-ENCHg/12d.35 .C85LA sheep-2-ENCHEN90g/22 goat-ENCHd.73 .C00LAEN
FigurFeig4u.rPe l4a.s Pmlaasmna-3 nH-3 UHFUAFAsc socroerseso off ssuubbjjeeccttss aaddmminiinstiesrteedre ddifdfeirfefenrt etrnetattmreeantmts:e (nAt)s C:R(ATL) CcoRnTtrLolc (o4n trol
g/day olive oil) and 4 g/day linseed oil (~2 g of ALA), n = 12/group; (B) 0.5 g/day, 1.0 g/day and 1.5
(4g/dayoliveoil)and4g/daylinseedoil(~2gofALA),n=12/group;(B)0.5g/day,1.0g/dayand
g/day of FO for 2wo weeks, n = 15/group; (C) 90 g/day of sheep-1-ENCH cheese for two and four
1.5g/dayofFOfor2woweeks,n=15/group;(C)90g/dayofsheep-1-ENCHcheesefortwoandfour
weeks, n = 15/group; (D) 50 g/day of different ENCH or CRTL cheeses for two months, n = 36/group.
weeks,n=15/group;(D)50g/dayofdifferentENCHorCRTLcheesesfortwomonths,n=36/group.
The data are expressed as mean ± SEM per group; * p < 0.05; *** p < 0.001 versus baseline (time 0) or
Thedataareexpressedasmean±SEMpergroup;*p<0.05;***p<0.001versusbaseline(time0)or
control group.
controlgroup.
3. Discussion
Recently, attention has been focused on dietary regimens aimed at meeting the optimal
nutritional lipid requirements to exert positive effects on health and to prevent the onset of chronic
diseases such as cardiovascular diseases, diabetes, metabolic syndrome and cancer [43]. The
beneficial effects of n-3 FA have been widely described within an optimal balance of all dietary FA
[15]. It is likely that the putative beneficial effects exerted by n-3 FA are dictated by the general
deficiency of EPA and DHA intake in Western European countries [44]. In particular, it has been
proposed to reduce the n-6/n-3 HUFA ratio to the desired optimal value of 4:1 by regulating the
dietary intake of the corresponding HUFA.
The aim of this study was to evaluate whether a functional food, such as a CLA-enriched cheese,
was able to address the imbalance between n-6 and n-3 fatty acid families in healthy subjects on a
regular diet. We previously observed that feeding sheep-1-ENCH cheese increased the plasma n-3
HUFA score, one of the more reliable indicators of n-3 FA status in humans [29,45] and in
hypercholesterolemic subjects [40]. In that study, we also found a significant reduction of LDL-
cholesterol and of the endocannabinoid anandamide [40]. In the present study, cheese intake was not
associated with changes in body weight, plasma lipid content or endocannabinoids (data not shown).
These data may suggest that the effects of ENCH cheese on LDL-cholesterol and endocannabinoids
levels are significant only in altered physiopathological conditions, such as those of
hypercholesterolemic patients, and are not due to a direct influence on cholesterol metabolism or
endocannabinoid biosynthesis.
We found that the intake of ENCH cheeses resulted in a significant increase in plasma DHA in
humans ingesting either 90 g per day of sheep-1-ENCH cheese for four weeks or 50 g per day of an
ENCH cheese from three ruminant species for two months. These data may suggest that not only the
amount but also the duration of the ENCH cheese intake are crucial to induce DHA biosynthesis. We
previously found that CLA intake in Zucker rats for 14 weeks increased the n-3 HUFA score in the
liver [46]. Several studies have examined the ability of CLA to increase the peroxisomal or
Int.J.Mol.Sci.2018,19,1730 8of16
3. Discussion
Recently,attentionhasbeenfocusedondietaryregimensaimedatmeetingtheoptimalnutritional
lipid requirements to exert positive effects on health and to prevent the onset of chronic diseases
suchascardiovasculardiseases,diabetes,metabolicsyndromeandcancer[43]. Thebeneficialeffects
of n-3 FA have been widely described within an optimal balance of all dietary FA [15]. It is likely
thattheputativebeneficialeffectsexertedbyn-3FAaredictatedbythegeneraldeficiencyofEPA
andDHAintakeinWesternEuropeancountries[44]. Inparticular,ithasbeenproposedtoreduce
then-6/n-3HUFAratiotothedesiredoptimalvalueof4:1byregulatingthedietaryintakeofthe
correspondingHUFA.
The aim of this study was to evaluate whether a functional food, such as a CLA-enriched
cheese, was able to address the imbalance between n-6 and n-3 fatty acid families in healthy
subjects on a regular diet. We previously observed that feeding sheep-1-ENCH cheese increased
theplasman-3HUFAscore,oneofthemorereliableindicatorsofn-3FAstatusinhumans[29,45]
and in hypercholesterolemic subjects [40]. In that study, we also found a significant reduction of
LDL-cholesterolandoftheendocannabinoidanandamide[40]. Inthepresentstudy,cheeseintake
wasnotassociatedwithchangesinbodyweight, plasmalipidcontentorendocannabinoids(data
not shown). These data may suggest that the effects of ENCH cheese on LDL-cholesterol and
endocannabinoidslevelsaresignificantonlyinalteredphysiopathologicalconditions,suchasthose
ofhypercholesterolemicpatients,andarenotduetoadirectinfluenceoncholesterolmetabolismor
endocannabinoidbiosynthesis.
We found that the intake of ENCH cheeses resulted in a significant increase in plasma DHA
in humans ingesting either 90 g per day of sheep-1-ENCH cheese for four weeks or 50 g per day
of an ENCH cheese from three ruminant species for two months. These data may suggest that
not only the amount but also the duration of the ENCH cheese intake are crucial to induce DHA
biosynthesis. We previously found that CLA intake in Zucker rats for 14 weeks increased the n-3
HUFA score in the liver [46]. Several studies have examined the ability of CLA to increase the
peroxisomal or mitochondrial activity of fatty acid oxidation, and CLA is a potent ligand of the
peroxisome proliferator-activated receptor alpha (PPAR-α) that regulates expression of the genes
involvedinperoxisomalbeta-oxidation[47].Theimportanceoftheperoxisomalbeta-oxidationprocess
waspreviouslydescribedinrelationtoDHAsynthesis[14]. WemayhypothesizethatCLAcould
inducehigherDHAformationviaPPAR-αactivation,butfurtherstudiesareneededtodemonstrate
a correlation between CLA and improved DHA synthesis. Indeed, we found that sheep-1-ENCH
cheese(90g/day)significantlyincreasedPPAR-αgeneexpression. Inaddition,ourdatasuggestthat
thisenhancedgeneexpressionisnotjustduetoDHA,sinceFO,inthisstudy,didnotdemonstrate
any significant effect on PPAR-α gene expression. Our data were supported by other studies in
experimental animals that detailed the ability of CLA to activate PPAR-α and thus to induce key
enzymesofperoxisomalbeta-oxidation[47,48].
Attar-Bashi and collaborators showed that CLA did not efficiently increase DHA levels from
ALA [49]. Experimental animals models and human studies have shown that high dietary ALA
partiallyinhibitsdelta-6desaturaseandthuspreventsthedesaturationof24:5n-3to24:6n-3inthe
DHAbiosynthesispathway[50].ALAcanbeendogenouslyconvertedintoitsmetabolitesataverylow
efficiency,5%toEPAandaround0.5%toDHA[51,52]. Ourpreviousdatademonstratedareductionof
DHAinthebrainofratsfedanALA-enricheddietandanincreaseoftheotherprecursor(22:5n-3)[53].
From our data, it appears that for CLA to exert its effect on circulating DHA levels, a relative but
notexcessiveintakeofALAisnecessary. WeproposethataratioofdietaryCLA/ALAof~3:1could
bedeterminanttoenhanceDHAproductionfromALA.TheroleofCLAatcertainconcentrations,
with respect to ALA, was further demonstrated by the prolonged intake of ALA-rich linseed oil,
whichonlyslightlyinducedanon-significantincreaseinthen-3HUFAscore. Moreover,theintakeof
thesheep-1-ENCHcheeseresultedinplasman-3HUFAscorescomparabletotheintakeoffishoil.
Thiseffectfurtherconfirmsthenotionthatfoodcompositionmaynotreflectfoodnutritionalvalue.
Int.J.Mol.Sci.2018,19,1730 9of16
Often,thenutritionalguidelinesfordietaryfatintakerelyonfoodfattyacidcompositionand,for
example,acertainconcentrationofALAissufficienttoclaimthatafoodisasourceofn-3. Ourdata
clearlydemonstratethatthisisnotthecaseandthatanincreaseincirculatingDHAmaybeefficiently
achievedwithdifferentstrategies,notonlybysupplyingdietaryn-3FA.Interestingly,wefounda
significantdecreaseofPA,themajorSFAinplasma,withtheintakeofENCHcheeses. Thismightbe
surprisingsincecheeseissometimesnegativelyperceivedbecauseofitselevatedSFAlevels[54]. Itis
knownthatdietaryPAdoesnotinfluencePAtissuelevelsinhumans,asitisregulatedbythebalance
betweenitsmainsources,i.e.,dietandbiosynthesis,throughdenovolipogenesis(DNL)[55],whichis
averywellconservedmechanismconvertingcarbohydratestolipids[56]. Wemayhypothesizethat
ENCHcheesesmayinhibitDNLprobablybyactivatingPPAR-α[57],therebyreducingthecirculating
PAlevels.
The activation of PPAR-α with the inhibition of DNL and the increase of the n-3 HUFA
score may, at least in part, explain the anti-neuroinflammatory effects exerted by CLA in
X-linked adrenoleucodystrophy, a genetic disorder characterized by an impaired peroxisomal
beta-oxidation[58,59], confirming that PPAR-α activation can also play an important role in the
prevention of neurodegenerative pathologies [60,61]. The development of innovative systems in
livestockbreedingaimedatincreasingmilkCLAcontentwouldproducedairyproductsrichinCLA,
which,ifincludedintoabalanceddiet,maycontributetothepreventionofsomeofthemajorchronic
pathologies[62]. ThesupposedhealthbenefitsofCLAwerediscoverednearlythreedecadesago[63].
Suchpositivehealtheffectswererelatedtocancersuppression,bodyfatreduction,delayoftheonset
of type II diabetes and of the development of atherosclerosis, improved bone mineralization, and
immunesystemmodulation[64–66]. Interestingly,thesebeneficialeffectsaresharedwithn-3HUFA.
Inconclusion,ourstudiesindicatethatENCHcheesesmaycontribute,withothernutritional
metabolic and genetic factors, to the modulation of fatty acid metabolism. Because of the limited
habitual consumption of seafood in most Western countries, if our data were confirmed in larger
multicenter studies, ENCH cheeses may be included in the guideline recommendations to ensure
a sufficient supply of n-3 HUFA. Future studies will be devoted to the evaluation of the possible
synergisticeffectsofCLA-enrichedproductsandotherdietarycomponents.
4. MaterialsandMethods
4.1. Subjects
All protocols involving human subjects were performed according to Good Clinical Practice
withpre-approvalfromtheappropriateEthicsCommittee. ThisstudywascarriedoutattheCatholic
UniversityoftheSacredHeart,Rome(Italy). Atotalof36apparentlyhealthyindividuals,16males
and20females,aged30–45years,withBodyMassIndex(BMI)20–26,andbloodglucoseandlipids
parametersshowninTable3,wereincludedinthestudy.Amongthe36participants,15wererandomly
assignedtoStudies1and2,24toStudy3,andall36wererecruitedforStudy4.
Table 3. Blood lipid parameters of the study populations: BMI = Body Mass Index; HDL-C =
High-densitylipoproteincholesterol;LDL-C=Low-densitylipoproteincholesterol.
BloodParameters Mean SEM
Age(years) 30–40
BMI(kg/m2) 20–26
Glycemia(mg/dL) 85.5 1.5
Triglyceride(mg/dL) 80.3 6.3
Totalcholesterol(C)(mg/dL) 190.5 6.7
HDL-C(mg/dL) 60.1 1.9
LDL-C(mg/dL) 113.8 6.1
TotalC/HDL-Cratio 3.3 0.1
Int.J.Mol.Sci.2018,19,1730 10of16
Theuseofalcohol,tobaccoandmedicinesthatmightinfluencetheabsorptionormetabolismof
lipids,wasnotpermittedduringthestudies,andthesubjectswereadvisedtomaintaintheircustomary
livinghabits,inparticularinrelationtodiet.
4.2. BloodSampling
Following overnight fasting, venous blood samples were taken from the antecubital vein
into anticoagulant-coated vacutainers (K3 EDTA) and centrifuged at 2000× g for 15 min at room
temperature. The plasma was separated from the red blood cells and stored at −80 ◦C until
quantificationoftotallipidsandfattyacidsprofiling. Thebuffycoatwasalsoseparated,stabilizedin
RNALater(Ambion,LifeTechnologies,Carlsbad,CA,USA)andstoredat−20◦CforRNAextraction.
4.3. StudyDesign
Study1: FeedingCLA-enrichedcheese(ENCH)
Fortwoandfourweeks,thedietof15healthysubjectswassupplementedwith90g/dayofa
sheepcheese,enrichedinALAandCLA(sheep-1-ENCH),kindlyprovidedbyArgiolasFormaggi
s.r.l(Dolianova,Italy). Thedailyintake(90g/day)ofsheep-1-ENCHcheesewasaspublishedinour
previouspaper[40]. CLA-enrichedmilkwasobtainedfromdairyewesonpasturewhenavailableor
bysupplementingthedietwithextrudedlinseeds.[40].
The subjects visited the clinic on three occasions: (a) for the base examination (time 0); (b) at
theendoftheintakeof90g/dayofsheep-1-ENCHcheesefortwoweeks;(c)followingtheintakeof
90g/dayofsheep-1-ENCHcheeseforfourweeks.
Study2: Feedingfishoil(FO)supplementswithn-3HUFA
Following a three-weeks washout (WO) period, the diet of the subjects from Study 1 were
supplementedwithstepwiseincreasingdosesofFOinpillform(containing14.6%EPA,7.8%DHA,
25.9%oleicacid(OA,18:1n-9),10.2%LAand0.9%ALA,withrespecttototalFA);kindlysuppliedby
AkerBiomarine(Oslo,Norway).
Thesubjectsconsumed: (a)0.5g/dayFOsupplementfortwoweeks;(b)1g/dayFOsupplement
fortwomoreweeksand(c)1.5g/dayFOsupplementforafurthertwo-weekperiod,correspondingto
one,twoandthreepillsperday,respectively.
Study3: Effectoffeedingα-linolenicacid(ALA),aprecursorofthen-3HUFAfamily
Twenty-fourhealthysubjectswererandomlydividedintotwogroups. Thedietofonegroup
wassupplementedfortwomonthswith4goflinseedoil(richinALA)correspondingtoanintakeof
2g/dayofALA,andthedietoftheothergroupwassupplementedwith4g/dayofoliveoil(control).
Study4: FeedingENCHcheesefromdifferentruminantsources
Thedietsof36healthyvolunteersweresupplementedfortwomonthswithdifferenttypesof
ENCHcheeseorwithacontrolcheese,producedfromthemilkofthreeruminantspecies(bovine,
ovineandcaprine).
TheENCHcheesesstudiedwereproducedfrom:
1. Cow’smilk(cow-ENCH);suppliedbyTeagasc,Moorepark,Fermoy,Co. Cork,Ireland. CLAwas
increasedinthemilkbysupplementationwithlinseedandsunfloweroils(unpublisheddata).
2. Sheep’smilk(sheep-2-ENCH);PecorinoSardoPDOcheesesuppliedbyAGRIS,Bonassai,Olmedo,
Italy,whichwasenrichedinCLAbyusingasheepfeedingregimencharacterizedbyextensive
grazingofgrass–legumepasturesandlowlevelsofsupplements[67].
3. Goat’smilk(goat-ENCH);suppliedbyINRA,Clermont-Ferrand-Theix,France.TheCLA-enriched
goatspread-typecheesewasmanufacturedbyActalia(Surgères,France). Themilkwasenriched