NMR-based Me abolomics and Fa y Acid P ofiles o Un a el
Bioma ke s in P eclinical Animal Models o Compulsi e Beha io
Ana C. Ab eu,
§
San iago Mo a,
§
Ana Isabel T is án, Elena Ma ín-González, Angeles P ados-Pa do,
Ma ga i a Mo eno,*and Ignacio Fe nández*
Ci e This: J. P o eome Res. 2022, 21, 612−622
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ABSTRACT: Compulsi i y is a key mani es a ion o inhibi o y con ol defici
and a ca dinal symp om o psychopa hological condi ions such as obsessi e-
compulsi e and a en ion-defici hype ac i i y diso de s, in which me abolic
al e a ions ha e aised a en ion as pu a i e bioma ke s o ea ly iden ifica ion.
The p esen s udy assessed he me abolic p ofile in a p eclinical model o a
compulsi e pheno ype o a s. We used he schedule-induced polydipsia (SIP)
me hod o classi y male Wis a a s in o high d inke s (HDs) o low d inke s
(LDs) acco ding o hei compulsi e d inking a e de eloped by exposu e o a
fixed- ime 60 s (FT-60) schedule o ein o cemen wi h wa e a ailable ad
libi um du ing 20 sessions. Be o e and a e SIP, blood samples we e collec ed
o subsequen se um analysis by nuclea magne ic esonance spec oscopy
coupled o mul i a ia e analysis. Al hough no diffe ences exis ed in he p e-SIP
se , he compulsi e d inking beha io induced ema kable me abolic al e a ions:
HD a s selec ed by SIP exhibi ed a hype lipidemic, hypoglycemic, and
hype glu amine gic p ofile compa ed wi h hei low-compulsi e coun e pa s. In e es ingly, hese al e a ions we e no a ibu able o
he me e exposu e o ewa d pelle s because a con ol expe imen did no show diffe ences be ween HDs and LDs a e 20 sessions
o pelle consump ion wi hou in e mi en ein o cemen . Ou esul s shed ligh owa d he implica ion o die a y and me abolic
ac o s unde pinning he ulne abili y o compulsi e beha io s.
KEYWORDS: compulsi e beha io , schedule-induced polydipsia, bioma ke s, NMR, me abolomics
1. INTRODUCTION
Compulsi i y is defined as a pe se e a ion o a esponse ha is
i esis ible, inapp op ia e, and una oidable despi e i s nega i e
consequences.
1
I is he co e ea u e obse ed in obsessi e-
compulsi e diso de (OCD), al hough i is p esen in o he
neu opsychopa hological condi ions such as schizoph enia,
au ism, a en ion-defici hype ac i i y diso de , and addic-
ion.
2−4
These a e conside ed as impulsi e compulsi e spec um
diso de s, wi h a high p e alence (1−3%) in Wes e n coun ies
and an app oxima e economic cos o $5 billion pe yea
acco ding o he Wo ld Heal h O ganiza ion.
5
Howe e , li le is
known abou he ela ion be ween me abolic ac o s and
inhibi o y con ol defici . Resea ch on he possible iden ifica ion
o me abolic bioma ke s unde lying inhibi o y con ol defici
and he effec o die on he ne ous sys em and beha io could
help scien is s and physicians o imp o e hei knowledge abou
new mechanisms o p e en ion and ea men in psychopa ho-
logical diso de s.
Me abolomics has been widely applied in biomedicine o
p o ide a p ecise analysis o small molecules (<1500 Da)
associa ed wi h human me abolism. Unlike DNA, RNA, o
p o eins, me aboli es can accu a ely eflec he mos di ec
me abolic changes in ou body unde a ce ain condi ion in a
sho ime pe iod and a e hus good indica o s o he onse and
p og ession o human diseases.
6
So, he analysis o me aboli es
ep esen s a sensi i e measu e o biological s a us in heal h o
unde disease.
7
Nuclea magne ic esonance (NMR) spec os-
copy offe s he unique p ospec o holis ically sc een se e al
me aboli es wi h a non-a-p io i selec ion in di e se ma ices as
biological fluids and e en in issue biopsies.
8
NMR-based
me abolomics analysis coupled o mul i a ia e s a is ical
echniques ha e been inco po a ed in o ag icul u al cul u e
p og ams and clinical disease esea ch o iden i y unique
me aboli e bioma ke s in he ques o nu i ional o o gano-
lep ic ad an ages o in a specific human disease, espec i ely.
When he ocus is placed on he iden ifica ion o disease-
associa ed bioma ke s, his could allow us o (1) p edic and
diagnose diseases and hei s ages, (2) p o ide insigh s in o
unde lying pa hways in he pa hogenesis and p og ession o he
Special Issue: Me abolomics Resea ch
Recei ed: No embe 3, 2021
Published: Feb ua y 10, 2022
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diseases, and (3) aid in disease ea men by assessing he
efficacy and mechanism o ac ion o he apeu ic solu ions.
6,9
The use o animal models wi h highe ansla ional powe ,
associa ed wi h neu obeha io al endopheno ypes based on
bioma ke s, has enabled us o b idge he gap be ween
(dys) unc ional neu onal ci cui s and psychological cons uc s
wi h bigge p edic i e s eng h han he cu en psychia ic
nosology.
10
Howe e , o he bes o ou knowledge, only a ew
s udies ha e in es iga ed me abolomic bioma ke s in animal
models. In a ecen s udy, Pe ez-Fe nandez e al.
11
ound a
hype lipidemic and hypoglycemic p ofile in animals exposed o
Chlo py i os, which also showed beha io al al e a ions, ha is,
impai men s in he eac ion o social no el y in he C awley
social es .
Schedule-induced polydipsia (SIP) is one o he mos well-
es ablished p eclinical models o he s udy o neu opsychopa-
hological diso de s p esen ing compulsi e beha io , such as
OCD, schizoph enia, and alcohol abuse. (Fo a e iew, see
Mo eno and Flo es).
12
The SIP p ocedu e is cha ac e ized by
he de elopmen o an adjunc i e beha io o excessi e and
pe sis en d inking, which is non egula o y and does no ely on
physiological demands, in ood-dep i ed animals exposed o
in e mi en ood- ein o cemen schedules in ope an chambe s
wi h wa e a ailable ad libi um.
13,14
Howe e , he e a e
impo an indi idual diffe ences in he de elopmen o
adjunc i e d inking a e 15−20 sessions in SIP. Ra s can be
sepa a ed in o wo g oups acco ding o hei a es o d inking,
one wi h high o compulsi e d inking (HD a s) and a second
g oup wi h low d inking o no SIP acquisi ion (LD a s). This
phenomenon esembles he key ea u es o human compulsi i y
and OCD, hus allowing us o iden i y ulne able popula ions
based on hei endopheno ype ( o a e iew, see Mo eno and
Flo es)
12
and en ailing an ou s anding chance o s udying
compulsi e beha io unde labo a o y condi ions.
In his sense, he p omising ole o me abolism as a pu a i e
con ibu ing ac o conce ning ulne abili y o inhibi o y
con ol defici necessi a es u he esea ch; specifically, he
use o bioma ke s o he ea ly iden ifica ion o such condi ions
c ea es he con ex o a be e unde s anding o he p oblem
and o ea ly de ec ion and, in he u u e, ea ly in e en ion. The
p esen s udy aims o add e idence in ha di ec ion wi h he aid
o SIP.
2. MATERIALS AND METHODS
2.1. Subjec s
Fo y male Wis a a s (En igo, Spain) we e used in his s udy
(40 o he SIP expe imen , 20 o hem o he con ol
expe imen ) and a i ed a he labo a o y weighing 200−250 g.
They we e housed in a ou a s pe cage (50 ×35 ×20 cm)
dis ibu ion a a empe a u e o 22 ±1°C wi h a 12:12 h ligh −
da k cycle wi h ligh s offa 08:00 h. They also had
en i onmen al en ichmen consis ing o PVC pipe ubes and
wooden blocks and ood and wa e p o ided ad libi um. Be o e
SIP, he animals’bodyweigh s we e g adually educed o 85% o
hei ee- eeding baseline le el h ough con olled eeding and
daily weighing and hen main ained h oughou he expe imen .
Food, consis ing o lab chow, was p o ided daily ∼30 min a e
each expe imen al session. All o he es ing occu ed be ween
9:00 am and 2:00 pm. All p ocedu es we e in acco dance wi h
he Spanish Royal Dec ee 53/2013 on he p o ec ion o
expe imen al animals and he Eu opean Di ec i e 2010/63/EU
and app o ed by he Animal Resea ch Commi ee o he
Uni e si y o Alme ia. We decla e ha he esea ch shows
commi men o he 3Rs p inciple ( eplacemen , educ ion,
efinemen ).
2.2. Expe imen al Design
Once all animals eached 85% bodyweigh compa ed wi h hei
baseline, blood samples (p e-SIP) we e aken (see as ollows) on
he day be o e he s a o he SIP p ocedu e. Then, a e SIP,
pos -SIP samples we e collec ed. A e 1 mon h o washou
(based on p e ious publica ions; see Mo a e al.),
15
hal o he
animals we e selec ed o a con ol es conce ning he die a y
impac on hei me abolism. Blood samples we e collec ed
be o e (p e-pelle ) and a e (pos -pelle ) exposu e o he es
pelle s wi h wa e a ailable ad libi um.Figu e 1 shows he
expe imen al imeline o ele an e en s.
2.3. Blood Sampling
Animals we e anes he ized using isoflu ane, and blood samples
(1 mL) we e collec ed om he la e al ail ein in 1.5 mL
au ocued plas ic ubes be ween 9:00 am and 12:00 pm (da k
cycle). The samples we e allowed o s and o 10 min be o e
cen i uging (Sigma 3-18KS, Ge many) a 3000 pm (800g) o
10 min a 23 °C, a e which se um was collec ed in o duplica e
0.5 mL au ocued plas ic ubes and s o ed a −80 °C un il assay.
2.4. SIP P ocedu e
Ra s we e es ed in 12 ope an SIP chambe s (32 ×25 ×34 cm)
(MED Associa es, S . Albans, VT). A p e ious desc ip ion o he
appa a us can be ound in Mo eno e al.
16
P og amming and
da a eco ding we e pe o med wi h he aid o a compu e and
comme cial so wa e Med PC (Cibe ec SA, Spain). P io o
SIP, wo baseline wa e inges ion es s on successi e days we e
pe o med, whe e he amoun (in mL) o wa e consumed by
each animal du ing a pe iod o ime o 60 min wi h ee access o
60 pelle s (Noyes 45 mg dus less ewa d pelle s; TSE Sys ems,
Ge many) was measu ed. A e 1 day o habi ua ion o he
chambe s session, a s unde wen 60 min daily sessions o a
fixed- ime 60 s (FT-60s) schedule o ood pelle deli e y, whe e
bo les con aining eshwa e ad libi um we e placed in he wall
opposi e o he pelle dispense . Measu es eco ded we e: (1)
o al amoun o wa e (in mL) consumed, (2) o al numbe o
licks o he bo le, and (3) o al numbe o ood magazine
en ies. A e 19 daily sessions o SIP acquisi ion, animals we e
selec ed in wo g oups, high and low d inke s (HDs and LDs, n=
Figu e 1. Expe imen al p ocedu e illus a ed in a imeline. A e habi ua ion o he lab, animals we e di ided in o high d inke s (HDs) and low d inke s
(LDs) by schedule-induced polydipsia (SIP)be o e (p e-SIP) and a e (pos -SIP) blood samples we e aken. A e SIP, animals emainedundis u bed
o 1 mon h. Then, an addi ional expe imen ega ding exposu e o ewa d pelle s ook place o assess i s die a y impac on me abolomic analyses;
again, blood samples we e collec ed be o e (p e-pelle ) and a e (pos -pelle ) exposu e.
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20 in each g oup), acco ding o i hei d inking a es du ing SIP
(a e age o wa e in ake on he las fi e sessions) we e abo e o
below he g oup median, espec i ely.
2.5. Exposu e o Die Pelle s
Hal o he animals o each g oup (HD and LD, n= 10 in each
g oup) we e exposed o 19 consecu i e exposu e sessions simila
o SIP o u he assess any die a y impac o he ewa d pelle s.
The animals ecei ed he same numbe o pelle s as du ing he
SIP p ocedu e (60 pelle s), bu in his case, he pelle s we e
p esen ed unde mass eeding condi ions wi hou a ood-
ein o cemen ime schedule; wa e was a ailable ad libi um.As
in SIP, he consump ion o all pelle s was assu ed by he
expe imen e s a e each session.
2.6. Sample P epa a ion o NMR
Fo NMR expe imen s, 150 μL o a blood se um was mixed
wi h 350 μLo D
2O con aining 0.9% NaCl and he sodium sal
o 3-( ime hylsilyl)p opionic-2,2,3,3-d4acid (TSP) a 0.01%
(w/w). The esul ing mix u e was o exed and cen i uged o 5
min a 13 500 pm, and 500 μL o supe na an s was ans e ed
in o o en-d ied 5 mm NMR ubes.
2.7. NMR Expe imen s
Acquisi ion o 1H NMR spec a o se um samples was conduc ed
as desc ibed by Pe ez-Fe nandez e al.
11
wi h some modifica-
ions. Measu emen s we e ca ied ou on a B uke A ance III
600 spec ome e ope a ing a 600.13 MHz, equipped wi h a 5
mm QCI quad uple esonance pulse field g adien c yop obe
and a SampleJe au osample , a 293 ±0.1 K and wi hou
o a ion. The wa e -supp essed Ca −Pu cell−Meibom−Gill
(CPMG) pulse sequence was applied wi h a o al spin echo
delay o 100 ms (τ−180°−τ, 400 μs−37 μs−400 μs) o
a enua e b oad signals om p o ein signals. The spec ome e
ansmi e was locked o D2O equency. Acquisi ion
pa ame e s we e se as ollows: NS = 60, DS = 16, size o fid=
32K, spec al wid h = 22.0 ppm, acquisi ion ime = 1.24 s,
elaxa ion delay = 3 s, numbe o loops = 120, line b oadening =
0.3 Hz, ecei e gain = 203. Spec a we e au oma ically phased,
baseline-co ec ed, and calib a ed o TSP signal a 0.0 ppm.
Acquisi ion and p ocessing o NMR spec a we e ca ied ou by
he TOPSPIN so wa e ( e sion 3.6.2). Me aboli e assignmen s
we e pe o med hanks o in o ma ion on scala couplings
ex ac ed om 1H−1HCOSY,1H−1HTOCSY,1H−13C
HSQC, and 1H−13C HMBC spec a, which we e eco ded
using s anda d B uke sequences, and wi h he help o he
Chenomx da abase (Chenomx, Edmon on, Canada), public
NMR da abases (HMDB), and li e a u e.
17−19
Quan ifica ion o
me aboli es was achie ed h ough he in eg a ed alues o he
ela ed peak a eas o nono e lapped signals in ela ion o he
inne s anda d (TSP).
2.8. Quan ifica ion o Fa y Acids
A e NMR acquisi ion, se um samples we e eeze-d ied o 72
h. Then, he a y acid con en and p ofile in se um samples and
also in he pelle s we e de e mined by gas ch oma og aphy
(Agilen Technologies 6890 N Se ies Gas Ch oma og aph,
San a Cla a, CA) a e di ec anses e ifica ion, as desc ibed by
Rod iguez-Ruiz e al.
20
2.9. S a is ical Da a Analysis
SIP acquisi ion da a we e analyzed using wo-way epea ed-
measu es analysis o a iance (ANOVA) wi h be ween-subjec
ac o (g oup: HD and LD) and wi hin-subjec ac o (session:
19 sessions). Pos hoc compa isons we e pe o med using he
Bon e oni co ec ion. The s a is ical significance was se a p<
0.05, and he effec size was epo ed when app op ia e: Pa ial
η2 alues a e epo ed and conside ed as small (0.01), medium
(0.06), o la ge (0.14) ollowing Cohen
21
ecommenda ions. All
analyses we e ca ied ou using S a is ica so wa e (S a so ,
e sion 6.0).
Wi h espec o chemome ics analyses o 1H NMR spec al
da a, AMIX 3.9.15 (B uke BioSpin) so wa e was used o
bucke ing NMR spec a using wo ypes o bucke ing p ocesses:
(1) egula bucke ing employing a bucke size o 0.04 ppm and
(2) a iable bucke ing o NMR peaks assigned o specific
me aboli es ( o uni a ia e s a is ical analyses). In bo h cases,
no maliza ion was achie ed by scaling he in ensi y o indi idual
peaks o he o al in ensi y eco ded in he egion om δH0.2 o
10.0 ppm, excep o he egion o δH5.2 o 4.74 ppm con aining
he esidual signal o H2O, which was emo ed. NMR egula
bucke ed da a was in es iga ed by means o p incipal
componen analysis (PCA) in explo a o y s udies and by pa ial
leas -squa es disc iminan analysis (PLS-DA) o o hogonal
pa ial leas -squa es disc iminan analysis (OPLS-DA) o
de e mine he exis ence o diffe ences be ween expe imen al
g oups and o iden i y he me abolic ea u es esponsible o
hei disc imina ion. This mul i a ia e da a analysis was
pe o med using SIMCA-P so wa e ( . 17.0, Ume ics). The
esul s o he c oss- alida ion o PLS-DA and OPLS-DA models
a e gi en by means o cumula i e R2and Q2 alues, whe e Q2
alues o >0.5 we e conside ed indica i e o a good p edic i e
model. Models we e also alida ed by being subsequen ly
subjec ed o pe mu a ion es s (a o al o 100), and he new
coefficien s R2and Q2gene a ed om he pe mu a ion es we e
Figu e 2. Means (±SEM) o (A) wa e in ake and (B) numbe o licks in FT-60s ac oss 19 sessions o SIP. S a is ical analyses indica ed significan
diffe ences be ween low d inke s (LDs, n= 20) and high d inke s (HDs, n= 20; ** p< 0.01) in bo h wa e in ake and o al licks. Significan diffe ences
om session 1 we e ound in HDs om session 6 in wa e in ake (## p< 0.01) and om session 8 (### p< 0.001) onwa d.
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compa ed wi h hose om he eal model. I in e cep R2and Q2
alues om he pe mu a ion es we e significan ly smalle han
Q2o he eal model, hen he model was ega ded as p edic able.
The s a is ical significance o he es ima ed p edic i e powe o
PLS-DA and OPLS-DA models was u he assessed wi h an
ANOVA es o he c oss- alida ed esiduals (c -ANOVA).
Models wi h p alues o <0.05 we e conside ed o ha e a good
p edic ion.
Impo an loadings (spec al egions) o he disc imina ion
obse ed om he p edic i e models we e selec ed by gene a ing
he a iable impo ance in p ojec ion (VIP) plo . Loadings wi h
VIP sco es o >1 we e conside ed ele an o he gene a ed PLS-
DA and OPLS-DA models. ANOVA (analysis o a iance)
analyses ollowed by leas significan diffe ence (LSD) pos hoc
es s we e employed o de e mine he significance o diffe ences
o he me aboli e a ios be ween g oups; p alues o <0.05 we e
conside ed s a is ically significan . Finally, me abolic changes
wi h alse disco e y a e (FDR)-adjus ed p alues (q alues) o
<0.05 we e conside ed.
3. RESULTS AND DISCUSSION
3.1. SIP Acquisi ion
LD and HD beha io is clea ly e idenced by no only he wa e
in ake bu also he numbe o licks, as p e iously men ioned.
Figu e 2 shows SIP acquisi ion and main enance du ing 19
sessions. One-way epea ed ANOVA measu es e ealed diffe -
ences in he SIP acquisi ion conce ning wa e in ake, as shown
by he in e ac ion be ween sessions and g oup (in e ac ion SIP
session ×g oup effec : (F18, 666 = 9.146, p< 0.001, pa ial η2=
0.198). This effec was also confi med by he significan
in e ac ion in o al licks (in e ac ion SIP session ×g oup effec :
F18, 576 = 8.445, p< 0.001, pa ial η2= 0.208).
Apos hoc compa ison e ealed ha SIP induced diffe en
d inking beha io s ac oss he 19 sessions in high and low
d inke s: LD and HD animals exhibi ed ema kable diffe ences
in wa e in ake om session 6 (p< 0.01) onwa d. Mo eo e ,
when compa ed wi h session 1, he HD g oup significan ly
inc eased i s wa e consump ion om session 4 (p< 0.01)
onwa d. A simila pa e n was ound conce ning o al licks,
whe e LD and HD g oups diffe ed om session 8 (p< 0.01)
onwa d, and HD showed an inc eased numbe o licks om
session 4 (p< 0.001) onwa d compa ed wi h session 1. No
significan diffe ences we e ound be ween LD and HD animals
conce ning o al magazine en ies.
3.2. Assignmen o Me aboli es in Blood Se um De ec ed
by 1H NMR Spec oscopy
The assignmen o each me aboli e p esen in se um samples
was achie ed and is illus a ed in Figu e 3.Table S1 p o ides ull
in o ma ion on chemical shi s, mul iplici y, and coupling
cons an s o each me aboli e o o each me aboli e- ype
compound.
The assigned me aboli es belong mos ly o he classes o
amino acids ( aline, isoleucine, leucine, alanine, lysine,
glu ama e, glu amine, aspa a e, y osine, and phenylalanine)
and de i a i es (e.g., 3-hyd oxybu y a e, c ea ine), o ganic acids
(ace ic acid, ace oace ic acid, ci ic acid, and py u ic acid),
ca bohyd a es (α-andβ-glucose), choline (Cho)-based
compounds, which a e essen ial componen s o cellula
memb anes, polyols (e.g., glyce ol), and a y acids. T ime hyl-
amine N-oxide is an osmoly e used by he body o coun e ac he
effec s o inc eased u ea concen a ion ha accumula es du ing
kidney ailu e and was also ound in he spec a. Ke one bodies
like 3-hyd oxybu y a e, ace a e, and ace oace a e, gene ally
induced by as ing,
17
could also be ound in a se um samples.
Figu e 3. Sub egions o a ypical 1H NMR spec um (600 MHz) o a blood se um sample aken o a a om p e-SIP g oup. Nume a ion: 1: LDL/
VLDL, 2: a y acids (FA), 3: leucine, 4: isoleucine, 5: aline, 6: 3-hyd oxybu y a e (3HB), 7: E OH, 8: lac a e, 9: alanine, 10: lysine, 11: ace a e, 12:
glu ama e, 13: glu amine, 14: ace oace a e, 15: py u a e, 16: ci a e, 17: PUFA, 18: aspa a e, 19: c ea ine, 20: choline, 21: ime hylamine N-oxide
(TMAO), 22: glyce ol, 23: glucose, 24: UFA, 25: py imidine, 26: uma a e, 27: y osine, 28: his idine, 29: phenylalanine, 30: o ma e.
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Figu e 4. (A) OPLS-DA sco e plo ob ained o 1H NMR da a o samples aken p io o (n= 40) and a e SIP (n= 38). (B) S-plo showing he mos
significan me aboli es o disc imina ion (me aboli es wi h VIP > 1 alues a e colo ed): Bo om le me aboli es we e significan ly dec eased (alanine:
loadings 1.46, 1.50; lac a e: loadings 1.34, 4.14; UFA: loadings 5.34, 2.06, 2.18, 2.26, 0.94), whe eas hose loca ed a he op igh we e inc eased pos -
SIP (choline: loading 3.22; ci a e: loadings 2.54, 2.70; ace a e: loading 1.94; ace oace a e: loading 2.22; glucose: loadings 3.34, 3.38, 3.42, 3.46, 3.54,
3.66, 3.78, 3.82, 5.26; e hanol: loadings 1.18, 3.66; and LDL/VLDL/sa u a ed a y acids: loadings 0.86, 1.26, 1.30, 2.14). Pa e o scaling was ca ied
ou . R2X= 0.639, Q2= 0.764, p(c -ANOVA) = 7.0 ×10−18. The model was alida ed by a pe mu a ion es (wi h 100 pe mu a ions).
Figu e 5. OPLS-DA (A) sco e and (B) con ibu ion plo s ob ained o 1H NMR da a o pos -SIP samples ha we e classified as LD (n= 20) and HD
(n= 18). Posi i e and nega i e ba s in he con ibu ion plo explain he spec al egions con aining me aboli es ha disc imina e o HD and LD
samples, espec i ely. These include glu amine (loadings 2.14, 2.46), a y acids/LDL/VLDL (loadings 0.86, 0.90, 0.94, 1.26, 1.30, 1.34, 2.02, 2.06,
2.26, 2.30), 3-hyd oxybu y a e (HB, loadings 2.32, 2.40, 4.16), choline (loading 3.22), and lac a e (1.34, 4.10), which inc eased in HD d inke s, and
e hanol (loadings 1.18, 3.66), alanine (loading 1.50), glyce ol (loadings 3.62, 3.66), and glucose (loadings 5.26, 4.66, 3.90, 3.86, 3.74, 3.46, 3.26),
which dec eased. Only he samples om e y low d inke s (<10 mL) and e y high d inke s (>20 mL) we e conside ed. Pa e o scaling was ca ied ou .
R2X= 0.68, Q2= 0.58, p(c -ANOVA) = 0.032.
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3.3. Chemome ics Analyses o 1H NMR Spec al Da a
Mul ipa ame ic s a is ical ools we e applied o NMR da a in
he analyses o a s se um samples o e alua e changes in he
me abolic p ofiles in he p esence o OCD. Blood samples we e
aken a ou diffe en imes du ing he expe imen : p e- and
pos -SIP and p e- and pos -pelle (as indica ed in Figu e 1). An
explo a o y analysis o NMR da a was fi s achie ed by means o
PCA ha clus e ed simila samples oge he based on he inpu
da a (Figu e S1). PCA is use ul o e ealing he majo ends in
he 1H NMR da a and he possible analy ical and biological
con ounde a iables. On he basis o he PCA esul s, a sligh
di ision o samples be ween p e- and pos -SIP g oups (Figu e
S1a) and be ween p e- and pos -pelle g oups (Figu e S1b)is
e iden , independen o he obsessi e-compulsi e beha io o
he a s.
3.3.1. Diffe en ia ion o P e-SIP and Pos -SIP Se um
Samples. To imp o e he disc imina ion obse ed be ween
p e-SIP and pos -SIP da a, we applied an OPLS-DA model o
he 1H NMR da a (Figu e 4A). The chosen p ep ocessing
me hod con ains he o hogonal signal co ec ion (OSC), which
Figu e 6. Box plo s showing he a e age, median qua iles, and ex emes o specific peak in eg a ion alues ( ela i e o he TSP peak in eg al and
no malized o o al spec a in ensi y) o 3-hyd oxybu y a e, glu amine, lac a e, choline, LDL/VLDL, and FA le els, excep o UFA, which inc eased
pos -SIP o HD a s, and glyce ol, alanine, and glucose, which dec eased pos -SIP o HD a s when compa ed wi h LD a s. All o hese me aboli es
p esen ed p alues o <0.05 using ANOVA analyses ollowed by he leas significan diffe ence (LSD) pos hoc es s in pos -SIP.
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allows one o elimina e unnecessa y in o ma ion. In he OSC
p ocedu e, he X ma ix was co ec ed by a sub ac ion o
a ia ion o hogonal o he y(con aining he classes o each
sample, in his case, p e-SIP e sus pos -SIP) ec o calib a ion.
The co esponding S-plo (Figu e 4B) e ealed he mos
ele an me aboli es o he disc imina ion (wi h VIP alues >1).
Me aboli es o he bo om le we e significan ly dec eased
(alanine, lac a e, unsa u a ed a y acids), whe eas hose loca ed
a he op igh we e inc eased pos -SIP (choline, ci a e, ace a e,
ace oace a e, e hanol, glucose, and LDL/VLDL/sa u a ed a y
acids).
The esul s indica e ha SIP induced changes in lipid-
me abolism- ela ed molecules ( a y acids and choles e ol le els,
choline, ace a e, ace oace a e, glu amine) and ene gy me abo-
lism ( ela ed o he ica boxylic acid (TCA) cycle). Ace a e is
o med in he body by he me abolism o ce ain subs ances, in
pa icula , in he li e in he oxida ion o lipids. I is a p ecu so
o ace yl-CoA, which is used by cells o he syn hesis o a y
acids and choles e ol. To u he in es iga e i a diffe en ial effec
is induced by he pa adigm in HD and LD g oups, we added his
a iable o he model in he ollowing analysis (de ailed in
Sec ion 3.3.2).
3.3.2. Diffe en ia ion be ween LD and HD G oups in
P e- and Pos -SIP Se um Samples. A alid disc imina ion
be ween LD and HD d inke s was ound only o pos -SIP
samples a e he applica ion o an OPLS-DA model (Figu e 5).
This means ha i was no possible o obse e me abolic changes
a p io i o he SIP p ocedu e, so i would no be possible o
“p edic ” he beha io o each a based on se um me abolic
p ofiles. To ob ain a alid model, we emo ed se um samples o
a s showing a d inking olume be ween 10 and 20 mL om he
model. So, his model ied o maximize diffe ences be ween
e y low d inke s (<10 mL) and e y high d inke s (>20 mL).
The blood se um o pos -SIP HD a s specifically showed
inc eased low-densi y lipop o ein (LDL)/ e y low-densi y
lipop o ein (VLDL), a y acid (excep unsa u a ed a y
acids), 3-hyd oxybu y a e, glu amine, choline, and lac a e le els,
accompanied by a dec ease in glyce ol, glucose, alanine, and
e hanol le els when compa ed wi h pos -SIP LD a s. Figu e 6
shows he box-and-whiske plo s o no malized in eg a ion
alues o peaks om hese al e ed me aboli es ( ela i e o TSP
signal in eg al) wi h an indica ion o he in eg a ion anges,
median qua iles, and ex emes.
These esul s sugges ha lipid-me abolism- ela ed molecules
(including o al choles e ol (TC) con en , glu amine, choline,
and glyce ol) migh be associa ed wi h a compulsi e beha io
pheno ype du ing SIP. To u he suppo he obse ed ends,
we conduc ed he iden ifica ion and quan ifica ion o a y acid
p ofiles in se um samples by gas ch oma og aphy wi h flame
ioniza ion de ec ion (GC-FID) (Figu e 7). The polyunsa u a ed
a achidonic acid (C20:4n6) was ound o be he majo a y acid
in he samples ollowed by he sa u a ed palmi ic acid (C16:0)
and by linoleic acid (C18:2n6) and oleic acid (C18:1n9). HD
a s showed an inc ease in he sa u a ed palmi ic acid (C16:0),
he monounsa u a ed oleic acid (C18:1n9), and he polyunsa-
u a ed linoleic acid (C18:2n6) compa ed wi h LD a s (p<
0.05) and a dec ease in a achidonic acid, he majo
polyunsa u a ed a y acid (PUFA) in se um samples. As
shown in Figu e 7, s a is ically significan p e-SIP diffe ences
be ween LDs and HDs exis and emain cons an pos -SIP.
These esul s ag ee well wi h NMR esul s ha de ec ed an
inc ease in a y acid/LDL/VLDL con en in HD a s, excep o
UFA. In e es ingly, GC-FID analysis showed ha he o al
PUFA con en was significan ly highe in LD a s compa ed wi h
HD a s al eady in p e-SIP samples (p< 0.05).
These esul s a e in iguing because some p eclinical s udies
ha e also ound a simila ou pu in compulsi e animals:
Inc eased TC, VLDL, and LDL le els we e ound in dogs
exhibi ing ail chasing (a s e eo yped beha io p oposed as a
alid p eclinical model o s udying OCD in animals)
22
in
compa ison wi h con ol subjec s,
23,24
whe eas dec eased
glucose se um le els we e ound in highly compulsi e a s
selec ed by SIP compa ed wi h noncompulsi e animals.
25
In
human s udies, OCD pa ien s ha e been shown o exhibi highe
high-densi y lipop o ein (HDL) se um le els han heal hy
con ols.
26
Mo eo e , B ennan e al.
27
pe o med a c i ical
e iew o 1H magne ic esonance imaging s udies on OCD in
b ain issues and concluded ha changes in glu ama e/
glu amine le els we e common among pa ien s e sus heal hy
indi iduals, al hough some inconsis encies ha e been un a eled.
Fo ins ance, some s udies epo ed a dec ease whe eas o he s
epo ed an inc ease in he glu ama e-glu amine (Glx) le el.
Also, choline was e e ed o be a c ucial bioma ke in OCD, and
mos s udies showed an inc ease in his me aboli e in OCD
indi iduals.
Simila ly, o he neu opsychia ic condi ions ela ed o
inhibi o y con ol ha e been s udied ega ding me abolic
changes. Schwa z e al.
28
epo ed significan al e a ions o
b ain issue ee a y acids and phospha idylcholine le els in
subjec s wi h schizoph enia and bipola diso de using a high-
h oughpu mass spec ome y app oach (UPLC−MS) and
sugges ed ha lipid abno mali ies may be an in insic ea u e o
bo h schizoph enia and bipola diso de . A maca e al.
29
obse ed dec eased se um choles e ol and lep in le els in
bipola diso de pa ien s, whe eas Ozbulu e al.
30
ound
dec eased se um gh elin and inc eased TC le els in eu hymic
pa ien s unde li hium ea men when compa ed wi h con ols.
In dep ession, Kaddu ah-Daouk and K ishnan
31
epo ed ha
a y acids, glyce ol, and γ-aminobu y ic acid (GABA) we e
al e ed in cu en ly dep essed pa ien s when compa ed wi h
con ols. Also, an inc ease in he concen a ion o he ke one 3-
hyd oxybu y ic acid was ound in emi ed pa ien s ela i e o
dep essed pa ien s. Fu he mo e, Nakaza o e al.
32
ound a
ela ionship be ween inc eased glu amine se um le els and
compulsi e-like beha io , as assessed by o al and pe se e a i e
e o s in se -shi ing asks in subjec s eco e ed om ano exia
ne osa, which also is adop ed in he impulsi e-compulsi e
spec um.
Figu e 7. Fa y acid quan ifica ion by GC-FID. Ba s wi h (*) and (**)
e ealed a significan inc ease and dec ease (p< 0.05 by uni a ia e
es s), espec i ely, in he lipid con en in he se um o HD a s
compa ed wi h LD a s.
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I could be a gued, howe e , ha hese me abolic effec s
migh ha e been induced by die due o he me e exposu e o
ewa d pelle s du ing SIP sessions. Indeed, obese indi iduals
ha e been epo ed o exhibi poo e beha io al inhibi ion
18
and
a s onge a en ional bias owa d ood
33,34
compa ed wi h
heal hy subjec s. Mo eo e , impulsi e beha io seems o be
co ela ed wi h a highe consump ion o as ood
35
and a highe
body mass index (BMI),
36
whe eas in p eclinical models,
exposu e o highly pala able, a - ich die s has been shown o
induce compulsi e ood-seeking beha io
37−39
and o affec
ma ble bu ying beha io .
40−42
Thus o explo e ha possibili y,
we planned and ca ied ou an addi ional expe imen whe e a
coho o animals we e subjec ed o 19 sessions o exposu e o
ewa d pelle s, as desc ibed in he Me hods sec ion.
3.3.3. Diffe en ia ion be ween P e-Pelle and Pos -
Pelle Se um Samples. A PLS-DA model was gene a ed o
in es iga e he me abolic diffe ences be ween se um samples
collec ed be o e and a e he con ol die expe imen . A alid
disc imina ion be ween p e- and pos -pelle g oups was ound in
he sco e plo (Figu e 8A), meaning ha he ewa d pelle s had
effec s on he se um me abolic composi ion. The loading plo
(Figu e 8B) highligh s he mos significan a iables o he
model by desc ibing he influence and ela ion among he
a iables in he model plane. The e o e, i is possible o conclude
ha he peaks in spec al zones should be significan ly inc eased
(uppe igh loadings) and dec eased (bo om le loadings) in
he blood se um due o he exposu e o ewa d pelle s in his
con ol expe imen (VIP alues >1) and also due o a
physiological me abolic e olu ion o e ime. An inc ease in
c ea ine, ci a e, glucose, lysine, glu amine/glu ama e, ace a e,
and PUFAs was de ec ed in he blood se um a e such exposu e.
The spec al zones in he bo om le o he loading plo e e ing
o he me aboli es ha dec ease in pos -pelle se um include
choline and o he spec al egions ha mos ly co espond o
noise and we e no ele an o analyses, so hese we e no
conside ed.
As in he p e ious se o expe imen s, he lipid p ofile o he
pelle s was in es iga ed and quan ified by GC-FID (Table S2).
The ewa d pelle s du ing he SIP expe imen showed a 2.3- old
inc ease in a y acid con en , including on PUFAs. Because he
model was no able o disc imina e be ween HDs and LDs ei he
p e- o pos -exposu e o ewa d pelle s, i is plausible o assume
ha such al e a ions affec ed bo h g oups equally, hus poin ing
o he idea ha he SIP pa adigm, and no he die , is esponsible
o he me abolic al e a ions obse ed in he ulne able subjec s,
he HD g oup. The e a e se e al unde lying mechanisms ha
should be in es iga ed in he u u e ega ding he me abolic
al e a ions obse ed in he compulsi e-d inking HD g oup
selec ed by SIP, o example, he possible ole o an al e ed
asop essin, a ho mone implica ed in he me abolic synd ome,
ha d i es a p oduc ion as a mechanism o s o ing me abolic
wa e .
43
Fu he mo e, he ele ance o gu mic obio a
dys egula ion in diffe en neu opsychopa hological diso -
de s
44−46
should be conside ed in he me abolic al e a ions
obse ed in compulsi e HD a s. In his sense, in ou labo a o y,
we ha e demons a ed ha compulsi e HD a s showed a lowe
bac e ial di e si y han LD a s, i espec i e o he die .
47
Howe e , he same s udy also demons a ed ha he
adminis a ion o a yp ophan-deple ed die educed bac e ial
e enness and showed a highly unc ionally o ganized
communi y in he compulsi e HD a s selec ed by SIP. This
poin s owa d a bac e ial communi y ha is agile o ex e nal
changes due o he dominance o a low numbe o species in
compulsi e HD a s compa ed wi h noncompulsi e LD a s.
The d ama ic effec ound in he p esen wo k adds e idence
no only o he s ess ul p ope ies o in e mi en ein o cemen
in SIP inducing compulsi e beha io bu also o se e e
physiological effec s, such as inc eased co icos e one le els,
25
inc eased amygdaloid and dec eased hippocampal olume,
15
and inc eased dend i ic spinal densi y in he do sal s ia um.
48
In
his sense, he a o emen ioned finding o inc eased glu amine
le els in HD in he p esen s udy is no su p ising gi en i s majo
ole in he b ain as he p ecu so o glu ama e, which is a key
ac o ega ding neu oplas ici y
49,50
and one o he al e ed
mechanisms
51
and pu a i e he apeu ic a ge s
52
in compulsi e
d inking in SIP.
4. CONCLUSIONS
The p esen s udy has in es iga ed he me abolomic p ofile by
means o NMR o high- and low-compulsi e a s selec ed by SIP
Figu e 8. (A) PLS-DA sco e plo ob ained o 1H NMR da a o samples be o e (n= 20) and a e (n= 20) changing he die o ewa d pelle s (p e- and
pos -pelle , espec i ely). (B) PLS-DA loading plo e ealing he mos significan me aboli es o disc imina ion (wi h VIP alues >1), which
co espond o c ea ine (loadings 3.06, 3.94), glucose (loadings 5.26, 3.94, 3.78, 3.74, 3.56, 3.50, 3.26), PUFA (loading 2.78), lysine (loadings 1.50,
1.74), ace a e (loading 1.94), o ma e (loading 8.46), and glu amine/glu ama e (loadings 2.14, 2.46, 2.36), which inc eased in pos -pelle samples, and
choline (loadings 3.22), which dec eased. Uni - a iance scaling was ca ied ou . R2X= 0.65, Q2= 0.90, p(c -ANOVA) = 6.5 ×10−9. The model was
alida ed by a pe mu a ion es (wi h 100 pe mu a ions).
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619
as a po en ial ool o iden i ying c i ical bioma ke s in
ulne able subjec s. We ound ha al hough SIP i sel induced
a change in he me abolomic p ofile, i affec ed he HD animals
diffe en ly, which showed a hype lipidemic, hypoglycemic, and
hypoglu amine gic p ofile, compa ed wi h he LD animals, in
line wi h he li e a u e ega ding bo h p eclinical models and
human pa ien s in OCD and ela ed diso de s. Mo eo e , me e
exposu e o ewa d pelle s did no esul in a alid model o
p edic ing he pheno ypic p ofile based on me abolomics, hus
leading us o disca d an effec due o die alone. Ou da a add
significan e idence o a c ucial opic in basic neu oscience and
po en ially clinical fields; howe e , u u e s udies a e ongoing in
ou labo a o ies o unsc amble his complex phenomenon and
o u he cha ac e ize he pu a i e ole o me abolism in he
igge ing and ea ly iden ifica ion o la en ulne abili ies. In
addi ion, he pu a i e impac o a high- a die on inhibi o y
con ol defici and he po en ial use o me abolomic bioma ke s
in he ea ly diagnose o compulsi e spec um diso de s a e
en isaged as well.
■ASSOCIATED CONTENT
*
sıSuppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/acs.jp o eome.1c00857.
Table S1. Peak assignmen o me aboli es iden ified.
Table S2. GC-FID a y acid p ofiles. Figu e S1. PCA
sco e plo s ob ained om 1H NMR da a (PDF)
■AUTHOR INFORMATION
Co esponding Au ho s
Ma ga i a Mo eno −Depa men o Psychology and Heal h
Resea ch Cen e CEINSA, Uni e si y o Alme ía, 04120
Alme ía, Spain; Email: [email p o ec ed]
Ignacio Fe nández −Depa men o Chemis y and Physics,
Resea ch Cen e CIAIMBITAL, Uni e si y o Alme ía, 04120
Alme ía, Spain; o cid.o g/0000-0001-8355-580X;
Phone: +34 950214465; Email: [email p o ec ed]
Au ho s
Ana C. Ab eu −Depa men o Chemis y and Physics, Resea ch
Cen e CIAIMBITAL, Uni e si y o Alme ía, 04120 Alme ía,
Spain
San iago Mo a −Depa men o Psychology and Heal h
Resea ch Cen e CEINSA, Uni e si y o Alme ía, 04120
Alme ía, Spain; P esen Add ess: San iago Mo a -
Depa men o Neu oscience and Facul y o Heal h and
Medical Sciences, Uni e si y o Copenhagen, 2200
Copenhagen, Denma k
Ana Isabel T is án −Depa men o Chemis y and Physics,
Resea ch Cen e CIAIMBITAL, Uni e si y o Alme ía, 04120
Alme ía, Spain
Elena Ma ín-González −Depa men o Psychology and
Heal h Resea ch Cen e CEINSA, Uni e si y o Alme ía,
04120 Alme ía, Spain
A
ngeles P ados-Pa do −Depa men o Psychology and Heal h
Resea ch Cen e CEINSA, Uni e si y o Alme ía, 04120
Alme ía, Spain
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acs.jp o eome.1c00857
Au ho Con ibu ions
§
A.C.A. and S.M. con ibu ed equally.
Funding
This esea ch has been unded by he S a e Resea ch Agency o
he Spanish Minis y o Science, Inno a ion and Uni e si ies
(CTQ2017-84334-R), Jun a de Andalucia (102C2000004,
UAL-FEDER 2020-AGR-B1781, UAL-FEDER 2020-CTS-
D2068, P20_01041, and CV20-78799), and he Gobie no de
Espana MCIN/AEI/10.13039/501100011033/Union Eu opea
“Nex Gene a ionEU”/PRTR (PDC2021-121248-I00 and
PLEC2021-007774) and MCIN/AEI/10.13039/
501100011033/FEDER “Unamane adehace Eu opa”
(PGC2018-099117-B-C21). A.C.A. hanks he Uni e si y o
Alme ia o he Hipa ia schola ship. We hank P o . Ma ia del
Ca men Ce on Ga cia (Uni e si y o Alme ia) o hei help ul
assis ance ega ding GC-FID measu emen s.
No es
The au ho s decla e no compe ing financial in e es .
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