Phytoene and Phytofluene Isolated from a Tomato Extract are Readily Incorporated in Mixed Micelles and Absorbed by Caco-2 Cells, as Compared to Lycopene, and SR-BI is Involved in their Cellular Uptake

RESEARCH ARTICLE Ca o enoids www.mn -jou nal.com Phy oene and Phy ofluene Isola ed om a Toma o Ex ac a e Readily Inco po a ed in Mixed Micelles and Abso bed by Caco-2 Cells, as Compa ed o Lycopene, and SR-BI is In ol ed in hei Cellula Up ake Paula Mapelli-B ahm, Cha les Desma chelie , Ma ielle Ma gie , Emmanuelle Reboul, An onio J. Mel´ endez Ma ´ ınez, and Pa ick Bo el* Scope: Abso p ion mechanisms o phy oene (PT) and phy ofluene (PTF) a e poo ly known. The main objec i es o he s udy a e o measu e hei micelliza ion and in es inal cell up ake efficiencies and o compa e hem o hose o commonly consumed ca o enoids. O he objec i es a e o assess he in ol emen o p o ein(s) in hei cellula up ake and whe he hey compe e wi h o he ca o enoids o micelliza ion and cellula up ake. Me hods and esul s: Toma o-ex ac -pu ified PT and PTF, mainly p esen as cis-isome s, a e much be e inco po a ed in syn he ic mixed micelles han pu e all- ans lycopene. PT impai s lycopene micelliza ion (−56%, P<0.05) while PT and PTF do no significan ly affec he micelliza ion o o he ca o enoids, and ice e sa. A low concen a ion, Caco-2 PTF up ake is highe (P<0.05) han ha o PT and lycopene (29%, 21%, and no de ec able). SR-BI, bu no CD36 nei he NPC1L1, is in ol ed in PT and PTF up ake. PT and PTF impai (p<0.05) β-ca o ene up ake (−13 and −22%, espec i ely). Conclusions: The high bioaccessibili y o PT and PTF can be pa ly explained by hei high micelliza ion efficiency, which is likely due o hei na u al cis isome iza ion and/o o hei high molecula flexibili y. SR-BI is in ol ed in hei cellula up ake, which can explain compe i ions wi h o he ca o enoids. 1. In oduc ion Phy oene (PT) and phy ofluene (PTF) a e ca o enes, i.e., non- oxygena ed ca o enoids, which a e ound in a wide a ie y o ui s and ege ables, e.g., in oma oes, ca o s, and ligh o - ange ap ico s (a concen a ions o a ound 1.4 and 0.4; 1.4 and 0.6; and 7.2 and 2.4 mg/100 g edible po ion, o PT and PTF, P. Mapelli-B ahm, D . A. J. Mel´ endez Ma ´ ınez Food Colou & Quali y Lab. A ea o Nu i ion & Food Science Uni e sidad de Se illa 41012, Se ille, Spain D .C. Desma chelie , M. Ma gie , D . E. Reboul, D . P. Bo el C2VN, Aix Ma seille Uni , INRA, INSERM Ma seille, F ance E-mail: [email p o ec ed] DOI: 10.1002/mn .201800703 espec i ely).[1,2] These ca o enoids con- ain h ee (PT) and fi e (PTF) conju- ga ed double bonds (CDB), while com- monly consumed ca o enoids con ain a leas en CDB (Figu e S1, Suppo ing In- o ma ion). This p o ides hem wi h a unique ea u e in he ca o enoid king- dom: hey do no abso b isible ligh and hus a e colo less o human.[3] In addi- ion, hei lowe numbe o CDB gi es hem a mo e wis ed shape compa ed o commonly consumed ca o enoids,[4,5] which has been sugges ed o affec hei bioa ailabili y and biological ac ions.[2] Mo eo e , i could also be expec ed ha hei endency o oxida ion would be lowe .[2] PT and PTF a e eadily abso bed by he human body, being ound in blood and se e al issues.[6,7] They ha e ecen ly ecei ed inc eased in e es because se - e al s udies ha e ound posi i e associa- ions be ween hei consump ion/blood concen a ion and some heal h benefi s. The in ake o PT and/o PTF could be ela ed o an imp o emen o he immune sys em and a educ- ion in he isk o de elop a ious diseases, including ce ain cance s.[2,8,9] Mo eo e , se e al s udies ha e indica ed ha hey could p o ec he skin agains UV-damage and p o ide cosme ic benefi s.[10–13] To each he bloods eam and hen a ge issues, ca o enoids mus fi s be eleased om he ood ma ix in which hey a e embedded and be inco po a ed in o mixed micelles.[14,15] PT and PTF ha e been shown o exhibi highe bioaccessibili y han o he ca o enoids p esen in he same ood ma ices.[16–19] Indeed, a bioaccessibili y anking o ca o enoid species seems o eme ge ega dless o he ood ma ix: PT and PTF >lu ein >β-ca o ene >lycopene. Howe e , a ailable da a do no allow us o conclude whe he he high bioaccessibili y o PT and PTF o igina es om a highe ex ac ion efficiency om ood ma ices, due o spe- cific in acellula localiza ion compa ed o o he ca o enoids,[20] o om a highe in insic solubili y in mixed micelles,[21] due o hei peculia chemical and physical p ope ies, o bo h. Once in mixed micelles, i is assumed ha PT and PTF a e aken up by Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (1 o 9) www.ad ancedsciencenews.com www.mn -jou nal.com en e ocy es, anspo ed o hei basola e al side, and inco - po a ed in o chylomic ons be o e being sec e ed in o he lymph.[14,15,22] These up ake and anspo p ocesses a e appa - en ly e y efficien o he colo less ca o enoids because, e.g., PT is a majo ca o enoid in a ious issues and i s bioa ailabili y has been shown o be nea ly iple han ha o lycopene.[23] Ye , hei in es inal abso p ion mechanisms ha e no been s udied and compa ed o hose o commonly s udied ca o enoids. Ne - e heless, s udies in he las decade ha e allowed expe s in his field o conclude ha en e ocy e up ake o commonly consumed ca o enoids is no only passi e bu acili a ed by memb ane p o eins.[24–27] Indeed, i has been shown ha CD36 molecule (CD36) is in ol ed in cell up ake o p o i amin A ca o enoids[24] and lu ein,[28] sca enge ecep o class B ype I (SR-BI) is in ol ed in cell up ake o p o i amin A ca o enoids,[24,29] lycopene,[28] and lu ein,[26] and NPC1 like in acellula choles e ol anspo e 1 (NPC1L1) is in ol ed in lu ein up ake.[30,31] Ye , i is no known whe he any o hese p o eins a e in ol ed in cell up ake o he colo less ca o enoids. Ou main objec i e was o ob ain undamen al da a on wo key s eps ha a e assumed o go e n he bioa ailabili y o he colo less ca o enoids, i.e., micelliza ion and apical up ake by in- es inal cells. Fo ha , we fi s measu ed he inco po a ion effi- ciency o oma o ex ac pu ified PT and PTF in syn he ic mixed micelles and compa ed i o ha o pu e commonly consumed ca o enoids. We nex assessed whe he p e iously men ioned p o eins in ol ed in up ake o commonly consumed ca o enoids we e also in ol ed in ha o hese colo less ca o enoids. Finally, because hese colo less ca o enoids migh be used as supple- men s in he u u e, we assessed in all expe imen s whe he hey compe e wi h he s udied commonly consumed ca o enoids. 2. Expe imen al Sec ion 2.1. Chemicals PT and PTF (99.6 and 99.8% pu e as checked by HPLC) we e isola ed om a oma o ex ac as desc ibed p e iously.[19] Pu- ified PT con ained 96% o 15-cis-isome and 4% o all- ans- isome and pu ified PTF con ained 94% o cis-isome s and 6% o all- ans-isome . No e ha in mos oods, human is- sues, and biological fluids, PT and PTF a e expec ed o be p esen as a mix u e o isome s, he cis isome s assumed o be p edominan .[5,32] All- ans α-ca o ene, lycopene, and lu ein (ࣙ95% pu e) we e a gi om DSM L d. (Basel, Swi ze - land). All- ans β-ca o ene (ࣙ97% pu e), 2-oleoyl-1-palmi oyl- sn-glyce o-3-phosphocholine (phospha idylcholine), 1-oleoyl- ac- glyce ol (monoolein), 1-palmi oyl-sn-glyce o-3-phosphocholine (lysophospha idylcholine), 3β-hyd oxy-5-choles ene ( ee choles- e ol), oleic acid, and sodium au ochola e we e pu chased om Sigma–Ald ich (Sain -Quen in-Falla ie , F ance). DMEM con- aining 4.5 g L−1glucose, ypsin-EDTA (500 and 200 mg L−1, espec i ely), nonessen ial amino acids, penicillin/s ep omycin, and PBS we e pu chased om Li e Technologies (Illki ch, F ance). Fe al bo ine se um (FBS) came om PAA (V´ elizy- Villacoublay, F ance). Block lipid anspo -1 (BLT1), used as chemical inhibi o o SR-BI, was pu chased om Sigma– Ald ich. Eze imibe β-d-glucu onide, used as chemical inhibi o o NPC1L1, was pu chased om Sequoia-Resea ch (Pangbou ne, UK). Sul o-N-succin-imidyl olea e (SSO), used as chemical in- hibi o o CD36, was syn hesized as p e iously published.[33] 2.2. P epa a ion o Ca o enoid-Rich Mixed Micelles Mixed micelles con aining ca o enoids we e syn hesized as p e- iously desc ibed,[34] wi h mino modifica ions. In summa y, sol- en solu ions o ca o enoids we e fi s mixed wi h sol en so- lu ions o micelle lipids and hen he mix u e was e apo a ed. Then, DMEM con aining 5 mm sodium au ochola e was added and mixed micelles we e syn hesized by sonica ion. The mixed micelle ac ion was op ically clea and s o ed a −20 °C un il Caco-2 cell expe imen s. 2.3. Micelliza ion Expe imen s 2.3.1. Measu emen o Ca o enoid Micelliza ion The amoun o ca o enoids ha could be inco po a ed in he mixed micelle ac ion was measu ed a h ee a ge ca o enoid concen a ions, 0.5, 2, and 10 µm. These concen a ions a e ex- pec ed o be ound in he human in es inal lumen a e ei he low die a y, high die a y, o pha macological in ake o hese ca o enoids. They we e es ima ed om a p e ious wo k[19] whe e ca o enoid concen a ions we e measu ed in gas o-in es inal fluid ollowing in i o diges ions o diffe en doses o hese com- pounds. 2.3.2. P o ocol o S udy Compe i ions be ween Ca o enoids o Micelliza ion To s udy his compe i ion, he amoun o ca o enoid eco e ed in mixed micelles was compa ed when only one ca o enoid was added a 0.5 µm du ing mixed micelle syn hesis (con ol) o he ca o enoid amoun eco e ed in micelles when 0.5 µm o ano he ca o enoid was added o he p e ious one du ing mixed micelle syn hesis. 2.4. Caco-2 Cell Expe imen s 2.4.1. Cul u e o Caco-2 Cells Caco-2 clone TC-7 cells we e a gi om D . M. Rousse (UMR- S872, Pa is, F ance). Cells we e hawed a passage numbe 70 o highe and we e cul u ed as p e iously desc ibed.[26] Be o e 3 weeks o each expe imen , he cells we e seeded on Millicell hanging cell cul u e inse s (1 µm po e size polyca bona e mem- b ane, Millipo e S.A.S., Molsheim, F ance) in six-well pla es a a densi y o 25 ×104cells pe well o allow o diffe en ia ion. Be- o e 12 h o each expe imen , media we e changed o FBS- ee medium a bo h sides. Based on p elimina y esul s (Figu e S2, Suppo ing In o ma ion) and unless o he wise s a ed, an incuba- ion ime o 2 h was selec ed o he ollowing Caco-2 cells expe - imen s. Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (2 o 9) www.ad ancedsciencenews.com www.mn -jou nal.com 2.4.2. P o ocol o E alua e he Maximal Amoun o Micella PT and PTF ha could be Theo e ically Taken Up by Caco-2 Cells The apical side o Caco-2 cell monolaye s ecei ed diffe en con- cen a ions o micella PT and PTF, mo e p ecisely om 0.3 o 6.9 µm. The amoun o PT and PTF aken up by he cells was mea- su ed a he end o he incuba ion ime (2 h). Qmax, which is he maximal amoun o ca o enoid ha could be heo e ically aken up by he cells, and appa en K, which is he micella ca o enoid concen a ion a which up ake is hal he Qmax, we e calcula ed. 2.4.3. Compa ison o Up ake o Diffe en Ca o enoid Species by Caco-2 Cells Pu ified 0.5 µm PT o PTF, o pu e lu ein, lycopene, o β-ca o ene was inco po a ed in mixed micelles and added o he apical side o Caco-2 cells monolaye s. The amoun o ca o enoids aken up by he cells was measu ed a e 2 h incuba ion. 2.4.4. Compe i ions be ween Micella Ca o enoids o hei Up ake by Caco-2 Cells Cells we e incuba ed wi h mixed micelles con aining one ca o enoid oge he wi h mixed micelles con aining ei he no ca o enoid (con ol) o ano he ca o enoid. These expe imen s we e ca ied ou wi h micella ca o enoid concen a ions o abou 1µm. 2.4.5. Apical Efflux o Micella PT and PTF by Caco-2 Cells Apical efflux was assessed as p e iously desc ibed.[34] Fi s , he apical side o he cells was incuba ed du ing 4 h wi h ca o enoid- ich mixed micelles ha con ained a ound 10 µm ca o enoids. Cells we e hen washed wi h PBS and incuba ed o 15 min wi h FBS- ee medium. Las ly, cells we e incuba ed du ing 30, 60, o 120 min wi h ca o enoid- ee mixed micelles a he apical side and he amoun o ca o enoid eco e ed in he apical medium was measu ed. 2.4.6. Effec o NPC1L1 and SR-BI Chemical Inhibi o s on micella PT and PTF Up ake by Caco-2 Cells Cells we e fi s p e-incuba ed wi h ei he 10 µmDMSO(con ol) o 10 µm o he co esponding chemical inhibi o (eze imibe glu- cu onide o NPC1L1 o BLT1 o SR-BI) o 1 h. The apical side hen ecei ed 1 mL o ca o enoid- ich mixed micelles (a 1.4 µm o PT o 1.2 µm o PTF) supplemen ed wi h ei he 10 µmDMSO (con ol) o 10 µm o he co esponding chemical inhibi o while he basola e al side ecei ed FBS- ee medium. The cellula up- ake o ca o enoids was measu ed a e 2 h incuba ion. 2.5. HEK Cell Cul u e Expe imen s To confi m p e ious esul s ob ained on he p o ein appa en ly in ol ed, o no , in PT and PTF up ake by Caco-2 cells, and o u he assess he po en ial in ol emen o CD36, which is no exp essed in Caco-2 TC-7 cells,[35] up ake s udies in G ipTi e cells, i.e., gene ically enginee ed Human Emb yonic Kidney cells (HEK 293-T cells), we e pe o med. HEK cells we e cul u ed and ans ec ed as p e iously desc ibed.[24] Fo ans ec ion, 3 µg o DNA was used, i.e., emp y pIRES plasmid o human CD36 in pIRES plasmid o s udy he in ol emen o CD36; and emp y pCDNA3.1 plasmid o human SCARBI in pCDNA3.1 plasmid o s udy he in ol e- men o SR-BI. The ans ec ions we e checked by Wes e n blo analysis.[33] Ca o enoids in mixed micelles we e no used in hese ex- pe imen s because bile sal s exe oxic effec s on HEK cells. The e o e, he ca o enoids ehicles we e p epa ed as ollows: fi s , ca o enoids in hexane we e inco po a ed in a glass ube and, a e e apo a ion o he sol en , 6 µL o e hanol we e added o acili a e he subsequen solubiliza ion o ca o enoids in FBS. Then, 1.2 mL o FBS and 10.8 mL o DMEM we e added and he final mix u e was o exed and sonica ed o wo min. Be o e each expe imen , ca o enoid concen a ion in he com- ple e medium was analyzed by HPLC. Th ee condi ions we e es ed: 1) HEK cells ans ec ed wi h he emp y plasmid (con- ol condi ion), 2) HEK cells ans ec ed wi h a plasmid con ain- ing ei he SCARB1, which encodes o SR-BI, o CD36,and3) HEK cells ans ec ed wi h a plasmid con aining ei he SCARB1 o CD36 oge he wi h an inhibi o o he co esponding p o ein (BLT1 a 10 µm o SSO a 400 µm,[36] espec i ely). Thus, he cells ecei ed 1 mL o comple e medium in which 5 µm o ei he PT o PTF was added, supplemen ed wi h ei he DMSO o he fi s and second condi ions, o wi h he co esponding inhibi o o he hi d condi ion. A e 3 h o incuba ion, ca o enoid concen- a ion was measu ed in he media and he sc aped cells. 2.6. Ca o enoid Ex ac ion and HPLC Analysis Ca o enoid ex ac ion was ca ied ou as p e iously desc ibed,[33] using α-ca o ene as an in e nal s anda d. Ca o enoid ex ac s we e e-dissol ed in 100 µL o e hyl ace a e and 10–80 µL we e injec ed. HPLC analyses we e ca ied ou on a Dionex sys em,[37] using a YMC-C30 column (5 µm, 4.6 ×250 mm) kep a 30 °C wi h a YMC-C30 p e-column (5 µm, 10 ×4 mm). The mobile phase consis ed o a mix u e o me hanol and me hyl e -bu yl e he wi h an elu ion g adien ha was desc ibed p e iously.[37] The quan ifica ion was pe o med by conside ing he da a ex- ac ed a 286 (PT), 350 (PTF), 450 (β-ca o ene, lu ein, and α- ca o ene), and 470 nm (lycopene), using Ch omeleon so wa e ( e sion 6.50 SP4 Build 1000, Dionex) and ex e nal calib a ion cu es. 2.7. Calcula ions and S a is ics Ca o enoid up ake efficiency by cells was exp essed as he pe - cen age o ca o enoids eco e ed in he sc aped cells a he end o he expe imen s ela i e o he sum o ca o enoids eco - e ed in he apical chambe plus hose eco e ed in he sc aped cells. Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (3 o 9) www.ad ancedsciencenews.com www.mn -jou nal.com Table 1. Pa ame e s o phy oene and phy ofluene up ake by Caco-2 cells. Ca o enoid Appa en Qmax (nmol) Appa en K(µm) R2 Phy oene 3.17 ±0.05a13.67 ±0.35a1.000 Phy ofluene 0.53 ±0.04b1.44 ±0.25b0.997 Caco-2 clone TC-7 cells we e hawed a passage numbe 67. Th ee weeks be o e each expe imen , he cells we e seeded on cul u e inse s (1 µm po e size polyca bona e memb ane) in six-well pla es a a densi y o a ound 25 ×104cells pe well o allow o diffe en ia ion. Twel e hou s be o e each expe imen , media we e changed o FBS- ee medium a bo h sides. Cells ecei ed 1 mL o ca o enoid- ich syn he ic mixed micelles a a ound 0.5 µM on he apical side. Ca o enoid up ake was measu ed a e 2 h incuba ion. Resul s a e shown in Figu e 1B. Bes fi ing cu es we e hype bolic ones: y=ax/(b +x). Appa en Qmax ep esen s he maximal amoun o ca o enoid ha could be aken up by cells. Appa en Kis he micella ca o enoid concen a ion a which he amoun aken up is hal he Qmax. Values ep esen means ±SEM o h ee eplica es. Mean alues wi h unlike supe sc ip le e s wi hin a column we e significan ly diffe en (p<0.05). Table 2. Ca o enoid up ake by Caco-2 cells a a micella concen a ion co - esponding o a low die a y in ake o ca o enoids. Ca o enoid Up ake (%) Phy oene 20.8 ±0.6b Phy ofluene 28.9 ±1.2a β-Ca o ene 30.6 ±0.7a Lu ein 25.8 ±2.1ab Caco-2 clone TC-7 cells we e hawed a passage numbe 92. Th ee weeks be o e each expe imen , he cells we e seeded on cul u e inse s (1 µm po e size polyca bona e memb ane) in six-well pla es a a densi y o abou 25 ×104cells pe well o allow o diffe en ia ion. Twel e hou s be o e each expe imen , media we e changed o FBS- ee medium a bo h sides. Cells ecei ed 1 mL o ca o enoid- ich syn he ic mixed micelles a a ound 0.5 µm on he apical side. Ca o enoid up ake was measu ed a e 2 h incuba ion. Values ep esen mean ±SEM o h ee eplica es. Lycopene up ake could no be accu a ely measu ed because i was lowe han he HPLC de ec ion limi . Mean alues wi h unlike supe sc ip le e s we e significan ly diffe en (p<0.05). Ca o enoid efflux efficiency by cells was calcula ed as he ela- i e amoun o ca o enoid eco e ed in he apical medium a he end o he expe imen compa ed o ha measu ed in he cells a e 4 h incuba ion. When micelliza ion and up ake expe imen s we e done using he same mixed micelles han hose used o measu e ca o enoid micelliza ion, he pe cen age o heo e ical bioa ailabili y o a ca o enoid was calcula ed as: micelliza ion efficiency (%) ×up- ake efficiency (%). All expe imen s we e done in iplica e, excep hose o s udy he implica ion o SR-BI and NPC1L1 in he up ake o PT and PTF by Caco-2 cells, which we e pe o med on wo diffe en days and included ou eplica es pe day. Resul s a e exp essed as mean ±SEM. S a is ical analyses we e pe o med using SPSS ( e sion 20, SPSS Inc., Chicago, IL, USA) s a is ical package. Be o e S u- den ’s - es o ANOVA, homogenei y o a iances was checked by Le ene’s es and no mali y o dis ibu ions by Q-Q plo s. When he F- es in ANOVA was significan , Tukey’s es was used as a pos hoc es o pai wise compa isons, bu Dunne ’s es was used when compa ing means om se e al expe imen al g oups agains a single con ol g oup mean. Fo all es s, he bila e al alpha isk was α=0.05. Rela ionships be ween wo con inuous a iables we e exam- ined by eg ession analysis on KaleidaG aph so wa e ( e sion 3.6, Syne gy so wa e, Reading, PA). 3. Resul s 3.1. Inco po a ion Efficiency o PT and PTF in Syn he ic Mixed Micelles as Compa ed o ha o Commonly Consumed Ca o enoids Ma ked diffe ences in inco po a ion efficiency o he in es iga ed ca o enoids we e obse ed (Figu e 1A). PT and lu ein displayed he highes inco po a ion efficiencies, which we e linea o e he h ee concen a ions es ed. PTF inco po a ion efficiency was simila o ha o PT and lu ein up o abou 2 µm, i.e., high die a y concen a ions, bu hen i appa en ly s a ed o pla eau when he concen a ion inc eased. Lycopene exhibi ed he lowes in- co po a ion efficiency wi h a maximum micella concen a ion o 0.06 µm a all h ee concen a ions es ed. 3.2. Compe i ion be ween Colo less Ca o enoids and O he Ca o enoids o Micelliza ion Nei he did PT o PTF compe e o hei micelliza ion when hey we e added concu en ly a 0.5 µm du ing mixed micelle syn he- sis. The addi ion o 0.5 µm PTF du ing mixed micelle syn he- sis did no significan ly impai lu ein o lycopene micelliza ion. Conce ning PT, i s addi ion did no significan ly impai lu ein micelliza ion whe eas i significan ly (p<0.05) impai ed ha o lycopene (−55.6%). Finally, he inco po a ion efficiencies o PT and PTF we e no significan ly affec ed by he simul aneous addi- ion o lu ein, β-ca o ene, o lycopene du ing mixed micelle syn- hesis (da a no shown). 3.3. Effec o he Concen a ion o Micella PT and PTF on hei Up ake Efficiency by Caco-2 Cells PT and PTF up ake by Caco-2 cells as a unc ion o hei micella concen a ion ollowed hype bolic cu es (Figu e 1B). Thus, hei up ake efficiency dec eased when hei micella concen a ion in- c eased. Mo e p ecisely, PT up ake efficiency dec eased om 23.4 o 14.6% (a 0.3 and 6.9 µm, espec i ely) and ha o PTF om 32.0 o 14.8% (a 0.4 and 2.2 µm, espec i ely). Calcula ed Qmax and Ko PT we e almos six- and en old highe han ha o PTF, espec i ely (Table 1). 3.4. Compa ison o Ca o enoid Up ake by Caco-2 Cells The up ake efficiency o lu ein, β-ca o ene, and PTF was no sig- nifican ly diffe en (p=0.121). Con e sely, PT up ake efficiency was significan ly lowe han ha o PTF and β-ca o ene (Table 2). The up ake o lycopene was oo low o be accu a ely measu ed and was he e o e ma kedly lowe han ha o he o he s udied ca o enoids. Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (4 o 9) www.ad ancedsciencenews.com www.mn -jou nal.com Figu e 1. Cha ac e iza ion o phy oene and phy ofluene micelliza ion and up ake by Caco-2 cells. A) Inco po a ion o phy oene, lu ein, phy ofluene, and lycopene in syn he ic mixed micelles. Mixed micelles wi h a ying concen a ions o pu e ca o enoids we e syn hesized and hei ca o enoid concen a ion was measu ed by HPLC. Linea end lines: Phy oene: y=0.874x(R2=0.999); lu ein: y=0.823x(R2=0.999). Cu ilinea end lines: Phy ofluene: y=−0.0734x2+0.899x(R2=1.000); lycopene: y=−0.0008x2+0.0137x(R2=0.836). B) Effec o micella phy oene and phy ofluene concen a ions on hei up ake by Caco-2 cells. The apical side o he cells ecei ed mixed micelles ha con ained diffe en concen a ions o phy oene and phy ofluene. Basola e al side ecei ed FBS- ee medium. Ca o enoid concen a ions we e measu ed in sc aped cells a e 2 h incuba ion. The bes -fi cu es we e hype bolic ones: y=ax/(x+b). Values ep esen means o h ee eplica es and e o ba s indica e s anda d e o o he mean. 3.5. Compe i ions be ween Micella Ca o enoids o hei Up ake by Caco-2 Cells The effec o he addi ion o ei he micella PT o PTF on he cel- lula up ake o commonly consumed ca o enoids is shown in Fig- u e 2A and B. Micella lu ein up ake was no significan ly affec ed by he addi ion o ei he micella PT o PTF (Figu e 2A). Con- e sely, micella β-ca o ene up ake was significan ly impai ed by PT and PTF (–12.9 and –21.6%, espec i ely) (Figu e 2B). The effec o he addi ion o ei he micella PT o PTF on lycopene up ake is no shown because i could no be accu a ely measu ed due o he e y low amoun o lycopene aken up by he cells. The effec o he addi ion o he o he s udied ca o enoids on PT and PTF up ake by Caco-2 cells is shown in Figu e 2C and D. The up ake o micella PT was significan ly impai ed when micel- la PTF, β-ca o ene, o lu ein we e added in he apical chambe (−30.8, −52.4, and −27.8%, espec i ely, p<0.001; Figu e 2C). Con e sely, only micella lu ein significan ly impai ed micella PTF up ake (–40%, p<0.001) (Figu e 2D). 3.6. Apical Efflux o PT and PTF by Caco-2 Cells The apical efflux o PT and PTF ollowing hei apical up ake was no significan ly diffe en , i.e., a ound 14 ±2% o bo h ca o enoids (p=0.649), and i did no significan ly a y om 30 o 120 min (da a no shown). 3.7. Effec o NPC1L1 and SR-BI Chemical Inhibi o s on Micella PT and PTF Up ake by Caco-2 Cells Eze imibe glucu onide, a chemical inhibi o o NPC1L1, did no significan ly affec PT o PTF up ake (Figu e 3A). Con e sely, up ake o PT and PTF was significan ly dec eased (−76.9 and −85.4%, espec i ely, p<0.001) when BLT1, a chemical inhibi o o SR-BI was added o he apical medium. 3.8. Effec o T ans ec ion o Memb ane P o eins on Micella PT and PTF Up ake by HEK Cells PT and PTF up ake was significan ly highe in HEK cells ans ec ed wi h SCARB1, which encodes o SR-BI, han in HEK cells ans ec ed wi h an emp y plasmid (p<0.01 and p<0.05 o PT and PTF, espec i ely). Fu he mo e, he addi ion o BLT1 o he SCARB1 ans ec ed cells led o abolish he highe up ake obse ed in hese cells (Fig- u e 3B). In addi ion, ans ec ion o HEK cells wi h CD36 did no significan ly change hei PT and PTF up ake efficiency (da a no shown). 4. Discussion This s udy was based on he hypo hesis ha he high bioa ail- abili y o PT and PTF ela i e o ha o o he ca o enoids ound in he same ood ma ices is due, a leas in pa , o hei peculia molecula p ope ies, which could lead o highe solubili y in mixed micelles and/o o highe up ake efficiency by in es inal cells. To e i y his hypo hesis, we fi s pu ified PT and PTF om oma o ex ac and we compa ed hei micelliza ion and hei cellula up ake efficiency o ha o pu e commonly consumed ca o enoids. We obse ed ha PT and PTF possess a much highe in insic abili y o be inco po a ed in o syn he ic mixed micelles han lycopene, ano he linea non-oxygena ed ca o enoid. In ac , a low and high die a y concen a ions, i.e., 0.5 and 2.0 µm, hei micelliza ion efficiency was simila o ha o Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (5 o 9) www.ad ancedsciencenews.com www.mn -jou nal.com Figu e 2. Compe i ions be ween micella ca o enoids o hei up ake by Caco-2 cells. A) Effec o phy oene and phy ofluene on lu ein up ake. B) Effec o phy oene and phy ofluene on β-ca o ene up ake. C) Effec o phy ofluene and commonly consumed ca o enoids on phy oene up ake. D) Effec o phy oene and commonly consumed ca o enoids on phy ofluene up ake. The apical side o he cells ecei ed 1 mL o mixed micelles ha con ained he ca o enoid o in e es plus ei he ca o enoid- ee mixed micelles (con ol) o mixed micelles loaded wi h ano he ca o enoid species. The a ge micella concen a ion o each ca o enoid in each compe i ion condi ions was 1 µm. Ca o enoid up ake was measu ed a e 2 h incuba ion. The effec o phy oene and phy ofluene on lycopene up ake could no be accu a ely measu ed because lycopene up ake was oo low o be accu a ely measu ed in ou expe imen al condi ions. Values ep esen mean o h ee eplica es and e o ba s indica e SEM. As e isks indica e significan diffe ences om he con ol (abso p ion o he ca o enoid o in e es alone): *p<0.05; ***p<0.001. Figu e 3. Implica ion o NPC1L1 and SR-BI on phy oene and phy ofluene up ake by cells. A) Effec o chemical inhibi o s o NPC1L1 and SR-BI on phy oene and phy ofluene up ake by Caco-2 cells. Cell apical sides we e p e-incuba ed o 1 h wi h ei he 10 µmDMSO(con ol)o 10µM chemical inhibi o (eze imibe glucu onide o NPC1L1 o BLT1 o SR-BI). Apical sides ecei ed he ea e phy oene- o phy ofluene-loaded syn he ic mixed micelles a 1.4 and 1.2 µm, espec i ely. Ca o enoid up ake was assessed a e 2 h incuba ion. The expe imen was ca ied ou wice, wi h 4 eplica es in each case. This figu e shows esul s o one expe imen . As e isks indica e significan diffe ences om he con ol (***p<0.001). B) Effec o ans ec ion o HEK cells wi h SR-BI gene and u he addi ion o SR-BI chemical inhibi o on phy oene and phy ofluene up ake by hese cells. Cells we e fi s ans ec ed wi h ei he an emp y plasmid (con ol) o a plasmid con aining SCARB1, i.e., he gene encoding he SR-BI p o ein. Then cells ecei ed comple e medium en iched wi h ei he micella phy oene o phy ofluene a 5 µm, supplemen ed o no wi h 10 µm DMSO o BLT1 ( he chemical inhibi o o SR-BI). Incuba ion ime was 3 h. Fo each ca o enoid ba s bea ing unlike supe sc ip le e s a e significan ly diffe en (p<0.05). In each figu e, alues ep esen means o h ee eplica es and e o ba s indica e SEM. Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (6 o 9) www.ad ancedsciencenews.com www.mn -jou nal.com lu ein, which is an oxygena ed ca o enoid acknowledged o ha e a highe micelliza ion efficiency compa ed o ca o enes.[38,39] We hypo hesize ha his high in insic solubili y in mixed micelles is due ei he o he ac ha PT and PTF we e mainly p esen in he o m o cis-isome s, which is simila o hei isome iza ion s a us in oods, and/o o he ac ha hese ca o enoids ha e a highe molecula flexibili y han he o he s udied ca o enoids. Conce ning he fi s hypo hesis, al hough i is no known whe he he cis-isome s o PT and PTF ha e highe solubili y in micelles han hei espec i e all- ans isome s, we hypo hesize ha his is e y likely because his has been shown o ano he linea ca o ene, i.e., lycopene.[39–41] Conce ning he second hypo hesis, i has been shown ha , due o hei lowe numbe o CDB (Figu e S1, Suppo ing In o ma ion), PT and PTF can old mo e eely and adop less igid shapes han commonly consumed ca o enoids.[32] Fu he mo e, he highe numbe o sigma bonds in hese molecules, whe e o a ion is possible,[42] leads o a mo e p onounced wis in he backbone o hese molecules.[4,5] In ac , o sional ene gies o he linea ca o enoids in es iga ed ank as ollows: PT (ࣈ57 kcal mol−1,3CDB)< PTF (ࣈ61 kcal mol−1,5CDB)<lycopene (ࣈ73 kcal mol−1,11 CDB).[32] This highe flexibili y and wis abili y a e assumed o ansla e in o be e inse ion o hese ca o enoids be ween lipid molecules composing mixed micelles. Howe e , we canno conclude whe he he high bioaccessibili y o PT and PTF is due o hei cis-isome iza ion, o hei high molecula flexibili y, o bo h. Second, we s udied he up ake o PT and PTF by in es inal cells. The fi s key obse a ion was ha hei up ake efficiency was much highe han ha o lycopene. In ac , he up ake efficiency o PTF was equi alen o ha o lu ein and β-ca o ene. The second key obse a ion was ha he sa u able up ake o PT and PTF s ongly sugges ed a p o ein-media ed up ake. Ano he in e es ing obse a ion was ha PT up ake was highe han ha o PTF a high die a y concen a ions, i.e. >2µm(Figu eS2, Suppo ing In o ma ion), while i was lowe a low die a y concen a ions (Table 2). This effec o he colo less ca o enoid concen a ion on hei ela i e cellula up ake efficiency was unexpec ed bu i was in ag eemen wi h a p e ious s udy.[43] In his clinical s udy, he bioa ailabili y o PTF was highe han ha o PT in he g oup o subjec s who inges ed he lowes concen a- ions o colo less ca o enoids (ࣈ0.9 mg o PT and PTF pe day o 12 weeks), while i was he opposi e in he g oup who inges ed he highes concen a ions (4.6 mg o PT and 3.2 mg o PTF pe day o 12 weeks). We hypo hesize ha his concen a ion effec can be due o diffe ences be ween PT and PTF ega ding hei ela i e affini y o memb ane anspo e (s). Indeed, he highe appa en Qmax o PT,ascompa ed o ha o PTF,couldbe explained by he hypo hesis ha PTF possesses a highe affini y o he main anspo e o hese colo less ca o enoids han PT. This las hypo hesis is suppo ed by i s lowe appa en Kand by he ac ha PTF significan ly inhibi ed PT up ake while he opposi e was no obse ed. The pe cen ages o heo e ical bioa ailabili y o PT and PTF a 0.5 µm, i.e., a a low die a y concen a ion, we e 18.3 and 26.1%, espec i ely, which was in ag eemen wi h he esul s ob ained in he p e iously men ioned s udy.[43] A e ob aining esul s ha sugges ed he colo less ca o enoid up ake is p o ein media ed, we e alua ed whe he p o eins ha ha e been shown o pa icipa e in he up ake o commonly con- sumed ca o enoids, i.e., SR-BI, CD36, and NPC1L1,[27] a e also in ol ed in PT and PTF up ake. O e all, ou esul s sugges ha SR-BI is in ol ed in he up ake o PT and PTF while CD36 and NPC1L1 a e no . The in ol emen o SR-BI is in ag eemen wi h he esul s ob ained o o he ca o enes, i.e., lycopene and β- ca o ene.[24,25,44] The lack o in ol emen o CD36 sugges s ha his p o ein is mo e specifically associa ed wi h he up ake o p o i amin A ca o enoids.[24] Ano he in e es ing finding was ha abou 14% o PT and PTF aken up by he cells was appa - en ly effluxed back o hei apical side. This is consis en wi h p e- ious da a sugges ing ha o he a -soluble mic onu ien s, such as ocophe ol, cholecalci e ol, o phylloquinone,[33,45,46] a e pa - ially effluxed by Caco-2. A e ha ing ob ained key in o ma ion on he mechanisms implica ed in abso p ion o hese ca o enoids and because hese phy ochemicals migh be used in he u u e as die a y supplemen s, whe he hey exhibi demons a ed benefi s o heal h, we assessed whe he hey compe e wi h commonly consumed ca o enoids o ei he hei micelliza ion o in es inal cell up ake. Indeed, significan compe i ions a hese key s eps o ca o enoid abso p ion could lead o a dec ease in abso p ion o ca o enoids ha possess well-acknowledged heal h effec s, e.g., β-ca o ene and lu ein. Conce ning micelliza ion, only one com- pe i ion was obse ed, i.e., PT significan ly impai ed lycopene micelliza ion. I seems logical o obse e ha he ca o enoid ha has he highes abili y o be inco po a ed in mixed micelles sig- nifican ly impai ed he micella inco po a ion o he one ha has he lowes abili y o be inco po a ed in micelles. Conce ning cel- lula up ake, ou esul s sugges ha PT and PTF can pa ially impai he in es inal up ake o β-ca o ene, and ice e sa. This is in ag eemen wi h p e ious esul s showing ha commonly consumed ca o enoids compe e o hei in es inal up ake,[47] and his is likely explained by he ac ha all hese ca o enoids sha e a leas one common memb ane anspo e , in ha case SR-BI. In summa y, his s udy has p o ided esul s allowing us o sugges why he bioaccessibili y o PT and PTF is unexpec - edly high as compa ed o ha o he o he main linea die a y ca o ene, i.e., lycopene. Indeed, his is likely because hese colo - less ca o enoids a e p esen mainly as cis-isome s in oods and/o because o hei high molecula flexibili y. This s udy has also p o ided us da a sugges ing ha SR-BI, which is in ol ed in up- ake o commonly consumed ca o enoids, is also in ol ed in cel- lula up ake o PT and PTF, which in u n explains compe i ions o cellula up ake. We ha e also ob ained da a sugges ing ha a ac ion o abso bed PT and PTF is effluxed back o he in es inal lumen. We acknowledge some limi a ions o his s udy. Fi s , we compa ed micelliza ion o mainly cis-isome s o PT and PTF wi h ha o all- ans isome s o commonly consumed ca o enoids. Thus, we canno conclude ha all- ans PT and PTF a e be e inco po a ed in micelles han all- ans common ca o enoids. Ne - e heless, his would ha e a low in e es o nu i ionis s because hese colo less ca o enoids a e na u ally p esen in oods as cis- isome s. Ye , his could be o in e es o people who would like o chemically syn hesize hese compounds. The second main limi- a ion is ha we did no use an in i o diges ion model o assess bioaccessibili y. Ne e heless, his model was used in p e ious s udies, and ou aim was o go u he by ob aining da a on he in insic solubili y o hese ca o enoids in micelles o unde s and why hey a e so bioaccessible. Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (7 o 9) www.ad ancedsciencenews.com www.mn -jou nal.com Suppo ing In o ma ion Suppo ing In o ma ion is a ailable om he Wiley Online Lib a y o om he au ho . Acknowledgemen s P.M.B. and C.D. a e join fi s au ho s. P.B. designed he esea ch p ojec wi h P.M.B., C.D., and A.M.M.; P.B., C.D., and E.R. designed he p o o- col; P.M.B. and V.M. conduc ed he micelliza ion s udies; P.M.B., M.M., V.M., and M.N. conduc ed he cell s udies; P.M.B. and C.H. measu ed ca o enoid concen a ions by HPLC; PMB analyzed he esul s wi h C.D. and E.R.; P.M.B. and C.D. pe o med s a is ical analyses; P.B., P.M.B., and C.D. w o e he pape wi h consul a ion om E.R.; P.B. had p i- ma y esponsibili y o he final con en o he manusc ip . All au- ho s ha e ead and app o ed he final manusc ip . The au ho s would like o hank Vincen Migno , Cha lo e Halimi, and Ma ion Nowicki (C2VN, Ma seille) o aluable echnical assis ance. P.M.B. ecei ed und- ing o ca y ou a Sho -Te m Scien ific Mission a PB’s human mi- c onu i ion eam in Ma seille om he Eu opean COST Ac ion EU- ROCAROTEN (CA15136, Eu opean ne wo k o ad ance ca o enoid e- sea ch and applica ions in ag o- ood and heal h, www.eu oca o en.eu, h p://www.cos .eu/COST_Ac ions/ca/CA15136). The Ca no S a Ins i u e p o ided unding o consumables. A.M.M. and P.M.B. acknowledged unding om he Andalusian Coun- cil o Economy, Inno a ion, Science and Employmen (p ojec e . CAROTINCO-P12-AGR-1287). Conflic o In e es A.M.M. is a membe o he ad iso y boa d o IBR-Is aeli Bio echnology Resea ch, L d. (Ya ne, Is ael). Keywo ds bioaccessibili y, bioa ailabili y, ca o enoids, lu ein, β-ca o ene Recei ed: July 16, 2018 Re ised: Augus 29, 2018 Published online: Sep embe 24, 2018 [1] E. Biehle , A. 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