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
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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
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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.
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