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. )ERPUSTAKAAN KAMPUS KESIHAf~ R UJ u KA N '· UNIVERSITI SAINS MALAYS~
USM SHORT-TEHM PRO.JECT FINAL REPORT
FF:BRUARV 2005
PRO.JF:CT TIT I , I~:
ACUTE EFFECTS OF AMMONIA ON 'CITI{ULLINE- NO
CYCLE ENZYMES', AH.GINASE AND !{ELATED
METABOLITES IN DIFFElillNT l{EGIONS OF RAT BRAIN
NAME OJI INVESTIGATOH:
Dr. Mnnunedy Swarny
( '( )- JN VESTH iATOR :
En. Chandran Gov indasa my
Department or C hemi ca l p8thology,
School or Medi ccll Scie nces,
USM
.,
USM SHORT-TERM PROJECT FINAL REPORT
FEBRUARY 2005
PHO.JECT TITLE:
ACUTE EFFECTS OF AMMONIA ON 'CITRULLINE- NO
CYCLE ENZYMES', AU.GINASE AND IillLATED
METABOLITES IN DIFFERENT REGIONS OF RAT BRAIN
NAME OF INVESTIGATOR:
Dr. Mnmmedy Swa1ny
CO-JNVESTJG/\TOR:
En. Chandran Govindasamy
Department of Chemical pathology,
School of Medical Sciences,
USM
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Laporan Akhir l)rojek Peyelidikan IUSM JP- 06)
0
1)
SAHAGIAN PENYELIDIKAN & PEMBANGUNAN CANSELORI UNIVERSITI SAINS MALAYSIA
Laporan Akhir Projek Penyelidikan _Janqka Pendek
Nama Penyelidik: DR. MUMMEDY SWAMY
USM J/P- 06
Nama Penyelidik-Penyelidik Lain (Jika berkaitan) En. CHANDRAN GOVINDASAMY
2) Pusat Pengajian/PusaUUnit Deparhnent of Chetnical pathology.
School of Medical Sciences, llealth Catnpus. lJSM
3) Tajuk Projek:
ACUTE EFFECTS Or AMMONIA ON "CITRULI JNE- NO CYCLE ENZYT\1ES ... ARGINASE AND RELATED METABOLITES IN DIFFERENT REGIONS OF RAT BRAIN.
USM J/P-06 - 1
p
4) (a) Penemuan Projek/Abstrak (Perlu disediakan makluman di flntara 100 - 200 perkataan di dalam Bahasa Malaysia dan Bahasa lnggeris. tni kemudiannya ak~:m dimuatkan ke dalam laporan Tflhunf!n Bahflgian Penyelidikan & Pembangunan sebagai satu c::~ra untuk menyampaikan dapat::~n projek tuanlpuan kepada pihak Universiti).
ACUTE EFFE~TS OF AMMONII\ ON 'CITRULLINE-NO CYCLE ENZYMES',
ARGINASE AND ltELATED META_BOLITES IN DIFFERENT REGJONS. OF RAT
BRAIN
ABSTRACT
Nitric oxide (NO) is involved in numy physiological and pathological processes in the
brain. NO is synthesized frmn arginine by nitric oxide synthase (NOS) enzytnes.
Citru11ine, which is fonned as a by-product. of the NOS reaction, can he recycled to
arginine by successive actions of argininosuccinnte synthetase (ASS) nnd
argininosuccinate lyase (ASL) via the citrulline-NO cycle. llypermnn1onetnia is kno\vn to
cause poorly understood perturbations of the citrulline-NO cycle. Both ASS and ASL
genes at·e reported to he induced in nstrocytes hut not in neurons of nggrcgates exposed to
5 mM arnn1oniun1 chloride, suggesting that hyperan1n1onen1ic hrain n1ight increase its
recycling of citrulline to arginine. To understand the role of citntlline-NO cycle in
hyperanltnonetnia, NOS, ASS, ASL and arginase activities, as well as nitrate/nitrite
(NOx), the stable end products of NO, nnd other related tnetabolites were estin1ated in
cerebral cortex (CC), cerebelltun (CB) and brain stenl (BS) of rats subjected to acute
ammonia toxicity (0.8n11nol of an11nonhun acetate per I OOg hody weight). N()x
concentration and NOS activity were found to increase in all the regions of brain in acute
amn1onia toxicity. The activities of ASS (CC. CB and BS) and ASL (CC and CB) also
showed an increase whereas the activity of arginase was not clmngcd. The concentrations
of arginine and ornithiue were increr.sed in nil the regions of hrain in acute mnrnonia
toxicity whereas citrulline concentration was not changed. Glutatnine concentration was
significantly increased in all regions of brain while glutatnate and GABA concentrations
were not changed. The results of this study clearly demonstrated the increased formation
of NO, suggesting the involvernent of NO in the pathophysiology of acute am1nonia
toxicity. The increased activities of ASS and ASL enzymes indicate the increased and
effective recycling of citrulline to arginine in acute mnn1onia toxicity, 111aking NO
production more effective and contributing to its toxic effects.
USM J/P-06 - 2
AKUT K.ESAN AMMONIA TERIIADAP 'ENZIM-ENZIM DALAM KITARAN
CITRULLINE-NO', ARGINASE DAN LAIN-LAIN METABOLIT DALAM
PELBAGAI SAHAGIAN OTAK TIKUS
ABSTRAK
Nitrik oksida (NO) terlibat dalan1 pelhagai proses fisiologi dan patologi di dalam otak.
NO disintesiskan daripada arginine oleh enzim-enzitn nitric oxide synthase (NOS).
Citrulline yang terbentuk sebagai hasi1 san1pingan daripada tindakbalas NOS, holeh
dikitar semula kepada arginine oleh tindakhalas-tindakbalas argininosuccinate synthetase ··
(ASS) dan argininosuccinate lyase (ASL) melalui kitaran citrulline-N(). Walau
bagaimanapun, kesan keadaan an1tnonia yang tinggi terhadap kitaran citrulline-NO ini
tidak dapat difahami dengan tepat. Dalam k~jian yang lepas~ kedua~dua gen ASS dan
ASL didapati teruja dalan1 astrocytes~ telapi tidak dalarn neurons. yang didedahkan
" kepada ammonium chloride pada kepekatan Sn1M. Ini menttt\jukkan kandungan amtnonja
yang tinggi di dalam otak menyebahkan peningkatan pembentukan citn11line kepada
arginine. Untuk men1ahami peranan kitaran citntl1ine-N() dalan1 keadaan atnmonia yang
tinggi, aktiviti-aktiviti NOS, ASS, ASL dan arginase serta kepekatan nitrat/nitrit (NOx)
dan lain-lain metabolit dianggarkan di dalam korteks serebral, serebel1un1 dan batang
otak tikus yang telah dikenakan ketoksikan amn1onia akut (O.Rtntnol an1n1onimn acetate '
bagi setiap I OOg berat badan). Kepekatan NOx dan aktiviti NOS didapati n1eningkat di
dalam semua bahagian otak tikus dalan1 keadaan ketoksikan an1n1onia akut. Aktiviti-
aktiviti ASS dan ASL juga menunjukkan peningkatan yang setara tetapi aktiviti arginase
tidak berubah. Kepekatan arginine dan ornithine dalatn pelhagai hahagian otak yang
c
mengalami ketoksikan ammonta akut menu11jukkkan peningkatan tetapi tidak pada
kepekatan citrulline. Kepekatan glutamine menuf1jukkan peningkatan yang ketara dalan1
semua hahagian otak tetapi kepekatan glutamate dan GABA tidak anenur1iukkan
perubahan yang ·ketara. Keputusan yang didapati dari eksperimen ini jelas n~enu11iukkan
penghasilan NO yang tinggi ini tnenyumbangkan kepada penglihatan NO dalam
patofisiologi ketoksikan atntnonia akut. Peningkatan dalan1 aktiviti-aktiviti ASS dan ASL
menur\iukkan peningkatan dan keberkesanan kitaran semula citrulline kepada arginine
dalam ketoksikan ammonia akut, juga rnenyebabkan penghasilan NO rner1jadi lebih
berkesan dan seterusnya menyutnbangkan kesan toksiknya. .,
5)
(b) Senaraikan Kata Kunci yang digunakan di dalam abstrak:
Bahasa Malaysia Bahasa lnqqeris
Otak tikus Rat brain
Ketoksikan ammonia akut Acute ammonia toxicity
Nitrik oxida Nitric oxide
Kitaran citrulline-NO Citrulline-NO cycle
Arginase Arginase
NitraUnitrit (NOx) Nitrate/Nitrite (Nox)
Output Dan Faedah Projek
(a) Penerbitan (termasuk laporan/kertas seminar) (Sila nyatakan jenis, tajuk, pengarang, tahun terbitan dan di mana telah diterbit/dibentangkan).
~ . 1) Adlin Zafrulan Zakaria, Chandran Govindasamy, H. A. Nadiger and Mummedy Swamy (2004) Effects of acute ammonia toxicity on Nitric oxide and ~~citrulline-NO cycle enzymes" in rat brain. 291
h
Annual conference of the Malaysian Society for Biochemistry and Molecular Biology (MSBMB). 28th & 29th Sept 2004, Nikko Hotel,~ Kuala Lumpur
2) Adlin Zafrulan Zakaria (2004) Effects of acute ammonia toxic_ity on Nitric oxide (NO), Citrulline-NO cycle enzymes, arginase and related metabolites in different regions of rat brain. A Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Pathology (Chemical Pathology) Universiti Sains Malaysia, November 2004.
3) M. Swamy, Adlin Zafrulan Zakaria, Chandran Govindasamy and H. A. Nadiger (2005) Effects of acute ammonia toxicity on Nitric oxide (NO), Citrulline-NO cycle enzymes, arginase and related metabolites in different regions of rat brain. Paper submitted for publication in Molecular Genetics and Metabolism.
USM J/P-06 - 3
·,,
(b) Faedah-Faedah Lain Seperti Perkembangan Produk, Prospek Komersialisasi Dan Pendaftaran Paten. (Jika ada dan jika perlu, sila guna kertas berasingan)
Nil
..................................................................................................................................... ,,
(c) Latihan Gunatenaga Manusia
i) Pelajar Si~wazah: Dr. Adlin Zafrulan Zakaria- A Student of M. Path
Chemical pathology trained to complete his dissertation
ii) Pelajar Prasiswazah: Nil.
······································································\··········· ············
iii) Lain-Lain: Nil
... ··················································· ············ ····························
····················· ... ······································································
···························································· ........................... ··········
USM .JIP-06 - 4
.. .• ,.:~·
.~ : .. ''H-·
6. Peralatan Yang Telah Dibeli:
Nil
····················································································································· I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I a I I I I I I I I I I I I a I I I I I I I II I I I I
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UNTUK KEGUNAAN JAWATANKUASA PENYELIDIKAN UNIVERSITI
···················································································································· ....................................................................................................................
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....................................................................................................................
Professor .)idi A?har Mohd. Hussin ~hairmsm r .e~-'3-rch & Ethic:J Committee
TfrANGAN PENGERUST s~h~i of !\·1cd~1 £..cie-nces l I JIK PENYELIDJKAN Hf.•:lhl~ Campus W PUSAT PENGAJIAN Ur.i,~:rsiti Suins 1v!atays.~t ~
1M SO f·~u":t:r:g R crinn, KELANTAN. IHALAYSIA.
USMJ/P-06 ~ 5
.....
Laporan l(omprehensif
•
LapOran Komprehensif
• Laporan Komprehensif:
Title:
ACUTE EFFECTS OF AMMONIA ON 'CI1,RULLINE - NO
CYCLE ENZYMES', ARGINASE AND RELATED
METABOLITES IN DIFFERENT REGIONS OF RAT BI{AIN
Introduction:
Name of Investigator: D.-. Mununedy Swa1ny
Co-investigator: En. Chandran Govindasan1y
Student: Dr. Adlin Zafrulan Zakaria
Department of Chemical pathology,
School of Medical Sciences,
USM
Nitric oxide (NO) is involved in numy physiological and pathological processes in the
brain. NO is synthesized fron1 arginine hy nitric oxide synthase (N<)S), and the citrulline
generated as a by-product can be recycled to arginine by argininosuc_cinate synthetase (/\S)
and argininosuccinate lyase (/\1 J) via the citrulline-NO cycle (7.hang et al 2000).
Generation of nitric oxide, N(), a versatile 1nolecule in signnling processt'S nnd unspeci lie
itntnune defense, is intertwined with synthesis. cataholisn1 and transport of arginine which
thus ultitnately participates in the regulation of a line-tuned balance between nonnal and
• pathophysiological conRequences of NO production (Wiesinger 2001 ). Co-induction of
inducible nitric oxide synthase and arginine recycling en7.ymes in cytokine-stinntlated
PC 12 cells and high output production of nitric oxide is reported by Zhang et al (2000).
Co-induction of argininosuccinate synthetase, cationic atnino acid transporler-2 .. and nitric
oxide synthase in activated n1urine tnicroglial cells was reported by Kavahara eta I (200 I).
The n1echanisrn of action of an1n1onia toxicity in brain is not clearly understood
(Sadasivudu et al 1983). However, alterations in tnetabolic and functional aspects of brain
such as depression in energy tnetahntistn • changes in neurotrfmstnitter levels,involvetnent
of glial cells,changes in the activity of Na+ ~K +-activated ATPase affecting the tnetnbranc
function(Sadasivudu et at 1977), and alterations in hlood-hrain harrier( I lorowitt7. 1981)
have been itnplicated. Hyperanunonetnia is known to cause poorly understood
perturbations of the citrulline:.NO cycle. It is reported by Braissant eta I ( 1999) that both AS
and AL genes are induced in astrocytes hut not in neurons of aggregates exposed to 5 n1M
NH4CI, suggesting the hypenm1n1onetnic brain n1ight increase its recycling of citrulline to
arginine. In order to understand the role of citrulline-No cyde in an1n1onia toxicity the
proposed study was undertaken.
•
Objectives:
General objectives:
The objective of the s_tudy was to understand the role of citrulline-nitric oxide cycle in three
different regions of rat brain; cerebral cortex, cerebellum and brain stem, subjected to acute
ammonia toxicity.
Specific objectives:
I. To determine the concentration of ammonia tn different regtons of rat brain
subjected to ammonia toxicity.
2. To determine the changes in the concentration of nitric oxide in different regions of
rat brain subjected to acute a1nmonia toxicity.
3. To determine the changes in the activities of the citrulline-NO cycle enzymes;
NOS, ASS, ASL, and activity of arginase in different regions of rat brain subjected
to acute ammonia toxicity.
4. To determine the changes in the concentrations of arginine, citrulline, ornithine,
glutamate, glutatnine and gamma-amino butyric acid (GABA) in different regions
of rat brain su~jected to acute ammonia toxicity.
• Materials and Methods:
Experimental design:
Sprague Dawley rats were used as subjects. Male rats weighing 200 - 350 grams were
used for the study and divided into two groups; the control group and the test group (acute
ammonia toxicity group). Ammonia toxicity was produced by the procedure of Besstnan &
Paul (1976) as described by Swatny & Sadasivudu (1983). This was done by adtninistrating
ammonium acetate at a dose of 0.8tntnol/1 OOgrmns body weight intraperitoneally. Acute
toxic effects of ammonia were then studied in these animals after 30 tninutes of that
injection. The control animals were given normal saline instead of anltnoniutn acetate and · ..
also sacrificed 30 minutes after the intraperitoneal ir1jection.
The rats were sacrificed using the guillotine and the brains were quickly removed
according to the procedure described by ·Sadasivudu & Lajtha (1970). This was done by
making an incision in the n1iddle of the skull followed by two rnore incisions laterally on
both-sides. Then the skull was flipped open and the brain was scooped out. Aficr that the
three different regions of the brain; cerebral cortex, cerebellun1 and hrain sten1 were
separated and put in nonnal saline to clear off the blood clots. Each brain region was then
wrapped in the aluminium foil, labelled and kept in ice box before hornogenization.
Each of the brain regions was weighed and used for preparation of hotnogenates in 0.05M
phosphate buffer. Homogenate preparation was done using a teffi?n pe~tle. Ten percent
(1 0%) weight/volurne homogenates were prepared for amn1onia, NO. NOS. arginase and
amino acids analysis while 20% hotnogenates were prepared for estitnation of ASS and
ASL activities. All the pararneters were assessed using 6 anin1als independently frorn each
group.
Estimation of ammonia:
Ammonia concentration in brain tissue homogenates was estimated using the enzymatic
assay with glutamate dehydrogenase, using the am1nonia kit frotn Randox. An1monia
combines with a.-ketoglutarate (a.-KG) and NADPH in the presence of glutamate
dehydrogenase (GLDH) to yield glutmnate and NADP+. The corresponding decrease in the
absorbance (A) ofNADP+ at 340nm is proportional to the atnmonia concentration.
a.-KG+ NI-h+ NADPH OI.DH Glutamate+ NADP+ ~
The concentration of atnmonia is calculated using the fonnula below,
Asample- Abtank X 294 Astandard- Ablank
and this would give the concentration of amrnonia in utnol/1. The an1n1onia concentrations
were however expressed as umol/g wet weight of tissue.
For the estimation of ammonia, 10% (weight/volume) tissue homogenates were used i.e.
using 1 Ograms of brain tissue in 1 OOrnls of phosphate buffer. Therefore, in a liter ( 1 OOOrnls)
of buffer, there should be 1 OOgrams of brain tissue. Frotn this we can conclude that umol/1
is equal to umol/1 OOgratns tissue:
utnol/1 = un1ol/g wet weight of tissue 100
· ..
.. Estimation of Nitric oxide (NO)
NO was estimated as nitrate/nitrite (NOx) by Griess reaction after conversion of nitrate to
nitrite by nitrate reductase, using the Nitric Oxide Assay Kit fron1 Calhiochen1 (Catalogue
number 482650). This kit used a colori1netric n1ethod in which nitrite \:vas tneasured to
detern1ine the concentration of nitric oxide. In aqueous solution, NO is rapidly converted to
nitrate and nitrite. The ratio of nitrate and nitrite concentrations produced from NO tnay
vary substantially depending on the biological fluids. Hence., for accurate assay of total NO
generated, both nitrate and nitrite levels must be monitored.
The Griess reaction is based on the chen1ical reaction, which uses sulfanilmnide
(Reagent I) and N-l-napthylethylenedian1ine eli hydrochloride (NED) (Reagent 2) under
acidic (phosphoric acid) conditions (Figure 2). The azo cmnpound fonned can be measured
spectrophoton1etrically at 540ntn. This systen1 detects N()2- in a variety of biological and
experitnental liquid tnatrices such as plastna. senm1. urine. tissue hotnogennte and tissue
cultui·e tnedimn. Spectrophotometric quantitation of nitrite using the <Jriess reagent. was a
straightforward method; however, it did not tneasure nitrate. Therefore, the NADH
dependent enzyme nitrate reductase (NR) was used to convert the nitrate to nitrile prior to
quantitation using the Griess reagent.
.,
.. 'JI I I . ,.
~J/'-..r
I I '·II . rJ/'.../'
Azo Compound
Figure 2: Chemical Reactions Involved in the Measuretnent of Nitrite (N()2-) Using the
Griess Reagents (Source: Internet [http: //www.promega.cmn/ths/tb229/tb229.pdf]).
The assay was performed in a tnicrotiter plate together with nitrate standard. 40ul of the
10% tissue homogenate and 45ul of buffer (given in the kit) was added so that the final
volume is 85ul. Then, 5ul of reconstituted nitrate reductase was added to each wel1, .•
followed by 1 Ou1 of 2mM NADII, before it was incubated for 20 tninutes at roon1
temperature in the dark. For the colour developn1ent., 20u1 each of Griess reagent 1 and 2
were added and after 5 minutes, the absorbance was read at 540nnl using the microliter
plate reader. The concentration of nitrite was then obtained frotn the standard curve
(Figure 3.1) by comparing the value of the absorbance. Finally, the concentration of NOx
was expressed in nmol/g wet tissue.
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Enzyme assays:
Nitre oxide synthase (NOS) activity:
NOS activity was estimated by the tnethod of Yui et al (1991) in which the stable end
products, NO~, were estimated using the Nitric Oxide Synthase Assay Kit frorn
Calbiochem (Catalogue Number 482702)~ This kit used colorimetric method in which
nitrite concentration was measured to determine the NOS activity. In biological systetn,
NOS acts on L-arginine to produce nitric oxide and citrulline. In this reaction NADPH acts
as an essential co-factor. Unfortunately, NADPH can interfere with Griess reagents that are
commonly used for nitrite detection, and lead to lower colour yield. In this kit, the ·~.
limitation was overcome by using lactate dehydrogenase (LDH) to retnove excess NADPH.
A heat-inactivated control was included for each NOS preparation to serve as a control to
measure endogenous nitrate and nitrite. In order to quantitate the ·nitrite concentration, a
nitrate standard curve was perforrned. The assay \Vas perfonned in a n1icrotiter plate
togetl1er with nitrate· standard and afier the colour developed, absorbance was read at
540nm using the microliter plate reader.
In the assay, 40u1 of the 1 Oo/o tissue hotnogenate was used and 20ul of distilled water was
added so that the final volume was 60u1. Then 1 Oul of freshly prepared NADPH solution
(1 mM) was added to each well, followed by I Oul of nitrate reductase, and the tnixture was
incubated for 1 hour at room ten1perature. After that 1 Oul co factors and 1 Oul I .DH were
added to each well, and the incubation was continued for another 20 rninutes. For the
colour development, SOul of Griess reagent I and SOul of Griess reagent 2 were added into
each well. After 10 minutes, the ahsorhance was read and the value of ahsorhance was then
compared with the standard curve (Figure 3.2) to determine the nitrite concentration.
;.1 . __ .., ·-i-_1 ·= ----T ... ·--- _ ~ i-~ t- ... ., .t ___ ,__ i _, _ :- ~ ~ , . _ i ~ ~ .. I .-~ • ·r·
.•. ~;: .~,: •··•· ;. •· •· --i- -!-~ • · ~- ;. •-1 . '.;.i .. -i- i· =··t··.! ·•- --t .: I ! ·• .1 :·.l'·. i • ~
- . ,._ · · 1 • · • 1 · - r ... 1 --:. ·r _·,·--:.··_:-__ ,:_:_: ·1 -~~·.,.l __ ._~ -.1.· -~- _· 1. :-1·7 +- i -~ -·. 1 • • _·irL._. Jl-.:,-:· .. r..~.!_.~J __ :t1:~ !~·-·--i- _ _ ___ _ • :.: . I., ~----+--~~--~~''·~·+-~rlr~~~+-~--+-·~~~r-;-~~;-~-r;-~··1·~~·-r~~-r1-1 : :·: ~~~-~~--+-·-~~
;.;, -~~~JI\.~--H ,.; .• ~-!---; ~!i· ~:.! ~1!: i.: 1 -·~-· .,_,_ •••• ;. -!-'-'· .:. .. t' ~----·.· !_·_ -·--•·j -~-~ ---- i 1' ..:.. ,. ~-..-• 'I ·_i-,_- '1 ' I i I I • t- I '-·t.; ;.. i i I I .; I-t I
0 • - .! ~ t ~- r-- ~ . -• ! ·_ ... _. ·-l· - •. I t · I· t
~-t•·· -~~:~ r:·;·; ·;: r!·:: -,:r.· ·•' !"i: • It n ....
0
ir I ..
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. i.- ; . I ·r J ; .... ; -- -- -t .. . . ' • • . • .... • - ; ' . , --1- • ' I , • ·I·, i .1· -·1 . ·r· ~ ~ i .. •. '! .· •• ···:++' •. -:. 1 !I :! : i I: --:.:. ',· 1
,• i ,,· . I . I '1, 't·.:_, ·, l l I: i .. ,. 't i. I • :: . 1'!! ,i; :, . : .. I. ' 1-t.Ll' I . .. I • : • : '· ,I '. • • • i_ • ' : I • • i i_ I. I, I l • ·I . I • • : I t' .. · ·.·:_,·_-·-- . . ' . I I I ---T-1-; , -~t· :.J.o _ .--· >. ·:-:~:-.:--:-:-~-~ -·-t' u-~-1 o--t-·l·· •---t-··--cg--i-•--+ 't-1· · -·~~~-: ~-~--! ·t··t •·1.-- ·t • • t
..... •. •·t 1--, I . ~--~-.-.- ·"-+····-- ., ~---~-~.· -1-~··-1- t: __ ~,-_,:·-~-1; '-··-~·:_:: _ _t · .. ~-~ ,:, ·. . ' ,._,_ l _t-f:.·"'i ·,-_r·.!_·~- i -.
·. ~- ·•• _,_' .·.·.·-t,:--_;_- .. 1-~i:_--+ __ : •. ~·j-- ..... !-~-·- -;- !'- . -. ~ . r t ., -T-1·-i • ·-I r., . ' ''! I ;. f - • -.. ' o·: '.o.,.,_ '! t·•· t-~ •-i 1. •• ·-· t·l·t • 1. ~ ·• ;·-·! : r_-t~·~,: ·t' I I -j·-4 It· t'·: .',: -~-~-: ~-!-~_, _ _.!., ~lL~ --~· ...... ;.~ ... ---~ ... -~ +~--;--·-•:--- -:r .... ~. --! ~, ---+--·- : .• __ -.:.o.
Finally, the enzyme activity was expressed in ntnol ofNOx formed per gratn wet weight of
tissue per hour (nmol NOx/g wet tissue/hr).
Argininosuccinate synthetase (ASS) activity:
Argininosuccinate synthetase activity was estimated by the n1odi fied method of Levin
( 1 971) as described by Sadasivudu and Indira Rao ( 1974). In the estitnation of ASS, the
incubation mixture contained 0.8n1l of 0.01 M each of citnt11ine, aspartic acid, ATP,
magnesium chloride, and 21 U of arginase, in 0.05M phosphate buffer (pi I 7.3). Reaction
was started with the addition .of 0.2tn1 of 20% homogenate and incubated at 37°C for 1 ··
hour. At the end of incubation period, 0.2rnl of 50% trichloroacetic acid was added to stop
the reaction. For controls, trichloroacetic acid was added before the incubation. The
mixture was then centrifuged and 0.5ml of supernatant was used for colour developanent.
The supernatant was tnixed with 1.5tnl of acid mixture (one part of concentrated sulphuric
acid and three parts of concentrated phosphoric acid) and 0.1 ml of isonitrosopropiophenone
(5% in absolute alcohol). It was kept in boiling water hath for 30 minutes, and then afler
cooling the tubes, absorbance was read at 540nm. Simultaneously, a urea standard was set
up by adding to the standard 0.5ml of substrate mixture and O.Sn1l of \Vater. The colour that
developed was read at 540nnl against the reagent blank. The net absorbance of the sample
was then compared with the standard urea curve (Figure 3.3) and th~ amount of urea was
determined. The enzyme activity was expressed as ~unol or urea fonned per gratn wet
weight of tissue per hour (~uno) urea/g wet tissue/hr).
•
tl
Arginininosuccinate layase (ASL) ~ctivity
Argininosuccinate layase activity was also assayed by the n1ethod of Levin ( 1971) as
described by Sadasivudu and Indira Rao (1974). The assay sysletn for ASL consisted of
0.3m1 of 1M phosphate buffer (pH 7.3), 0.6ml ofargininosuccinate (6.0tnM), 0.2ml of20%
homogenate, and 0.1 ml of arginase (1 0.5U). At the end of 1 hou·r incubation at 37°C, the
reaction was stopped by the addition of 0.3ml of 50% trichloroacetic acid. Controls were
identical, except that trichloroacetic acid was added before incubation. After incubation,
the mixture was centrifuged at 3000rpm for 5 minutes and 0.5tnl of the supernatant was
used for urea estimation.
Urea was estimated by the tnodified diacetylmonoxin1e (DAM) tnelhod. In this tnethod,
0.5ml of the supernatant was added with 1.0ml of acid reagent and 1.0tnl of DAM reagent,
and kept boiling for 1 0 minutes in water bath. At the end of 1 0 tnini.ttes., the test tubes were
cooled under running lap water and the absorbance was read against hlank reagent at
540nin wavelength. By using the standard urea curve (Figure 3.4), the atnount of ur~a can
be estimated by comparing the absorbance value. The enzyn1e activity was then expressed
as J.lmol of urea formed per gram wet weight of tissue per hour (J.Lnlol urea/g wet tissue/hr).
· ..
------
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!: :t. ~:.:.! ;j, I 1 ~: ••. r ': ~~ t ~; .• ~i'l :• j.
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,
Arginase activity:
Arginase activity was assayed according to the n1ethod of Herzfeld & Raper ( 1976) as
described by Swamy et al (1983). The first step is to activate the enzyn1e in the sample.
This is done by incubating 300ul of the 10% homogenates with equal vo1utne of imidazole
buffer, containing 56mM imidazole and 56mM MnC12 at ph 7.4, for 10 minutes at 50°C.
The activated preparations were then centrifuged at 3000rptn for 5 minutes~ and the
supernatants were used for measuretnent of enzyme activity. Enzyme assay was carried out
in a total of 0.8ml of incubation mixture consisting of 1 00urno1 of L-arginine and 60un1ol
of glycine buffer (both adjusted to pH 9.5 with l.ON NaOH), and 0.21nl of supen1atant afier ·~.
activation. After incubation at 37°C for 10 minutes, the reaction was stopped with the
addition of 0.2ml 50% trichloroacetic acid. For the controls, the trichloroacetic acid was
added first together with the incubation 1nixture. The mixture was then centrifuged again at
3000rpm for 5 minutes and 0.5m1 of supernatant was taken for estitnation of urea. Urea
was estimated using the DAM method as described for ASL activity above and the activity
was expressed as J.tmol of urea formed per gram wet weight of tissue per hour (J..lmol urea/g
wet tissue/hr).
,.
Amino acid analysis:
Analysis of amino acid was done using the Biochrom 20 Plus Amino Acid Analyzer, which
uses a PC-controlled liquid ion exchange system. This analyzer separates atnino acids on a
high-pressure ·temperature-control1ed liquid exchange colutnn which was continuously
analyzed by ninhydrin photometric detection. Chromatographic controL detection and
safety systems were perfonned via PC with ful1y integrated sofiware. Atnino acids were
continuously analyzed by a fast and precisely controlled in-line reactor after mixing with
ninhydrin, which was the most specific reagent available for flow reactions.
Ten percent (I 0%) tissue homogenate was used for this analysis and the satnple was ·~.
deproteinised first before analysis. This is accomplished using the con1pound 5-
sulphosalicylic acid (SSA) which needs to be the highest purity to avoid contamination of
the resin. For this step, 200ul of satnple· was mixed with 200ul of SSA. Then~ 1 OOul of
internal standard was tnixed in the treated satnple n1aking it diluted 2.5 tin1es. From this
diluted sample, 20ul was it,jected into the ana1yzer cohnnn and the chrontatogram was
generated automatically. The concentration of amino acids was then calculated using the
computer software based on the internal standard concentration. The printed-out results in
the unit of J.lmol/1 have to be multiplied hy dilution factor (2.5) and finally calculated as
J.Lmollgram wet tissue. Figure 4 shows a representative of amino acids chrotnatogran1.
~ -· (fQ
= .... (1)
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] -('D
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met cysth
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phser
· ---- · -- · argsuccinic acid ( . ---- ... ------·- ~~--- .. ---.:.·.~ -· ~·~I~ ..
- --------· --~-------- _ .. ---·· - phe 1.. ----- aaiba
l~
l .. ".- -- ... --( .: ·-- -------------'-- - homocystine
.- _:_:: ~- gab a ( (. --~. ethan l · .. ~-~- -----~------ amm + d·aminolev a
L_ · _· --------~~ hylys L-.. -·. =---- -·----------· . --t_-- ------·----~- ... ..;.-·- ---·· .. --- lys
( :- -~:o:C :;~- : ~- -~ :::-: _ 1 h,Jl'is --.. --·----- 3-mhh;
t c: ans --~ car
l : -·~- ::. ---~- arg
~ 0 Ol
orn
p' 0
· \ Statistical analysis:
Results were reported as mean ± standard deviation (SO) from 6 animals for each
parameter calculated. Statistical analysis of results was done using independent t-test,
using the SPSS ·software (version 11.5). p value of < 0.05 was taken as statistically
significant at 95% confidence interval.
Results:
Results at a glance:
1) The concentration of ammonia increased significantly in all the three brain
regions in rats subjected to hyperammonemic model. ....
2) NO concentration and NOS activity were significantly increased in all the
regions of rat brain studied.
3) ASS activity was significantly increased in all the three brain regions in rats
subjected to ammonia toxicity while ASL activity increased significantly in
cerebral cortex and cerebellum.
4) Arginase activity was not significant in all the regions of rat brain studied.
5) Arginine, ornithine and glutamine concentrations increased significantly in all
the three regions in rats su~jected to ammonia intoxication whereas citrulline
levels increased significantly only in cerebellum and glutamate concentration
increased significantly only in brain sten1. GABA levels were unaltered in all '
the three brain regions in rats subjected to hyperamrnonernia.
Ammonia concentration: The concentration of ammonia was signifi cantly increased in
all three regions of rat brain subjected to ammonia toxicity suggesting that the animals in
the test group were having high ammonia level in the brain (Table 1 ). It c<tn be clearly
seen that there was a uniform increase in ammonia concentration in a ll the three brain
regions in the experime ntal group (Figure 5. 1 ).
Tablet: Ammonia Concentration in Different Regions of Rat 1J1·ain n
BRAIN CONTROL TEST %CHANGE 11
REGIONS Cerebra l Cortex 3.37 ± 0.70 5.49 ± 1.29 + 62.91
Cerebell um 3.05 ± 0.44 4.54 ± o.oo + 48.85
Brain Ste m 2 .44 ± 0.39 4.05 ± 0.65 + 65.98
a concentration is expressed as ~uno I per gra,m wet weight of ti ssue b +=increase, -= decrease
resu lts are mean ± S .D., n=6
8 <1>
7 ::J en en 6 :p +-'
5 ...c:: 0> ·~ 4
l 3
2 0>
1 -0 E 0 ::J
p VALUE
p < 0.005
· -:---p < 0.001
p < 0.001
0 Control
oTest
Cerebral Cerebellum Brain Stem
Cortex
Figm·e 5.1 : Ammonia Concentration in Diff('t·ent He~ions nfHa( n1·ain
Nitric oxide concentration:
The concentration of nitrate/nitrite (NOx) increased significantly in a ll three brain regions
in ammonia toxicity group indicating that there was an increase in the nitric oxide
concentration (Table 2). The increase of NOx in cerebel lum was the highest when
compared to the rise in cerebral cortex and brain stem (rigure 5.2).
Table 2: Nitric Oxide Concenh·ation in Different Regions of Rat Bra inc
BnAIN CONTROL Tft:ST % CIIANGF: 11
REGIONS Cerebral Cortex 6 14 + 74 - RJ2 ± 5R + 35.50
Cerebellum 761 ± 53 11 5J + Ill + 5 1.51
Brain Stem 640 ± 67 909 ± 90 + 42.03
c concentration is expressed as rynol per gram wet weight of tissue h += increase,- = decrease
Values are mean ± S.D., n=6
<lJ ::l
1400 (/) 1200 :2 -..c: 1000 0')
"(i) 3 800 -<lJ 3 0')
x 600 0 z 400 ...... 0
0 200 E c
0
p VALUE:
p < 0.00 1
p < 0.001
p < 0.001
10Control1 ~Test
Cerebral Cerebellum Brain Stem Cortex
Figure 5.2: Nitric Oxide Concentration in Different Regions of Rat Brain
\ ,
Enzyme activities:
The activities of the citrulline-NO cycle enzymes were noted to he higher in the rat brain
su~jected to ammonia toxicity (Table 4.1, Table 4.2 and Table 4.3). The activities of
NOS and ASS were significantly increased in all three brain regions afler amtnonia
administration whereas activity of ASL was only significant in the cerebral cortex and
cerebeJlum but not in brain stem. The increase in NOS activity was highest in the
cerebellum compared with the other two regions (Figure 6).
There was very minimal change in the activity of arginase in all regions of rat brain
subjected to ammonia toxicity, which was statistically not significant, cotnpared to the "·
control animals (Table 5 and Figure 7).
Table 4.1: NOS Activity in Different Regions of Rat Brain d
BRAIN CONTROL TEST o/o CHAN(~E b pVALUE REGIONS Cerebral Cortex 119.17 ± 11.29 161.33± + 35.38 p < 0.001
15.53 Cerebellum I 59.00 ± 23.43 216.50 ± + 36.16 p < 0.003
28.59 Brain Stem 150.33 ± 16.46 209.50 ± + 39.36 p < 0.001
21.27
Table 4.2: ASS Activity in Different Regions of Rat Brain e
BRAIN CONTROL TEST 0/o CI-IANGE b pVALUE REGIONS Cerebral Cortex 1.44 ± 0.17 1.94 ± 0.26 + 34.72 p < 0.001
Cerebellum 1.82 ± 0.19 2.40 ± 0.17 + 31.87 p < 0.001
Brain Stem 1.15±0.13 1.48 ± 0.25 + 28.70 p < 0.02
--------- ---··
Table 4.3: ASL Activity in Different Regions of Rat Brain e
BRAIN CONTROL TEST 0/o CI-IANGE b p VALUE REGIONS Cerebral Cortex 2.62 ± 0.20 3.11 ± 0.25 + 18.70 p < 0.004
Cerebellum 2.34 ± 0.14 2.96 ± 0.23 + 26.49 p < 0.001
..
Brain Stem 2.67 ± 0.25 2.83 ± 0.22 +5.99 NS
d activity expressed as nmol ofNOx (nitrate/nitrite) formed per gram wet weight of tissue per hour
e activity expressed as umol of urea fonned per gratn wet weight of tissue per hour b +=increase,-= decrease
values are mean± S.D., n=6 NS =statistically not significant
Figure 6: Citrulline-NO Cycle Enzymes Activities in Differ·ent Regions of Rat Brain
NOS Activity ....: ..c m 300 r 1
:J neontrot (/)
250 ~ B Tesl
l 200 Ol 150 ..._
6 100 z 50 '+-0 (/) 0 0 E Cerebral Cerebellum Brain Stem c
Cortex
ASS Activity
3 0 IIIII Test
~ 2.5 OControl ::>
"' "' 2.0 ., ~
1.5 0'1 <u !!! 1.0 ::>
0 "' ~
0.5
::> 00
Brain Stem Cerebellum Cerebral Cortex
ASL Activity
€ 4 .5 In Co;,lrot .,
::> l!llesl en 35 "' ·;.
a; 3 ~ 2.5 ~ 2 I!! ::> 1 5 0 "' 0 0 .5 E ::> 0
Cerebral Cerehelhrm Br11in Stem Cortex
..
· Table 5: Arginase Activity in Different Regions of R~t Br~in c
BRAIN CONTROL TEST %CHANGE" p VALUE REGIONS Cerebral Cortex 23.73 ± 3.95 23.92 + 5.32 + 0.80 NS
Cerebellum 32.35 ± 6.4 1 33.85 ± 3.62 +4.64 NS
Brain Stem 19. 10 ±4.65 21.72 ± 3.32 + 13.72 NS
e activHy expressed as umol of urea formed per gram wet weight of ti ssue per hour 11 + = increase - = decrease
' values are mean± S.O, n=6 NS = statistically not significant
50
45 ~
~ 40 (i)
:::) (/) 35 (/) ...,
...... 30 Q)
~ 25 0> --co 20 Q) ,_ :::1 15 ._ 0
0 10 E 5 :::1
0 Cerebral Cerebellum Cortex
~stati ~t ica lly not significant, n- 6
IOControl E!:!Test
Brain Stem
Figure 7: Arginase Activity in Different Regions of Rat Brain
Amino acids concentrations:
Tables 6.1 to 6.6 show the concentrations of amino acids in different regions of rat brain
in both the control and test groups. Concentrations of arginine and ornithine were
significantly increased in all three brain regions after adrninistration of amrnonia.
However, the concentration of citrulline was only significant in cerebellmn (Figure 8.1 ).
Glutamine concentration was increased significantly in all three regions of rat brain in
ammonia toxicity group. However~ the concentration of glutatnate was noted to he
significantly increased only in the brain stem. GABA concentration, on the other hand,
was not significant in all three regions of the brain but it was observed that there were
reduced levels in cerebral cortex and cerebellum (Figure 8.2).
·.,