\ -..

. )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

·····················································································································

····················································································································· .....................................................................................................................

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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,

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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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).

·.,