Whey proteins: targets of oxidation, or mediators of redox protection

F ee Radical Resea ch ISSN: 1071-5762 (P in ) 1029-2470 (Online) Jou nal homepage: www. and online.com/jou nals/i a20 Whey p o eins: a ge s o oxida ion, o media o s o edox p o ec ion Linda Giblin, A. Süha Yalçın, Gökhan Biçim, Anna C. K äme , Zhi ei Chen, Michael J. Callanan, Elena A anz & Michael J. Da ies To ci e his a icle: Linda Giblin, A. Süha Yalçın, Gökhan Biçim, Anna C. K äme , Zhi ei Chen, Michael J. Callanan, Elena A anz & Michael J. Da ies (2019) Whey p o eins: a ge s o oxida ion, o media o s o edox p o ec ion, F ee Radical Resea ch, 53:sup1, 1136-1152, DOI: 10.1080/10715762.2019.1632445 To link o his a icle: h ps://doi.o g/10.1080/10715762.2019.1632445 © 2019 The Au ho (s). Published by In o ma UK Limi ed, ading as Taylo & F ancis G oup Published online: 12 Sep 2019. Submi you a icle o his jou nal A icle iews: 4960 View ela ed a icles View C ossma k da a Ci ing a icles: 15 View ci ing a icles Full Te ms & Condi ions o access and use can be ound a h ps://www. and online.com/ac ion/jou nalIn o ma ion?jou nalCode=i a20 REVIEW ARTICLE Whey p o eins: a ge s o oxida ion, o media o s o edox p o ec ion Linda Giblin a ,A.S € uha Yalc¸ın b ,G € okhan Bic¸im b , Anna C. K € ame c , Zhi ei Chen c , Michael J. Callanan d , Elena A anz a and Michael J. Da ies c a Teagasc Food Resea ch Cen e, Moo epa k, Fe moy, Co k, I eland; b Depa men o Biochemis y, School o Medicine, Ma ma a Uni e si y, _ Is anbul, Tu key; c Depa men o Biomedical Sciences, Panum Ins i u e, Uni e si y o Copenhagen, Copenhagen, Denma k; d Depa men o Biological Sciences, Co k Ins i u e o Technology, Co k, I eland ABSTRACT Bo ine whey p o eins a e highly alued dai y ing edien s. This is p ima ily due o hei amino acid con en , diges ibili y, bioac i i ies and hei p ocessing cha ac e is ics. One o he epo ed bioac i i ies o whey p o eins is an ioxidan ac i i y. Nume ous die a y in e en ion ials wi h humans and animals indica e ha consump ion o whey p oduc s can modula e edox bioma ke s o educe oxida i e s ess. This bioac i i y has in pa been assigned o whey pep ides using a ange o biochemical o cellula assays in i o.Supe imposingwhey pep ide sequences om gas oin es inal samples, wi h whey pep ides p o en o be an ioxi- dan in i o, allows us o p opose pep ides om whey likely o exhibi an ioxidan ac i i y in he die . Howe e , whey p o eins hemsel es a e a ge s o oxida ion du ing p ocessing pa - icula ly when exposed o high he mal loads and/o ex ensi e p ocessing (e.g. in an o mula manu ac u e). Oxida i e damage o whey p o eins can be selec i e wi h ega d o he esidues ha a e modi ied and a e associa ed wi h he deg ee o p o ein un olding, wi h a-Lac albumin mo e suscep ible han b-Lac oglobulin. Such oxida i e damage may ha e ad e se e ec s on human heal h. This e iew summa ises how whey p o eins can modula e cellula edox pa hways and con e sely how whey p o eins can be oxidised du ing p ocessing. Gi en he ex ensi e p ocessing s eps ha whey p o eins a e o en subjec ed o, we conclude ha oxida ion du ing p ocessing is likely o comp omise he posi i e heal h a ibu es associ- a ed wi h whey p o eins. ARTICLE HISTORY Recei ed 4 Ma ch 2019 Re ised 5 June 2019 Accep ed 6 June 2019 KEYWORDS An ioxidan ; bioa ailable; gas oin es inal diges ion; glyca ion; in an o mula; oxida i e damage; p ocessing; acemised amino acids; whey p o eins In oduc ion Milk p o eins (whey p o eins (20%) and caseins (80%)) a e high-quali y sou ces o amino acids (AA) in he human die . Bo ine milk p o eins and pep ides play impo an oles in human heal h no jus in e ms o nu i ion bu also in e ms o hei no able bioac i i ies. The majo bo ine whey p o eins a e a-Lac albumin (a-Lac) and b-Lac oglobulin (b-Lg) wi h immunoglobulins, bo ine se um albumin (BSA) and lac o e in as mino p o eins (Table 1). Whey p o eins p o ide a comple e p o ein sou ce and a e ich in bo h sulphu -con aining and b anched-chain AAs. Whey p o eins do no coagula e unde he acidic con- di ions p esen in he s omach, and a e conside ed o be “ as p o eins”since hey each he jejunum sho ly a e en e ing he gas oin es inal ac and ha e a diges ible indispensable AA sco e o 1.09 [1]. Whey p o eins exhibi a wide ange o bioac i i ies including an ioxidan , an ibac e ial, an i ungal, an i i al, an ihype ensi e, an i h ombo ic, opioid and immunomodula o y p ope ies [2]. Consequen ly, whey p oduc s a e ecognised as alue-added ing e- dien s and a e commonly used in he spo s nu i ion ma ke , nu i ional be e ages o he elde ly and in an o mula (IF) [3]. These p oduc s a e also p ized in ood o mula ion as hey imp o e p oduc quali y by wa e - binding, s abilising ae a ed ood p oduc s and ac ing as emulsi ying agen s [4]. Whey p oduc s can be sup- plied as ing edien s o ood o mula ions as whey p o- ein concen a e (WPC), whey p o ein isola e (WPI) o as whey hyd olysa es (WH) all o which di e in he deg ee o p ocessing and p o ein con en (Table 2). Howe e , nume ous p ocessing s eps om milk o whey powde o ood o mula ion inhe en ly expose whey p o eins hemsel es o edox modi ica ions. This e iew summa ises how whey p o eins can modula e cellula edox pa hways and con e sely how whey p o- eins can be oxidised du ing p ocessing. CONTACT Linda Giblin [email p o ec ed] Teagasc Food Resea ch Cen e, Moo epa k, Fe moy, Co k, I eland ß2019 The Au ho (s). Published by In o ma UK Limi ed, ading as Taylo & F ancis G oup This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/Licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. FREE RADICAL RESEARCH 2019, VOL. 53, NO. S1, 1136–1152 h ps://doi.o g/10.1080/10715762.2019.1632445 Whey p o eins and hei abili y o modi y edox pa hways –s udies in animal models and humans The an ioxidan and de oxi ying bioac i i ies o whey p o eins a e mos likely linked o hei con ibu ion o glu a hione (GSH) syn hesis, ecen ly e iewed in Co ochano e al. [5]. Whey p o eins a e ich in Cys wi h b-Lg con aining 5 Cys esidues, a-Lac has 8 Cys, BSA has 35 Cys and lac o e in con ains 34 Cys, al hough in each case he majo i y o Cys is p esen as disulphide bonds. The hiol (R-SH) g oup o Cys eac s apidly wi h many oxidan s. This AA when p esen wi h Gly and Gln in he ipep ide, GSH, is an impo an co ac o and an ioxidan in mammalian cells and issues. Reduced GSH, is eadily oxidised o he disulphide species, oxi- dised glu a hione (GSSG), wi h he la e hen eadily ecycled by he enzyme GSH educ ase, a he expense o nico inamide adenine dinucleo ide phospha e (NADPH), back o i s educed o m. O he oxidised spe- cies can howe e also be o med om GSH including GSH sulphonamide and oxy acids, wi h he la e spe- cies being i e e sible p oduc s. GSH de oxi ies a num- be o endogenous and exogenous oxins including oxic me als, pe oleum dis illa es, lipid pe oxides, qui- nones, bili ubin and p os aglandins h ough di ec con- juga ion. Cell lines (e.g. C2C12, MRC-5, PC12, Caco 2 , HUVEC, 9HTEo, HepG2, and REPE-1) exposed o a ious whey p oduc s (WPC and WPI) ha e documen ed inc eases in GSH le els wi h some excep ions [5]. Whey p oduc s ha e also been epo ed o inc ease ac i i ies o he p o ec i e enzymes supe oxide dismu ase (which emo es supe oxide adicals) and ca alase (which educes H 2 O 2 o wa e ) and dec ease le els o eac i e oxygen species, lipid pe oxida ion and DNA damage in cellula assays [5]. Howe e using cell lines wi h whole p o eins has i s limi a ions, no leas o which is he al e ed edox homeos asis o immo alised cell lines [6] and he nonphysiological exposu e o cells o in ac die a y p o eins. Human o animal in e en ion ials wi h die s ha include whey p oduc s a e he bes assessmen o impac on cellula edox pa hways al hough eliable and consis en da a wi h well-de ined bioma ke s is a he limi ed. In a signi ican numbe o cases gene ic and non-speci ic assays o an ioxidan ac i i y ha e been used, which is no ideal. Table 3 lis s a and mouse s udies wi h a ocus on edox pa ame e s, whe e animals consumed di e en whey p oduc s (WPC and WPI) a a dosage o 0.02–1 g/kg body mass o e a pe iod o 7–84 d. Redox bioma ke s we e measu ed in li e , b ain, e y h ocy e, muscle, se um, kidney, colon, sali a y gland o pa a hy oid gland in he p esence o absence o a ious s esso s (exe cise, diabe es, hea , b ain inju y, hepa o oxici y, dyslipidaemia, schizoph e- nia o a la oxin). Al hough con lic ing da a ha e been epo ed [7], he majo i y o ials ha e epo ed inc eases in GSH and o he an ioxidan ma ke s. Howe e exac ly which species, and wha concen a- ions, a e esponsible o hese changes is unclea in mos cases. Human die a y whey in e en ion ials wi h a ocus on edox eadou s ha e been e iewed p e iously by Co ochano e al. [5], wi h addi ional ials de ailed in Table 4. These in e en ion ials gene ally ec ui pa icipan s o , on a e age, a 3-week s udy wi h o wi hou an exe cise ou ine, measu ing plasma GSH as an indica o o whole body edox s a us. Many s ud- ies epo inc eases in plasma GSH om ime ze o wi h whey in e en ion bu o he s obse e no e ec on plasma GSH le els (Table 4)[8,9]. Whe he o no a sin- gle edox analy e de ec ed a e y low le els in he plasma is a good bioma ke o edox s a e a a global o local issue le el is deba able [10,11]. The e is also some e idence om in e en ion ials ha whey p o eins may dec ease plasma GSH le - els, cause hea damage and li e inju y [12,13]. O al Table 1. Bo ine whey p o eins. Bo ine whey p o ein Concen a ion (g/L) in bo ine milk Molecula mass (kDa) AAs To al 7 b-Lac oglobulin 3.5 18.3 162 a-Lac albumin 1.2 14.2 123 Immunoglobulins 0.7 150–900 Va iable Bo ine se um albumin 0.4 66.4 583 Lac o e in 0.02–0.35 80 700 Table 2. Composi ion o di e en bo ine whey p oduc s. Whey powde ype P o ein (%) Fa (%) Lac ose (%) Whey p o ein concen a e (WPC) 34–80 1–74–52 Whey p o ein isola e (WPI) 90–95 0.5–1 0.5–1 Hyd olysed whey p o ein (WH) 80–90 0.5–8 0.5–10 FREE RADICAL RESEARCH 1137 Table 3. Animal s udies wi h di e en whey p oduc s and he an ioxidan esponses epo ed. Bo ine whey p oduc , sou ce a Dose and ime Species/ issue/o gan/disease Resul s b Re e ence WPC, Dai y Coope a i e Poland 0.3 g/kg; 7–14 d Ra ; sali a y gland, pa o id gland "GSH; "GSH-Px; "SOD; WPC imp o ed edox homeos asis in sali a y glands [131] WPC, Dai y Coope a i e Poland 0.3–0.5 g/kg; 7–21 d Ra ; li e "hepa ic GSH; "hepa ic MDA [132] WPC, Da isco 0.02–0.04–0.06–0.08–0.1 g/kg bw (30 d) Ra ; CCl 4 hepa o oxici y; li e #MDA; WPC p e en ed li e damage induced by CCl 4 [133] Un-dena u ed WP, P o he V R , I aly 18%; 3 weeks Ra ; CCl 4 hepa o oxici y; li e "GSH [134] WP, G een Land o Food Indus ies, Egyp 10%; 28 ds Ra ; CCl 4 hepa o oxici y; li e , #MDA; "GSH [135] WPC, Da isco 0.1 g/mL; 30 d Ra ; CCl 4 hepa o oxici y; li e , #MDA; "CAT; "GST [136] WPI, Da isco 0.1 g–0.2 g/kg; Ra ; lipolysaccha ides/ li e , se um "SOD; #DNA damage; #Caspase-3 (apop osis) [137] WPC, Da isco 0.3 g/kg; 30 d Ra ; s la oxin-con amina ed die ; li e , es es "GSH, #LP, #DNA damage; WPC p e en s geno oxici y o a la oxins [138] WPC, Da isco 0.3 g/kg; 30 d Ra ; a la oxin-con amina ed die ; li e , es es "GSH, #LP, #DNA damage; WPC educed oxida i e s ess induced by a la oxins [139] WPC, P obio ica 0.15 g/kg; 8 weeks Ra ; exe cise model; muscle, li e WP inc eased hepa ic GSH and p o ec ed agains exe cise induced muscle p o ein oxida ion [140] WPI, Diamond Whey, I aly 20% Mouse; exe cise model; muscle, li e #LP; #GSH/GSSG [141] WPC, Camillo ek, India 0.3 g/kg; 28 d Ra ; e y h ocy e om young and aged animals #T-SH; #sialic acid; "ipid hyd ope oxides; "p o ein ca bonyls; WPC es o es edox s a us in e y h ocy es [142] WPI, Da isco 0.15 g/kg; 28 d Ra ; non-alcoholic a y li e "hepa ic GSH, #hepa ic MDA [143] WP, Immunocal 3.3% wice daily; 28 d Mouse; auma ic b ain inju y "GSH/GSSG; p e en ed educ ion o GSH/GSSG in b ain [144] WPI, Bioplex Nu i ion, USA 100 g; 12 weeks Mouse; b ain mi ochond ia #MDA; #4-hyd oxyalkenals; inc eased b ain mi ochond ia ac i i y [145] WP, Immunocal 0.66 g/kg; 60 d Mouse; ALS model T ea men p e en ed disease- associa ed educ ions in whole blood and spinal co d issue GSH [146] WPC 0.3 g/kg; 28 d Ra ; neu odegene a ion; b ain "Beclin-1; "A g-3 (Au ophagy); "FRAP; #PC; "T-SH; #ROS; #NO; "ace ylcholines e ase [142] WPI, Immunocal 0.33 g/mL; 6 weeks Mouse; schizoph enia model; b ain "GSH/GSSG a ios; "GSH [147] WP, Tu key WP supplemen ed die ; 21 d Ra ; bu n inju y model; li e , kidney Whey p o ein supp essed bu n-induced changes in hepa ic and enal issue [148] WP, Tu key WP supplemen ed die ; 21 d Ra ; expe imen al lapa o omy and colonic anas omosis; li e , abdominal wall, colon Whey p o ein supplemen a ion inc eased GSH and supp essed MDA in di e en issues [149] WPI, Balance Muscle Technologies, New Zealand 150—250 g/kg; 4 weeks Ra ; HLAP; colon No change DNA damage [150] WP, mozza ella cheese 10 g/100 g; 6 weeks Ra ; i on o e load; oxici y; plasma, e y h ocy e "SOD, "GSH, no change GSH- Px in e y h ocy es; no change plasma Vi E; WP had an ioxida i e and an igeno oxic e ec s [7] WP, Immunocal 0.1 g; 4 weeks Mu ine; i on o e load/ oxici y #MDA; #Hexanal; "GSH- Px; "GSH [151] a WPI: whey p o ein isola e; WP: whey p o ein, ype no speci ied; WPC: whey p o ein concen a e. b GSH: glu a hione; GSH –Px: glu a hione pe oxidase; CAT: ca alase; GPT: glu amic-py u ic acid ansaminase; ALP: alkaline phospha ase; SOD: supe oxide dismu ase; GST: glu a hione S- ans e ase; GSSG: oxidised glu a hione; LP: plasma lac a e; T-SH: o al hiol; PC: p o ein ca bonyl; ROS: eac i e oxygen species; NO: ni ic oxide; MDA: malondialdehyde, CCl 4 : ca bon e achlo ide. FRAP: e ic- educing an ioxidan powe . 1138 L. GIBLIN ET AL. Table 4. Human in e en ion ials wi h whey p oduc s and physiological esponse. Bo ine whey p oduc a Desc ip ion o he s udy Resul s b Re e ence WPI undena u ed Open-labeled clinical ial, 38 pa ien s wi h non-alcoholic s ea ohepa i is we e gi en 20 g whey p o ein isola e o 12 weeks. Imp o emen s in li e biochemis ies, inc eased plasma GSH, o al an ioxidan capaci y, and educed hepa ic mac o esicula s ea osis in pa ien s compa ed o ime ze o (s udy s a ). [152] WPI 23 cance pa ien s ecei ed 4 0g WPI plus 2.64 mg zinc and 0.76 mg selenium o al snack o 12 weeks compa ed o a con ol g oup (n¼19) who ecei ed 40 g mal odex in. Baseline assessmen s a 6 and 12 weeks. Con ols showed a signi ican ly lowe % change in plasma GSH le els whe eas he e was a signi ican ime-dependen inc ease in he in e en ion g oup om ime ze o o 12 weeks and be ween whey and mal odex in in e en ion a bo h 6 weeks and 12 weeks. [153] WP A pilo open-label s udy o die a y supplemen a ion wi h p essu ised whey in 27 cys ic ib osis pa ien s. Whey dosage: 20 g/d in pa ien s <18 yea s and 40 g/d in olde pa ien s o 1 mon h. Whole blood glu a hione le els did no change om ime ze o o 1 mon h. O al supplemen a ion wi h p essu ised whey imp o es nu i ional s a us and can ha e addi ional bene icial e ec s on in lamma ion in pa ien s wi h cys ic ib osis. [8] WPC immunocal 10 cys ic ib osis pa ien s ecei ed whey p o ein isola e (20 g/d) o 3 mon hs compa ed o a cys ic ib osis con ol g oup (n¼11) who ecei ed a casein placebo. 46.6% inc ease om ime ze o was obse ed in he lymphocy e GSH le els wi h whey. [154] WPI 5 d o ene gy balance, ene gy de ici , and esis ance exe cise a e ene gy de ici . 8 males and 7 emales heal hy esis ance- ained subjec s comple ed esis ance exe cise and consumed ei he placebo o 30 g whey p o ein immedia ely pos -exe cise. Muscle biopsies we e ob ained a 1 and 4 h in o eco e y in each ial. Res ing p o ein le els o au ophagy- ela ed gene p o ein 5 dec eased a e ene gy de ici compa ed wi h placebo. [155] WPI Acu e s udy 4 h: Heal hy, 9 seden a y male subjec s ed wo doses (0.8 o 1.6 g/kg body mass). No e ec o he supplemen a ion a ei he dose o e he 4-h sampling pe iod on blood glu a hione concen a ion. [9] Ch onic s udy: 18 males subjec ed o 6-week ae obic (bike) aining pe iod and whey supplemen a ion The ae obic aining pe iod esul ed in signi ican ly lowe glu a hione concen a ions in whole blood, an e ec ha was mi iga ed by WPI supplemen a ion. WPI WPI (40 g/d) we e supplemen ed o 31 o e weigh people wi h impai ed as ing glucose/DM2 o 12 weeks. An inc ease in glu a hione pe oxidase, a dec ease in u ic acid and no change in glu a hione educ ase, o al an ioxidan s a us, oxida i e damage, in lamma ion and glucose ma ke s we e obse ed a 12 weeks compa ed o ime ze o. [156] Signi ican imp o emen s in an h opome ic pa ame e s and a mass we e also de ec ed. WPI Whey p o ein supplemen a ion (30 g, h ee imes pe day) and esis ance aining in 10 o e weigh young men compa ed o a con ol g oup and esis ance aining only g oup. Inc eased le els o o al an ioxidan capaci y and GSH was obse ed a e whey supplemen a ion compa ed o o he ea men g oups and compa ed o p e- es . Plasma i amin C le els we e signi ican ly inc eased wi h whey supplemen a ion compa ed o p e- es . Al hough exe cise can lead o an ioxidan sys em imp o emen and educe some ca dio ascula isk ac o s among o e weigh subjec s, he combina ion o esis ance aining and whey consump ion was mo e e ec i e. [157] WPI immunocal The e ec s o WPI supplemen a ion (20 g/d) o 6 mon hs on 15 Pa kinson’s disease pa ien was compa ed o 16 pa ien s on soy p o ein supplemen a ion. Signi ican inc eases in plasma concen a ion o educed GSH and he a io o educed o oxidised glu a hione we e ound in he 15 WPI-supplemen ed pa ien s compa ed o baseline. This was associa ed wi h a signi ican dec ease o plasma le els o homocys eine. [158] WPI immunocal o i o ec amin O al supplemen a ion wi h wo di e en Cys- ich whey p o ein o mulas o 30 HIV- in ec ed pa ien s. Pa ien s we e andomised o a supplemen al die wi h a daily dose o 45 g o whey p o eins. O al supplemen a ion wi h P o ec amin signi ican ly inc eased plasma GSH le els in pa ien s wi h ad anced HIV-in ec ion compa ed o baseline. [159] WPC A double-blind clinical ial o 4 mon hs wi h 9 HIV-in ec ed child en (6 yea s) who ecei ed whey p o ein (mon h 1 ¼20% RDA p o ein, mon h 2 ¼30% RDA, mon h 3 ¼40% RDA, WPC supplemen a ion signi ican ly inc eased e y h ocy e GSH le els and signi ican ly dec eased CD8þcells compa ed o baseline. [160] (con inued) FREE RADICAL RESEARCH 1139 ga age o adul male Sp ague Dawley a s (n¼6) wi h he en i onmen al pollu an ac olein (0.005 g/kg body weigh /d) o 30 d esul ed in a signi ican dec ease in GSH le els (8.38 ± 1.17 nmol/mg p o ein) in ed blood cells compa ed o he con ol g oup (11.31 ± 1.63 nmol/mg p o ein) (p<.05) [12]. Addi ion o whey p o ein a a dosage o 0.2 g/kg body weigh /d did no hal he loss o GSH. In addi ion, his co- ea - men wi h whey p o ein exace ba ed an obse ed inc ease in plasma homocys eine le els and c ea ine kinase le els induced by ac olein, such ha he le els o hese ma ke s in he co- ea men g oup we e signi i- can ly highe han in he con ols. This led he au ho s o hypo hesise ha in ake o ac olein oge he wi h whey p o eins may cause hea damage in a s [12]. Li e may also be ad e sely a ec ed by whey ea - men . G€ u gen e al. [13] in es iga ed li e heal h in Wis a albino male a s (n¼10) a e a whey p o ein die o 5 d (sho - e m) o 4 weeks (long- e m). Hepa ic inju y was obse ed by abno mal hepa ocy e his ology and signi ican ly inc eased le els o se um aspa a e amino ans e ase and hepa ic in e luekin-1bin a s ha consumed whey compa ed o he con ol g oup, wi h he ma ke s o li e inju y wo sening wi h inc eas- ing ime o exposu e o whey p o ein die (p<.05). Bioa ailable an ioxidan whey pep ides In addi ion o p o iding essen ial AAs and educed hiols (Cys esidues), whey p o eins also con ibu e pep- ides wi h po en ial an ioxidan ac i i y [5,14,15]. Howe e o be bioac i e beyond he gu , whey pep i- des mus su i e gu ansi and be bioa ailable o hei a ge [16]. The ha sh condi ions o he uppe gas o- in es inal ac unc ion o hyd olyse p o eins in o indi- idual AAs o anspo ac oss he in es inal ba ie . Se e al ecen s udies ha e acked he a e o whey p o eins du ing uppe gas oin es inal diges ion using in i o diges ion models [14,17–20] o gas ic [18,21] and jejunal e luen s [22,23] om pigs [18,21,24] and humans [19,22,23] pos consump ion o a ious dai y oods (IF, WPI, skim milk powde , unpas eu ised milk, whey powde , and lac o e in). Based on hese s udies, Table 5 lis s loca ions wi hin indi idual whey p o eins om which pep ides ha e been iden i ied in he in es- inal phase. b-Lg has ou gu - esis an “ho spo s;”con- sis ing o agmen s ( ) wi h he ollowing amino acid esidues om he p ima y sequence (41–58), (92–100), (126–138) and (149–154). a-Lac also has 4 ho spo s; (17–27), (63–68), (80–90) and (97–102). BSA has 5; (11–18), (107–114), (219–224), (489–495) and (514–518). Lac o e in has 7; (67–77), (140–145), (216–228), (289–295), (309–318), (332–337) and (592–594). This sugges s ha hese egions o he p i- ma y sequence a e somewha esis an o gas oin es- inal diges ion and pep ides om hese ho spo s may su i e he gu long enough o be anspo ed ac oss he in es inal ba ie . Ce ainly, he p esence o p oline and/o aspa ic acid, o glu amic acid esidues wi hin a pep ide appea s o con e a esis ance o gas oin es- inal diges ion [23]. I should also be no ed ha pep ide p o iles di e acco ding o deg ee o p ocessing [24]. Table 5 also de ails whey pep ides ha ha e been epo ed o ha e po en ial edox ac i i y enc yp ed wi hin hese ho spo s [5,25–35]. These whey pep ides ha e been epo ed o show edox ac i i y in he e ic- educing an ioxidan powe assay (FRAP), 2,20-azino- bis(3-e hylbenzo hiazoline-6-sul onic acid) (ABTS) assay, oxygen adical abso bance capaci y assay (ORAC) o he 1,1-diphenyl-2-pic ylhyd azyl (DPPH) adical assay. I should howe e be no ed ha all o hese assays a e gene ic in i o assays conduc ed in he absence o al e na i e a ge s, and hence he da a canno be ead- ily ansla ed o mo e complex sys ems. In some cases, howe e , hey ha e also been shown o boos cellula an ioxidan s a us, albei in i o [5,15]. C oss ma ching gas oin es inal esis an ho spo s o an ioxidan bio- ac i i y allows us o p opose a lis o po en ial an ioxi- dan whey pep ides likely o a i e in he bloods eam, pos whey consump ion. Li le is known abou pep ides de i ed om whey ci cula ing in he bloods eam. Jakobsson e al. [36] quan i ied a-Lac (140–250 mg/L se um/L human milk/kg body weigh ) by adioimmuno- assay in blood plasma om 1 mon h old b eas ed Table 4. Con inued. Bo ine whey p oduc a Desc ip ion o he s udy Resul s b Re e ence mon h 4: 50% RDA) compa ed o con ol g oup. WPI, p essu ised 2-week pe iod, 18 heal hy males and 18 heal hy emales we e andomised in o h ee di e en g oups. Each g oup inges ed 15, 30, o 45 g/d p essu ised whey p o ein in he mo ning in ba o ma o 14 d. Inc eases in lymphocy e GSH le els om p e o pos supplemen a ion was a ec ed by he amoun o whey p o ein inges ed, wi h 45 g/d o whey supplemen a ion o e 2 weeks inc easing lymphocy e GSH by 24% om baseline. [161] a WPI: whey p o ein isola e; WP: whey p o ein, ype no speci ied; WPC: whey p o ein concen a e. b GSH: glu a hione. 1140 L. GIBLIN ET AL. in an s (n¼3), 30–60min a e eeding. Kui unen e al. [37] also epo ed ha plasma om 20 ull e m in an s was posi i e o a-Lac and b-Lg, le els o which declined o e ime (almos 60% educ ion by 8 mon hs o age) as he in an gu ba ie ma u ed. No in o ma- ion on he sequences o he bioa ailable pep ides was p o ided. Howe e , we ha e ecen ly iden i ied a num- be o an ioxidan whey pep ides pos in i o gas o- in es inal diges ion capable o a e sing he igh junc ions o Caco 2 -HT29 monolaye s (a widely accep ed model o he in es inal ba ie ) and a i ing in he basola e al compa men [15]. Redox modi ica ion o whey du ing p ocessing As whey p oduc s ypically unde go se e al p ocessing s eps om milk o inal ood ma ix, he e is subs an ial e idence ha p ocessing can induce o exace ba e edox eac ions ha modi y whey p o eins. A he ou - se , milk p oduced o consume consump ion is hea - ea ed o kill bac e ia and inc ease shel li e. Mul iple me hods a e used including low (e.g. 15 s a 74 C), high (e.g. 15 s a 90C), and ul ahigh (e.g. 145 C o a ew seconds) empe a u e ea men s. Mos comme cial milk is also homogenised by high p essu e ea men o educe a globule size. Bo ine liquid whey is hen p o- duced by ei he he enzyma ic ea men o milk (swee whey) o by he addi ion o acids o mine als (acid whey) bo h o which esul in he p ecipi a ion, and he e o e emo al, o caseins. Signi ican modi ica ions on whey p o eins occu wi h p ocessing, due o hea ea men (pas eu isa ion and sp ay d ying), exposu es o high p essu es (e.g. du ing homogenisa ion), ligh exposu e, he use o s e ilisa ion/disin ec ion agen s such as H 2 O 2 (pe mi ed a concen a ions 16 mM in he USA [38]), high pH alues (employed o gi e hyd o- lysed p o ein samples e.g. o IF) and long- e m expos- u e o educing suga s (e.g. lac ose, glucose, galac ose. and p oduc s om hese). Oxida ion, glyca ion, and acemisa ion o whey p o eins The mal ea men o milk and milk p oduc s can esul in a signi ican inc ease in he le el o oxida i e modi i- ca ion and o ma ion o p o ein ca bonyls (wi h he Table 5. Whey pep ide sequences esis an o uppe gas oin es inal diges ion and an ioxidan pep ides enc yp ed wi hin hese sequences. Whey p o ein a,b Ho spo s: loca ion o pep ide sequences esis an o gas oin es inal diges ion c An ioxidan whey pep ides wi hin hese ho spo s b,c,d b-Lg [14,20,22,23,162] (41–58) (40–48), (42–46), (42–47), (43–49), (43–51), (45–57), (50–56), (52–61), (55–61), (56–62), (58–61) [26–28,32,33] (92–100) (89–96), (92–100), (94–100), (95–101), (95–110), (96–105), (96–100) [25,27–29] (126–138) (122–131), (122–134), (123–131), (123–134), (123–135), (124–131), (125–135) [25,30] (149–154) (145–149), (149–156), (150–160), (151–162) [26,27,31] a-Lac [14,20,22,23] (17–27) (13–19), (15–22), (16–23), (19–20), (25–26) [27,33] (63–68) (80–90) (82–88) [27] (97–102) (99–102), (101–104) [15,34] BSA [14,22] (11–18) (107–114) (219–224) (489–495) (514–518) Lac o e in [14,19] (67–77) (140–145) (216–228) (228–229) [35] (289–295) (309–318) (332–337) (592–594) a Whey p o eins: b-Lac oglobulin (b-Lg), a-Lac albumin (a-Lac), and bo ine se um albumin (BSA). b Re e ences a e in squa e b acke s. c Pep ide loca ion ( agmen ) on p ima y sequence o whey p o ein is s a ed as name o whey p o ein, agmen (posi ion om amino acid o amino acid) o example b-Lg (41–58). d Amino acid in i alic indica es he s a o end o he an ioxidan pep ide sequence alls ou side he gu esis an ho spo s. FREE RADICAL RESEARCH 1141 si es o some o hese cha ac e ised [39]) and c osslinks. The le els o hese ma e ials ha e been p oposed as a ma ke o milk powde quali y [40]. The highes le els o p o ein oxida ion p oduc s ha e been epo ed o be p esen in powde ed IF [39,41–44]. a-Lac has been epo ed o show enhanced oxida ion compa ed o b-Lg [45]. A ecen s udy has epo ed ha he mal ea men can induce educible (disulphide) c osslinks in isola ed b-Lg (100 mM), bu no isola ed a-Lac (150 mM) in bo h he absence and p esence o H 2 O 2 (500 mM), which appea s o be associa ed wi h he p o- ein un olding and he accessibili y o he ee Cys-121 esidue on b-Lg [46,47]. Blocking his hiol p e en ed c oss-link o ma ion, unde lining he impo ance o his esidue. Mixed c oss-links be ween b-Lg and a-Lac we e de ec ed when bo h p o eins we e co- ea ed [47]. Disulphide c oss-links can also be gene a ed ia hiol- disulphide exchange (i.e. nonoxida i e) eac ions, wi h hese occu ing ia a ack o a ee hiol (and usually he mo e eac i e anion o m) on a disulphide bond, wi h his esul ing in an exchange o pa ne s, and hence c oss-link o ma ion [48,49].Thisp ocessoccu smo e ap- idly a highe empe a u es and on p o ein un olding, as his inc eases he accessibili y o eac ion si es [48]. These eac ions can be enhanced by he addi ion o ee hiols [50] and highe Ca 2þ concen a ions [51]. Exposu e o bo h hea and H 2 O 2 appea s o ha e a g ea e e ec han ei he agen alone, wi h his asc ibed o he occu ence o a mechanism in ol ing he o ma ion o a sul enic acid (RSOH species) a Cys121 in b-Lg media ed by H 2 O 2 , once his esidue is made accessible by he mal un olding [46,47]. Limi ed loss o Me and T p (a e y high H 2 O 2 concen a ion) has also been de ec ed wi h bo h b-Lg and a-Lac on ea men wi h hea and H 2 O 2 [46,47]. Inc eased expos- u e o hea (longe imes and highe empe a u es) and highe concen a ions o H 2 O 2 ha e been shown o enhance he ex en o modi ica ion, as do combina ions o hese wo ac o s [46,47]. No p o ec ion agains dam- age was de ec ed when adical apping agen s we e included, indica ing ha hese a e molecula ( wo elec- on) and no adical (one elec on) eac ions [46]. These da a indica e ha oxida i e damage can be selec i e wi h ega d o he esidues ha a e modi ied, and ha un olding o he p o eins is a c i ical ac o wi h ega d o he ex en o damage. Dec easing hea loads and oxidan exposu e would he e o e be expec ed o minimise whey p o ein modi ica ion. Pho o-oxida ion a ising om ligh exposu e o milk p epa a ions con aining ibo la in ( i amin B 2 ,an endogenous componen o milk) can induce changes in milk p o ein s uc u e (e.g. polyme isa ion, seconda y and e ia y s uc u e o speci ic p o eins), as well as inducing he o ma ion o p o ein ca bonyls, he Ty oxida ion p oduc s, di- y osine (di-Ty ) and 3,4-dihy- d oxyphenylalanine (DOPA), and he T p-de i ed spe- cies, N- o mylkynu enine (NFK) and kynu enine (Kyn) [52,53]. Di e en oxidan sys ems can he e o e gene - a e di e en pa e ns and ex en s o damage, and in ol e al e na i e mechanisms, hough he o e all e ec s (e.g. agg ega ion) may be simila . Ligh exposu e has been used as a non he mal echnology o con ol pa hogens and ex end p oduc shel -li e bu his may esul in inc eased o ma ion o ca bonyls and hence p o ein damage [54]. P esence o oxida ion, glyca ion, and acemised whey p o eins in IF Whey p o ein modi ica ion is a po en ial heal h isk o in an s ed wi h IF, and he e is con incing da a ha indica es ha b eas milk has conside able heal h bene i s ( e iewed [55]). Table 6 summa ises he la ge numbe o di e en ypes and le els o p o ein modi- ica ions de ec ed in IF samples. A a mac omolecula le el, hese include bo h educible (p esumed o be disulphide-linked species, due o hei loss on dio hio- h ei ol ea men ) and (mul iple ypes o ) non educi- ble p o ein agg ega es [56]. E idence has been p esen ed o he c oss-linked species di-Ty (a species a ising om adical eac ions), as well as lysinoalanine (LAL), lan hionine (LAN), and he p ecu so species o he la e wo p oduc s, dehyd oalanine (DHA) (Table 6)[56,57]. Whe he he DHA a ises om base-ca a- lysed o adical-media ed eac ions a e unknown. Signi ican le els o p o ein ca bonyls a e also p esen in he samples, as de ec ed by assaying o al ca bonyl con en and also by using immunoblo ing on p o- eins sepa a ed by SDS-PAGE, wi h he la e expe i- men s indica ing ha a signi ican quan i y o he ca bonylsisp esen onhigh-molecula -massagg e- ga es [56]. The exac na u e o he p o eins in ol ed and he si es wi hin hese p o eins emain o be de e mined, hough i is clea ha he e a e signi i- can s uc u al changes o he IF p o eins induced by p ocessing [58]. Modi ica ions ha e also been de ec ed on IF p o eins a he AA le el, wi h e idence epo ed o he o ma- ion o T p oxida ion p oduc s (N- o mylkynu enine, NFK; kynu enine, Kyn; and 3-hyd oxykynu enine, 3OHKyn), di-Ty , Phe-de i ed ma e ials (3-hyd oxyPhe, m-Ty ) and he Me oxida ion p oduc , me hionine sul - oxide [56,59]. Some o hese species a e consis en wi h he species de ec ed on isola ed whey p o eins 1142 L. GIBLIN ET AL. exposed o speci ic oxidan sys ems (e.g. hea /H 2 O 2 and also ligh in he p esence o ibo la in) [46,47,52,60,61]. A numbe o he species de ec ed a e consis en wi h he occu ence o adical eac ions (e.g. di-Ty , which appea s o be only o med by adical eac ions), bu o he s (e.g. me hionine sul oxide) can be o med by bo h adical and molecula ( wo elec on) p ocesses (e.g. di ec oxida ion by H 2 O 2 )[62]. A numbe o hese ma e ials may unde go addi ional eac ions (e.g. edox cycling) ha may exace ba e damage [63]. Fu he mo e, some o hese p oduc s (e.g. hose om T p) may ha e mul iple biological ac i i ies and con ib- u e o disease, i aken up [64]. IF also con ain signi ican le els o ad anced glyca ion endp oduc s (AGE) ma e ials including he ea ly s age p oduc u osine, and well-cha ac e ised AGEs including N E -(ca boxyme hyl)lysine (CML), N E -(ca boxye hyl)lysine (CEL), py aline, and p o ein c osslinks, such as pen osi- dine ( om Lys and A g esidue linked by a pen ose), glyoxal lysine dime (GOLD), me hylglyoxal lysine dime (MOULD), and A g-de i ed p oduc s such as a gpy imidine and hyd oimidazolone isome s [56,65–72]. The le els o hese ma e ials ha e been examined using a wide a ie y o bo h di ec and indi ec me hodologies [66,73,74]. AGE is a chemically he e ogeneous g oup o compounds. AGE o ma ion in dai y p oduc s in ol e Mailla d eac ions, bu Table 6. Selec ed alues o concen a ions (nmol/mg p o ein excep whe e o he wise indica ed) o p o ein oxida ion p oduc s, ad anced glyca ion endp oduc s (AGEs) and acemised AAs p esen in powde ed in an o mula samples. Concen a ions o oxida ion p oduc s in in an o mula samples P oduc s In ac p o ein IF Hyd olysed p o ein IF P o ein ca bonyls 2.88 a 8.6–60.9 [41] 19.4 [42] 6.5 [40] 0.85 b 18.3 [42] Dehyd oalanine (DHA) 0.47 a 0.58 b Lan hionine (LAN) 0.01 a 0.09 b Lysinoalanine (LAL) 0.03 a Below limi o de ec ion [57] 18.2 ng mg 1 p o ein [42] 150–920 ng mg 1 p o ein [163] Highe alues de ec ed in liquid IF samples [164,165] 0.29 b 63.3 ng mg 1 p o ein [42] Me a- y osine 0.97 a 1.83 b Kynu enine 1.58 a 4.54 b 3-hyd oxykynu enine 2.82 a 7.40 b Me hionine sul oxide 64.97 a 24.82 b Di y osine 3.12 a 2.95 a 1.3 [42] 6.46 b 1 b 6.8 [42] Ca boxyme hyllysine (CML) 36.3 a ng mg 1 p o ein 60.1 ng mg 1 p o ein [42] 12 and 145 ng mg 1 o low- empe a u e ea ed, and high- empe a u e hea ed IF espec i ely [125] 1.2 ng mg 1 p o ein [66] 81.0 b ng mg 1 p o ein 212.4 ng mg 1 p o ein [42] Fu osine 2.3 a mgmg 1 p o ein 5.6 mgmg 1 p o ein [42] 2.7 mgmg 1 p o ein [66] 8.0–19.4 mgmg 1 p o ein [74] 13.215.5 mgmg 1 p o ein [166] 0.21–0.81 mgmg 1 p o ein [167] 0.251.05 mgmg 1 p o ein [168] 7.0 mgmg 1 p o ein [169] 3.5 b (mgmg 1 p o ein) 2.4 mgmg 1 p o ein [42] 2.6 mgmg 1 p o ein [74] 0.52–0.81 mgmg 1 p o ein [167] 0.60.9 mgmg 1 p o ein [168] D-His 2.7 a,c 2.5 b,c D-Lys 1.6 a,c 4.3 b,c D-Phe 1.4 a,c 3.4 b,c D-Ty 1.1 a,c d D-A g 5.5 a,c 32.0 b,c D-Ala 1.3 a,c d D-Ile 0.3 a,c d D-Leu 0.7 a,c 1.4 b,c D-Me 7.2 a,c d D-Se 0.5 a,c d D-Val 4.3 a,c 0.3 b,c Da a om [113] excep whe e o he wise indica ed. a Mean da a om h ee comme cial b ands. b Da a om h ee eplica e measu emen s om a single comme cial b and. c Da a exp essed as % o D-isome ela i e o o al (D þL) isome s. d No de ec ed. 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