Plasmatic Oxidative and Metabonomic Profile of Patients with Different Degrees of Biliary Acute Pancreatitis Severity

an ioxidan s A icle Plasma ic Oxida i e and Me abonomic P o ile o Pa ien s wi h Di e en Deg ees o Bilia y Acu e Panc ea i is Se e i y Ped o Sil a-Vaz 1,2,3,4,* , I ana Ja ak 5, Luís Ra o 6, Ped o F. Oli ei a 7, Sa a Mo gado-Nunes 4,8 , Aida Paulino 2,3,4, Miguel Cas elo-B anco 1,3,4 , Ma ia Filomena Bo elho 9,10,11,12 , JoséGuilhe me T alhão9,10,11,12,13, Ma co G. Al es 14 and Ana Ma ga ida Ab an es 9,10,11,12   Ci a ion: Sil a-Vaz, P.; Ja ak, I.; Ra o, L.; Oli ei a, P.F.; Mo gado-Nunes, S.; Paulino, A.; Cas elo-B anco, M.; Bo elho, M.F.; T alhão, J.G.; Al es, M.G.; e al. Plasma ic Oxida i e and Me abonomic P o ile o Pa ien s wi h Di e en Deg ees o Bilia y Acu e Panc ea i is Se e i y. An ioxidan s 2021,10, 988. h ps://doi.o g/ 10.3390/an iox10060988 Academic Edi o : Joseph Cullen Recei ed: 16 May 2021 Accep ed: 15 June 2021 Published: 21 June 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 il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Heal h Sciences Resea ch Cen e, Uni e si y o Bei a In e io (CICS-UBI), 6200-506 Co ilhã, Po ugal; [email p o ec ed] 2Gene al Su ge y Depa men , Hospi al Ama o Lusi ano, Unidade Local de Saúde de Cas elo B anco, 6000-085 Cas elo B anco, Po ugal; [email p o ec ed] 3Facul y o Heal h Sciences, Uni e si y o Bei a In e io , 6200-506 Co ilhã, Po ugal 4Clinical Academic Cen e o Bei as (CACB), 6200-506 Co ilhã, Po ugal; [email p o ec ed] 5Depa men o Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal; [email p o ec ed] 6Heal h School o he Poly echnic o Gua da, 6300-559 Gua da, Po ugal; [email p o ec ed] 7QOPNA & LAQV, Depa men o Chemis y, Uni e si y o A ei o, 3810-193 A ei o, Po ugal; [email p o ec ed] 8Poly echnic Ins i u e o Cas elo B anco, Escola Supe io de Ges ão, 6000-084 Cas elo B anco, Po ugal 9Biophysics Ins i u e, Facul y o Medicina, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal; [email p o ec ed] (M.F.B.); [email p o ec ed] (J.G.T.); [email p o ec ed] (A.M.A.) 10 Coimb a Ins i u e o Clinical and Biomedical Resea ch (iCBR) A ea o En i onmen Gene ics and Oncobiology (CIMAGO), Facul y o Medicina, Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal 11 CNC.IBILI Conso ium/Cen e o Inno a ion Biomedicine and Bio echnology (CIBB), Uni e si y o Coimb a, 3000-548 Coimb a, Po ugal 12 Clinical Academic Cen e o Coimb a (CACC), 3000-561 Coimb a, Po ugal 13 Su ge y Depa men , Cen o Hospi ala e Uni e si á io de Coimb a (CHUC), Facul y o Medicina, Uni e si y Hospi al, 3000-075 Coimb a, Po ugal 14 Depa men o Ana omy and Uni o Mul idisciplina y Resea ch in Biomedicine (UMIB), Ins i u e o Biomedical Sciences Abel Salaza (ICBAS), Uni e si y o Po o, 4050-313 Po o, Po ugal; [email p o ec ed] *Co espondence: [email p o ec ed]; Tel.: +351-966-498-337 Abs ac : Acu e panc ea i is (AP) is an in lamma o y p ocess o he panc eas wi h a iable in ol e- men o he panc ea ic and pe ipanc ea ic issues and emo e o gan sys ems. The main goal o his s udy was o e alua e he in lamma o y bioma ke s, oxida i e s ess (OS), and plasma me abolome o pa ien s wi h di e en deg ees o bilia y AP se e i y o imp o e i s p ognosis. Twen y-nine pa ien s wi h bilia y AP and 11 heal hy con ols we e en olled in his s udy. We analyzed se e al in lamma o y bioma ke s, mul i ac o ial sco es, eac i e oxygen species (ROS), an ioxidan s de enses, and he plasma me abolome o bilia y AP and heal hy con ols. Hepcidin (1.00), CRP (0.94), and SIRI (0.87) we e he mos accu a e se ological bioma ke s o AP se e i y. OS played a pi o al ole in he ini ial phase o AP, wi h signi ican changes in ROS and an ioxidan de enses ela ing o AP se e i y. Phenylalanine (p< 0.05), h eonine (p< 0.05), and lipids (p< 0.01) showed signi ican changes in AP se e i y. The ole o hepcidin and SIRI we e con i med as new p ognos ic bioma ke s o bilia y AP. OS appea s o ha e a ole in he onse and p og ession o he AP p ocess. O e all, his s udy iden i ied se e al me aboli es ha may p edic he onse and p og ession o bilia y AP se e i y, cons i u ing he i s me abonomic s udy in he ield o bilia y AP. Keywo ds: acu e bilia y panc ea i is; in lamma ion; p ognos ic; se e i y; hepcidin; sys emic in lam- ma o y esponse index; oxida i e s ess; me abonomics An ioxidan s 2021,10, 988. h ps://doi.o g/10.3390/an iox10060988 h ps://www.mdpi.com/jou nal/an ioxidan s An ioxidan s 2021,10, 988 2 o 18 1. In oduc ion Acu e panc ea i is (AP) is an in lamma o y diso de o he exoc ine panc eas wi h high mo bidi y and mo ali y i associa ed wi h local and sys emic complica ions [ 1 , 2 ]. Mos pa ien s p esen wi h mild sel -limi ed AP wi h a good p ognosis [ 3 ]. Howe e , 15–20% p og ess o a se e e AP wi h panc ea ic nec osis and high mo bidi y and mo ali y associa ed wi h sys emic in lamma o y esponse synd ome (SIRS) and mul iple o gan ailu e [ 4 , 5 ]. The e has been an inc easing incidence in he las decades, al hough wi h an unchanged mo ali y a e [ 6 ]. The o e all mo ali y o AP is less han 5%, hough se e e AP is associa ed wi h a mo ali y up o 30–50% [ 1 , 7 ]. The mos common causes o AP a e galls ones and alcohol [ 6 ]. In he Medi e anean and o he sou he n Eu opean coun ies, including Po ugal, galls ones a e he dominan e iology [8]. Se e al heo ies associa ed wi h he ea ly pa hogenesis o AP ha e been desc ibed, such as he in a-acina and duc al ac i a ion o p o eoly ic enzymes, leukocy e chemo axis, he elease o p o- and an i-in lamma o y cy okines, oxida i e s ess (OS), mi ochond ial dys unc ion, galls one mig a ion, mic oci cula ion inju y, as well as bac e ial ansloca ion o he panc eas and sys emic ci cula ion [ 9 , 10 ]. Ne e heless, he molecula mechanisms by which hose e ec s a e media ed emain unknown. AP is associa ed wi h a high a iabili y o se e i y, and al hough an ea ly iden i ica ion o pa ien s who will de elop se e e AP is c ucial, his iden i ica ion emains a g ea challenge. De e mina ion o AP p ognosis is c ucial o op imize he ini ial he apeu ic app oach and dec ease mo ali y. Se e al se ological bioma ke s and sco es ha e been desc ibed, namely C- eac i e p o ein (CRP) [ 11 ], hepcidin [ 12 , 13 ], p ocalci onin (PCT) [ 14 ], he sys emic in lamma o y esponse index (SIRI) [ 13 ], he bedside index o se e i y in acu e panc ea i is (BISAP) [15], SIRS [16], and he modi ied Ma shall sco e (MMS) [17]. The ole o eac i e oxygen species (ROS) in he pa hophysiology o AP has been demons a ed [ 18 , 19 ] and occu s in he ea ly s ages o his in lamma o y p ocess [ 20 ]. Howe e , i is no clea whe he ROS ac as media o s o ini ia e he complex cascade ha leads o AP and, hus, he exac ole o OS in he de elopmen o AP emains unclea [ 21 ]. OS is p esen ly conside ed a c i ical media o o he ea ly local e en s ela ed o AP and he associa ed SIRS [ 22 ]. Mi ochond ia a e suscep ible o OS, and mi ochond ial dys unc ion is o en used as a speci ic bioma ke o oxidan exposu e [ 23 ]. In expe imen al models o AP, acina cells we e shown o die h ough nec osis and apop osis, pa hways linked o mi ochond ial dys unc ion [ 24 ]. O he s sugges ha mi ochond ial dys unc ion is associa ed wi h sys emic in lamma o y p ocesses [ 25 ], bu he mechanisms emain la gely unknown. Thus, OS-associa ed e en s and bioma ke s in AP emain o be de ined. The de e mina ion o me aboli e changes ha desc ibe a biological pheno ype based on 1 H nuclea magne ic esonance (NMR) spec oscopy has been widely applied o de- ine p ognos ic bio luid p o iles o physiological o pa hological s a es [ 26 ]. In in lam- ma o y diso de s, such as AP, only a ew s udies o me abolome ha e been epo ed. Villaseño e al. [ 27 ] s udied he me abolic pheno ype in u ine and plasma samples o 15 pa ien s wi h AP. The e iologies o he AP g oup we e galls ones (6/15) and alcohol (9/15). They ound changes in alanine, aline, and hippu a e in pa ien s wi h AP and concluded ha hese me aboli es ha e he po en ial o se e as diagnos ic ools. Lusczek e al. [ 28 ] e alua ed he po en ial o u ina y 1 H-NMR me abonomics in he diagnosis o AP. The au ho s analyzed i e pa ien s wi h AP and concluded ha i is possible o ob ain a dis inc me abolome in AP pa ien s’ u ine samples compa ed o heal hy con ols. Tha s udy iden i ied ci a e as he p ima y me aboli e associa ed wi h he in lamma o y AP s a e and alcohol consump ion. Xu e al. [ 29 ] s udied he se um me abolomics o mild AP in 38 pa ien s. Al hough he au ho s did no speci y he e iology o AP pa ien s, by using he ch oma og aphy-high- esolu ion mass spec ome y (UPLC-HRMS) echnique, hey iden i ied decanyl choline, dodecanol, and 2- e adecanone as diagnosis me aboli es, and sphinganine, L- hy onine, glycocholic acid and 2- e adecanone as he apeu ic esponse me aboli es. Xiao e al. [ 30 ], using gas-ch oma og aphy/mass spec ome y (GS-MS), iden- i ied se e al me aboli es, such as 3-hyd oxubu y ic, D-glucose, and hexadecenoic acid, An ioxidan s 2021,10, 988 3 o 18 wi h po en ial clinical ele ance o diagnosis in 40 pa ien s wi h AP. I was sugges ed 3-hyd oxybu y ic acid and ci ic acid ha e a p ognosis alue. Huang e al. [ 31 ] s udied h ee AP e iologies (29 pa ien s wi h hype lipidemia, 20 wi h alcoholic AP, and 27 wi h bilia y AP). They concluded ha L- y osine, oc adecanoic acid, choles e ol, glyce ol 1- hexadecanoa e, and L-lac ic acid a e me aboli es wi h po en ial diagnosis ele ance and (R)-3-hyd oxybu y ic acid and manni ol could dis inguished AP om he heal hy g oup. Howe e , his s udy did no e alua e he cha ac e is ics be ween each g oup o AP pa ien s, no was he p ognosis o AP se e i y aken in o accoun . These i e pape s desc ibed se - e al me aboli es, mainly as diagnos ic ools, bu only one speci ically s udied he subg oup o pa ien s wi h bilia y AP, compa ing i wi h o he AP e iologies. This s udy aimed o e alua e he pa hophysiology o AP by analyzing he in lam- ma o y bioma ke s and mul i ac o ial sco es, OS, and plasma me abolome o bilia y AP pa ien s and heal hy con ols. I is in ended o con ibu e o a be e unde s anding o bilia y AP’s pa hophysiology and hus imp o e p ognosis. 2. Ma e ials and Me hods 2.1. Pa icipan s S udy pa icipan s we e ec ui ed among pa ien s wi h bilia y AP admi ed o he Depa men o Gene al Su ge y o he Hospi al Ama o Lusi ano o Unidade Local de Saúde de Cas elo B anco, Uni e si y Teaching Hospi al, Cas elo B anco, Po ugal. Twen y-nine pa ien s and 11 heal hy con ols we e conside ed o he s udy. The s udy p o ocol was app o ed by he local e hics commi ee ( e e ence numbe 10294/15) and was conduc ed in compliance wi h he Decla a ion o Helsinki. Signed in o med consen was ob ained om all pa icipan s. 2.2. S udy Design Subjec s wi h AP ha accep ed he eques o pa icipa e and me he eligibili y c i e ia o admission we e ec ui ed be ween No embe 2015 and Janua y 2016. AP was de ined acco ding o he e ised A lan a classi ica ion (RAC). A leas wo o he ollowing h ee ea u es mus ha e been p esen : (1) abdominal pain consis en wi h AP; (2) se um lipase ac i i y (o amylase ac i i y) a leas h ee imes g ea e han he uppe limi o a no mal alue; (3) and cha ac e is ics indings o AP on con as -enhanced compu ed omog aphy (CECT) and less commonly on magne ic esonance imaging (MRI) o ansabdominal ul asonog aphy (US) [17]. All adul pa ien s ( ≥ 18 yea s old) wi h bilia y AP we e included. Pa ien s wi h o he causes o AP excep bilia y, ecu en AP, ch onic panc ea i is, panc ea i is due o malignancy, p egnan pa ien s, pa ien s wi h ime om onse o disease o p esen a ion in he eme gency oom g ea e han 24 h, and pa ien s being hospi alized o mo e han 24 h a he ime o ec ui men we e excluded. Fo an e iological con i ma ion o AP, he pa ien s unde wen US wi h iden i ica ion o galls ones o mic oli hiasis. A e he US, pa ien s wi h e iological doub unde wen endoscopic ul asonog aphy o exclude mic oli hiasis and cha ac e ize he bilia y ee. Local and sys emic complica ions we e de ined acco ding o he RAC [ 17 ]. The se e i y o AP was de ined acco ding o he RAC: mild (no o gan ailu e o no local o sys emic complica ions), mode a ely se e e (o gan ailu e ha esol es wi hin 48 h and he p esen local o sys emic complica ions o bo h wi hou pe sis en o gan ailu e), and se e e (p esence o pe sis en o gan ailu e o SIRS). O e all, his s udy included 40 indi iduals: mild (n= 10), mode a ely se e e (n= 9), and se e e (n= 10) AP pa ien s and heal hy con ols (n= 11). All subjec s included in his s udy we e e alua ed o como bidi ies by applying he Cha lson como bidi y index (CCI). This index is he mos equen ly used ool o measu e co-exis ing diseases and i has been alida ed o p edic ing he isk o mo ali y, disabili y, hospi aliza ion, and leng h o hospi al s ay in se e al clinical se ings [ 32 ]. CCI is An ioxidan s 2021,10, 988 4 o 18 age-dependen and e alua es se e al pa hological condi ions, including ca dio ascula , espi a o y, li e , and pep ic diseases as well as diabe es and AIDS [33]. 2.3. Analysis o Bioma ke s and Sco es Sys ems Blood samples we e collec ed om each pa ien ha had been as ing o a leas 6 h on admission and a e 48 h o onse o symp oms, d awn in o 5 mL hepa in- ea ed ubes and cen i uged o 10 min a 1500 × g. The plasma was hen collec ed and s o ed a − 80 ◦ C un il analysis. Lipase (U/L), amylase (U/L), CRP (mg/dL9, PCT (ng/dL), whi e blood cells (WBC, 10 3 / µ L), neu ophil (N, 10 3 / µ L), lymphocy e (L, 10 3 / µ L), monocy e (M, 10 3 / µ L), calcium (mg/dL), albumin (g/dL), o al p o eins (g/dL), and hepcidin (ng/mL) we e analyzed ollowing ce i ied analysis a he Depa men o Clinical Pa hology, Hospi al Ama o Lusi ano o he Unidade Local de Saúde de Cas elo B anco. Neu ophil/lymphocy e (N/L) a io and SIRI we e calcula ed. The BISAP sco e was desc ibed in 2008 and consis s o a simple, ea ly assessmen o mo ali y isk in pa ien s wi h AP. I e alua ed i e pa ame e s: blood u ea ni ogen (BUN), men al s a us, SIRS, age, and pleu al e usion. The cu -o used o assess he mo ali y isk and se e i y o AP was ≥ 3. SIRS is a simple and widely used sco e in a clinical se ing. I e alua es ou pa ame e s: empe a u e, espi a o y a e, pulse, and WBCs. This sco e is de ined by he p esence o ≥ 2 o he desc ibed pa ame e s. The MMS allows o he assessmen o mul io gan ailu e in AP. O gan ailu es includes one o mo e o he ollowing pa ame e s: ca dio ascula , espi a o y, and kidney ailu e. Mul io gan ailu e is p esen o a cu -o o ≥ 2. In he p esen s udy, all h ee sco es we e assessed on admission and 48 h a e onse o symp oms. 2.4. Oxida i e S ess E alua ion Ca bonyl g oups, p o ein ni a ion, and lipid pe oxida ion a e usually used as bioma k- e s o OS- ela ed al e a ions in p o eins and lipids. We e alua ed, on admission: 2,4- dini ophenyl (2,4-DNP), 3-ni o y osine (3-NO), and 4-hyd oxynonenal (4-HNE), espec- i ely. The con en o hese adduc s in plasma samples was e alua ed using speci ic an ibodies ia slo -blo as p e iously desc ibed [ 34 , 35 ]. The ac i i y o se e al an ioxidan de ense enzymes was also e alua ed, namely glu a hione pe oxidase (GPx), glu a hione educ ase (GR), supe oxide dismu ase (SOD), and ca alase (CAT). GPx and GR concen a- ions in plasma samples we e de e mined using p e iously desc ibed me hods [ 36 ]. The assay o SOD ac i i y was based on he eac ion in which SOD educes he supe oxide anion o hyd ogen pe oxide and oxygen, using a me hod p e iously desc ibed [ 37 ]. The CAT ac i i y assay was based on he measu emen o hyd ogen pe oxide p oduced by he ac ion o CAT. 2.5. P o on NMR (1H-NMR) Spec oscopy 1 H-NMR spec a o plasma samples we e acqui ed and quan i ied as p e iously desc ibed [ 38 ]. Sodium uma a e ( inal concen a ion o 1 mM) was used as an in e nal e e ence (6.50 ppm) o quan i y he ollowing me aboli es p esen in plasma samples media (mul iple , ppm). Rela i e a eas o 1 h-NMR esonances and me aboli es concen a ions we e quan i ied as p e iously desc ibed [ 38 ]. In b ie , plasma samples composed o pa ien s wi h bilia y AP, collec ed on admission, and heal hy subjec s o he con ol g oup we e analyzed. These samples we e hawed, homogenized using a o ex, and cen i uged (9200 pm, 5 min). NMR spec a we e acqui ed on a Va ian Ino a 600 MHz (14.1 T) spec ome e equipped wi h a 3 mm QXI p obe wi h a z-wa e p esa u a ion. Wa e sa u a ion equencies we e op imized o each sample. Chemical shi s we e in e nally e e enced o a glucose double a 5.23 ppm. Me aboli e assignmen was based on a compa ison wi h p e iously published da a and e e ence spec a a ailable in public da abases such as HMBD [ 39 ]. Addi ionally, 2D homonuclea TOCSY spec a we e eco ded o help wi h spec al assignmen [40]. An ioxidan s 2021,10, 988 5 o 18 Da a ma ices o mul i a ia e analysis we e c ea ed in AMIX-Viewe ( e sion 3.9.15, B uke Biospin, Rheins e en) using all in ensi y alues in he 0.75–8.5 ppm egion wi h he exclusion o wa e and uma a e egions as well as he egions wi hou signal. P ocessed spec a we e aligned o minimize shi a ia ions [ 41 ] and no malized by in eg al a ea o accoun o ma ix dilu ion e ec s and expe imen al condi ions [ 42 ]. Mul i a ia e analysis was pe o med in Simca 14 (Ume ics, Umea, Sweden) on uni - a iance scaled da a ma ices. P incipal componen analysis (PCA) e alua ed he ini ial da a s uc u e, ollowed by pa ial leas squa es disc iminan analysis (PLS-DA) o iden i y me aboli e con ibu ion o class sepa a ion. PLS-DA loadings we e calcula ed by mul iplying he a iable weigh s (w) wi h he espec i e s anda d de ia ions and colo -coded acco ding o he size o a iable impo ance o p ojec ion alues (VIP). Me aboli es wi h VIP > 1 we e conside ed ele an o g oup sepa a ion. De aul se en old c oss- alida ion and pe mu a ion es s [ 43 ] we e used o alida e he obse ed a ia ion (R 2 ) and p edic i e po en ial (Q 2 ) o PLS-DA models. Addi ionally, well- esol ed peaks o ele an me aboli es we e in eg a ed (AMIX-Viewe , e sion 3.9.15, B uke Biospin, Rheins e en) and no malized by in eg al a eas. Biological e ec s o no malized a eas we e es ima ed by calcula ing e ec size. Addi ionally, hey we e analyzed ia wo-way ANOVA ollowed by Tukey’s pos hoc es (G aph-Pad P ism 6 o Windows, G aph Pad So wa e, La Jolla, Cali o nia, USA, www.g aphpad.com, accessed on 15 Decembe 2020). 2.6. S a is ical Analysis Resul s a e exp essed in mean (SD) ± s anda d e o o he mean (SEM), median (Q1, Q3), o n (%). No mali y was assessed using he Shapi o–Wilk es . Rega ding he quan i a i e a iables, he cen al endency was compa ed using S uden ’s o Mann– Whi ney es s (2 ca ego ies) o ANOVA o he K uskal–Wallis es (>2 ca ego ies). In quali a i e a iables, associa ions we e e i ied using he Chi-squa ed es , wi h Fishe ’s co ec ion when necessa y. Recei e ope a ing cha ac e is ics (ROC) cu es we e calcula ed o assess he p ognos ic accu acy and de e mine he bes cu -o poin s. A p< 0.05 indica ed s a is ical signi icance. S a is ical analysis was pe o med using SPSS 25.0 (SPSS, Chicago, IL, USA). 3. Resul s 3.1. Clinical Da a The baseline cha ac e is ics o heal hy con ols (n= 11) and bilia y AP pa ien s: mild AP (n= 19), mode a ely se e e AP (n= 9), and se e e AP (n= 10) a e shown in Table 1. The e we e no signi ican di e ences in he demog aphic ea u es be ween he con ol g oup and bilia y AP pa ien s. The e we e also no di e ences be ween he demog aphic cha ac e is ics in he h ee deg ees o AP se e i y, as exp essed in Table 1. Table 1. Baseline cha ac e is ics o heal hy con ols and bilia y AP pa ien s included in he s udy. ( ‡ Mann–Whi ney es ; ‡‡ Chi-squa ed es ; ‡‡‡ K uskal–Wallis es ). Con ol (n= 11) Sample (n= 29) pMild AP (n= 10) Mode a ely Se e e AP (n= 9) Se e e AP (n= 10) p Age (yea s) 69 ±14.6 65 ±18.9 0.65 ‡54 ±16.8 69 ±17.2 73 ±18.7 0.53 ‡‡‡ Female sex 6 (54.5%) 20 (69%) 0.41 ‡‡ 8 (80%) 6 (66.7%) 6 (60%) 0.39 ‡‡ BMI (kg/m2)26.7 ±3.0 26.7 ±3.8 0.98 ‡27.0 ±2.9 26.4 ±4.6 26.6 ±4.0 0.92 ‡‡‡ BMI ≥30 kg/m22 (18.2%) 6 (20.7%) 0.91 ‡‡ 2 (20%) 2 (22.2%) 2 (20%) 0.91 ‡‡ CCI 3 (1–4) 3 (1–4) 0.49 ‡1 (0–2) 3 (2–4) 3 (1–4) 0.49 ‡‡‡ Hospi al s ay (days) 12 ±7.8 - 6 ±2.5 11 ±5.0 18 ±8.7 - Mo ali y 3 (10.3%) 3 (30%) AP: acu e panc ea i is; BMI: body mass index; CCI: Cha lson como bidi ies index. Se e al biochemical ma ke s and mul i ac o ial sco es we e assessed, bo h on admis- sion and 48 h a e onse o symp oms, which a e exp essed in Table 2. An ioxidan s 2021,10, 988 6 o 18 Table 2. Se e al biochemical ma ke s and mul i ac o ial sco es o heal hy con ols and bilia y AP pa ien s included in he s udy. (‡K uskal–Wallis es ). Con ols (n= 11) Time Sample (n= 29) Mild AP (n= 10) Mode a ely Se e e AP (n= 9) Se e e AP (n= 10) p‡ WBC 6.5 ±1.6 Admission 14.5 ±5.4 11.2 ±4.5 15.0 ±5.6 17.4 ±4.1 0.017 48 h 12.1 ±6.3 6.9 ±3.5 12.7 ±7.5 14.9 ±2.8 0.002 Neu ophils 4.8 ±0.9 Admission 12.4 ±5.5 8.6 ±4.5 13.2 ±5.3 15.7 ±3.6 0.011 48 h 10.0 ±6.2 4.7 ±3.2 10.4 ±6.9 13.2 ±2.7 0.001 SIRI Aission 7.4 ±6.3 3.6 ±3.9 8.8 ±7.5 11.4 ±4.9 0.022 48 h 6.6 ±6.4 1.6 ±2.3 5.9 ±6.4 10.6 ±5.7 0.001 PCT Admission 2.9 ±6.9 0.7 ±0.9 4.5 ±5.8 5.9 ±10.9 0.001 48 h 4.1 ±7.3 0.6 ±0.1 6.6 ±9.1 7.9 ±8.8 <0.001 CRP 5.3 ±1.1 Admission 42.8 ±82.7 5.4 ±1.2 33.0 ±51.5 88.9 ±22.0 0.032 48 h 170.7 ±152.6 15.9 ±14.0 180.6 ±107.2 316.6 ±107.9 <0.001 Hepcidin Admission 64.2 ±69.6 20.4 ±12.2 69.9 ±37.9 100.7 ±90.5 0.047 48 h 136.7 ±138.7 26.4 ±27.2 56.6 ±51.9 286.7 ±87.3 <0.001 Calcium Admission 9.0 ±0.6 8.9 ±0.5 9.1 ±0.4 8.9 ±0.9 NS 48 h 8.5 ±0.8 8.9 ±0.3 8.7 ±0.5 7.9 ±1.0 0.014 Albumin Admission 3.7 ±0.5 3.8 ±0.4 3.7 ±0.4 3.5 ±0.5 NS 48 h 3.1 ±0.5 3.5 ±0.3 3.2 ±0.3 2.7 ±0.6 0.001 To al p o eins Admission 6.5 ±0.6 6.7 ±0.5 6.7 ±0.5 6.3 ±0.6 NS 48 h 5.9 ±0.6 6.4 ±0.4 6.0 ±0.3 5.3 ±0.6 0.001 BISAP Admission 1 (1–3) 1 (0–1) 1 (1–2) 3 (2–3) <0.001 48 h 2 (1–4) 1 (0–1) 1 (1–3) 4 (4–5) <0.001 SIRS Admission 1 (1–2) 1 (0–1) 2 (1–2) 2 (1–2) 0.045 48 h 1 (0–2) 0 (0–1) 1 (0–2) 3 (2–3) 0.001 MMS Admission 0 (0–1) 0 (0–0) 0 (0–2) 1 (0–2) 0.017 48 h 0 (0–2) 0 (0–0) 0 (0–2) 3 (2–3) <0.001 AP: acu e panc ea i is; BISAP: bedside index o se e i y in acu e panc ea i is; BMI: body mass index; CRP: C- eac i e p o ein; NS: no signi ican ; MMS: modi ied Ma shall sco e; SIRS: sys emic in lamma o y esponse synd ome; WBC: whi e blood cells. Th ough he analysis o Table 2, bo h on admission and 48 h a e onse o symp oms, all se ological bioma ke s excep calcium (p= 0.82), albumin (p= 0.21), and o al p o eins (p= 0.34) on admission show signi ican changes be ween he di e en deg ees o AP se e i y. All he mul i ac o ial sco es show signi ican changes be ween he deg ees o se e i y o AP. On admission, PCT (0.83), BISAP (0.83), and SIRI (0.82) had he highes p edic i e alues o se e e AP. Rega ding mo ali y, BISAP (0.95), SIRI (0.85), and PCR (0.85) had he highes p edic i e alues. Fo y-eigh hou s a e onse o symp oms, o se e e AP, he se ological ma ke wi h he mos signi ican p edic i e powe was hepcidin (1.00) ollowed by BISAP (0.98), CRP (0.94), and SIRI (0.87). When analyzing he p edic i e powe ela ed o mo ali y, BISAP (0.94) was ound o ha e he highes p edic i e alue ollowed by hepcidin (0.90), CRP (0.88), and SIRI (0.86) as ep esen ed in Figu e 1. 3.2. Pa ien s wi h Bilia y AP P esen ed Inc eased Le els o Plasma OS-Rela ed Ma ke s and Lowe CAT and SOD Ac i i ies Than Heal hy Subjec s and Inc eased Le els o Lipid Pe oxida ion and Lowe Le els o CAT and SOD in Se e e Bilia y AP As ep esen ed in Figu e 2A, a signi ican inc ease on admission was e i ied ela ed o he le els o lipid pe oxida ion (p< 0.05), p o ein ni a ion (p< 0.001), and p o ein ca bonyla ion (p< 0.05) in AP pa ien s when compa ed wi h heal hy indi iduals. In addi ion, as shown in Figu e 2B, he e was a signi ican inc ease in lipid pe oxida ion when compa ed o mild and se e e AP (p< 0.05) and mode a ely se e e and se e e AP (p< 0.05). A endency o inc eased p o ein ni a ion le els in se e e AP and in se e e o ms o AP in p o ein ca bonyla ion, al hough wi hou s a is ical signi icance, was also demons a ed. An ioxidan s 2021,10, 988 7 o 18 An ioxidan s 2021, 10, x FOR PEER REVIEW 7 o 19 Figu e 1. Recei e ope a ing cha ac e is ics (ROC) cu es o de e mining he cu -o alues o WBC, SIRI, PCT, CRP, hepcidin, BISAP, SIRS, and MMS o (a) se e e AP and (b) mo ali y on admission and (c) se e e AP and (d) mo ali y 48 h a e onse o symp oms. AP: acu e panc ea i is; BISAP: bedside index o se e i y in acu e panc ea i is; BMI: body mass index; CRP: C- eac i e p o ein; MMS: modi ied Ma shall sco e; SIRS: sys emic in lamma o y esponse synd ome; WBC: whi e blood cells. 3.2. Pa ien s wi h Bilia y AP P esen ed Inc eased Le els o Plasma OS-Rela ed Ma ke s and Lowe CAT and SOD Ac i i ies Than Heal hy Subjec s and Inc eased Le els o Lipid Pe oxida ion and Lowe Le els o CAT and SOD in Se e e Bilia y AP As ep esen ed in Figu e 2A, a signi ican inc ease on admission was e i ied ela ed o he le els o lipid pe oxida ion (p < 0.05), p o ein ni a ion (p < 0.001), and p o ein ca bonyla ion (p < 0.05) in AP pa ien s when compa ed wi h heal hy indi iduals. In addi ion, as shown in Figu e 2B, he e was a signi ican inc ease in lipid pe oxida ion when compa ed o mild and se e e AP (p < 0.05) and mode a ely se e e and se e e AP (p < 0.05). A endency o inc eased p o ein ni a ion le els in se e e AP and in se e e o ms o AP in p o ein ca bonyla ion, al hough wi hou s a is ical signi icance, was also demons a ed. Figu e 1. Recei e ope a ing cha ac e is ics (ROC) cu es o de e mining he cu -o alues o WBC, SIRI, PCT, CRP, hepcidin, BISAP, SIRS, and MMS o ( a ) se e e AP and ( b ) mo ali y on admission and ( c ) se e e AP and ( d ) mo ali y 48 h a e onse o symp oms. AP: acu e panc ea i is; BISAP: bedside index o se e i y in acu e panc ea i is; BMI: body mass index; CRP: C- eac i e p o ein; MMS: modi ied Ma shall sco e; SIRS: sys emic in lamma o y esponse synd ome; WBC: whi e blood cells. An ioxidan s 2021, 10, x FOR PEER REVIEW 8 o 19 Figu e 2. Plasma le els o OS- ela ed ma ke s o acu e panc ea i is (AP) and heal hy con ol g oups. (A.1) 4-HNE (lipid pe oxida ion), (A.2) 3-NO (p o ein ni a ion), (A.3) DNP (p o ein ca bonyla ion) in he con ol g oup and bilia y AP pa ien s. (B.1) 4-HNE (lipid pe oxida ion), (B.2) 3-NO (p o ein ni a ion), (B.3) DNP (p o ein ca bonyla ion) in he di e en deg ees o bilia y AP se e i y on admission. AP: acu e panc ea i is; 3-NO: 3-ni o y osine; 4-HNE: 4-hyd oxynonenal; DNP: 2,4- dini ophenyl-hyd azone. Resul s a e exp essed as mean ± SEM. The di e ences e sus con ol a e ma ked by *, whe e * ep esen s p < 0.05 and *** p < 0.001. Mann–Whi ney es : (A.1–A.3). Compa ison o Fishe ’s leas signi ican di e ence pos hoc es : (B.1–B.3). The ac i i y o enzymes ela ed o he an ioxidan sys em such as GR, GPx, CAT, and SOD we e e alua ed. As shown in Figu e 3A, a endency owa d an inc ease in GR ac i i y was de ec ed in he plasma o AP pa ien s compa ed o he heal hy con ol indi iduals bu wi hou s a is ical signi icance. CAT and SOD p esen ed wi h a signi ican dec ease in AP pa ien s’ ac i i ies compa ed o he heal hy con ol g oup (p < 0.01 and p < 0.001, espec i ely). Figu e 2. Plasma le els o OS- ela ed ma ke s o acu e panc ea i is (AP) and heal hy con ol g oups. ( A.1 ) 4-HNE (lipid pe oxida ion), ( A.2 ) 3-NO (p o ein ni a ion), ( A.3 ) DNP (p o ein ca bonyla ion) in he con ol g oup and bilia y AP pa ien s. ( B.1 ) 4-HNE (lipid pe oxida ion), ( B.2 ) 3-NO (p o ein ni a ion), ( B.3 ) DNP (p o ein ca bonyla ion) in he di e en deg ees o bilia y AP se e i y on admission. AP: acu e panc ea i is; 3-NO: 3-ni o y osine; 4-HNE: 4-hyd oxynonenal; DNP: 2,4- dini ophenyl-hyd azone. Resul s a e exp essed as mean ± SEM. The di e ences e sus con ol a e ma ked by *, whe e * ep esen s p< 0.05 and *** p< 0.001. Mann–Whi ney es : ( A.1 – A.3 ). Compa ison o Fishe ’s leas signi ican di e ence pos hoc es : (B.1–B.3). An ioxidan s 2021,10, 988 8 o 18 The ac i i y o enzymes ela ed o he an ioxidan sys em such as GR, GPx, CAT, and SOD we e e alua ed. As shown in Figu e 3A, a endency owa d an inc ease in GR ac i i y was de ec ed in he plasma o AP pa ien s compa ed o he heal hy con ol indi iduals bu wi hou s a is ical signi icance. CAT and SOD p esen ed wi h a signi ican dec ease in AP pa ien s’ ac i i ies compa ed o he heal hy con ol g oup (p< 0.01 and p< 0.001, espec i ely). An ioxidan s 2021, 10, x FOR PEER REVIEW 9 o 19 Figu e 3. The ac i i y o an ioxidan enzymes in plasma o acu e panc ea i is (AP) and heal hy con ol g oups. GR: glu a hione educ ase; GPx: glu a hione pe oxidase; CAT: ca alase; SOD: supe oxide dismu ase om he heal hy con ol g oup and bilia y AP pa ien s (A) and in he di e en deg ees o bilia y AP pa ien s (B). Resul s a e exp essed as mean ± SEM. The di e ences e sus con ol a e ma ked by *, whe e * ep esen s p < 0.05, ** p < 0.01 and *** p < 0.001. Mann– Whi ney es : (A.1–A.4). Compa ison o Fishe ’s leas signi ican di e ence pos hoc es : (B.1–B.4). Acco ding o he se e i y, as shown in Figu e 3B, when analyzing he ac i i y o he an ioxidan enzymes in he plasma o pa ien s wi h di e en deg ees, he e was a s a is ically signi ican dec ease in CAT ac i i y in pa ien s wi h se e e AP compa ed wi h mild bilia y AP (p < 0.05). Plasma SOD ac i i y was also signi ican ly dec eased when compa ing pa ien s wi h mild and mode a ely se e e bilia y AP and be ween mild and se e e bilia y AP pa ien s (p < 0.001, o bo h). Figu e 3. The ac i i y o an ioxidan enzymes in plasma o acu e panc ea i is (AP) and heal hy con ol g oups. GR: glu a hione educ ase; GPx: glu a hione pe oxidase; CAT: ca alase; SOD: supe oxide dismu ase om he heal hy con ol g oup and bilia y AP pa ien s ( A ) and in he di e en deg ees o bilia y AP pa ien s ( B ). Resul s a e exp essed as mean ± SEM. The di e ences e sus con ol a e ma ked by *, whe e * ep esen s p< 0.05, ** p< 0.01 and *** p< 0.001. Mann–Whi ney es : ( A.1 – A.4 ). Compa ison o Fishe ’s leas signi ican di e ence pos hoc es : (B.1–B.4). Acco ding o he se e i y, as shown in Figu e 3B, when analyzing he ac i i y o he an ioxidan enzymes in he plasma o pa ien s wi h di e en deg ees, he e was a An ioxidan s 2021,10, 988 9 o 18 s a is ically signi ican dec ease in CAT ac i i y in pa ien s wi h se e e AP compa ed wi h mild bilia y AP (p< 0.05). Plasma SOD ac i i y was also signi ican ly dec eased when compa ing pa ien s wi h mild and mode a ely se e e bilia y AP and be ween mild and se e e bilia y AP pa ien s (p< 0.001, o bo h). 3.3. NMR-Based Me abonomics Analysis o Plasma Samples om AP Pa ien s To analyze he changes in plasma me abolome ela ed o panc ea ic in lamma ion, we applied a non a ge ed mul i a ia e analysis. Bo h explo a o y unsupe ised me hod PCA and supe ised me hod PLS-DA sco es sca e plo s o all se e i y deg ees and analyzed a he same ime e ealed he signi ican in luence o he in lamma o y p ocess on plasma me abolome composi ion, which can be obse ed in clus e ing ends o indi idual bilia y AP g oups. In Figu e 4a, PCA was applied o es he in e nal da a s uc u e and sea ch o clus e - ing ends, possible g oup sepa a ion, and ou lie s. Ob ained da a demons a ed clus e ing acco ding o es ed g oups. O e lap was obse ed be ween con ols and mild bilia y AP and mode a ely se e e and se e e bilia y AP, indica ing simila i ies o me abolomes. Addi ionally, da a we e analyzed by PLS-DA, as ep esen ed in Figu e 4b, which is used o maximize he di e ence be ween g oups. An ioxidan s 2021, 10, x FOR PEER REVIEW 10 o 19 3.3. NMR-Based Me abonomics Analysis o Plasma Samples om AP Pa ien s To analyze he changes in plasma me abolome ela ed o panc ea ic in lamma ion, we applied a non a ge ed mul i a ia e analysis. Bo h explo a o y unsupe ised me hod PCA and supe ised me hod PLS-DA sco es sca e plo s o all se e i y deg ees and analyzed a he same ime e ealed he signi ican in luence o he in lamma o y p ocess on plasma me abolome composi ion, which can be obse ed in clus e ing ends o indi idual bilia y AP g oups. In Figu e 4a, PCA was applied o es he in e nal da a s uc u e and sea ch o clus e ing ends, possible g oup sepa a ion, and ou lie s. Ob ained da a demons a ed clus e ing acco ding o es ed g oups. O e lap was obse ed be ween con ols and mild bilia y AP and mode a ely se e e and se e e bilia y AP, indica ing simila i ies o me abolomes. Addi ionally, da a we e analyzed by PLS-DA, as ep esen ed in Figu e 4b, which is used o maximize he di e ence be ween g oups. Figu e 4. Sco es sca e plo s ob ained ia PCA (a) and PLS-DA (b) o 1D cpmg spec a o plasma om heal hy con ol g oup and bilia y AP pa ien s. Deg ees o bilia y AP se e i y a e indica ed in he legends abo e he plo s. Pai -wise compa isons be ween bilia y AP se e i y deg ees and he heal hy con ol g oup we e pe o med (PCA and PLS-DA) o iden i y he me aboli es con ibu ing o g oup sepa a ion. In PCA sco es, sca e plo clus e ing acco ding o es ed g oups and p ecise sepa a ion be ween he g oups can be obse ed in Figu e 5a. PLS-DA analysis u he con i med he g oups’ sepa a ions. A se en old in e nal c oss- alida ion was applied o assess he explained a iance (R2) and p edica i e powe (Q2) o PLS-DA models, as ep esen ed in Figu e 5b. Al hough he in e nal alida ion yielded sa is ac o y model pa ame e s, addi ional alida ion was pe o med ia he pe mu a ion es . The PLS-DA quali y assessmen pa ame e s, goodness o i o he da a R2, and p edic i e alue o he model Q2 we e >0.8 in all he cons uc ed models and co obo a e model eliabili y and hei sui abili y o da a mining, as ep esen ed in Figu e 5c. The esul s can also be p esen ed as a hea map (Figu e 6) o biologically ele an e ec sizes (ES > 0.7) [44] conce ning he esul s a ained in he plasma o he g oup o pa ien s wi h bilia y AP se e i y (mild, mode a ely se e e, o se e e) when compa ed wi h he heal hy con ol g oup. This ep esen a ion shows ha plasma aline le els had an in e io di e ence be ween heal hy and mild bilia y AP, bu i was g adually inc eased om mode a ely se e e and se e e AP. The same happens o isoleucine, h eonine, his idine, and lipids con en s. When phenylalanine was analyzed, he e was a ma ked inc ease in heal hy and se e e AP le els. In u n, mala e showed a ma ked inc ease be ween heal hy and mode a ely se e e AP pa ien s. Figu e 4. Sco es sca e plo s ob ained ia PCA ( a ) and PLS-DA ( b ) o 1D cpmg spec a o plasma om heal hy con ol g oup and bilia y AP pa ien s. Deg ees o bilia y AP se e i y a e indica ed in he legends abo e he plo s. Pai -wise compa isons be ween bilia y AP se e i y deg ees and he heal hy con ol g oup we e pe o med (PCA and PLS-DA) o iden i y he me aboli es con ibu ing o g oup sepa a ion. In PCA sco es, sca e plo clus e ing acco ding o es ed g oups and p ecise sepa a ion be ween he g oups can be obse ed in Figu e 5a. PLS-DA analysis u he con i med he g oups’ sepa a ions. A se en old in e nal c oss- alida ion was applied o assess he explained a iance (R 2 ) and p edica i e powe (Q 2 ) o PLS-DA models, as ep esen ed in Figu e 5b. Al hough he in e nal alida ion yielded sa is ac o y model pa ame e s, addi ional alida ion was pe o med ia he pe mu a ion es . The PLS-DA quali y assessmen pa ame e s, goodness o i o he da a R 2 , and p edic i e alue o he model Q 2 we e >0.8 in all he cons uc ed models and co obo a e model eliabili y and hei sui abili y o da a mining, as ep esen ed in Figu e 5c. The esul s can also be p esen ed as a hea map (Figu e 6) o biologically ele an e ec sizes (ES > 0.7) [ 44 ] conce ning he esul s a ained in he plasma o he g oup o pa ien s wi h bilia y AP se e i y (mild, mode a ely se e e, o se e e) when compa ed wi h he heal hy con ol g oup. This ep esen a ion shows ha plasma aline le els had an in e io di e ence be ween heal hy and mild bilia y AP, bu i was g adually inc eased om mode a ely se e e and se e e AP. The same happens o isoleucine, h eonine, his idine, and lipids con en s. When phenylalanine was analyzed, he e was a ma ked inc ease in heal hy and se e e AP le els. In u n, mala e showed a ma ked inc ease be ween heal hy and mode a ely se e e AP pa ien s. An ioxidan s 2021,10, 988 16 o 18 Re e ences 1. 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