Phosphate-activated glutaminase activity is enhanced in brain, intestine and kidneys of rats following portacaval anastomosis

wi h p o ein con en we e signi ican ly highe in PCA g oup han in con ol o sham-ope a ed a s (duodenum PAG ac i i y was 976.95±268.87 µka /g o p o ein in PCA a s s 429.19±126.92 µka /g o p o ein in sham- ope a ed a s; kidneys PAG ac i i y was 1259.18 ± 228.79 µka /g p o ein in PCA a s s 669.67± 400.8 µka /g o p o ein in con ols, P < 0.05; duodenal p o ein con en : 173% in PCA s sham-ope a ed a s; in kidneys he con- en o p o ein was 152% in PCA s sham-ope a ed a s). PAG ac i i y and p o ein exp ession in PCA a s we e highe in co ex and basal ganglia han hose in sham- ope a ed a s (co ex: 6646.6 ± 1870.4 µka /g o p o ein s 3573.8 ± 2037.4 µka /g o p o ein in con ol a s, P < 0.01; basal ganglia, PAG ac i i y was 3657.3 ± 1469.6 μka /g o p o ein in PCA a s s 2271.2 ± 384 μka /g o p o ein in sham ope a ed a s, P < 0.05; In he ce - ebellum, he PAG ac i i y was 2471.6 ± 701.4 μka /g o p o ein s 1452.9 ± 567.8 μka /g o p o ein in he PCA and sham a s, espec i ely, P < 0.05; con en o p o ein: ce eb al co ex: 162% ± 40% s 100% ± 26%, P < 0.009; and basal ganglia: 140% ± 39% s 100% ± 14%, P < 0.05; bu no in ce ebellum: 100% ± 25% s 100% ± 16%, P = ns). CONCLUSION: Inc eased PAG ac i i y in kidney and duodenum could con ibu e significan ly o he hype am- monaemia in PCA a s, animal model o encephalopa hy. PAG is inc eased in non-synap ic mi ochond ia om he co ex and basal ganglia and could be implica ed in he pa hogenesis o hepa ic encephalopa hy. The e o e, PAG could be a possible a ge o he ea men o HE o li e dys unc ion. © 2006 The WJG P ess. All igh s ese ed. Key wo ds: Hype ammonaemia; Minimal hepa ic encephalopa hy; Glu amine; P o ein con en ; Sham- ope a ed Rome o-Gómez M, Jo e M, Díaz-Gómez D, de Te án LC, Rod igo R, Camacho I, Eche a ía M, Felipo V, Bau is a JD. Phospha e-ac i a ed glu aminase ac i i y is enhanced in b ain, in es ine and kidneys o a s ollowing po a- ca al anas omosis. Wo ld J Gas oen e ol 2006; 12(15): 2406-2411 h p://www.wjgne .com/1007-9327/12/2406.asp RAPID COMMUNICATION Phospha e-ac i a ed glu aminase ac i i y is enhanced in b ain, in es ine and kidneys o a s ollowing po aca al anas omosis Manuel Rome o-Gómez, Ma ía Jo e , Daniel Díaz-Gómez, Lau a Collan es de Te án, Regina Rod igo, Inés Camacho, Mi iam Eche a ía, Vicen e Felipo, Juan D Bau is a www.wjgne .com Manuel Rome o-Gómez, Daniel Díaz-Gómez, Uni o he Clinical Managemen o Diges i e Diseases, Hospi al Uni e si- a io de Valme, Se illa, Spain Ma ía Jo e , Lau a Collan es de Te án, Juan D Bau is a, De- pa men o Biochemis y, B oma ology and Toxicology, Facul y o Pha macy, Uni e si y o Se ille, Spain Regina Rod igo, Vicen e Felipo, Labo a o y o Neu obiology, Fundación Valenciana de In es igaciones Biomédicas, Valencia, Spain Inés Camacho, Biochemis y Uni , Hospi al Uni e si a io de Valme, Se illa, Spain Mi iam Eche a ía, Ins i u o de In es igaciones Biomédicas, De- pa amen o Physiology, Uni e si y o Se ille, Spain Suppo ed by unding om he Spanish Minis y o Heal h (g an s # PI040384 and # 03/155-2002) awa ded o he Spanish Ne wo k o Hepa ic Encephalopa hy Resea ch and a g an om PAI (CTS-532) Co espondence o: D . Manuel Rome o-Gómez, Uni o Clinical Managemen o Diges i e Diseases, Hospi al Uni e si a io de Valme, c a Cádiz s/n, 41014 Se ille, Spain. [email p o ec ed] Telephone: +34-95-5015799 Recei ed: 2005-10-25 Accep ed: 2005-11-18 Abs ac AIM: To assess whe he po aca al anas omosis (PCA) in a s a ec s he p o ein exp ession and/o ac i i y o glu aminase in kidneys, in es ines and in h ee b ain a eas o co ex, basal ganglia and ce ebellum and o explain he neu ological al e a ions ound in hepa ic en- cephalopa hy (HE). METHODS: Six een male Wis a a s weighing 250-350 g we e g ouped in o sham-ope a ion con ol ( n = 8) o po aca al shun ( n = 8). Twen y-eigh days a e he p ocedu e, he animals we e sac ificed. The duodenum, kidney and b ain we e emo ed, homogenised and mi- ochond ia we e isola ed. Ammonia was measu ed in b ain and blood. Phospha e-ac i a ed glu aminase (PAG) ac i i y was de e mined by measu ing ammonia p o- duc ion ollowing incuba ion o one hou a 37ć wi h O-ph halaldehyde (OPA) and specific ac i i y exp essed in uni s pe g am o p o ein (µka /g o p o ein). P o ein exp ession was measu ed by immunoblo ing. RESULTS: Duodenal and kidney PAG ac i i ies oge he PO Box 2345, Beijing 100023, China Wo ld J Gas oen e ol 2006 Ap il 21; 12(15):2406-2411 www.wjgne .com GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG Wo ld Jou nal o Gas oen e ology ISSN 1007-9327 [email p o ec ed] © 2006 The WJG P ess. All igh s ese ed. INTRODUCTION Hype ammonaemia plays a majo ole in he pa hogenesis o hepa ic encephalopa hy (HE). Phospha e-ac i a ed glu aminase (PAG) ca alyzes he hyd olysis o glu amine (Gln) o yield glu ama e (Glu), ene gy, nucleo ide syn hesis and ammonia. Two main iso o ms o PAG ha e been desc ibed: he kidney- ype (K-PAG) and he li e ype (L-PAG). The K-PAG has been ound in kidney, b ain and illi en e ocy es, while L-PAG is es ic ed o he li e . Hype ammonaemia is la gely conside ed o be de i ed om u ea b eakdown by in es inal bac e ia, hus mos ea men s ha e been ocussed on educing ammonia p oduc ion om colon bac e ia[1]. Howe e , some da a sugges ha small in es ine plays an impo an ole in ammonia p oduc ion du ing he pa hogenesis o hepa ic encephalopa hy[2]. Indeed, duodenal PAG ac i i y has been ound o be nea ly ou imes highe in ci ho ic pa ien s han in heal hy con ols, and mo eo e i is closely ela ed o minimal hepa ic encephalopa hy[3]. In a s, he highes PAG ac i i y along he leng h o gas oin es inal ac has been measu ed in he small in es ine[4]. Lowe bu s ill subs an ial ac i i y (15% o o al PAG ac i i y) has been ound in la ge in es ine. PAG ac i i y dis ibu ion along he gas oin es inal ac in humans is simila o ha in a s[5]. PAG plays ano he impo an ole a enal le el. Glu amine is il e ed and eabso bed in he p oximal con olu ed ubule whe e i is deamida ed by PAG. Two hi ds o his ammonia p oduc ion is exc e ed in u ine. This p ocess is pH sensi i e and helps o main ain acid-base homeos asis and o exc e e ni ogen[6]. Howe e , PAG ac i i y in kidney emains la gely unexplo ed in po aca al shun ed a s. Dis ibu ion and loca ion o PAG in b ain as well as i s ole in he pa hogenesis o HE, a e widely con o e sial, while PAG ac i i y in po aca al shun ed a s s ill emains unknown. Po aca al anas omosis (PCA; o po aca al shun PCS) in a s is widely accep ed as a model o ype B hepa ic encephalopa hy ha mimics minimal hepa ic encephalopa hy in humans. The aim o his s udy was o assess whe he po aca al anas omosis in a s a ec s he exp ession and/o ac i i y o glu aminase in kidney, in es ines and he as ocy es o h ee b ain a eas (co ex, basal ganglia and ce ebellum) in ol ed in he neu ological al e a ions o hepa ic encephalopa hy. MATERIALS AND METHODS Animal su ge y Six een male Wis a a s (n = 16) weighing 250-350 g we e andomly alloca ed in o sham ope a ion con ols (n = 8) o po aca al shun (n = 8). All animals we e pai ed wo weeks be o e and ou weeks a e ope a ion and kep unde s anda d labo a o y condi ions. Bo h PCA and sham-ope a ed a s we e as ed o e nigh in single wi e- ne loo cages wi h ee access o ap wa e . All animal p ocedu es we e app o ed by ou ins i u ion and me he guidelines o Spain (RD 223 e ased 14 h Ma ch 1998) and Eu opean (Di ec i e 86/609/CEE) Union o ca e and managemen o expe imen al animals. Ra s we e ope a ed unde gene al anaes hesia wi h iso luo ane o a oid li e me abolism o in a enous Rome o-Gómez M e al. Phospha e-ac i a ed glu aminase ac i i y in a s 2407 www.wjgne .com agen s and end- o-side po aca al shun was pe o med as p e iously desc ibed[7]. B iefly, a e middle lapa o omy o isce a ex e io iza ion, in e io ena ca a and po al eins we e exposed, dissec ed and clamped la e ally oge he wi h a Sa insky clamp. Longi udinal incisions in bo h eins and la e al anas omosis wi h unning su u e as usual we e pe o med. Finally, po al unk was ied and cu nex o li e hilum, u ning he po aca al la e al shun in unc ionally e minal. Sham-ope a ion was pe o med ollowing lapa o omy, he in e io ena ca a was isola ed and clamped o 30 s. Sham-ope a ed animals se ed as con ols. De e mina ion o ammonia in b ain and blood Ammonia was measu ed in ce eb al co ex and blood as p e iously desc ibed[8]. Ce eb al co ex was homogenized and dep o einised in 5 olumes o ice-cold 100 g/L ichlo oace ic acid, and kep on ice o 15 min. A e cen i uga ion a 12 000 /min o 10 min a 4ć, he supe na an s we e collec ed, neu alized wi h 2 mol/L KHCO3 and cen i uged a 12 000 /min o 10 min a 4ć. The neu alized supe na an s we e used o measu e ammonia in µmole/g issue. Blood (150 µL) was aken om he ail ein he hi d week a e su ge y. Blood samples we e dep o einized wi h one olume o ice-cold 100 g/L ichlo oace ic acid and kep on ice o 15 min. A e cen i uga ion a 12 000 /min o 10 min a 4ć, he supe na an s we e collec ed, neu alized wi h 2 mol/L KHCO3 and cen i uged a 12 000 /min o 10 min a 4ć. The neu alized supe na an s we e used o measu e ammonia. In a final olume o 100 µL, he eac ion mix- u e con ained 50 µL o 60 µL o sample, 30 mmol/L α-ke oglu a a e, 0.5 mmol/L nico inamide adenine dinu- cleo ide ( educed o m) in po assium phospha e bu e (pH 8.0). A e eco ding o he ini ial fluo escence, eac ions we e s a ed by he addi ion o 5 µg o glu ama e dehyd o- genase (Boeh inge Mannheim, Ge many) and moni o ed by he luo ime e (Fluo oskan Ascen ; Labsys ems; Oy, Helsinki, Finland) o a leas 70 min. S anda ds con aining up o 25 nmol o ammonia we e included in each assay. Assays we e pe o med in Cos a 96-well UV pla es (ca . No. 3635; Co ning Cos a Co po a ion, Camb idge, MA). Animal handling and b ain issue p epa a ion All p ocedu es we e ca ied ou in he cold oom a 2ć - 4ć. The a s we e killed by ce ical disloca ion. The skull o each a was opened o emo e blood om he su ace o he issue and his p ocedu e needed o be pe o med in <30 s. The issue was placed in 5 mL o isola ion medium in a Pe i dish main ained in an ice ba h. The b ain was chopped wi h ine scisso s and he chopped ma e ial was washed equen ly wi h isola ion medium o emo e blood. Meanwhile, he fi s po ion o small in es ine (3 cm o leng h) was emo ed and placed in 5 mL o isola ion medium in Pe i dish main ained in an ice ba h. The duodenum was washed and mucosa was emo ed by glass ilm and quickly ozen in liquid ai . Samples o kidney we e simila ly ob ained. Tissue samples we e hen homogenized manually in 1 mL o isola ion medium pe 100-150 mg o issue using a Dounce www.wjgne .com homogenize i ed wi h a Te lon pes le ha ing a o al clea ance o 0.1 mm. Usually, six up- and down-s okes we e su ficien o gene a e a ough homogena e which was hen dilu ed wi h isola ion medium o a final olume o 1.2 mL and homogenized u he wi h ou up- and down- s okes. P epa a ion o mi ochond ia The p ocedu e o isola ion o b ain mi ochond ia was based on p e iously epo ed me hods[9] excep o 1 mmol/L e hylene glycol-bis (β-aminoe hyl e he ) e a- ace ic acid (EGTA) being used in he homogenisa ion medium ins ead o EDTA. The homogena e was cen i uged a 2 000 /min o 3 min, he pelle was washed wi h 400 μL o homogenisa ion medium and e- cen i uged a 2 000 /min o 3 min. Bo h supe na an s we e pooled and cen i uged o 8 min a 12 000 /min o ob ain he c ude mi ochond ial pelle . The pelle was suspended in 300 μL o he 30 g/L Ficoll medium (see below) and laye ed on o 1.2 mL o 60 g/L Ficoll medium and cen i uged a 12 000 /min o 30 min. The 60 g/L Ficoll medium con ained 6% (w/w) Ficoll, 0.24 mol/L manni ol, 0.06 mol/L suc ose, 0.05 mmol/L K-EDTA and 10 mmol/L T is-HCl, pH 7.4. The 30 g/L Ficoll medium was he 60 g/L Ficoll medium dilu ed 1 : 1 wi h glass e-dis illed wa e . The loose, flu y, whi e uppe laye o he pelle was emo ed, he emaining b own pelle was e-suspended in isola ion medium wi hou EGTA and he suspension was cen i uged a 12 000 /min o 10 min. The pelle was e-suspended in incuba ion medium (isola ion medium wi h 2.5 mL/L bu e con aining p o ease inhibi o , 7 g/L T i on X-100, 5 mmol/L β-me cap o-e hanol) o ob ain a p o ein concen a ion in he ange o 5-10 g/L. A e incuba ion on a mixing wheel o 30 min a 4ć, he samples we e ozen a -80 ć o ba ched ac i i y measu emen . PAG ac i i y assay Mi ochond ial p o ein was measu ed by he me hod o B ad o d e al[10] wi h bo ine se um albumin (BSA) as s anda d. B ie ly, 25 μL o mi ochond ial solu ion was added o 35 μL o eac ion medium (150 mmol/L K2HPO4, pH 8; 171 mmol/L L- GLn; 1 mmol/L NH4Cl; pH 8). A e incuba ion o 60 min he eac ion was s opped wi h 10 μL o 100 g/L ichlo oace ic acid (TCA). Blanks we e p epa ed sepa a ely ollowing he incuba ion o he eac ion medium and samples we e mixed be o e he addi ion o TCA. When he sample- mix u e eac ion was s opped, he eac ion mix u e was placed in ice o 15 min and hen cen i uged a 12 000 /min o 5 min a 4 ć. The mic o- i e pla e was loaded wi h 5 μL o supe na an and 150 μL o OPA eagen (0.2 mol/L K2HPO4, pH 7.4; 56 mL/L e hanol; 10 mmol/L O-ph haldialdehyde; 0.4 mmol/L β-me cap o-e hanol). The pla e was incuba ed in da k a oom empe a u e o 45 min. S anda ds o NH4Cl we e p epa ed o concen a ions o 50-300 mg/L. Abso bance was measu ed a 405 nm wi h a spec opho ome e (R&D Sys em, Palo Al o, USA). Speci ic ac i i ies o enzymes we e exp essed in in e na ional uni s pe g am (μka /mg) o mi ochond ial homogena e p o ein. Analysis o glu aminase p o ein con en by immunoblo ing Samples om sham-ope a ed a s o a s wi h PCS we e homogenized in medium con aining 66 mmol/L T is- HCl (pH 7.4), 10 g/L SDS, 1 mmol/L EGTA, 100 ml/L glyce ol, 1 mmol/L sodium o ho anada e and 1 mmol/L sodium luo ide and he p o ein concen a ion was de e mined by he bicinchonic acid me hod (Pie ce, Rock o d, IL, USA). Samples we e subjec ed o gel elec opho esis and immunoblo ing as p e iously desc ibed[11] using iso o m-speci ic polyclonal an ibodies aised in abbi s agains K- glu aminase p o eins dilu ed a 1 : 1000. A e incuba ion wi h an i- abbi IgG conjuga ed wi h alkaline phospha ase (Sigma, Ge many) and de elopmen wi h alkaline phospha ase colou de elope (Sigma, Ge many), he image was cap u ed using he Gel P in e Plus Sys em (TDI, Mad id, Spain) and he densi ies o he spo s we e measu ed using he In elligen Quan ifie ™ so wa e Ve sion 2.5.0 (BioImage®, Mad id, Spain). Resul s we e ela i ized o he op ical densi y espec o con ols. S a is ical analysis Da a we e exp essed as mean ± SD. S a is ical analyses we e pe o med using he SPSS 11.0 so wa e (spss, Chi- cago, IL). Di e ences in glu aminase ac i i y o p o ein con en we e analysed by S uden - es . P İ 0.05 was conside ed s a is ically significan o all es s applied. RESULTS Ammonia measu emen Ammonia was significan ly highe in PCA g oup han in con ol a s. Plasma ammonia le el was 166 ± 51 µmol/L in PCA a s and 83 ± 12 µmol/L in con ol a s (P < 0.05). In he co ex, b ain ammonia was 0.9±0.4 µmol/g o issue in PCA a s and 0.3 ± 0.1 µmol/g o issue in sham ope a ed a s (P < 0.05). PAG ac i i y in duodenum and kidneys Duodenal and kidney PAG ac i i ies we e signi ican ly highe in PCA g oup han in con ol a s. In he duodenum, PAG ac i i y was 976.95 ± 268.87 μka /g o p o ein in PCA a s and 429.19 ± 126.92 μka /g o p o ein in sham-ope a ed a s (P < 0.05). In mi ochond ia om kidneys, PAG ac i i y was 1259.18 ± 228.79 μka /g p o ein in PCA a s and 669.67 ± 400.8 μka /g o p o ein in con ols (P < 0.05). Glu aminase p o ein con en in in es ine and kidneys The e was a signi ican e ec o PCA on PAG p o ein in duodenum. The con en o p o ein was 173% in PCA compa ed o sham-ope a ed a s and in kidneys he con- en o p o ein was 152% in PCA compa ed o sham- ope a ed a s (Figu e 1). PAG ac i i y in b ain The PAG ac i i y was 6646.6 ± 1870.4 μka /g o p o ein in he co ex o PCA a s and 3573.8 ± 2037.4 μka /g o p o ein in ha o con ol a s (P < 0.01). The PAG ac i i y was 3657.3 ± 1469.6 μka /g o p o ein in basal ganglia 2408 ISSN 1007-9327 CN 14-1219/ R Wo ld J Gas oen e ol Ap il 21, 2006 Volume 12 Numbe 15 o PCA a s and 2271.2 ± 384 μka /g o p o ein in ha o sham-ope a ed a s (P < 0.05). In he ce ebellum, he PAG ac i i y was 2471.6 ± 701.4 μka /g o p o ein and 1452.9 ± 567.8 μka /g o p o ein in he PCA and sham- ope a ed a s, espec i ely (P < 0.05). In PCA a s, he PAG ac i i y was inc eased up o 186% in co ex, 161% in basal ganglia and 170% in ce ebellum compa ed o sham- ope a ed a s. The highes ac i i y was ound in he co ex (Figu e 2). Glu aminase p o ein con en in ce eb al co ex and basal ganglia The con en o glu aminase was significan ly inc eased in ce eb al co ex o PCA a s compa ed o sham-ope a ed animals (162% ± 40% and 100% ± 26%; P < 0.009) and he con en o glu aminase was significan ly highe in basal ganglia o PCA a s han in ha o sham-ope a ed animals (140% ± 39% and 100%±14%; P < 0.024), bu he e was no significan di e ence be ween he g oups a e PCA in ce ebellum (100% ± 25% and 100% ± 16%; P = NS) (Figu e 3). DISCUSSION The PCA pe o med in a s is widely accep ed as a model o li e dys unc ion. In his s udy, PAG ac i i y was inc eased in kidney and duodenum, which could con ibu e significan ly o sys emic hype ammonaemia. PAG ac i i y was inc eased in co ex and basal ganglia, which migh be esponsible o b ain hype ammonemia and in a- mi ochond ial ammonia le els de i ed om glu amine hyd olysis ha a e main ained because success ul de oxifica ion by glu amine-syn he ase is p ecluded. Du ing he 2nd hal o he 20 h cen u y, hype am- monaemia was conside ed o be de i ed om u ea b eakdown by in es inal bac e ia and he majo i y o ea men s a e a ge ed agains bac e ia-de i ed ammonia om he colon[1]. Howe e , he hypo hesis was no uni e sally accep ed[12]. Hype ammonaemia ollowing po aca al shun ing in a s has been ound o be simila in ge m- ee as well as in non-ge m- ee animals [13, 14], p o iding suppo o he concep ha hype ammonaemia and encephalopa hy could de elop wi hou pa icipa ion o bac e ia[15]. The highes hype ammonaemia has been ound in po al-d ained isce a and de i ed mainly om glu amine deamida ion[16]. Hence, inc eased PAG ac i i y in small in es ine could explain hese obse a ions a leas in pa . Mo eo e , inc eased PAG in duodenum has been demons a ed in ci ho ic pa ien s su e ing om minimal hepa ic encephalopa hy[3]. Recen ly, enal ammoniagenesis has been implica ed in se e al o ms o hepa ic encephalopa hy. In pa ien s su e ing om hepa ic encephalopa hy due o a iceal bleeding o o e dose o diu e ics, ammonia p oduc ion om kidney seems o be he main ac o in ol ed in he de elopmen o hepa ic encephalopa hy[17]. Glu amine is il e ed in he glome uli and en e s he lumen o he neph on. The il e ed glu amine is eabso bed in he p oximal ubule whe e glu amine is deamida ed by PAG. The main ac o in he egula ion o kidney PAG ac i i y seems o be pH. Du ing me abolic acidosis, PAG ac i i y inc eases which induces highe exc e ion o ammonia[18]. In hepa ic encephalopa hy, me abolic alkalosis is obse ed mo e equen ly han acidosis and inc eased PAG ac i i y in kidney could be a p o ec i e mechanism. This PAG inc ease could be e en mo e p o ec i e han he e en associa ed wi h he sys emic hype ammonaemia. Howe e , his possibili y needs o be explo ed in g ea e de ail in u u e s udies. Ammonia eaching he b ain can be de oxi ied o ShamPCA A B Duodenum Kidney ShamPCA Figu e 1 Rep esen a i e immunoblo ing om duodenum (A) and kidneys (B). Glu aminase con en in en e ocy es om a s wi h po aca al shun (PCA) and 6 con ol a s (sham) homogenised and subjec ed o immunoblo ing, 10 µg o p o ein applied in each lane. 7000 6000 5000 4000 3000 2000 1000 0 Duodenum Kidney Co ex Basal ganglia Ce ebellum PAG ac i i y (µka /g o p o ein) PCA Sham Figu e 2 PAG ac i i y (ka /g o p o ein) in duodenum, kidneys and b ain o PCA and con ol a s (sham). Co ex Sham PCA Basal ganglia Ce ebellum Sham PCA Sham PCA Figu e 3 Glu aminase con en in ce eb al co ex and basal ganglia. Ce eb al co ex basal ganglia o ce ebellum om 7 a s wi h po aca al shun (P) and 6 con ol a s (C) was homogenised and subjec ed o immunoblo ing, 10 µg o p o ein was applied in each lane. The codes unde he bands o ep esen a i e blo s indica e he di e en a s wi h po aca al shun (P) o con ol a s (C). Rome o-Gómez M e al. Phospha e-ac i a ed glu aminase ac i i y in a s 2409 www.wjgne .com glu amine only in as ocy es due o he p edominance o glu amine syn he ase in hese cells. Glu amine accumula ion as a by-p oduc o ammonia me abolism has been implica ed in he pa hogenesis o hepa ic encephalopa hy[19]. Glu amine is an osmo ic amino acid and plays a majo ole in he egula ion o cell olume. A aised peak o glu amine-glu ama e/c ea ine a io is a ypical ea u e o b ain spec oscopic magne ic esonance imaging in hepa ic encephalopa hy. Howe e , glu amine syn he ase has no been ound o be inc eased in b ain o po aca al shun ed a s[20]. Indeed, adminis a ion o amino acid mix u es induces hype ammonaemia, aises glu amine peak in he b ain and is associa ed wi h impai men in neu opsychological unc ion[21]. Also, use o me hionine- sul oxamine blocks he ac i i y o glu amine syn he ase and imp o es abno mali ies induced by ammonia, such as seizu es o as ocy es swelling[22]. Ne e heless, accumula ion o glu amine ollowing ea men wi h d ugs ha a e able o block N-me hyl-D-aspa a e (NMDA)- ecep o s has no been shown o be associa ed wi h neu ological impai men [23]. Indeed, in as ocy e cul u e, glu amine me abolism is linked o ee adical p oduc ion and oxida i e s ess and his may ep esen a key mechanism in ammonia neu o oxici y [24]. Thus, glu amine accumula ion in he b ain is nei he a pa hological e en pe se no a sa e ammonia de oxifica ion pa hway. Glu amine accumula ing in he as ocy es can be conside ed as a “T ojan ho se” leading ci cui ously o neu ological impai men . In cul u ed as ocy es, glu aminase inhibi o s such as 6-diazo-5-oxo-L-no leucine (DON) induce a comple e blockade o glu aminase ac i i y and p eemp ee adical p oduc ion and neu o oxici y induced by ammonia[25]. Mos o he glu amine in as ocy es is me abolized by mi ochond ial PAG[26]. Since ammonia induces ee adical p oduc ion, he PAG can be implica ed in ee adical p oduc ion. The inhibi ion o his enzyme could be a new he apeu ic a ge . Howe e , DON has also been epo ed o inhibi γ-glu amyl- anspep idase, inc ease glu amine elease, inhibi anspo o glu amine in o cells and block he anspo o glu amine in o mi ochond ia. All hese mechanisms could induce a dec ease in he amoun o glu amine a ailable o hyd olysis[27]. The dis ibu ion o K- ype PAG in he b ain has been s ongly deba ed. Some s udies ha e epo ed ha PAG is absen in as ocy es o ce ebellum[28]. In he cu en s udy, PAG ac i i y in non-synap ic mi ochond ia om he ce ebellum showed he lowes ac i i y in he b ain and no di e ences we e obse ed be ween PCA and sham-ope a ed a s using immunoblo ing. Thus, PAG could be de ec ed in ce ebellum, bu a a low-le el in compa ison o o he a eas such as basal ganglia o co ex. In summa y, PAG is enhanced in he in es ine and kidney o PCA a s and induces hype ammonaemia and hepa ic encephalopa hy. Fu he mo e, PAG ac i i y and glu aminase con en a e inc eased in as ocy es om co ex and basal ganglia. Mi ochond ial glu aminase ac i i y in as ocy es could be implica ed in he p oduc ion o ammonia. The induc ion o he mi ochond ial pe meabili y ansi ion and ee adicals p oduc ion as he end p oduc o glu amine me abolism could be esponsible a leas in pa o he pa hogenic e ec obse ed in hepa ic encephalopa hy. Hence, PAG migh be a new he apeu ic a ge in he managemen o hepa ic encephalopa hy. Fu he s udies using PAG inhibi o s o PAG knock-ou mice help cla i y he ole o inc eased PAG exp ession in he pa hophysiology o hepa ic encephalopa hy. REFERENCES 1 She lock S. Ch onic po al sys emic encephalopa hy: upda e 1987. Gu 1987; 28: 1043-1048 2 Rome o Gómez M, Bau is a JD, G ande L, Ramos Gue e o RM, Sánchez Muñoz D. [New concep s in he physiopa hology o hepa ic encephalopa hy and he apeu ic p ospec s]. Gas oen e ol Hepa ol 2004; 27 Suppl 1: 40-48 3 Rome o-Gómez M, Ramos-Gue e o R, G ande L, de Te án LC, Co pas R, Camacho I, Bau is a JD. In es inal glu aminase ac i i y is inc eased in li e ci hosis and co ela es wi h minimal hepa ic encephalopa hy. J Hepa ol 2004; 41: 49-54 4 James LA, Lunn PG, Elia M. Glu amine me abolism in he gas oin es inal ac o he a assess by he ela i e ac i i ies o glu aminase (EC 3.5.1.2) and glu amine syn he ase (EC 6.3.1.2). B J Nu 1998; 79: 365-372 5 James LA, Lunn PG, Middle on S, Elia M. Dis ibu ion o glu aminase and glu amine syn he ase ac i i ies in he human gas oin es inal ac . Clin Sci (Lond) 1998; 94: 313-319 6 an de Poll MC, Soe e s PB, Deu z NE, Fea on KC, Dejong CH. Renal me abolism o amino acids: i s ole in in e o gan amino acid exchange. Am J Clin Nu 2004; 79: 185-197 7 Numa a M. A modified echnique o make a po aca al shun in a s. Mic osu ge y 1983; 4: 243-244 8 He menegildo C, Mon o P, Felipo V. Ac i a ion o N-me hyl-D-aspa a e ecep o s in a b ain in i o ollowing acu e ammonia in oxica ion: cha ac e iza ion by in i o b ain mic odialysis. Hepa ology 2000; 31: 709-715 9 Lai JC, Cla k JB. P epa a ion o synap ic and nonsynap ic mi ochond ia om mammalian b ain. Me hods Enzymol 1979; 55: 51-60 10 B ad o d MM. A apid and sensi i e me hod o he quan i a ion o mic og am quan i ies o p o ein u ilizing he p inciple o p o ein-dye binding. Anal Biochem 1976; 72: 248-254 11 Felipo V, Miñana MD, G isolía S. Long- e m inges ion o ammonium inc eases ace ylglu ama e and u ea le els wi hou a ec ing he amoun o ca bamoyl-phospha e syn hase. Eu J Biochem 1988; 176: 567-571 12 Shawc oss D, Jalan R. Dispelling my hs in he ea men o hepa ic encephalopa hy. Lance 2005; 365: 431-433 13 Nance FC, Kline DG. Eck’s fis ula encephalopa hy in ge m ee dogs. Ann Su g 1971; 174: 856-862 14 Wa en KS, New on WL. Po al and pe iphe al blood ammonia concen a ions in ge m- ee and con en ional guinea pigs. Am J Physiol 1959; 197: 717-720 15 Webe FL J , Veach GL. The impo ance o he small in es ine in gu ammonium p oduc ion in he as ing dog. Gas oen e ology 1979; 77: 235-240 16 Olde Damink SW, Jalan R, Redhead DN, Hayes PC, Deu z NE, Soe e s PB. In e o gan ammonia and amino acid me abolism in me abolically s able pa ien s wi h ci hosis and a TIPSS. Hepa ology 2002; 36: 1163-1171 17 Jalan R, Kapoo D. Re e sal o diu e ic-induced hepa ic encephalopa hy wi h in usion o albumin bu no colloid. Clin Sci (Lond) 2004; 106: 467-474 18 Cu hoys NP. Role o mi ochond ial glu aminase in a enal glu amine me abolism. J Nu 2001; 131: 2491S-2495S; discussion 2496S-2497S 19 Alb ech J, Dolińska M. Glu amine as a pa hogenic ac o in hepa ic encephalopa hy. J Neu osci Res 2001; 65: 1-5 20 Desja dins P, Rao KV, Michalak A, Rose C, Bu e wo h RF. E ec o po aca al anas omosis on glu amine syn he ase p o ein and gene exp ession in b ain, li e and skele al muscle. Me ab B ain Dis 1999; 14: 273-280 21 Bala a S, Olde Damink SW, Fe guson K, Ma shall I, Hayes 2410 ISSN 1007-9327 CN 14-1219/ R Wo ld J Gas oen e ol Ap il 21, 2006 Volume 12 Numbe 15 www.wjgne .com PC, Deu z NE, Williams R, Wa dlaw J, Jalan R. Induced hype ammonemia al e s neu opsychology, b ain MR spec oscopy and magne iza ion ans e in ci hosis. Hepa ology 2003; 37: 931-939 22 Rama Rao KV, Jayakuma AR, No enbe g MD. Induc ion o he mi ochond ial pe meabili y ansi ion in cul u ed as ocy es by glu amine. Neu ochem In 2003; 43: 517-523 23 Kosenko E, Llansola M, Mon oliu C, Mon o P, Rod igo R, He nandez-Viadel M, E ceg S, Sánchez-Pe ez AM, Felipo V. Glu amine syn he ase ac i i y and glu amine con en in b ain: modula ion by NMDA ecep o s and ni ic oxide. Neu ochem In 2003; 43: 493-499 24 Jayakuma AR, Rama Rao KV, Schousboe A, No enbe g MD. Glu amine-induced ee adical p oduc ion in cul u ed as ocy es. Glia 2004; 46: 296-301 25 No enbe g MD, Rama Rao KV, Jayakuma AR. Ammonia neu o oxici y and he mi ochond ial pe meabili y ansi ion. J Bioene g Biomemb 2004; 36: 303-307 26 Sonnewald U, The ien G, Bu e wo h RF. Po aca al anas omosis esul s in al e ed neu on--as ocy ic me abolic a ficking o amino acids: e idence om 13C-NMR s udies. J Neu ochem 1996; 67: 1711-1717 27 Rama Rao KV, Jayakuma AR, No enbe g MD. Di e en ial esponse o glu amine in cul u ed neu ons and as ocy es. J Neu osci Res 2005; 79: 193-199 28 Laake JH, Takumi Y, Eide J, To gne IA, Robe g B, K amme E, O e sen OP. Pos embedding immunogold labelling e eals subcellula localiza ion and pa hway-speci ic en ichmen o phospha e ac i a ed glu aminase in a ce ebellum. Neu oscience 1999; 88: 1137-1151 S- Edi o Pan BR L- Edi o Wang XL E- Edi o Ma WH Rome o-Gómez M e al. Phospha e-ac i a ed glu aminase ac i i y in a s 2411 www.wjgne .com