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.