Butylated hydroxytoluene can protect polyunsaturated fatty acids in dried blood spots from degradation for up to 8 weeks at room temperature

Transcription

1 Butylted hydroxytoluene n protet polyunsturted ftty ids in dried lood spots from degrdtion for up to 8 weeks t room temperture Metherel et l. Metherel et l. Lipids in Helth nd Disese 213, 12:22

2 Metherel et l. Lipids in Helth nd Disese 213, 12:22 RESEARCH Open Aess Butylted hydroxytoluene n protet polyunsturted ftty ids in dried lood spots from degrdtion for up to 8 weeks t room temperture Adm H Metherel, Ryn C Hogg, Lindy M Buzikievih nd Ken D Strk * Astrt Bkground: Dried lood spots (DBS) from fingertip prik lood n enle high throughput ftty id profiling ut my e prone to lipid peroxidtion during storge. The use of utylted hydroxytoluene (BHT) on hromtogrphy pper n prevent polyunsturted ftty id (PUFA) loss ut exmintions on the length of storge times possile re not omprehensive. Method: In the first study, venous whole lood ws sturted on pper strips pre-soked with, 2. or. mg/ml BHT nd exposed to ir for up to 28 dys. In seond study, the effet of seling DBS on. mg/ml BHT-soked hromtogrphy strips in pped test tues or vuum seled polypropylene gs with nd without nitrogen purging ws exmined over eight weeks. The ftty id omposition of the DBS were determined y gs hromtogrphy nd the effet of smple storge on omeg-3 iomrkers were exmined. Results: PUFA nd omeg-3 iomrkers in DBS stored without BHT were drmtilly redued y dy 3. In generl, BHT delyed dereses in eiospentenoi + dooshexenoi id from seline (3.2 ±.2 wt%) to 28 dys (2.6 ±.3 wt%) of storge. In the % n-3 highly unsturted ftty ids (HUFA) in totl HUFA iomrker, BHT ws more effetive t preventing hnges, prtiulrly with. mg/ml BHT where no differenes were deteted up to 28 dys. Seled storge with BHT tended to inrese the stility of the PUFA in DBS nd nitrogen purging did not pper to provide dditionl enefits. The % n-3 HUFA in totl HUFA iomrker lso ppered to e more stle in the seled storge study. Conlusions: The storge of DBS in seled ontiners with BHT my prevent PUFA degrdtion for up to 8 weeks. The % n-3 HUFA in totl HUFA iomrker ppers to provide more onsistent ssessment of omeg-3 sttus throughout storge s ompred with other omeg-3 lood iomrkers. Keywords: Butylted hydroxytoluene, Dried lood spots, High-throughput, Omeg-3 iomrker, Peroxidtion Bkground Dried lood spots (DBS) from fingertip prik lood does not require trined phleotmist nd n enle highthroughput sreening of lood omeg-3 iomrker sttus when omined with diret trnsesterifition nd fst gs hromtogrphy. Omeg-3 lood iomrkers re ssoited with hroni disese [1-3] nd my e useful for helth promotion, disese detetion nd disese * Correspondene: kstrk@uwterloo. Deprtment of Kinesiology, University of Wterloo, 2 University Avenue West, Wterloo, Ontrio N2L 3G1, Cnd prevention strtegies in humn popultions. Previously, DBS nlysis hs een vlidted for the determintion of ftty id omposition in whole lood [4-7], nd hs een used to ssess ftty id profiles from elderly individuls [8], young Cndin dults [9], Itlin nd Tietn dults [1], nd mothers nd their neworns [11,12]. Omeg-3 lood iomrkers suh s the sum of the perentge of eiospentenoi id (EPA, 2:n-3) nd dooshexenoi id (DHA, 22:6 n-3) (% EPA + DHA)[1] in erythroytes nd the perentge of omeg-3 highly unsturted ftty ids (HUFA, 2 rons, Metherel et l.; liensee BioMed Centrl Ltd. This is n Open Aess rtile distriuted under the terms of the Cretive Commons Attriution Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited.

3 Metherel et l. Lipids in Helth nd Disese 213, 12:22 Pge 2 of 9 ron-ron doule onds) in totl HUFA (% n-3 HUFA in totl HUFA)[3] utilize qulittive ftty id vlues in their lultions, nd s suh DBS nlysis remins suitle s lood volumes re often unknown. However, peroxidtion of polyunsturted ftty ids (PUFA) in lood stored on hromtogrphy pper n limit the utility of high-throughput DBS smpling. Previously, dereses in the PUFA ontent of erythroytes re prevented for up to 4 weeks when stored t 2 C [13,14], nd for up to 2 yers t 8 C [1] in the sene of n ntioxidnt. However, ess to 8 C storge is often limited outside of reserh institutions. In 1979, it ws demonstrted tht utylted hydroxytoluene (BHT) improves PUFA reovery in the ftty id nlysis of lipids [16], nd it is typilly inluded in routine ftty id nlyses [17]. The ddition of BHT to erythroytes for storge extends PUFA stility from 4 to 17 weeks [13]. Previously, PUFA in DBS with BHT tretment were shown to e stle for etween 3 weeks [6] nd 3 months [18] t 4 C, nd for less thn 2 months [18] t room temperture [18]. The im of the present study is to ssess the vlidity of using different levels of BHT dded to hromtogrphy pper to prevent PUFA degrdtion in DBS smples stored openly t room temperture for one month. In ddition, the effet of storing DBS smples with BHT in vuum seled polypropylene gs nd in pped test tues with nd without nitrogen purging for up to 8 weeks t room temperture on PUFA degrdtion ws exmined. This single individul study will provide sis for the ssessment of these storge methods in more extensive storge stility protools tht utilize lrger smple sizes. Methods Study design All proedures nd protools reeived prior pprovl from the Humn Reserh Ethis Committee of the University of Wterloo. In study one, hromtogrphy pper ws soked in three seprte onentrtions of 2,6-ditert-utyl-4-methylphenol (utylted hydroxytoluene, BHT, Sigm-Aldrih, St. Louis, MO, USA) in methnol. Conentrtions of BHT in methnol inluded mg/ml (ontrol), 2. mg/ml nd mg/ml [6]. The BHT/ methnol mixture ws llowed to trvel to the top of one sheet of hromtogrphy pper (Whtmn Ltd., Snford, ME, USA) in thin lyer hromtogrphy tnk efore the pper ws removed nd ir dried. The ftty id ompositions of DBS from eh BHT ondition were immeditely nlyzed in triplite y gs hromtogrphy t time. The remining smples were stored in open test tues nd nlyzed in triplite fter 1, 3, 7, 14, 21 nd 28 dys to trk hnges in ftty id omposition due to PUFA degrdtion. In study two, ll strips of hromtogrphy pper were treted with BHT in methnol t onentrtion of. mg/ml. At time, DBS were olleted in triplite nd immeditely trnsesterified for gs hromtogrphy determintion of ftty id omposition. This time determintion served s seline for ll four storge onditions, s the smple olletion proedures were similr ross storge onditions. The remining smples were rndomly ssigned to e stored in; losed srew p test tue, losed srew p test tue following nitrogen purging, vuum seled polypropylene g seled with ommerilly ville vuum seler (Foodsver V284; Sunem, Mississug, ON), or vuum seled polypropylene g fter nitrogen purging. The ftty id omposition of three smples from eh storge ondition were determined weekly for eight weeks. Blood olletion Blood ws otined y venipunture from single fsting mle on two osions orresponding to study one nd study two. Venous lood ws olleted from the nteuitl vein into 1 ml evuted tues (Vutiner; Beton Dikinson, Frnklin Lkes, NJ) y trined tehniin nd ethylene diminetetreti id (EDTA) ws dded to prevent ogultion. Blood ws sored onto the thin strips (1 m 3 m) of hromtogrphy pper suh tht n re of pproximtely 1 m 2 ws sturted using psteur pipette. Ftty id nlysis Ftty id omposition of eh DBS ws determined using method previously desried [4]. Ftty id methyl esters were prepred from DBS y diret trnsesterifition in 14% oron trifluoride in methnol (Piere Chemils, Rokford, IL, USA) with hexne using onvetionl lok heter set t 9 C for 6 minutes. The orgni lyer ontining the ftty id methyl esters ws olleted for nlysis on Vrin 39 gs hromtogrph (Vrin In., Mississug, ON) s previously desried [19]. The gs hromtogrph ws equipped with DB-FFAP 1 m.1 mm i.d..1 μm film thikness, nitroterephthli id modified, polyethylene glyol, pillry olumn (J&W Sientifi from Agilent Tehnologies, Mississug, ON) with hydrogen s the rrier gs. Smples (2 μl) were introdued y Vrin CP-84 utosmpler into the injetor heted to 2 C with split rtio of 2:1. The initil temperture ws 1 C with.2 min hold followed y 3 C/min rmp to 2 C, n 8 C/min rmp to 22 C with 3.2 min hold nd then n 8 C/min rmp up to 24 C with 1 min hold t the end. The flme ioniztion detetor temperture ws 3 C with ir nd nitrogen mke-up gs flow rtes of 3 ml/min nd

4 Metherel et l. Lipids in Helth nd Disese 213, 12:22 Pge 3 of 9 2 ml/min, respetively, nd smpling frequeny of Hz. Ftty id ompositions of ll smples re expressed s perent weight of individul ftty ids in totl ftty ids. The sum of EPA nd DHA (EPA + DHA) nd EPA, DHA nd n-3 doospentenoi id (DPAn-3) (EPA + DHA + DPA n-3), the perentge of n-3 HUFA in totl HUFA, nd the rtio of totl omeg-3 ftty ids to omeg-6 ftty ids (n-3/n-6) were lulted to illustrte hnges in the omeg-3 sttus in the smples nd the differentil rtes of degrdtion in the ftty id sulsses. Sttistil nlysis Qulittive vlues for individul ftty ids nd ftty id groups re expressed s men ± SD. All sttistil nlyses were performed with the SPSS System (SPSS In., Chigo, IL). Vrious ftty ids nd ftty id groups were exmined y one-wy ANOVA with individul mens ompred following signifint F-vlue y Tukey s Honestly Signifint Different (HSD) post ho proedure. Signifine ws inferred t p <.. Results The effet of different levels of BHT during open ir storge t room temperture on ftty id omposition Totl PUFA perentges deresed y 49% without BHT, 1% with 2. mg/ml BHT nd 6% with. mg/ml BHT (Tle 1). In the HUFA sulss, dereses were 62% without BHT, 34% with 2. mg/ml BHT, nd 13% with. mg/ml BHT. These results indite inresing BHT onentrtions provided my redue the degrdtion of PUFA nd HUFA of DBS. The. mg/ml BHT ondition lso ppers to protet levels of rhidoni id (ARA), EPA nd DHA in DBS. In ontrst, sturted ftty id (SFA) perentges inresed y 3% during storge without BHT nd y 1% with 2. mg/ml BHT. Monounsturted ftty id (MUFA) perentges inresed y 24% during storge without BHT nd y 9% with 2. mg/ml BHT. There ws no hnge in SFA nd MUFA perentges with. mg/ml BHT y dy 28 of storge. Without BHT, lood iomrkers of omeg-3 ftty id sttus (% n-3 HUFA in totl HUFA, EPA + DHA, n-6/n-3 rtio nd EPA + DHA + DPAn-3) hnged signifintly (p <.) fter only 3 dys of storge (Figure 1). EPA + DHA nd EPA + DHA + DPAn-3 levels deresed from seline (2.84 ±.17 nd 3.8 ±.21, respetively) to dy 3 (1.1 ±.3 nd 1.73 ±.14) nd remined deresed until dy 28 (.94 ±. nd 1.31 ±.12). Conversely, the immedite derese in the % n-3 HUFA in totl HUFA from seline (27. ±.6) to dy 3 (22.9 ± 1.6) returned to levels not different (p >.) from seline t dy 28 (24.4 ± 2.). The n-6/n-3 rtio inresed from seline (8.2 ±.4) up to dy 14 of storge (14.2 ± 1.) ut strted to derese to dy 28 (12. ± 1.8), lthough not signifintly (p >.). Storing DBS in the presene of BHT delyed nd in ertin iomrkers prevented the hnges in omeg-3 sttus tht re oserved when DBS re stored without BHT. With 2. mg/ml BHT, seline % n-3 HUFA in totl HUFA (2.7 ±.) ws mintined for up to 21 dys (2.8 ±.9). With. mg/ml BHT, the % n-3 HUFA in totl HUFA did not hnge from seline during 28 dys. A similr pttern ws oserved in the rtio of n-6/n-3, ut the hnge from seline (8.17 ±.24) to dy 28 (11.4 ±.3) ws more drmti in the 2. mg/ml BHT ondition. There ws lso grdul nd signifint inrese in the n-6/n-3 rtio with. mg/ml BHT. Conversely, EPA + DHA nd EPA + DHA + DPAn-3 perentges in the DBS smples were deresed signifintly fter only 14 dys with 2. mg/ml BHT nd 21 dys with. mg/ml BHT. Ftty id omposition nd omeg-3 iomrkers during seled storge DBS smples stored with. mg/ml BHT under seled onditions were exmined for up to 8 wks (Tle 2). In generl, there ws tendeny for dereses in the perentges of n-3 PUFA nd inreses in the perentge of MUFA. These differenes were signifint in oth the vuum seled propylene g onditions nd the nitrogen purged seled test, ut surprisingly not in the seled test tue without nitrogen purging. Chnges in the % n-3 HUFA in totl HUFA of only.4 2.8% for ll seled storge onditions throughout the entire study were not signifint (p >.) (Figure 2). The rtio of n- 6/n-3, nd the sums of the perentge of EPA + DHA nd EPA + DPAn-3 + DHA demonstrted onsiderle vrition ross the weekly mesures with signifint differenes in t lest one of the storge onditions. Disussion Results of the urrent study indite tht BHT n provide sustntil protetion ginst PUFA degrdtion in DBS smple. Totl HUFA omposition in DBS remins stle in the presene of BHT for up to 21 dys in open ir lthough signifintly lower levels re oserved fter 3 nd 14 dys of storge. Storge stility n e extended to t lest 8 weeks when stored nd pped in test tue. These results re not in greement with previous study demonstrting tht DBS smples wrpped nd seled in foil were unstle during 2 month storge period [18]. However, the speifi BHT onentrtion used in this previous study is not reported, nd s suh. mg/ml of BHT in the present study my explin the improved stility. In ddition, our smples were stored in test tues or vuum pked

5 Metherel et l. Lipids in Helth nd Disese 213, 12:22 Pge 4 of 9 Tle 1 Weight % of ftty ids in dried lood spots t vrying BHT onentrtions over 28 dys Nme d 1 d 3 d 7 d 14 d 21 d 28 d mg/ml BHT SFAs 42.1 ± ± ± ± ± ±. 4.9 ±.9 MUFAs 18. ± ± ± ± ± ± ±.7 N-6 FAs 34. ± ± ± ±. d 18.3 ±.1 e 19. ±.3 de 18.2 ±.6 e N-3 FAs 4.17 ± ± ± ± ± ± ±.1 ARA 8.7 ± ± ± ± ± ± ±.22 EPA.6 ±.1.64 ±.4.29 ±.2.26 ±.3.2 ±..2 ±.2.17 ±.2 DHA 2.19 ± ±.3.8 ±..63 ±.4 d. ±.2 d.73 ±.2 d.77 ±.6 d PUFAs 38.1 ± ± ± ±. d 19.6 ±.1 e 2.6 ±. de 19.6 ±.4 e HUFAs 14.1 ± ± ±.1.6 ±.24 d.2 ±.22 d.8 ±.8.4 ±.19 d 2. mg/ml BHT SFAs 37.1 ± ± ± ± ± ± ±.7 d MUFAs 19. ± ± ± ± ± ±.2 d 2.7 ±.1 d N-6 FAs 36.7 ± ± ± ± ± ± ±.3 N-3 FAs 4.43 ± ± ± ± ± ± ±.6 d ARA 9.12 ± ± ± ± ± ± ±.13 d EPA.71 ±.8.73 ±.6.68 ±.6.66 ±.3.62 ±.2.63 ±.7.2 ±.1 DHA 2.4 ± ± ± ± ±. 1.9 ± ±. PUFAs 41.1 ± ±.3 4. ± ± ± ± ±.2 d HUFAs 1.7 ± ± ± ± ± ±. d 1.4 ±.2 e mg/ml BHT SFAs 36.6 ± ± ± ± ± ± ±.7 MUFAs 18.6 ± ± ± ± ± ± ±.1 N-6 FAs 36. ± ± ± ± ± ± ±.2 N-3 FAs 4.9 ± ± ± ± ± ± ±.13 ARA 8.68 ± ± ± ± ± ± ±.9 EPA.82 ±.8.71 ±.3.62 ±.1.66 ±.4.6 ±.1.62 ±.2.61 ±.4 DHA 2.41 ± ± ± ± ± ± ±.6 PUFAs 4.6 ± ± ± ± ± ± ±.2 HUFAs 1. ± ± ±.2 1. ± ±.7 d 14.8 ± ±.1 d Vlues re mens ± SD, n = 3. BHT, utylted hydroxytoluene; SFA, sturted ftty id; MUFA, monounsturted ftty id; FA, ftty id; n-6, omeg-6; n-3, omeg-3; ARA, rhidoni id; EPA, eiospentenoi id; DHA, dooshexenoi id; PUFA, polyunsturted ftty id; HUFA, highly unsturted ftty id. Vlues with different supersript letters within row re signifintly different y Tukey s HSD post ho proedure (P <.) fter signifint F-vlue y one-wy ANOVA (P <.). polypropylene gs tht my provide more protetive sel thn foil wrpping. BHT medited protetion of PUFA is most likely due to free rdil svenging y BHT. The phenol group in BHT is thought to donte proton to free rdils, thus neutrlizing the free rdils nd preventing them from epting hydrogen protons from the methylene groups in PUFA nd therey preventing degrdtion [2]. The results of the present study support BHT protetion ginst PUFA loss s inresing onentrtions of BHT on the hromtogrphy pper demonstrtes inresed ility to prevent PUFA degrdtion. Additionlly, storing DBS in selle ontiners further prevents PUFA loss regrdless of storge under n inert gs suh s nitrogen. During open ir storge without BHT, omeg-3 lood iomrkers responded differently tht pper to e dependent on how eh iomrker is lulted (Figure 1). Dereses in individul ftty ids from seline were 23% for 18:2n-6 (dt not shown), 33% for 18:3n-3 (dt not shown), 47% for ARA, % for EPA nd 61% for DHA (Tle 1) tht is in greement with known kineti rtes of degrdtion. Omeg-3 PUFA hve greter suseptiility for degrdtion s ompred with omeg-6 PUFA, s omeg-3 PUFA generlly ontin more doule onds for given hin length. The kineti rtes of degrdtion re 1.4 M -1 s -1 for 18:1n-9, 62 M -1 s -1 for 18:2n-6, 11 M -1 s -1 for 18:3n-3, 197 M -1 s -1 for ARA, 249 M -1 s -1 for EPA, nd 334 M -1 s -1 for DHA [21,22].

6 Metherel et l. Lipids in Helth nd Disese 213, 12:22 Pge of 9 rtio % n-6 PUFA to n-3 PUFA % EPA + DHA % EPA + DHA + DPAn % n-3 HUFA in totl HUFA BHT Conentrtion (mg/ml) 2. BHT Conentrtion (mg/ml) 2. BHT Conentrtion (mg/ml) d d d 2. BHT Conentrtion (mg/ml) d d d 1 d 3 d 7 d 14 d 21 d 28 d Figure 1 Omeg-3 lood iomrkers in dried lood spot smples stored with, 2. or mg/ml BHT over 28 dys t room temperture. BHT, utylted hydroxytoluene; HUFA, highly unsturted ftty id; PUFA, polyunsturted ftty id; EPA, eiospentenoi id; DHA, dooshexenoi id; DPAn-3, omeg-3 doospentenoi id; n-3, omeg-3; n-6, omeg-6. Brs represent mens with error rs representing the S.D., n = 3. Brs with different letters within BHT ondition re signifintly different y Tukey s HSD post ho proedure (P <.) fter signifint F-vlue y one-wy ANOVA (P <.). The signifint drop in the % n-3 HUFA in totl HUFA in DBS fter 1 nd 3 dys in open ir without BHT, with susequent return to seline levels eginning t 7 dys of storge is intriguing. This my reflet higher degrdtion rte of suseptile omeg-3 HUFA, with degrdtion of suseptile n-6 HUFA eventully thing up nd resulting in slight nd sutle reound of the % n-3 HUFA in totl HUFA. In ontrst, the sum of the % of EPA + DHA deresed rpidly nd did not return under the sme onditions. The % of EPA + DHA is lulted reltive to the entire ftty id pool inluding lrge mounts of ftty ids with reltively low degrdtion rtes inluding plmiti id (16:), olei id (18:1n-9) nd 18:2n-6. Chnges in the n-6/n-3 rtio support the hypothesis of differing degrdtion rtes etween omeg-3 nd omeg-6 PUFA. The n-6/n-3 rtio demonstrtes n inverse pttern s ompred with the % n-3 HUFA in totl HUFA with rpid inrese followed y slight derese s time psses. The n-6 PUFA with slower degrdtion rte is in the numertor while the more rpidly degrding omeg-3 PUFA is in the denomintor. The differenes in the n-6/n-3 rtio nd % n-3 HUFA in totl HUFA re likely due to the inlusion of 18:2n-6 in the n-6/n-3 rtio. The degrdtion rte of 18:2n-6 is muh slower in omprison with 18:3n-3 nd HUFA, nd the mount of 18:2n-6 in whole lood is high (23.2 ±.4% of totl ftty ids) s ompred with the other PUFA (38.1 ±.8% of totl ftty ids) sed on seline vlues. Although ftty id peroxidtion my still e ourring, the utiliztion of the % n-3 HUFA in totl HUFA lood iomrker my provide more useful nd roust mesure of omeg-3 sttus during storge tht n msk the effets of HUFA peroxidtion nd still provide n urte omeg-3 ssessment. Our results suggest this my not e the se with the other omeg-3 iomrkers ssessed. In the present study, purging with nitrogen did not provide dditionl protetion ginst PUFA degrdtion during the 8-week storge period. It is unler whether nitrogen purging would hve provided dditionl enefits during longer storge period. Previously, PUFA omposition in O. int (springtil hexpod) hs een mintined when sponifition nd trnsesterifition ws performed in nitrogen-filled hedspe ir [23]. In ddition, diret gssing of diry everge enrihed with 2% flxseed oil with nitrogen proteted ginst PUFA degrdtion [24], nd nitrogen hs lso een used to slow oxidtive degrdtion of perishle food produts [2]. In lood, nitrogen prevents ftty id degrdtion in phospholipids for t lest four weeks in plsm nd less thn four weeks in erythroytes during storge t 2 C [14], nd in erythroytes for t lest two yers when stored t 8 C [1]. Chnges in HUFA omposition during

7 Metherel et l. Lipids in Helth nd Disese 213, 12:22 Pge 6 of 9 Tle 2 Weight % of ftty ids in dried lood spots fter BHT tretment nd storge y test tue or vuum seling Week * Week 1 Week 2 Week 3 Week 4 Week Week 6 Week 7 Week 8 Test tue seled SFAs 37.2 ± ± ± ± ± ± ± ± ± 2.3 MUFAs 24.6 ±.3 2. ±. 2.2 ± ± ± ± ± ± ± 1.3 N-6 FAs 29.2 ± ± ± ± ± ± ± ± ± 1.7 N-3 FAs.11 ±.12.1 ± ± ±.33.4 ± ± ± ± ±.39 ARA 6.4 ± ± ± ± ± ± ± ± ±.69 EPA.86 ±.6.8 ±..8 ±.6.86 ±..8 ±.11.8 ± ±.4.87 ±.6.81 ±.7 DHA 1.64 ± ±.7 1. ± ± ± ± ± ± ±.18 PUFAs 34.3 ± ± ± ± ± ± ± ± ± 2. HUFAs 11.6 ± ± ± ± ± ±. 12. ± ± ± 1.2 Test tue seled under nitrogen SFAs 37.2 ± ± ± ± ± ± ± ± ± 1.4 MUFAs 24.6 ± ± ±.4 d 26. ±.2 d 26. ±.2 d 2.8 ± ±.2 d 26.9 ±.7 d 27.4 ±. d N-6 FAs 29.2 ± ± ± ±. 3. ± ± ± ± ± 1.2 N-3 FAs.11 ± ±.4.44 ± ±.9 d 4.61 ±.14 d.1 ± ± ±.12 d 4.41 ±.18 d ARA 6.4 ± ± ± ± ± ± ± ± ±.21 EPA.86 ±.6.81 ±.2.8 ±.6.76 ±.4.84 ±..91 ±.1.89 ±.4.78 ±.3.78 ±. DHA 1.64 ± ± ± ±. 1.6 ± ± ± ±. 1.1 ±. PUFAs 34.3 ±.1 3. ± ± ± ±. 3.7 ± ± ± ± 1.4 HUFAs 11.6 ± ± ± ± ± ± ± ± ±.4 Vuum seled SFAs 37.2 ±. 36. ± ± ± ±. 3.3 ± ± ± ± 2.2 MUFAs 24.6 ± ± ±. 26. ± ± ± ± ± ±. N-6 FAs 29.2 ± ± ± ± ± ± ± ± ± 1.7 N-3 FAs.11 ±.12.3 ± ± ± ± ± ± ± ±.32 ARA 6.4 ± ± ± ± ± ± ±.2 6. ±.8.87 ±.4 EPA.86 ±.6.78 ±.2.77 ±..8 ±.4.76 ±.6.81 ±.2.76 ±.4.73 ±.1.72 ±.6 DHA 1.64 ± ± ± ± ± ±. 1.1 ± ± ±.1 PUFAs 34.3 ± ± ± ± ± ± ± ± ± 2. HUFAs 11.6 ± ± ± ± ± ± ± ± ±.7 Nitrogen dried vuum seled SFAs 37.2 ± ± ± ± ± ±. 36. ± ± ±.7 MUFAs 24.6 ± ±.1 d 27. ± ± ± ±.3 de 27.7 ±. d 28.2 ±.3 de 28.7 ±.3 e N-6 FAs 29.2 ± ± ± ± ± ± ± ± ±.3 N-3 FAs.11 ±.12.1 ±.17.1 ± ± ± ± ± ± ±.13 ARA 6.4 ± ± ± ± ± ± ±.29.7 ± ±.14 EPA.86 ±.6.8 ±.4.76 ±.2.77 ±.2.73 ±.4.77 ±.2.7 ±.4.68 ±.8.7 ±.3 DHA 1.64 ± ± ±.2 1. ± ± ± ± ± ±.4 PUFAs 34.3 ± ± ± ± ± ± ± ± ±. HUFAs 11.6 ± ±. 1.9 ± ± ± ± ±. 1.1 ± ±.3 Vlues re mens ± SD, n = 3. *Week dt seline is the sme nlysis for ll four storge onditions. SFA, sturted ftty id; MUFA, monounsturted ftty id; FA, ftty id; n-6, omeg-6; n-3, omeg-3; ARA, rhidoni id; EPA, eiospentenoi id; DHA, dooshexenoi id; PUFA, polyunsturted ftty id; HUFA, highly unsturted ftty id. Vlues with different supersript letters within row re signifintly different y Tukey s HSD post ho proedure (P <.) fter signifint F-vlue y one-wy ANOVA (P <.).

8 Metherel et l. Lipids in Helth nd Disese 213, 12:22 Pge 7 of 9 % n-3 HUFA in totl HUFA % EPA + DHA + DPAn-3 % EPA + DHA rtio % n-6 PUFA to n-3 PUFA TT NTT VS NVS Storge Method d d d TT NTT VS NVS Storge Method TT NTT VS NVS Storge Method TT NTT VS NVS Storge Method wk 1 wk 2 wk 3 wk 4 wk wk 6 wk 7 wk 8 wk Figure 2 Omeg-3 lood iomrkers in dried lood spot smples fter storge for 8 weeks in either test tue, test tue under nitrogen, vuum seled g or nitrogen dried in vuum seled g. PUFA, polyunsturted ftty id; HUFA, highly unsturted ftty id; EPA, eiospentenoi id; DHA, dooshexenoi id; DPAn-3, omeg-3 doospentenoi id; n-3, omeg-3; n-6, omeg-6; TT, test tue storge; NTT, test tue storge fter nitrogen purging; VS, vuum seling in polypropylene g; NVS, vuum seling in polypropylene g fter nitrogen purging. Brs represent mens with error rs representing the S.D., n = 3. Brs with different letters within storge ondition re signifintly different y Tukey s HSD post ho proedure (P <.) fter signifint F-vlue y one-wy ANOVA (P <.). 8 weeks of nitrogen storge suggest tht oxygen exposure my not e the only ontriutor towrds peroxidtion in lood smples during storge. In previous studies, erythroyte exposure to ir nd pure oxygen in vitro for 48 hours t either 37, 26 or 4 C did not result in inresed lipid peroxidtion s mesured y mlondildehyde formtion [26]. Plsm uled with nitrogen nd stored t 2 C lso did not prevent PUFA degrdtion [27]. In helthy ells, pproximtely 3% of Hemogloin(H)-(Iron)Fe 2+ is onverted to H-Fe 3+ resulting in prodution of superoxide rdils ( O 2 ) [28-3] tht n generte lipid peroxyl rdils nd lipid hydroperoxides. If the hydroperoxides re not effiiently removed, they n deompose to form more free rdils in the presene of iron tht n further exerte oxidtive dmge [31]. Thus, PUFA degrdtion in the present study my e result of Fe 2+ -indued peroxidtion mehnisms, nd BHT hs een shown to ompletely prevent this mehnism of PUFA peroxidtion [31-33]. Other potentil mehnisms of degrdtion my inlude light exposure, therml degrdtion nd humidity. Although humidity ws not ontrolled for, ll smples were stored in the sene of light nd t onstnt room temperture. The present study hs limittions. Our onlusions re limited to single individul. A single, homogenous lood smple ws used to enle extensive time point hrteriztion of the PUFA degrdtion nd restrit vrition to the degrdtion proess. Further ssessment of the stility of PUFA in DBS smples need to e onduted on lrger ohorts where the influene of other pro- nd nti-oxidnt lood mterils on ftty id degrdtion n e ddressed. The urrent study however provides vlule informtion nd timepoints nd storge onditions to e exmined. An dditionl limittion is tht the seline PUFA nd HUFA levels were higher in study one s ompred with seline vlues in study two. Although lood smples for study one (different BHT levels) nd study two (seled storge omprisons) were tken from the sme individul, they were olleted t different time points. As suh, ompring results etween study one nd study two must e mde with ution s the potentil for PUFA degrdtion my hve een

9 Metherel et l. Lipids in Helth nd Disese 213, 12:22 Pge 8 of 9 inresed in study one. Totl omeg-3 PUFA mesures of pproximtely 4% in study one nd % in study two re higher thn verge DBS vlues determined in young Cndin dults [9], ut similr to vlues determined in n elderly Cndin popultion [8]. The higher omeg-3 ftty id omposition of lood with the potentil for inresed peroxidtion in the present study my therefore underestimte the PUFA stility of DBS smples from the generl North Amerin popultion. Conlusions Pretretment of hromtogrphy pper with BHT for DBS smpling my protet ginst PUFA degrdtion in onentrtion dependent mnner for up to 21 dys t room temperture, nd susequent seling of DBS in test tues or vuumed polypropylene gs n further protet ginst PUFA degrdtion for minimum of 8 weeks. The % n-3 HUFA in totl HUFA iomrker ppers more resistnt to hnges due to PUFA degrdtion given it is lultion sed on ftty ids with reltively similr peroxidtion rtes. The % n-3 HUFA in totl HUFA my hve the potentil to provide more urte determintions of omeg-3 ftty id sttus following extended DBS storge. Future studies on the mehnisms of PUFA degrdtion during DBS storge to determine the min determinnt of oxidtion re required. Improving storge methods for DBS smples to llow for storge t room temperture would e enefiil for popultion sreening for omeg-3 iomrker determintions of disese risk nd nutritionl intke, nd in field studies, prtiulrly in developing ountries nd ommunities where linil resoures my e limited. Arevitions ARA: Arhidoni id; BHT: Butylted hydroxytoluene; DBS: Dried lood spot; DHA: Dooshexeoni id; EPA: Eiospentenoi id; HUFA: Highly unsturted ftty id; MUFA: Monounsturted ftty id; NTT: Nitrogen test tue; NVS: Nitrogen vuum seled; PUFA: Polyunsturted ftty id; SFA: Sturted ftty id; TT: Test tue; VS: Vuum seled. Competing interests The uthors delre tht they hve no ompeting interests. Authors ontriutions AHM drfted the mnusript nd ws involved in study design nd dt nlysis. RCH rried out smple olletion nd storge nd prtiipted in smple preprtion nd nlysis. LMB rried out smple olletion nd storge nd prtiipted in smple preprtion nd nlysis. KDS oneived of the study nd prtiipted in its design nd oordintion nd helped to drft the mnusript. All uthors red nd pproved the finl mnusript. Aknowledgements Operting funds were provided y grnt from the Helth Tehnology Exhnge to KDS. Infrstruture ws purhsed through Cnd Foundtion of Innovtion nd the Ontrio Reserh Fund mthing grnts. Reeived: 3 Novemer 212 Aepted: 13 Ferury 213 Pulished: 2 Ferury 213 Referenes 1. Hrris WS, von Shky C: The Omeg-3 Index: new risk ftor for deth from oronry hert disese? Prev Med 24, 39: Lnds B: A ritique of prdoxes in urrent dvie on dietry lipids. Prog Lipid Res 28, 47: Strk KD: The perentge of n-3 highly unsturted ftty ids in totl HUFA s iomrker for omeg-3 ftty id sttus in tissues. 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10 Metherel et l. Lipids in Helth nd Disese 213, 12:22 Pge 9 of vn DC, Pel R, Ellers J: Mximized PUFA mesurements improve insight in hnges in ftty id omposition in response to temperture. Arh Inset Biohem Physiol 29, 72: Giroux HJ, Ateu G, Sik H, Britten M: Influene of dissolved gses nd het tretments on the oxidtive degrdtion of polyunsturted ftty ids enrihed diry everge. J Agri Food Chem 28, 6: Byes AL: Investigtions on the use of nitrogen for the stiliztion of perishle food produts. Food Tehnol 19, 4: Stoks J, Dormndy TL: The utoxidtion of humn red ell lipids indued y hydrogen peroxide. Br J Hemtol 1971, 2: Hirsh EZ, Slivk S, Gions AP: Stility of ftty ids in hyperlipoproteinemi plsm during long-term storge. Clin Chem 1976, 22: Clemens MR, Wller HD: Lipid peroxidtion in erythroytes. Chem Phys Lipids 1987, 4: Heel RP, Eton JW: Pthoiology of heme intertion with the erythroyte memrne. Semin Hemtol 1989, 26: Misr HP, Fridovih I: The genertion of superoxide rdil during the utoxidtion of hemogloin. J Biol Chem 1972, 247: vn den Berg JJ, Op den Kmp JA, Luin BH, Roelofsen B, Kuypers FA: Kinetis nd site speifiity of hydroperoxide-indued oxidtive dmge in red lood ells. Free Rdi Biol Med 1992, 12: Dvies KJ, Golderg AL: Proteins dmged y oxygen rdils re rpidly degrded in extrts of red lood ells. J Biol Chem 1987, 262: Dvies KJ, Golderg AL: Oxygen rdils stimulte intrellulr proteolysis nd lipid peroxidtion y independent mehnisms in erythroytes. J Biol Chem 1987, 262: doi:1.1186/ x Cite this rtile s: Metherel et l.: Butylted hydroxytoluene n protet polyunsturted ftty ids in dried lood spots from degrdtion for up to 8 weeks t room temperture. Lipids in Helth nd Disese :22. Sumit your next mnusript to BioMed Centrl nd tke full dvntge of: Convenient online sumission Thorough peer review No spe onstrints or olor figure hrges Immedite pulition on eptne Inlusion in PuMed, CAS, Sopus nd Google Sholr Reserh whih is freely ville for redistriution Sumit your mnusript t