Hyperammonemia induced oxidative stress in cirrhotic rats without promoting differential protein expression in the brain cortex: A 2D-DIGE analysis. In press

Ad ances in Bioscience and Bio echnology, 2012, 3, 1116-1123 ABB h p://dx.doi.o g/10.4236/abb.2012.38137 Published Online Decembe 2012 (h p://www.SciRP.o g/jou nal/abb/) Hype ammonemia induced oxida i e s ess in ci ho ic a s wi hou p omo ing di e en ial p o ein exp ession in he b ain co ex: A 2D-DIGE analysis* Pila Ca bone o-Aguila 1, Mª del Ma Diaz-He e o1,2, José A. del Campo2, Olga C emades1, Manuel Rome o-Gómez2, Juan Bau is a1# 1Depa amen o de Bioquímica y Biología Molecula , Facul ad de Fa macia, Uni e sidad de Se illa, Se illa, Spain 2UCM Diges i e Diseases and CIBERehd, Hospi al Uni e si a io de Valme, Se illa, Spain Email: #[email p o ec ed] Recei ed 20 Sep embe 2012; e ised 27 Oc obe 2012; accep ed 19 No embe 2012 ABSTRACT Oxida i e s ess induced by a high ammonia concen- a ion could modi y p o ein exp ession in he b ain. This s udy was unde aken in o de o in es iga e he impac o hype ammonemia, caused by hioace mide (TAA) in a s, on b ain co ex p o ein exp ession us- ing 2D-DIGE, and analyzing i s ole in he pa ho- genesis o HE. Hype ammonemia was induced wi h TAA. Ammonia and ac i e oxidan s we e measu ed by L-glu ama e dehyd ogenase and dichlo odihyd o- luo escein diace a e me hods, espec i ely. Lipid pe - oxida ion and p o ein oxida ion bioma ke s we e also s udied. Di e en ial p o ein exp ession in he co ex o TAA- and con ol- a s was s udied by 2D-DIGE. Image analysis was pe o med using he DeCyde ™ So wa e. Ammonia concen a ion in plasma and b ain issue was highe in TAA- a s compa ed o con ol- a s, 3.12 and 2.43 old highe , espec i ely. Ac i e oxidan s p oduc ion in TAA- a s was inc eased by 2.7 old compa ed o con ol- a s. Measu emen s o MDA, HNE and ca bonyl g oups, bioma ke s o lipid pe oxida ion and p o ein oxida ion espec i ely, we e ound o be s a is ically signi ican ly inc eased in TAA- a s compa ed o con ol- a s (3.16-, 2.44- and 1.95- old, espec i ely), e lec ing he p esence o oxida i e s ess in he b ain o TAA- a s. 2D-DIGE analysis o b ain co ex p o ein allowed he de ec ion o 2896 spo s, howe e , image analysis showed no s a is ically signi ican di e en ial p o ein exp ession be ween he p o eins exp essed in TAA- and con- ol- a s. No s a is ical signi ican di e en ial p o ein exp ession in he co ex o TAA- a s was obse ed, al hough oxida i e s ess bioma ke s o lipid and p o eins we e highe in he b ain o TAA- a s han in con ol- a s. These esul s suppo he idea ha oxi- da i e pos - ansla ional modi ica ions a e implica ed in HE physiopa hology. Keywo ds: Oxida i e S ess; Hype ammonemia; Ci hosis; Hepa ic Encephalopa hy; P o ein Exp ession 1. INTRODUCTION Hepa ic encephalopa hy (HE) is he majo neu ologic diso de occu ing in pa ien s wi h se e e li e disease which p esen s in ch onic and acu e o ms. Ch onic HE is a neu opsychia ic diso de which commonly occu s in he se ing o alcoholic ci hosis and is o en associa ed wi h changes in pe sonali y, al e ed mood, decline in in ellec ual capaci y and abno mal muscle one. Acu e HE (acu e li e ailu e) has a high mo ali y a e (80% - 90%) due o he de elopmen o b ain edema and in- c eased in ac anial p essu e, and o en p esen s wi h he ab up onse o deli ium, seizu es and coma [1]. Al- hough he pa hophysiology o HE emains unclea , i is well es ablished ha ammonia plays a majo ole [2,3]. Ammonia accumula es in he b ain in bo h acu e and ch onic li e ailu e, leading o a se ies o me abolic, ene ge ic and signal ansduc ion- ela ed al e a ions [4]. Howe e , i appea s ha o he ac o s may also con ib- u e. In pa icula , mic oglial ac i a ion and b ain in- lamma ion co ela e wi h he p og ession o encephalo- pa hy and he onse o b ain edema in a s wi h acu e li e ailu e [5]. Mo eo e , inc eased exp ession o he endo helial and inducible iso o ms o ni ic oxide syn- hase as well as heme oxygenase-1 [6] and di ec oxida- i e bioma ke s [7,8] ha e been epo ed in animal mod- els, sugges ing ha oxida i e/ni osa i e s ess also plays a signi ican ole in he pa hogenesis o b ain complica- ions obse ed in li e ailu e, and pa icula ly in HE. *This wo k was suppo ed by a g an (Accion in eg ada: DE2009-0098) o he minis y o Inno ación y Ciencia o Spain. #Co esponding au ho . All hese da a indica e ha oxida i e s ess is impli- OPEN ACCESS P. Ca bone o-Aguila e al. / Ad ances in Bioscience and Bio echnology 3 (2012) 1116-1123 1117 ca ed in he de elopmen o HE, bu hey a e no able o gi e any p ecise in o ma ion abou he mechanisms and p o eins/enzymes in ol ed in he physiopa hology o HE. The e o e, he aim o he p esen wo k was o s udy he e ec o oxida i e s ess, induced by high ammonia con- cen a ion, on a b ain p o eins, using an animal model o HE. As a model o ch onic HE, TAA- a s a e o en used because hey allow s udying longe exposi ion o high ammonia concen a ions han he PCS- a model [9]. In an a emp o gain knowledge on he pa hogenesis o ch onic HE, di e en ial p o eins exp ession was s udied using a wo dimensional di e en ial in gel elec opho e- sis (2D-DIGE) app oach, which allowed i s iden i ica ion and o s udy i s ela ion wi h HE. 2. MATERIALS AND METHODS 2.1. Chemicals Chemicals, p o eases, and an ibodies used in hese s ud- ies we e mainly pu chased om Sigma-Ald ich (Mad id, Spain). Elec opho esis and DIGE ma e ials we e pu - chased om GE Heal hca e (Ba celona, Spain). Supe - Signal Wes Pico Chemiluminiscen Subs a e was pu - chased om The mo Scien i ic Inc. (Rock o d, USA). 2.2. Animals Male Wis a a s (n = 20) weighing 230 - 260 g a he beginning o he expe imen s, we e used in he p esen s udy. Animals we e supplied by he Cen e o P oduc- ion and Expe imen al animal o Espa inas, Uni e si y o Se ille, Spain. The animals we e housed ou pe cage, and main ained a cons an oom empe a u e (22˚C ± 2˚C), wi h a i icial ligh -da k cycle o 12 h and a ela i e humidi y o 65% - 70%, and ee access o ood and wa- e du ing 2 weeks o acclima ion. All animal p oce- du es we e pe o med wi h app o al om he Se illa Uni e si y Animal E hics Commi ees, unde he guide- lines o he di ec i e 86/609/CEE o he Eu opean Com- muni y. 2.3. Animal Model o HE (Thioace amide T ea men ) A e he 2 weeks o acclima ion, he animals we e di- ided in o wo g oups, and wo animals pe cage: con ol g oup (n = 10) and hioace amide (TAA) ea ed g oup (TAA- a s, n = 10). The con ol- a s ecei ed ood and no mal wa e and se ed as a con ol o 16 weeks. The TAA- a s ecei ed ood and con inuous adminis a ion o 0.03% TAA (Sigma, Mad id, Spain) in he d inking wa- e as an ini ial concen a ion ha was subsequen ly modi- ied acco ding o weigh changes in esponse o TAA du ing he induc ion o ci hosis. The ea men was main ained du ing 12 weeks [10]. A he 12 h week he TAA- a s g oup had 8 animals (n = 8) and he con ol- a g oup had 10 animals (n = 10). Animals we e weighed weekly. Animals we e killed by an o e dose o pen o- ba bi al and b ains apidly emo ed. Di e en b ain loci (co ex, ce ebellum, hippocampus) we e dissec ed, o- zen wi h liquid N2, and s o ed a –80˚C un il use. In his s udy he co ex was used. 2.4. Sample P epa a ion o Analy ical P ocedu es B ain samples (co ex) (100 mg) we e homogenized in 1 ml homogeniza ion bu e (10 mM Hepes, 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 0.6 mM MgSO4, 0.5 μg/ml leupep in, 0.7 μg/ml peps a in, 0.5 μg/ml ype II S soybean ypsin inhibi o , 40 μg/ml PMSF), using a man- ual glass homogenize (~50 passes). Homogena es we e o exed and sonica ed o 10 s a 20% powe in a soni- ca ion ba h (B aun, Be lin, Ge many), and cen i uge a 2500 × g in a mic o uge (Mik o 200R, He ich, Tübingen, Ge many) o emo e in ac cells and nuclei. P o ein con- cen a ions we e de e mined acco ding o he B ad o d me hod [11]. 2.5. Measu emen o Ammonia Concen a ion Ammonia concen a ions we e measu ed in a e ial plasma and b ain issue using he L-glu ama e dehyd ogenase me hod [12]. Plasma concen a ions o ammonia we e measu ed be o e he TAA- ea men and a he ime o killing. Ammonia concen a ion was calcula ed and ex- p essed as μmol/l. B ain issue was homogenized as de- sc ibed abo e, and dep o einized in 5 olumes o ice- cold ichlo oace ic acid (TCA) (100 g/l), and kep on ice o 15 min. A e cen i uga ion a 12,000 × g o 10 min a 4˚C, he supe na an was collec ed, neu alized wi h 2 M KHCO3 and cen i uged a 12,000 × g o 10 min a 4˚C. The neu alized supe na an was used o measu e ammonia by he glu ama e dehyd ogenase me hod as desc ibed abo e. 2.6. Measu emen o Ac i e Oxidan s P oduc ion B ain ac i e oxidan s p oduc ion was measu ed in b ain homogena es ob ained as desc ibed abo e by he Tho - bu ne and Juu link me hod [13] using 5-(and-6)-ca boxy- 20,70-dichlo odihyd o luo escein diace a e (DCFDA), wi h mino modi ica ions [14]. 2.7. Measu emen o Low Molecula Dialdehydes Malondialdehyde (MDA) was de e mined by HPLC ac- co ding o Ma eos e al. [15] as desc ibed in [8]. 4-Hy- d oxy-2-noneal (HNE) was de e mined acco ding o s an- da d immunode ec ion p ocedu es [16] as desc ibed in [8]. Copy igh © 2012 SciRes. OPEN ACCESS P. Ca bone o-Aguila e al. / Ad ances in Bioscience and Bio echnology 3 (2012) 1116-1123 1118 2.8. Measu emen o Ca bonyl G oups The o al p o ein ca bonyl le els in homogena es om b ain issue (co ex) ob ained in homogeniza ion bu e , as desc ibed abo e, o TAA- and con ol- a s we e as- sayed ollowing he E ans me hod [17] as desc ibed in [8]. 2.9. Sample P epa a ion o Elec opho e ic Sepa a ions Co ex samples (in con ol o TAA- ea ed a s) we e made powde wi h liquid N2 and kep a –80˚C. Ap- p oxima ely 10 mg o powde we e mixed wi h 100 µl o lysis bu e (30 mM T is-Cl pH 8.5, 2 M Thiou ea, 7 M U ea, 4% (w/ ) Chap). The homogena e we e le on ice 10 minu es and sonica ed 5 min. The homogena es we e cen i uged in QIAsh edde (Qiagen, Hilden, Ge many) o elimina e he DNA and hen cen i uged 15 min a 18,000 g a 4˚C in a mic o uge (Mik o 200 R, He ich, Tübingen, Ge many). The supe na an was aken and p o ein con en was de e mined acco ding o he me hod o B ad o d [11]. 2.10. Con en ional 2D-Elec opho esis P o ein samples (150 µg) we e loaded on o IPG s ips (pH 4 - 7, 13 cm) p e iously hyd a ed o e -nigh wi h ehyd a ion bu e (2 M Thiou ea, 7 M U ea, 4% (w/ ) Chaps, 0.2% IPG Bu e pH 3 - 10, 0.2% (w/ ). The i s dimension was pe o med using IPGpho 3 IEF sys em (GE Heal hca e, Buckinghamshi e, UK) a 20˚C. The condi ions o he IEF we e pe o med acco ding o he manu ac u e ’s ins uc ions (300 V du ing 3 hou s, g a- dien o 1000 V du ing 3 h, g adien o 8000 V du ing 6 h, 8000 V du ing 6 h). The ocused s ips we e equilib a ed in equilib a ion bu e (50 mM T is-Cl, 6 M U ea, 30% ( / ) glyce ol, 2% (w/ ) SDS) wi h 1% (w/ ) di hio- h ei ol (DTT) o 15 min and ans e ed o 2.5% (w/ ) iodoace amide o 15 min. The SDS-polyac ilamide gel elec opho esis (SDS-PAGE) was un on 12% T and 2.7% C, 20˚C, 450 V, 10 W, 15 mA/gel 10 min, 25 mA/gel 2 h using a C i e ion Cell uni (Bio-Rad Labo a- o ies Inc., Munich, Ge many). Two gels we e s ained wi h SYPRO-Ruby p o ein gel s aining o isualize o al p o eins and o p o ein iden i ica ion. 2.11. Di e en ial in Gel Elec opho esis 2.11.1. Sample P epa a ion and P o ein Labeling wi h Cydye P o eins samples (100 mg co ex) o con ol and TAA- ea ed a s we e ea ed wi h 2-D Clean-Up Ki (GE Heal hca e) ollowing he manu ac u e ’s ins uc ions. The p o ein pelle was esuspended in lysis bu e (30 mM T is, 7 M u ea, 2 M hiou ea, and 4% (w/ ) CHAPS pH 8) and adjus ed o pH 8. P o ein concen a ion was de e mined using he B ad o d me hod. P io o labeling he p o eins wi h sa u a ion dye, he amoun s o T is (2- ca boxye hyl) phosphine hyd ochlo ide (TCEP; In i o- gen, USA) and dye we e op imized acco ding o he manu ac u e ’s p o ocol. T adi ional h ee-dye sys em was employed o educe a iabili y associa ed wi h he mul iple samples being compa ed [18]. Con ol and TAA- ea ed a samples we e labeled wi h N-hyd oxy succinimidyl es e de i a- i es o he cyanine dyes Cy2, Cy3, and Cy5 ollowing a s anda d p o ocol [19]. 2.11.2. 2-D Gel Elec opho esis Immobilised 24 cm linea pH g adien (IPG) s ips, pH 3-11NL, we e ehyd a ed in ehyd a ion bu e (7 M U ea, 2 M Thiou ea, 4% CHAPS, 0.5% IPG Bu e , 50 mM DTT) o e nigh , acco ding o he s anda d guide- lines. Iso-Elec ic-Focusing (IEF) was pe o med using an IPGpho TM 3 appa a us (GE Heal hca e) o a o al o 40 kV/hou a 20˚C. Equilib a ed IPG s ips we e ans- e ed on o 12.5% uni o m polyac ylamide gels pou ed be ween low luo escence glass pla es. S ips we e o e - laid wi h 0.5% w/ low-mel ing-poin aga ose in unning bu e con aining b omphenol blue. Gels we e un using he E an Dal 12 de ice (GE Heal hca e) a 2.5 W/gel o 30 minu es hen 100 W in o al a 10˚C un il he dye on had un o he bo om o he gels. 2.11.3. Image Acquisi ion and S a is ical Analysis A e second-dimension SDS-PAGE, gels we e scanned using he Typhoon 9400 a iable mode image (GE Heal hca e). Cy2 p o ein maps we e scanned using a 488-nm lase wi h a 530-nm emission il e ; Cy3 was scanned using a 532-nm lase wi h a 605-nm emission il e ; Cy5 was scanned using a 635-nm lase wi h a 695-nm emission il e . Gels we e scanned a 100 μm esolu ion. Image analysis was pe o med using he De- Cyde ™ So wa e e sion 7.0 (GE Heal hca e). Gel- o- gel ma ching o he s anda d p o ein maps om each gel, ollowed by s a is ical analysis o di e en ial p o ein abundance be ween samples, was de e mined using he biological a ia ion analysis module (BVA) o DeCy- de ™. Analysis o a iance (ANOVA) was applied o ma ched spo s and he da a we e il e ed o e ain p o ein spo s wi h p ≤ 0.05 de e mined by 1-way ANOVA and a old-change o ≥1.2. Using he EDA Ve sion 1.0 (ex- ended da a analysis) module wi hin he DeCyde so - wa e (GE Heal hca e), da a analysis was pe o med us- ing p incipal componen s analysis (PCA) on p o eins o in e es [20]. 2.12. S a is ical Analysis Resul s a e exp essed as mean ± SEM. S a is ical analy- Copy igh © 2012 SciRes. OPEN ACCESS P. Ca bone o-Aguila e al. / Ad ances in Bioscience and Bio echnology 3 (2012) 1116-1123 Copy igh © 2012 SciRes. 1119 sis was ca ied ou using S uden ’s es . A alue o p < 0.05 was conside ed signi ican . 3. RESULTS In his wo k we ha e s udied he e ec o oxida i e s ess induced by hype ammonemia in an animal model (TAA- a s) o HE. Ou aim has been he analysis o p o- ein exp ession in he b ain o TAA- a s and con ol- a s, and he co ela ion be ween hese al e a ions and he physiopa hology o HE. OPEN ACCESS p Fi s o all, we ha e con i med ha he animal model used, TAA- a s, was a sui able model o s udy HE-asso- cia ed hype ammonemia. Ra s ea ed wi h TAA showed symp oms o b ain inju y such as le ha gy, lack o spon- aneous mo emen , loss o igh ing e lex, lack o e- sponse o pain; and as shown in Table 1, he le el o ammonia in plasma (303.98 ± 13.56 μmol/l e sus 97.50 ± 9.23 μmol/l) and in b ain co ex (8.39 ± 1.07 μmol/g issue e sus 3.44 ± 0.56 μmol/g issue) we e highe in TAA- a s compa ed wi h con ol- a s. 3.1. Oxida i e S ess Bioma ke s Once i was es ablished ha TAA- a s we e hype am- monemic, and ha i s beha io was ela ed wi h some o he hallma ks o HE and aken in o accoun ha oxida- i e s ess has been implica ed as possible cause o HE [7-9,14], we es ed he hypo hesis ha high ammonia concen a ion could p omo es oxida i e s ess in he b ain o TAA- a s. Fo his pu pose he p oduc ion o ac i e oxidan s (see Figu e 1) and bioma ke s o lipid and p o ein oxida ions (see Table 1) ha e been quan i- ied. Measu emen s o global ac i e oxidan s p oduc ion we e ca ied ou in a he e ogeneous mix u e o neu ons, glial and mic oglial cells om TAA- and sham- a b ain using DCFDA as a p obe. As Figu e 1 shows, ac i e oxidan s p oduc ion in TAA- a s was 2.47 imes highe han ha o he con ol- a s (247% ± 16% e sus 100%, espec i ely). Since DCFDA me hod mainly measu ed eac i e oxygen species (ROS) we also in es iga ed he p oduc ion o eac i e ni ogen species using ni ic oxide (NO) as an app op ia e ma ke o eac i e ni ic species (RNS). The p oduc ion o NO was signi ican ly highe in TAA- a s han in con ol- a s (21.80 ± 1.57 μmol/mg p o ein e sus 14.76 ± 1.26 μmol/mg p o ein, espec- i ely, p < 0.01) (Figu e 2). Measu emen o low molecula dialdehydes (MDA and HNE) and ca bonyl g oups, bioma ke s o lipid pe - oxida ion and p o ein oxida ion, espec i ely, we e ca - ied ou by s anda dized me hods as desc ibed in Ma e i- als and Me hods. Resul s a e shown in Table 1. The oxida ion o lipids has been analyzed using wo di e en me hods: malondialdehyde (MDA) and 4-hyd oxy-2- nonenal (HNE). In bo h cases, we ound highe le els in TAA- a s han in con ol- a s (0.062 ± 0.006 μmol MDA/mg p o ein e sus 0.021 ± 0.004 μmol MDA/mg p o ein, p < 0.01; and 0.018 ± 0.003 μmol HNE/mg p o- ein e sus 0.008 ± 0.002 μmol HNE/mg p o ein, p < 0.01, espec i ely). Table 1. Ammonia concen a ion in plasma and co ex. Lipid pe oxida ion and p o ein oxida ion in he co ex o con ol- and TAA- a s. Con ol- a s TAA- a s Ammonia: Plasma (µmol/l) Co ex (µmol/g issue) 97.50 ± 9.23 3.44 ± 0.56 303.98 ± 13.56 8.39 ± 1.07 <0.01 <0.01 Lipid pe oxida ion: MDA (µmol/mg p o ein) HNE (µmol/mg p o ein) 0.026 ± 0.004 0.016 ± 0.002 0.082 ± 0.010 0.039 ± 0.004 <0.01 <0.01 P o ein oxida ion: C = O (nmol/mg p o ein) 2.72 ± 0.24 5.32 ± 0.47 <0.01 -20 30 80 130 180 230 280 con ol- a s TAA- a s DCF-Fluo escence (%) Figu e 1. Ac i e oxidan s p oduc ion in con ol- and TAA- a s. P. Ca bone o-Aguila e al. / Ad ances in Bioscience and Bio echnology 3 (2012) 1116-1123 1120 0 5 10 15 20 25 con ol‐ a s TAA‐ a s NOp oduc ion(umol/mgp o ein) Figu e 2. NO p oduc ion in con ol and TAA- a s. P o ein oxida ion has been s udied by quan i ica ion o ca bonyl g oups, by 2,4-dini ophenylhid azine (DNPH) me hod, obse ing a highe le el o p o ein oxida ion in TAA- a s compa ed o sham- a s (5.30 ± 0.57 nmol/mg p o ein e sus 2.72 ± 0.24 nmol/mg p o ein, p < 0.01, espec i ely). These esul s show ha hype ammonemia p omo es oxida i e s ess in he b ain o TAA- a s, bu hey do no e eal he mechanisms and p o eins in ol ed in he physiopa hology o HE. 3.2. 2D-DIGE Analysis o Hype ammonemic Ra B ain Co ex P o eins In o de o iden i y p o eins in ol ed in he physiopa- hology o HE, we ha e s udied di e en ial p o ein ex- p ession in b ain co ex om TAA- and con ol- a s. Di e en ial p o ein exp ession pa e n has been analyzed by 2D-DIGE echnique using minimal labeling wi h h ee luo opho es (Cy2, Cy3 and Cy5). Con a y o con en ional p o ein s aining such as Coomassie b illian blue (CBB) o sil e s aining, luo escen agging o he en i e b ain co ex p o eins d as ically inc eases he num- be o de ec able p o ein spo s in s anda d 2-DE. A p e- ious p o eomic s udy o b ain a b ain co ex p o eins using CBB G-250 esul ed in he de ec ion o 636 indi- idual 2-D spo s ( esul s no shown). The luo escen DIGE analysis o b ain co ex p o ein om TAA- and con ol- a s desc ibed he e esul ed in he isualiza ion o 2896 ± 18 spo s (Figu e 3). Hence, he DIGE-based p o eomic p o iling s udy o b ain co ex a p o eins is app oxima ely 4.5- old mo e sensi i e as compa ed o con en ional p o ein labelling echniques. In addi ion, gel- o-gel a ia ions in he 2-D p o ein dis ibu ion pa - e n a e g ea ly educed due o he simul aneous analysis o wo di e en p o eomes and a pooled s anda d on he same slab gel [21-23]. Thus, a ound 3000 di e en ially labelled b ain co ex p o eins can be analyzed pe gel using 2D-DIGE echnology, which is a supe io o con en ional non luo escen me hods. In o de o p e en he in oduc ion o po en ial a i ac s due o subcellula ac iona ion, his s udy employed o al b ain co ex ex- ac s as s a ing ma e ial o ou compa a i e p o eomic p o iling s udy. Consequen ly, he b ain co ex p o eins analyzed he e ep esen s majo soluble p o eins and he e o e does no ake in o accoun po en ial changes in in eg al ecep o s and o he non-soluble componen s. Figu e 3 illus a es he ep esen a i e p o ein spo pa - e n ob ained o a 2D-DIGE expe imen , showing he spo s ob ained o he con ol- a s co ex p o eins (Cy3 exci ed, image A) and he TAA- a s co ex p o eins (Cy5 exci ed, image B), espec i ely. As shown in he supe - posi ion o p o ein spo pa e n ob ained by labeling wi h Cy3 and Cy5 (image C). The analysis o hese esul s by he DeCyde ™ So wa e, e eals ha no s a is ical di - e ences be ween p o ein exp essions in he co ex o TAA- and con ol- a s we e obse ed. 4. DISCUSSION To e i y ha a e 12 weeks TAA- ea ed a s (n = 8) we e hype ammonemic, ammonia concen a ion in plasma and b ain o con ol- and TAA- a s was assessed; ob- aining he ollowing esul s: 96.6 ± 12.3 μmoles/l and 356.6 ± 32.3 μmoles/l, p < 0.01, in plasma and 0.36 ± 0.05 μmoles/g issue and 0.97 ± 0.13 μmoles/g issue, p < 0.01, in he b ain, espec i ely. These esul s show ha ammonia le els in plasma and o al b ain issue we e highe in TAA- a s compa ed o con ol- a s, 3.12- and 2.43- old highe , espec i ely. The p esence o oxida i e s ess in he b ain o 12 weeks-old TAA- a s was con i med by measu emen o global ac i e oxidan s p oduc ion, in a he e ogeneous mix u e o neu ons, glial and mic oglial cells om TAA- and con ol- a b ain. Ac i e oxidan s p oduc ion was measu ed using DCFDA as a p obe. Ac i e oxidan s Copy igh © 2012 SciRes. OPEN ACCESS P. Ca bone o-Aguila e al. / Ad ances in Bioscience and Bio echnology 3 (2012) 1116-1123 1121 pH 4.2 6.9 pH 4.2 6.9 6.9 pH 4.2 MW 14.20 20.10 29.00 24.00 36.00 45.00 65.00 55.00 (a) (b) (c) Figu e 3. 2-D gel images o a co ex labeled wi h minimal CyDye DIGE luo opho es. Con ol- a s sample labeled wi h Cy3, (a); TTA ea ed- a s sample labeled wi h Cy5, (b); Supe posi ion o images A and B, (c). p oduc ion in TAA- a s was 2.7- old highe han con- ol- a s (Figu e 1). The measu emen o low molecula dialdehydes (MDA and HNE) and ca bonyl g oups, bio- ma ke o lipid pe oxida ion and p o ein oxida ion, e- spec i ely, we e ound o be signi ican ly inc eased in TAA- a s compa ed wi h con ol- a s (Table 1). This esul e lec s he p esence and ac ion o oxida i e s ess in he b ain o 12 week-old TAA- a s, whe e lowe en- zyma ic an ioxidan de ense has been desc ibed oo [24, 25]. These esul s ag ee wi h o he s udies ha show ha high ammonia concen a ion exposu e induces oxida i e s ess in a as ocy e cul u es and mouse b ain slices in i o [14,26,27], and in i o in he a b ain [24]. Me abolic, ene ge ic and signal ansduc ion- ela ed al e a ions obse ed in HE and hype ammonemic a s [4] has been ela ed wi h al e ed mo emen s and psycho- in ellec ual ac i i ies. These al e a ions migh be ex- plained in pa by di e en ial exp ession o p o eins and enzymes in he co ex o hype ammonemic a s [28-30], a b ain a ea ela ed wi h psychomo o ic dis u bances. The e o e, we s udy p o ein exp ession in he co ex o TAA- a s, an animal model o ch onic HE, and sham- a s by a p o eomic app oach based on 2D-di e en ial in gel elec opho esis (2D-DIGE). 2D-elec opho esis is an impo an p o eomic ool, whe e housands o p o eins spo s can be isualized, e- sul ing in a global iew o he s a e o a p o eome [31], and compa ing he 2D spo pa e ns om di e en sam- ples (con ol- and TAA- a s co ex, o example), changes in indi idual p o eins abundance can be de ec ed and quan i ied; allowing he iden i ica ion o p o ein ma ke s ha a e cha ac e is ic o a speci ic physiological o pa hological s a e o a cell o a issue [32]. Howe e , a limi a ion on compa a i e 2-D analysis is he high deg ee o gel- o-gel a ia ion in spo pa e ns ha makes i di - icul o dis inguish any ue biological a ia ion om expe imen al a ia ion [33]. A me hod ha minimized hese d awbacks is di e en ial in gel elec opho esis (DIGE), we e samples a e labeled p io o elec opho e- sis wi h spec ally esol able dyes CyTM2, Cy3 and Cy5. Samples a e hen mixed p io o EIF and esol ed on he same gel [21]. The luo opho es a e s uc u ally simila and unde go nucleophilic subs i u ion wi h he ε-amino g oup o lysine esidues o ming an amide. The luo es- cen dyes ha e e y simila molecula masses and a e posi i ely cha ged o mach he cha ge ha is eplaced on he lysine esidue. This cha ge/mass ma ching ensu es ha all he samples essen ially comig a e o he same poin du ing elec opho esis. This ensu es ha , when he dye:p o ein a io is low, p o ein molecules a e only la- belled wi h a single dye molecule [22], e ining g ea ly he de ec ion o changes a he p o ein le el be ween samples. Va ia ion in spo in ensi y due o expe imen al ac o s, such as p o ein loss du ing sample en y in o he s ips, will be he same o each sample wi hin a single DIGE gel. The e o e he ela i e amoun s o a p o ein be ween samples in a gel will be unchanged. Wi h con en ional “one sample pe gel” 2D echniques, samples o be com- pa ed a e sepa a ed independen ly in di e en gels, and consequen ly, spo mig a ion and in ensi y will be di e o each gel and sample in an expe imen adding o he o e all expe imen al a ia ion. The analysis o he spo s (Figu e 3) showed no s a is- ically signi ican di e ences in exp ession among he 2896 esol ed spo s by DIGE me hod in he co ex o TAA- ea ed a s and con ol- a s. Al hough hese esul s clea ly indica e ha no s a is ically signi ican di e - ences (p < 0.05) we e obse ed be ween TAA- ea ed Copy igh © 2012 SciRes. OPEN ACCESS P. Ca bone o-Aguila e al. / Ad ances in Bioscience and Bio echnology 3 (2012) 1116-1123 1122 a s and sham- a s, i should be s a ed ha sys ema ic di e ences o 14 spo s we e obse ed by DIGE s udy. Al hough hese sligh ly di e ences could no be consid- e ed as he main causes o HE, i s ole should also no be excluded. These esul s suppo he idea ha oxida i e pos - ansla ional modi ica ions a e implica ed in HE physiopa- hology, and ha e leaded us o conside he hypo hesis ha he cause o HE lies in pos - ansla ional modi ica- ions a he han in di e en ial exp ession o p o eins. We a e cu en ly wo king on his hypo hesis. REFERENCES [1] Mon o , P., Cauli, O., Mon oliu, C., Rod igo, R., Llansola, M., Pied a i a, B., el Mlili, N., Boix, J., Agus í, A. and Felipo, V. (2009) Mechanisms o cogni i e al e a ions in hype ammonemia and hepa ic encephalopa hy: The a- peu ical implica ions. Neu ochemis y In e na ional, 55, 106-112. doi:10.1016/j.neuin .2009.01.021 [2] Bu e wo h, R.F. (2002) Pa hophysiology o hepa ic en- cephalopa hy: A new look a ammonia. Me abolic B ain Disease, 17, 221-227. doi:10.1023/A:1021989230535 [3] Rome o-Gomez, M. (2010) Pha maco he apy o hepa ic encephalopa hy in ci hosis. Expe Opinion on Pha ma- co he apy, 11, 1317-1327. doi:10.1517/14656561003724721 [4] Kosenko, E., Kaminsky, Y. and G au, E. (1994) B ain ATP deple ion induced by acu e ammonia in oxica ion in a s is media ed by ac i a ion o NMDA ecep o s and Na1, K1-ATPase. Jou nal o Neu ochemis y, 63, 2172- 2178. doi:10.1046/j.1471-4159.1994.63062172.x [5] Jiang, W., Desja dins, P. and Bu e wo h, R.F. (2009a) Di ec e idence o cen al p oin lamma o y mechanisms in a s wi h expe imen al acu e li e ailu e: P o ec i e e ec o hypo he mia. Jou nal o Ce eb al Blood Flow & Me abolism, 29, 944-952. doi:10.1038/jcb m.2009.18 [6] Jiang, W., Desja dins, P. and Bu e wo h, R.F. (2009b) Hypo he mia a enua es oxida i e/ni osa i e s ess, en- cephalopa hy and b ain edema in acu e (ischemic) li e ailu e. Neu ochemis y In e na ional, 55, 124-128. doi:10.1016/j.neuin .2009.02.007 [7] No enbe g, M.D., Jayakuma , A.R. and Rama Rao, K.V. (2004) Oxida i e s ess in he pa hogenesis o hepa ic encephalopa hy. Me abolic B ain Disease, 18, 313-329. doi:10.1023/B:MEBR.0000043978.91675.79 [8] Ca bone o-Aguila , P., Diaz-He e o, Mª.M., C emades, O., Rome o Gomez, M. and Bau is a, J. (2011) B ain biomolecules oxida ion in po oca al shun ed a s. Li e In e na ional, 31, 964-969. doi:10.1111/j.1478-3231.2011.02515.x [9] Reddy, P.V.B., Mu hy, C.R.K. and Reddanna, P. (2004) Fulminan hepa ic ailu e induced oxida i e s ess in nonsynap ic mi ochond ia o ce eb al co ex in a s. Neu- oscience Le e s, 368, 15-20. doi:10.1016/j.neule .2004.06.046 [10] Li, X.N., I ing, S.B. and Ba y, A. (2002) Rep oducible p oduc ion o hioace amide-induced mac onodula ci - hosis in he a wi h no mo ali y. Jou nal o Hepa ology, 36, 488-493. doi:10.1016/S0168-8278(02)00011-9 [11] B ad o d, M.M. (1976) Rapid and sensi i e me hod o he quan i a ion o mic og am quan i ies o p o ein u iliz- ing he p inciple o p o ein-dye binding. Analy ical Bio- chememis y, 72, 248-254. [12] Be gmeye , H.U. and Beu le , H-O. (1996) Me hod o enzyma ic analysis, Vol. VIII. 3 d Edi ion, Academic P ess, New Yo k, 454-461. [13] Tho bu ne, S.K. and Juu link, B.H. (1996) Low glu- a hione and high i on go e n he suscep ibili y o oli- godend oglial p ecu so s o oxida i e s ess. Jou nal o Neu ochemis y, 67, 1014-1022. doi:10.1046/j.1471-4159.1996.67031014.x [14] Mu hy, C.R., Rama Rao, K.V., Bai, G. and No enbe g, M.D. (2001) Ammonia-induced p oduc ion o ee adi- cals in p ima y cul u es o a as ocy es. Jou nal o Neu- osciene Resea ch, 66, 282-288. [15] Ma eos, R., Lecumbe i, E., Ramos, S., Goya, L. and B a o, L. (2005) De e mina ion o malondialdehyde (MDA) by high-pe o mance liquid ch oma og aphy in se um and li e as a bioma ke o oxida i e s ess: Ap- plica ion o a a model o hype choles e olemia and e alua ion o he e ec o die s ich in phenolic an ioxi- dan s om ui s. Jou nal o Ch oma og aphy B, 827, 76-82. doi:10.1016/j.jch omb.2005.06.035 [16] Ku isaki, E. and Hi aiwa, K. (2009) Wes e n blo analysis o 4-hyd oxy-2-nonenal (HNE)-modi ied p o eins in pa a- qua - ea ed mice. Legal Medicine, 11, S431-S433. doi:10.1016/j.legalmed.2009.01.082 [17] E ans, P., Ly as, L. and Halliwell, B. (1999) Measu e- men o p o ein ca bonyls in human b ain issue. Me hods in Enzymology, 300, 145-156. doi:10.1016/S0076-6879(99)00122-6 [18] Ka p, N.A. and Lilley, K.S. (2005) Maximising sensi i - i y o de ec ing changes in P o ein exp ession: Expe i- men al design using minimal CyDyes. P o eomics, 5, 3105-3115. doi:10.1002/pmic.200500083 [19] Alban, A., Da id, S.O., Bjo kes en, L., Ande son, C., Sloge, E., Lewis, S. and Cu ie, I. (2003) A no el ex- pe imen al design o compa a i e wo-dimensional gel analysis: Two-dimensional di e ence gel elec opho esis inco po a ing a pooled in e nal s anda d. P o eomics, 3, 36-44. doi:10.1002/pmic.200390006 [20] Pe ez-Pe ez, R., O ega-Delgado, F.J., Ga cía-San os, E., Lopez, J.A., Cama ei a, E., Rica , W., Fe nendez-Real, J.M. and Pe al, B. (2009) Di e en ial p o eomics o omen al and subcu aneous adipose issue e lec s hei unalike biochemical and me abolic p ope ies. Jou nal o P o eome Resea ch, 8, 1682-1693. doi:10.1021/p 800942k [21] Unlu, M., Mo gan, M.E. and Minden, J.S. (1997) Di e - ence gel elec opho esis: A single gel me hod o de ec - ing changes in p o ein ex ac s. Elec opho esis, 18, 2071- 2077. doi:10.1002/elps.1150181133 [22] Tonge, R., Shaw, J., Middle on, B., Rowlinson, R., Rayne , S., Young, J., Pognan, F., Hawkins, E., Cu ie, I. and Da ison, M. (2001) Valida ion and de elopmen o luo- Copy igh © 2012 SciRes. OPEN ACCESS P. Ca bone o-Aguila e al. / Ad ances in Bioscience and Bio echnology 3 (2012) 1116-1123 Copy igh © 2012 SciRes. 1123 OPEN ACCESS escence wo-dimensional di e en ial gel elec opho esis p o eomics echnology. P o eomics, 1, 377-396. doi:10.1002/1615-9861(200103)1:3<377::AID-PROT377 >3.0.CO;2-6 [23] Ma ouga, R., Da id, S. and Hawkins, E. (2005) The de- elopmen o he DIGE sys em: 2D luo escence di e - ence gel analysis echnology. Analy ical and Bioanaly i- cal Chemis y, 382, 669-678. doi:10.1007/s00216-005-3126-3 [24] Kosenko, E., Kaminsky, Y., Kaminsky, A., Valencia, M., Lee, L., He menegildo, C. and Felipo, V. (1997) Supe - oxide p oduc ion and an ioxidan enzymes in ammonia in oxica ion in a s. F ee Radic Resea ch, 27, 637-644. doi:10.3109/10715769709097867 [25] Widme , R., Kaise , B., Engels, M., Jung, T. and G une, T. (2007) Hype ammonemia causes p o ein oxida ion and enhanced p o easomal ac i i y in esponse o mi ochon- d ia-media ed oxida i e s ess in a p ima y as ocy es. A chi es o Biochemis y and Biophysics, 464, 1-11. doi:10.1016/j.abb.2007.03.027 [26] Schliess, F., Gö g, B., Fische , R., Desja dins, P., Bidmon, H.J., He mann, A., Bu e wo h, R.F., Zilles, K. and Häussinge , D. (2002) Ammonia induces MK-801-sensi- i e ni a ion and phospho yla ion o p o ein y osine esidues in a as ocy es. FASEB Jou nal, 16, 739-741. [27] Reineh , R., Gö k, B., Becke , S., Q a skha a, N., Bid- mon, H.J., Selbach, O., Haas, H., Schliess, F. and Häussinge D. (2007) Hypoosmo ic swelling and ammo- nia inc ease oxida i e s ess by NADPH Oxidase in cul- u ed as ocy es and i al b ain slices. Glia, 55, 758-771. doi:10.1002/glia.20504 [28] Cauli, O., Lopez-La ubia, P., Rod igues T.B., Ce dan, S. and Felipo, V. (2007) Magne ic esonance analysis o he e ec s o acu e ammonia in oxica ion on a b ain. Role o NMDA ecep o s. Jou nal o Neu ochemis y, 103, 1334-1343. doi:10.1111/j.1471-4159.2007.04878.x [29] Cauli, O., Mlili, N., Rod igo, R. and Felipo, V. (2007) Hype ammonaemia al e s he mechanisms by which me- abo opic glu ama e ecep o s in nucleus accumbens modula e mo o unc ion. Jou nal Neu ochemis y, 103, 38-46. [30] Cauli, O., Rod igo, R., Pied a i a, B., Boix, J. and Felipo, V. (2007) In lamma ion and hepa ic encephalopa hy: Ibup o en es o es lea ning abili y in a s wi h po aca al shun s. Hepa ology, 46, 514-519. doi:10.1002/hep.21734 [31] Gö g, A., Obe maie , C., Bogu h, G., Ha de , A., Scheibe, B., Wildg ube , R. and Weiss, W. (2000) The cu en s a e o wo-dimensional elec opho esis wi h immobi- lized pH g adien s. Elec opho esis, 21, 1037-1053. doi:10.1002/(SICI)1522-2683(20000401)21:6<1037::AI D-ELPS1037>3.0.CO;2-V [32] F iso, G., Kaise , L., Raud, J. and Wiks öm, L. (2001) Di e en ial p o ein exp ession in a igeminal ganglia du ing in lamma ion. P o eomics, 1, 397-408. doi:10.1002/1615-9861(200103)1:3<397::AID-PROT397 >3.0.CO;2-Z [33] Voss, T. and Habe l, P. (2000) Obse a ions on he e- p oducibili y and ma ching e iciency o wo-dimensional elec opho esis gels: Consequences o comp ehensi e da a analysis. Elec opho esis, 21, 3345-3350. doi:10.1002/1522-2683(20001001)21:16<3345::AID-EL PS3345>3.0.CO;2-Z