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<:Br i ni vasNag a r ajDhan a:' o ti

A thesis submittedto the School of Graduate Studies in partial fulfilmentof the

requirements for the degree of Doctor of Philosophy

Department of Biochemistry Memorial Univ<"lrsityofNewfoundland

July 1991

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DEDICATED TO MY LATE FATHER

NAGARAJ SRINIVASAIH DHANAKOTI

ABSTRACT

Kidneys of normal animals remove citrulline from blood and convert it, stoichiometricallyto arginine.This citrulline arises from the intestinal metabolism of glutamine. This intestinal-renal path....ay constitutes the major endogenous source of arginine. The objectives of the present studies are to determine the location of arginine synthesis in kidney and its response to citrulline concentrations in vitro and.!D...:i.UQ and to different arginine or proteinintakes in rats.

Investigations on the localization of enzymes of arginine synthesis (argininosllccinate synthetase and argininosuccinate lyase) and of breakdown (arginaseand ornithine

aminotransferase), revealed that the enzymes of arginine synthesis are exclusively present in the cytosol of the cells of the proximal convoluted tubuleand that of arginine degradation are enriched in other kidney regions.

Arginine synthesis from citrulline in isolated kidney cortical tubuteswa s found tobe high lysensitive to citrul line concentrations inthe physiological plasmarange (0.06roM), suggesting that renal arginine synthesis.i.n...Y..l..could be regUlated by cirCUlatingcitrullinelevels. Thus, instudIes on renal arginine synt.hesis.i.n....Y..iY2, it was found that kidneys of rats infusedwIthcitrulline (saline-infused, as controls) responded to the elevated plasma citrullinele vels by increasing its uptake and producinginc r e a s e d quantities of arginine.

U In studies withra ts feddifferentlevelsofarqini ne (0.0 , 0.5, 2.0\)or protein(5, 12and50\) for 1 we ek, i t wasfo u n d that th e re n aluptakeof ci trullineand release of arginineand alsocircu l at i ng citru l li ne leve l s weresimilar in all these ani mals.This sugl;Jes ted that rena l argin inesy n t h esisis Ind e p e nd e n t ofdi e t a ryarginine orprote inint a ke. The resu lts sug g e s t tha t availabil ityof citrulli n eis ali mit i ng factor ro r renal argin ine synthesisin rats.

i i i

ACItNOWLEDGEMEN'l'B

I would like to express my sincereappre c iationtoDr. J. T.

Brosnan for his supervision, guidanceand encouragement throughout thecou rse of this study. I would also liketo thank Drs.G.R. Herzberg and M.E. Brosnan for actingboth as co- superv isorsand membersof supervisory committeeand forth e i r helpful discu ssionsand support.

si nc ere thanks toMr. Doug HallandMrs .SoniaBanfieldfor performing amino ac id ana l ys is. Thanks ar e due to Mrs. Bea Ha ll, Steve Ewar t , oalongQian,stevesqu i r e s , SusanSkanes, Vaughn Collett,Paul Warren, DaveHall, steve Dayand JaneQia n for cr e a t i ng anexcellent atmosphere inthe laboratory.

Aspec i a l thank you to Dr. Markandeya Jois and his fa mil y fortheir support and enco uragement.

I wouldlike to thank Dr.K.M.W. Keough (Deptartment Head) for givingmean oppo r t uni t y to demons trat ebioch e mistryla bs . Thanksare due to all members of the Bi oc hemistry Department for creatinga very freindly atmosphere.

I would like totha nk Ms.Jackie Daltonand Ms.Betty-Ann Williamsfor typing the manuscript.

I wouldalsolike to thank my family members in Indiafo r theirsupport and encouragement duringthe course of my stud ies.

I would like to thank the Dean of Graduate studies for the financial assistance in the form of fellowship.

Iv

TABLE OFCONT ENT S

~.

Abstract .... ... .... ... ...• •... . ... .. . Acknowledgements... .... ... . ... ... . ... . iij

List of Tables... ... .. . ... ... ... vii

List of figures... ... .. .. . ... ... . . .... ix

Cha p t e r 1 Introduction . Arginine requirements in differentspecies.... ...•... Ar g i n i nere qu i r e me nt s inphysiologi calconditions.... Species whichdo not require dietary arginine for growthor maintenance of nitrogenbalance . Beneficiale r rect.s of arginine indifferent pa t holog ica l co nd i t i o ns... . . ... ... 10

Biochemicalfunctionsof arginine... . ... ... . 17

Need for endogenous arginine synthesis... . 20

Minordrain onendogenousargininepools... . ... . 21

Enzymes involved in arginine util i zat ion. ... .. .. . 22

Transport of arg inine... ... .. ...•.. . 28

Roleof differentorgansand theimpo r ta nceof endoge nous arg i n i ne synthes is. . . ... .... .. ... .... 29

Sourcesof cirCUlatingcitru lline. .. .... .... .. . ... . 31

Importa nceofint est i na l citrullinesynt he s is. .... .. . 34

Whftot is knowneoout; renal arginine synthesis?... 35

Una ns we r e d questions. ... ... ... .. .. ... 36

Objectives... ... ... . ... .. ... . ... 36

Chapter 2 Materials andMethods ... ... .. . .... . .... ... . 38

Animals... . . . . ... . ... . .. ... ... 38

Diets and feedingprotocol.... .. . ... .... .. ... 38

Preparation of homogenates from diffe r e nt kidney regions . ... ... .. .... ... ... ... 38

Preparationof cortex. homogenateand subcellUlar fractionation ... . ... .. ... ... ... 39

Prepa rationof kidneyco r t i c a l tubu l e fractions... 41

Fractionationof kidneyco r t i c a l tubules ona Percoll^Rgradient. .... ... . .. . ... ... .... ... 43

Arginineproductioninki d ne y cortical tubules and its fractions... ... ... .... . ... ... . .. 45

Determina t ionof arginineby high-pressureliquid chromatography.. ....•. . ..•.... .... . . . ... .• ... .. . 46

Renal flux of citrUllineand arginine tin vivo studies)... ... ...•.. .... 47

Enzymeassays. .. .. .. .. ... . .... . .... ... 54

Protein and DNA measurement... •... 61

Chemica ls. ... ... . ... ... .. .... ... . 61

Calculati ons , .

Statist icalanalys is .

Chapt er 3 Cellul a r and subce l lula r loc al i za tion of enzymes of argininemptabol i smin rat ki dne y .

Synopsis .

Int r od uc t i o n .

Objectives .

Results .

Distribut i o nof enz ymes of arginine metaoolls m in

different ki d n eyre gio ns .

Distributionofenz yme sofargi n inemp-tabo l ism in ki dn e ycart.leal tubul e fractions . Argini neproduc tion in kidn e y cortical tubule r rectn ons•... .•. ..•..•• ..••..... ...••... •......••. •• • Subce llu la r localizat ionof enzymes of arginine

synthesis inkidne y cortex .

Discussion .

Conclud ingremarks .

Chapter4Rena l argininesynthesis: studiesinY.i..t..l:Q and 1n~.•. . .•... ... . ... ...

sy nopsis .

Introductio n ~., .

Objectives .

Arginine synthe s i s inis ol ate d cort i ca l tubul e s . Argini nesy nt hesisas a functionof time andtubu le

amount .

Meta boli s m ofnewly synthesizedarg i ni ne . Ar g i n i ne synthesis in the presenceof various

sUbstrates .

Depe ndenc eon citru ll ine concent ra tion .

~i~~~~~~o~~~~~~~~~.~.~: :: :: : :: : :: :: :: : : : : : : :: : :: :

Chapter 5Is rena l argin inesyn t hesisaf fected bydie t ary argi ni ne or dietaryprote in intake? .

synopsis .

Introduction .

Objectives .

Experimental pr ot oc o l .

Dietsandfee d ingprotocol .

Results .

Varying di etaryarg in i ne .

Varying dieta ry pro tein .

Discus s ion .

v Eogo 61 62

6 .

6S 66 67 67 67

'0

.0

8. 87

88

8. 8. .0

.2 'S

• •

10 7

112 11' 113 11' 11' 116 11.

11'121 12 .

vi

Chapter6 Summary and ge nera l Discussion.... . . ........ . ... 135 SUT.lmary . • • ••••• •.•••.• •••• •••.• . • ••. .•• .•..• .•..• •••• 136 Gen e ral Dis cus s i on .... .. .. . ... . .. . . . ... .. . . ... .. . . 137 Re ferenc e s .... . . .. . ... .. .. . ..... ..... .. . ....... ......... 143 Appendix A.. . ........ . ....... ... .. .... . ........ . . . ...... . . 157

vii LIST OF TABLES

Table 1.1 Arginine requirements for growth andminimal orotate excretionin differentspe,~ies•. ... ..

Ta b l e 1.2 Beneficialerrec t.s of argininein different pathological conditions... ... . ... ... 11 Table 1.3 Biochemical functions of arginine... ... ... ... 18 Table 1.4 Enzymes and reactions contributingto arginine

re mo v a l. ... ... ... ... .. .. ... .... . ... 23 Table1.5 Distribution of enzymes of arginine metabolism

in odult male rat tissues.. ...• 24 Table 1.6 snavmee and reactions of net arginine synthesis.. 30 Table2.1 Plateau forHe - i nu l i n and citrulline....•.•.. .• 52 Table2.2 Recovery ('t;lof added citrulline, arginine and

ornithinein Wholeblood or plasma... 53 Ta b l e 2.3 Assay conditions for marker enzymesand enzymes

of argin inemetabolism... ... 55 Table 3.1 Distributionofenzymes of arginine metabolism

indifferent regions of the rat kidney... 72 Ta b l e 3.2Distributionof enzymes of arginine metabolism

in rat kidney cortical tubule fractions... 76 Tab le4.1Arginine productionin kidney cortical tubules

in the presence of various substrates. ... 97 Table4.2 Arteriovenousdifferences acrossthe kidneyin

blood,plasma and blood cells forcitrulline and ar g i n i n e in normal and citrulline-infusedrats.. 100 Table 4.3 Basic physiologicaldata for saline- and

citrulline-in fusedrats... ... ... ... 102 Table4.4 Arterialplasmalevels and net renal fluxof

citrulline andarginine in saline- and

citrulline-infusedrats.... ... .. ... . .... . ... 103 Table 5.1 COlllposition of arginine diets... .. .••. ..•... 117 Table 5.2 Composition of proteindiets...•...•.•.. 118

viil

£a.qg Table 5.3 Body weight,di e t consu medand weightgain of

adul t Sprague-Dawley ra t s fed ar.gininediets.... 120 Tabl e5. 4Phys iol og ica ldata for rats fed ar g i n in e diets.. 122 Table5.5Arterial plasma le ve l s and net rena),flux of

citrullineand argininein rats fed arginine diets... . ... ... .. .. ... .. .. ... 123 Ta ble 5.6Bodyweight, diet consumed and weight gainof

adu l t Sprague-Da wleyratsfed pr ot e i n diets.. .. . 125 Ta bl e 5. 7Phy s i olog i c a l datafo r rats fed protei ndiets... 126 Table 5.8 Arterial plasmalevelsand netre na l flux of

cit rull ineand argin inein ra t s fed protein diets... ... ... .... ... ... 127

ix LISTOF FIGURES

Figure 2.1 Cros s-sectionof the kidney (A) and different regionsof kidney... .. . ... ... .... .... 40 Figure 2.2 Confimationofar g ini n'"peak by spiking the

sa mplewith arginine (AI or treatmentwith ar-ginase

(B) ... .. .. ... ... . ... .. .... 48

Figure 2.3 Argininosllcoinatesynthetase acti v i t y inrat ki dne y homogenateas a funct ir-.Iof time (A) and prote inconc en t ration... ... .. 56 Figure2.4 ArqininoSllccinate ly a se activityinrat kidney

homogena t e as a runct.Lcnofti me CAl and protei n concentra tion

(B) .. .... .. .... .... ... .... .... .. 5 7

Figure2.~ Arginase activity in ra t kid neyhomogenate as a func t i on of time (AI an~protein

concentration

(0). ... .. ... ... .. ... ... . 58

Figure2.6 Ornithine aminotransferaseactivityin rat kidney homogen ate as a functionof t";'.~... (A)and pro tein concent ra tion

CD) .. .... ... ... ... ....

in differentkidney regions... ... 69 Fi gur e 3.2 Cross-sectionof thekidne yCA)andstructural

featuresof a co rticalnephron(B)... 70 Figure 3.3 Distributionof enzymes of arginine metabolism

in different regionsofthera t kidney. ... 73 Figure3.4 Isola tionof cortical tU!:"ll sfr ac tio ns . ... 75 Figure 3.5 Dist r ibutionof enzymesof arginine metabolism

in rat kidney cortIcat tubule fr a c t i ons .... ... T' Figure 3.6 Argi nine prod uctioninkidneycort i c a l tubule

fractio ns.."" .•... .••...••.••.• .•• .•.. 81 Figure3.7 Subc e llularlocalizationof enzymesof arginine

synthesisin ratki dney cortex... ... ... . 83 Fi g uz.a4.1 Arg ininesynthesis in rat renal cortica l

tubulesas a function of tillle (A) and tubule qua nt i1:y (b) ... ... ... ... ... ... 93 Filure 4.2 Rea c tions invol vedinthesynthesisand

bceakdovnofarg inine .... . . .... ..•.•...•• ••.. 94

Figure4.3 Fiqure4.4 Figure 4.5

riq ur e4.6 riqu r e 5.1

Fiqure 5.2

. . .

Het ..;"u:i.i&~of ne wl ysy n t hes izedargi n i ne•.. .~.

Depende nc e of ar qininesynthesisinrat re nal cort ical tu bules on citrull ine conc e ntr ation.. Relat i onsh i p between the A-Vdlffe L·enceac ross the kid ne ys for citru l l ineand forarginine in saline- and ci t ru l line -infused rats... • .•. .. .. Figure depictscitrullinere mo v a lin YiY2:

Dependenc e on fittered citrulli ne.•... .. Thefigure plotstheci rcula t i n g levels of citr ulline (A)or arfJinine (9 ) aga i n s t arg i n i n e ingested... ...•... •..•.. ...•... The figure depictsthe renalargini ne release agains t renalcit rulli neuptake (A) and . argi n ine in ges t e d.. . ..•....•. ... •... . . . .

96

98

10 .

106

"9

130

CHAPTER1

INTRODUCTI ON

CRAPTER.1 INTRODUCTION

Theco ncep t of ess en tia lit yof a nutri e ntwas fir s t int r oduc e d byOs borne andMendel (19 16).However , ito.wasRose and coworkers intheea r ly1930'swhoes t abl i sh ed the conce ptof essentialityot ea tne acids in the di etat animals and huaens. Thei r approachprimarily involvedde l eting a spe c if icami noacid from the diet and determi n i ngthe effectof this on thegr owth of weanling ratsandon ni t r ogen (N) balancein humans. Fromsuch st u d i e s , it was conc lude dtha t arginineis a"d i e t a r y non- ess en t i a l " eafncacid for the mai ntenanceof growthinweanling rats and nit rogen ba l a nc ein adulthuma ns (Scull& Ros e, 1930;

Ros e et at, 1954).From these observati ons , it couldbededuced thatar gini ne is syn t h esize d endoge nously in ratsandhumans .

Borsook and Dubnof f (19 41) we re the firs t torepo r t that arginine isformed from cit rullinein ra t kidney sttccs. Howe ver, it was notuntil1973tha t it bec ame clear from the studiesof Feat he r s t o n and hisassociates(197 3) thatthe kidneyisa_a j o r endogenous sourceof arg inine . An obv i o us que s t i onthatarises is th is : does the endogenous synthesisat arg i nine meet the die t ary requi reme ntswithinand acrossdifferent animalspecies , and in di f fe r e nt altered physio log i c al and pathol og i c al states? Bas edon theev iden c e intheli te r atu r e , it can besa i dthatendogenousl y

~yntheslzedarginine doesnotmeet thedietary re q u i r ement in",11

sit uations.

Accordingto Rose (1937)", it'elimination of an amino acid from the diet resu l ts ingrowthfailure and negative nitr oge n balance, then it is conside redas essent ial , otherwiseas non- essential. The s e cri te r iaalonemay notbe SUfficientto define theessentiality of an aminoacid. Byemployingother indicators suchas ceccLc acid excretionand blood ammonialevels,arginine has been shown to bees s en t i al ina number of species and in differentconditions. Incre a s e d orot icacid excretionis a unique indicatorinthat it is speci ficonly toar g i n ine deficiency (Milnerand Visek, 1973 ). In arginine deficiency, impairment in ur e acycle functionnot onlyresultsin increasedbloodammonia leve l s but also ca us e s ac cumul a t i o nof carbamoyl phosp hate in mitochondria follo wedby itsleakage intothe cyt os ol le a di ng to the fo r ma tion oforoti c acid (xcener, 1965). Arginineis known toha ve powerfUl secretogogueand thymotropicproperties (Barbul, 1986) . Therefore, it may be worthwhileto includeindicators such asci r CUlatinghormon ele vel s and immune responsiveness to assess argininedeficiency.

Arginine requirements in different species

The arginine requirementfor growthorformi ni ma lorota t e excretionin different sp eciesis summari ze d in Table 1.1. In this section, I willdiscus s the ebud Les carriedoutby various investigatorson argininereq uirementsin differen t species.

Also, itsrequirement in ce rta inphys i olog i c al conditionswill be

Table1.1

Arg i n ineRe qu ire ments for Growt hand Mi n i ma l Or o ta te Ex cretion in Different sp e c ies'

Animal spe cies Dietary Arginine (\ of diet, by weight)

Ref e r e nc e

Rat Dog Cat Pig Gu i n e a Pig Chi cken Ra b bit Turkey Kh i ma l Orotat.

~ Rat Dog Cat

>0.2 6< 0.5 6

0.'

0.8 0.48 1.7 2.0 0•• 1. 35

>0.5 6 <0. 8 4 0.53 1.05

(Mil ne r &visek,1974 ) (Cza r n e c k i & Bak e r , 1984 ) (Cos tell oet aI, 198 0 ) (S ou thern &Baker, 1983) (O'De ll &Re g an , 19 6 3 ) (Sa v age&O' Dell, 19 60 ) (Adamson& Fisher. 1976) (Kratzer et e L, 1947)

(Milne r ' Vise);, 1974) (Cza r nec ki " Bak er, 1984) (Costello etal, 1980)

•Ferre t require s arg inin e (0. 4\) to mainta in lo wblood ammon ia 1evels ( De s hmukh " Sh op e. 1983).

Housedoesnotrequ irearginine fo r gr owth (Ba uer' Berg,19 4 3) . Dietaryargin ine is not required for growth in human infants (Snydermanet aI , 1959), children (Nakag awa et aL, 19 63 ) and for neithe r th e maintenance of positive nitrogen bala nc e (Roseet aI, 1954) norforminimalor ota t e excretion(Care y et aI,1981) in ad u lts.

addressed.

ScullandRose (19 30 ) werethe fi rsttoreport that arg inine is notan essential amino acid for ....ea nl i ng rats basedsolely on growth . In their st udy, rats which were fed di ets (hydrolyzed cas e i n treatedwith arginase and ur e as e ) containing lowargin i ne (0.4 %)gainedbody weight similar tothose fed 1.3%argini ne (hyd rol yzed casein).Furthermore,their results showedthat the incr ementsin tissuearginine were2to 3 timesas large as may be ac c ounted for bytheing e s t e d arginine, ind icati ng tha t ar g in ine is synth es i ze d endogeno usly.Howeve r,st ud i es fromthe samelaboratory (Borman et aI,1946) which involved feeding highlypurified amino acid diets (wi t h or without arginine) to weanling rats concludedthat arginineis a necessarydietary compone nt foroptimumgrowth. Theseresults fromth esa me lab oratory ar eco nt r a dic t o r y. In theea r lierstUdies (Sc ull&

Rose, 1930), the presenceof ornithinein the protein hydrolysates (ge ne r a t e d by theactionof arginase on arginine) was nottakenintoaccount. The r e f o t"e , i t is possible that ornithine ma y have been converted toar g i n ine and thus met tha requireme ntrcr growth.

Dietaryargi n i neis required for optimal mammarygrowth in

rats (Pau&Milner, 1982). IllIllIature (35-40g) and maturerats

(150-175g) fed purifiedamino aciddi e t s withoutarginine had depressedfood intake, weight gain,lower positivenitrog en balance and showed marked increases in urinaryexcret i on of

or-ocate andtric a r boxylic acidcyc l e intermedia te s (Milneret aI, 1974).St u diesin rat s havedeaon s t ratedthe arq in i ne re quir emen ts (' of di et ) fo rma xilla l bodywe i ght gainandllIin\..'1 or otate excreti o ntobe>0.26<0.56and >0. 5 6<0.84 , respe ctiv e l y (Milner&V! s e k,1974).

The "re sponse of dog s toarg i nine deficiency isdi fferen t

fromthat of rats. Immature Lab rador Ret ri e verdogs(J Kg) fed ami noacid die tswithnoargi ni n e (0' )and contai ni ngvarying amount of dietary N(14, 21 or 28\;)showed signs of emes i s , ex c e s s i v e saliv~t io nand mu sc le tremo r s.Ani mals an argi nine- devoid diets cons umed les s food,los t b~y weigh t and had elevated level s of bloodorot ate and ammonia co mpa red to thos e fed0.56end1.12\arginin e (Ha eta L, 1978 ).MIld hype r u mone ili a an d inc re as ed ee eeeeeexcretion occu rredin young (7-wk) and old (17 -wk) Englis hPointe rdogsfeddietsdevoid of arginine (Czarnecki" Baker, 1984). There sul ts from thi s study als o su g g e sted that youngdogsneed 0.4\ arqininetomaxi mi ze we ight gain,whilere qui ring 0.53\toIlinimizeorotic ac i d ex cretion.On the contra ry, earlier studies by Rose ' Rice, 193 9 demons trated thatfo r the adul t dog, argi n ine isa dispensabl e aminoacidas theywe re able to maintai nbody weight andnitrogenba lance of maturedogsvne n fed arginin e-fre e (p u r i fie d eef n c aci d )die t s.Thus , theabov e twore ports are appar e nt lyin co nfl ict .Thisconflictmaybe largel ydueto experimenta lditterence s. Rose ' Rice (1939) employedadult dogs

instead of young dogs and also theydid not measure orotate excret ionand blood ammonia lev el s . However, it has been sho!in that even inadul t dogs. argini ne is an indisp e nsab leaminoacid ba s ed on orotate excret ion andblood ammonia le ve l s (BurnsetaI, 1981)•

Arginine deficiency in the ca t is the most rap idly induced nut rient deficiencyobserved in anymamma l.Th e consumptio nby thene a r ~ a du ltcat of a sing lemeal devoid of argin ineled to serious cl i nica l symptoms(frothi ng at the mouth,ataxia, emesis and te tanicspasms) or evendeath within a coup leof hours from acuteammonia intoxica t ion(Morris &Rogers, 19 78) . Costel loet al (1980) havereport ed thatthe cat needs 0.8% argi nine to support growthan d 1.05%to minimizeurina r y orotic acid excre tion.

Ferrets

Yo ung , fasted fer r e tsdevelop hypera mmonem i a and enceph alopa t hysoonafte r a si ngle fe edingof dietde f i ci e n t in arginine(Deshmukh and Shop e , 1983).Fer r ets (S-wkol d ) develop hype r ammonem i a wit h in 2-3 h whe n fe d diets containi ng le s s than 0.3\ argini ne.The seanirn~lsdev e lop seizures and coma accomp a nied by sh i ve ri ng when the i r serumammo nia leve l s increase lO-f old.Ferre t s req ui re 0. 4%argi ni nein thei r diet to maintain normal se r umammoniale v els (2 5 0~9/100ml ).Thi s ammonia concent r a tionis abou t 0.15roMwhich wouldbe very high in a rat orhuma n .

Inyoun gpigs (7-9 Kg) fedsem i purified di e t s , su pp lemented withdrgi nlne to achievefina l concent rationsof 0.18 -0.6B\. bodywei ghtgai nandefficie n c yof feedutilizatio n'Were ma xi mizedat0.48\dieta ryar g inine . No impr ov e lllent inthe se parlrllete rs 'Wa s observed with Dore than0.48\ar gini ne .Amode st increasein orotic aci d excret ionwasobs erved in pigs when fed le s s than0.4 8\arginine(South e rn& Baker, 1983).Thewea n li ng pigresemb lesthe rat in that it cansynthesiz e argininebut needs a dietarysu ppl y of arginine fo rmaximumgrowth (Mert z ot aI . 19 52) .

Guinea Pi g s

Theguineapig, unlikethe rat, has an unu su a lly high requirement for arginine which isnot clearl yundersto od. Th e argininerequirelllentof thegrowing quineapig isno t Dot by 25 \ ca s e in (Heini ck eetai, 19 55 ) . Maximullgrowth was observed in the se an I-als when fed 30\ casein (1.2\ arginine) supp lemente d with0.5\ arginine (O'De l l " Reqan, 1963).

Rabbit

Heward etal (1967) were thefirst toreportthe re quire ment of arginine torthe young rabbit.Theopt i mulII arginine level for the qrowthof New Zealan drabbits (5-wkold) was foundto be 0.98 and 1.2 5 \ of the diet for 2.75\and 3.15\nit roqenintake s re s pect ive l y. Adamson an dFisher(19 7 6 )ha ve reported alowe r re qu i re ment (0.6\ argini ne)tobe suf fici ent fo r optimal growth and for mainte nanc e of serumargi nine leve ls inyoung femal e

rabbi t s .Theseworkersals o repo rted tha t adult lIIalerabbitsdo no t require di e t a ry argi n ine.

~andDU:l.U

Alt hough, the essentiality of argininefor the chickwas esta b l ished by Klose et at, (1 9 3 8 ), it was Savage andO'Dell (1960 ) ...,hore po rte d tha t the ne wlyhatchedWhite Legho rnchi c k has a high requirement fo r argi n ine. TheChickne eds2.0' argini ne in the diet tosupport maximullIgrowth. Brrmz e turkey poults (l-wkold) requ i reapproximately1.35' arginine inthe diet tosupp o r t ma ximumgrowth (Kratz er at aI , 1947) .There a sons for the high argini ne req ui r eme n t .in the Chickand turkeypoult are not known.

Arg ini n ereauingeptain certain phy s i o l og i c al conditions

Pregn a n c y andlact ation

Arginine supplellenta tion (1\) of casein dietsfed to pregna n t rats red uc ed their orot a teexcretionand increasedthe birthwei ght andweaningweight of thepups . It alsored uced oro tic acid excretionduri nglatege sta t ion (d16-18 ) andearly lactatio n.t·'a ilureof endoge nous argini nesynthes is tome e t th e requirementofthe preg nant rat in redu cing oro t a t e excret i on , sugge ste dth a t dietaryarginine isrequired during gestati on and lac t a t i o nfo r opti malreproductive re spons e and nurs ing perform ance (pau&Milne r, 1981).

spermatogenesis(reproduction)

Th e roleof argi ninein spermatogenesisis still unclear. In thestudiesof Hol t et<11 (1942 ), in young men, ther e wa s a striki ngdi minution in spermatozoa count after nine days of argini nede-privation. Theyalso confirmed this finding in rats.

Degenerativechangesinthetesteswere note dwith i n 3 weeks of argini newi thdraw a L None of the menstudiedby Roseand his associ a tes (1955) showedany reduc tion inspe r m countwhe n argin inewaswithhe ld for as long as 64da y s.Although the effect ofdi eta ry argini ne deficiency onspermatogenesisis at best unclear, thereis a la r g e body of cl inical wor k reporti ngthe us c of supp leme nta l argin ine invariousmalein f e r t i li tycond i tio n s , pa r t i cul a rl y oligospermia (Barb ul , 19 8 6 ).

species 'Which 40 not require dietary arginine for growth or maintenance of N-balance;

Mice fedpuri f ieddiets la c king in ar gininegrew comparabl y totho s e fed di ets conta i ni ng ar gini n e.Asthe r e were no differen c es in the rate of wei g h t gain,Baue r&Berg (1943) conc l udedthat themouse has theabil ityto synthesizear gi ni ne at a fai r ly rap id rate.

~

Human in f a nt s , (Sny derm a n et ai, 1959 ) child re n(10-12year ol d ) (Na kaq...a et aL,1963 ) and adults (Ros e et al , 195 4 ) donot

10

requiredieta ryar g'i ni ne tor the llai nte na nc eof pos i t i ve nitrogen balance. Recently , Careyet':!: (1987) reported th a t an llIrqinine- deficientdiet in nOOlal adult hUllAnsdoes notLn e r e a s e bIO':)d lIl111110nia level s ororotic acid excretionover ashod '.periodof time (1 0 d). Thu. au t hors concl uded thatth e !ldu l thU1D.anhasthe ca p a city to synt hes i z e arginine.l1I.~,su f fi c i e n t tor the mainte na nce of nonnalcel lul ar metabolism.Thus th eabo v e stUdies sugge s t that argini n e isa dis p e nsableaminoacid forgro wthin in fant s, chil drenandadu l t s.

Benefi ci al. freotlofargininein dif f erent patho l oqicll1

The beneficial effect sof ar gininein differ entpathological conditionsare sUUlarized in Tab le 1.2.

Ur •• cycle4180;4'[1

Anu mberof geneticabnormal i t i esofthe urea cycle have beendescribed(Walser, 1983), each involving a deficiencyor defectof apartiCUl a r enzymein thecy c l e. TheenzYIlopathie s can lea dto hy pera rnmone mia , or nithinemia , c:itrullinemia ,

argininosucc inic acidur ia and argi nine mi a depen d i ngon the spe c ifi c enzyme deficienc y. Someot the s eureacyc l ediso rders have been tre :t.t.edby feedinglow nitrogendiets,often sup pleme n t e d with ar g in i ne orcit ru ll i ne (Shi h ' Efron, 1972 ).

Brusilow (1984) and coworkersha ve showntha t arg i nine andsodilJu benzoat e supp lementscan improve the ClinicalSymp tOIlSof

11

TABLE 1.2

aener tc r ai. Eff ectsof Ar ginine inDiffere ntPathologicalCondit ions

pat h ological condition Refere nce

Ure a cycle dis o rders (Walse r, 19 8 3; Br us!low,1984) Chronic renalins Uff i c i enc y (a vene e t c et aL, 1975,Tiziancllo

et ai ,1980) TPN so lutions (Hos pitali ze d (Fahey , 19 57) patients )

Loss ofle an bodymaea (We ins i e r at al , 197<:) ; Ba r'bu Let eL, (catabo licst a t esasobs e rve d 19 8 4 ; aaecui,1986;vr e e x, 19 8 6 ) duringsur gery andp:r:olange d

hospitaliz at i on)

Protein maln utrition (V!seket aL, 1986)

Sepsis, Tr auma and Injury (Barbu lt 19 86 : KirK& Bar bu L, 1990) (wou nd he a l i ng)

Thermal burns (Saito et; aj , 19 8 7 )

Reyelssynd rome (Sinatraet ak ,19 7 5 ; Delo ng &Glick, 1982)

Cancer (?) (Levy et aI,1954; Milner&

Stepanovich,1979)

12 patients (children) with carbamoylphosphate synthetase-X (CPS-I), ornithine transcark'lamoylase (OTC), axqininosuccinate synthetase (AS ) and argininosuccinate lyase (AL) deficiency. These children developed hyperamrnonemiawithin 15-68h after arginine deprivation .Nitrogen accumulated as ammonia or glutamine or both. Benzoic acid (sodium salt) supplementation helps in reducing nitrogen load often seen in patients with urea cycle disorders. Excess nitrogenin the form of glycine can conjugate with benzoic acid to form hippuric acid which can easily be excreted.Arginine intake (or citrulline inth e case of OTC deficiency) proved effective in reverf:d.ng acute hyperarnmonemic episodes. ornithine was unable to prevent nitrogenaccumulation inthe s e enayee-cer.tctencystates. Thus, these workers concluded that arginineis an indispensable amino acid forchildren with urea cycle enzyrnopathies, whereascitrullineis indispensable for CPS-I or OTe deficiency.

ReyeIS syn\3rome

In children withth i s rare disease, there are abnormalities in cerebral and hepatic functions (Reye et aI, 19 6 3 ;Snodgross&

DeLong,1976).One of the abnormalities that is commonlyseenin these children is elevatedblo od am1;lonialevels. This is mainly due to decreasedactivitiesof the mitochondrial enzymes carbamoyl phosphatesynthetase-I and ornithine transcarbamoylase (Sinatra et at , 19 7 5 ) . Serum arginine and citrullinele vel s are markedly depressed in these children . citrullineand arginine supplementation ha v e been proven beneficial in reducing the

13 elevat edbloodammonialevel s (DeLong' Glick , 19 82 ) . 'atientson1;0 1:&1paren teral DUtrit,1oD (TPN)

In somehos p i tal i z ed patients , thereare situa tionsin whic h their nutrition Is prov idedby TPNsoluti ons which usuall y co nt a i n eit her proteinhydrolysates or purealllino acids.Pr ote in hydrolysates freque ntlyconta inappreciablequantitiesof

~1Il)Ilonia. If suchsol utions , withinadequate amoun tsof arq!n! ne, are admi nisteredto patients, hyperammonemia may ensue. Fa he y

(1957) demons tra te d thatinfu s i ons oflarg e quantitiesof amino acid s (ca s ein or fibrinhyd roly sate s) inhumans caused hype r ammone mi a and evencoma. These effects werereversedbythe additionof argininetotheaminoac i d solutions. Heirdet a1 (1972) re po r t e d theoccurrenceof hyperalllmone mia and incr eased oroticacidu riainthreeinfants who were in fus edwithasol ut ion ofcrystallineamin oacids withoutargini ne that was relati vel y freeof ammoni a. Infus i o n ofla rge dosesof essentialaatnc acids without argini neled to depressedle ve lsof ur e a cycl e intermediatesin the blood and hypera mmonemi ain two children

",ith ac ut e renal failure (MotiletaI, 1980). Rep letionof hanbodyma ss

One of the major cl inicalcomplicationstha t aris e s during pro lo nge d hospital i zat i on ofpatients is the los s of lean body mass (Wei nsi er et aI , 1979 ). Thi smay bedue to reducedpr ote i n int akeduetolos s of appe ti t e and increasedmus clepro t e ol y s is or decre a s edpro teinsynthe s is. It is knownthat endogenously synthe s izedargini ne is ut ili zed fCor muscleprot", insynthes is

"

(Featherstonet aI , 1973) . It has been shown tha t ra t s fed a ve r y low pro tein (O.S%:la c t al bumi n) diet for14weeks lost 40\

of theirbod y weight and were in negative nitrogenbalance (Karl at aI , 198 1 ).When the s e animals were rafed amino acid diets containing varyingamountsof arginine(0.0,0.75&1.5%:) for14 d, the y gainedbody weight and werein posi t i ve nitrogenbalance.

This increase in bodyweig ht gainand ni t r og e n balance was far higher in animals fed 0.75and1.5 %: arginine thanin tmose fed0%

arginine.'l'he s e cesuttis suggest that arginineis requiredfor replenishmentof leanbody mass and forth e maintenanceof nitrogenbalance .No attempts have been made to examinethe effect of arginine inre pleti nq lean body mass inhospitalized patients.

ChronicrenalinsUfficiency

Implloirme nt of endogenous arginine synthesis canoc cur in patientswit hchr on icrenal insufficiency. In adultrats with chronic re na l insufficienc ybotha decrease in plasmaarginine le ve l s anda decrease intheincorporationof argi nine inmuscle protein have been observed (Cha net aI, 197 4 1 Swenseid et e L, 1975;Wang et aI , 1977).The s e resul tssuggest tha t there is decreased end ogenousarginine synthesis and thusprovidethe basis for essent iality of argin inein patients withchronic re nal failure. In patients with chro n icrena l LnauffLcLe nc y, re nal argininerelease is greatlydepressed (Tizianelloet a L,1980).

Th i s suggestsanee d for dietaryarginine.

seps i s. trauma. the rma l burnsandenhancementot immune

~

The beneficialeff ectsofar g inine suppl ementatio n in clinical situationssuch as surgery (p re-and post-ope rati ve ), sepsis, trauma, thermal burns, andals o insti mul a t i ng the responseof the immune sys t e mha vebeen recentl y de s c r ibed (Barbu l, 1986; Kirk&Barbul, 1990;Saitoet ei , 1987; Dal yet aI, 1988).

The importanceof argininesupplementation on recove r yfrom tra uma was firstreportedby Seifter et al (1978).These workers wounded rats (200-300g) by makinga7-cmpa ra verte bral inc isi o n throughthe skinand thepa nnic u l us carnosus•The ratswere then placedon amino aciddietscontai n i ng either1%arginine or devoidofar gin i ne. The controlswerefedlabo r ato ry cho w(1.8%

arginine) .Animals on arginine-deficientdietgrew poorly (pre- (1.8va,7. 0 g/d ) and post-(lve , 4.3 g/d)in jury ) and showed impairedwound healing compared tothos e fe d arginine.Additi on a l supplementationof arginine (1%argininein drinkingwater) to in j ur e d animalS , decreasedthe post-operative weight loss and inc r e a sed hydroxyproline deposition (aninde xof collagen synt he s is ) at specificwound sites . From the s e observations, they concludedthatar g i nineisessentialfor thesynt he s isof the increased qua nti tyof collagenrequired for woundhealingand it decreasessome of theneg ativ e aspectsofthe metabolic responses toinj ur y . Similarly,Pui andFisher (1979)

demonstratedthe benefic ia l er recc of arginine supplementation on

bodywe i g ht gain and nitrogen retention in traumatizedrats.

Barbul and hi s associates (1977) have demonstratedthat l%:

argi ni ne:HC l supplem e nta tio nincreasedthymic weightand the numberofth ymi c sma lllymphocytesin normal rats and preve nted or reducedthe thynlicinvolutionthat occursinmice and rats sUbjected totrauma.

Th ebe n eficialrole of ar gini neinwound he al ing in humans has alsobeen re c e ntl ydemo nst ra t ed by Ba r bul andhis associates (1990). Humanvoluntee rswere sUbjected first to wounding (subcutaneous incision (S-cm) in the rightdeltoid re g i on ) and thenwe r e givendailysupplements of 30 g argin ine:aspartate

(n=1 2 ) or 30 g argin i ne:HcI (n=12) or placebo (n=12) for 14 d.

The dep os iti on ofhyd r oxy pro line at the wounded site (ond14) and alsothe immunerespo nse of peri phe ral bloodlymphocytes (in Y..i:t..J:2)tomitoge n i c stimU l i (concanava linA and

phyto hema gglut ini n ) was assessedon do, 7, and 14. Increased hyd r oxy pr ol ine depos itionatthe wound edsite and enhanced lymph oc yt e mit ogenesis we r eOb s e rve d only inthose given arg i nine supplements. It shouldbe no ted tha t in their studies (Barbul et aL, 199 0 ) , ar g i ni ne intake (30 g) can contributesi g n if i c ant l y toni t r og en loa d.They didnotemp l oy proper controls andshould ha v ecompa r e dthe ir resul tsto thosegivenan is onitr oge nolls loa d. Argin inesupplementsha v e proven effectiveinimproving ni t rogen balance,elevat i ngplasmasomatomedin Clevels (an indicato rofgr owt h hormone secretion) andenhancing immune responses in surgica l patients (Dal y et ei,1988). Intakesor

arginine (2' of total energyintake) havebeen showntoimprove me t a bo l ic and bmunefunctionin post-burncondi tio ns (Sai toat at, 1987).

Barbul andcovo r ke r s (1983 )fr omthe i r studie s with hypo phy s ec t.omi zed ratssuggested thatan int.a c t. hyp othal a lllic- pit.uitary axis isnec e s saryfortheben efi ci al ef f e c tsof argininese e n in traumatizedanimal s . studie s ofth eef f ects of excessarginineon t.he immune system have indicatedthat arg in ine act.lonmay be mediatedvi a the enhancement of nat ura l killer activi t.yandmonocyte-mediatedcytotoxi cityintheho s t and its di rect effect onthe tumor itself (Moriguchi et aL, 1987). In tumor-be ar ing mice, arginine supplementat i on (1')enhanced int erfer on- i nd uc ed na t ur al killerce ll activ ity, lymphok ine - activatedkillercellge ne r a t i o n,lIa crophage cytotoxicity and extendedthemediansurvival ti me of the s e anima ls (Re yno l ds et al, 1990).

FroIlthe discus sio nthus far, it is clear thatendog en ou sl y synthesized argininedoes no t ee e c the re qu ir ement for growt h, tormaint enan c e of nitrogen ba l a nce, and forJli nim a l orota te excretioninvarioussituations. Also, it isevident that argininesupplementation ha s a bene fi cialrole incertain sit ua tions.

Biochelllical function, cf arg inine

Argini neserves manyimport ant me ta boli c functions andthe s e arc summarized inTable1.3.

TABLE 1.J Bioche mical Functionsof Arginine

1. Arg in i ne is re qu ired forprotein synthesis.

2. Arg i ni ne is re qu ire dfor ammonia detoxificat i on (a n intermediateof ur ea cycle).

3. Ar g i ni neis a pre curs or fo rcr e a t ine synthesis (role in energymetabolism).

Arginineserves asaprecursorfor the synt hesisof polyamlnes (rolein celldivision, tissuegrow thand different iation)•

5. Argi ni ne hasthymotrop i c activity (role ininjury or trauma ,wou nd heal i ng, sepis and thermal bur ns) . 6. Argininem~diat esthe cytot oxiceffectsof macrophages

andpolymorpho nu c learneutrophils .

7. Endo t he lium-der ive d re l a x ing factoris nitricoxide(NO) which is synthesized fromargi nine (NO activatessoluble guanylate cyclase leadingtoa var ietyof functionsin different cells).

18

..

Argi ni ne is a norm a l co nstitue ntat proteins.ateeene s, whichare involved\lithnucleic acid interactions, are highly basic pro tei ns asthey areenrichedin arginineand lys ine.

Ar g ini ne isre quired for proteinsynthesis inbothprokaryotic and eUkaryoticcells.

Arg i n i n e alsose rv e sas anin t ermedia t e Inthe ur e a cycle and is involved. inammoniade t o xifi c a t i o n . Theimpo r t an ce of argi ni neinammoniadetoxifica tionis clearlyevident in patients with inhe ri tedurea cycl e dis orders as pre v iouslydi s cu s s ed. Arginine servesas a amid i no gr oup donor forthesynt hesis of cre atine, a majorbuffe r re s erveof highenergyphospha tefor rege neration ofATPin muscl e. Ar g i ni ne alsofunc tion s asa pre cursorfor thesynt hesis of polyami nes, whichappe arto play animpor tant rol e in cel l division, tissue growth and differ entiation (Pe gg&Mc Ca nn, 1982).

The endotheliulIl-derivedrela xing factor(EORF) is NO andis synthe siz ed fromL-arginine (Ignarroat aI,1987; Palmeretat, 1987). Nitricoxideisknown to have profoun dbiological ef fec ts

(Monc a da etaI , 1989; Igna rro , 198 9 ) . The variou s biological actions of NO inc lude endot he l ium-dependentrelaxa tion, cytotoxicity of macrophages(Hi bbset aI, 1987) ,

polymorphonu cl ear neut rophi l s (Rime leet aI,198 8) and cell-to- cell communicationinthe centra l ne rvous sys t em (xne v r esat aI, 1989 ;Bredt , snyder , 198 9) . Nitri c oxid e is kno wn tomod ul a te vasod ilat ionandarterial blood pr e ssur e andalso to inhiblt plate l e t agg regati onand adhesionof pla telo;!ts to endothe lia l

20 surface(Radomski et ea,19 9 0 ) . Thus, NO mimics the actions of vasodilato rssuch as glycer y l tri n itra t e whic hhasbe e n inuse cl inica l ly to decr easevasc u la r re sistan c e and blo od pressure.

Ev i denc e thatha sbeen presentedinthel i te r ature ind i c ate sthat the L-argini ne : NO pathwa y is awi d e s p r e a d transduc tionmecha nism fortheactiv a tion of the sol uble9ulmylat e cyclase le adingtoa var i etyof functio n s indifferent cel ls (vascu l ar endothel i al cel ls, neu trop hil e , macropha qes, brainsynaptasomes ) (Monca daet aI , 1989).

The fo cusof thi s re v iewha s beenon the importa n ce of ar gi n i ne indifferent sp ec i esand in differen t conditio nsandan itsfun c tions inmetab oli s m. There f ore, itis clea rlyimportan t to unde r s ta nd theneedfor endoge nous arg i n inesynthesis . Also, it isimpo rtantto understan d the me t a bol icfa t e of argini ne.The remai nd er of thisrev i ewwillfocus on the biosynth e s i s and brea kdown of argi n ine.

Need forendoge nou s arginine synthesis

The syn t hesis of the prec ursor of polyamines, put r e s c i ne fromor n i t h i ne (pegg&McCann, 198 2) , doe s not have an overall ef f ect on ar g i n i ne utilizat ion.Also, the ureacycl e doesnot ha ve anet eff e c t on ar gin i nedegrad ati on . Onemolecul eof ar gini ne isproducedforev e r y o ne removed in the opera t i on of th i s cyc le (Kreb s &Henseleit, 193 2). However ,ornithine

21

generated by theactionofar gi naseon ar gi ni ne canbe:fur t he r me ta bol i z e dviaornithine am.inotransfe ra s e (OAT) . In fact, the ma j o r meta bo l ic fateof ar g in i ne inthe liver iscat.aeofLsavia arginaseand or nithineamino t rans f era s e. It has beensugge s ted in the litera ture (MeGiv anetaI, 197 7;He ns le e ' Jones, 19 82 ) that OATispri mar ily involvedinthe catabol1 t'1l11of orni t hineinthe liver. Re cen t ly, Alo n so &Ru bio (1989) demonstrat e d that even un d e r conditi on s of arginine depriv ation,th ere is cataboli sm of or ni thine inthe li ver, kidneyandmusc leof mice. In the ir st Udie s, tis sueornI th ine co nce ntrationswe remeasur ed (o ndB.

over a lO-hperiod) aft e r the admi nist ratio nof gabacu line (a po te ntia l suicide inhi b i t or of OAT) to miceadap t e d (7 d) to aminoaciddie tswithandwit hou t argini ne . It was observedthat theornit hinein thetis sue s accumulated, ev e n inanimal sgiv en arqinine -devoiddie ts.Theyestimated that atle ast 45 Jl.llolesof ornithineissynthe s ized andcata boli ze d dallyvia OAT in the mous edep r ived of arginine. Thi ssuggests that anet dr a i nof the endoge no us argini nepool can occurvia theOAT reactio n.Sucha drllinon the endogeno usargini ne poo lmayresultin impa innent In ur e acyciefunc tio n. The r efore , in orderto replenish liver ar gin i nepools, there isaneed for endoge no us arginine sy nthes is .

Minor drain on the endogenousargininepools

Ami nor drainonthe endoge nou sargininepool can occurvia the formati onofmetabolite s suchas creatine, ni t ric oxide and

22 polyamines.Thesemet21bolites have importantmetabolic functions. It shou ldbe noted thatornithine and citrullinegeneratedin the formation of guanid inoaceticacid (precursorof creatine) and nitricoxide, t-espectidveIy, canbe recycledto arginine. Also,in thetIver, the carbonskeleton of arginine ca n be either metabolizedto glucoseor completelyoxidized to CO2 and water. This depends on the fate ofth e glutamatethatis generated by the combinedac t i o ns ofar g i n a s e and ornithi neaminotransferase onarginine.

Enzymes inv o l ve dinarginine utilization

It is appropriate at this point to discuss the reactions and the enzymes involved in the utilizationof arginine (Ta bl e 1.4). I have discussed five enzyme s : arginase,ornithine

aminotransferase, L-arginine:glycineamidinotransferase (or transamidinase), ornithi nedecar boxylaseand nitric oxide synthetase . The activities and the distribution of these enzymes are presentedin 'l'a})1e 1.5 ).

Arginase [E.C.3.5.3.1} catalyzesthe conversionof arginine to urea and ornithine.The la r ge negative freeenergy associated withthisre ac t i on makes it irreversible.The reportedJ<.,values for arginasefr omra t liver and kidneyra nge anywhere from 1-20 roM.The enzyme hasan absolute re qu i r e me nt forMn+z (Garqanta&

Bond, 1986). Ar g i na s e is widelydistributed(Herzfeld&Rape r, 1976). It is abundant in the liv e r, primarilyfunctioning inthe

23 TABLE 1.4

Enzymesand Reac tion s co nt ributing to Arginine RelllOYal.

1. Arginase (Al

Arginine+"zO

--->

Arginine+Glycine<- - - -> Guanidinoacetic acid-+Ornithine 3. Ornithineaminotransferase (OAT)

Ornithine+a-ketoglutarate

< --->

4. Or ni thinedecarboxylase (ODC) Ornith ine

--->

Argini ne -->--> -- >--> --> --> Nitric oxide_c+ citrulline

• Convertedto creatineby guanidl.noacetate rnethyltransferase . bCan be conver tedto spermidineand spermine(p o l y a mi ne s j .

0;Formationof nit ricoxidefr om arginine involvesmanysteps.

-3':t'

~= ; ::;

--- ..

an

gi ^2.

:!:l=

~~~

- ^{~ =}

~!ll

~.:

~ _~O'!^~~

~

l~

l

~

:

~~ ~

~ 5~

:s

..

~

l~ ^: _~ ^{3 i! ~} _{!i ]}

~i.:.1 t~:.

Ii

^{~~ ~}

^{.. ~ !jg']}

f~ c:~i:

t:·~

! ~:;~l

;:

1 m

!l

.. _{~ cc ~}

~ i

~

i~i ~

.

.

l~ .

~m

r

i

f" :~'g ~

~ g~~.ll

i~!

! ::E:

~!j ^{e !i '"J}

! ~~!

^{!h H}

j~! ~~i~

2S fot1llation of urea in ureotelicanimals .However, it is also present insignIfIcant amounts in other tissues (Herzfeld&

Raper, 197 6 ; Table 1.5 ).

Recent lyLevillain and coworkers (1989) have shown that re n a l arginaseis almostexclusively located inthe rat proximal straight tubule (6 3 segment). They suggested a role for arginase inth e maintenance of the medullaryur e a concent rationgradient.

Arginaseexistsin multipleforms in rat, mouse and human tissues (Skrzypek-Osieckaet aI, 1983; spolarics&Bond, 1988,Zamecka&

Porembska, 1988 ). There are two main forms of a rq Lnaaein rat kidney andli v e r. In kidney, arginase A4 (acidic protein) is the mainform, accountingfor95-98% of the activity whileA1 (alkalineprotein) is present in trace quantities.The A4form ofthe enzyme is almost exc.luaIveLy present inmitochond:-ial matri x and Alis localized in the cytosol (Skrzypek-Osil~ckaet aI, 198 3 ). In contrastto the kidney, arginaseA1 is themajor form in theli v e r and is cytosolic .Immunological studies have demonstrated tha t the kidneymajor form (A4l does not cross react with live r argi naseAI' The differencesbetweentheto.wo forms of the enzyme in theirpro pe r t i e s and subcellUlar location may indicate differe nt functions for the enzyme . The most 1 ikely metabolicrole of arginase in kidney is to provideornithine for the formationof pr oline or glutamateor po Lyaminea or all three.

ornithine amino transfe rase (OAT) [E.e. 2.6.1.13 )is involved in there v e r s ibl e conversionof ornithineanda-ketoglutarateto L- gl ut a mate-y- s emi alde hyd e and glutamate(Ta b l e 1.4 ).

"

L-glutamate-y-semialdehydeis an unstable intermediate that undergoes spontaneous conversion to Al-L- p y r r ol l n e - 5- c a r bo xy l a t e (P5C) which in turn can be converted to proline by P5C-reductase (Jones, 1983).The formation of ornithine, glutamate or proline in different tissues varies depending on a specific tissue requirement. In the liver, it is Lnvo rved in the net catabolism of ornithine as discussed~arlier. Synthesis of ornithine from glutamate occurs in the intestinal mucosa, wl1ile no synthesis occurs in liver or kidney homogenates (Henslee&Jones, 1982).

The activity of OAT is highly dependent on pyridoxal phosphate and is inhibItedbythe general aminotransferase inhibitor arninooxyacetate. Activity is high in the kidney, liver, and small intestine of the rat (Her2.feld&Knox, 1968 ;'I'able1. 5 ) . OAT is localized in the mitochondrial matrix in rat kidney (Passarella et aI, 1989). However, its cellular location in the kidney has not been identified.

L-arginine:glycinellmidinotransferase (or transamidinase) [EC 2.1.4.1] is involvad in the formation of guanidinoacetic acid, a precursor for creatine synthesis, and ornithine trom arginine and glycine in the kidney, and was first id e n t if i e d by Borsook&Oubnoff (1941a).Guanidinoacetic acid synthesized in kidney is then transported to the liver where it is methylated by guanidinoacetate methyltransferase(E.C. 2.1.1.2]to yieldthe final product .creatlne (Borsook&Oubnoff, 1940) which plays an important role in energy metabolism. It was mentioned earlier that creatinine excretion may serve as a drain on endogenous

27 a;rginine pool (V!sek, 198 6). This assumption is not correctin th e sensethat orni thinegeneratedinthere a c tion catalyzedby glycineamidlnot ra nsferase ca n be recycledfor arginine synthesis.

L-arginine:g lycineamidinotransferaseis predominantly found in kidneyand pancrea s of ratwith some activity inot he r ti s sues (Van Pilsumet ak ,1972) . Multipleforms of the enzymeexi s t in rat and human kidne y andthey are similarin their properties (Gr o s s at ai, 1988). Glycine amidinotransferaseis localizedin a subpopulationof proximal convolutedtubules,probably pr oxi ma l st r a i g ht tub u les (MCGuire et aI, 1986). It ssu b cellular Io ce c Ion is in the innermitochondrialmembrane, probablyon the external side (Magriet al, 1975). TheK", for arginine and VIM~of pu r ifi e d kidney enzyme are 2. 8mMand 0.39~mole/min/gprotein, respectively (MCGu ire at al ,1980).

The firEcstepin the formation ofpolyami nes iscatal yzed by ornithinedeca rboxylase(ODC) [E.C. 4.1.1.l'f ] .It catalyzes the irrevers iblecon vexs Icn of ornithineto putrescine. Putrescine formedin the reactioncan be furtherconvertedto spermidine and sperminewhich areUb i q u itou s l y present (Pegg &

McCann, 198 2 ). Thewide distribution ofar g i n a s e andODC (only intraces), suggests that the true precursorfor polyamine formation is arg i nine.The ar gininerequirementfor polyamine formation is veryminimaL DOC has an absoluterequirementfor pyridoxa l phosph ate(Pogg &McCann, 1982 ). The rat liver DOC has a half-life of10-15mi n and the enzyme islo c a t e d inthe

28

cytosol.The cellularloc a t i o nofODe in the kidneyha s not been determined.ODe can be inhibited irreversiblYby its analog, DFMO

(a-difluro-methylornithine) (Ta b or &Tabor,1984; Peqg &McCann , 1982 ) •

Theformation of ni t ricoxide (NO) andcit r u l li ne in a ser ies of reactions fr om arginine iscatalyzedby acomplex enzyme,NO;Jynthetase[E.e. 1.1 4.23] (Tabl e 1.4 ). Nitricox i d e formed in the reaction ishighly unstable (h a l f-l i f e of only3-5 s) and is rapidlyinactivated by molecular oxyge n leading to the fo r ma t ion of nitrite(NOz") and nitrate (N03") (Ignarr o, 1989) . Nitrat e exc r e t i on (Leafet aI,1989 ) does notac t asadrainon endogenous argininepools , as citrullineformed in the reaction can be recycled to arginine (a e e ea et ai , 19901Hecker etal, 1990).

Nitric oxide synthetasehas an absoluterequirementfor NADPH and is lIcti v a t e d by ca+2/calmodulin. It s activity has been reported ina widenumbe r of cells and tissues (Tab l e 1.5). The formation of nitric oxide is specifically inhibitedbyan arginine-analog ,NG-monome thyl-L-arginine (L-NMMA) whichhas been usedas a toolto understandL-argini ne:NOpathway inva r i ous biological systems.

Transportof argin ine

Transportof arginineintocel lsof differenttissue s has beenstudied. It sharesthet:r a ns por t system wi t h other cationic

2. aminoacids (lysineand ornithine) and the system involved isy', which is not dependent on sodium.This system has been characterized in hepatocytes and in the brush border membranesof the kidney and intesti ne(White, 1985). There exists a clinical condition in which transport of arginine is defective. Lysinuric- protein intolerance is a rare inherited disease with a defect in basic diamino acidtransport, including intestinal, re na l and hepatic transport ofly s i ne , arginine and ornithine. Elevated blood ammoniaand decreased plasmalevels of arginineand ornithine and also an inc r e a s e d oroticacid exc r-etiicn have been observed in the s e pat.Lerrt.a (Rajantie, 198 1).

Role of di f f erentorgansand the impo rt a n ce of endoge n ous arginine synthes is

From the discussion thus far, it is clear that in some animal species.argini necanbe synthesizedendogenously and that theli ve r does not releasearginine for extra-hepatic utilizationas arginine is catabolizedby the actionsof argina se and ornithine aminotransferase, respectively . The question that arisesnow is this: whatis theimpo r t a nc e of thenet endogenous argininesynthesisand wh e r e does it occur? In thisrespect, I ha ve discussedtherole of kidneysand int e s t i ne . The enzymes and the reactions involved in argi ninesynthesis are presented in Table 1.6.

It is clear from the studies of Featherston and cowor ke r s

30

TABL E1. 6

Enzymes and The Reactions Involved in Net Argininesynthesis

Enz ymes andReac tionsof Net J\.rgini ne synt h esi s 1. Carbamoyl phosphate synthetase-I{a mmo n i a ] (CPS-I) Ammonia+Bicarbonate+2ATP-- - >Car b amoy l ph o s ph at e +2ADP +P_t+2H"

2. Ornithinetranscarbamoylase(OTC)

carbamoylphosphate + Ornithine<-- -- ->Citrulline+ PI 3. Argininosuccinatesynthetase (AS)

citrulline+Asparta te+ATP--->Argininosllccinate+AMP+PPI 4. Argininosllccinatelyase (AL)

ArgininoSllccinate+H20

--- >

Jl (197 3 ) th atthe kid ney is a majorendogenoussou rc e of arginine. Inthei r experiments ,ani malswere in j e c t edfirst with t'4 C] Ure i d ocitru ll i neandthe n thekidi-eysorlive r s orbothwere ligated . They the nme a s ure d the incorporatio nof {14c] Gua n i di no- arginine into muscle pr o t e i n. The incorporationoflabel l e d ar g i nine intomuscl e protei n was greatly red uce d inanima lswhen kidneyswer eligatedwhi le liga tion ofli vers was withoutany effect.From theseobser v at i on s they concl u d edth atthe ki dn e y is a maj or biosyn t hetic sou rceof arginine. It is knowntha t ki d ne ys of norma l an imals re move citrul linefrom the blood and release it as argini ne (Windmuelle r &Spaeth,1981 ; Brosnan et aI, 1983) . Th e ac t ivities of carbamo y l phos patesynthetase-I [E. C.6.3 .4.16 J (CP S- I ) and ornithi necar ba moyl transferase [E.C . 2.1.3.3] (OTC) areei ther lo w or absent inthe kidney (Joneset aL, 1961; Raij man , 1974;Table1.5). Thissungeststha t the ki d ne ys ca nnot synthesi zecitr u ll i ne.An obviousq...eatIon thatarisesis this:

what isthe source ofcircUla tingcitrulline forren a l arginine synt hes is?

Sources of oirculating citrulline

Theevidencepresen tedinth el~teratureclear lysuggests th at the intestineplaysanimportant roleinthe contributionto cir cUlatingcitru l line . Wind muelle r&spaet h (1974; 1978 ; 1980 ) have demons tratedin studies with isolated, va s c u l a r l y perfused prepara t ions of rat inte s t i n e and with autoperfused segmentsof

32 rat jejunum.inY.J..x.Q. that thesmall intes ti ne rele a s e s citru lline (6.5 J.lmoles/h/ l OO 9body ....ei gh t) intoth e bl ood. This cit rulli ne isnot appreci ably takenupby theliver . Th i s conc lusion isbas edon the studies by Windmuel l e r&spaeth (1981).In the irperfu si on ex p erimentswit h rats , a oontInuous por t al inf usion ofci t r ulline (38.0nmoles/min ,overa per i odof 150minand about 40pas s e s ). accumulatedinthe perfusate ata linea r rat eof35.2 na cte eymdn , Thushepati cuptake wasonly about n.Although there is no dataavailablein the lite ratur e on ci t ru ll inetra ns po r t inthe liver, itcanbe specu lated that liver cells tr an spor t this aminoacid poor ly.

Ki d ne y sre adilytake up citrUlline. By emp loying themeth od of continuous tub ul ar microperfusion (inYiY.Qet in~l of rat proxi mal convo l utedtu bules, Ke t t ner and Silbernagl (1985) ha ve shown the existenceof two transpor t systems for citru l li ne in thebrushborderofth eproxima l tubule, onewi t h a high ca pacity and lo waffini ty, andthe othe rwithLew ca pa c i tyand high affini ty . Thecooperat i ve inte ractionbet wee n the s etwo systems provid efo r avi r t ual l ycomp l e terea bsorpt i on of filt e r ed citr u l line at physiological as wel l as elevat e dlevelsof citru l line.

Theque s tion thataris e s nowis this : what isthe sourceof int est i na l cit rUlline and howisitsynt hesized? Studiesby Windmueller and spae t h (1974, 1978 &198 0 )have demon strate d that citrul line is formed fromgluta min e , a maj o r meta bolic fuel for theintes tine . Inthe i r stUdi e s , perfusion of isol a ted rat

JJ small intesti ne withrat blo od (conta ining gluta mi ne, 150 /-I1I\01es ofN), gl ut amin e nitrogen was incorporated int ocitrul line (34%), alani ne (32%)I ammonia (23%) and proline (10%). From th e s e observ a t i o n s ,they concludedthat the major me ta bolic fat eof glutami n e ni t r ogen in th e int estineis the formation of citrull i ne.

Th epat hway of cit ru l linesynthesis from gluta mine has been describedbyJones (1983).Glutami ne ishydroly z e d to gluta mate by glut a mi n a s e. Glutama teis the n convertedto

'l'-g l utamate-y -semia ldehydeby th e action of pyrroline -5 - carboxylate synthase (Wakabayashi&Jones 1983). L-g l u tamate-y- semial de hy deandglutama teareconvertedto ornithineand a- ketogl uta r a t e , a revers i b le re a ct i o n catalyzedbyorni th i ne aminotransfera s e (Hens lee&Jones, 198 2).Orni th ineformedcan be conver tedto citrulline by the action of ornithi ne

tr an s c arba mo yl ase (OTC) pr ovide d carbamoy lphos phate is available.

Th i s is provided bycarbarnoy l ph opha t e synthe tase-I. BothCPS-I andOTC are mit oc hond ria l mat ri xenzymesandth e re a ct i ons catalyzed by the s e enzyme sare shownin 'I'able1.ti.These enzymes ar epre s e nt in significantquantities in the intesti ne ('r ab l e 1.S) . Cohe net al (198 5 ) havedemonstratedtha t the true subst ra te forCPS-I is ammoniaand itsK...for the purified enzyme and for the is ol a t ed intactmi tochondria (rat liver) are 0.038 and 0.013mM,respe cti ve l y . CPS- Iha s an absol ute re q u i r emen t fo r a cofact or . N-acety l glutarnate (NAG). N-acetylqlutamate is syn t hesized fromacettl CoA and gl utamateca ta lyzed by another

34 mitocho ndr i almat r ixenzyme, NAGsy nt het a s e [E.C. 2.3.1.1.]. Ar gi n ine al l os t e ricallyregu latesthe syn th esisofN- acety lgluta mate (Wals er , 1983).UnlikeCPS-I, CPS-I I util i zes glutamineas a subs trate and is involved in thesyn thes isof pyr i mi d i ne s (Ke sner,19 6 5).The~of OTe fo r carbamoylphosphate andorn ithine are 0.2and 0.09 mM,re s p e c t i vely (Marsha l l Ii Cohen , 1972).

Importanoeof intestinaloi t r u l li n e syn t n e s is

Ha vin g discussed th e sou rceand the pat hwa yof intest inal citrullinesynthesis, it isessential to understand the importa nce of its role in different metabolicand patho l o gical situations . Evidencein thel i ter at ur e suggestsan impor t a nt rol e of the intestine inci t r u ll i nesynthesis.Stud iescarried out by Hoogenraadandhisassociates (1985)demonstrated thatthe gr owt h of ratswas severely depr e s sed whentheir intestinal citrul l inesyn th es iswas specifica l lyinhibi ted.They brought aboutth i s in hib i t ionby admi nis te ringGly-Gly-PALO

(qlycylqlycine -&- N-(phosphonacety l)-L-ornithin e ),a powerful and specificinhi bi t o r ofintes tina l ornithi ne transcarbamoylase ,in the d.:inki ng water of ratsfed dietscon tainingeit herar g i nine or ornithi ne (ar ginine -deficient). In animalsadminis te redwith the inhibi to r, serumar ginine andcitrul line le v el s wer e also gr Gatly red u ced.Theinhibitionof growth was easilyreversedby dieta r y citrulline . ca r nivoreslikecatsandferre ts ,cannot

synthesizecitru lline due to thelow act i vi tie s of theintes t i na l enzymes,py r r oline-5- c a r boxylate synthase(Rogers&Phang, 1985 ) and ornithi neaminotransferase(Morris,1985).Therefore, the s e anima lsstrictlydependondietaryarginine.The importance of dietarycitrulline in correctinghype ra mmonemi aince r t ai n enzyrnopathies(CPS-!&OTC). in neyete synd romeandin lysinuri c patients was discussed earlier.Fr om the discuss ionsso far, it can be concluded that the int e s t i n al /renal systemis es senti al for endogenousargininesynt hes i s .

whatisknownabout renal ar ginine synt hes is?

The conversionof citrulline to arg ininein the kidneyis dueto the action of argininosuc cinate synt he ta s e (AS) and argininosucc i natelyase(AL) (Ta b le1.6). Argininosuc cinate synthetase (E.C. 6.3.4 . 5] catalyze sthe conversion of citrUlline , aspa r t a t e and ATPto argininosuccinate, AMP and pyrophosphate

(PP_j) . This reactionis dr i ve n effectivelyin the for ward directionby the removalof PPjby cyt o s olic inorga ni c pyrophospha tase. The conversi onof argininosuccinatetoarginine and fumarateisca t a l y z ed by argininosuc cinate lya s e [E.C.

4.3.2.1]andthe negativefree ene rqy cha nge associated with this reactiondrives i tinthe direction of arginineformation. The apparent~va l u e s of AS(hog kidne y ) for ci t ru lli ne . aspartate are 0.044 and0.0 38mM, respectively(Rochovansky&Ratner, 1967 ) andthat of AL(b e e f liver) for argininosuccinateis0.15 10M

3.

(Ratner et aI, 19 '3). 80t h AS and Atare predominan tlyfoundin thelive r but are als o found in signif icantquanti tiesin k.idney.

SOllie activityot thes e enzymesis alsoobservedin brainand spleen(x ae c et aI,19 7 6: Ratner&Murakaml-Murofushi, 1980: 'I'&ble 1.5). Both ASand AL arepre sentinthecytosolof li ver cel ls,but their cellu lar andsubc e llular location in the kidney is not well characterized.Szepesl at a1 (1970) reporte dtha t argininesynthetaseactivi t y (measuredas ASplusALI vas predom i nantl yfou ndwi t h i ntherenal med u lla . Mo rr isetal (1989) have recentl y sho wn that the mRNA's for AS and At arelocalized inthe re nalcortex. These tworep ortsar einconfl ictand thus definitive in f orm a t i on is neededon their exact loc a tionin the kidney .

upansyeredquntion.

Frollthe discussions sofa r, it can bese e n thatno previ ous attemptshavebeenmade to localizeprec i s e l yth e site of argininesyn thes isin the kid ney.No informationis availab leon there gu lati on of renal argininesynthesis.inx.iY2by citrul l ine concentra tio ns or by dietary arginine.

L To deter1llinethe cellul a r and subcel l u la r localiza tion ot theenzyseeof arginine synthes isintherat kidney.

2. To eeeemtne the dependence of arginine synthesison citrulline conc e n t r a t i o ns.i.n..-Y.i.t.!;:QandinY..iY.2.

3. Todetermine whethe r re na l argininesynt hesiscan be regulatedeithe r bythelevels of dietaryarginine or dietary protein.

J7

"

KATER:IALS AND METHODS

Adult ma le spraque-nawjey rat s [Cha r leli Ri ver, Montreal, Canada)initially....eighing 300-4509 were usedforallexpe rime n ts.

They were ho u s ed individuallyincag e s in a roomkept at 24± 1°C witha12:12 h light:darkcycle. The li ghts we reoff between2000 hand 0800h. 'rho animalGhadAd.li!ti..t.wnacce ssto tap wate r and fo od .

Diets andfe edin g protoc ol

Animalsusedfor experimentsinCHAPTER3 and CHAPTER 4, we r e fed Purina rat chowiUt~. For the experimentsin CHAPTER 5, theanimals were fed for 7d, dietscontainingvarying amounts of arg inineor protein.Thecompositionof the ar g i n i ne (0, 0.5 and 2.0%) and protein (5, 12 and 50%) dietsarepresentedinTable 5.1 andTableS~2 ,respectively . The preparationand thefe e di ng cr these diets to ratsare expl ained in detail inCHAPTER5.

preparation of homogenatesfrom.different kidnet re gi ons

Ra t s weresacrificed by cervical dislocationandthe kidne ys were ra pi dly re moved , decapsulated and placed in ice-cold homog en izatio n medium (0.33Msucrose, 5mMHEPES, 1 mM EGTA(pU