Dynamics and Considerations in the Determination of the Excretion of Gluten Immunogenic Peptides in Urine: Individual Variability at Low Gluten Intake

Nu ien s 2021, 13, 2624. h ps://doi.o g/10.3390/nu13082624 www.mdpi.com/jou nal/nu ien s A icle Dynamics and Conside a ions in he De e mina ion o he Exc e ion o Glu en Immunogenic Pep ides in U ine: Indi idual Va iabili y a Low Glu en In ake Lau a Co o 1,2, Ca olina Sousa 3 and Angel Cebolla 1,* 1 Biomedal S.L., 41900 Se ille, Spain; lau a.co [email protected] 2 Human Nu i ion and Food Science Doc o al P og am, Uni e si y o G anada, 18011 G anada, Spain 3 Depa men o Mic obiology and Pa asi ology, Facul y o Pha macy, Uni e si y o Se ille, 41012 Se ille, Spain; csoum[email p o ec ed] * Co espondence: aceboll[email p o ec ed]; Tel.: +34-955-983-215 Abs ac : Backg ound: A li elong s ic glu en- ee die is he only a ailable ea men o celiac dis- ease, bu o al exclusion o glu en is di icul o achie e. The aim o his s udy was o de e mine he ange o ime and he amoun o glu en immunogenic pep ides (GIP) exc e ed in u ine a e speci ic glu en inges ions. Me hods: 20 heal hy pa icipan s ollowed he same die o 12 days in which 50 mg and 2 g o glu en we e inges ed and all he u ina ions we e collec ed. GIP we e analyzed by la e al low immunoassay (LFIA) es s and quan i ied using an LFIA eade . Resul s: GIP we e de- ec ed in 15% and 95% o pa icipan s a e 50 mg and 2 g glu en in akes, espec i ely. The highe equency and concen a ion o GIP was ound be ween 6 and 9 h a e bo h glu en inges ions. The anges o de ec ion we e 3–12 h (50 mg) and 0–15 h (2 g). Conclusions: An inc ease in he equency o u ine es s may be a sui able app oach o a oid alse nega i e esul s. The use o he LFIA es in h ee u ine samples collec ed a di e en imes may show a sensi i i y o 19.6% o a glu en inges- ion like 50 mg, inc easing o 93% a e 2 g consump ion. Keywo ds: glu en immunogenic pep ides; glu en exc e ion u ine; glu en- ee die moni o ing; celiac disease 1. In oduc ion Celiac disease (CD) is a ch onic sys emic immune-media ed disease igge ed by he inges ion o die a y glu en in gene ically p edisposed indi iduals wi h he human leuko- cy e an igen, HLA-DQ2, and/o HLA-DQ8 haplo ypes [1]. The clinical p esen a ion o CD is ex emely a iable, anging om ypical gas oin es inal symp oma ology o ex ain es- inal symp oms o ha e no symp oms a all. Impo an ly, ex ain es inal symp oms com- p ise a subs an ial p opo ion o he clinical mani es a ions o CD such as de ma i is he - pe i o mis, a h i is, neu ological symp oms, anaemia, os eopenia, os eopo osis, oo h enamel de ec s, aph hous s oma i is, hype ansaminasemia, e c. [1–3]. The pa hogenesis o CD in ol es s uc u al changes in he small in es inal mucosa and in aepi helial lym- phocy e in il a ion when glu en immunogenic pep ides (GIP) esis an o diges i e en- zymes c oss he epi helial ba ie o he lamina p op ia, leading o he ac i a ion o bo h inna e and adap i e immune esponses [2,4]. Cu en ly, he only ea men a ailable o CD is a li elong glu en- ee die (GFD). S ic adhe ence o he GFD is c ucial o e e se he clinical mani es a ions and o p e en long- e m complica ions [1,3,5–7]. Howe e , a die wi h he o al exclusion o glu en is challenging o mos pa ien s, who need high le els o discipline and mo i a ion [1]. Mo eo e , GFD is mo e expensi e, less pala able, and imposes social cons ain s, such as when dining ou and a eling [8–10]. Consequen ly, a subs an ial numbe o pa ien s Ci a ion: Co o, L.; Sousa, C.; Cebolla, A. Dynamics and Conside a ions in he De e mina ion o he Exc e ion o Glu en Immunogenic Pep ides in U ine: Indi idual Va iabili y a Low Glu en In ake. Nu ien s 2021, 13, 2624. h ps://doi.o g/ 10.3390/nu13082624 Academic Edi o : A min Alaedini Recei ed: 7 July 2021 Accep ed: 26 July 2021 Published: 29 July 2021 Publishe ’s No e: MDPI s ays neu- al wi h ega d o ju isdic ional claims in published maps and ins i u- ional a ilia ions. Copy igh : © 2021 by he au ho s. Li- censee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and con- di ions o he C ea i e Commons A - ibu ion (CC BY) license (h p://c ea- i ecommons.o g/licenses/by/4.0/). Nu ien s 2021, 13, 2624 2 o 16 wi h CD, especially hose who a e asymp oma ic, commi die ansg essions and hey a e a isk o de eloping his ological lesions and complica ions as a esul o hei condi ion [11]. The epo ed a es o GFD adhe ence ange be ween 12% and 90% in adul s [11–13] and be ween 23% and 98% in child en [14]. Al hough i has been desc ibed in he li e a u e ha a daily inges ion o less han 50 mg appea s o be sa e o mos pa ien s wi h CD [15], o he au ho s ha e dec eased his le el o 30 mg o a oid in es inal mucosal abno mali ies [16]. As he e is a g ea di e si y in glu en sensi i i y among indi iduals [15], he es ablishmen o a ha mless h eshold o daily glu en in ake o he celiac popula ion emains a oublesome ask. Glu en is an alcohol-soluble mix u e o s o age p o eins, known as p olamins, o ce- eals such as whea , ye, and ba ley [17]. These p o eins a e undamen al o dough o - ma ion in bake y p oduc s because o hei iscoelas ici y; howe e , hei applica ions in he ood indus y a e b oade [18]. Whea glu en p olamins, called gliadins and glu enins, a e cha ac e ized by being ich in p oline and glu amine amino acids, which make hem esis an o hyd olysis by gas ic and panc ea ic enzymes [17]. As a esul , an innume able di e si y o GIP is p oduced in he gas oin es inal ac , igge ing an immune esponse in indi iduals wi h CD. In any case, mos o he immunogenici y could be assigned o a limi ed numbe o glu en epi opes [19]; among he GIP con aining he mos ac i e T cell epi opes o CD, he α-gliadin 33-me pep ide has been desc ibed as a pa adigm o immu- nodominance [20]. The e is limi ed e idence ega ding glu en diges ion, me abolism, and exc e ion mechanisms. As a die a y p o ein, glu en hyd olyza ion occu s mainly in he small in es- ine by panc ea ic enzymes, which b eak polypep ides in o small pep ides and amino ac- ids ha a e anspo ed h ough he in es inal ba ie [21,22]. Fu he mo e, i has been desc ibed ha a ac ion o longe pep ides esis an o he ac ion o he pep idases can also c oss he basola e al memb ane o he en e ocy es and each po al ci cula ion [21]. Se e al au ho s ha e epo ed he de ec ion o GIP in he u ine o pa ien s wi h CD and heal hy indi iduals using mass spec ome y and an ibody-based me hods [11,13,23–28]. Thus, hey demons a ed ha glu en-de i ed pep ides en e he kidneys, and a e he ul a il a ion p ocess hey a e pa ially o o ally exc e ed in he u ine. I emains un- known i a p opo ion o hese pep ides is also eabso bed and hen me abolized o ex- c e ed using al e na i e pa hways. The use o GIP de ec ion in u ine has been de eloped as a di ec es o GFD moni- o ing in con as o he classical me hods, a he han only de ec ing he consequences o die ansg essions [11,23]. U ine is an ad an ageous sample o disease moni o ing, as i can be collec ed ully non-in asi ely, in la ge amoun s, and epea edly o e long pe iods o ime [29]. U ine is a complex ma ix o di e en componen s, such as wa e , glucose, p o eins, amino acids, and ino ganic sal s [29]. Howe e , he usual low concen a ion o p o ein in u ine and i s he e ogenei y wi hin and be ween indi iduals complica e he de- e mina ion o he speci ic momen o analy e exc e ion [29]. The aim o his s udy was o de e mine he indi idual a iabili y and he dynamics and limi o de ec ion (LoD) o u ine GIP exc e ion a e wo di e en amoun s o low/mode a e glu en inges ion (50 mg and 2 g) by moni o ing a signi ican numbe o pa icipan s wi h minimized die a ia- ions. 2. Ma e ials and Me hods 2.1. S udy Popula ion Be ween Janua y 2020 and Ma ch 2020, 20 heal hy olun ee s we e en olled om ci cles o ela i es o Biomedal S.L. (Se ille, Spain) employees in collabo a ion wi h he esea ch g oup o he Uni e si y o Se ille (Se ille, Spain). The c i e ia o inclusion as heal hy olun ee s we e: (1) pa icipan s who we e >18 yea s old; (2) no been diagnosed wi h CD, non-celiac glu en sensi i i y, and no ood alle gies, ood in ole ances, and o he kinds o gas oin es inal diseases; (3) pa icipan s who we e p epa ed o ollow a s ic Nu ien s 2021, 13, 2624 3 o 16 die ; and (4) o ha e he de e mina ion and abili ies o daily u ine and s ool collec ion. The exclusion c i e ia we e as ollows: (1) pa icipan s wi h associa ed pa hologies o se- e e psychia ic diseases; and (2) pa icipan s who did no collec he samples p ope ly on a leas 70% o occasions. All he subjec s p o ided w i en in o med consen o pa icipa e in he s udy, which was app o ed by he local e hics commi ee (n. 2381-N-19). 2.2. S udy Design The s udy in ol ed all pa icipan s o e a 19-day pe iod. The i s week was he wash-ou s age, in which he pa icipan s had o ollow a s ic GFD. Two days be o e he i s glu en inges ion, hey we e asked o collec one sample each o u ine and eces o con i m he absence o die a y glu en (Figu e 1). A e he wash-ou pe iod, pa icipan s we e p o ided wi h equi alen glu en- ee lunch and dinne menus and glu en- ee b ead, which we e supplied daily by he esea ch eam. The meals we e consumed wi hin he p esc ibed GFD. Two doses o glu en (50 mg and 2 g) we e inges ed in he mo ning (9:00) on days 8 and 12, espec i ely, and one sample o all he o dina y indi idual u ina- ions and deposi ions (da a published sepa a ely) we e collec ed du ing he whole pe iod (12 days in o al). F om he beginning o he end o he s udy, a ood- ecall ques ionnai e was used o assess GFD adhe ence and luid in ake, and he pa icipan s had o eco d he name and he quan i y o he dishes ha hey consumed daily. Figu e 1. S udy imeline. 2.3. Glu en Adminis a ion Glu en inges ions consis ed o wo doses o 50 mg and 2 g o powde ed whea glu en (El G ane o In eg al™; Biog an S.L., Mad id, Spain) encapsula ed in “000” size gela in caps (You Supplemen s™, B edbu y, S ockpo , England). The quan i y selec ion was based on he minimum amoun o glu en ha , when ea en daily, could p o oke his olog- ical changes in pa ien s wi h CD [15] and an amoun conside ed app op ia e o obse e he dynamic o exc e ion o GIP in u ine. The gela in caps we e analyzed using Glu- enTox® ELISA Sandwich ki (Hygiena, Se ille, Spain), based on G12 and A1 an ibodies, o con i m he absence o glu en. Glu en es ima ion was calcula ed by analyzing se e al samples o maize s a ch Maizena™ (Unile e , London, England) spiked wi h he pow- de ed glu en a di e en concen a ions and analyzed using he Glu enTox® ELISA Sand- wich ki (Hygiena, Se ille, Spain). Conside ing he esul s ob ained (nea 100% eco e y), glu en doses o each subjec we e p epa ed using he o al weigh o he powde ed glu- en: 50 ± 5 mg and 2000 ± 5 mg in 1 and 4 caps, espec i ely. An equi alence calcula ion o he glu en dosages o b ead po ions was pe o med using he me hodology desc ibed by Biagi e al. [30]. The slice o b ead was 11 cm × 12 cm Nu ien s 2021, 13, 2624 4 o 16 and weigh ed 30 g. Based on he nu i ional composi ion gi en by he manu ac u e , he whole slice con ained 2.48 g o glu en. The co esponding amoun o glu en in he b ead slice was 0.6 g o slice o 50 mg o glu en (Figu e 2a) and 24 g o 2 g o glu en (Figu e 2b). A ba e y (AAA) was used as he s anda d o size compa ison. Figu e 2. Small piece o b ead om he slice ep esen ing 50 mg o glu en (a) and slice o b ead co esponding o 2 g o glu en (b). 2.4. Meal Adminis a ion All pa icipan s ollowed he same GFD du ing he glu en exc e ion pe iod and we e p o ided wi h eady- o-ea meals o lunch and dinne in addi ion o glu en- ee ce i ied b ead (Beike ™, D . Schä , Pos al BZ, I aly) o comple e meals and o b eak as ime. The die was isocalo ic and he inges ion o esh ui s, unp ocessed nu s, and glu en- ee be e ages was ee o choice, depending on he ene gy equi emen s and habi s o each pa icipan . The meals we e o de ed om a ca e ing company and we e analyzed daily by he ISO17025 ce i ied labo a o y se ices o Biomedal S.L. (Se ille, Spain), using Glu- enTox® ELISA Sandwich ki (Hygiena, Se ille, Spain) o con i m he absence o glu en. 2.5. U ine Collec ion De ailed ins uc ions we e gi en o all pa icipan s a he beginning o he s udy. The subjec s we e p o ided wi h all ma e ials o u ine collec ion, including speci ic plas ic sc ew-capped con aine s, labels, cool bags, iso he mal boxes, and cool packs. The pa ici- pan s we e ins uc ed o collec be ween 30 and 60 mL o each mic u i ion and o w i e down he da e and ime o when hey pass u ine. All u ine samples we e p ese ed in iso he mal boxes wi h cool packs a 4–8 °C and d opped o wi hin 48 h o collec ion. All samples we e s o ed a −20 °C un il p ocessing. 2.6. U ine Analysis GIP quali a i e esul s in u ine we e measu ed using a la e al low immunoassay (LFIA) (iVYCHECK GIP U ine ki , Biomedal S.L., Se ille, Spain) ollowing he manu ac- u e ’s ecommenda ions. De os ed u ine samples we e homogenized and mixed wi h a condi ioning solu ion. The ea e , 100 μL o he mix u e was added o he immunoch o- ma og aphic casse e and isual in e p e a ion o he esul s was ca ied ou a e 30 min ( ecommended ime o samples con aining a low amoun o GIP). A posi i e esul was conside ed when he es line showed a ed colo , and he con ol line showed a g een colo . A nega i e esul was conside ed when only he con ol line showed a g een colo . The LoD o he echnique de e mined by isual inspec ion was 2 ng/mL. The concen a ion o GIP in u ine was also measu ed in he immunoch oma og aphic s ips a e 30 min using he iVYCHECK Reade (Biomedal S.L., Se ille, Spain). The a- Nu ien s 2021, 13, 2624 5 o 16 lidi y o his me hod was p e iously desc ibed by Mo eno e al. [23]. The eade was cali- b a ed p io o u ine analysis using he α-gliadin 33-me pep ide as a s anda d. The meas- u ing ange es ablished o his me hod was: 1.56–25 ng GIP/mL u ine. The esul s a e exp essed as ng GIP pe mL o u ine. Each sample was un in duplica e, and a leas wo di e en aliquo s o each sample we e es ed. 2.7. S a is ics The esul s o he quan i a i e a iables we e exp essed using he mean (SD) and median (IQR o ange), and hose o he ca ego ical a iables we e exp essed as absolu e (N) and ela i e (%) equencies. The goodness-o - i o no mali y was calcula ed using he Shapi o–Wilk es . The Mann–Whi ney U es was employed o compa e quan i a i e a iables in independen g oups and o pai ed quan i a i e a iables, he Wilcoxon es was used. Only u ines no la e han 24 h pos glu en inges ion we e included o s a is ical analysis due o la e u ines om all pa icipan s gi ing a nega i e esul . Ranges o ime we e es ablished o he s udy o he dynamics o GIP exc e ion in in e als o 3 h. All samples om each pa icipan collec ed in each ange we e clus e ed o ob ain one esul pe pa icipan . Any GIP+ sample indica ed a o al posi i e esul . Spea man’s co ela ion was used o calcula e he associa ion be ween he liquid con- sump ion a e glu en inges ion and he concen a ion o GIP in u ine. Basic p obabili y ules we e used o ob ain he diagnos ic sensi i i y o he s udied echniques o e a p e- de e mined ange o ime wi h he di e en samples collec ed. S a is ical analyses we e pe o med wi h IBM SPSS S a is ics 25.0 o Windows (IBM Co p, A monk, NY, Uni ed S a es). S a is ical signi icance was se a P < 0.05. 3. Resul s 3.1. Subjec s and Samples A o al o 20 indi iduals, including 13 (65%) emales and 7 (35%) males, comple ed he s udy a e 10 d opou s om he p eselec ion p ocess due o un o eseen e en s (n = 6) and COVID-19 mobili y es ic ions (n = 4). The median age o pa icipan s was 30.5 yea s (IQR 24.7–34.0) (Figu e 3). Figu e 3. Flowcha o he s udy pa icipan s. None o he pa icipan s we e decla ed o be diagnosed wi h a ele an disease o had been aking any p obio ics o ibe supplemen s. One pa icipan epo ed ollowing Nu ien s 2021, 13, 2624 6 o 16 a special i ness die be o e he s udy. Acco ding o he ood- ecall comple ed, all pa ici- pan s we e complian wi h he p esc ibed GFD and he glu en dose inges ion. The a e age luid in ake pe pa icipan du ing he s udy pe iod was 1.5 ± 0.6 L/day. 3.2. GIP De ec ion in U ine Samples A o al o 290 u ine samples we e collec ed om all pa icipan s du ing he 24 h a e glu en inges ion, 142 co esponding o he 50 mg glu en dose, and 148 o he 2 g glu en dose. The emaining samples o he s udy we e excluded o s a is ical analysis as hey ob ained GIP nega i e esul s. The medians o he numbe o samples collec ed pe pa - icipan in he i s 24 h we e 7 (IQR 5–8) o he 50 mg in ake and 7 (IQR 5.5–8.5) o he 2 g in ake (Table 1). Table 1. Indi idual cha ac e is ics o GIP exc e ion in u ine wi hin 24 h a e 50 mg and 2 g glu en inges ions. U ine GIP Exc e ion in 24 h 50 mg Glu en 2 g Glu en Pa ici- pan Samples LFIA+ Time Median Time Range Peak Max GIP GIP Me- dian GIP Range Samples LFIA+ Time Median Time Range Peak Max GIP GIP Median GIP Range n n h h h ng/mL ng/mL n n h h h ng/mL ng/mL 1 7 0 6 1 4.50 0.00 (4.50) 4.50 1.80 0.00 (1.80) 2 7 0 8 6 6.00 5.00 (4.00–9.00) 5.00 9.10 13.23 (2.93 – 16.17) 3 4 0 6 3 7.88 3.25 (6.25–9.50) 9.50 2.20 0.80 (1.80– 2.60) 4 8 0 9 4 8.54 6.50 (5.50–12.00) 7.00 3.50 5.43 (2.30– 7.73) 5 6 0 4 1 14.67 0.00 (14.67) 14.67 2.50 0.00 (2.50) 6 4 1 3.33 0.00 (3.33) 3.33 4.40 0.00 (4.40) 4 3 6.33 5.00 (3.83–8.83) 8.83 4.92 5.63 (2.10– 7.73) 7 15 0 18 1 8 10 0 10 5 7.67 3.00 (7.00–10.00) 7.00 4.17 4.60 (2.77– 7.37) 9 7 0 7 3 6.00 2.33 (5.00–7.33) 6.00 3.47 2.40 (2.20– 4.60) 10 10 0 7 4 7.96 8.75 (6.00–14.75) 6.00 6.50 11.43 (1.77– 13.20) 11 6 0 7 2 3.21 3.42 (1.50–4.92) 1.50 1.92 0.37 (1.73– 2.10) 12 6 2 8.29 2.42 (7.08– 9.50) 7.08 2.69 0.23 (2.57– 2.80) 5 2 9.04 5.92 (6.08–12.00) 6.08 4.60 2.47 (3.37– 5.83) 13 5 0 7 1 7.83 0.00 (7.83) 7.83 1.95 0.00 (1.95) 14 5 0 3 0 16 7 0 10 1 8.00 0.00 (8.00) 8.00 3.23 0.00 (3.23) 17 10 0 10 2 7.50 0.00 (7.50) 7.50 2.60 0.00 (2.60) 19 7 0 5 2 6.32 1.97 (5.33–7.30) 5.33 3.07 2.73 (1.70– 4.43) 21 8 0 8 4 8.04 7.07 (4.42–11.48) 4.42 3.12 5.53 (1.73– 7.27) 22 5 1 7.67 0.00 (7.67) 7.67 2.30 0.00 (2.30) 8 1 5.13 0.00 (5.13) 5.13 1.70 0.00 (1.70) 23 5 0 6 3 7.65 3.50 (6.30–9.80) 7.65 2.43 0.97 (1.60– 2.57) TOTAL 7 0 7.67 0 (3.33– 9.50) 7,08 2.57 2.20 (2.20– 4.40) 7 2 7.00 13.25 (1.50–14.75) 6,54 2.68 14.83 (1.33– 16.17) GIP: glu en immunogenic pep ides; LFIA: la e al low immunoassay. GIP we e de ec ed in 4/142 (2.8%) o he u ine samples up o 24 h a e 50 mg glu en inges ion, co esponding o 3/20 (15%) pa icipan s. F om hese pa icipan s, GIP we e Nu ien s 2021, 13, 2624 7 o 16 de ec ed in only one sample o wo subjec s and in wo samples o one subjec . Rega d- ing he 2 g dose, 33.1% (49/148) o he u ine samples we e GIP+ du ing he 24 h o collec- ion, co esponding o 19/20 (95%) o he pa icipan s. GIP+ samples we e ob ained in only one o wo u ina ions o 10/19 pa icipan s (52.6%), in h ee o ou u ina ions o 7/19 pa icipan s (36.8%), and in i e o six u ina ions o 2/19 pa icipan s (10.5%). GIP+ samples we e ound om he i s o he ou h collec ed samples a e 50 mg glu en inges ion. The de ec ion o GIP in u ine could be ex ended up o he eigh u ina- ions a e he 2 g dose, wi h he hi d sample being whe e mos pa icipan s (16/20; 80%) had GIP+ u ine. 3.3. Time Cou se o GIP Exc e ion GIP we e de ec ed in u ine samples collec ed in he i s 3 h a e 2 g glu en in ake and be ween 3–6 h a e 50 mg glu en inges ion (Figu es 4 and 5). The majo i y o GIP+ u ine samples we e ound in he ange o 6–9 h a e bo h glu en doses (18.8% and 78.8%, espec i ely) (Figu es 4 and 5). As expec ed, he 2 g inges ion esul ed in signi ican p o- po ions o posi i e samples o a longe pe iod (3–15 h) wi h a es be ween 41.2% and 78.8% (Figu e 5). No posi i e esul s we e ound a e 12 h and 15 h pos inges ion o he 50 mg and he 2 g doses, espec i ely (Figu es 4 and 5). Figu e 4. Resul s o quali a i e analysis o GIP exc e ion in u ine a e 50 mg o glu en inges ion using a LFIA es . The end o he GIP de ec ion dynamics is ep esen ed by he dashed line. Nu ien s 2021, 13, 2624 8 o 16 Figu e 5. Resul s o quali a i e analysis o GIP exc e ion in u ine a e 2 g o glu en inges ion using a LFIA es . The end o he GIP de ec ion dynamics is ep esen ed by he dashed line. Despi e he a iabili y obse ed among indi iduals, bo h glu en inges ions showed a compa able pe iod o ini ial GIP de ec ion (7.1 h ( ange 3.3–7.7)) o he 50 mg dose and 5.8 h ( ange 1.5–14.7) o he 2 g dose) (P = 0.285). A longe ange o ime o de ec able GIP pe pa icipan was ound in he la ge glu en dose (0 ( ange 0–2.4) s. 3.1 ( ange 0– 8.8)) bu wi hou s a is ical signi icance (P = 0.180). 3.4. GIP Quan i ica ion in U ine In line wi h he ime o GIP de ec ion a e glu en inges ion, highe concen a ions o GIP we e measu ed in he u ine o mos pa icipan s in he same pe iod (6–9 h) using an LFIA eade . The median o GIP in his pe iod was 0 ng GIP/mL u ine ( ange 0–2.8) o he 50 mg dose and 2.57 ng GIP/mL u ine ( ange 0–13.2) o he 2 g in ake (Figu e 6, Table 2). Figu e 6. Dynamic o GIP exc e ion in u ine a e 2 g o glu en in ake using a LFIA eade . Po en- ial ou lie s a e ep esen ed as do s. Nu ien s 2021, 13, 2624 9 o 16 Table 2. U ine GIP de ec ion in 3-hou pe iods a e 50 mg and 2 g o glu en in akes. 50 mg Glu en 2 g Glu en Time Pa icipan s GIP+ Pa ici- pan s GIP [ng/mL] Pa icipan s GIP+ Pa ici- pan s GIP [ng/mL] h n n Median (Range) n n Median (Range) 0–3 15 0 0.00 (0) 15 1 0.00 (0–2.10) 3–6 15 1 0.00 (0–4.40) 15 10 1.70 (0–16.17) 6–9 16 3 0.00 (0–2.80) 18 14 2.57 (0–13.20) 9–12 18 1 0.00 (0–2.57) 16 8 0.00 (0–4.83) 12–15 18 0 0.00 (0) 17 7 0.00 (0–3.37) 15–18 8 0 0.00 (0) 6 0 0.00 (0) 18–21 6 0 0.00 (0) 4 0 0.00 (0) 21–24 14 0 0.00 (0) 12 0 0.00 (0) GIP: glu en immunogenic pep ides. Howe e , he peak le els o GIP in u ine we e obse ed a di e en ime pe iods; o he 50 mg dose, i was 4.4 ng GIP/mL, de ec ed 3.3 h pos inges ion and o he 2 g dose i was 16.17 ng GIP/mL, de ec ed 5 h a e glu en inges ion. Conside ing he pe iod o GIP de ec ion a e bo h glu en doses (3–12 h) he median o GIP was 0 ng GIP/mL u ine ( ange 0–4.4) o he 50 mg dose and 1.73 ng GIP/mL u ine ( ange 0–16.17) o he 2 g in ake wi h s a is ical di e ences be ween inges ions (P < 0.001) (Figu e 7). Figu e 7. GIP de ec ed in u ine samples wi hin 24 h a e 50 mg and 2 g glu en inges ions. We obse ed a signi ican nega i e co ela ion be ween he liquid consump ion 12 h a e glu en consump ion and he le els o GIP in u ine de ec ed using an LFIA eade in he same pe iod ( ho = −0.79, 95% CI [−0.91, −0.53]; P < 0.001) (Figu e 8). In con as , no co ela ions we e ound be ween liquid consump ion and u ina ion equency ( ho = 0.119, 95% CI [−0.34, 0.53]; P = 0.62) and GIP concen a ions and u ina ion equency ( ho = −0.004, 95% CI [−0.45, 0.44]; P = 0.99). Nu ien s 2021, 13, 2624 16 o 16 35. Comino, I.; Fe nández-Baña es, F.; Es e e, M.; O igosa, L.; Cas illejo, G.; Fambuena, B.; Ribes-Koninckx, C.; Sie a, C.; Rod í- guez-He e a, A.; Salaza , J.C.; e al. Fecal Glu en Pep ides Re eal Limi a ions o Se ological Tes s and Food Ques ionnai es o Moni o ing Glu en-F ee Die in Celiac Disease Pa ien s. Am. J. Gas oen e ol. 2016, 111, 1456–1465, doi:10.1038/ajg.2016.439. 36. Ména d, S.; Leb e on, C.; Schumann, M.; Ma ysiak-Budnik, T.; Duga e, C.; Bouhnik, Y.; Malamu , G.; Cellie , C.; Allez, M.; C enn, P.; e al. Pa acellula e sus T anscellula In es inal Pe meabili y o Gliadin Pep ides in Ac i e Celiac Disease. Am. J. Pa hol. 2012, 180, 608–615, doi:10.1016/j.ajpa h.2011.10.019. 37. Camine o, A.; Nis al, E.; He án, A.R.; Pé ez-And és, J.; Fe e o, M.A.; Vaque o Ayala, L.; Vi as, S.; Ruiz De Mo ales, J.M.G.; Albillos, S.M.; Casquei o, F.J. Di e ences in Glu en Me abolism among Heal hy Volun ee s, Coeliac Disease Pa ien s and Fi s - Deg ee Rela i es. B . J. Nu . 2015, 114, 1157–1167, doi:10.1017/S0007114515002767.