List of abbreviations

Transcription

1 Contents List of bbrevitions... 4 Summry... 5 Összefogllás... 6 Introduction... 7 Rte of lipid peroxidtion in brin nd liver tissues nd the totl ntioxidnt sttus of blood plsm in developing chicks... 9 Introduction... 9 Mterils nd methods:... 1 Results... 1 Discussion Summry Chnges of some biochemicl nd ntioxidnt prmeters in plsm nd erythrocytes of pentthlon horses before nd fter exercise Introduction Mterils nd methods: Results Discussion Summry Effect of deferoxmine nd L-rginine tretment on lipid peroxidtion in n intestinl ischemireperfusion model in rts Introduction Mterils nd methods Results Discussion Summry Evlution of cid-bse nd ntioxidnt indices in horses operted on for colic Introduction Mterils nd methods Results Discussion Summry Overview of the new scientific results Publictions References... 4 Acknowledgements... 46

2 List of bbrevitions ABE ADP AMP ANOVA ATP CAT CK cnos DMSO EDTA FADH FR FRAP G6P GPX GSH GSH-Red GSSG H2O2 Hb - HCO 3 HOCl I/R inos IPPV LDH L-NAME LP MDA M4HN ctul bse excess Adenosine diphosphte Adenosine monophosphte Anlysis of vrince Adenosine triphosphte ctlse cretine kinse Constitutive nitric oxide synthse Dimethyl sulfoxide Ethylendimine tetrcetic cid Flvine denine dinucleotide free rdicl ferric reducing bility of plsm Glucose 6 phosphtse glutthione peroxidse reduced glutthione glutthione reductse oxidised glutthione Hydrogen peroxide hemoglobin stndrd bicrbonte concentrtion hypochlorite ischemi-reperfusion Inducible nitric oxide synthse Intermittent positive pressure ventilltion lctte dehydrogense L-nitrorrginine methyl ester lipid peroxidtion mlondildehyde Mlondildehyde+4hydroxi nonenl MnCl 2 MPO NADH NO NOS O2.- OFR OH..- ONO 2.- ONO 2 pco 2 po 2 PUFA RBC ROI SAT SOD SOP SV TAS TBARS TCO 2 TP TRAP UA vvs WBC XDH XO Mngnese chloride myeloperoxidse Nicotinmide denine dinucleotide nitric oxide nitric oxide synthse Superoxide rdicl oxygen free rdicl Hydroxil rdicl Peroxynitrit nion rdicl peroxynitrit nion rdicl prtil crbon dioxyde tension prtil oxygen tension polyunsturted ftty cid red blood cell rective oxygen intermediers Oxygen sturtion of hemoglobine superoxide dismutse shm operted Spontneuos ventilltion totl ntioxidnt sttus thiobrbituric cid rective substnces totl crbon dioxyde concentrtion totl protein Totl peroxide rdicl trpping cpcity Uric cid vörösvérsejt White blood cell Xntine dehydrogense Xnitne oxidse 4

3 Summry Free rdicl induced nd medited processes re present in gret vriety of physiologicl nd pthologicl pthwys. The bsic source of FR production is the respirtory chin where different oxygen derived free rdicls re trnsformed finlly to wter nd crbon dioxide. On the other hnd it ws observed tht FRs my induce such oxido-reductive cscde mechnisms tht cn dmge protein, nucleic cid nd ft molecules of the living cells therefore there re defence mechnisms tht involve numerous molecules nd enzymes (the ntioxidnt system) to control their production nd elimintion. The ction of certin bsic components of the ntioxidnt system is investigted in 4 trils in this study. In chpter one ge relted chnges of tissue lipid peroxidtion (LP) of liver nd brin, s well s plsm ntioxidnt cpcity of broiler chicken cockerels were investigted. Rte of LP ws found to be comprtively low in the liver nd high in the brin of the 1-dy-old broiler chicks. Incresed LP ws observed in the liver tissue on the 1 th nd in the brin tissue on the 21 st dy of life, the former ws ccompnied by concomitnt decrese of plsm ntioxidnt cpcity. The im of the study detiled in chpter 2 ws to exmine exercise-induced chnges of some plsm nd red blood cell biochemicl nd ntioxidnt prmeters in pentthlon horses. Blood smples were tken from horses before, immeditely nd 24 hours fter competing two runs of 1 minute of intense exercise over jumps. The pek intensity periods were preceded by 2-minute wrming up nd seprted by 2-minute brek There were elevted concentrtions of plsm TP, lctte, uric cid, CK, LDH nd FRAP (p<.5) in the post exercise smples compred to the preexercise smples. All prmeters returned pproximtely to the initil vlues fter 24 hours rest. Similr tendencies were observed in the chnge of plsm TAS nd RBC GPX vlues. Erythrocyte GSH nd TBARS concentrtions did not show ny chnge immeditely fter the exercise but decresed TBARS nd incresed GSH concentrtions were observed fter 24 hours rest (p<.5). Plsm UA nd FRAP vlues showed good correltion in liner model. There were opposite chnges in FRAP nd TAS vlues clling ttention to the fct tht ssessing the ntioxidnt cpcity by different prmeters cn give highly different results. In chpter 3 LP chnges during intestinl ischemi-reperfusion (I/R) in rt model with nd without desferrioxmine nd L-rginine tretment re discussed. The only significnt chnge observed in the shm-operted group ws the incresed intestinl SOD ctivity fter the ischemic period. In the I/R group significnt increse of intestinl M4HN concentrtion ws observed during hypoxi tht ws followed by similr chnges in intestinl nd RBC TBARS nd plsm FRAP vlues upon reperfusion. In the deferoxmine treted group the intestinl TBARS nd M4HN concentrtions were significntly lower while FRAP nd NO concentrtions were significntly higher compred to the I/R group. At the sme time RBC TBARS concentrtion nd GPX ctivity significntly decresed within group D. In group A the intestinl M4HN concentrtion ws significntly lower thn in the I/R group. Plsm FRAP nd NO concentrtion showed similr chnges to group D. It is concluded tht I/R incresed the LPO in the intestinl tissue nd ltered some prmeters of plsm nd RBCs, too. Desferrioxmine tretment prevented these effects, while the usefulness of L-rginine remins doubtful. The study described in chpter 4 ws undertken to evlute lipid peroxidtion chnges in red blood cells nd plsm of horses tht were operted on for ileus. No significnt chnge over time ws observed in ny prmeter in the control horses. Horses ffected with colic were presented with metbolic cidosis s red from significntly lower ph nd BE thn the control ones. Upon nesthesi horses hving intestinl disorders developed mrked mixed type cidosis. In spite of tht oxygen tension nd sturtion of the mixed venous blood showed significnt increse during nesthesi with pek t two hours fter the. Plsm uric cid concentrtions were some 1,5 times higher in the control group during nesthesi. Lte post-nesthetic vlues show opposite picture due to mrked increse seen in the colic horses. Though there re no significnt chnges over time of FRAP vlues in either group colic horses exhibited pproximtely twice higher 5

4 concentrtions thn did the control ones. Plsm NO concentrtion ws significntly higher in the colic horses before the opertion nd showed constnt decrese therefter. Erythrocyte TBARS concentrtion ws higher in the colic group t ll smpling times during the observed period. The highest TBARS concentrtion ws found 1 hour fter (ie.: minutes fter reperfusion). There were no significnt ltertions observed in the GSH, GSSG, concentrtions nd GPX nd SOD ctivities of RBCs though the ltter one followed tendency opposite to the TBARS concentrtion in the colic ptients. It ws concluded tht the intestinl I/R resulted in mrked metbolic cidosis nd ltered severl ntioxidnt prmeters of plsm nd erythrocytes. This indictes tht mximl effort should be tken to correct metbolic cidosis during the opertion of colic horses. Furthermore chnges in the blood LP prmeters cn serve s n indiction for ntioxidnt therpy during the opertion of horses with ileus. Összefogllás A szbdgyökök számos élettni és kóros folymt meghtározó tényezői. Az élő szervezetekben szbdgyökök keletkezésének legfőbb forrás légzési lánc, melyben különböző oxigén eredetű szbdgyökök lkulnk soroztos elektron felvétellel széndioxiddá és vízzé. Ugynkkor z is bizonyítást nyert, hogy szbdgyökök kifejezett rekciókészségükből ddón károsítni képesek sejteket felépítő fehérje, nukleinsv és lipid molekulákt. Fentiek okán káros htások megelőzésére z élő rendszerekben bonyolult, számos molekulát és enzimet egyesítő ún. ntioxidáns rendszer működik. Jelen dolgozt z ntioxidáns rendszer egyes elemeinek működésének vizsgáltát célozz, melyet négy kísérlet keretében végeztem. Az első fejezetben broilercsirkékben vizsgáltm máj- és gyszövet lipidperoxidációjánk, vlmint vérplzm ntioxidáns kpcitásánk életkorrl kpcsoltos változását. Megállpítottm, hogy npos korú broilercsirkékben lipidperoxidáció mgsbb szintű z gyszövetben mint májbn. Fokozott LP-t figyeltem meg májszövetben tíz npos, míg z gyszövetben 21 npos korú álltokbn. A májszövet fokozott LP-jével egyidőben vérplzm ntioxidáns kpcitás jelentősen csökkent értéket muttott. A második fejezetben bemuttott vizsgált keretében zt vizsgáltm, hogy öttus lovkbn, versenyterhelés htásár milyen biokémi és ntioxidáns-rendszerbeni változások lkulnk ki. A vizsgáltot lovk torkolti vénájából gyűjtött vérből végeztem, mit kétszer egyperces intenzív, kdálypályán teljesített ugrássor előtt, illetve után közvetlenül, vlmint 24 ór elteltével gyűjtöttem. A csúcsintenzitású munkvégzéseket 2 perces bemelegítés előzte meg, illetve kötötte össze. A nyuglmi helyzethez képest mgsbb (p <,5) TP, CK, LDH, húgysv és FRAP értékeket mértem versenyterhelés után közvetlenül levett vérmintákból. A 24 ór elteltével ismételt vizsgált lklmávl jelzett muttók ismét kiindulási értékhez közeli trtománybn voltk mérhetők. Az fent leírthoz hsonló tendenciát tpsztltm vérplzm TAS értékének és vvs-ek GPX ktivitásánk változásábn is. A vérplzm TAS és FRAP értékei ellentétes irányú változását figyeltem meg, mi rr figyelmeztet, hogy z össz-ntioxidáns kpcitást különböző módszerekkel mérve jelentősen eltérő eredmények dódhtnk. A hrmdik fejezetben z intestinlis I/R-nek LP-s muttókr kifejtett htását vizsgáltm deferoxmin és L-rginine kezelés mellett, illetve kezelés nélkül ptkány modellben. Az áloperáción átesett álltok (SOP) esetében z egyetlen szignifikánsnk muttkozó változás bélszövet SOD ktivitásánk z ischemiás periódus utáni emelkedése volt. A csk intestinlis I/Rn átesestt (I/R) csoportbn bélszövet M4HN koncentrációj szignifikáns emelkedést muttott hypoxiás periódus végén, mit bél és vvs TBARS és vérplzm FRAP értékének hsonló irányú változás követett reperfúzió során. A deferoxminnl kezelt csoportbn bélszöveti TBARS és M4HN értékek szignifikáns lcsonybbk, míg vérplzm FRAP és NO koncentrációk lcsonybbk voltk z I/R-csoportbn mérteknél. Ugynkkor vvs-ek TBARS koncentrációj és GPX ktivitás szignifikánsn csökkent D-csoportbn. Az L-rgininenl kezelt csoportbn z I/R csoporthoz képest szignifikánsn lcsonybb bélszöveti M4HN értékek voltk mérhetők. A 6

5 vérplzm FRAP és NO koncentrációj D-csoporthoz hsonló változást muttott. Fentiekből zt következtetést vontm le, hogy z I/R megemelte LP-t bélszövetben és mérhető változásokt okozott vérplzm és vvs-ek megfelelő muttóibn is. Deferoxmin kezeléssel fenti htások kivédhetők voltk, míg z L-rginine kezelés htás kérdéses mrdt. A negyedik fejezetben vérplzm és vvs-ek LP-s változásit vizsgáltm kólikás lovk ileus műtétje során gyűjtött mintákból. A kontroll álltokbn egyik mért változó sem muttott szignifikáns idő szerinti változást. Az ileus mitt műtétre került álltok klinikár érkezésükkor metbolikus cidózisr jellemző lcsony ph és BE értékekkel rendelkeztek. Az nesztézi során z ileus műtéten átesett álltoknál kifejezett vegyes típusú cidózis lkult ki. Ennek ellenére pvo2 és vénás SAT értékek szignifikánsn emelkedtek z nesztézi során, hsüreg megnyitásától számított második óránál érve el legmgsbb szinteket. A vérplzm húgysv szintje mintegy 1,5-szer mgsbb volt kontroll álltokbn z nesztézi során. Az lttást követően 12, 24 órávl gyűjtött mintákbn zonbn fordított helyzetet tláltm kólikás lovknál tpsztlhtó jelentős koncentrációemelkedésnek köszönhetően. Bár FRAP értékek idő szerinti változás egyik csoportbn sem volt szignifikáns, kólikás lovkbn mintegy kétszer mgsbb értékeket figyeltem meg mint kontrollokbn. A kólikás csoportbn műtétet megelőzően mgs, de későbbi mintvételeknél folymtos csökkenést muttó vérplzm NO koncentrációt tláltm. A vvs-ek TBARS koncentrációj megfigyelt periódus ltt mindvégig mgsbb volt kólikás álltokbn mint kontrollokbn. A legmgsbb TBARS értéket hsüreg megnyitás után 1 órávl ( reperfúziót követő percben) vett mintákból mértem. A kólikás betegekben vvs-ek GSH és GSSG koncentrációjábn vlmint GPX és SOD ktivitásábn nem muttkozott szignifikáns eltérés, bár ez utóbbi TBARS koncentráció változásávl ellentétes tendenciát muttott. Fentiek figyelembevételével elmondhtó, hogy z intestinlis I/R kifejezett metbolikus/vegyes típusú cidózist idézett elő és megváltozttt vérplzm és vvs-ek bizonyos ntioxidáns muttóit. Ennek lpján fontos, műtétek során törekedni z cidózis kompenzálásár, továbbá vérplzm és vvs-ek LP-s prmétereinek változás z ntioxidáns terápi lklmzásánk szükségességére hívj fel figyelmet. Introduction There is incresing mount of both clinicl nd bsic reserch done on free rdicl induced or medited processes in living systems. It ws observed tht molecules hving unpired electrons on their outmost moleculr orbitl my induce such oxido-reductive cscde mechnisms tht cn dmge protein, nucleic cid nd ft molecules of the living cells. These molecules re clled free rdicls (FR) (Del Mestro, 198, Demopoulos 1973, Pryor 1973, Pcifici nd Dvies, 1991). On the other hnd the genertion of free rdicls is considered physiologic process, still they do not cuse ny dmge s there re regultory mechnisms to control their ction (Hornsby nd Crivello, 1991, Slter, 1981). The bsic source of FR production is the respirtory chin where different oxygen derived free rdicls re trnsformed finlly to wter nd crbon dioxide. These nd some other FRs like moleculr, sigm nd delt singlet oxygen, hydroxil rdicl nion, superoxide nion rdicl, perhydroxi rdicl, hydrogen- nd lipid peroxides re clled rective oxygen intermediers (ROI) or oxygen free rdicls (OFR) (Fridovich, 1978). Any process tht leds to the ccumultion of these substnces nd thus exhusts the cpcity of the defence system cn cuse serious consequences often recognized s specil diseses or syndromes. Free rdicl induced dmges most esily occur in the ftty cids of the biologicl membrnes especilly in the polyunsturted ftty cids (PUFA). During the oxidtion of these, the structure of the membrnes is prtilly or totlly disrupted, cells undergo degenertion nd my die while different ctbolic enzymes nd meditors re relesed into the interstitil spce nd to the circultion, which by reching other orgns cn worsen the bsic processes (Pryor 1973, 1982). Free rdicl induced dmge of membrne lipids is clled lipid peroxidtion (LP). Mny other gents or conditions prt from OFRs cn ply role in the induction of LP (e.g.: chemicls like prqut, crbon tetrchloride, hlothne etc.), systemic diseses (inflmmtion, septicemi, dibetes) diseses of different 7

6 orgns, opertions (reperfusion of hypoxic tissues, orgn trnsplnttion), ging, physicl exercise (McCy et l., 1983, Prks e l 1983, 19, Shw et l., 1983, Hunt et l., 1988, Reynolds et l., 198). Besides OFRs there is outstnding importnce of nitrogen centred free rdicls in biologicl systems. Among these nitric oxide (NO) hs the most complex effect. Its bsic function is to mintin the resting vsodiltor tone of vessels in most orgns nd lso cts s neurotrnsmitter in the brin nd in the non-cholinergic non-drenergic enterl nervous system (Slzmnn, 1995). The physiologicl source of NO production is the mino cid L-rginine, the gunidino group of which is utilized by the nitric oxide synthse enzyme (NOS) to produce NO while the originl molecule is converted to L-citrulline. There re two bsic types of this enzyme. The constitutive one (cnos) is found in the endothel cells (endothelil NOS) nd neurons (neuronl NOS) while the inducible form plys role in certin inflmmtory processes nd found in the following cells: mcrophges, smooth muscle cells, fibroblsts. Besides being meditor, NO itself cn ct s FR, nd plys crucil role in mny FR rections. Combining with superoxide it cn form the hrmful peroxynitrit nion rdicl (ONO 2.- ), which cn brek down to nitrogen dioxide nd OH. (Cristol et l., 1995). The ltter one is considered s one of the most potent FRs. Furthermore peroxynitrit cn dmge proteins forming nitrotyrozine with the tyrosine moieties of protein chins (Beckmn et l., 199). As it ws mentioned before FR production is bsiclly physiologicl process. The ccelertion of FR production or the ccumultion of FRs cn led to the exhustion of the defence mechnisms. These defence mechnisms involve numerous molecules nd enzymes Generlly, molecules tht cn neutrlize FRs by ccepting them re clled scvengers (Slter et l., 1981). Antioxidnts protect the cells ginst OFRs. There re numerous nturl ntioxidnts tht cn be further clssified on which step of the LP chin rection they cn inhibit. Thus vitmin E inhibits the initition LP, vitmin E nd C hinders the production of lipid hydroperoxides, thiols cn brek down the ltter, deferoxmin, d-penicillmin prevents the Fenton-rection by metl cheltion. Besides vitmin A nd E nd other crotenoids cn directly eliminte FRs by scvenging them. One of the most importnt ntioxidnt enzymes is glutthione peroxidse (GPX), selenium dependent enzyme. The selenium independent form of this molecule is clled glutthione S trnsferse. This enzyme elimintes lipid peroxides or H 2 O 2 while oxidizing the reduced form of glutthione (GSH) to glutthione disulfide (GSSG). The ltter molecule is converted bck to GSH by glutthione reductse (GSH-Red) (Dormndy 1978). Superoxide dismutses (mngnese nd copper/zinc dependent forms) cn neutrlize O.- 2 by converting it to the less hrmful H 2 O 2 tht cn be further metbolised to wter by ctlse or GPX (Fridovich, 1975, 1978). Other enzymes cn brek down the products of LP or repir the moleculr dmge cused by FRs (eg.: epoxid-hydrolse, ldehydereductses, some cytochroms, DNA-repir enzymes). When trying to understnd LP processes in certin pthologic conditions one hs to consider tht there re mny other biologicl processes going on tht cn interfere with the LP rections which re usully very quick mking it otherwise difficult to revel true cuses nd consequences. As seen from the introduction LP processes re involved in lmost if not ll physiologic nd pthologic process. Out of this gret vriety I hve studied three phenomenons in four trils. Chpter/Experiment I.: Age relted chnges of LP nd ntioxidnt sttus in developing chicken Chpter/Experiment II.: - Chnges of LP nd ntioxidnt indices in pentthlon horses during exercise Chpter/Experiment III.: - Role of LP nd ntioxidnts in experimentl intestinl ischemireperfusion (I/R) in rts Chpter/Experiment IV: -. Evlution of cid bse nd lipid peroxidtion indices in horses with nturlly cquired intestinl I/R Relevnt literture to ech experiment is summrized in the corresponding chpters. 8

7 Rte of lipid peroxidtion in brin nd liver tissues nd the totl ntioxidnt sttus of blood plsm in developing chicks Act Veterinri Hungric 49 (2), pp (21) Introduction Age-relted chnges of the ntioxidnt system hve been studied in mny species previously. Mny observtions were tken on the embryonic development of the ntioxidnt system of chicks. It ws estblished tht the trnsport of lipids cross the yolk sc membrne occurs only on the third week of incubtion (Noble nd Cocchi, 199) nd is ccompnied by the trnsport of the ntioxidnt Vitmin E (Gál. et l., 1995). The trnsport of vitmin A precedes this process vi n ctive trnsport mechnism nd reches its mximum level on the second week of incubtion when the development of the retin is going on. Antioxidnt defence mechnisms my vry in the orgns due to different concentrtions of the mjor ntioxidnts. Concentrtion of vitmin A, E, reduced glutthione (GSH) nd ctivities of ntioxidnt enzymes superoxid-dismutse (SOD) nd glutthione-peroxidse (GPX) ws found to be significntly higher in the embryonic liver tissue thn in the brin, wheres concentrtion of scorbic cid ws higher in the ltter orgn (Suri et l., 1996; Gál et l., 1995). Some of the ntioxidnt pthwys in the developing chick embryo is regulted by the incresing oxygen consumption nd cuses GSH ccumultion in the liver, nd higher SOD ctivity in the brin (Wilson, 1992). In newly htched chicks the brin tissue is the most cpble one to spontneous nd to in vitro iron induced lipid peroxidtion (LP), nd is chrcterised by low GPX nd ctlse (CAT) ctivity (Suri et l., 1999). The susceptibility of the brin tissue to LP is further exggerted by the increse in its polyunsturted ftty cid content during embryonic development (Mldjin et l., 1996). Other investigtions reveled mny nutritionl diseses of poultry tht re connected with disturbnces of the ntioxidnt system. The most well known disorder is the nutritionl encephlomlci of poultry tht develops on the 7-21 st dy of life due to bsolutely or reltively low concentrtion of vitmin E in the feed (Slmnn et l., 1991; Fuhrmnn et l., 1996). Evidence ws rised tht incresing the n-3 PUFA content in the diet of chicken elevtes liver LP nd depresses plsm ntioxidnt cpcity. These chnges could be wekened by vitmin E supplementtion of the diet (Husvéth et l, 2). Other nutritionl diseses like vitmin A, selenium, monensin, nd slinomycin-timulin toxicosis, tibil dyschondroplsi nd pulmonry hypertension (scites) syndrome re lso closely connected with impired ntioxidnt mechnisms or incresed rte of free rdicl formtion (Mézes et l, 1992, Enkvetchkul et l., 1993, Bottje nd Widemn, 1995, Whitehed et l., 1994, Mézes et l., 1997). Ontogenetic chnges in the ntioxidnt mechnisms hve been observed in mny other species for exmple rts, cttle nd swine (Ledwozyw nd Kdziolk, 1989, Crillo et l., 1992, Gunther et l., 1993, Gál et l., 1996). These findings support the LP theory of geing (Hrmn, 1969) nd underline the necessity of pproprite ntioxidnt defence in newborn nimls. Recently new methods were developed to chrcterise the ntioxidnt sttus of biologicl systems with single prmeter insted of mesuring the levels of certin ntioxidnts seprtely. One of these methods is the totl ntioxidnt sttus (TAS) ssy tht ws developed by Miller et l (1993) for the evlution of ntioxidnt cpcity of humn plsm. The method ws pplied to detect chnges of ntioxidnt power of chicken plsm by Gál nd Kopál (1997). In our study we imed to use this method for the ssessment of the plsm ntioxidnt cpcity of growing chickens. As there re few dt vilble on the ntioxidnt system chnges in newly htched developing chicks, we crried out this study to evlute LP in the orgns nd ntioxidnt sttus of plsm of newly htched chicks from the 1 st to the 21 st dy of life. We hypothesised tht the tmospheric oxygen creted significnt oxidtive stress for the nimls cusing mesurble chnges in the rte of LP in brin nd liver tissues nd in the totl ntioxidnt sttus of plsm. 9

8 Mterils nd methods: Animls Twenty-one one-dy-old Ross cockerel chicks were purchsed from commercil htchery (HE-ROSS Htcheries Co., Ócs, Hungry). The nimls were kept together during the experimentl period in cges in btteries with rised floors nd were fed commercil diet ( strter diet -meeting the Hungrin nd NRC recommendtions for vitmin nd nutritionl substnce content of feed) for broilers. The birds were kept within controlled environment t 2 to 25 C. Additionl heting ws used during the initil 2-week period. Lighting ws provided 24 hours dy. The chicks hd free ccess to feed nd wter. The experiment ws pproved by the Locl Supervising Committee for the Use nd Welfre of Experimentl Animls (25-9/2 ) t the Fculty of Veterinry Science, Szent István University, Budpest. Smple collection At ech smpling time (1, 1, nd 21 dys of life) 7 chicks were slughtered, brin, liver nd heprinized blood smples were collected. After blood smpling plsm ws seprted from the blood cells by centrifugtion (3 rpm for 5 minutes) nd pproximtely.5 grm of ech tissue smple ws homogenised with physiologicl (16 mmol/l) sline solution. Prepred smples were stored t -2 C to wit the nlyses for mximum five dys. Chemicl nlyses Rte of LP ws determined by the modified thiobrbituric cid rection (TBARS) (Plcer et l., 1964). The totl ntioxidnt sttus (TAS) of plsm ws mesured by commercil test kit (TAS kit, Rndox Lbortories, Crumlin, UK). All regents except the Rndox TAS kit were purchsed from Sigm-Aldrich Ltd. Budpest, Hungry. Sttisticl nlysis Dt were nlysed by mens of one-wy nlysis of vrince (ANOVA) nd Student's t-test with the help of the STATGRAPHICS sttisticl softwre. Significnce ws ccepted t 5% confidence level. Results re expressed s mens ± stndrd error of men (SEM). Results One wy ANOVA reveled significnt chnges ccording to time in ll prmeters mesured (Tble 1). Tble 1: Concentrtion of TBARS in brin nd liver tissues nd TAS of plsm in chicks Prmeters TBARS, nmol/g wet tissue TAS, mmol/l Age in dys brin liver plsm 1 73 ± 7 26 ± ± ± 2 5 b ± 4.46 b ± b ± 6 46 b ± ±.6 Note: results in the sme column with different letters in superscript men significnt (p<,5) differences The concentrtion of TBARS ws lmost three times higher in the brin of the newly htched chicks thn in their liver. At dy 1 the TBARS level ws lmost twice higher in the liver thn in the previous smples nd there ws no significnt difference between the two orgns. Another ten dys lter the brin TBARS concentrtion showed mrked increse while the level of 1

9 this substnce in the liver remined lmost constnt throughout the investigted period. The TAS vlue of plsm of 1 dys-old-chicks ws less then hlf of those mesured in ny other smple, which showed lmost the sme level. Discussion Our dt equivoclly with those of Husvéth et l., (2) confirm tht the TAS method cn be pplied to evlute chick plsm ntioxidnt sttus. TAS vlues of the newborn nd developing chick were very similr to those (round 1 mmol/l) mesured in other species (Miller et l., 1993, Gál nd Kopál, 1997) except t dy 1 when significntly lower TAS vlue ws found. This result suggests tht the ntioxidnt mechnisms of plsm re temporrily exhusted t this period of development. It cn be presumed tht the ntioxidnt system is developed well enough to cope with the cute effects of chnging from the gs exchnge cross choriollntois membrne for the pulmonry ventiltion during htching but lter on becomes exhusted by the incresed rte of peroxide formtion. The bove findings re supported by the results of Mézes et l. (1997), who found low ctivities of CAT nd GPX nd high concentrtion of TBARS in plsm of chicks of similr ge. The mrked post-htch increse in the liver TBARS concentrtion could hve been cused by the incresing lipid content of the orgn due to ft rebsorption from the yolk sc. After this process completed nd lipid trnsport from the liver cesed no further increse in LP could be observed. It seems tht the liver my ply crucil role in the regultion of the ntioxidnt mechnisms of plsm, too s the increse of liver TBARS concentrtion ws ccompnied by decresed plsm TAS vlue. The brin hs different ntioxidnt defence system compred to liver s the level of the mjor ntioxidnts like vitmin E, GSH CAT, GPX is low in this orgn but there is reltively high vitmin C concentrtion which is thought to ply importnt role in ntioxidnt pthwys of the embryonic chick brin (Suri et l., 1999). In this study we observed mrked increse of TBARS concentrtion in the brin from the 1 th to 21 st dy of development. Though neither concentrtion nor ctivity of individul ntioxidnts were mesured in this experiment one my speculte tht these mechnisms re impired t this developmentl stge in the brin tissue. Others found incresed LP nd decresed GPX-ctivity in the brin tissue of chicks only t 35 dys of ge (Mézes, 1988). These results cll ttention to the fct tht the brin hs decresed ntioxidnt cpcity round the third week of life in which ge the nutritionl encephlomlci, which thought to be the most importnt disese connected with impired ntioxidnt sttus, most often occurs (Slmnn et l.1991, Fuhrmnn et l., 1996). Similr tendency ws observed in the developing rt brin (i.e.: incresing LP till the third week of life) by Gunther et l. (1993). In conclusion it is stted tht newly htched chicks hve similr plsm ntioxidnt cpcity to other species. Rte of LP is comprtively low in the liver nd high in the brin of the 1-dy-old broiler chicks. Incresed LP ws observed in the liver tissue on the 1 th nd in the brin tissue on the 21 st dy of life, the former ws ccompnied by concomitnt decrese of plsm ntioxidnt cpcity. Summry Age relted chnges of tissue lipid peroxidtion (LP) of liver nd brin, s well s plsm ntioxidnt cpcity of broiler chicken cockerels were investigted. Tissue LP ws chrcterised by the spectrophotometric ssessment of thiobrbituric cid rective substnces (TBARS). Plsm ntioxidnt power ws evluted by the mesurement of totl ntioxidnt sttus (TAS). The newly htched broiler chicks hd similr TAS vlue (1.19 mmol/l) to new-borns of mmmlin species. There were significnt chnges (p<.5) observed in the time course of ll prmeters. Tissue TBARS concentrtion ws higher in the brin thn in the liver t htching, noting tht the ltter orgn hs more effective ntioxidnt defence during the embryonic life. The concentrtion of TBARS incresed to 1 th dy in the liver but only to the 21 st dy in the brin, nd the former ws ccompnied by pproximtely 5% decrese of plsm ntioxidnt cpcity. This suggests tht the liver plys importnt role in forming the ntioxidnt defence mechnisms of plsm of broiler chicks. 11

10 Chnges of some biochemicl nd ntioxidnt prmeters in plsm nd erythrocytes of pentthlon horses before nd fter exercise Veterinry Clinicl Pthology 3 (4), pp (21) Introduction Response to exercise in horses hs been studied from severl spects. Mny studies were tken to evlute chnges of physiologicl, biochemicl s well s lipid peroxidtion prmeters in thoroughbred (Snow et l. 1985, Ono et l., 199, Mills et l., 1996, Hrris et l., 1997) nd stndrdbred rcehorses (Lindholm nd Sltin, 1974, Keenn, 1978, Rose et l., 1983, Gottlie-Vedi et l., 1995, Art et l., 1995, Rsnen et l., 1996 nd Avellini et l., 1997) three-dy event horses (Willimson et l., 1996) nd in endurnce horses (Lucke nd Hll 1978, Rose et l., 1977, Deldr et l., 1982, Rose et l., 1983). There is only one rticle on exercise-induced chnges in polo horses (Crig et l., 1985) nd few ppers bout show jumping horses (Art et l., 199, 199b). The uthor is not wre of studies on biochemicl nd ntioxidnt chnges in pentthlon horses. During exercise ATP is ctbolised to ADP nd further to AMP nd IMP in the muscle fibres. This process is ccompnied by glycogen consumption nd incresing glucose-3-phosphte, glycerol nd lctte concentrtion (Snow et l, 1985, Hrris et l., 1997, Lindholm nd Sltin, 1974,). Exercise induced processes re well reflected in chnges of blood constituents. Numerous uthors found incresed pcked cell volume, Hb-concentrtion, RBC nd WBC count s well s incresed concentrtion of plsm totl protein (TP), lbumin, glucose, ure, cretinine, ketones, bilirubin, iron, phosphte, free ftty cids, cholesterol, lctte, uric cid (UA) cortisol nd glucgon. Similrly, higher ctivities of cretine-kinse, lctte-dehydrogense lkline-phosphtse, sprtte-mino-trnsferse nd gmm-glutmil-trnsferse were documented (Lindholm nd Sltin, 1974, Keenn, 1978, Rose et l., 1983, 1983b, Lucke nd Hll 1978,, Deldr et l., 1982). Some other components, for exmple potssium, clcium nd bicrbonte, were found to be decresed by the sme uthors. There is growing evidence supporting the theory tht incresed oxygen consumption during exercise cretes oxidtive stress to the nimls. Exercise cused elevtion of plsm lipid peroxide (LP) level, nd decresed red blood cell (RBC) glutthione peroxidse (GPX) ctivity while no chnge in ctlse nd superoxide dismutse (SOD) ctivities were observed. In ddition, combintion injection of ntioxidnt vitmin E nd Selenium given prior to work decresed resting plsm LP level nd could slightly ttenute the effect of exercise on the forementioned prmeters (Ono et l., 199). Direct proof ws produced tht during exercise muscle- nd RBC-phospholipids re peroxidised nd metstbile end product of lipid peroxidtion, mlondildehyde (mostly protein-bound) ccumultes in the muscle fibres nd in the erythrocytes (Mtsuki et l., 1991). In nother tril the increse of plsm LP nd oxidised glutthione level in RBCs ws observed in horses running in hot nd humid environment (Mills et l., 1996). Repeted bouts of exercise in horses were reported to result in incresed plsm xntinoxidse ctivity, lctte nd UA concentrtion nd lso in higher plsm totl peroxyl rdicltrpping (TRAP) bility (Rsnen et l., 1996). While single bout of exercise ws generlly found to decrese ntioxidnt power of different biologicl systems in horses, regulr trining incresed the erythrocytes in vitro resistnce to LP, lymphocyte GPX ctivity nd decresed resting plsm TBARS concentrtion. Furthermore, trining ttenuted plsm vitmin E nd TRAP loss in cute exercise (Avellini et l., 1995). In this study we imed to evlute biochemicl nd ntioxidnt prmeter chnges in plsm nd RBCs s no previous study described them in horses competing in pentthlon contests. We lso wished to introduce the recently developed ferric reducing bility of plsm (FRAP) for the mesurement of horse plsm ntioxidnt cpcity. 12

11 Mterils nd methods: Animls Altogether fourteen horses: 5 mres, 7 stllions nd 2 gelded ones between the ge of 5-1 yers were used in this tril tht took plce on preliminry pentthlon contest preceding the 1999 World Pentthlon Chmpionship in Budpest. At the time of the competition the wether ws sunny, temperture ws C with reltive humidity of 45%. Ech horse hd to complete 2 minutes of wrming up followed by 1-minute contest run consisting of 12 jumps of 12 cm height, involving one double nd one triple obstcle s well, then fter 2 minutes (while the horses were doing the sme work s in the wrming up) the contest run ws repeted by the sme pentthlet riding the sme horse. Averge speed in the contest runs ws pproximtely 7 m/s. Smple collection Blood smples were tken from the jugulr vein into heprinized collecting tubes 24 hours prior to the competition, immeditely fter the 2 nd contest run nd 24 hours fter the competition. After smpling, plsm ws seprted nd RBC hemolystes were prepred. Chemicl nlysis The following biochemicl prmeters of plsm were determined spectrophotometriclly: totl protein (TP) (biuret method) uric cid (UA) (Uric Acid Dignostic Regent Kit, Renl Fine Chemicls Co. Budpest, Hungry, Ct. No.: ) nd lctte concentrtion (Lctte Regent test kit, Sigm Dignostics Inc. U:SA. Ct. No.: 735-1), lctte-dehydrogense (LDH) nd cretine-kinse (CK) ctivities (LDH nd CK dignostic test kits, both from Dignosztikum Ltd., Budpest, Hungry, Ct. No.: 4211 nd 4411, respectively). The totl ntioxidnt sttus (TAS) of plsm, erythrocyte glutthione-peroxidse (GPX) nd superoxid-dismutse (SOD) ctivities were determined using commercilly vilble kits (TAS, Rnsel, nd Rnsod kits, respectively; ll mnufctured by Rndox, Cork, Irelnd),. The ferric reducing bility of plsm (FRAP) ws ssessed s described by Benzie nd Strin (1996). In brief this method is bsed on the reduction of ferric-tripyridyltrizin regent to the ferrous form, i.e. by ntioxidnts in the smple, which hs n intense blue colour nd cn be mesured spectrophotometriclly t 593nm wvelength. For vlidtion of the method liquots of horse plsm smples in two different concentrtion levels were pooled nd frozen t -2 C. Within-run precision ws clculted from 1 ssys done on the sme dy. Between-run precision ws determined from 1 ssys done over 2 dys. The concentrtion of thiobrbituric cid rective substnces (TBARS) nd reduced glutthione (GSH) in RBCs were mesured ccording to Plcer (1964) nd Sedlk (1968), respectively. All chemicls for ntioxidnt prmeters except the Rndox kits were purchsed from Sigm-Aldrich Ltd. Budpest, Hungry. Sttisticl nlysis The sttisticl nlysis of the lbortory dt ws completed by using the Sttgrphics softwre. Results Anlyticl performnces of the FRAP ssy: Within-run coefficients of vrition (CV) were less thn 1, % t ll vlues tested (.284 mmol/l nd.365 mmol/l). Between run CVs were less thn 3.2%. Plsm nd RBC results re shown in Tble 2. 13

12 Tble 2. Biochemicl nd ntioxidnt prmeters before, immeditely nd 24 hours fter exercise in horse plsm nd RBC hemolystes (men ± stndrd error) Vlues on the sme line with different letters re significntly different, p<.5 (ANOVA) Prmeters Before exercise Immeditely fter exercise 24 hours fter exercise TP, g/l 69±1 73 ±1 b 68 ± 1 Plsm Lctte, mmol/l.86 ± ±.6 b 1.1±.5 CK, U/l 16 ± ± 21 b 12 ± 6 LDH, U/l 364 ± ± 34 b 417 ± 34 b Uric cid, µmol/l 58 ± 1 72 ± 13 b 58 ±1 b FRAP, mmol/l.31±.8.37 ±.16 b.33 ±.12 TAS, mmol/l.73 ±.4.34 ±.2.53 ±.24 RBC TBARS, nmol/g 395 ± ± ± 21 b protein GSH, nmol/g protein 445 ± ± ± 9 b b GPX, U/g protein 47 ± 4 38 ±4 4 ± 3 SOD, U/g protein 72 ± ± ± 116 After the exercise horses showed elevted levels of plsm TP, lctte, CK, LDH nd FRAP (p<.5) compred to the pre-exercise vlues nd ll these returned pproximtely to the initil vlues fter 24 hours rest. Similr tendencies were observed in the chnge of plsm TAS nd RBC GPX vlues. GSH nd TBARS concentrtions did not show ny chnge immeditely fter the exercise but decresed TBARS nd incresed GSH concentrtions were observed fter 24 hours rest (Tble 2.). Plsm UA nd FRAP vlues showed good correltion in liner model. (p<.1, R 2 =.68, correltion coefficient:.78) (Figure 1) 14

13 FRAP (µmol/l) Uric cid concentrtion (µmol/l) Figure 1. Liner correltion between the ferric reducing bility of plsm (FRAP) nd plsm uric cid concentrtion in 14 horses smpled prior to, immeditely fter, nd 24 hours fter exercise (y = 3.2x , r =.78). The solid line indictes the line of eqution, the inner pir of dshed lines indictes the 95% probbility of the line of eqution, the outer pir of dshed lines indictes the 95% probbility of the plotted points. Discussion All the mesured routine plsm biochemicl prmeters showed significnt increse due to the type of exercise used in this study indicting first of ll the incresed lod on muscles leding to enzyme lekge, lctte nd uric cid ccumultion in plsm. Note tht the elevtion in TP concentrtion ws though significnt but negligible compred to other prmeters indicting tht not hemoconcentrtion but metbolic effects plyed primry role in these chnges. FRAP vlues were lso elevted fter physicl stress. These findings re in good correltion with those of Mcmenimn et l. (1992) nd Rsnen et l. (1996). The ltter ones lso found incresed lctte, uric cid concentrtion nd totl TRAP levels in trotters. Their suggestion is tht the increse of the TRAP vlue is minly cused by the ccumultion of uric cid in plsm due to purine degrdtion. According to the originl publiction of Benzie nd Strin (1996) pproximtely 6 percent of the humn plsm FRAP vlue is given by the uric cid content of the blood. So we cn deduct tht the rise of the FRAP concentrtion in this tril ws bsiclly cused by the incresed UA concentrtion. On the other hnd we hve found opposite chnges in TAS vlues, nother prmeter showing the generl plsm ntioxidnt sttus. This my seem to contrdict the forementioned results but we hve to consider the differences in the two methods we used. In the FRAP method there is direct reduction of the colour forming gent (ferric-tripyridyltrizin) so the ntioxidnt cpcity is proportionl to the reducing bility, while in the TAS method complex free rdicl ction (2, 2 - zinobis benzothizoline-6-sulphonic cid) is generted by the mens of pseudoperoxidse (metmyoglobin) nd hydrogen peroxide, so the ntioxidnt cpcity will be proportionl to the inhibitory bility of the smple on the formtion of the coloured complex. In tht sense ny ntioxidnt present in the plsm including enzymes s well, cn tke prt in the ltter rection. This deduction is further ugmented by the finding of strong correltion (r=,91) of TAS nd ctlse ctivity of liver cells in toxicologicl model in rts (O Brien et l., 1999). 15

14 The decresing tendency of GPX ctivity due to exercise cn denote the effect of oxidtive stress on red cells nd my explin the lte elevtion of GSH concentrtion. The decresed GPX ctivity in long term trining period reported by Avellini et l (1995), is certinly of other origin (Se-deficiency, prolonged physicl stress) thn we found in our cute exercise model. Our results re in line with those of Ono et l (199) who lso found decresed GPX nd unchnged SOD ctivity in RBC of exercised horses. It is uncler why the erythrocyte TBARS level did not correlte with other LP indices. We suspect tht the method we use ws not sensitive enough s LP produces minly protein bound mlondildehydes in the erythrocytes, the detection of which requires hydrolysis of proteins prior to nlyses (Mtsuki et l 1991). All prmeters except GSH nd TBARS returned pproximtely to the initil vlues fter 24 hours rest tht gree the findings of Mills et l (1996). We cnnot exclude tht the chnges we hve found in the erythrocytes were prts of longer-term processes due to the continuous trining of the horses. It lso hs to be mentioned tht in this study blood ws collected from the jugulr vein tht my not be representtive of centrl circultion for some prmeters like ph, pyruvte concentrtion nd prtil crbon dioxide nd oxygen tension (Miller-Grber et l., 1988). The ltter suggests tht LP indices lso might be ffected by the site of smpling. On the other hnd the mentioned study proved tht other importnt biochemicl prmeters like lctte did not differ between the jugulr vein nd the pulmonry rtery so we cn ccept tht our dt re representtive for the centrl circultion. Since in our study blood ws collected from horses in contest, ccess to other smpling sites like the pulmonry rtery ws impossible nd we could not perform comprison of prmeters mesured in blood smples from different vessels. It is concluded tht the type of the pplied exercise, which cn be considered quite usul for pentthlon horses, cused detectble biochemicl nd LP chnges in the plsm of the horses tht were not unequivoclly reflected in the erythrocytes. Attention should be pid to the evlution of results of plsm ntioxidnt cpcity described by different prmeters s they cn differ significntly. Summry The im of this study ws to exmine exercise-induced chnges of some plsm nd red blood cell biochemicl nd ntioxidnt prmeters in pentthlon horses. Blood smples were tken from fourteen horses before, immeditely nd 24 hours fter competing two runs of 1 minute of intense exercise over jumps. The pek intensity periods were preceded by 2-minute wrming up nd seprted by 2-minute brek The following plsm biochemicl prmeters were determined: totl protein (TP), uric cid (UA) nd lctte concentrtion, lctte-dehydrogense (LDH) nd cretine-kinse (CK) ctivities. The totl ntioxidnt sttus (TAS) nd the ferric reducing bility of plsm (FRAP) were lso mesured. Thiobrbituric cid rective substnces (TBARS), reduced glutthione (GSH), TP levels, glutthione-peroxidse (GSH-Px) nd superoxide-dismutse (SOD) ctivities of red blood cell hemolystes were determined. There were elevted concentrtions of plsm TP, lctte, CK, LDH nd FRAP (p<.5) in the post exercise smples compred to the preexercise smples. All prmeters returned pproximtely to the initil vlues fter 24 hours rest. Similr tendencies were observed in the chnge of plsm TAS nd RBC GSHPx vlues. Erythrocyte GSH nd TBARS concentrtions did not show ny chnge immeditely fter the exercise but decresed TBARS nd incresed GSH concentrtions were observed fter 24 hours rest (p<.5). Plsm UA nd FRAP vlues showed good correltion in liner model. It is concluded tht the type of the pplied exercise, which cn be considered quite usul for pentthlon horses, cused detectble biochemicl nd lipid peroxidtive chnges. There were opposite chnges in FRAP nd TAS vlues clling ttention to the fct tht ssessing the ntioxidnt cpcity by different prmeters cn give highly different results. 16

15 Effect of deferoxmine nd L-rginine tretment on lipid peroxidtion in n intestinl ischemi-reperfusion model in rts Act Veterinri Hungric 5 (3), pp (22) Introduction Intestinl ischemi-reperfusion (I/R) induces series of processes tht led to severe gut wll injury finlly resulting in tissue necrosis nd shock. The role of free rdicls in these situtions ws investigted from severl spects. There re mny pthwys involved tht cn generte free rdicls in the I/R. In the smll intestine the xnthine-oxidse mechnism is one of the most importnt source of oxygen free rdicls while in the colon ldehyde-oxidses re thought to ply similr role (Grnger et l., 1981, McCord et l., 1982, Prks et l., 1983). Free rdicl-cused injury forces endothelil cells to relese pltelet ctivting fctor nd vrious kinds of leukotriens tht trigger neutrophil grnulocyte migrtion towrds the dmged mucos (Grnger et l., 1989, Suzuki et l., 1989, Zimmermnn et l., 199, Kubes et l., 199). Neutrophils vi their myeloperoxidse ctivity contribute to the production of free rdicls (Otmiri et l., 1988). Intestinl I/R cn lso dmge the function of distnt orgns like the lung nd hert vi the relese of different meditors (eg.: tumour necrosis fctor, pltelet ctivting fctor, leukotriens, prostglndins) (Cty et l., 199, Horton et l., 1991). There were mny trils to prevent the deleterious effects of I/R with different drugs like enzyme inhibitors, free rdicl scvengers, ntioxidnt enzymes, nti-inflmmtory compounds, nti-neutrofil gents nd metl-cheltors. Deferoxmine is known s the most prominent representtive of the ltter group nd ws used successfully to prevent I/R injury (i.e. incresed mucosl permebility) in ct model nd improved survivl rtes fter n experimentlly induced gstric diltion-volvulus in dogs (Hernndez et l., 1987, Lntz et l., 1992). Furthermore if deferoxmine ws dded to crdioplegi solution 93% of the left ventriculr contrctility of the isolted perfused rt herts ws preserved (Ely et l., 1992). There is growing evidence tht supports tht nitric oxide (NO) nd its derivtes ply importnt role in the pthogenesis of intestinl I/R. Nitric oxide is smll lipophylic molecule continuously produced by constitutive enzyme (constitutive NO synthse, cnos) expressed in mny cells in the intestine nd plys importnt role in the regultion of epithelil permebility (Kubes, 1992). Administrtion of NO donor (C ) ttenuted endothelil dysfunction nd improved short-term survivl of experimentl cts fter intestinl I/R (Crey et l., 1992). If rts were given L-rginine (,5g /kg bw per orlly) prior to the estblishment of I/R the process of reprtion nd cell prolifertion ws more pronounced nd levels of polymines nd cgmp lso incresed. These effects were ntgonized by simultneous dministrtion of NO-synthse inhibitor NG-nitrorginine-methyl-ester (Rul et l., 1995). If cts undergoing intestinl I/R were given NO synthse inhibitor (N- nitro L-rginine methyl ester) the blood to lumen clernce of Cr 51- EDTA nd I 125 -lbumin ws significntly higher thn in the control ones. Furthermore L-rginine tretment ws ble to ttenute these chnges (Kubes, 1993). On the other hnd NO cn tke prt in different biochemicl rections especilly with superoxide to form the hrmful peroxynitrite rdicl (Beckmn et l., 199). There re lso huge number of studies tht re focused on the effects of NO, NO-donors, NO-synthesis inhibitors, nd peroxynitrite in crdic, lung nd brin I/R, but their results re quite conflicting bout the effects of these compounds. We hypothesized tht LP-chnges in the intestines undergoing I/R could be reflected in plsm nd red blood cell prmeters s well. We lso imed to evlute effects of iron cheltion with deferoxmine nd NO synthesis enhncement by L-rginine on the forementioned processes. 17

16 Mterils nd methods Experimentl design Altogether 56, 3-month-old femle White Wistr rts were utilized in this study. Before the experiment the nimls were weighed nd divided into 4 groups s follows: The experimentl ischemi reperfusion group (Group I/R, n=14): The nimls were nesthetized by intrperitonel injection of sodium pentobrbitl (5 mg/kg bw). The jugulr vein ws ccessed surgiclly nd blood smples were collected into heprinized collecting tubes. Afterwrds, midline lprotomy ws performed nd the crnil mesenteric rtery ws ligted using trumtic surgicl clmps. After 3 minutes 1 ml of physiologicl sline solution (Slsol A infusion, Bxter, Toronto, Cnd) ws injected into the jugulr vein of the nimls s the vehicle of the ctive ingredients used in the treted groups. Another 15 minutes lter blood smple ws tken from the jugulr vein gin nd full thickness specimens were hrvested from the ischemic jejunum from 7 nimls. In the other hlf of the nimls in this group, blood flow ws restored in the crnil mesenteric rtery nd blood smpling ws repeted nd jejunl smpling ws completed fter 45 minutes. After collection of the intestinl smples the nimls were euthnized by overdosing sodium pentobrbitl. L-rginine (Group A, n=14) nd Deferoxmine (Group D, n=14) treted groups: The nimls underwent the sme procedure s the ones in group I/R, except tht L-rginine (3 mg/kg bw, dissolved in physiologicl sline solution) or deferoxmine (5 mg/kg bw, dissolved in physiologicl sline solution) ws injected to the nimls 15 minutes prior to reperfusion. Both chemicls were supplied by Sigm-Aldrich Ltd. Budpest, Hungry. Shm operted group (Group SOP, n=14). Four rts were nesthetized nd jejunl smples were collected immeditely fter nesthesi to serve s negtive controls for the operted groups. Smples were collected from the rest of the nimls ccording to the sme scheme s in group I/R except no ligture of the crnil mesenteric rtery ws performed. The experiment ws pproved by the Locl Supervising Committee for the Use nd Welfre of Experimentl Animls (25-9/2 t the Fculty of Veterinry Science, Szent István University, Budpest). Smple hndling After smpling blood smples were immeditely centrifuged t 3 rpm for five minutes, plsm ws removed nd red blood cell hemolystes (1:9 v/v with redistilled wter) were prepred. Jejunl specimens were flushed with physiologicl sline solution then pproximtely hlf grm ws homogenized with 4,5 ml of physiologicl sline solution. All smples were frozen nd stored t -2 C to wit the biochemicl nlysis for mximum 5 dys. Anlyticl procedures Erythrocyte nd intestinl glutthione-peroxidse (GPX) nd superoxide-dismutse (SOD) ctivities were determined using commercilly vilble kits (Rnsel, nd Rnsod kits, respectively; mnufctured by Rndox, Cork, Irelnd). The concentrtion of thiobrbituric cid rective substnces (TBARS) in RBCs nd intestinl homogentes were mesured ccording to Plcer et l. (1966). Overll concentrtion of lipid peroxidtion end products mlondildehyde nd 4-hydroxinonenl in the jejunl smples ws ssessed by the LPO-586 kit (Oxis Int. Inc. Bioxytech LPO- 586 Ct. No.: 2112D). The ssy is bsed on the rection of chromogenic regent N-metyl-2- phenylindole with mlondildehyde nd 4-hydroxylkenls t 45 C. Plsm nitric oxide (NO) concentrtion ws mesured vi the detection of its metbolites nitrite nd nitrte (reduced to nitrite with nitrte-reductse) with the Griess-Ilosvy regent ccording to Grishm et l. (1996). The ferric reducing bility of plsm (FRAP) ws ssessed s described by Benzie nd Strin (1996). In brief, this method is bsed on the reduction of ferric-tripyridyltrizin regent to the ferrous form, i.e. by ntioxidnts in the smple, which hs n intense blue colour tht is mesurble spectrophotometriclly t 593 nm wvelength. Most prmeters (except plsm FRAP nd NO) re given per grm protein content of the smples. Totl protein concentrtion ws determined spectrophotometriclly with the biuret regent (purchsed from Dignosztikum Ltd. Budpest, 18

17 Hungry). All other chemicls for ntioxidnt prmeters were purchsed from Sigm-Aldrich Ltd. Budpest, Hungry. Sttisticl nlysis of the lbortory dt ws completed by the help of Microsoft Excel 5. nd the Sttgrphics 6. (Mnugistics Inc. 2115, Rockwille, MD, US.) softwre progrms. Dt of the intestinl smples nd differences of results between ech smpling time of plsm nd RBC indices (between groups) were compred by two smples Student's t-tests, plsm nd RBC results were checked by pired t-tests (within groups). A P vlue less thn.5 ws considered significnt. Results There ws no significnt chnge of intestinl TBARS concentrtion in group SOP nd D whilst some 2-3 times higher levels (p<.5) were seen in groups I/R nd A from the 45th to the 9th minute (i.e. during reperfusion) (414±172 to 941±312 nd 378±9 to 1252±415 nmol/g protein, respectively). Furthermore intestinl TBARS concentrtion ws significntly higher fter reperfusion in these two groups thn in group SOP nd D (941±312 nd 1252±415 nmol/g protein compred to 433±117 nd 27±5 nmol/g protein, respectively (Figure 2). TBARS nm ol/g prote in b b. min. 45. min. 9. min. c c SOP I/R L-Arginin Deferoxmin Groups Figure 2. Intestinl TBARS concentrtion (nmol/g protein). Averge ± SD. Columns mrked with different letters indicte significnt difference of results Overll concentrtion of intestinl mlondildehyde nd 4-hydroxi-lkenls (M4HN) is shown on Figure 3. There were no chnge reveled in groups SOP, A nd D, however there ws 77-87% higher M4HN concentrtion observed in group I/R fter 45 minutes of ischemi stying pproximtely t the sme level fter reperfusion s well (98±29 vs.177±43 nd 187±64 nmol/g protein, respectively)(p<.5). Furthermore post-reperfusion vlues in group I/R were significntly higher thn the corresponding ones in ny other group (Figure 3). 19

18 MDA + 4-O H none n l nm ol/g prote in b b. m in. 45. m in. 9. m in. S OP I/R L-Arginin De fe rox min Gro ups Figure 3. Intestinl MDA nd 4-OH nonenl concentrtion (nmol/g protein). Averge ± SD. Columns mrked with different letters indicte significnt difference of results The only significnt chnge of the intestinl SOD ctivity ws n increse from 27±54 to 531±174 U/g protein tht ws found in the SOP group during the first 45 minutes of nesthesi. Then the vlues showed bout t the sme levels for the rest of the experiment (Figure 4). The sme tendency ws observed in ll the other groups.. min. S O D U/g prote in b b 45. min. 9. min. 1 S OP I/R L-Arginin De fe rox min g ro ups Figure 4. Intestinl SOD ctivity. Averge ± SD. Columns mrked with different letters indicte significnt difference of results Intestinl GPX ctivity showed no significnt difference in ny group (Figure 5) 2

19 GPX U/g prote in min. 45. min. 9. min. 1 SOP I/R L-Arginin De fe rox min g ro ups Figure 5. Intestinl GPX ctivity. Averge ± SD FRAP vlues did not chnge in the SOP group. Significntly higher FRAP concentrtions mesured were in groups I/R, A nd D in smples tken t 9 minutes of nesthesi (1.13±.27, 2.9±1.64 nd 2.25±.85 mmol/l, respectively) thn in those hrvested t 45 minutes. In ddition FRAP concentrtion lso incresed significntly during the first 45 minutes in Group D from.7±.17 to 1.29±.39 mmol/l (Figure 6). 5 b. min. 45. min. FRAP m m ol/l b b c 9. min. SOP I/R L-Arginin Deferoxmin groups Figure 6. Plsm FRAP vlues. Averge ± SD. Columns mrked with different letters indicte significnt difference of results within groups During ischemi, FRAP vlues showed significntly higher increse in Group D compred to group I/R. By the end of reperfusion the chnge of FRAP concentrtion in group I/R (+.61±.18 mmol/l) ws significntly higher thn in the Group SOP (-.35±.33 mmol/l) but ws significntly lower thn in group D (+1.5±.57 mmol/l). Compring the chnges of FRAP concentrtion during 21

20 the whole experimentl period only Groups I/R nd D differed significntly (.69±18 nd 1.95±.95 mmol/l). Plsm NO concentrtion did not show ny chnge between smpling times in Groups SOP nd I/R (Figure 7). However, there ws significnt increse upon reperfusion from 18.±3.1 to 28.9±7.2 µmol/l in Group D. Similrly, differences of NO concentrtion were significnt only between Group I/R nd Group D, considering either 45 or 9 minutes smpling. 8. min min. NO mol/l b 9. min. 1 SOP I/R L-Arginin Deferoxmin gro ups Figure 7. Plsm NO concentrtions. Averge ± SD. Columns mrked with different letters indicte significnt difference of results within groups Erythrocyte TBARS concentrtion ws significntly higher (43±73 nmol/g protein) thn the bsl vlue (197±33 nmol/g protein) fter 9 minutes in Group I/R; however no significnt chnge ws observed t 45 minutes fter ischemi compred to the bsl vlues of this group. Similr but not so mrked elevtion ws observed in Group A s well (31±26) vs. (258±3 nmol/g protein) (p<.5; Figure 8). TBARS nmol/g protein b b SOP I/R L-Arginin Deferoxmin. min. 45. min. 9. min. groups Figure 8. Erythrocyte TBARS concentrtions. Averge ± SD. Columns mrked with different letters indicte significnt difference of results within groups 22