UC 577,1; 61 ISSN 03543447 JUGOSLOVENSKA ......JUGOSLOVENSKA MEDICINSKA BIOHEMIJA YUGOSLAV MEDICAL BIOCHEMISTRY Adresa uredni{tva Jugoslovenska medicinska biohemija, Dru{tvo medicinskih

UC 577,1; 61 ISSN 0354-3447

Jugoslov Med Biohem 23 (4): 333’406, 2004

Volume: 23 Belgrade, October – December 2004 No: 4

Godi{te: 23 Beograd, oktobar – decembar 2004 Broj: 4

PREGLEDNI ^LANAK – REVIEW ARTICLE

Danijela Vu~evi}, Tatjana Radosavljevi}, Gordana \or|evi}-Deni}ULOGA EOZINOFILNIH LEUKOCITA U PATOGENEZI BRONHIJALNE ASTME . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

ORIGINALNI NAU^NI RAD – ORIGINAL PAPER

Vesna Vu~i}, Miroslav Adì}, Ana Ni}iforovi}, Nevena Ti{ma, Sabera Ru`diji}, Marija B. Radoj~i}CELL DEATH IN IRRADIATED PROSTATE CANCER CELLS ASSESSED BY FLOW CYTOMETRY . . . . . . . . . . . . . . 343

Aleksandra Nikoli}, Aleksandra Divac, Nada Bogdanovi}, Marija Miti}-Miliki}, Dragica Radojkovi}CFTR GENE ANALYSIS IN PATIENT WITH ATYPICAL CYSTIC FIBROSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351

Aleksandra Peri}-Popadi}, Mirjana Bogi}, @ikica Jovi~i}, Sanvila Ra{kovi}, Vesna Tomi}-Spiri}, Sneàna Kova~evi}, Jasna Bolpa~i}, Miodrag ôli}THE INFLUENCE OF ATOPY ON sICAM-1 SERUM LEVELS IN PATIENTS WITH ALLERGIC RHINITIS AND BRONCHIAL ASTHMA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Jelena Poznani}, Ljubica Peri{i}, Jelena Uro{evi}, Branka Petru~ev, Tatjana \ureinovi}, Nata{a To{i}, Lidija Krivokapi}-Dokmanovi}, Dragana Jani}, Milica ^vorkov-Draì}, Gordana Bunjeva~ki, Sonja Pavlovi}BIOCHEMICAL PHENOTYPE AND ORIGIN OF THE THREE MOST COMMON BETA-THALASSEMIA MUTATIONS IN SERBIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

Zorica S. Sai~i}, Dejan N. Mijalkovi}, Aleksandra L. Nikoli}, Du{ko P. Blagojevi}, Mihajlo B. Spasi}, Vojislav M. Petrovi}EFFECT OF THYROXINE ON GLUTATHIONE-DEPENDENT ANTIOXIDANT ENZYME ACTIVITIES AND GLUTATHIONE CONTENT IN THE INTERSCAPULAR BROWN ADIPOSE TISSUE OF DIFFERENT MATURATED RATS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

Ljiljana Petrovi}-Rackov, Nada Pejnovi}, Zoran Miju{kovi}, Gordana Ercegovi}INFLAMMATORY RESPONSE IN RHEUMATOID ARTHRITIS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

Radmila Maksimovi}, Ljuba Mandi}, Slavica Spasi}THE BASIC HAEMATOLOGICAL MEASUREMENTS IN PERIPHERAL BLOOD FROM WORKERS EXPOSED TO MERCURY VAPOURS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

Y U G O S L A V M E D I C A L B I O C H E M I S T R Y

J U G O S L O V E N S K AMEDICINSKA BIOHEMIJAâsopis Dru{tva medicinskih biohemi~ara Srbije i Crne GoreOfficial Journal of the Society of Medical Biochemists of Serbia and Montenegro

SADR@AJ – CONTENTS

Nastavak na pole|ini korica (continued on back cover)

Jugoslovensku medicinsku biohemijureferi{u: Bowker International Serials Database, Bulletin Scientifique, Chemical Abstracts Service, EMBASE / Excerpta Medica, Elsevier BIOBASE / Current Awareness in Biological

Sciences, Current Awareness in BiomedicineJugoslovenski Bibliografsko Informacijski Institut, Referativnyi Zhurnal.

Jugoslovenska medicinska biohemijais covered by the following indexing and abstracting service: Bowker International Serials Database, Bulletin Scientifique, Chemical Abstracts Service, EMBASE / Excerpta Medica,

Elsevier BIOBASE / Current Awareness in Biological Sciences, Current Awareness in Biomedicine, Referativnyi Zhurnal,

Yugoslav Institute for Bibliography and Information (YUBIN)

Nastavak sadràja (continued from front cover)

STRU^NI RAD – PROFESSIONAL PAPER

Dragica Milenkovi}, Aleksandar Vuksanovi}, Nata{a Lali}, Sanja Simi}-Ogrizovi}, Violeta DopsajNOVI PROTOKOL ZA LABORATORIJSKO ISPITIVANJE PACIJENATA SA KALKULOZOM URINARNOG TRAKTA. . . 387

Nada Kosti}, Branislava Brki}, Zorica âparevi}, Verica Milo{evi}SOMATOSTATIN U OBOLJENJIMA GASTROINTESTINALNOG TRAKTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

OBAVE[TENJA – TECHNICAL REPORTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

JUGOSLOVENSKA MEDICINSKA BIOHEMIJA

YUGOSLAV MEDICAL BIOCHEMISTRY

Jugoslovenska Medicinska Biohemijais electronic available on http://www.dmbj.org.yu/jugoslovmedbiohem



Adresa uredni{tvaJugoslovenska medicinska biohemija,

Dru{tvo medicinskih biohemi~ara Srbije i Crne Gore, Farmaceutski fakultetVojvode Stepe 450, FAH 146, 11221 Beograd, Srbija i Crna Gora

Tel. / Fax: 011’36 15 631e-mail:dmbj¤eunet.yu

www.dmbj.org.yu

Editorial Office Society of Medical Biochemists of Serbia and MontenegroPharmaceutical Faculty, Vojvode Stepe 450, P.O. Box 146

11221 Belgrade, Serbia and MontenegroTel. / Fax: +381’11’36 15 631

e-mail:dmbj¤eunet.yuwww.dmbj.org.yu

Izdavanje ýJugoslovenske Medicinske Biohemijeû finansira Ministarstvo za nauku, tehnologije i razvoj Republike Srbije

Yugoslav Medical Biochemistry is published by financial support of Ministry of Science, Technology and Development of Republic of Serbia

âsopis Dru{tva medicinskih biohemi~ara Srbije i Crne Gore

Official Journal of the Society of Medical Biochemists of Serbia and Montenegro



Jovan Kavari}Gordana Mati}Ivanka Mileti}Jasmina Mimi}-OkaVesna Star~evi}

Branislava Brki} Bogomir Dimitrijevi}

Vidosava B. \or|evi}Svetlana Ignjatovi}Jelena Joksimovi}

Effi Anagnostou-CacarasAthens, Greece

Stoyan DanevSofia, Bulgaria

Robert RejAlbany, USA

Armando D'Angelo Milan, Italy

Dolphe KutterLuxemburg

Susan SchiffmanDurham, USA

GLAVNI I ODGOVORNI UREDNIKEDITOR - IN - CHIEF

Nada Majki}-Singh

REDAKCIONI ODBOREDITORIAL BOARD

ME\UNARODNI REDAKCIONI ODBORINTERNATIONAL ADVISORY BOARD

Editorial Office

Nada Majki}-Singh, Editor-in-ChiefSociety of Medical Biochemists of Serbia and Montenegro, Pharmaceutical Faculty,

Vojvode Stepe 450, 11221 Belgrade, P.O. Box 146, Serbia and Montenegro

Jose M. Queralt†Barcelona, Spain

Marina Stojanov



Vol. 23 (2004)

Informacije o ~asopisu: âsopis izlazi tromese~no. Svako godi{te sadrì ~etiri broja.Rukopisi se dostavljaju na adresu uredni{tva. Autori se izve{tavaju o prijemu rukopisa.Svi radovi se recenziraju.

Informacije o pretplati: Pretplata za organizacije i inostranstvo iznosi 50 US $. Pretplatase {alje na teku}i ra~un Dru{tva medicinskih biohemi~ara Srbije i Crne Gore. 255-0006390101000-02, Privredna banka Beograd a.d., Beograd (sa naznakom »pretplataza JMB«).

Informacije o ogla{avanju: Svi zahtevi koji se odnose na ogla{avanje u Jugoslovenskojmedicinskoj biohemiji dostavljaju se na adresu Uredni{tva.

Informations for contributors: »Jugoslovenska medicinska biohemija« publishes review articles, original research papers and preliminary communications dealing withclinical chemistry, medical biochemistry and related fields. All manuscript should con-firm to generally accepted usage in composition of scientific papers. For technicalrequirements, authors should consult a current issue. Contributors in two double-spaced typewritten copies should be addressed to The Editor, Yugoslav Medical Bio-chemistry, Pharmaceutical Faculty, Department of Medical Biochemistry, VojvodeStepe 450, P.O. Box 146, 11221 Belgrade, Serbia and Montenegro.

Information to subscribers: Subscribers rate per Volume (containing 4 issue) US $ 50including surface postage. Subscription orders and correspondance should be addres-sed to the Editorial Office, Jugoslovenska medicinska biohemija, Pharmaceutical Fa-culty, Department of Medical Biochemistry, Vojvode Stepe 450, P.O. Box 146, 11221Belgrade, Serbia and Montenegro.

Grafi~ko ure|enje: [tampa:Graphic design: Printed by:

Milena Mijailovi} REPROGRAF

Jugoslov Med Biohem 2004; 23 (4) 333

Bronhijalna astma i hroni~ne opstruktivne bolesti plu}a

Bronhijalna astma je inflamacijska bolest kojukarakteri{e preosetljivost vazdu{nih puteva sa po-vremenim periodima bronhospazma. Radi se o spaz-mu ili duìm kontrakcijama bronhijalne i bronhiolarneglatke muskulature. Exstrinsic astma (atopijska, aler-gijska astma) je mnogo ~e{}a od intrinsic astme(nealergijske, neatopijske, inflamacijske astme).Bronhijalna astma je veoma sloèno oboljenje kojepodrazumeva biohemijske, autonomne, imunske,infektivne, endokrine i psihi~ke faktore razli~itog ste-pena u razli~itih osoba (1).

Smatra se da je inflamacija koja nastaje kao po-sledica preosetljivosti ili hiperosetljivosti vazdu{nihputeva osnovni patofiziolo{ki razlog u svim tipovimaastme. Osloba|anje medijatora inflamacije dovodi dospazma glatkih mi{i}a bronhija, vaskularne kongesti-je, pove}ane propustljivosti krvnih sudova, edema,stvaranja guste, lepljive sluzi i prestanka funkcije (1).

Po mnogima eozinofilni leukociti su klju~ne efektorne}elije u patogenezi ove inflamacijske bolesti vazdu{-nih puteva (2–4, 9, 47, 48–59). Aktivirani eozinofilisekretuju {iroki spektar preformiranih i novosinteti-sanih medijatora koji razaraju integritet bronhijalnesluznice, zaustavljaju pokretanje cilija i dovode doo{te}enja i jake sekrecije epitelnih }elija. O{te}enjeepitelnih }elija je u korelaciji sa preosetljivo{}u vaz-du{nih puteva (1, 51, 57, 58). Brojni citokini koje pro-dukuju eozinofili obezbe|uju lokalni mehanizamkojim se poja~ava i modulira postoje}i zapaljenskiproces (5, 55, 56). Metaplazija peharastih }elija, kojanastaje kao posledica inflamacije disajnih puteva,tako|e umnogome doprinosi klini~koj simptoma-tologiji, opstrukciji disajnih puteva i mortalitetu (6, 47,51). Bitne patomorfolo{ke karakteristike bronhijalneastme su i subepitelna fibroza, hipertrofija glatkihmi{i}a i formiranje novih krvnih sudova, {to u krajn-jem ishodu dovodi do remodelovanja zida disajnihputeva (5, 7, 51, 56).

Osim bronhijalne astme, inflamacija je zna~ajnaza razvoj hroni~nih opstruktivnih bolesti plu}a (HOBP).Ipak, inflamacijski odgovor u HOBP se zna~ajno raz-likuje od odgovora u astmi, {to je prikazano na tabeli I.Me|utim, neki bolesnici imaju istovremeno i HOBP iastmu, pa inflamacija u njihovim plu}ima moè pokazi-vati karakteristike obe bolesti (8).

UC 577,1; 61 ISSN 0354-3447

Jugoslov Med Biohem 23: 333–341, 2004 Pregledni ~lanakReview article

ULOGA EOZINOFILNIH LEUKOCITA U PATOGENEZI BRONHIJALNE ASTME

Danijela Vu~evi}, Tatjana Radosavljevi}, Gordana \or|evi}-Deni}

Institut za patolo{ku fiziologiju, Medicinski fakultet, Beograd

Kratak sadràj: Patogeneza bronhijalne astme nije do kraja razja{njena. U plu}ima, perifernoj krvi i spu-tumu astmati~ara prisutan je pove}an broj eozinofila. Eozinofilija je identifikovana kao faktor rizika za razvojopstrukcije vazdu{nih puteva. Izrazit eozinofilni zapaljenski infiltrat u bronhijalnoj sluznici i korelacija izme|ubroja eozinofilnih leukocita i teìne bolesti podràva hipotezu prema kojoj su eozinofili glavne inflamacijske }elijesposobne da izazovu patofiziolo{ke promene karakteristi~ne za astmu. Aktivirani eozinofili sekretuju {iroki spek-tar preformiranih i novosintetisanih medijatora koji dovode do o{te}enja bronhijalnog epitela, spazma glatkihmi{i}a bronhija, pove}anja sekrecije sluzi i vazodilatacije. Dokazano je da se u toku astmati~nog napadapove}ava proizvodnja oksidanasa. Brojna istraìvanja ukazuju da eozinofili u krvi i vazdu{nim putevima osobaobolelih od bronhijalne astme stvaraju ve}u koli~inu oksidanasa u odnosu na zdrave osobe.

Klju~ne re~i: eozinofilni leukociti, bronhijalna astma, inflamacija, oksidansi

Adresa autora:

Danijela Vu~evi}Institut za patolo{ku fiziologiju Medicinskog fakulteta u BeograduDr Suboti}a 911000 Beograd

334 Jugoslov Med Biohem 2004; 23 (4)

U nekih bolesnika s hroni~nom bronhijalnomastmom jasno razlikovanje ove bolesti od HOBP nijemogu}e kori{}enjem savremenih metoda za imidìngplu}a i ispitivanje plu}ne funkcije. Znaci karakteris-ti~ni za bronhijalnu astmu i HOBP, koji su va`ni zadiferencijalnu dijagnozu ovih bolesti prikazani su natabeli II. Me|utim, ovi znaci se ne sre}u kod svihbolesnika. Na primer, osoba koja nikad nije pu{ilamoè da se razboli od HOBP. Tako|e, astma moèda se razvije u odraslih, pa ~ak i u starijih osoba (8).

Eozinofilni leukociti i zapaljenje mukoze disajnih puteva

Postoje dokazi o akutnoj i hroni~noj inflamacijikoje su nepravilno raspore|ene u disajnim putevimaastmati~ara (9). Op{te je prihva}eno da su eozinofilidominantne efektorne }elije u hroni~noj inflamaciji(2’ 4, 9, 47, 48 ’59). Svoju ulogu ostvaruju u tokuprocesa degranulacije osloba|anjem ~itavog nizamedijatora i faktora rasta (Tabela III–V).

Table I Karakteristike inflamacije u bronhijalnoj astmi i HOBP

Bolest plu}a

Inflamacijske }elije

Medijatori inflamacije

Posledice

Odgovor na terapiju

Bronhijalna astma

EozinofiliMali porast broja makrofagaPorast broja CD4+ T limfocitaAktivacija mastocita

Leukotrien D4 (LTD4)Interleukin-4 (IL-4)Interleukin-5 (IL-5)Mnogi drugi medijatoriFragilan epitelZadebljanje bazalne membraneMetaplazija sluziUve}anje `lezdaGlikokortikoidi inhibiraju inflamaciju

Table II Diferencijalna dijagnoza bronhijalne astme i HOBP

Bolest plu}a

Bronhijalnaastma

HOBP

Znaci koji ukazuju na bolestPo~etak u mladosti (~esto u detinjstvu)Simptomi se menjaju iz dana u danSimptomi se javljaju no}u ili rano ujutruêsta pridruènost alergije, rinitisa i/ili ekcemaPozitivna porodi~na anamneza za astmuUglavnom reverzibilno ograni~enje protoka vazduhaPo~etak u srednjem ìvotnom dobuSpora progresija simptomaAnamneza o dugotrajnom pu{enjuDispnoja pri fizi~kom naporuUglavnom ireverzibilno ograni~enje protoka vazduha

Table III Proteinski sadràj eozinofilnih granula

ProteinGlavni osnovni protein(MBP-major basic protein)Eozinofilni katjonski proteinEozinofilni neurotoksinEozinofilna peroksidazaLizozimKisela fosfatazaArilsulfataza BKatalazaEnoil-CoA hidrataza3-ketoacil-CoA tiolazab-glukuronidazaKatepsin DElastazaGranulocitno-monocitni faktor rasta (GM-CSF)Interleukin-2 (IL-2)Interleukin-4 (IL-4)Interleukin-5 (IL-5)Interleukin-6 (IL-6)Faktor nekroze tumora a(TNFa)RANTESTip II fosfolipaza A2Baktericidni protein kojipove}ava permeabilnostKisela fosfatazaArilsulfataza B KatalazaCitohrom b558ElastazaEozinofilni katjonski proteinLizofosfolipaza (Charcot--Leyden kristalni protein)Ciklooksigenaza5-lipoksigenaza15-lipoksigenazaLeukotrien C4 sintetazaEozinofilna peroksidazaEsteraza

Vrsta granulaSekundarne (specifi~ne) granule

Sekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granule

Sekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granule

Sekundarne (specifi~ne) granuleSekundarne (specifi~ne) granuleSekundarne (specifi~ne) granule

Male granuleMale granuleMale granuleMale granuleMale granuleMale granulePrimarne (nespecifi~ne granule)

Lipidna tela{caLipidna tela{caLipidna tela{caLipidna tela{caLipidna tela{caLipidna tela{ca

HOBP

NeutrofiliVeliki porast broja makrofagaPorast broja CD8+ T limfocitaLeukotrien B4 (LTB4)

Interleukin-8 (IL-8)Faktor nekroze tumora a (TNFa)Mnogi drugi medijatoriSkvamozna metaplazija epitelaDestrukcija parenhimaMetaplazija sluziUve}anje `lezda

Glikokortikoidi imaju mali efekat ili ne ispoljavaju svoje dejstvo


Iz granula eozinofila osloba|aju se katjonskiproteini (eozinofilni katjonski protein, eozinofilna per-oksidaza, eozinofilni neurotoksin, i dr.), enzimi (fos-folipaza A2, eozinofilna kolagenaza, katalaza, kiselafosfataza, histaminaza, itd.) i neuropeptidi (supstancaP, vazoaktivni intestinalni peptid ’ VIP, i dr.). Najzastu-pljeniji protein u granulama eozinofila (MBP ’ majorbasic protein), deluje toksi~no na epitel vazdu{nihputeva. Pokazano je da MBP izaziva o{te}enje pneu-

Table IV Eozinofilni lipidni medijatori

Faktor aktivacije trombocita

(PAF-platelet activation factor)

Leukotrien B4 (LTB4)

Leukotrien C4 (LTC4)

Tromboksan A2 (TXA2)

Prostaglandin E2 (PGE2)

Prostaglandin G2 (PGG2)

Prostaglandin F2a (PGF2a)

Prostaglandin I2 (PGI2)

5-hidroksieikosatetraenoi~na kiselina (5-HETE)



5,15-dihidroksieikosatetraenoi~na kiselina (5,15-diHETE)

8,15-dihidroksieikosatetraenoi~na kiselina (8,15-diHETE)

14,15-dihidroksieikosatetraenoi~na kiselina

(14,15-diHETE)

Lipoksin A4 (LXA4)

Lipoksin C4 (LXC4)

13-hidroksilinoleinska kiselina (13-HODE)

Table V Eozinofilni faktori rasta

Faktor nekroze tumora a (TNFa – tumor necrosis factor a)Faktor nekroze tumora b (TNFb – tumor necrosis factor b)Faktor rasta poreklom iz trombocita (PDGF – platelet-derived growth factor)Faktor rasta vaskularnog endotela (VEGF – vascular endothelial growth factor)Heparin vezuju}i epidermski faktor rasta (HB-EGF → heparin-binding epidermal growth factor)Nervni faktor rasta (NGF – nerve growth factor)Endotelin (ET – endothelin)

Tabela VI Eozinofilni receptori za hemokine i njihovi endogeni ligandi

Subfamilija hemokina

CXC hemokini

CXC hemokini

CXC hemokini

CXC hemokini

CC hemokini

CC hemokini

CC hemokini

CC hemokini

CC hemokini

CC hemokini

CC hemokini

CC hemokini

CC hemokini

CC hemokini

CC hemokini

CC hemokini

Nomenklatura

CXCR1

CXCR1

CXCR2

CXCR2

CCR1

CCR1

CCR1

CCR1

CCR1

CCR1

CCR3

CCR3

CCR3

CCR3

CCR3

CCR3

Endogeni ligandi

Interleukin-8 (IL-8)

Granulocitni protein hemotakse-2

(GCP-2 → granulocyte chemotactic protein-2)

Interleukin-8 (IL-8)

Granulocitni protein hemotakse-2

(GCP-2 → granulocyte chemotactic protein-2)

Makrofagni inflamacijski protein 1a (MIP1a)

RANTES

Monocitni protein hemotakse-2

(MCP-2 → monoocyte chemotactic protein-2)


(MCP-3 →monoocyte chemotactic protein-3)



Leukotaktin-1

Eotaksin I

Eotaksin II

Leukotaktin-1





RANTES


mocita (10, 11) i deskvamaciju epitela respiracijskogtrakta (12, 13). MBP naru{ava transport jona u epitelutraheje, {to moè da izmeni zapreminu i sastavte~nosti koja oblaè vazdu{ne puteve (14).

Nezaobilazni element aktivacije i efektornefunkcije eozinofila su i novosintetisani lipidni medija-tori ciklooksigenaznog puta (prostaglandini i trom-boksan A2) i lipoksigenaznog puta (leukotrieni,lipoksini i dr.). Osim toga, eozinofili stvaraju i citokine(eokine), i to pre svega interleukin-3 (IL-3), IL-5 igranulocitno-monocitni faktor rasta (GM-CSF), kojisvojim autokrinim dejstvom odràvaju i intenzivirajuinflamaciju. U granulama eozinofila utvr|eno je i pris-ustvo IL-10, IL-11, IL-12, IL-16, interferona g (INFg) ifaktora koji inhibira migraciju makrofaga (MIF ’ ma-crophage migration inhibitory factor). Zapaljenskiproces moduliraju i eokini akutne inflamacije (IL-1a,IL-6, i faktor nekroze tumora a ’ TNFa), faktor akti-vacije trombocita (PAF), kao i hemokini (IL-8, makro-fagni inflamacijski protein 1a ’ MIF1a, i dr.) (2’4, 9,47, 48’59).

Inflamacijski odgovor predstavlja kaskadu do-ga|aja koja se manifestuje sekvencionalnom akcijomintegrina, selektina, superfamilije imunoglobulina,kadherina, adresina i drugih familija adhezivnih mo-lekula i njihovih receptora. Zapaljenske }elije se kotr-ljaju preko endotela posredstvom mehanizama kojeobezbe|uju selektini (L, E i P selektini). Zahvaljuju}iintegrinima ostvaruje se ~vrsto vezivanje inflamacij-skih }elija za endotel. Naime, na neaktiviranim leuko-citima integrini se nalaze u miruju}em stanju i eks-primiraju se na bazalnom nivou. Aktivacijom }elijastimuli{e se i ispoljavanje i aktivacioni status ovihglikoproteinskih membranskih molekula, kao i sekre-cija L-selektina. Integrini su uklju~eni i u kontakteleukocita sa proteinima ekstracelularnog matriksa.Najzna~ajniji ligandi integrina me|u proteinima eks-tracelularnog matriksa su kolagen, laminin, fibronek-tin i vitronektin (15, 67).

Eozinofili na svojoj povr{ini ispoljavaju adhe-zivne molekule va`ne za povezivanje ovih leukocita unastanku imunskog odgovora, za usmeravanje nji-hovog kretanja kroz krvne sudove i za interakciju saekstracelularnim matriksom. Subfamiliji b1 integrina(raniji naziv VLA antigeni ’ very late activation) pri-padaju VLA-4, VLA-5 i VLA-6. U b2 subfamiliju inte-grina svrstani su Mac-1 (macrophage-1 antigen kojise jo{ ozna~ava i kao CD11b/CD18), leukocitni funk-cionalni antigen-1 (skra}eno ozna~en kao LFA-1 iliCD11a/CD18) i p150/95 (CD11c/CD18). a4b7 je ta-ko|e integrin }elijske membrane eozinofila. Na povr-{ini eozinofila ispoljeni si u L-selektin i sijaloglikopro-tein (Sialil Lewis X). PECAM-1, koji je dobio naziv odpo~etnih slova engleskih re~i platelet endotheliumcellular adhesion molecule-1, i intercelularni adhe-zivni molekul-1 (ICAM-1) predstavljaju adhezivne mo-lekule iz superfamilije imunoglobulina tako|e ekspri-mirane na eozinofilima (2, 4, 67).

Eozinofili vezuju imunoglobuline (Ig) preko povr-{inskih receptora. Tako se IgE i IgG vezuju za FcgRI,FcgRII i Mac-2 receptore, dok se IgA vezuje za FcaR(4).

Svoje specifi~ne receptore na povr{ini eozinofilaimaju i C5a, CR1, CR3 i C1q komponenta komple-menta (4).

Brojni povr{inski receptori eozinofila olak{avajuinterakciju ovih }elija sa citokinima. Na ovaj na~insvoje delovanje ostvaruju IL-2, CD25, IL-3, IL-5, IL-13, GM-CSF, interferon a (INFa), INFb, INFg, TNFa,i transformi{u}i faktor rasta b (TGFb) (4, 16).

Na povr{ini eozinofila se mogu uo~iti i receptoriza hemokine. Vezivanjem za ove receptore hemokiniu~estvuju u migracijskim kretanjima eozinofila, akti-vaciji integrina, indukciji respiracijskog praska, tran-skripciji citokina (neki od njih), angiogenezi, stvaranjukolagena i proliferaciji hematopoetskih prekursora.Najnovija istraìvanja pokazuju da je RANTES (hemo-kin ~iji naziv predstavlja kovanicu dobijenu odpo~etnih slova engleskih re~i regulated upon acti-vation normal T cell expressed and secreted) pravihemotakti~ki faktor u bolesnika sa alergijskom ast-mom, dok je IL-5 neophodan kofaktor (4, 17).

Eozinofili i njihovi produkti su prisutni u krvi, di-sajnim putevima, pljuva~ki i u bronhoalveolarnom la-vatu (BAL) osoba obolelih od bronhijalne astme. Uastmati~ara eozinofilija je u korelaciji sa stepenomopstrukcije i teìnom bolesti (2, 4).

O procesima koji dovode do aktivacije eozinofi-la in vivo jo{ uvek se relativno malo zna. Osnovnakarakteristika eozinofilne funkcije je da je za nju neo-phodan »priming« eozinofila, {to prevedeno sa engle-skog jezika zna~i »prvi sloj, prvo premazivanje«. Pri-ming bi mogao da se posmatra kao me|usobni uti-caj modulacijskih i aktivacijskih signala. Aktivacijaeozinofilnih receptora preko adhezivnih molekula,komponenti komplementa i imunoglobulina bi moglada bude uklju~ena u ove doga|aje. Receptori eozi-nofila su potentni signalni molekuli in vitro, ali svojuoptimalnu funkciju postiù tek nakon priminga sacitokinima i hemotaksinima. IL-5 i GM-CSF primingeozinofila neophodan je za odgovore ovih }elija, uklju-~uju}i sintezu i osloba|anje bioaktivnih medijatora,{to doprinosi pove}anoj bronhijalnoj reaktivnosti(18). Smatra se da cirkuli{u}i eozinofili dobijaju pove-}anu sposobnost da sekretuju IL-5 kad stignu u plu-}a, gde su stimulisani i drugim citokinima, kao {to suIL-2 i IL-4 (19).

Migracija eozinofila iz cirkulacije u plu}no tkivose odigrava nakon njihove adhezije za vaskularneendotelne }elije, komponente ekstracelularnog ma-triksa i tkivne }elije. Kretanje }elija u zapaljensko pod-ru~je se sastoji iz rolovanja (kotrljanja), ~vrste adhezi-je i transendotelne migracije. Eozinofilna dijapedezana mesto inflamacije je regulisana citokinima pod-staknutom i poja~anom ekspresijom endotelnih ad-


hezivnih molekula. Na povr{ini endotelnih }elija utvr-|eno je prisustvo vaskularnog adhezivnog molekula-1 (VCAM-1), ICAM-1, ICAM-2, PECAM-1, E-selektina,P-selektina, fibronektina i laminina (20). Svaki od ovihmolekula ima svoj odgovaraju}i ligand na eozinofili-ma i bazofilima (LFA-1, Mac-1, VLA-4, L-selektin)(21). Specifi~na ekspresija VLA-4 na eozinofilima ilimfocitima, i njeno odsustvo na neutrofilima dovelisu do hipoteze da je VCAM-1 predominantni endotel-ni regulator hroni~ne inflamacije bronhijalne mukoze(22). IL-4 dovodi do pove}ane ekspresije VCAM-1 naendotelnim }elijama (23). Pove}ana endotelna eks-presija VCAM-1, E-selektina i ICAM-1 je povezana saalergijskim zapaljenjem plu}a (2). Lutman i saradnici(24) su eksperimentalno pokazali da IL-4 i IL-13 po-ja~avaju efekat TNF? na eozinofilnu aktivaciju, kao isinergisti~ki efekat ovih citokina sa IL-5 na eozinofil-nu aktivaciju.

IL-5 poja~ava adheziju humanih eozinofila zavaskularni endotel i dovodi do hiperreaktivnosti don-jih disajnih puteva (25). Receptor IL-5 pripada famili-ji tipa 1 citokinskih receptora. a subjedinica (bc lanackoji koriste i IL-3 i GM-CSF za signalnu transdukciju)ovog visokoafinitetnog receptora se aktivira nakonvezivanja IL-5 za receptorski IL-5 a lanac (IL-5Ra), itime zapo~inje serija intracelularnih doga|aja. Naovaj na~in tirozinskom fosforilacijom Janus kinaze 2(JAK2) i proteina STAT1a (signal transducer andactivators of transcription 1a) aktivira se JAK-STATput. Familiju STAT proteina ~ini sedam ~lanova(STAT 1a, STAT 1b i STAT 2’6). Osim JAK2, IL-5aktivira i druge tirozin kinaze (lyn, hck, yes, btk, tec,c-fes). JAK2 i c-fes su direktno udruène sa bc subje-dinicom GM-CSF/IL-3/IL-5 receptora, sugeri{u}i daje njihova aktivacija rani doga|aj citokinske signali-zacije (26, 27). JAK kinaze su neophodne za aktivaci-ju STAT proteina koji se nalaze u latentnom obliku ucitoplazmi. Nakon aktivacije, odnosno fosforilacije,STAT proteini formiraju homodimere i heterodimere,translociraju se u jedro, vezuju za odre|enu sekvencudezoksiribonukleinske kiseline (DNK) i reguli{u tran-skripciju. U humanim eozinofilima IL-5 indukuje dvaDNK-vezuju}a kompleksa koji sadrè tirozin fosfori-lisane proteine. Jedan od ova dva DNK-vezuju}akompleksa sadrì STAT1a verovatno kao dimer. Me-|utim, IL-5 pored indukcije STAT proteina indukuje imnoge druge nuklearne proteine. Dugotrajna stimu-lacija ovim eokinom dovodi do indukcije transkrip-cionog faktora c-myc u humanim eozinofilima. IL-5preko ras puta indukuje transkripcione faktore c-fos ic-jun, p21 ras, raf, MAPKK (p41 i p45) i MAPK (p44).S obzirom da do sada nije identifikovan apsolutnoeozinofilni specifi~ni regulacijski DNK element i tran-skripcioni faktor, mogu}e je da je specifi~na signa-lizacija rezultat specifi~ne kombinacije transkripcionihfaktora (25).

Eozinofilna apoptoza u hroni~noj inflamaciji bronhijalne mukoze

Broj eozinofila in vivo je regulisan ne samo pro-dukcijom eozinofila u kostnoj srì, ve} i stepenomeozinofilne apoptoze, koja predstavlja naj~e{}i oblikfiziolo{ke }elijske smrti. Bcl-2 (B-cell leukemia onco-gene-2) familija gena kodira heterogenu grupu pro-teina koji predstavljaju klju~ne intracelularne modula-tore (regulatore) }elijskog umiranja po tipu apoptoze(28, 29). Otkri}em Bcl 2 gena, Bcl 2 familija proteinapostaje posebno istraìvana oblast zbog izuzetnogzna~aja za razumevanje molekulsko-biolo{kih meha-nizama koji leè u osnovi procesa apoptoze. Posled-njih godina naro~ito se istraùju antiapoptozni ~lanoviBcl 2 familije proteina (29’34).

Odloèna eozinofilna apoptoza se smatra jed-nim od najodgovornijih mehanizama koji doprinoseeozinofiliji. U ljudi izgleda da je IL-5 specifi~an faktorpreìvljavanja eozinofila, pa nije iznena|uju}a ~injeni-ca da su eozinofilija i visoka ekspresija IL-5 udruèneu hroni~noj inflamaciji bronhijalne mukoze. Eozino-filna apoptoza moè biti odloèna IL-om 5 kojeg se-kretuju susedne }elije, ali i autokrinom produkcijomovog citokina. Izgleda da stimulacija eozinofila IL-om5 dovodi do indukcije Bcl-xl (Bcl-2-regulated factorxl), koji je vaàn antiapoptotski gen. Postoje istraì-vanja gde je verifikovana pove}ana ekspresija Bcl-2nakon tretiranja eozinofila IL-om 5 (35). Antiapoptoz-no dejstvo Bcl 2 proteina zasniva se na mogu}nostida veè Bax (Bcl 2-associated x) protein u formi hete-rodimera i tako onemogu}i stvaranje proapoptoznihBax/Bax homodimera. Tokom indukcije procesa apo-ptoze, homodimeri Bax proteina formiraju kanale namembranama mitohondrija. Putem ovih kanala mito-hondrije napu{taju molekuli citohroma c, koji su odposebne va`nosti za aktivaciju izvr{ne faze procesaapoptoze (36). Na osnovu ovih saznanja, ve}ina auto-ra kao glavnu biolo{ku ulogu Bcl 2 proteina u inhibi-ciji procesa apoptoze smatra njegovu sposobnostvezivanja za Bax protein i heterodimerizacionu neu-tralizaciju njegovog proapoptoznog dejstva (37’39).U in vitro uslovima je dokazano da je preìvljavanjeeozinofila produèno kada se inkubiraju u kulturamasa eozinofilnim citokinima kao {to su IL-3, IL-5 i GM-CSF (35).

Eozinofilni leukociti i oksidacijskoo{te}enje u bronhijalnoj astmi

Plu}a predstavljaju primarno ciljno tkivo za oksi-dacijsko o{te}enje zbog svoje lokalizacije, anatomije ifunkcije. Kod naglog pove}anja broja i koli~ine oksi-danasa, u uslovima tzv. oksidacijskog stresa, prirodniza{titni mehanizmi popu{taju. Dokazano je da se utoku astmati~nog napada pove}ava proizvodnja oksi-danasa. Brojna istraìvanja ukazuju da eozinofili u krvii vazdu{nim putevima osoba obolelih od bronhijalneastme stvaraju ve}u koli~inu reaktivnih kiseoni~kih

vrsta (RKV) u odnosu na zdrave osobe (4, 8, 46, 50,51, 57, 58, 61’66, 68, 69). Metaboliti kiseonika ~ijiizvor su i eozinofili, direktno o{te}uju proteine plu}-nog matriksa i/ili slabe funkciju antiproteaza i/ili inak-tiviraju enzime uklju~ene u sintezu elastina i obnovuplu}nog tkiva. RKV eozinofila podsti~u sekreciju sluzii bronhokonstrikciju. Tako npr., vodonik peroksid(H2O2) kontrahuje glatki mi{i} disajnih puteva invitro, a izoprostan F2a-III, koji se stvara delovanjemslobodnih radikala u toku peroksidacije arahidonskekiseline, je snaàn konstriktor disajnih puteva ~oveka(8, 40, 41).

U izdahnutom vazduhu astmati~ara zabeleènesu povi{ene vrednosti H2O2 i azot monoksida (NO)(42, 43). NO je veoma reaktivan i nestabilan oksi-dans. U sintezi NO u~estvuje enzim NO sintetaza(NOS). Inducibilna forma ovog enzima (iNOS), koja jeodgovorna za citotoksi~ne i biocidne efekte NO,prisutna je u eozinofilnim leukocitima. Aktivira se bak-terijskim lipopolisaharidima i odre|enim citokinima(INFg, TNFa i IL1b). Inducibilnoj NOS je za maksi-malnu produkciju NO potrebno 8’12 sati, a koli~ineNO koje se tom prilikom produkuju su znatne (15,44).

Oksidansi eozinofila mogu da reaguju i sa lipidi-ma i nukleinskim kiselinama, {to moè da dovede dodisfunkcije ili smrti }elije. Tako|e, u uslovima oksi-dacijskog stresa aktivacijom transkripcionog faktoraNF-kappaB koji upravlja ekspresijom razli~itih infla-

macijskih gena va`nih za bronhijalnu astmu, kao {tosu IL-8 i TNFa, dodatno se podsti~e zapaljenski pro-ces (8, 60).

Singlet kiseonik (1O2), superoksid anjon (O2.-),

H2O2 i hidroksil radikal (OH.) su RKV koje eozinofilistvaraju u toku respiracijskog praska. U prisustvuH2O2 kataliti~kim dejstvom eozinofilne peroksidazedolazi do oksidacije halogenih elemenata i stvaranjareaktivnih kiselina (hipobromne, bromovodoni~ne ijodovodoni~ne kiseline) (16). Istraìvanje na mi{evi-ma inficiranim Toksokarom kanis (Toxocara canis)je pokazalo da se eozinofilna peroksidaza taloì uplu}nom parenhimu i miokardu, {to moè da dovededo o{te}enja srca i plu}a (45).

Zaklju~ak

Patogeneza bronhijalne astme nije do krajarazja{njena. Ono {to se sa sigurno{}u zna je da eozi-nofili bitno doprinose njenom nastanku. Kompleksanpatofiziolo{ki supstrat ove bolesti otkriva nova poljaistraìvanja interakcija eozinofilnih medijatora i cito-kina sa medijatorima i citokinima drugih inflamacij-skih }elija. Tako|e, name}e se potreba za dodatnimprou~avanjem kako inhibicije aktivnosti IL-4 i IL-5,tako i poja~avanja aktivnosti IL-10, IL-12 i IFNg, tj.{to uspe{nijeg imitiranja njihovih dejstava u cilju pro-nalaènja efikasnijih lekova u terapiji bronhijalneastme.


Slika 1 Reaktivne kiseoni~ke vrste i plu}ni antioksidacijski mehanizmi

GLUKOZA

G6PD6PGD

NADP+

NADPHGSH

SOD

Fe++

KATALAZA

superoksidanjon

vodonikperoksid

GSSG

EPO

eozinofilna peroksidaza

HOBrhipobromna

kiselina

O2–

⋅OHhidroksilradikal

H2O2 H2O

+

GSHreduktaza

GSHreduktaza

RIBULOZA-6FOSFAT


Literatura

1. \or|evi}-Deni} G. Patolo{ka fiziologija respiracijskogsistema. U: Beleslin BB., Proti} S, \or|evi}-Deni} G(ured.) i saradnici. Specijalna patolo{ka fiziologija, Za-vod za ud`benike i nastavna sredstva, Beograd, 2003:119’42.

2. Ulfman LH, Kuijper PHM, Van der Linden JAM, Lam-mers JJ, Zwaginga JJ, Koenderman L. Characteriza-tion of eosinophil to TNF-a-activated endotheliumunder flow conditions: a4 integrins mediate initial atta-chment and E-selectin mediates rolling. J Immunol1999; 163: 343’50.

3. Nakajima H. CD4-positive T-lymphocytes and inter-leukin-5 mediate antigen-induced eosinophil infiltrationinto the mouse trachea. Am Rev Respir Dis 1992; 146:374.

4. Adolphson CR, Gleich GJ. Eosinophils. In: Holgate S,Church K (eds). Allergy. Gower Medical, London, 1993:6.1’6.12.

5. Savi} N. Odre|ivanje koncentracije interleukina-4 iinterleukina-5 u serumu bolesnika sa bronhijalnom ast-mom i njihov dijagnosti~ki i prognosti~ki zna~aj. Magi-starska teza, Beograd, Univerzitet u Beogradu, 2001.

6. Hawker KM, Johnson PRA, Hughes JM, Black JL.Interleukin-4 inhibits mitogen-induced proliferation ofhuman airway smooth muscle in culture. Am J Physiol1998; 275: 469 ’77.

7. \or|evi}-Deni} G. Medijatori anafilakti~ke reakcije utoku plu}nih bolesti. De~ Pulm 1993; I, 1’2: 11’16.

8. Lenfant C, Khaltaev N. Global strategy for the diagno-sis, management and prevention of chronic obstructivepulmonary disease: National heart, lung and bloodinstitute/World health organization (NHLBI/WHO)Workshop 2001; 2701: 28’57.

9. Jovi~i} @. Chronic inflammation in bronchial asthma.Medicinska istraìvanja 1999; 33 (3): 15’20.

10. Ayars GH. Eosinophil-and eosinophil granule-mediatedpneumocyte injury. J Allergy Clin Immunol 1985; 76:595.

11. Hisamatsu K. Cytotoxicity of human eosinophil granulemajor basic protein to the human nasal sinus mucosain vitro. J Allergy Clin Immunol 1990; 86: 52.

12. Frigas E, Loegering DA, Gleich GJ. Cytotoxic effects ofthe guinea pig eosinophil major basic protein on tra-cheal epithelium. Lab Invest 1980; 42: 35.

13. Motojima S. Toxicity of eosinophil cationic proteins forguinea pig tracheal epithelium in vitro. Am Rev RespirDis 1989; 139: 801.

14. Jacoby DB. Effect of human eosinophil major basicprotein on ion transport in dog tracheal epithelium. AmRev Respir Dis 1988; 137: 13.

15. Maravi}-Stojkovi} V, Radak \, Dimkovi} S. Endotel uaterosklerozi. U: Radak \, Maravi}-Stojkovi} V. ured. Ii-munologija u genezi i terapiji ateroskleroze. Beograd,2004: 31’41.

16. Litchfield MT, Lee TH. Asthma cells and cytokines. JAsthma 1992; 29(3): 181’91.

17. Venge J, Lampinen M, Hakansson L, Rak S, Venge P.Identification of IL-5 and RANTES as the major eosi-nophil chemoattractants in the asthmatic lung. J AllergClin Immunol 1996; 97: 1110’15.

18. Bracke M, Dubois GR, Bolt K, Bruijnzeel PLB, VaermanJP, Lammers JWJ. Differential effects of the T helpercell type 2-derived cytokines IL-4 and IL-5 on ligandbinding to IgG and IgA receptors expressed by humaneosinophils. J Immunol 1997; 159: 1459’65.

THE ROLE OF EOSINOPHILIC LEUKOCYTES IN PATHOGENESIS OF BRONCHIAL ASTHMA

Danijela Vu~evi}, Tatjana Radosavljevi}, Gordana \or|evi}-Deni}

Institut za patolo{ku fiziologiju, Medicinski fakultet, Beograd

Summary: Pathogenesis of bronchial asthma has not been completelly understood. Eosinophilic leuko-cytes accumulate in high numbers in the lungs, blood and sputum of asthmatic patients. Peripheral bloodeosinophilia has been identified as a risk factor for the development of airway obstruction. Prominenteosinophilic inflammatory infiltrate in the bronchial mucosa and correlation between eosinophil numbers anddisease severity supports the hypothesis that eosinophils are central inflammatory cells capable of inducingpathophysiological features of asthma. Activated eosinophils secrete a wide range of preformed and newly ge-nerated mediators that damage the bronchial epithelium, contract smooth muscle, increase mucous secretionand cause vasodilation. There is ample evidence that oxidants generation is increased during an asthma exa-cerbation. Many investigations indicate that airway and blood eosinophils produce more oxidants in asthmaticpatients compared with control subjects.

Key words: eosinophilic leukocytes, bronchial asthma, inflammation, oxidants

19. Lai CKW, Ho ASS, Chan CHS, Tang J, Leung JCK, LaiKN. Interleukin-5 messenger RNA expression in peri-pheral blood CD4+ cells in asthma. J Allergy ClinImmunol 1996; 97: 1320’6.

20. Bochner BS, Schleimer RP. The role of adhesion mo-lecules in human eosinophil and basophil recruitment.J Allergy Clin Immunol 1994; 94: 427’38.

21. Wardlaw AJ, Symon FS, Walsh GM. Eosinophil adhe-sion in allergic inflammation. J Allergy Clin Immunol1994; 94: 1183’9.

22. Barks JL, McQuillan JJ, Iadermarco MF. TNF-a andIL-4 synergistically increase vascular cell adhesion mo-lecule-1 expression in cultured vascular smooth musclecells. J Immunol 1997; 159: 4532’8.

23. Dickensheets HL, Donnely PR. IFN-g and IL-10 inhibitinduction of IL-1 receptor type I and type II gene expre-ssion by IL-4 and IL-13 in human monocytes. J Immu-nol 1997; 159: 6226’33.

24. Lutmann W, Matthiesen T, Matthys H, Virchow JCJr.Synergistic effects of interleukin-4 or interleukin-13 andtumor necrosis factor-alpha on eosinophilic activationin vitro. Am J Resp Cell Mol Biol 1999; 20(3): 474’80.

25. Wang P, Wu P, Cheewatrakoolpon G, Myers JG, EganRW, Billah MM. Selective inhibition of IL-5 receptora-chain gene transcription by IL-5, IL-13 and granulo-cyte-macrophage colony stimulating factor in humanblood eosinophils. J Immunol 1998; 160: 4427’32.

26. Abbas KA. Cellular and molecular immunology, W.B.Saunders Company, 1997: 249’78.

27. Van der Bruggen T, Koenderman L. Signal transduc-tion in eosinophils. Clin and Exp Allergy 1996; 26:880’91.

28. Adams JM, Cory S. The Bcl-2 protein family: arbiters ofcell survival. Science 1998; 281 (5381): 1322’26.

29. Braju{kovi} G, [karo Mili} A, Cerovi} S, Marjanovi} S,Kneèvi} U{aj S, îzmi} M, et al. Familija Bcl 2 proteinakod malignih bolesti. Vojnosanit Pregl 2004; 61(3):305’10.

30. Makin G, Hickman JA. Apoptosis and cancer chemo-therapy. Cell Tissue Res 2000; 301 (1): 143’52.

31. Thompson CB. Apoptosis in the pathogenesis andtreatment of disease. Science 1995; 267 (5203):1456’62.

32. Katoch B, Sebastian S, Sahdev S, Padh H, Hasnain SE,Begum R. Programmed cell death and its clinical impli-cations. Indian J Exp Biol 2002; 40(5): 513’24.

33. Rutledge SE, Chin JW, Schepartz A. A view to a kill: lig-ands for Bcl-2 family proteins. Curr Opin Chem Biol2002; 6 (4): 479’85.

34. Mareel M, Leroy A. Clinical, cellular and molecularaspects of cancer invasion. Physiol Rev 2003; 83(2):337’76.

35. Adachi T, Motojima S, Hirata A, Fukuda T, Kihara N,Kosaku A. Eosinophil apoptosis caused by theophy-lline, glucocorticoids and macrolides after stimulationwith IL-5. J Allergy Clin Immunol 1996; 6 (98): 207’15.

36. Korsmeyer SJ. Bcl-2 gene family and the regulation ofprogrammed cell death. Cancer Res 1999; 59 (7 suppl):1693s’700s.

37. Korsmeyer SJ, Shutter JR, Veis DJ, Merry DE, OltvaiZN. Bcl 2/Bax: a rheostat that regulates an anti-oxidantpathway and cell death. Semin Cancer Biol 1993; 4(6):327’32.

38. Reed JC, Miyashita T, Takayama S, Wang HG, Sato T,Krajewski S. Bcl-2 family proteins: regulators of celldeath involved in the pathogenesis of cancer and resist-ance to therapy. J Cell Biochem 1996; 60(1): 23’32.

39. Murphy KM, Ranganathan V, Farnsworth ML, KavallarisM, Lock RB. Bcl-2 inhibits Bax translocation fromcytosol to mitochondria during drug-induced apoptosisof human tumor cells. Cell Death Differ 2000; 7(1):102’11.

40. Pratico D, Basili S, Vieri M, Cordova C, Violi F,Fitzgerald GA. Chronic obstructive pulmonary diseaseis associated with an increase of isoprostane F2alpha-III, an index of oxidant stress. Am J Respir Crit CareMed 1998; 158:1709’14.

41. Montuschi P, Collins JV, Ciabattoni G, Lazzeri N,Corradi M, Kharitonov SA, et al. Exhaled 8-isoprostaneas an in vivo biomarker of lung oxidative stress inpatients with COPD and healthy smokers. Am J RespirCrit Care Med 2000; 162:1175’7.

42. Dekhuijzen PN, Aben KK, Dekker I, Aarts LP, WieldersPL, Van Herwaarden CL, et al. Increased exhalation ofhydrogen peroxide in patients with stable and unstablechronic obstructive pulmonary disease. Am J RespirCrit Care Med 1996; 154: 813’16.

43. Maziak W, Loukides S, Culpitt S, Sullivan P, KharitonovSA, Barnes PJ. Exhaled nitric oxide in chronic obstruc-tive pulmonary disease. Am J Respir Crit Care Med1998; 157: 998’1002.

44. Jukema JW. New insights into atherosclerosis. Cardio-logie 2000; 7: 37’40.

45. Dimayuga E, Stober M, Kayes SG. Eosinophil peroxi-dase levels in hearts and lungs of mice infected withToxocara canis. J Parasitol 1991; 77: 461.

46. Bowler RP, Crapo JD. Oxidative stress in allergic respi-ratory diseases. J Allergy Clin Immunol 2002; 110:349’56.

47. Lawrence T, Willoughby DA, Gilroy DW. Anti-inflam-matory lipid mediators and insights into the resolutionof inflammation. Immunology 2002; 2: 787’95.

48. Domachowske JB, Bonville CA, Easton AJ, RosenbergHF. Pulmonary eosinophilia in mice devoid of inter-leukin-30. J Leukoc Biol 2002; 71: 966’72.


49. Kips JC, O’Connor BJ, Langley SJ, Woodcock A,Kerstjens HAM, Postma DS, et al. Effect of SCH55700,a humanized antihuman interleukin-5 antibody in se-vere persistent asthma. Am J Res Crit Care Med 2003;167: 1655’59.

50. Williams TJ. The eosinophil enigma. J Clin Invest 2004;113 (4): 507’9.

51. Tobin MJ. Asthma, airway biology, and nasal disordersin AJRCCM 2003. Am J Res Crit Care Med 2004; 169(2): 265’76.

52. Flood-Page P, Menzies-Gow A, Phipps S, Ying S, Wan-goo A, Ludwig MS, et al. Anti-IL-5 treatment reducesdeposition of ECM proteins in the bronchial subepithe-lial basement membrane of mild atopic asthmatic. JClin Invest 2003; 112 (7): 1029’36.

53. Kay BA, Menzies-Gow. Eosinophils and interleukin-5:the debate continues. Am J Respir Crit Care Med 2003;167 (12): 1586’7.

54. Flood-Page PT, Menzies-Gow AN, Kay AB, RobinsonDS. Eosinophil’s role remains uncertain as anti-inter-leukin-5 only partially depletes numbers in asthmaticairway. Am J Respir Crit Care Med 2003; 167: 199’ 204.

55. Kanazawa H, Nomura S, Yoshikawa J. Role of micro-vascular permeability on physiologic differences inasthma and eosinophilic bronchitis. Am J Respir CritCare Med 2004; 169 (10): 1125’30.

56. Grootendorst DC, Rabe KF. Mechanisms of bronchialhyperreactivity in asthma and chronic obstructive pul-monary disease. Proceedings of the ATS 2004; 1 (2):77’87.

57. Brightling CE, Pavord ID, Flood-Page PT, Menzies-GowAN, Kay AB, Robinson DS. Eosinophils in asthma andairway hyperresponsiveness. Am J Respir Crit Care Med2004; 169 (1): 131’3.

58. Busse WW, Kelly AEB. Is the eosinophil a »humptydumpty« cell in asthma? Am J Respir Crit Care Med2003; 167 (2): 102’3.

59. Liu LY, Sedgwick JB, Bates ME, Vrtis RF, Gern JE, KitaH, et al. Decreased expression of membrane IL-5 re-ceptor a on human eosinophils: I. Loss of membraneIL-5 receptor a on airway eosinophils and increased so-luble IL-5 receptor a in the airway after allergen. JImmunol 2002; 169: 6452’58.

60. Gagliardo R, Chanez P, Mathieu M, Bruno A, Costanzo G,Gougat C, et al. Persistent activation of nuclear factor-kappaB signaling pathway in severe uncontrolled asthma.Am J Respir Crit Care Med 2003; 168: 1190’8.

61. Rahman I. Oxidative stress, transcription factors andchromatin remodelling in lung inflammation. BiochemPharm 2002; 64: 935’ 42.

62. Chow CW, Abreu MTH, Suzuki T, Downey GP. Oxida-tive stress and acute lung injury. Am J Respir Cell MolBiol 2003; 29: 427’31.

63. Urso ML, Clarkson PM. Oxidative stress, exercise andantioxidant supplementation. Toxicology 2003; 189:41’54.

64. Heunks LMA, Dekhuijzen PNR. Respiratory musclefunction and free radicals: from cell to COPD. Thorax2000; 55: 704’16.

65. Del Donno M, Verduri A. Oxidants and antioxidants inpulmonary diseases. European Respiratory News sup-plemental issue 2000: 1’48.

66. Dr–ge W. Free radicals in the physiological control ofcell function. Physiol Rev 2002; 82: 47’930.

67. Popper HH, Pailer S, Wurzinger G, Feldner H, Hesse C,Eber E. Expression of adhesion molecules in allergiclung diseases. Virchows Arch 2002; 440 (2): 172’80.

68. Piotrowski WJ, Marezak J. Cellular sources of oxidantsin the lung. Int J Occup Med Environ Health 2000; 13(4): 369’830.

69. Klings ES, Farber HW. Role of free radicals in thepathogenesis of acute chest syndrome in sickle cell dis-ease. Respir Res 2001; 2: 280’5.


Rad primljen: 9. 8. 2004

Prihva}en za {tampu: 25. 8. 2004


Introduction

Despite the significant advances in the area ofcancer chemotherapy, radiotherapy, applied eitheralone or as adjuvant, still remains a method of choicefor treatment of many malignant diseases. One ofthem is prostate cancer, the most frequent cancer inmen population, which unfortunately shows a contin-ual casualty increase (1). The ultimate aim of radio-therapy is to efficiently eradicate tumor cells with min-imal deleterious effects to the surrounding normal tis-sues and to the whole organism. In that view, induc-tion of apoptosis is very desirable therapeutic endpoint

(2, 3). However, much is yet to be learned about eithersystemic or individual biological effects of both con-ventional (gamma-, x-ray) or accelerated particle (pro-ton, etc.) ionizing radiation in order to optimize clinicalresults of treatment of human prostate cancer.

Regardless of the routine use of simple test forprostate specific antigen in sera, that can detect dis-ease at an early stage (4, 5), the number of men withmetastatic prostate cancer is still high. The early sta-ges of disease are usually managed by ionizing radia-tion and/or hormone therapy, but there is no success-ful therapy for metastatic prostate carcinoma. Advan-ced disease is mostly treated by radiation therapy,sometimes in combination with hormone or chemo-therapy, but hormone withdrawal often leads to selec-tion of hormone-independent clones (6). Dose-esca-lated (i.e. 70–80 Gy) radiotherapy is an importanttreatment option especially for men with intermediate-risk prostate cancer (7). On the other hand, radiothe-rapy is often inefficient due to radioresistance of pro-state cancer cells.

UC 577,1; 61 ISSN 0354-3447

Jugoslov Med Biohem 23: 343 –350, 2004 Originalni nau~ni radOriginal paper

CELL DEATH IN IRRADIATED PROSTATE CANCER CELLS ASSESSED BY FLOW CYTOMETRY

Vesna Vu~i}1, Miroslav Adì}1, Ana Ni}iforovi}1, Nevena Ti{ma2, Sabera Ru`diji}3, Marija B. Radoj~i}1

1Laboratory of Molecular Biology and Endocrinology, VINÂ Institute of Nuclear Sciences, Belgrade, Serbia and Montenegro

2Institute of Oncology and Radiology of Serbia, Belgrade, Serbia and Montenegro3Laboratory of Molecular Neurobiology, Department of Neurobiology and Immunology,

Institute for Biological Research, Belgrade, Serbia and Montenegro

Summary: Despite the significant advances in cancer chemotherapy, radiotherapy still remains a methodof choice for treatment of metastatic human prostate cancer. This study presents quantitative analysis of 60Cogamma-radiation effects on cell growth and cell death of metastatic human prostate cancer PC-3 cell line, per-formed in time (24–72h) and dose (2–20 Gy) dependent manner. The irradiated PC-3 cells were mostly dying bynecrosis at late time intervals (72h), while apoptotic cell death was negligible. The EC50 or 50% of cytotoxicity wasnot achieved within the radiation doses used (2–20 Gy), but significant cell growth inhibition with IC50 of 10.4 Gywas observed. It is concluded that the increase in the radiation dose may have an important cytostatic effect, butfor the complete eradication of metastatic prostate cancer novel cytotoxic drugs and radiosensitizers should beintroduced as adjuvant.

Key words: human prostate cancer, cell death, gamma-rays, flow-cytometry

Address for correspondence:

Marija B. Radoj~i}, Ph.D., Res.Assoc.VINÂ Institute of Nuclear SciencesP.O. Box 522– 090, 11001 Belgrade, Serbia and Montenegrotel. +381 (11) 245–82–22 ext.304fax +381 (11) 344–01–[email protected]


Recent studies suggest that some prostate can-cer cells can undergo apoptosis (8). The response toionizing radiation, depends on a number of factorssuch as the stage of differentiation, mutations in spe-cific genes (such as p-53 and bcl-2) that will determinethe ability of the target cells to enter apoptosis (9, 10).For clinical purposes (i.e. eradication of the tumor,but prevention of undesired inflammatory sequelae,radiation sickness and fibrosis), it is useful to investi-gate whether the cells of certain types are susceptibleto apoptosis or necrosis, as well as to determine thetime and dose dependence of the process. In additionto cell killing, radiation can also lead to cell cycle arrestand stopping of proliferation with significant decreasein cell growth (11).

The purpose of this study is to investigate radia-tion induced cell death in PC-3 prostate cancer cell linein time- and dose-dependent manner. The changes incell growth following irradiation were also determined.

Materials and Methods

Cell lines. Human prostate cancer cell line PC-3was purchased from American Type Culture Collec-tion (CRL 1435, Rockville, MD). They are androgenindependent and were established from bone metas-tasis, which is the most usual place for metastaticprostate cancer. PC-3 were maintained in RPMI 1640medium supplemented by 10% heat inactivated fetalcalf serum, 100 IU/mL penicillin/streptomycin and 2mmol/L L-glutamine (Sigma Aldrich Chemie GmbH,Germany), at 37 °C under 5% CO2 atmosphere. Cellswere grown as monolayers in 75 cm2 culture bottlessupplied with 15 mL RPMI, and after a few passagescells were transferred in 25 cm2 culture bottles (Nunk,Nalgene, Danmark).

Cell Irradiation. For investigation of radiationinduced effects on PC-3, 3×105 cells were seeded in25 cm2 culture flasks, and after 72 hours were irradi-ated at room temperature with 2, 10 or 20 Gy gamma-rays from 60Co gamma-source, at the dose rate of 20Gy/h. The effects of irradiation on cell viability, mor-phology and genomic DNA structure were determined24-, 48-, and 72 h after irradiation.

Trypan blue exclusion assay. For analysis ofcell growth and spontaneous cell death in culture, cellswere seeded at a density of 12×103 cells/cm2 in 25cm2 culture flasks. Cell growth, viability and morpho-logy were followed for 8 consecutive days, by trypanblue exclusion (TBE) assay. Medium from each bot-tle was collected, cells were harvested by trypsinization(1 mL 0.25% / 0.02% trypsine/EDTA, Sigma Aldrich,per bottle) and pooled with the medium. Cells werewashed twice in phosphate buffered saline (PBS) andpelleted at 1800 rpm for 5 min at room temperature.Pellets were resuspended in fresh media and the num-ber of viable (trypan blue negative), dead (trypan bluepositive) and total cells (viable + dead) was counted in

five squares at 320× magnification using Neubauerhaemocytometer and Leitz-Wetzlar Orthoplan micro-scope. Cell viability was determined as % of cells thatexcluded trypan blue stain. The doubling time (td) wascalculated according to the following formula:

td =ln2µ

µ = (ln x – ln x0) / t

where x represents cell number in time t, and x0cell number in time t0.

The same assay was used for determination ofviable, dead and total cell number in irradiated sam-ples 24-, 48- and 72 h post-irradiation. The viabilityindex (Vi) of each sample was calculated related toappropriate, unirradiated control, which was used asviability index 1 (100%).

Cell death analysis by flow cytometry. Doublestaining of cells by Annexin and propidium iodide (PI)enabled estimation of cell viability after irradiation anddiscrimination between two ways of cell death – apop-tosis and necrosis. After trypsinization and centrifuga-tion of cells, approximately 105 cells of each samplewere mixed with 100 µL of Annexin V-FITC reagent(Travigen Inc., Gaithersburg, MD, USA) containing5 mg/mL Annexin V-FITC and 5 µg/mL propidiumiodide, and incubated at room temperature for 15minutes in dark and then diluted with 400 µL of bind-ing buffer. Multiparameter measurement of the cellsample in order to detect radiation-induced cell deathwas performed using a FACS-calibur flow cytometer(Becton Dickinson, San Jose, CA, USA) with 488 nm,15 mW argon-ion laser. Staining of the cells withAnnexin V-FITC (Annexin) permitted identification ofcells in early apoptosis, while staining of the cells withAnnexin and PI permitted quantification of cells in thelate apoptosis and necrosis. Data were acquired imme-diately after staining by analyzing about 20,000 cells/sample. The data were further processed by BectonDickinson LYSIS II software.

DNA fragmentation assay. This assay was usedfor confirmation of necrosis, detected by double stain-ing and flow cytometryc analysis. It was performed aspreviously described with minor modifications (12).Cells were incubated in one volume of digestion buffer(100 mmol/L NaCl, 25 mmol/L EDTA, 10 mmol/LTris-HCl, pH 8.0, 0.5% SDS and 0.5 mg/mL RNA-seA) for 2 h at 50 °C, followed by the addition of pro-teinase K (0.6 mg/mL) and digestion was continuedovernight. DNA was deproteinised using the phenol/chloroform/isoamil-alcohol reagent, for three times.The aqueous layer was transferred to a new tube andprecipitated with one volume of isopropanol and 1/10volume of ammonium acetate overnight at 4 °C. TheDNA pellet was washed three times with ice-cold 95%ethanol and dried at room temperature. The final DNApellet was resuspended in 20 mL TE buffer (10 mmol/LTris, pH 7.4, 1 mmol/L EDTA) and the concentration


of DNA was determined spectrophotometrically.Electrophoresis of 4’10 mg of each DNA sample, wascarried out for 60 min at 60 V at room temperature on1% agarose gel containing 1 mg/mL ethidium bromi-de. Gels were scanned by GelDoc apparatus.

Statistical analysis. For statistical analysis oftime- and dose- dependent changes in viability indexand flow-cytometric determination, two-way ANOVAwas used. If a statistical significance was found, Tukeypost-hoc test was used to determine which groups di-ffer from each other. Statistical significance wasaccepted if p<0.05.

Results

Analysis of cell growth and spontaneous cell death in culture

PC-3 cells were plated in 25 cm2 culture flasks,at the density of 12 × 103 cells/cm2 (e.g. 3 × 105 cellsper bottle), and cell growth, viability and morphologywere monitored for 8 consecutive days (Figure 1).During the first day post plating the viable cell numberincrease from 12 × 103 cells/cm2 to almost 13.5 ×103 cells/cm2, suggesting that plating efficiency wasvery high. The log phase of cell growth occurredbetween 2nd and 6th day post plating. The confluencewas reached on the 6th day at the cell density of about116 × 103 cells/cm2. Further incubation led to theslightly decrease in cell number. TBE assay showedthat approximately 2’8% of all cells were TB positiveat all time points, indicating that cell viability was highduring the whole experiment. We also calculated PC-3cell doubling time, which was 32.9 ± 2.8 h under con-ditions maintained in our laboratory.

Cell growth of irradiated PC-3 human prostate cancer cells

Effects of gamma-ionizing radiation on PC-3growth were evaluated by TBE assay. The test is con-venient for determination of Viability (V) and Viabilityindex (Vi) after radiation treatment. TBE assay wasperformed 24-, 48- and 72 h after irradiation by 2-,10- and 20 Gy. The obtained results (Table I andFigure 2) showed significant decrease in cell numberand Viability Index (Vi), comparing with appropriatecontrol, both by dose (F 190.1, p<0.001) and time (F45.2, p<0.001) as determined by two-way ANOVA.This effect was most pronounced 72 h after treatmentwith 20 Gy, when the Vi decreased from 1, establishedin control, to 0.35. The radiation dose which causeddecrease in cell Vi from 1 to 0.5, termed IC50 (mitoticcell death dose), was 10.4 ± 0.4 Gy. The dose depen-dent Vi (e.g. viable cell number) decrease was signifi-cant for all experimental points (Table I). The statisti-cal differences between groups were analyzed byTukey post hoc test, comparing irradiated sampleswith the control from the same time point, and signi-ficance was established at *p<0.05. The number ofTB positive cells, indicating cytotoxicity (actual celldeath), was relatively low (up to 20 %, data not shown),which was in agreement with cytometric quantificationof necrotic cells determined after double Annexin V-FITC/PI staining (Figure 3).

Radiation-induced cell death analysis by flow cytometry

All samples analyzed by TBE, were also stainedby Annexin V-FITC and PI, and analyzed by FACS-Calibur flow cytometer. The results of quantification ofcells in different states, such as viable cells, cells inearly apoptosis, cells in late apoptosis or necrotic cells,and necrotic cells or cell aggregates, are presented inFigure 3 A-D. Figure 3A represents a flow cytometryscatter plot and the dots represent cells in different

Figure 1. Growth and viability of PC-3 cells in culturedetermined by TBE assay. Data are the mean SEM

from 2 distinct experiments.

24 48 72 96 120 144 168 192

Time in culture (h)

140000

120000

100000

80000

60000

40000

20000

0

Cel

l num

ber/c

m2

viabledeadtotal

Table I Number of viable cells in the control and 60Cogamma-irradiated human prostate cancer cells PC-3

measured by TBE assay 24–72 h post-irradiation. Data are the mean ± SEM from 3 distinct experiments.Statistical time- and dose-dependent differences were

determined by two way ANOVA and Tukey post hoc test.** p<0.01, *** p<0.001.

Dose →Time (h)↓

24

48

72

control (0 Gy)

57.3±1.6

92.4±4.9

84.6±3.9

2 Gy

** 43.7±2.8

***49.9±9.7

***69.6±1.4

10 Gy

***40.9±4.4

***46.8±5.9

***44.7±5.3

10 Gy

***40.9±4.4

***46.8±5.9

***44.7±5.3


states, depending on the place in scatter plot. Cells inearly apoptosis were Annexin positive, while PI stainingindicated late apoptosis or necrosis (Figure 3A). As itmay be observed from Figure 3B, cell viability of thesamples decreased with the increase in the radiationdose and with the period of incubation. Radiation-induced decrease of viability showed dose-dependentsignificance which was judged by two-way ANOVAanalysis at p<0.05 (F 9.12, p<0.001), but not time-dependent significance (F 2.06, p=0.142). The Tukeypost hoc test, used for comparison of irradiated sam-ples with appropriate control, showed that only radia-

tion dose of 20 Gy induced significant viability decre-ase, 48 and 72 h post irradiation. The evidence forapoptosis was minimal in either control or irradiatedPC-3 cells. With the increase in the radiation dose, thepercentage of cells in the state of early apoptosis in-creased from 0.2% up to 0.8 % (Figure 3C), but therewere neither time- nor dose-dependent significance (F1.35, p=0.33 and F 1.42, p=0.33 respectively).

The radiation dose and time dependent increasein number of PI positive cells in the late apoptosis ornecrosis was also observed. The dose dependentincrease in necrotic cells was significant (F 7.85,p<0.001), but time-dependence was not statisticallysignificant (F 1.9, p=0.17). The Tukey post hoc analy-sis showed significant increase in number of dead cellsafter irradiation with 20 Gy, 48 and 72 h after treat-ment (Figure 3D).

Electrophoretic analysis of purified genomic DNA from irradiated cells

The genomic DNA from control and irradiatedsamples, including attached and floating cells, wasanalyzed on 1% agarose gel containing 1 mg/mL ethi-dium bromide. As it may be observed in Figure 4 theinitial fragmentation of PC-3 cell DNA to a high mole-cular size band (>10 Kb) was visible in all samples. Inthe case of irradiated samples, in addition to the frag-mentation of PC-3 cell DNA to a high molecular sizeband (>10 Kb), smaller fragments (1<Kb) also appe-ared. The observed smear is most pronounced 72 hpost irradiation with doses of 10 and 20 Gy. It corre-lated well with the highest percent of dead cellsobtained by cytometry (Figure 3D). DNA ladder wasnot observed in examined samples, confirming flowcytometric data, that PC-3 do not die by apoptosisafter gamma-irradiation.

Discussion

The hormone-independent metastatic prostatecancer is incurable at present. In the lack of efficientchemotherapeutic agents, ionizing radiation therapystill remains as a method of choice for the disease cure.It is known that apoptotic cell death plays an importantrole in the death of both normal prostate and andro-gen-dependent malignant prostate tissue followingandrogen withdrawal. Cancer cell death is leading to adecrease in either glandular or tumor volume, respec-tively. However, recent data indicate that apoptosismay not be the dominant form of cell death followingradio- and chemotherapy in epithelial tissues (7, 13).Disruption of the pathways that lead to apoptosis is oneof the major mechanism by which cancer cells becomeresistant to radiation or chemotherapy (14).

In this paper, we have analyzed 60Co gamma-radiation-induced death of PC-3 human prostate can-cer cells. PC-3 cells originates from epithelial cells.

Figure 2. Time course of viable cells number in the control and 60Co gamma-irradiated human prostate cancer cells PC-3 (A) and Viability index calculated

according to the related control from the same time point (B) as measured in TBE assay. Error bars represent

standard error of the mean (SEM). Data are the mean SEM from 3 distinct experiments.

24 48 72

Post-irradiation period (h)

120

100

80

60

40

20

0

100

90

80

70

60

50

40

30

20

10

024 48 72


Cel

l per

cm

2 (×

103 )

Viab

ility

inde

x (%

)

0 Gy

2 Gy

10 Gy

20 Gy

2 Gy

10 Gy

20 Gy

A

B


They are hormone-refractory cells derived from hu-man bone metastasis of prostate adenocarcinoma,representing advanced prostate cancer (15). For theseexperiments, we chose doses of 2 and 10 Gy, to berepresentative of the 1.8’2 Gy daily clinical fractionsgiven during curative radiotherapy and the 8’10 Gysingle doses given in palliative radiotherapy, as well asthe dose of 20 Gy which is in the range of cumulativecurative dose for prostate carcinomas (70’80 Gy).

The growth curve of PC-3 cells showed sigmoid-like shape, with the population doubling time about 33h. The viability of PC-3 cells was 92’98 % throughoutthe log phase of growth. The culture reach confluence6th days post plating, at the density of 120 × 103

cells/cm2, which is considerable less than other epithe-lial prostate cancer cell line, DU 145 (180 × 103

cells/cm2), suggesting that PC-3 are bigger than DU

145 (to be published). After day 6th, number of cellsslightly decreased, retaining surprisingly high viability(97’98%). Based on data from cell growth curve, allirradiation experiments were performed in the logphase of cell growth. Cells were irradiated with 2’20Gy from 60Co-source at the dose rate of 20 Gy/h. Theirradiated PC-3 cell cultures were followed for threeconsecutive days i.e. through approximately two pro-liferation cycles. The data obtained by the TBE assay,showed significant decrease in cell Viability index, e.g.in the number of viable cells in irradiated samples rel-ative to the control from the same time point. Theprocess was dependent both on the radiation doseand on the incubation time. On the other hand, thenumber of TBE positive cells remained relatively low,indicating that radiation caused cell cycle arrest andblocking of cell proliferation, rather than actual cell

Figure 3. Time course of percent changes in PC-3 cell viability and death after 60Co gamma-irradiation as determined by flow cytometry. Viable cells (A), early apoptosis (B) or late apoptotic/necrotic cells (C). Data are the mean (n=3) and theerror bars represent SEM. Statistical differences were determined by two-way ANOVA followed by Tukey post hoc analysis.

** p<0.01, *** p<0.001.

100 101 102 103 104

Annexin V-FITC

control2 Gy10 Gy20 Gy

Prop

idiu

m Io

dide

100

10

1 10

2

10

3

104

100

80

60

40

20

0

Cel

l num

ber/c

m2

*****

24 48 72


50

40

30

20

10

0

Nec

rosi

s (%

)

** ***

24 48 72


10

9

8

7

6

5

4

3

2

1

0

Apop

tosi

s (%

)

24 48 72


A

C

B

D

death. The obtained data enabled determination ofdoses which induced 50% of cell growth inhibition(named IC50) and 50% of cytotoxicity, i.e. dose whichinduced 50% of trypan blue positive cells (EC50). IC50was achieved at the dose of 10.4 Gy, but EC50 washigher than applied doses, as 20 Gy induced only 20%of trypan blue positive cells. This indicated that theradiation treatment in the clinically relevant dose inter-val would predominantly inhibit PC-3 growth ratherthan induce cell killing. The similar results were obtai-ned for other hormone-refractory epithelial prostatecancer cell line DU 145, derived from brain metastasis(to be published), but in the case of cervix epithelialcancer cells HeLa S3, the same doses of ionizing radi-ation induced cell death in up to 45% of irradiated cells(16). This comparison suggests that prostate cancercells are much radioresistant than HeLa S3 cell line.

In order to determine the form of radiation-indu-ced PC-3 cell death two different techniques were used:flow-cytometry analysis of cell morphological featuresafter double staining with propidium iodide and Anne-xin-V-FITC, and DNA electrophoresis of purified DNA.Simultaneous staining of cells with Annexin V-FITCand propidium iodide enabled distinction of early apo-ptosis from late apoptosis and/or necrosis. After dou-

ble staining and flow-cytometryc analysis of controland irradiated samples, the most of dead cells were inlate apoptosis or necrosis (Annexin V+, PI+ cells). Theearly apoptosis (Annexin V+, PI- cells) occurred ininsignificant number of cells, less than 1% of total cellnumber in each sample, and there were no significantchanges in percent of apoptotic cells depending ontime or dose. On the contrary, the cell necrosis was do-se dependent, as determined by two-way ANOVA, andwas most pronounced 72 hours post treatment. Theseresults suggest that the prevailing form of 60Co gammaradiation-induced PC-3 cell death was necrosis.

As it was not possible to distinguish the necrosisfrom the late apoptosis by double staining, it was nec-essary to perform the gel electrophoresis of purifiedgenomic DNA from PC-3 cells. DNA fragmentationassay confirmed the presence of the necrosis process,showing the absence of DNA ladder characteristic forapoptosis in control (17), as well as in irradiated sam-ples. One explanation for the absence of apoptosis fol-lowing radiotherapy in PC-3 cell line is that these cellshave mutant p53 gene. However, the impairment ofother mechanisms necessary for initiation of the apop-totic process is not excluded. Namely, recent investi-gation showed that gamma-radiation activates acidicsphingomyelinase to produce ceramide, a catabolicproduct of membrane sphingolipids that is a cell deathsignal (14, 18, 19). It was suggested that the otherepithelial prostate carcinoma LNCaP cells are highlyresistant to induction of apoptosis by gamma-radia-tion due to a defect in ceramide generation (14, 20,21). Likewise, resistance to apoptosis involves a defectin ceramide generation in the PC-3 prostate cancercell line (20, 22).

In summary, the obtained results suggests that60Co gamma-ionizing radiation caused notable humanprostate cancer PC-3 cell killing. The irradiated PC-3cells were mostly dying by necrosis, while apoptoticcell death was negligible. Although within the radiationdoses used in this study (2’20 Gy) the EC50 i.e. 50%of cytotoxicity was not achieved, we found significantcell growth inhibition with IC50 of 10.4 Gy. Thus, thisin vitro study suggests, that the increase in radiationdose may have an important cytostatic effect, ratherthan eradicating the advanced prostatic carcinoma. Italso suggests that, in addition to gamma irradiation,current antitumor strategies should introduce novelcytotoxic adjuvant or radiosensitizers, in order to achi-eve complete eradication of metastatic human pro-state cancer.

Acknowledgment. This study was supported bythe Ministry for Science, Technology and Developmentof Serbia, grant No. BOI-1953


Figure 4. Agarose gel electrophoresis of genomic DNAextracted from 60Co gamma-irradiated PC-3 cells. Lane S:

DNA standard molecular size markers; lanes 1–12: genomic DNA pattern of samples irradiated with 0, 2-,

10 and 20 Gy respectively, isolated 24 hours (lines 1–4),48 h (lines 5–8) or 72 h (lines 9–12) post-irradiation.

S 1 2 3 4 5 6 7 8 9 10 11 12

Kb

108643

21.5

10.75

0.50.40.20.1

References

1. Jemal A, Thomas A, Murray T, Thun M. Cancer statis-tics, 2002. CA Cancer J Clin 2002; 52: 23’47.

2. Crompton NEA. Programmed Cellular Response inRadiation Oncology. Acta Oncol 1998; Suppl 11: 1’49.

3. Ross GM. Induction of cell death by radiotherapy.Endocr Relat Cancer 1999; 6: 41’4.

4. Marinovi} V, ûperlovi} M, Hajdukovi}-Dragojlovi} Lj.Prostate-specific antigen: biochemical characteristics,biological functions and diagnostic potential in prostatecancer screening. Jug Med Biohem 1997; 16: 129’36.

5. Marinovi} V, Nedi} O, Stanojevi} N, Bari~evi} I, PavlicaS. Investigation of the relationship between two majorprostate tumour markers. Jug Med Biohem 2000; 19:407’10.

6. Raghavan D. Non-hormone chemotherapy for prostatecancer: principles of treatment and application to thetesting of new drugs. Semin Oncol 1998; 15: 371’89.

7. Bromfield GP, Meng A, Warde P, Bristow RG. Cell deathin irradiated prostate epithelial cells: role of apoptoticand clonogenic cell kill. Prostate Cancer and ProstaticDiseases 2003; 6: 73–85.

8. Algan O, Stobbe CC, Helt AM, Hanks GE, Chapman JD.Radiation inactivation of human prostate cancer cells:The role of apoptosis. Radiation Res 1996; 146: 267’75.

9. Szumiel I. Ionizing radiation- induced cell death. Int JRadiat Biol 1994; 66: 329’41.

10. Kyprianou N, King DE, Bradbury D, Rhee J. Bcl-2 over-expressio delays radiation-induced apoptosis withoutaffecting the clonogenic survival of human prostate can-cer cells. Int J Cancer 1997; 70: 341’8.

11. Hartwell LH, Kastan MB. Cell cycle control and cancer.Science 1994; 266: 1821’ 8.

12. Armstrong K, Isaacs JT, Ottaviano YL, Davidson NE.Programmed cell death in an estrogen independenthuman breast cancer cell line, MDA-MB-468. CancerRes 1992; 52: 3418’24.

13. Olive PL, Vikse CM, Vanderbyl S. Increase in the fractionof necrotic, not apoptotic, cells in SiHa xenografttumours shortly after irradiation. Radiother Oncol 1999;50: 113’9.

14. Kimura K, Bowen C, Spiegel S, Gelmann EP. TumorNecrosis Factor- sensitizes prostate cancer cells toGamma-Irradiation-induced apoptosis. Cancer Res1999; 59: 1606’14.

15. Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, JonesLW. Establishment and characterization of a human pro-static carcinoma cell line (PC-3). Invest Urol 1979; 17:16’23.

16. Ni}iforovi} A, Zari} B, Daki} A, Ti{ma N, Radoj~i} MB.Flow Cytometry Evaluation of HeLa S3 Cell DeathInduced by Gamma-Radiation. Jug Med Biohem 2004;23: 1’8.

17. Oberhammer F, Wilson JW, Dive C, Morris ID, HickmanJA, Wakeling AE. Apoptotic death in epithelial cells.cleavage of DNA to 300 and/or 50 kb fragments prior toor in the absence of internucleosomal fragmentation.EMBO J 1993; 12: 3679’84.

18. Haimovitz-Friedman A, Kan CC, Ehleiter D, Persaud RS,McLoughlin M, Fuks Z et al. Ionizing radiation acts oncellular membranes to generate ceramide and initiateapoptosis. J Exp Med 1994; 180: 525’35.

19. Lee JM, Bernstein A. p53 mutations increase resistance


]ELIJSKA SMRT U OZRAÊNIM ]ELIJAMA KANCERA PROSTATE ANALIZIRANA PROTO^NOM CITOMETRIJOM

Vesna Vu~i}1, Miroslav Adì}1, Ana Ni}iforovi}1, Nevena Ti{ma2, Sabera Ru`diji}3, Marija B. Radoj~i}1

1Laboratorija za molekularnu biologiju i endokrinologiju, VINÂ Institut za nuklearne nauke, Beograd, Srbija i Crna Gora

2Institut za onkologiju i radiologiju Srbije, Beograd, Srbija i Crna Gora3Laboratorija za molekularnu neurobiologiju, Institut za biolo{ka istraìvanja, Beograd

Kratak sadràj: Uprkos zna~ajnom napretku u hemoterapiji kancera, radioterapija ostaje metod izbora utretmanu metastaziranog kancera prostate. Ovaj rad predstavlja kvantitativnu analizu efekata 60Co gama zra~enjana }elijski rasti i }elijsku smrt PC-3 }elijske linije humanog kancera prostate, pri ~emu je pra}ena vremenska(2–72h) i dozna zavisnost (2–20 Gy). Ozra~ene PC-3 }elije su uglavnom umirale nekrozom u kasnijem vremen-skom intervalu (72h), dok je apoptoza bila zanemarljiva. Vrednost EC50 odnosno 50% citotoksi~nosti nije dosti-gnuta primenjenim dozama, ali je ustanovljena zna~ajna inhibicija }elijskog rasta, sa vredno{}u IC50 od 10.4 Gy.Zaklju~eno je da pove}anje doze moè imati zna~ajan citostati~ki efekat ali da je za kompletno odstranjivanje me-tastaziranog kancera prostate neophodno uvo|enje novih citotoksi~nih agenasa ili radiosenzitera kao adjuvanata.

Klju~ne re~i: humani kancer prostate, }elijska smrt, gama zra~enje, proto~na citometrija

to ionizing radiation. Proc Natl Acad Sci USA 1993; 90:5742’6.

20. Nava VE, Cuvillier O, Edsall LC, Kimura K, Milstien S,Gelmann EP et al. Sphingosine enhances apoptosis ofradiation-resistant prostate cancer cells. Cancer Res2000; 60: 4468’74.

21. Garzotto M, White-Jones M, Jiang Y, Ehleiter D, LiaoWC, Haimovitz-Friedman A, et al. 12-O-Tetradecanoyl-

phorbol-13-acetate-induced apoptosis in LNCaP cells ismediated through ceramide synthase. Cancer Res 1998;58: 2260’4.

22. Wang XZ, Beebe JR, Pwiti L, Bielawska A, Smyth MJ.Aberrant sphingolipid signaling is involved in the resist-ance of prostate cancer cell lines to chemotherapy.Cancer Res 1999; 59: 5842’8.


Received: April 1, 2004

Accepted: August 5, 2004


Introduction

Cystic fibrosis is one of the most common life-threatening autosomal recessive disorders that is usu-ally estimated to affect 1 in 2000–3000 Caucasiannewborns, with a carrier frequency of 1 in 26 individu-als (1). In its classic and most common form, cysticfibrosis manifests with chronic obstructive lung dis-ease, exocrine pancreatic insufficiency, elevated sweatchloride concentration and in males infertility due toobstructive azoospermia (2).

Cystic fibrosis is caused by mutations in the Cy-stic Fibrosis Transmembrane Conductance Regulator(CFTR) gene, spanning 250kb at chromosomal region7q31.3 and consisting of 27 exons. The gene was dis-covered in 1989, and it encodes a protein expressed atthe apical membrane of exocrine epithelial cells (3).

CFTR protein functions principally as a cAMP-inducedchloride channel and appears capable of regulatingother ion channels.

The most common mutation in the CFTR geneis F508del located in exon 10, and it is present onapproximately two-thirds (66%) of all cystic fibrosischromosomes. However, there is great mutational he-terogeneity in the remaining one-third of all alleles.Nearly 1300 mutations within CFTR have been identi-fied to date, and reported to Cystic Fibrosis GeneticAnalysis Consortium (4). Although these mutationsvary greatly in their frequency and distribution, the vastmajority are present in either single individual or smallnumber of individuals.

Mutations affect CFTR through a variety of mole-cular mechanisms which can produce little or no func-tional CFTR at the apical membrane. The phenotypicspectrum associated with mutations in the CFTR geneextends beyond the classically defined cystic fibrosis.Besides patients with atypical cystic fibrosis, there arelarge numbers of so-called monosymptomatic disea-ses, such as various forms of obstructive azoospermia,idiopathic pancreatitis or disseminated bronchiectasisassociated with CFTR mutations uncharacteristic forcystic fibrosis (5).

UC 577,1; 61 ISSN 0354-3447

Jugoslov Med Biohem 23: 351–354, 2004 Originalni nau~ni radOriginal paper

CFTR GENE ANALYSIS IN PATIENT WITH ATYPICAL CYSTIC FIBROSIS

Aleksandra Nikoli}1, Aleksandra Divac1, Nada Bogdanovi}2, Marija Miti}-Miliki}2, Dragica Radojkovi}1

1Institute of Molecular Genetics and Genetic Engineering, Belgrade2Institute of Tuberculosis and Lung Disease, Clinical Center of Serbia, Belgrade

Summary: This paper reports a case of a patient presenting with atypical cystic fibrosis whose sweat testshowes borderline values. In vast majority of cases the sweat test is essential diagnostic tool for establishing thediagnosis of cystic fibrosis, but only after the molecular genetic testing the diagnosis can be confirmed. Thepatient was found to be compound heterozygote for two CFTR mutations, F508del and D1152H. The presenceof F508del mutation was analyzed by PSM method, while the screening for the second mutation was performedusing DGGE. The strategy of mutation detection in cystic fibrosis patients, especially those with atypical presen-tations who carry less frequent mutations, should include both direct and indirect methods of molecular diag-nostics.

Key words: atypical cystic fibrosis, CFTR gene, DGGE, molecular diagnostics


Aleksandra Nikoli}Institute of Molecular Genetics and Genetic EngineeringVojvode Stepe 444ap. fah 44611001 BelgradeSerbia and MontenegroPhone: +381 11 3976658Fax: +381 11 3975808E-mail: [email protected]


This paper reports a case of atypical cystic fibro-sis that was diagnosed by the combination of directand indirect mutation detection methods. It is shownthat the patient is a compound heterozygote for twoCFTR mutations.


Case history

Patient is a 38 year-old woman presented with adiagnosis of bronchiectasis. Her past medical historywas noteworthy for the onset of respiratory symptomssuch as: recurrent pneumonia and periods of coughand haemoptysis. Sweat test performed at the age of6 showed borderline values. At the age of 33, com-puted tomography (CT) has shown the presence ofbronchiectasis. At the age of 38, she presented withlung disease progression, which was observed by CT,and was referred for molecular testing for cystic fibro-sis.

Methods

DNA was extracted from peripheral blood usingGFXTM Genomic Blood DNA Purification Kit (Amer-sham Biosciences).

The presence of the most frequent CFTR muta-tion – F508del was detected by PCR-Mediated Site-Directed Mutagenesis (PSM) method (6). The 219bplong fragment was amplified with the following pri-mers: 5'-GCACCATTAAAGAAAATATGAT-3' and 5'-CATTCACAGTAGCTTACCCA-3', and digested withMboI. Products were analyzed on 10% polyacrilamidegel.

The screening for the presence of variations inCFTR exons was performed by Denaturing GradientGel Electrophoresis (DGGE) method, as previouslydescribed (7). Exon 18 was amplified with the follow-ing primers: 5'-GTAGATGCTGTGATGAACTG-3' and5'-GTGGCTATCTATGAGAAGGA-3' and sequencedwith the primer 5'-TGCCCTAGGAGAAGTGTG-3'using Thermo Sequenase CyTM5 Dye Terminator Kit(Amersham Pharmacia Biotech).

Results and Discussion

The presence of CFTR F508del mutation wasanalyzed by PSM method. After digestion with MboInormal allele is digested giving fragments 202bp and17bp long, while mutant allele remains undigested.This analysis has shown that the patient is heterozy-gous for F508del mutation (Figure 1).

The screening for the second mutation was per-formed using DGGE. After DGGE analysis was per-formed for several CFTR exons, altered band patternwas seen in exon 18. Mixing of the patient sample with

control samples heterozygous for two mutations inexon 18, has shown that the mutation present inpatient's sample is D1152H. Mixing with M1137V/Ncontrol gives extra heteroduplex bands, while no extrabands are seen after mixing with D1152H/N control(Figure 2).

The presence of D1152H mutation was confir-med by direct DNA sequencing (Figure 3).

Figure 1. PSM analysis of patient's CFTR genefor the presence of F508del mutation:

1. Heterozygote for F508del2. Homozygote for normal allele

3. Patient (heterozygote for F508del) 4. Homozygote for F508del

1 2 3 4

heteroduplex →219bp →202bp →

Figure 3. The part of the patient's CFTR gene exon 18 sequence containing D1152H mutation

in heterozygous state (S=C/G)

120 125 130 135 140

A C T C C A G C A T A S A T G T G G A T A G C

Figure 2. DGGE analysis of patient's CFTR genefor the presence of variations in exon 18:

1. patient's sample2. control D1152H/N (heterozygote for D1152H)3. control M1137V/N (heterozygote for M1137V)4. mixed patient's sample and control D1152H/N5. mixed patient's sample and control M1137V/N

1 2 3 4 5

heteroduplexes


The described patient, presenting with atypicalcystic fibrosis, was found to be compound heterozy-gote for two CFTR mutations, F508del and D1152H.It has been previously reported that the F508del/D1152H genotype is associated with mild CF pheno-type (8).

The CFTR mutation D1152H is caused by pointmutation G to C at position 3586. Patients carryingD1152H mutation are usually diagnosed at advancedage, present mild pulmonary disease and pancreaticsufficiency. The mutation D1152H was found to beassociated with normal sweat chloride values (9). Cha-racterization at the protein and at the electrophysio-logical level has shown that this mutation does notalter the permeability sequence of the CFTR channels(10). However, it significantly reduces the whole cellcAMP activated chloride currents, indicating that thismutation interferes with the proper gating of the chlo-ride channels.

In vast majority of cases, the sweat test remainsthe essential diagnostic tool for establishing the diag-nosis of CF. Although the threshold of 60 mmol/L for

the sweat chloride concentration has proven to be dis-criminating and useful in clinical practice, in describedpatient a borderline value was observed. Only after themolecular genetic testing, the diagnosis of cystic fibro-sis was confirmed. In our opinion in borderline sweatchloride results, clinician should consider moleculargenetics testing for cystic fibrosis. Further exhaustivegenetic analysis is justified in patients with symptomssuggestive of CF and borderline sweat chloride con-centration.

Although methods for direct detection of themost frequent CFTR mutations remain essential, met-hods for the screening of the whole gene are increa-singly used for the purposes of cystic fibrosis molecu-lar diagnostics. In our experience, the denaturing gra-dient gel electrophoresis is a method of choice, due toits reliability and sensitivity. It is followed by direct DNAsequencing, used for characterization of the detectedaberrant pattern. The strategy of mutation detection inanalysis of CF patients, especially those with atypicalpresentations who carry less frequent mutations, sho-uld include both direct and indirect methods of mole-cular diagnostics.

ANALIZA CFTR GENA KOD PACIJENTA SA ATIPI^NOM CISTI^NOM FIBROZOM

Aleksandra Nikoli}1, Aleksandra Divac1, Nada Bogdanovi}2, Marija Miti}-Miliki}2, Dragica Radojkovi}1

1Institut za molekularnu genetiku i geneti~ko inènjerstvo, Beograd2Institut za plu}ne bolesti i tuberkulozu, Klini~ki centar Srbije, Beograd

Kratak sadràj: U ovom radu je prikazan slu~aj atipi~ne cisti~ne fibroze sa grani~nom vredno{}u znojnogtesta. U ve}ini slu~ajeva znojni test je glavni dijagnosti~ki parametar za dijagnostikovanje cisti~ne fibroze, ali sedijagnoza moè potvrditi samo na osnovu rezultata molekularno-geneti~kog testiranja. Utvr|eno je da je pacijentsloèni heterozigot za dve CFTR mutacije, F508del i D1152H. Prisustvo mutacije F508del detektovano je PSMmetodom, dok je za analizu prisustva druge mutacije kori{}ena DGGE metoda. Strategija detekcije mutacija kodpacijenata sa cisti~nom fibrozom, naro~ito onih sa atipi~nim prezentacijama bolesti koji nose re|e mutacije, tre-balo bi da uklju~uje i direktne i indirektne metode molekularne dijagnostike.

Klju~ne re~i: atipi~na cisti~na fibroza, CFTR gen, DGGE, molekularna dijagnostika

References

1. Bobadilla JL, Macek MJr, Fine JP, Farrell PM. Cysticfibrosis: A worldwide analysis of CFTR mutations – cor-relation with incidence data and application to screen-ing. Hum Mutat 2002; 19: 575–606.

2. Zielenski J. Genotype and phenotype in cystic fibrosis.Respiration 2000; 67: 117–33.

3. Riordan JR, Rommens JM, Kerem BS, Alon N, Roz-mahel R, Grzelczak Z, et al. Identification of the cysticfibrosis gene: Cloning and characterization of comple-mentary DNA. Science 1989; 245: 1066 –73.

4. Cystic Fibrosis Genetic Analysis Consortium: www.ge-net.sickkids.on.ca/cftr

5. Noon PG, Knowels MR. »CFTR-opathies«: Disease phe-notypes associated with cystic fibrosis transmembraneconduntance regulator gene mutations. Respirationresearch 2001, 2: 328–32.

6. Friedman KJ, Highsmith WEJr, Silverman LM. Detec-ting multiple cystic fibrosis mutations by PolymeraseChain Reaction-Mediated Site-Directed Mutagenesis.Clin Chem 1991; 37 (5): 753–5.

7. Fanen P, Ghanem N, Vidaud M, Besmond C, Martin J,Costes B et al. Molecular characterization of cystic fibro-sis: 16 novel mutations identified by analysis of the who-le Cystic Fibrosis Conductance Transmembrane Regu-


lator (CFTR) coding regions and splice site junctions.Genomics 1992; 13: 770– 6.

8. Lebecque P, Leal T, De Boeck C, Jaspers M, CuppensH, Cassiman JJ. Mutations of the cystic fibrosis geneand intermediate sweat chloride levels in children. Am JRespir Crit Care Med 2002; 165: 757– 61.

9. Feldmann D, Couderc R, Audrezet MP, Ferec C, Bien-

venu T, Desgeorges M et al. CFTR genotypes in patientswith normal or borderline sweat chloride levels. HumMutat 2003; 22 (4): 340.

10. Vankeerberghen A, Wei L, Teng H, Jaspers M, CassimanJJ, Nilius B et al. Characterization of mutations locatedin exon 18 of the CFTR gene. FEBS Letters 1998; 437(1–2): 1–4.

Received: March 19, 2004


Introduction

Preliminary studies have suggested that intercel-lular adhesive molecule 1 (ICAM-1; CD54) may beinvolved in pathogenesis of asthma (1). Soluble formof the intercellular adhesive molecule 1 (sICAM-1) wasdetected in elevated levels in the sera of adult patientswith some inflammatory, immune or malignant dise-ases (1). It originates from proteolytic cleavage ofmembrane ICAM-1 (m ICAM-1) (2). In asthmatic pa-tients epithelial cells of airways may express increasedamounts of ICAM-1 molecule, which is one of theresponses to different stimuli such as local cytokines,infectious agents, air pollutants, allergens. Their res-

ponse to these stimuli alters the immune and inflam-matory environment that is critical in diseases such asbronchial asthma.

The aim of the study was to measure and estab-lish the possible difference in sICAM-1 levels in sera ofpatients with atopy (patients with allergic rhinitis andpatients with allergic rhinitis and bronchial asthma) incomparison with the group of patients without atopy(patients with asthma without rhinitis) as well as possi-ble differences in sICAM-1 levels between groups ofpatients with allergic rhinitis and asthma in compari-son with patients with allergic rhinitis only.

Material and Methods

Our study comprised patients with allergic rhini-tis and bronchial asthma. The diagnosis of bronchialasthma was established following the guidelines fordiagnosis and treatment of asthma of the internationalexpert group of the National Institute of Health and


UC 577,1; 61 ISSN 0354-3447

Jugoslov Med Biohem 23: 355 –359, 2004 Originalni nau~ni radOriginal paper

THE INFLUENCE OF ATOPY ON sICAM-1 SERUM LEVELS IN PATIENTS WITH ALLERGIC RHINITIS AND BRONCHIAL ASTHMA

Aleksandra Peri}-Popadi}1, Mirjana Bogi}1, @ikica Jovi~i}1, Sanvila Ra{kovi}1, Vesna Tomi}-Spiri}1,Sneàna Kova~evi}2, Jasna Bolpa~i}1, Miodrag ôli}3

1Institute of Allergology and Immunology, Clinical Center of Serbia, Belgrade, Serbia and Montenegro2Internal Medicine, »Dr Laza Lazarevi}« General Hospital, [abac

3Institute of Medical Research, MMA, Belgrade

Summary: It has been shown that adhesive molecules are involved in inflammatory diseases of the lungssuch as bronchial asthma. The purpose of the study was to measure and establish possible difference in serumlevels of soluble ICAM-1 in 42 atopic patients (patients with allergic rhinitis and patients with bronchial asthma) incomparison with 28 patients without atopy (patients with asthma without rhinitis); whether there is a difference insICAM-1 levels between groups of 26 patients with allergic rhinitis and asthma in comparison with group of 16patients with allergic rhinitis only and also in comparison with 10 healthy controls. Results of the study have sub-stantiated statistically significant difference in sICAM-1 levels between all groups of patients in comparison tohealthy control, but no statistically significant difference in sICAM-1 levels between patients with and without atopy(Z=’1.738) or between patients with allergic rhinitis and bronchial asthma in comparison with group of patientswith allergic rhinitis only (Z=0.00). ICAM-1 is an important marker of inflammation in patients with allergic rhini-tis as well as in those with bronchial asthma. Atopic status does not influence differences in sICAM-1 levels.Although mean sICAM-1 levels were higher in patients with allergic rhinitis and bronchial asthma (312.71 ng/mL)in comparison with mean sICAM-1 levels in patients with allergic rhinitis only (279.69 ng/mL), no statistically sig-nificant difference was noted in sICAM-1 levels between these groups of subjects, i.e. asthma itself did not con-tribute to statistically significant increase of sICAM-1 levels.

Key words: intercellular adhesive molecule 1, allergic rhinitis, bronchial asthma, atopy


Aleksandra Peri}-Popadi} Institute of Allergology and Immunology, Clinical Center of Serbia, Koste Todorovi}a 2, 11000 Belgrade, Serbia and MontenegroPhone: 361 7777 37/00e-mail: [email protected]

National Institute of Heart, Lungs and Blood, Bet-hesda, May 1997, suppl. 2002 (3). All patients hadpositive results of metacholine and/or Ventolin test.Sensitization to standard inhaling allergens was testedby skin prick test in all patients.

All patients were subjected to physical medicalexamination. Pulmonary function before blood sam-pling was measured using Autospir Discom-14 ChestCorporation Tokyo, Japan. In addition to history, skintests and determination of serum IgE, patients withallergic rhinitis were subjected to in vivo specificrhino-provocative tests to evidence the presence ofallergic rhinitis.

The sICAM-1 levels were measured in bloodsamples obtained after the cubital vein puncture usingvacutainer, without EDTA addition. The sample wasleft to coagulate at room temperature for 60’120 minu-tes. After that the samples were centrifuged at 1300 gfor 10 minutes at room temperature. The separatedserum was stored at ’20 °C until the actual analysis.ELISA was used for the sICAM-1 serum level determi-nation. The commercial Parameter human sICAM-1immunoassay (ELISA, R&D Systems Inc. Minneapolis,USA) was used. The sICAM-1 serum levels were ex-pressed in ng/mL. The lowest detectable value ofserum sICAM-1 was 0.35 ng/mL (following the ma-nufacturers instructions).

The subjects were classified into followinggroups: 16 with allergic rhinitis, 26 with allergic rhinitisand bronchial asthma (all of them combined com-posed the group with atopy); 28 patients bronchialasthma without allergic rhinitis (composing the groupwithout atopy). The control group was composed of10 healthy volunteers, mean age 37 years without his-tory of allergy, asthma, allergic rhinitis, atopic der-matitis or any other significant disease.

Statistical analysis was conducted using EPIINFO ver. 10 program package. Statistical differenceswere calculated using non-parametric Mann-Whitneyand Kruskal-Wallis tests. Correlations among differentparameters were conducted using the Spearman'srank correlation coefficient (4, 5).

Results

The control group of healthy subjects had meansICAM-1 values of 226.64 ng/mL, coinciding with con-trol values stated by the manufacturer (R&D Systems).The mean sICAM-1 levels in patients with and withoutatopy were 300.10 ng/mL and 315.00 ng/mL, respec-tively. The correlation coefficient of sICAM-1 levelsbetween groups of healthy subjects and patients withand without atopy was H=12.072, p<0.01, suggest-ing statistically significant difference of sICAM-1 levelsbetween the groups of subjects.

The correlation coefficient of sICAM-1 levels bet-ween groups with atopy (patients with allergic rhinitis

and patients with asthma and allergic rhinitis) andhealthy subjects was Z1=’2.670, p<0.01, suggestinghighly statistically significant difference between thegroups, i.e. sICAM-1 level was significantly higher inpatients with atopy than in healthy controls.

The correlation coefficient of sICAM-1 levelsbetween groups without atopy (patients with bronchialasthma without allergic rhinitis and negative skin pricktest) and healthy subjects was Z2=’3.166, p<0.01,suggesting highly statistically significant differencebetween the sICAM-1 levels in the groups, i.e sICAM-1level was significantly higher in patients with asthmawithout atopy than in healthy controls. The correlationcoefficient of sICAM-1 levels between groups with andwithout atopy was Z3=’1.738, p>0.05, suggesting nostatistically significant difference between the sICAM-1levels in the groups, i.e. sICAM-1 serum levels weresimilar in patients with atopy (allergic rhinitis only andallergic rhinitis with bronchial asthma) and thosewithout atopy (patients with bronchial asthma only),(Table I).

The mean value of sICAM-1 levels in patientswith allergic rhinitis was 279.62 ng/mL, while inpatients with allergic rhinitis and asthma the level of312.71 ng/mL was recorded. The mean sICAM-1 level


Table II Mean values of sICAM concentrations in our studies groups

Group

ICAM-1(ng/mL)

Valid NMeanStd Dev.Min.Max.

healthy

N=10 226.632.94197.0301.9

rhinitis

N=16279.62 136.34183.14376.63

asthma andrhinitis

N=26312.71168.66167.05930.65

H=12.072 p<0.01Z1=’2.670 p<0.01Z2=’3.166 p<0.01Z3=’1.738 p<0.05

Table I Mean values of sICAM concentrations in our studies groups

Group

ICAM-1(ng/mL)

Valid NMeanStd Dev.Min.Max.

healthy

N=10 226.632.94197.0301.9

atopy

N=42300.10 136.34167.05930.65

atopy free

N=28315.0079.26190.04488.89

H=12.072 p<0.01Z1=’2.670 p<0.01Z2=’3.166 p<0.01Z3=’1.738 p<0.05

in healthy subjects was 226.64 ng/mL. The correlationcoefficient of sICAM-1 levels between the groups wasH=7.172, p<0.05, suggesting the statistically signifi-cant difference in sICAM-1 levels between the groups.The correlation coefficient of sICAM-1 levels betweenthe groups of patients with allergic rhinitis and healthycontrols was Z4=’2.821, p<0.01, suggesting the sta-tistically significant difference between the groups, i.e.patients with allergic rhinitis had statistically signifi-cantly higher sICAM-1 levels than the healthy controls.The correlation coefficient of sICAM-1 levels betweenthe groups of patients with allergic rhinitis and asthmaon one hand and healthy controls on the other wasZ5=’2.172, p<0.05, suggesting the statistically signif-icant difference between the groups, i.e. patients withallergic rhinitis and asthma had higher sICAM-1 levelsthan the healthy controls. The correlation coefficientof sICAM-1 levels between the groups of patients withallergic rhinitis on one hand and bronchial asthma andallergic rhinitis on the other was Z6=0.00, p>0.05,suggesting no statistically significant differencebetween the groups, i.e. sICAM-1 levels were similarin patients with allergic rhinitis only and those suffe-ring from both allergic rhinitis and bronchial asthma(Table II).

Discussion

Nowadays, there is a general consensus thatinflammation is crucial in pathophysiology of respira-tory allergic diseases (6). The level of soluble adhesivemolecules in the serum reflects the degree of systemicinflammation, but the dynamics of these molecules inthe pathogenesis of allergic diseases and their evolu-tion in the course of treatment remain to be evi-denced. ICAM-1 may be induced on various cell typesusing miscellaneous inflammation stimuli (7). Allergicinflammation is associated with ICAM-1 expression onthe surface of different cells such as the endothelium,bronchial epithelium and eosinophillic leukocytes ((8-11). Induction of expression of these molecules takesplace under the influence of various proinflammatorycytokines such as IFN-gamma, IL-1, TNF-alpha andothers.

The purpose of the study was to measure andcompare levels of soluble ICAM-1 in the sera of atopicpatients non-atopic patients and to compare the levelswith the sICAM-1 levels in healthy controls. The meansICAM-1 levels in healthy controls was 226.64 ng/mL.Shiota and associates reported somewhat highermean sICAM-1 value in healthy volunteers, i.e. 260.90ng/mL. It is possible that the difference in the numberof healthy volunteers (10 vs. 39) contributed to the dif-ference in the value obtained.

The mean value of sICAM-1 levels in our atopicpatients (all patients with allergic rhinitis and patientswith both allergic rhinitis and bronchial asthma) was300.10 ng/mL as compared with 315.00 ng/mL meas-

ured non-atopic patients (bronchial asthma withoutrhinitis). Similar values in asthmatic patients werereported by Shiota and associates (12). Analysis of thelevels of sICAM-1 between the groups of atopic pa-tients and healthy controls as well as the differencebetween non-atopic group and healthy controls hasrevealed the presence of statistically significant differ-ence. This actually means that atopic and non-atopicpatients had statistically significant levels of sICAM-1 inthe sera in comparison with healthy controls. This fin-ding is in concert with referential data (1, 13, 14), sub-stantiating the hypothesis on ICAM-1 as a marker ofinflammation in respiratory diseases. However, analy-sis of correlation of sICAM-1 levels between groups ofatopic vs. non-atopic patients failed to substantiate thestatistically significant difference, i.e., both atopic andnon-atopic patients had similar sICAM-1 levels in therespective sera. Laan and associates (15) obtained si-milar values in pediatric population, and Figen Do’Eguand associates (16) failed to identify any statisticallysignificant differences in sICAM-1 levels between ato-pic and non-atopic children.

Results of our study have substantiated the pres-ence of statistically significant difference in sICAM-1values of patients with allergic rhinitis in comparisonwith healthy controls. In a study on a group of patientswith allergic rhinoconjunctivitis Turgay et al. (17) re-ported no statistically significant difference betweentheir sICAM-1 levels and healthy controls, but menwith allergic rhinoconjunctivitis had higher values ofsICAM-1 levels than in women, and in comparisonwith healthy controls. The sICAM-1 levels were higherin patients with allergic rhinoconjunctivitis that hadhigher symptom score (17). Kato and associates (18)reported that the level of sICAM-1 in the sera ofpatients with polynosis was up-regulated in the earlyseason in comparison with healthy control. However,Chia-Ming Liu and associates (19) found overlappingvalues in sICAM-1 levels of patients with allergic peren-nial rhinitis and healthy controls. He explained thephenomenon by the target organ size (the nose) thatis substantially lower in comparison with target sur-faces of other organs such as the lungs and skin.

The reason for the differences in sICAM-1 levelsin our allergic rhinitis patients in comparison withhealthy control may lie in the »minimum persistentinflammation« present in patients hypersensitive tomite, that have it all the year round even in absence ofsymptoms (20). Also, the minimum exposure to aller-gens in natural conditions may lead to the occurrenceof differences in sICAM-1 levels.

Our study has also substantiated the presence ofstatistically significant difference in sICAM-1 levelsbetween groups of patients with atopy (allergic rhinitisand allergic rhinitis combined with asthma) and groupof healthy volunteers, complying with referential data.However, in spite of the notable difference in meansICAM-1 levels in patients with asthma versus patients


with allergic rhinitis only (312.71 ng/mL vs. 279.62ng/mL) there was no statistically significant differencein sICAM-1 levels between these groups of patients.The reason may lie in heterogeneous composition ofthe groups where patients with bronchial asthmamainly used the therapy of inhaling glycocorticos-teroids that may affect the down-regulation of ICAM-1molecules (13, 16, 21’23).

The results of the study have substantiated thatsICAM-1 is a marker of inflammation in both patientswith bronchial asthma and those with allergic rhinitis,but that atopy itself does not influence the highersICAM-1 levels in comparison with non-atopic patients.Also, in our study asthma did not contribute signifi-cantly to the total sICAM-1 levels. Naturally, future stu-dies are necessitated for better elucidation of the fun-ction and regulatory mechanisms of serum ICAM-1.


UTICAJ ATOPIJE NA NIVO sICAM-1 U SERUMU KOD PACIJENATA SA ALERGIJSKIM RINITISOM I BRONHIJALNOM ASTMOM

Aleksandra Peri}-Popadi}1, Mirjana Bogi}1, @ikica Jovi~i}1, Sanvila Ra{kovi}1, Vesna Tomi}-Spiri}1, Sneàna Kova~evi}2, Jasna Bolpa~i}1, Miodrag ôli}3

1Institute za alergologiju i imunologiju, Klini~ki centar Srbije, Beograd2Interna medicina, Op{ta bolnica »Dr Laza Lazarevi}«, [abac

3Institut za medicinska istraìvanja, VMA, Beograd

Kratak sadràj: Poznato je da su adhezivni molekuli uklju~eni u inflamacione bolesti plu}a kao {to je bron-hijalna astma. Cilj ovog rada je bio da se izmeri i utvrdi da li postoji razlika u koncentracijama solubilnog ICAM-1u serumu 42 bolesnika sa atopijom (bolesnici sa alergijskim rinitisom i bolesnici sa bronhijalnom astmom) uodnosu na grupu od 28 bolesnika bez atopije (bolesnici sa astmom bez rinitisa) da li postoji razlika u koncen-tracijama sICAM-1 me|u grupama od 26 bolesnika sa alergijskim rinitisom i astmom u odnosu na 16 bolesnikasamo sa alergijskim rinitisom kao i u odnosu na 10 zdravih kontrola. Rezultati rada su pokazali da postoji statisti~kizna~ajna razlika u koncentracijama sICAM-1 svih pomenutih grupa ispitanika u odnosu na zdravu kontrolu, ali dane postoji statisti~ki zna~ajna razlika koncentracija sICAM-1 me|u grupama bolesnika sa atopijom i bez atopije(Z = ’1,738) kao ni me|u grupama ispitanika sa alergijskim rinitisom i bronhijalnom astmom u odnosu na grupuispitanika samo sa alergijskim rinitisom (Z = 0,00). sICAM-1 predstavlja zna~ajan marker inflamacije i kod bolesni-ka sa alergijskim rinitisom kao i kod onih sa bronhijalnom astmom. Atopijski status ne uti~e na razlike u koncen-tracijama sICAM-1. Iako su srednje vrednosti koncentracija sICAM-1 bile vi{e kod bolesnika sa alergijskim riniti-som i bronhijalnom astmom (312,71 ng/mL) u odnosu na srednje koncentracije sICAM-1 kod bolesnika samo saalergijskim rinitisom (279,62 ng/mL), nije postojala statisti~ki zna~ajna razlika u koncentracijama sICAM-1 me|uovim grupama ispitanika, tj. sama astma nije doprinosila statisti~ki zna~ajnijem pove}anju koncentracija sICAM-1.

Klju~ne re~i: intercelularni adhezivni molekul 1, alergijski rinitis, bronhijalna astma, atopija

References

1. El-Sawy-IH, Badr-El-Din-Om, El-Azzouni-OE, Motawae-HA. Soluble intercellular adhesion molecule-1 in sera ofchildren with bronchial asthma exacerbation. Int ArchAllergy Immunol 1999; 119 (2): 126’32.

2. Van de Stolpe A, van der Saag PT. Intercellular adhesionmolecule-1 (ICAM-1). J Mol Med 1996; 74 (1); 13’33.

3. Global Strategy for Asthma Management and Preven-tion. National Heart Lung and Blood Institute NationalInstitutes of Health. NHLBI/WHO Workshop Report NIHPub No 95’3659, 1995, Revised 2002, 2.

4. Jekel JF, Elmore JG, Katz DL. Epidemiology Biostaticsand Preventive Medicine. Philadelphia, WB SaundersCompany, 1996.

5. Eric-Marinkovi} J, Dotli} R, Jano{evi} S, Kocev N, Gaji}M, Ille T, Stanisavljevi} D, Babi} D. Statistics for rese-archers in the field of medical science, Belgrade, Schoolof Medicine 2001.

6. Howarth PH, Bradding P, Montefort S, et al. Mucosalinflammation and asthma. Am J Resp Crit Care Med1994; 150: s 18’22.

7. Wegner CD, Gundel RH, Reilly P, Haynes N, Letts G,Rothlein R. Intercellular adhesion molecule-1 (ICAM-1)in the pathogenesis of asthma. Science 1990: 247:456’9.

8. Springer T. Adhesion receptors of immune system.Nature 1990; 346: 425’33.

9. Smith CW, Marlin SD, Rothlein R, Toman C, AndersonDC. Cooperative interactions of LFA-1 AND Mac-1 withintercellular adhesion molecule-1 in facilitating transen-dothelial migration of human eosinophils in vitro. J ClinInvest 1989; 83: 2008’17.

10. Paolieri F, Battifora M, Riccio AM, Pesce G, CanonicaGW, Bagnasco M. Intercellular adhesion molecule-1 oncultured human epithelial cell lines: influence of proin-flammatory cytokines. Allergy 1997; 52: 521’31.

11. Ciprandi G, Buscaglia S, Pesce GP, Bagnasco M,Canonica GW. ICAM-1/CD54 expression on conjunctivalepithelium during pollen season. Allergy 1995; 50:184’7.

12. Shiota Z, Wilson JG, Marukawa M, Ono T, Kaji M.Soluble intercellular adhesion molecule 1 (ICAM-1) anti-gen in sera of bronchial asthmatics. Chest 1996; 109(1): 94’9.

13. Kobayashi T, Hashimoto S, Imai K, Amemiya E, Yama-

guchi M, Yachi A, Horie T. Elevation of serum solubleintercellular adhesion molecule-1 (sICAM-1) and sE-se-lectin levels in bronchial asthma. Clin Exp Immunol1994; 96 (1): 110’5.

14. Kokuludag A, Sin A, Terzioglu E, Saydam G, Sebik F.Eleveation of serum eosinophil cationic protein, solubletumor necrosis factor receptors and soluble intercellularadhesion molecule-1 levels in acute bronchial asthma. JInvestig Allergol Clin Immunol 2002; 12 (3): 211’ 4.

15. Laan MP, Koning H, Baert MR, Oranje AP, BuurmanWA, Savelkoul HF, Neijens HJ. Levels of soluble inter-cellular adhesion molecule-1, soluble E-selectin, tumornecrosis factor-alpha, and soluble tumor necrosis factorreceptor p55 and p75 in atopic children. Allergy 1998;53 (1): 51’8.

16. Do'Egu F, Ikincio Egullari A, Eegin Y, Babacan E.Circulating Adhesion Molecule Levels in ChildhoodAsthma. Ind Pediatr 2002; 39: 1017’21.

17. Turgay M, Keskin G, Kminkli G, Senturk T, Tutkak H,Duman M. Intercellular adhesion molecule-1 (sICAM-1)in patients with allergic rhinoconjunctivitis. AllergolImmunopathol 1996; 24 (3): 129’31.

18. Kato M, Hattori T, Matsumoto Z, Nakashima I. Dy-namics of soluble adhesion molecule levels in patientswith pollinosis. Arch Otolaryngol Head Neck Surg 1996;122 (12): 1398’400.

19. Liu C-M, Shun C-T, Cheng Y-K. Soluble adhesion mole-cules and cytokines in perennial allegic rhinitis. Annals ofAllergy, Asthma & Immunology 1998; 81: 176 ’180.

20. Passalacqua G, Ciprandi G, Canonica GW. United air-ways disease: therapeutic implication. Thorax 2000; 55:26’7.

21. Bogi} M, Peri}-Popadi} A, Jovi~i} Z, Kova~evi} S,Ra{kovi} S, Tomi}-Spiri} V, Savi} N, ôli} M. Is solubleintercellular adhesion molecule-1 a marker of diseaseactivity in bronchial asthma? Jugoslov Med Biohem2004; 23 (1): 55’58.

22. Shiota Z, Sato T, Ono T. Serum levels of soluble ICAM-1in asthmatic patients. Arerugi 1993; 42 (12): 1782’7.

23. Van der Saag PT, Caldenhoven E, van de Stolpe A.Molecular mehanisms of steroid action a novel type ofcross-talk between glucocorticoids and NF-kappa Btrancription factors. Eur Respir J Suppl. 1996; 22:146s ’153s.



Accepted: May 5, 2004


Introduction

The b-globin gene cluster is located on chro-mosome 11. The alignment of genes in humanb-globin locus represents the order of gene expres-sion during ontogenic development: embryonalgenes (e) are located at the 5’end, then fetal genes(Ag, Gg) and finally, at the 3’end, adult genes (d, b).Thalassemia syndromes are a group of hereditarydisorders generally caused by mutations in globingenes. In Serbia, thalassemia syndromes are less fre-quent than in adjacent Mediterranean countries. Theoverall frequency of thalassemia syndromes in Serbiais 1.9% (1).

Since 1998 thalassemia syndromes have beensystematically characterized on molecular level in thepopulation of Serbia (2). Nine different mutationshave been detected: seven b-thalassemic mutationsand two thalassemic hemoglobin (Hb) variants (HbLepore, Hb Sabine), among which 70% were Hb Le-pore, b°39 and b+ IVS-I-110 (2). Two studies showedthat Hb Lepore is the most common cause of tha-lassemia in Serbia (30%) (1, 2).

Hb Lepore is an abnormal thalassemic hemo-globin variant, which consists of normal a-globinchains and fused db-globin chains. The db-globinchain is a product of an unequal recombination thatjoins the proximal end of the d-globin gene with thedistal end of the b-globin gene (3). Three types of HbLepore, that differ in the position at which the transi-tion from d to b DNA occurs, have been described:Hb Lepore-Hollandia, Hb Lepore-Baltimore and HbLepore-Boston-Washington (Hb Lepore BW) (3).

Substitution (C→T) alters codon 39 to a stopcodon. Such chain termination (nonsense) mutation

UC 577,1; 61 ISSN 0354-3447


BIOCHEMICAL PHENOTYPE AND ORIGIN OF THE THREE MOSTCOMMON BETA-THALASSEMIA MUTATIONS IN SERBIA

Jelena Poznani}1, Ljubica Peri{i}1, Jelena Uro{evi}1, Branka Petru~ev1, Tatjana \ureinovi}1, Nata{a To{i}1, Lidija Krivokapi}-Dokmanovi}2, Dragana Jani}2, Milica ^vorkov-Draì}3,

Gordana Bunjeva~ki3, Sonja Pavlovi}1

1Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia and Montenegro2University Children’s Hospital, Belgrade, Serbia and Montenegro

3Mother and Child Health Care Institute of Serbia »Dr Vukan ûpi}«, Belgrade, Serbia and Montenegro

Summary: Molecular (DNA) characterization of thalassemia is the most reliable methodology for the diag-nosis of this group of diseases. As thalassemias are very heterogeneous, hematological data and additional bio-chemical analysis are essential for their differential diagnosis. In this paper we present hematological and bio-chemical characteristics of the carriers of three most common beta-thalassemia mutations in Serbia (Hb Lepore,b°39 and b+IVS-I-110), to be taken into consideration as the initial step of the diagnostic approach to the tha-lassemia patients. Also, this paper represents a detailed survey of the diversity of b-globin gene haplotypes in car-riers of the most common b-thalassemia mutations and normal betaA/betaA individuals of Serbian descent. A novelhaplotype associated with Hb Lepore-Boston-Washington gene has been identified in Serbian population. Thesedata support the hypothesis of multicentric origin of this mutation. The mutation has arised de novo in the chro-mosomal background characteristic for Serbian population. Additionally, we have shown that two most commonMediterranean mutations, b°39 and b+ IVS-I-110, have probably been introduced into Serbian population from Italyand Turkey, respectively, through historically documented migrations and settlements.

Key words: b-thalassemia, haplotype, Hb Lepore


Sonja Pavlovi}Institute of Molecular Genetics and Genetic EngineeringVojvode Stepe 444a, 11000 Belgrade, Serbia and MontenegroTel: +381 11 3976 445Fax: +381 11 3975 808E-mail: [email protected]


aborts mRNA translation and leads to synthesis of atruncated polypeptide (4). Therefore, codon 39 re-sults in b° thalassemic phenotype. Substitution(G→A) at position 110 in intron I (IVS-I-110) createsan alternative splice site. The incorrectly splicedmRNA leads to premature termination of translationbut allows accumulation of some amount of normalglobin mRNA (b+ thalassemic phenotype) (5).

In this study we have analyzed hematologicaldata and Hb content (Hb A, Hb A2 and Hb F) in car-riers of three most common beta-thalassemia muta-tions in Serbia. Also, we present a detailed study ofthe diversity of b-globin gene haplotypes in normalbetaA/betaA and thalassemic individuals of Serbiandescent, conducted with the aim of defining the ori-gin of beta-thalassemia in Serbia.

The b-globin gene cluster is highly polymorphic.The pattern or combination of polymorphic restric-tion sites in b-globin locus for any chromosome iscalled a haplotype (6). There are nine common hap-lotypes (I-IX) known to be present in different Medi-terranean populations with significant percentage (6).Additionally, four beta globin intragenic single basepolymorphisms have led to the definition of three dif-ferent beta globin gene frameworks (6). Haplotypesand frameworks of b-globin gene cluster are widelyused nowadays to determine the chromosomal back-ground on which a mutation arose, as well as its geo-graphical pattern.

Material and Methods

Forty-five Serbian patients from 20 unrelatedfamilies affected with b-thalassemia, were studied: 21Hb Lepore BW b-thalassemic variant carriers (from 8unrelated families), 14 b°39 b-thalassemia mutationcarriers (from 7 unrelated families) and 10 b+IVS-I-110 mutation carriers (from 5 unrelated families).Healthy subjects related to the carriers were also ana-lyzed.

Hematological parameters were obtained usingan automated cell counter. Abnormal hemoglobinwas detected by electrophoresis on cellulose acetatein Tris-borate-EDTA (pH 8.4) (7). Hb F was quantifiedby standard methods (8, 9) and Hb A2 and Hb Leporewere estimated by elution from celogel electrophoret-ic strips.

DNA was extracted from peripheral blood col-lected with sodium citrate or EDTA as an anticoagu-lant (10). DNA samples were used as templates inpolymerase chain reaction (PCR). For each sampleeight PCR-RFLPs were performed, one for each poly-morphism (HincII/e, XmnI/5’Gg, HindIII/Gg, HindIII/Ag, HincII/yb, HincII/3’yb, AvaII/b, BamHI/3’b). Theconditions for PCR amplification were as describedpreviously (11, 12), with changes in temperature ofannealing for e, 5’Gg, Gg, Ag, yb, 3’yb, b, 3’b DNA

fragments containing b-globin gene cluster polymor-phic sites: 55, 55, 60, 55, 61, 61, 57 and 53 °C res-pectively. For haplotype analysis the polymorphic res-triction enzyme sites were determined by digestion ofPCR amplified fragments using the methodology ofSutton et al (12). Polymorphic sites were detected byagarose gel electrophoresis. The framework basesubstitutions were identified by dideoxy chain termi-nation method using fluorescently labeled dideoxynucleotides (Pharmacia Biotech, Uppsala, Sweden)on an automated DNA sequencer (ALFexpress DNASequencer, Pharmacia Biotech) using the primer 5’-AA TCA TTC GTC TGT TTC CCA-3’.

Results

All carriers of Hb Lepore had a clinical pheno-type of thalassemia trait. Their MCV and MCH valueswere reduced (Table I). Hb A2 level was normal ordecreased, while Hb F was slightly increased. Hb ana-lysis by electrophoresis on cellulose acetate revealedthe presence of an abnormal Hb fraction, later char-acterized as Hb Lepore by PCR analysis. Heterozy-gous carriers of b° 39 mutation had evident anemia,but unusually mild microcytosis and hypochromia forthis type of mutation (b°). As expected, Hb A2 and HbF were markedly elevated (5% and 3.2%, respective-ly). All carriers of b+ IVS-I-110 mutation were clini-cally asimptomatic. They presented with relativelymild anemia and moderately reduced MCV and MCHvalues. Both Hb A2 and Hb F levels were slightly ele-vated.

In order to elucidate the origin of the most com-mon thalassemic mutations in the population ofSerbia, haplotype analysis of Hb Lepore BW, b° 39and b+ IVS-I-110 mutation carriers was performed.We have studied eight linked polymorphic restrictionsites along b-globin gene cluster (Figure 1). In orderto establish the haplotype of thalassemic chromoso-mes with certainty, nonaffected relatives were inclu-ded in the study.

Four different haplotypes associated with b-tha-lassemia mutations were detected: two of them weretypical of Mediterranean countries (I and V), and twoautochthonous haplotypes, specific for Serbian po-pulation, were discovered as well (haplotypes A andB) (Table II).

Table I Hematological data (average values and standarddeviations), Hb A2 and Hb F values for b-thalassemia

heterozygotes

MutationHb Lepore(db fusion)Codon 39(C→T)IVS I-110(G→A)

GenderMF

MFMF

No.1110

7755

Hb (g/L)114.5 ± 9.02108.5 ± 5.22

109.0 ± 1.5091.0 ± 0.73

128.5 ± 0.50126.0 ± 0.90

MCV (fL)66.2 ± 3.5166.0 ± 1.53

67.4 ± 3.9067.0 ± 0.3366.1 ± 1.6664.1 ± 3.35

MCH (pg)20.35 ± 1.5020.55 ± 1.75

22.6 ± 0.9822.1 ± 0.3320.8 ± 0.5020.9 ± 1.05

Hb A2 (%)1.95 ± 0.821.84 ± 0.62

5.0 ± 0.254.6 ± 0.504.3 ± 0.154.2 ± 0.20

HbF (%)2.15 ± 0.602.16 ± 0.51

4.20 ± 4.083.87 ± 0.432.33 ± 0.772.30 ± 0.60


All analyzed Lepore chromosomes were of theidentical haplotype: haplotype A. Comparing this ha-plotype with Hb Lepore BW haplotypes reported todate showed that it differs from all of them. Thus, inthis study a novel haplotype associated with Hb Lepo-re BW mutation was identified. The specificity of thishaplotype is the presence of HindIII/Ag restriction site(Figure 2).

Haplotype analysis revealed that b° 39 thalasse-mia mutation is mostly associated with haplotype Iand framework 1. However, in one family, b° 39 chro-mosomes were found to be associated with frame-work 2. Two different haplotypes were associatedwith b+IVS-I-110 thalassemic mutation: haplotype Iand haplotype B. Also, we have detected the pres-ence of haplotypes I and V, as well as haplotypes Aand B specific for Serbian population, in normalbetaA/betaA individuals.

Discussion

The results presented in this paper may help theclinicians as a guideline for differential diagnostics ofthalassemia trait. The hematological values of partic-ular importance are: MCV lower than 70fL and MCHlower than 24pg. If these values are consistently de-creased (despite Fe supplementation therapy) in ane-mic patient, hemoglobin electrophoresis is recom-mended, followed by DNA analysis.

Haplotype analyses we present in this paperhave been carried out on b-thalassemia carriers ofcommon Mediterranean mutations (Hb Lepore BW,b° 39, b+IVS-I-110). A number of similar studieshave already provided insights into origin and distri-bution of these mutations. Beta-globin gene haplo-types associated with thalassemic mutations weresporadically studied in former Yugoslavia, too (13).However, chromosomal background of b-thalas-semia mutations in Serbian population demonstratesspecificity, not previously reported.

In this paper we present a novel haplotype asso-ciated with Hb Lepore BW gene. Hb Lepore BW iscommon in Mediterranean countries. Up to date, 6haplotypes associated with Hb Lepore BW chromo-somes were identified: haplotype V in 34 families ofSpanish, Yugoslavian and Italian origin (14’16), hap-lotype I in 23 Italian families (15), haplotypes VI andVII in Italian families (15) and two unique haplotypes(in a Hungarian individual and in one Black family)(15). A multicentric origin of this mutation has beensuggested (16). In order to elucidate the origin of HbLepore BW mutation in Serbia, the 5’ and 3’ subhap-lotypes were taken into consideration as independententities (see Figure 1). We have detected two diffe-rent 5’ subhaplotypes: the first one, typical for both Iand V haplotypes, and the second, autochthonous 5’subhaplotype present in both haplotypes A and B.There were also two 3’ subhaplotypes: the one typical

Table II Haplotypes detected in the healthy and thalassemic population of Serbia

Polymorphic restriction sites: HincII/e, XmnI/5'GgHindIII/Gg, HindIII/Ag, HincII/yb, HincII/3'yb,

AvaII/b, BamHI/3'b

Haplotype I [4]Haplotype V [4]Haplotype AHaplotype B

e++++

5'Gg––––

Gg––––

Ag––++

yb––––

3'yb––––

b++++

3'b+––+

5'subhaplotype 3'subhaplotype

Figure 2. Detection of HindIII/Ag polymorphic site by 1% agarose gel electrophoresis. 1. l/HindIII/EcoRI DNAmarker; 2. undigested PCR fragment; 3. homozygote

for HindIII/Ag polymorphism; 2690bp indicates pUC19 plasmid DNA digested with HindIII (control of the restriction enzyme activity)

Figure 1. The location of eight polymorphic restrictionsites in the b-globin gene cluster. Sites are numbered from5' to 3': 1-HincII/e, 2-XmnI/5'Gg, 3- HindIII/Gg, 4-HindIII/Ag,

5-HincII/yb, 6-HincII/3'yb, 7-AvaII/b, 8-BamHI/3'b; 5' and 3' subhaplotypes are defined by restriction sites

1–6 and 7, 8, respectively.

1 2 3

← 2690 bp← 1842 bp← 1060 bp← 755 bp

5’ 3’

3’ subhaplotype5’ subhaplotype

e

↑1

↑↑ ↑ ↑↑ ↑ ↑2 3 4 5 6 7 8

Gy Ay yb1 d b


for haplotype I and the other typical for haplotype V(see Table II).

All Serbian Hb Lepore BW genes are associatedwith autochthonous 5’ subhaplotype and 3’ subhap-lotype typical for haplotype V. We have previouslyshown that they are also associated with framework 2(17). The Hb Lepore BW gene associated with haplo-type V and framework 2 was virtually the only typefound in Eastern Italy and former Yugoslavia (13, 16).Considering the fact that two different haplotypescontaining autochthonous 5’ subhaplotype (A and B)as well as haplotype V were detected in healthy Ser-bian population, two possible hypotheses could ex-plain the origin of Hb Lepore BW mutation on a novelchromosomal background.

There is a possibility that unequal recombina-tion or gene conversion occurred between two chro-mosomes with haplotypes A or B and Hb Leporemutation associated with haplotype V, respectively,producing new chromosomal background for Hb Le-pore mutation. The fact that the probable site of re-combination could have been in the 9,1 kb segment,which has been defined by Chakravarti et al. (18) asa hot spot region for nonuniform recombination inb-globin gene cluster, supports this hypotesis.

However, the mutation might have arisen denovo, by a single independent mutational event in ahealthy individual with haplotype A and spread in thepopulation. The high frequency of Hb Lepore BWhemoglobin variant in Serbian population, the homo-geneity of Hb Lepore BW haplotype (no haplotype Vwas detected), as well as its uniqueness, suggest itsindependent origin. The finding that Hb Lepore BWchromosomes are concentrated in particular geogra-phical region of Serbia also supports this hypothesis.

It has been proposed that b°39 mutation origi-nates from Italy (19). Our data suggest Western Me-

diterranean (Italian) origin of this mutation in Serbia.Chromosomal background of b°39 mutation in ourpopulation is similar to the one found in other Medi-terranean countries. However, in one family b°39chromosomes are found to be associated with frame-work 2. Although mutation spread almost never invol-ves a change of b-globin gene framework, the excep-tion has been detected (19). To the best of our know-ledge, this is the first report of the association bet-ween framework 2 and b°39 mutation. This exampleof mutation spread from one beta-globin gene frame-work to another, within a population, may be due tointer-allelic gene conversion events or recurrent mu-tation (19).

b+IVS-I-110 mutation is previously reported tobe of Eastern Mediterranean (Turkish) origin andassociated with haplotype I (20). In this study, b+ IVS-I-110 mutation is found to be associated with two dif-ferent haplotypes. Actually, these two haplotypes dif-fer only in their 5’ subhaplotypes: one is typical forhaplotype I, and the other is 5’ subhaplotype fromhaplotypes A or B. The likelihood that this haplotypedimorphism associated with the same b-thalassemiamutation is a consequence of random mutation isvery low. The diversity of haplotypes associated withb+ IVS-I-110 mutation, would then be generated byrecombination events (crossing-over or gene conver-sion) between the original b-thalassemic chromo-some (haplotype I) and the 5’ subhaplotype charac-teristic for normal population of Serbia.

Although thalassemia syndromes are sporadicdisorders in Serbia, they reflect numerous historicallydocumented migrations over Balkan Peninsula andyet show autochthonous features.

Acknowledgements. This work was supportedby grant 1417 from Ministry for Science and Techno-logy of Serbia.


References

1. Beksedi} D, Cuharska T, Stojimirovi} E, Dini} B. In He-moglobin and Hemoglobinopathies, (ed) SerbianBlood Transfusion Centre, 1980: 122–7.

2. Pavlovi} S. Molecular genetics of thalassemia syndro-mes: genotype-phenotype correlation. Doctoral Thesis.University of Belgrade, Belgrade, FR Yugoslavia, 2001:136–7.

3. Baglioni C. The fusion of two peptide chains in He-moglobin Lepore and its interpretation as a genetic de-letion. Proc Natl Acad Sci USA 1962; 48: 1880–6.

4. Trecartin RF, Liebhaber SA, Chang JC. Beta°-tha-lassemia in Sardinia is caused by a nonsense mutation.J Clin Invest 1981; 68: 1012–15.

5. Spritz RA, Jagadeeswaran P, Choudary PW. Base sub-stitution in an intervening sequence of a beta+-tha-lassemic human globin gene. Proc Natl Acad Sci USA1981; 78: 2455–62.

6. Orkin SH, Kazazian HH Jr, Antonarakis SE , Goff SC,Boehm CD, Sexton JP, Weber PG, Giardina PJV. Lin-kage of b-thalassemia mutations and b-globin poly-morphisms with DNA polymorphisms in human b-glo-bin gene cluster. Nature 1982; 296: 627–31.

7. Marengo-Rowe AJ. Rapid electrophoresis and quantita-tion of haemoglobin on cellulose acetate. J Clin Pathol1965; 18: 790–2.

8. Pembrey ME, McWade P, Weatherall DJ. Reliable rou-tine estimation of small amounts of foetal haemoglobin

by alkali denaturation. J of Clin Pathol 1972; 25: 738–40.

9. Molden DP, Alexander NM, Neely WE. Fetal hemoglo-bin: optimum conditions for its estimation by alkali de-naturation. Am J Clin Pathol 1982; 77: 568–72.

10. Poncz M, Solowiejczk D, Harpel B, Mory Y, Schwartz E,Surrey S. Construction of human gene libraries fromsmall amounts of peripheral blood: analysis of b-likeglobin genes. Hemoglobin 1982; 6: 27–36.

11. Simjanovska L, Petkov GT, Stojanovski N, Basak AN,Efremov GD. The origin of Hb O-Arab in the Balkancountries. Balkan Journal of Medical Genetic 1998; 1(1): 8–12.

12. Sutton M, Bouhassira EE, Nagel RL. Polymerase chainreaction amplification applied to the determination ofb-like globin gene cluster haplotypes. Am J Hematol1989; 32: 66–9.

13. Efremov GD. Hemoglobinopathies in Yugoslavia: anupdate. Hemoglobin 1992; 16: 531– 44.

14. Ribeiro ML, Cunha E, Goncalves P, Matrin Nunez G,Fernandez Galan MA, Tamagnini GP, Smetanina NS, GuL-H, Huisman THJ. Hb Lepore-Baltimore and HbLepore-Washington-Boston in Central Portugal and Spa-nish Alta Extremadura. Hum Genet 1997; 99: 669–73.

15. Lanclos KD, Patterson J, Efremov GD, Wong SC, Ville-gas A, Ojwang PJ, Wilson JB, Kutlar F, Huisman THJ.Characterization of chromosomes with hybrid genes for

BIOHEMIJSKI FENOTIP I POREKLO TRI NAJÊ[]E BETA-TALASEMIJSKE MUTACIJE U SRBIJI

Jelena Poznani}1, Ljubica Peri{i}1, Jelena Uro{evi}1, Branka Petru~ev1, Tatjana \ureinovi}1,Nata{a To{i}1, Lidija Krivokapi}-Dokmanovi}2, Dragana Jani}2, Milica ^vorkov-Draì}3,

Gordana Bunjeva~ki3, Sonja Pavlovi}1

1Institut za molekularnu genetiku i geneti~ko inènjerstvo, Beograd, Srbija i Crna Gora

2Univerzitetska de~ja klinika, Beograd, Srbija i Crna Gora3Institut za zdravstvenu za{titu majke i deteta »Dr Vukan ûpi}«,

Beograd, Srbija i Crna Gora

Kratak sadràj: Molekularna (DNK) karakterizacija talasemija je najpouzdaniji metod za dijagnostikuove grupe oboljenja. Kako su talasemije vrlo heterogene, hematolo{ki podaci i dodatne biohemijske analize suod esencijalne va`nosti za njihovu diferencijalnu dijagnostiku. U ovom radu su prikazani hematolo{ki parametrii biohemijske karakteristike nosilaca tri naj~e{}e b-talasemijske mutacije u Srbiji (Hb Lepore, bº39 i b+IVS-I-110) koje je neophodno uzeti u obzir u po~etnom koraku postavljanja dijagnoze kod talasemi~nih pacijenata.Pored toga, ovaj rad predstavlja detaljnu analizu diverziteta haplotipova b-globinskog lokusa kod nosilacanaj~e{}ih b-talasemijskih mutacija i zdravih betaA/betaA osoba u Srbiji. Identifikovan je novi haplotip asociransa Hb Lepore-Boston-Va{ington genom u srpskoj populaciji. Ovi podaci idu u prilog hipotezi o multicentri~nomporeklu ove mutacije. Mutacija je nastala de novo u hromozomskom kontekstu koji je karakteristi~an za popu-laciju u Srbiji.Tako|e je pokazano da su dve naj~e{}e mediteranske mutacije, bº39 i b+IVS-I-110, verovatno»uvezene« u populaciju Srbije iz Italije, odnosno Turske, istorijskim migracijama i seobama naroda.

Klju~ne re~i: b-talasemija, haplotip, Hb Lepore

Hb Lepore-Washington, Hb Lepore-Baltimore, Hb P-Nilotic and Hb Kenya. Hum Genet 1987; 77: 40–5.

16. Fioretti G, De Angioletti M, Masciangelo F, Lacerra G,Scarallo A, de Bonis C, Pagano L, Guarino E, De RosaL, Salvati F, Carestia C. Origin heterogeneity of Hb Le-pore-Boston gene in Italy. Am J Hum Genet 1992; 50:781–6.

17. Uro{evi} J, \ureinovi} T, Poznani} J, ^vorkov-Draì}M, Bunjeva~ki G, Jani} D, Krivokapi}-Dokmanovi} L,Popovi} Z, Pavlovi} S. Homogeneity of Hb Lepore genein FR Yugoslavia. Balkan Journal of Medical Genetics2001; 4 (1&2): 29–32.

18. Chakravarti A, Buetov KH, Antonarakis SE, Water PG,Boehm CD, Kazazian HH. Nonuniform recombinationwithin the b-globin gene cluster. Am J Hum Genet1984; 36: 212–7.

19. Kazazian HH Jr, Orkin SH, Markham AF, Chapman CR,Youssoufian H, Waber PG. Quantification of the closeassociation between DNA haplotypes and specific ?-thalassemia mutations in Mediterranean. Nature 1984;310: 152–4.

20. Tadmouri GO, Garguier N, Demont J, Perrin P, BasakAN. History and origin of beta-thalassemia in Turkey:Sequence haplotype diversity of the beta-globin gene.Hum Biol 2001; 73: 661–74.


Received: June 22, 2004



Introduction

Thermogenesis is the major function of inter-scapular brown adipose tissue (IBAT) which is foundin small mammals. Thyroid hormone (TH) is essen-tial for normal development in vertebrate species.Normal thyroid gland activity is concerned mainlywith energy metabolism in nearly all tissues of thebody. TH is a major regulator of energy homeostasiswith hyperthyroidism increasing basal metabolic rateand body temperature. The development of a hyper-thyroid state in vertebrates elevates basal metabolicrate due to increments in the rate of O2 consumptionin target tissues (1) an effect accomplished by both

(a) short-term mechanism activating mitochondrialcytochrome c oxidase and (b) long-term pathwayinvolving changes in nuclear and mitochondrial geneexpression through 3,3,5-triiodothyronine signaling.In the latter mechanism, respiratory genes may beupregulated through liganded TH receptor whichbinds to a TH-responsive element in the promoterregions (2). In IBAT, as well as in other aerobic tis-sues acceleration of aerobic metabolism by thyroxineenhances the generation of reactive oxygen species(ROS) in mitochondrial and microsomal sites (3).These conditions determine a higher consumption ofcellular antioxidants (4, 5) and inactivation of antioxi-dant enzymes (6) thus inducing oxidative stress (7)with the concomitant increase in lipid peroxides andprotein oxidation (1).

The data concerning changes in the amount oflow molecular antioxidants such as glutathione (GSH),(8, 9), as well as the activity of antioxidative enzymes indifferent hyperthyroid rat tissues were investigated byPetrovi} et al. (10), Asayama et al. (8), Mijalkovi} (11),Sai~i} et al. (12, 13).

UC 577,1; 61 ISSN 0354-3447


EFFECT OF THYROXINE ON GLUTATHIONE-DEPENDENT ANTIOXIDANTENZYME ACTIVITIES AND GLUTATHIONE CONTENT IN THE INTERSCAPULAR

BROWN ADIPOSE TISSUE OF DIFFERENT MATURATED RATS

Zorica S. Sai~i}, Dejan N. Mijalkovi}, Aleksandra L. Nikoli}, Du{ko P. Blagojevi}, Mihajlo B. Spasi}, Vojislav M. Petrovi}

Institute for Biological Research »Sini{a Stankovi}«, Department of Physiology, Belgrade, Serbia, Serbia and Montenegro

Summary: Effect of thyroxine on glutathione-dependent antioxidant enzyme activities and glutathione(GSH) content in the interscapular brown adipose tissue (IBAT) of different aged rats were studied. Male MillHill hybrid hooded rats aged 15, 45 and 75 days were treated with L-thyroxine, T4 (40 mg/100 g body mass),s.c., one dose per day, 14 days (finally aged 30, 60 and 90 days, respectively). Effect of T4 on GSH-dependentantioxidant enzyme activities in the IBAT differs with respect to age. T4 treatment gradually decrease activitiesof all GSH-dependent antioxidant enzymes in 60 and 90 days old rats in comparison to young ones. GSH con-tent in animals of 30 and 60 days old rats are lower in comparison with 90 days old rats, but the effects areoppposite. L-thyroxine treatment significantly increase GSH content in 30 days old rats (p<0.001) in respectwith coresponding controls, while decrease in 60 and 90 days old animals were detected (p<0.01). Differentresponse of non-mature rats to thyroxine comparing to older rats could be attributed to the difference in thy-roxine metabolism and developmental phase of regulatory physiological systems maturation including antiox-idative.

Key words: thyroxine, glutathione-dependent antioxidant enzymes, glutathione, interscapular brown adi-pose tissue, rats


Dr Zorica S. Sai~i}, Leading scientistInstitute for Biological Research »Sini{a Stankovi}«Department of Physiology Bulevar despota Stefana 142, 11060 Belgrade, Serbia, Serbia and MontenegroTel: (+ 381) 11 2078 325Fax: (+ 381) 11 761 433E-mail: [email protected]


GSH represents a major non-enzymatic antioxi-dant and the most abundant non-protein thiol sourcein the cell (14, 15). GSH serves as a substrate for glu-tathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) and under physiological condi-tions, glutathione reductase (GR) will rapidly reduceany oxidized glutathione (GSSG) to reduced form(GSH). Glutathione has several major functions: itdetoxifies ROS under normal and impaired home-ostasis, detoxifies drugs and maintains an essentialthiol status of proteins and other molecules and pro-vides the main molecular form in which cysteine canbe stored within the organism and used for transferbetween tissues (16, 17).

TH are known to act directly on multiple sites invertebrate cells, but it seems that mitochondria arethe most striking target. IBAT is very rich with mito-chondria and potentially important source of free rad-icals.

In the present work we examine the effect ofL-thyroxine, T4 on total glutathione content (GSH, re-duced + GSSG,oxidized) and glutathione-dependentantioxidant enzyme activities: glutathione peroxidase(GSH-Px, EC 1.11.1.9), glutathione-S-transferase(GST, EC 2.5.1.18) and glutathione reductase (GR,EC 1.6.4.2.) in IBAT of different aged rats, 30, 60 and90 days.


The experiments were carried out with the maleMill Hill hybrid hooded rats. Animals were housedfrom birth to 30th day of age near by their mothers.After 30 days they were transfered to individual cages(four animals per cage). All animals were held undercontrolled conditions of illumination (lights on: 5 a.m.-5 p.m.) and temperature (23 °C) and were allowedfree access to water and food. Animals at the 15th,45th and 75th day of age were treated with L-thyrox-ine, T4 (40 mg dissolved in 9 mmol/L NaOH/100 gbody mass), s.c., one dose per day, during the next14 days before sacrificing (finally aged 30, 60 and 90days, n=31) as performed earlier by Wooten andCascarino (18). The study was performed using dou-ble control group protocol. One control group wasconsisted of non-treated (intact) animals (n=29). Thesecond control group received 9 mmol/L NaOH/100g body mass, the same way as T4 treated animals(n=26).

All animals were sacrificed by decapitation al-ways between 8 and 10 a.m. to avoid any possiblerhytmic variations in the antioxidant enzyme level.Immidiately after the decapitation IBAT were extract-ed and washed out with saline solutions (154 mmol/LNaCl). Homogenization was performed with a Jankeand Kunkel (Staufen,Germany) Ika-Werk Ultra-Turraxhomogenizer at 0–4 °C in 0.25 mol/L sucrose, 1mmol/L EDTA and 0.05 mol/L TRIS-HCl solution, pH

7.4 (19, 20). The homogenates were sonicated for 30sat 10 kHz on ice to release enzymes (21) and used todetermine the content of total glutathione (GSH +GSSG). The remaining sonicates were centrifuged (90min, 85000 × g, 4 °C) and the supernatant was usedfor GSH-dependent antioxidant enzyme activity assaysand total protein determination. All chemicals wereSigma (St. Louis, MO, U.S.A.) products.

GSH-Px activity was assayed using t-butylhydroperoxide as substrate (22, 23) and the activitywas expressed in nanomoles of NADPH oxidized/min/mg protein. For the determination of GST activity, 1-chloro-2,4-dinitro benzene (CDNB) was used as asubstrate (24) and the activity was expressed in nmolGSH used/min/mg protein. GR activity was measuredas suggested by Glatzle et al. (25) and expressed innmol oxidized NADPH/min/mg protein. For the GSHassay sonicated samples were deproteinized by 10%sulfosalycilic acid (2:1, v/v) and centrifuged 10 minon 3020 × g. Content of total GSH (GSH,reduced +GSSG,oxidized) was determined by enzymatic met-hod suggested by Tietze (26) as modified by Griffith(27) and expressed as nmol GSH/g wet mass. AllGSH-dependent antioxidant enzyme assays were per-formed at 25 °C and expressed as specific activity(units per mg protein) and as total activity (units perg wet mass). Protein content was measured by themethod of Lowry et al. (28) using bovine serum albu-min as a reference.

Statistical analysis was performed using proto-cols suggested by Hinkle et al. (29). In experimentaldesign here applied treatment was performed on dif-ferent maturated rats, thereby the effects were statis-ticaly analyzed considering two factors: treatmentand age using two-way analysis of variance (two-wayANOVA).

Results

The GSH-dependent antioxidant enzyme activi-ties after T4 treatment were presented in Table I(activity expressed both per mg protein – as specificand per g wet mass – as total). Statistical data arepresented in Tables II and III.

GSH-Px specific activity was significantly inrea-sed (p<0.01) in T4 treated 90 days aged rats (33.3 ±3.2) in comparison with corresponding controls (22.4± 2.0). At the same time, the activity of GSH-Px in 30and 60 days aged rats did not show any differencesbetween compared groups (Table I). On the otherhand, when activity of GSH-Px expressed both asspecific and as total (Table I) decrease during deve-lopment in rats (significant age effect- (A); p<0.001;Tables II and III). This effect is evident in 30 daysaged rats in respect to old ones.

We observed the similar effect in GST activitywhich is not statistically significant, but shows the


same trend, e.g. decreasing during development (sig-nificant age effect – (A); p<0.001; Tables II and III).

At the same time, specific GR activity appearedto be lower (p<0.05) in T4 treated 30 days aged rats(91.7 ± 9.3) in comparison with the correspondingcontrols (113.5 ± 10.6). GR activity also decrease(p<0.001) during development in rats (significantage effect - (A), Tables II and III).

The effect of treatment is presented in Tables IIand III as (T) effect. Treatment is strongly age depen-dent (interaction A × T, in Tables) only in GSH con-tent (Tables II and III; Figure 1).

Our results showed, that T4 treatment had oppo-site effects on GSH content. While in 30 days old ani-mals we found a significant increase (p<0.001) in

Figure 1. Glutathione content (GSH) in the IBAT of 30,60 and 90 days old rats treated with L-thyroxine (T4),

internal controls (Ki) and controls (K) expressed in nmolGSH per g wet mass. Columns represent mean values

and vertical bars are S.E.M. ** p < 0.01 *** p < 0.001

0 30 60 90Age (days)

KKiT4

350

300

250

200

150

100

50

0

nmol

GSH

/ g

wet

mas

s

Table I The activities of glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST) and glutathione reductase (GR)in the IBAT of 30, 60 and 90 days old rats treated with L-thyroxine (T4), internal controls (Ki) and controls (K).

Enzyme activities are expressed in units per mg protein as specific and in units per g wet mass as total.The results are presented as Mean ± SD.

Specific activityGSH-PxGSTGRTotal activityGSH-PxGSTGR

Controls(K)

34.4 ± 16.453.9 ± 20.9

113.5 ± 38.3

548 ± 305966 ± 532

1749 ± 714

InternalControls (Ki)

44.0 ± 14.354.3 ± 15.398.9 ± 29.0

880 ± 625951 ± 603

1740 ± 748

Treatment(T4)

44.2 ± 14.553.5 ± 13.191.7 ± 34.8

873 ± 3131072 ± 3381632 ± 311

Controls(K)

12.8 ± 4.231.3 ± 7.750.5 ± 5.9

576 ± 2231380 ± 2552156 ± 252


22.4 ± 7.741.1 ± 11.251.8 ± 16.3

940 ± 1811586 ± 3452078 ± 720

Treatment(T4)

14.0 ± 2.937.3 ± 7.251.1 ± 9.4

424 ± 911133 ± 2571523 ± 135

Controls(K)

22.4 ± 4.436.6 ± 3.461.1 ± 8.5

807 ± 1781314 ± 322281 ± 152


23.4 ± 6.936.5 ± 7.769.4 ± 5.9

798 ± 2011304 ± 2392392 ± 236

Treatment(T4)

33.3 ± 6.543.7 ± 11.360.4 ± 8.7

1244 ± 2641681 ± 6072240 ± 260

30 days 60 days 90 days

Table III Two-way analysis of variance (ANOVA) for GSH-dependent antioxidant enzyme activities and GSH

content in rats of different age treated with T4 or corresponding controls. Results are presented as total

activity and expressed in units per g wet tissue. Df – degreeof freedom, MS – mean square. *** p < 0.001 * p < 0.05

GSH-Px

GST

GR

GSH

DfMSFDfMSFDfMSFDfMSF

Age (A)2

4039703.38*

213922457.75***

220727607.95***

244867

37.6***

Treatment (T)2

3350152.82

337310.19

2542947

2.082

106058.88***

A × T4

3758313.14*

4295041

1.644

2333550.94

4468637.4**

Error65

119516

62179740

74260631

361194

Table II Two-way analysis of variance (ANOVA) for GSH-dependent antioxidant enzyme activities in rats

of different age treated with T4 or corresponding controls.Results are presented as specific activity and expressed

in units per mg protein. Df – degree of freedom, MS – mean square. *** p < 0.001

GSH-Px

GST

GR

DfMSF

DfMSF

DfMSF

(A)2

393528.4***

22304

13.2***

220656

29.9***

(T)2

3532.54

21060.61

23290.48

A × T4

1381.00

478.90.45

45120.74

Error65139

62175

71692

*****

**


GSH content between T4 treated (300.7 ± 20) andcorresponding controls (45.7 ± 5.9) in 60 and 90 daysold rats we found decrease in GSH content (p<0.01).Detailed analysis of grand means reveiled, that T4direct effects should be viewed as clear change of T4treated animals in comparison with control and T4 dis-olving buffer treated animals. Furthermore, animals at30 days of age responded different to treatment inrespect to old animals. These results suggest endoge-nous developmental pathern of antioxidant enzymeexpression which could be modified by external fac-tors.

Discussion

From experimental studies, as well as an epi-demiological data, it can be inferred that hyperthy-roidism is associated with a general increase in tissueoxidative stress. On the other side, great controversyexists as to whether hyperthyroidism is associatedwith an increase or decrease in the activity of antioxi-dant enzymes (8, 30). It was shown, that antioxidativedefence system is endogenous dynamic systemincorporated in homeostatic regulation lead by inter-nal regulatory signals (31).

ROS have been related with many physiologicaland pathophysiological processes. Under physiologi-cal conditions, it is estimated that approximately 80%of stationary oxygen uptake depends on mitochon-drial respiratory chain activity (32). However, it’s beenshown that mitochondrial respiratory chain generatessuperoxide anion radical (O2

.–) at two differentplaces, such as in the proximity of NADH dehydroge-nase and of ubiqinon-cytochrome b. Superoxideanion radical is free radical species which is hydrogenperoxide precursor during its generation in mitochon-dria (33). In that regard, treatment with TH increaseactivity of several enzymes coupled with mitochondr-ial respiratory chain, content of cytochrome c, as wellas the size and number of mitochondria. Thereforewe may conclude, that TH influence on respiration inmitochondria by changing the concentration of somecomponents in electron-transport chain, as well asredox state of its components (34–36).

BAT is anatomically distinct from white adiposetissue and is located in a number of regions of thebody. It is particularly abundant in the interscapular,axillary and perirenal regions. The brown adipocytecontains several lipid droplets and the fat-free cyto-plasm is occupied almost exclusively with mitochon-dria packed with cristae (37). Proliferation and hyper-trophy of BAT occurs in response to increased ther-mic need (i.e. cold exposure). This growth is accom-panied by increases in total protein, amount of mito-chondria (37–39) and alteration in mitochondrialultrastructure (40). The factors controlling brown fatproliferation are not clearly defined. Norepinephrine(or an intact sympathetic nervous system) is required,

but is not a sufficient agent alone. Thyroid hormoneis required as a permissive synergistic agent (41–43)for the BAT adaptive thermic response, but only atlow concentrations (44, 45).

Therefore, we choose to examine the effects ofinduced hyperthyroidism on GSH-dependent antioxi-dant enzymes, as well as GSH content in the IBAT ofdifferent maturated rats, since this tissue, as we men-tioned before, are rich with mitochondria and there-fore might be significant source of ROS generationunder T4 stimulation.

Changes in GSH-Px and GST activity in 90 dayold rats suggest that in maturated rats enzymaticantioxidant activities in the IBAT depend on age(p<0.001, Tables II and III) more than treatmentitself. The slight rise in activity of GSH-Px and GST in90 day old rats is followed with statistically significantdecrease in total amount of GSH in T4 treated ani-mals compared with corresponding controls. Thisdistinct fall in GSH content is age and treatmentdependent (p<0.001, Table III).

There were no changes in GSH-Px and GSTactivity in 30 day old rats. On the other side, GSHcontent in 30 day old rats in T4 treated animals weresignificantly higher than corresponding controls(p<0.001, Table III). The diminished levels of tissueGSH have generally been correlated with the covalentbinding of xenobiotics to tissue macromolecules(46). The occurrence of lower concentrations of GSHin older animals can be explained by the followingpossibilities. Firstly, the actual loss of GSH may be aresult of increased rate of oxidation due to higherconsumption of oxygen an concomitant higher gen-eration of hydrogen peroxide and hydroperoxides.Secondly, the diminished GSH concentration may bedue to either increase degradation or decreased syn-thesis of GSH. In fact, activity of GSH-dependentantioxidant enzymes have been found to be higher inthe T4 treated animals in comparison to controls,which implies higher consumption of reduced GSH.In the same time, GR activity was not changed. Thir-dly, the lower concentration of GSH may be due toincreased utilization of GSH in the removal of lipidand other peroxides.

Also, we must take in consideration that duringmaturation there may be an accumulation of toxicsubstances which would elevate the activity of enzy-mes such as GSH-Px and GST, resulting in the intra-cellular depletion of reduced GSH. Higher concentra-tions of TH itself in T4 treated animals, may inducetheir own removal by GSH-dependent antioxidantenzymes, particularly with GST.

On the other side, quite opposite effects wereobserved in 30 day old T4 maturated animals. Statis-tically significant rise in GSH content (p<0.001,Table III) may be explained different in comparisonto old rats. Elevated GR activity in T4 treated 30 day


old rats is probably adaptive response to overallbiochemical and physiological processes within thecells, rather than direct effect of antioxidaive regula-tory elements. This suggests, increased turnover bet-ween GSSG and GSH and maintaining of stable re-dox environment. It has been postulated that redoxenvironment obtained by redox couples is one ofdevelopmental determinant (47). One of cellular re-dox couples is GSH/GSSG and its optimal ratio isconsidered as developmental causal.

In this investigation, we have demonstrated thatduring maturation of rats T4 treated animals exhibit adiminshed reducing potential. This observations sug-gests, that during the period of lower GSH concen-trations in hyperthyroid rats IBAT becomes suscepti-ble to oxidative damage due to higher generation ofoxidative molecules such as H2O2, hydroperoxidesetc. Thus, older animals could be at risk and morevulnerable to deleterious effects of hyperthyroid state.

It can be concluded from presented results,which under normal conditions there are a delicatebalance between the rate of formation and the break-down of ROS in the IBAT which is partially under thecontrol of TH. Alteration in the thyroid state of thebody influences the antioxidative defence in the IBATand can lead to a pathophysiological state. Differentresponse of non-mature rats to thyroxine comparingto older rats, could be attributed to the difference inthyroxine metabolism and developmental phase ofregulatory systems maturation including antioxida-tive. Direct effects of T4 on mature rats might besummarized as part of its overall catabolic role.

Acknowledgements. This work was supportedby the Ministry of Sciences, Technology and Develop-ment, Republic of Serbia, Grant No 1669.

EFEKAT TIROKSINA NA AKTIVNOST ANTIOKSIDACIONIH GLUTATION-ZAVISNIH ENZIMA I KOLIÎNU GLUTATIONA

U INTERSKAPULARNOM MRKOM MASNOM TKIVU PACOVARAZLIÎTE STAROSTI

Zorica S. Sai~i}, Dejan N. Mijalkovi}, Aleksandra L. Nikoli}, Du{ko P. Blagojevi}, Mihajlo B. Spasi}, Vojislav M. Petrovi}

Institut za biolo{ka istraìvanja »Sini{a Stankovi}«, Odeljenje za fiziologiju, Beograd

Kratak sadràj: Ispitivan je efekat tiroksina na aktivnost antioksidacionih glutation-zavisnih enzima i ko-li~inu glutationa (GSH) u interskapularnom mrkom masnom tkivu (IBAT) pacova razli~ite starosti. Mu`jaci MillHill hybrid hooded pacova starih 15, 45 i 75 dana tretirani su sa L-tiroksinom, T4 (40 mg/100 g telesne mase),s.c., jedna doza dnevno, tokom 14 dana (do finalne starosti 30, 60 i 90 dana). Efekat T4 na aktivnost GSH-za-visnih antioksidacionih enzima u IBAT-u se razlikuje u odnosu na starost.Tretman sa T4 smanjuje aktivnost svihGSH-zavisnih antioksidacionih enzima kod pacova 60 i 90 dana starosti u pore|enju sa mladim jedinkama.Koli~ina GSH kod ìvotinja starih 30 i 60 dana je nià u pore|enju sa pacovima starih 90 dana. Tretman tiroksi-nom zna~ajno pove}ava koli~inu GSH kod pacova starih 30 dana (p<0,001) u odnosu na odgovaraju}e kont-role, dok kod pacova starosti 60 i 90 dana izaziva smanjenje (p<0,01). Razli~it odgovor ne-maturiranih pacovana tiroksin u odnosu na maturirane ìvotinje moè se pripisati razlikama u metabolizmu tiroksina i razvojnoj faziregulatornih fiziolo{kih sistema uklju~uju}i i antioksidacioni.

Klju~ne re~i: tiroksin, glutation-zavisni antiksidacioni enzimi, glutation, interskapularno mrko masnotkivo, pacovi

References

1. Videla LA. Energy metabolism, thyroid calorigenesis,and oxidative stress: functional and cytotoxic conse-quences. Redox Report 2000; 5: 265–75.

2. Goglia F, Moreno M, Lanni A. Action of thyroid hor-mones at the cellular level: the mitochondrial target.FEBS Let 1999; 452: 115–20.

3. Fernandez V, Videla LA. Influence of hyperthyroidismon superoxide radical and hydrogen peroxide produc-

tion by rat liver submitochondrial particles. Free RadRes Commun 1993; 18: 329–35.

4. Huh K, Kwon TH, Kim JS, Park JM. Role of the hepa-tic xanthine oxidase in thyroid dysfunction: effect ofthyroid hormones in oxidative stress in the rat. ArchPharm Res 1998; 21: 236–40.

5. Giavarotti KAS, Rodrigues L, Rodrigues T, JunqueiraVBC, Videla LA. Liver microsomal parameters related

to oxidative stress and antioxidant systems in hyperthy-roid rats subjected to acute lindane treatment. FreeRad Res 1998; 29: 35–42.

6. Fernandez V, Llesuy S, Solari L, Kipreos K, Videla LA,Boveris A. Chemiluminescence and respiratory respon-ses related to thyroid hormone-induced liver oxidativestress. Free Rad Res Commun 1988; 5: 77–84.

7. Sies H. Biochemistry of oxidative stress. Angew ChemInt Ed Engl 1986; 25: 1058 –71.

8. Asayama K, Dobashi K, Hayashibe H, Megata Y, KatoK. Lipid peroxidation and free radical scavengers in thy-roid dysfunction in the rat; a possible mechanism ofinjury to heart and skeletal muscle in hyperthyroidism.Endocrinology 1987; 121: 2112–8.

9. Morini P, Casalino E, Sblano C, Landriscina C. The res-ponse of a rat liver lipid peroxidation, antioxidant enzy-me activities and glutathione concentration to the thy-roid hormone. Int J Biochem 1991; 23: 1025–30.

10. Petrovi} VM, Spasi} M, Sai~i} Z, Mili} B, Radoji~i} R.Increase in superoxide dismutase activity induced bythyroid hormones in the brains of neonate and adultrats. Experientia 1982; 38: 1355–6.

11. Mijalkovi} ND. Efekat tiroksina na za{titu od oksida-cionih o{te}enja u ciljnim tkivima pacova razli~ite staro-sti. Magistarska teza. Beograd. Hemijski fakultet, 2002.

12. Sai~i} ZS, Mijalkovi} D, Blagojevi} D, Nikoli} A, Spasi}MB, Petrovi} VM. Effect of thyroxine on the antioxidantenzyme activities and glutathione content in the heart ofrats. Proceedings of the Second International Conferenceof the Society for Free Radical Research-Africa, Mauritius:2001 July 15–19, Plenary lecture. 2001; 27–8.

13. Sai~i} ZS, Mijalkovi} D, Nikoli} A, Blagojevi} D, Spasi}MB. Efekat tiroksina na antioksidacioni za{titni sistem uskeletnim mi{i}ima pacova razli~ite starosti. Medicus2003; 16: 10–6.

14. Dröge W. Aging-related changes in the thiol/disulfideredox state: implications for the use of thiol antioxi-dants. Exp Gerontol 2002; 37: 1331–43.

15. Warner HR. Superoxide dismutase, aging and degener-ative disease. Free Rad Biol Med 1994; 17: 249–58.

16. Kannan R, Kuhlenkamp JF, Ookhtens M, Kaplowitz N.Transport of glutathione at blood-brain barrier of therat: inhibition by glutathione analogs and age-depend-ence. J Pharmacol Exp Ther 1992; 263: 964–70.

17. Julius M, Lang CA, Gleiberman L, Harburg E,Difranceisco W, Schork A. Glutathione and morbidity ina community-based sample of elderly. J Clin Epidemiol1994; 47: 1021–6.

18. Wooten LW, Cascarano J. The effect of thyroid hor-mone on mitochondrial biogenesis and cellular hyper-plasia. J Bioenerg Biomemb 1980; 12: 1–11.

19. Rossi MA, Audi GS, Dianzani MU. Glutathione peroxi-dase, glutathione reductase and glutathione transferasein regenerating rat liver. Med Sci Res 1987; 15: 109.

20. De Waziers I, Albrecht R. The effects of vitamin A, nutri-tional status on glutathione, glutathione transferaseand glutathione peroxidase activities in rat intestine.Experientia 1987; 43: 394–5.

21. Takada Y, Oschino N, Okabe T, Kayiyama M. Super-oxide dismutase in various tissues from rabbits bearingthe Vx-2 carcinoma in the maxillary sinus. Cancer Res1982; 42: 4233–5.

22. Paglia DE, Valentine WN. Studies on the quantitativecharacterization of erythrocyte glutathione peroxidase.J Clin Lab Med 1967; 70: 74–7.

23. Tamura M, Oschin N, Chance B. Some characteristicsof hydrogen and alkyl-hydroperoxides metabolizing sys-tems in cardiac tissue. J Biochem 1982; 92: 1019-31.

24. Habig WH, Pabst MJ, Jakoby WB. Glutathione-S-trans-ferases. J Biol Chem 1974; 249: 7130–9.

25. Glatzle D, Vuilleumier JP, Weber F, Decker K.Glutathione reductase test with whole blood a conven-ient procedure for the assessment of the riboflavin sta-tus in humans. Experientia 1974; 30: 665–8.

26. Tietze F. Enzymic method for quantitative determina-tion of nanogram amounts of total and oxidized glu-tathione: applications to mammalian blood and othertissues. Anal Biochem 1969; 27: 502–22.

27. Griffith OW. Determination of glutathione and glu-tathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 1980; 106: 207–12.

28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.Protein measurement with the Folin phenol reagent. JBiol Chem 1951; 193: 265–75.

29. Hinkle DE, Wiersma W and Jurs SG. Applied Statisticsfor Bihavioral Sciences. 5th ed, Boston: HoughtonMifflin Company, 2003.

30. Fernandez V, Barrientos X, Kipreos K, Valenzuela A,Videla LA. Superoxide radical generation, NADPH oxi-dase activity, and cytochrome P-450 content of rat livermicrosomal fractions in an experimental hyperthyroidstate: relation to lipid peroxidation. Endocrinology 1985;117: 496–501.

31. Blagojevi} D, Buzadì} B, Kora} B, Sai~i} ZS, Radoji~i}R, Spasi} MB, Petrovi} VM. Seasonal changes in theantioxidative defense in ground squirrels (Citellus cite-llus): possible role of GSH-Px. J Environ Pathol ToxicolOncol 1998; 17: 241–50.

32. Scholz R, Bucher TH. Hemoglobin-free perfusion of ratliver. In: Chance B, Easterbrook RW, Williamson JMeds. Control of energy metabolism. Academic Press:New York, 1965: 393–414.

33. Turrens JF, Boveris A. Generation of superoxide anionby the NADH dehydrogenase of bovine heart mito-chondria. Biochem J 1980; 191: 421–7.

34. Roodyn DB, Freeman KB, Tata JR. The stimulation bytreatment in vivo with triiodothyronine or amino acid


incorporation into protein by isolated rat liver mito-chondria. Biochem. J 1965; 94: 628–41.

35. Bronk JR. Thyroid hormone: effects on electron trans-port. Science 1966; 153: 638–9.

36. Nishiki K, Erecinska M, Wilson DF, Cooper S. Evalu-ation of oxidative phosphorylation in hearts from euthy-roid, hypothyroid and hyperthyroid rats. Am J Physiol1978; 235: C212–9.

37. Barnard T, Skala J. The development of brown adiposetissue. In: LINDBERG eds. Brown adipose tissue.Elsevier: New York, 1970: 33–72.

38. Desautels M, Zaror-Behrens G, Himms-Hagen J.Increased purine nucleotide binding, altered polypep-tide composition, and thermogenesis in brown adiposetissue mitochondria of cold-adapted rats. Can J Bio-chem 1978; 56: 378–83.

39. Desautels M, Himms-Hagen J. Roles of noradrenalineand protein synthesis in the cold-induced increase inpurine nucleotide binding by brown adipose tissuemitochondria. Can J Biochem 1979; 57: 968–76.

40. Desautels M, Himms-Hagen J. Parallel regression ofcold induced changes in ultrastructure, composition,and properties of brown adipose tissue mitochondriaduring recovery of rats from acclimation to cold. Can JBiochem 1980; 58: 1057–68.

41. Barnard T, Mory G, Nechad M. Biogenic amines and thetrophic response of brown adipose tissue. In: PARVEZeds. Biogenic amines in development. Elsevier/North-Holland, Amsterdam, 1980; 391–493.

42. Himms-Hagen J. Thyroid hormones and thermogene-sis. In Girardier S, eds. Mammalian thermogenesis.Chapman and Hall, London, 1983; 141–77.

43. Sundin D. GDP binding to rat brown fat mitochondria:effects of thyroxine at different ambient temperatures.Am J Physiol (Cell Physiol 10) 1981; 241: C134–9.

44. Silva JE, Larsen PR. Adrenergic activation of triiodo-thyrine production in brown adipose tissue. Nature,London 1983; 305: 712–3.

45. Triandafillou J, Gwilliam C, Himms-Hagen J. Role ofthyroid hormone in cold induced changes in rat brownadipose tissue mitochondria. Can J Biochem 1982; 60:530 –7.

46. Farooqui MYH, Ahmed AE. The effects of acrylonitrileon hemoglobin and red cell metabolism. J ToxicolEnviron Hlth 1983; 12: 695–707.

47. Schafer FQ, Buettner GR. Redox environment of thecell as viewed through the redox state of the glutathionedisulfide/glutathione couple. Free Rad Biol Med 2001;30: 1191–212.


Received: June 22, 2004


Introduction

Rheumatoid arthritis (RA) is chronic inflammato-ry disease of unknown etiology and multifactor patho-genesis, of changing clinical course and unpredictableprognosis in which base is the progressive destructivesynovitis and the impairment of the joint structure (1).Symmetric pains and swelling of small peripheral jo-ints, long-term morning stiffness, general symptoms,the occurrence of rheumatoid nodules, rheumatoidfactor (RF) in blood and characteristic radiologicalchanges in joints are the primary clinical features ofthe disease. The appearance of extraarticulary mani-festations, autoimmunoglobulin antibodies and immu-ne complexes in the circulation with activation of thecomplement system imply to systemic nature of thedisease, which justifies the term rheumatoid disease(2). The most epidemiological, pathophysiological,immunological and genetic aspects of the disease arewell known, but the precise starter and factors, whichmaintain the destructive inflammatory process, stillhaven't been determined. Because of that it is not

unusual to claim that RA is the disease with manycauses or many diseases with one cause (2, 3).

Immunopathological process in synovial tissuedevelops in five phases (4): in the first phase the pres-entation and recognition of the unknown antigen isbeing done by T lymphocyte; in the second phaseoccurs the proliferation of T and B lymphocytes, mi-gration of inflammatory cells in to the joint and angio-genesis. The cells components of the joint react to theinjury by changing the functional profile. Changesoccur in endothelium of small blood vessels and hyper-plastic reaction of synovia with proliferation of synovialcells – the third immunological phase of the disease. Inthe fourth phase of the disease the panus is formed. Itis metabolically active and autonomous joint tissue.Chondrocytes and osteoclasts are activated. Cytokinesinterleukine (IL)-1, the tumor necrosis factor-alpha(TNF-alpha), interferon-gamma (INF-gamma) andmany others, which are produced by macrophages andactivated T cells, stimulate synovial cells to createhydrolytic enzymes, proteinases. The initial damage ofthe joint occurs. In the fifth immunological phase manymediators such as prostaglandins, activated compo-nents of complement, proteases, free oxygen and nitro-gen radicals bring to the definitive destruction of jointcartilage, bones and surrounding structures (5).

The tumor necrosis factor-alpha and IL-1 havealmost identical biological activity, mutually stimulate


UC 577,1; 61 ISSN 0354-3447


INFLAMMATORY RESPONSE IN RHEUMATOID ARTHRITIS

Ljiljana Petrovi}-Rackov, Nada Pejnovi}, Zoran Miju{kovi}, Gordana Ercegovi}

Clinic of Rheumatology and Clinical ImmunologyInstitute of Medical Biochemistry

Military Medical Academy, Belgrade

Summary: The aim of the research is to determine the clinical significance of cytokines TNF-alpha, IL-12,IL-15 and IL-18 in evaluation of the activity of rheumatoid arthritis (RA). By comparing the concentrations in 30patients with high, 14 patiens with moderate and 20 patiens with mild activity of RA it is established that thepatients with high degree of disease activity, have significantly high (p<0.01; p<0.05) concentrations of examinedcytokines and rheumatoid factor in blood and synovial fluid as well as C-reactive protein in serum in relation topatients with moderate and mild active disease. We have concluded that the cytokines concentrations can begood indicators of the degree of the general activity of RA. This research can contribute to interpretation of insuf-ficiently well known views of pathogenesis role of cytokines in active disease.

Key words: cytokines, arthritis, rheumatoid factor, C-reactive protein

Address for corespondence:

Dr sc. med. Ljiljana Petrovi}-Rackov 11000 BeogradSvetozara Markovi}a 43tel: 011/3611 788mobil: 063/361 004e-mail: ljrackov¤eunet.yu

production and act synergistic in the induction of infla-mmatory reaction (6, 7). Proinflammatory effects IL-18, IL-15 and IL-12 have been examined and demon-strated in experimental conditions in vitro and in vivowith mice on model of arthritis induced by collagen(8’13). According to us available literature great num-ber of research with patients with RA has not beenconducted. It is unknown if the production ofcytokines in blood, and especially in synovial fluid (SF)is connected with the severity of RA and if their levelscan reflect the disease activity, to imply to progressionand foresee the course of the disease. These have justbeen the basic motives to conduct the research withthe aim to determine clinical significance of cytokinesfor the evaluation of activity of rheumatic arthritis. Inthis region such research has not been done.

Methods

In four year period, total of 89 patients have beenanalyzed, 64 patients with RA, newly formed or in thephase of deterioration of the disease. The diagnosis ofall patients has been set according to revised criteriafor classification and diagnosis of RA (ARA/ACR,1987). We have analyzed clinical manifestations ofpatients (P group) and have grouped the in relation ofthe disease activity in three groups: patients with high-ly active RA (HiA), 30 patients, patients with mode-rately active RA (MoA), 14 patients and patients withmildly active RA (MiA), 20 patients. The control groupwas consisted of 25 subjects with the arthritis of theknee during the deterioration of osteoarthrosis (OA),for whom we have presumed not to have derangedimmunological parameters in blood and SF. The totalevaluation of activity of rheumatic arthritis is deter-mined on the basis of total number of swollen joints(SJ), total number of tenderness joints (TJ), values onvisible analogical scale for pain (VAS) and rapidity oferytrocite sedimentation rate (ESR) by means of is cal-culated index Disease Activity Score 28 (DAS 28),numerical indicator of the degree of disease activity(14). High disease activity have represented the givenvalues DAS >5.1, moderate >3.2, and mild diseaseactivity have showed given values of DAS>2.6. In bothexamined groups the analyzes of the samples of se-rum (S) and SF has been done and the concentrationsof C-reactive protein (CRP) (mg/L) have been determi-ned by immunonephelometry method, IgM-RF(IU/mL) Latex nephelometriy (DADE Behring, Ger-many) and concentrations of cytokines (pg/mL) TNF-alpha, IL-12, IL-15 and IL-18 immunoenzymatic (ELI-SA) methods by using kits for humane interleukines(R&D, USA). The value of ESR (mm/h) was determi-ned by standard laboratory procedure according toWestergreen.

For evaluation of statistical significance of diffe-rence between characteristics of observation in controlgroup and group of patients with RA Mann-Whitney Utest was administered. For comparing between groups

of patients divided according to the degree of activityboth Kruskal-Waliss test and Mann-Whitney U testswere used. The level of significance in all administeredmethods was on in the limit of 0.05.

Results

Comparing the number of subjects in controlgroup and group of patients with RA in relation to sex,showed that there is highly significant difference,caused by larger number of female patients in thegroup of patients with RA (p<0.01) (Table I).

In groups with highly and moderately active RAexamined patients have had significant increase ofserum values RF in relation to the group of patientswith mild case of RA (p<0.01) (Table II).

Activity of RA in the group of patients was deter-mined and numerically presented on the basis of val-ues of DAS 28 index. Clinical manifestations havebeen compared between the two groups of exami-nees, and between groups of HiA, MoA and MiA, bycomparing clinical parameters, individual factors ofDAS: average values of number of tenderness joints,swollen joints and average values ESR and VAS.

Values of all examined clinical parameters (SJ,TJ, VAS, DAS), have high significantly differentiatedbetween the groups of patients with different activity ofRA, with the highest values in HiA group, smaller inMoA and the least in the group of patients with mildRA activity (Table III). The examined subjects in con-trol group have had significantly lower (p<0.01) aver-age values clinical parameters in relation to patientswith RA (results are not show).


Characteristics

Number patients (% of total number examiness)Age (yr.) Female Male Mean duration of disease (m.)Mean duration of presently hardships (m.)

Control25 (28)

59.21510

45.25.4

Patients64 (72)

57.84519

74.46.0

Group

Table I General characteristics of the study population

Group

Number of »seropositive« pat.

Number of »seronegative« pat.

Total number patients

%

89

11

100

N

30

0

30

%

100

0

100

N

14

0

14

%

100

0

100

N

13

7

20

%

65

35

100

N

57

7

64

P HiA MoA MiA

Table II The division of the group of patients in relation to disease activity and seropositivity

Comparing mean values of CRP, RF and ESRbetween control group and group of patients with RA,indicated significant differences in the concentrationsCRP, RF and values ESR (p<0.01). RF and CRP val-ues were higher in the samples of synovial fluid andsamples of RA patients' serums as well as ESR values(Table IV).

Comparing mean values of TNF-alpha, IL-18, IL-15, IL-12 has showed that the values of thesecytokines are significantly higher (p< 0.01) in serumsamples and synovial fluid of the patients with RA inrelation to samples of the control group (Table V).

Average serum values of CRP were the highest inHiA group, significantly lower (p<0.01) in MoA groupand the lowest (p<0.01) in MiA group. Average valuesof CRP in synovial fluid were the highest in MoA group,then in MiA group while the lowest were in the groupwith the highest RA activity, but without any significantdifferences (Figure 1). Mean values of RF in serumsamples and in the synovial fluid samples were highestin HiA group, significantly lower in MoA group (p<0.01) and again significantly the lowest in MiA group( p<0.01) (Figure 2).

Comparing the mean values of cytokines of thepatients with RA according to disease activity indicatesthat with all observed parameters exist highly statisti-cally significant differences (p<0.01), which appearbecause mean concentrations of cytokines werealways higher in HiA group in relation to MoA and MiAgroups. Only for the IL-12 concentrations in syno-


GroupParameters

TJSJ

ESRVASDAS

* p < 0.05; ** p < 0.01

HiA⎯x ± SD

9.8 ± 6.18.4 ± 5.7

78.0 ± 24.970.3 ± 13.810.1 ± 5.2

MoA⎯x ± SD

4.2 ± 0.92.8 ± 0.7

47.4 ± 10.731.4 ± 4.6

4.7 ± 0.5

MiA⎯x ± SD

2.4 ± 0.51.8 ± 0.6

27.2 ± 11.312.0 ± 5.7

2.9 ± 0.1

p0.00**0.00**0.00**0.00**0.00**

Table III Clinical parameters values in groups of patients with RA in relation to disease activity

Group

Patients

Control

zp

MeanSD

MeanSD

* p < 0.05; ** p < 0.01

CRP (mg/L)

S43.9538.726.677.155.790.00**

CRP (mg/L)

SF25.4121.264.774.465.740.00**

RF (IU/mL)

S489.96546.71

14.0810.026.220.00**

RF (IU/mL)

SF316.14327.82

12.366.985.950.00**

SE (mm/h)

55.529.314.8

9.66.440.00**

Table IV Comparing of mean values CRP and RF in serum and synovial fluid and ESR between control group and group of patients with RA

Group

Patients

Control

zp

MeanSD

MeanSD

* p < 0.05; ** p < 0.01

IL-18 (pg/mL) IL-15 (pg/mL) IL-12 (pg/mL)

Table V Cytokines concentrations (TNF-alpha, IL-18, IL-15, IL-12) in the serum samples and synovial fluid of the control group and group of patients with RA

S156.6990.3989.6817.234.40

0.00**

SF281.11317.23112.1884.833.24

0.00**

S1.773.030.590.203.88

0.00**

SF10.399.783.341.175.13

0.00**

S11.808.094.922.144.77

0.00**

SF10.527.586.082.113.73

0.00**

TNFa (pg/mL)S

5.593.532.320.804.66

0.00**

SF13.9619.363.451.225.18

0.00**

Figure 2. Concentrations of RF in serum and synovial fluid samples of the patients

according to degree of RA activity

S SF

887.43900800700600500400300200100

0

263.93

51.99

504.37

277.28

HiAMoAMiA

60.99

Figure 1. Concentrations of CRP in serum and synovial fluid samples of the patients

according to degree of RA activity

S SF

69.1670

60

50

40

30

20

10

0

30.44

15.5923.58

HiAMoAMiA

CR

P, m

g/L

RF

, IU

/mL

26.3328.01

vial fluid the difference was statistically significant(p<0.05) (Figure 3–5).

Intergroup comparisons between three groupsaccording disease activity within the group of patientswith RA, has showed the existence of statistically sig-nificant difference in all cases and concentration of

cytokines in serum and synovial fluid have always beenthe highest in HiA and the smallest in MiA. Only situa-tions when the intergroup differences haven't been sig-nificant (p>0.05) are with TNF-alpha in serumbetween HiA and MoA, IL-12 in serum and in synovialfluid where the difference haven't been significantbetween HiA and MoA.

Discussion

In pathogenesis of RA TH1 immune responseprevails. TH1 cells create special profile of cytokines,which together with products of synovicites disturbsnatural balance in cytokines net inside the synovial tis-sue. It leads at last to immunoinflmmatory reactionand joint damage. Many up today researches showedthat IL-12 has major role in the differentiation of TH1cells response, by stimulating differentiation of T cell'sprecursor (TH0) in TH1 phenotype. In the arisen TH1reaction IL-12 increases the proliferation of T cells andproduction of IFN-gamma and other T cells cytokinesin synergism with Il-18. Two cytokines create synergis-tic effect by reciprocal increase expression of T cellsreceptor and by different mechanism of IFN-gammagenetic expression (on transcription level). They areproduced by synovial macrophages. Lately in synovialtissue of patients with RA has been discovered the IL-15, mediator with pleitropic effects on many immunesystem cells. In RA IL-15 activates T cells and stimu-lates their intercells contact with macrophages. That'show it indirectly induces large synthesis of TNF-alphaand other cytokines of the macrophages. With thehelp of IL-15 activated synoivial T cells secrete TNF-alpha directly. Interleukine-15 stimulates the prolifera-tion of T cells, induces the expression of adhesive mol-ecules and has significant effect of chemotaxis. It stim-ulates the proliferation of B cells with the consequen-tial production of immunoglobulin including RF. Itintensifies the cytotoxicity of natural killer cells, acti-vates neutrophiles and stimulates the differentiation ofosteoclasts and angiogenesis. Interleukine-15 is pro-duced mainly by T cells and macrophages.

The discovery of new proinflammatory cytokineIL-18 is added to the list of mediators which stimulateactivity and development of TH1 cell response withability to induce the production of IFN-gamma in Tcells and NK cells. Synergistic effect with IL-12 and IL-15 in the induction of IFN-gamma defines it as themember of the cytokine family, which induce TH1(IFN-gamma, IL-2, IL-12 and IL-15). Interleukine-18 isthe member of IL-1 family, because it shows the struc-tural and functional similarity with this cytokine. It alsoowns pleiotropic effects and the role in innate andacquired immunity. Interleukine-18 is synthesized bymacrophages, dendritical cells, chondrocytes, osteo-clasts, keratinocytes, Kupffers cells, adrenal gland cor-tex cells and pituitary gland cells. The presence of IL-18 is proved in many diseases in human population,but his role in stimulating or repression the disease is


Figure 3. Concentrations of TNF alpha, IL-15 and IL-12in serum samples of patients according

to the degree of RA activity

TNFa IL-15 IL-12

7.46

1614121086420

5.75

2.56 2.97

16.00HiAMoAMiA

8.84

Figure 4. Concetrations of TNF-alpha, IL-15 and IL-12 insynovial fluid samples of patients according to the degree

of RA activity

TNFa IL-15 IL-12

23.0125

20

15

10

5

0

7.74

3.71

15.84

7.08

3.81

13.01

HiAMoAMiA

Figure 5. Concentrations of IL-18 in serum and synovialfluid samples of patients according to the degree

of RA activity

S SFIL-18 IL-18

500

400

300

200

100

0

209.67

465.40

146.15

82.4794.63

HiAMoAMiA

Cyt

okin

es, p

g/m

LC

ytok

ines

, pg/

mL

IL-1

8, p

g/m

L

0.650.78

7.86

8.717.58

115.70

different. In RA it contributes to the development ofTH1 response and induces the IFN-gamma and TNF-alpha synthesis in T and NK cells with consequentialstimulation of production and secretion of proinflam-matory cytokines of monocytes. The most importantrole of IL-18, pleiotropic proinflammatory cytokine insynovitis is most probably to facilitate and enable thecreation of IFN-gamma dominant T cell response,which is induced by IL-12. There will be necessary fur-ther research to establish how much is the real contri-bution of IL-18 in process of immunopatogenesis ofRA. The dilemma about the positive and negativeeffects of IL-18 in inflammation and synovitis and ifthere is a need to block or not to his effects becauseof the potentially positive effects still remains. Theseand other unknown facts invite us for further research-es concerning IL-18.

In our research first was done the analyze of clin-ical manifestations of diseases inside the group of thepatients with active RA, by comparing several clinicalparameters which form DAS (average values of thenumber of tenderness joints and number of swollenjoints and average values of ESR and VAS). Patients inthe group with highly active disease had significantlyhigher number of tenderness and swollen joints inrelation to group with moderate and mild activity ofRA. The mean values of ESR and VAS of patients withthe most severe case of the disease were significantlyhigher in relation to mean values in groups with lowerdegree of RA activity. Average values of DAS index ofRA patients were 6.7. The highest DAS value was inHiA group (10.1) while the patients in MoA group (4.7)and MiA (2.9) had significantly lower DAS values.Patients with RA were divided according to the degreedisease activity measured by values of DAS index,were significantly different in relation to the severity ofclinical manifestations of the disease.

Between the patients with RA and OA as a con-trol group and between the groups of patients dividedaccording to degree of disease activity inside thegroup of patients with RA we have compared concen-trations of TNF-alpha, IL-18, IL-15 and IL-12 in thesamples of S and SF. The average values of all 4cytokines were high significantly different between thecontrol group and group of patients with RA. Patientswith erosive arthritis have higher average concentra-tions of examined cytokines in blood as well in fluid ofjoint in relation to examinees with OA. This indicatesthat pathophysiological mechanism and the degree ofinflammatory reaction is different in RA and OA. Wehave confirmed that TNF-alpha, IL-18, IL-15 and IL-12 have significant role in pathogenesis of rheumatoidsynovitis.

Comparing the results of patients with differentdegree of RA activity significant difference was achie-ved. Average concentrations of TNF-alpha, IL-18, IL-15 and IL-12 in S and SF were highly and statisticallydifferent between the groups of patients with high,

moderate and mild active RA. Patients with high activeRA had higher average concentrations of all examinedcytokines in serum and synovial fluid in relation topatients with moderate and mildly active RA. When wewant to look at the intergroup comparisons betweengroups of HiA and MiA patients, high and statisticallysignificant differences were established in cytokinesconcentrations, except for the average values of IL-12in S (p=0.62) and SF (p=0.43). Concentrations ofTNF-alpha of HiA and MoA patients' serum have alsobeen approximately equal (p=0.11), while for otherexamined parameters highly significant differencescame up. Comparing mean values of cytokines con-centrations between HiA and MiA groups, highly andstatistically significant differences came up. Highercytokines concentrations in more severe disease casesand their lower values in the disease of the lesser activ-ity indicate that levels of TNF-alpha, IL-18, IL-15 andIL-12 in S and in SF influence clinical manifestationsand severity of RA and can be good indicators of gen-eral activity of RA as well as parameters to evaluationof the disease severity. We can expect high values ofcytokines in S as well in SF in the patients with activeRA. We have concluded that the concentrations ofcytokines can predict with great accuracy the severityof RA. Concentrations of TNF-alpha, IL-18 and IL-15in SF reflect the degree of general activity of the dis-ease and severity better than their serum values.

In active disease in laboratory analyzes acutephase parameters of inflammation CRP, fibrinogenand speeded ESR were usually increased. For nowthere isn't specific biohumoral indicator of diseaseactivity, except of RF. On the basis of comparison ofconcentrations of CRP and RF in serum and synovialfluid, we have tried to estimate if there is general andlocal activity of the disease. Patients in HiA group hadhigher average concentrations of CRP and RF in S andRF in SF in relation to MoA and MiA patients. It impliesthat RF concentrations in blood and ST and CRP con-centrations in S were good indicators of the generalactivity degree and severity of the disease. Local con-centrations of CRP in the knee joint haven't showedany difference among patients groups and it isn't thesystem inflammatory reaction indicator.

Our research has indicated to the importance ofdetermining several cytokines concentrations in bloodand synovial fluid in patients with active RA and cancontribute to explanation of insufficiently known viewsof role in pathogenesis and significance of TNF-alpha,IL-12, IL-15 and especially IL-18 cytokines in the acti-ve disease. This research confirmed that examined cy-tokines could be indicators of the degree of RA activi-ty of the same value and specific quality as up to nowused standard biochemical parameters. Concen-trations of cytokines can be useful for the early assess-ment of disease activity. The research can serve as thebasis for further studies and evaluation of the adminis-tered therapy's effect.


References

1. Firestein SG. Etiology and pathogenesis of rheumatoidarthritis. In: Kelley WN, Ruddy S, Harris ED and SledgeCB, editors. Textbook of Rheumatology. Philadelphia:W.B. Saunders Co 2001; 921’66.

2. van Riel LCMP, Wijnands JHM, van de Putte BAL.Rheumatoid arthritis. Evalulation and management ofactive inflammatory disease. In: Klippel HJ, Dieppe AP,editors. Rheumatology, 2nd ed. London: Mosby 1998;14.1’14.12

3. Budd CR, Fortner AK. T lymphocytes. In: Kelley WN,Ruddy S, Harris ED and Sledge CB, editors. Textbook ofRheumatology. Philadelphia: W.B. Saunders Co 2001;113’29.

4. Gaston HJ. Cellular immunity in RA. In: Klippel HJ,Dieppe AP, editors. Rheumatology, 2nd ed. London:Mosby 1998; 10.1’10.6

5. Szekanecz Z, Koch AE. Cytokines. In: Kelley WN, RuddyS, Harris ED and Sledge CB, editors. Textbook ofRheumatology. Philadelphia: W.B. Saunders Co 2001;275’90.

6. Dinarello AC, Moldawer LL. Proinflammatory and anti-inflammatory cytokines in rheumatoid arthritis. In:Dinarello AC, Moldawer LL., editors. A Primer forClinicians. Florida:College of Medicine 1999; 1’88.

7. Kollias G, Douni E, Kassiotis D. The function of tumournecrosis factor and receptors in models of multi-organinflammation, rheumatoid arthritis, multiple sclerosisand inflammatory bowel disease. Ann Rheum Dis 1999;58: 132’9.

8. Cordero OJ, Selgado FJ, Mera-Valera A, Nogueira M.Serum interleukin-12, interleukin-15, soluble CD26, andadenosine deaminase in patients with rheumatoid arthri-tis. Rheumatol Int 2001; 21 (2): 69 ’74

9. Plater-Zyberk C, Joosten AL, Helsen MM, Sattonnet-Roche P, Siegrfried C, Alouani S et al. Therapeutic effectof neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis. J Clin Invest 2001; 108:1825’32.

10. Joosten LA, Helsen MM, van den Berg WB. Blockade ofendogenous interleukin 12 results in suppression ofmurine streptococcal cell wall arthritis by enhancementof interleukin 10 and interleukin 1Ra. Ann Rheum Dis2000; 59: 196’205.

11. Ortiz AM, Laffon A, Gonzalez AI. CD69 expression onlymphocytes and interleukin-15 levels in synovial fluidsfrom different inflammatory arthropathies. RheumatolInt 2002; 21: 182’8.

12. Wei XQ, Leung PB, Arthur MLH, McInnes BI, Liew YF.Reduced incidence and severity of collagen-inducedarthritis in mice lacking IL-18. J Immunol 2001; 166:517’21.

13 Liew YF, McInnes. Role of interleukin 15 and interleukin18 in inflammatory response. Ann Rheum Dis 2002; 61:ii100’ii102.

14. Prevoo LM, Hof AM, Kuper HH, Leeuwen AM, Putte BL,Riel LP. Modified disease activity scores that includetwenty-eight-joint counts. Arthritis Rheum 1995; 38:44’8.


INFLAMATORNI ODGOVOR U REUMATOIDNOM ARTRITISU

Ljiljana Petrovi}-Rackov, Nada Pejnovi}, Zoran Miju{kovi}, Gordana Ercegovi}

Klinika za reumatologiju i klini~ku imunologijuInstitut za medicinsku biohemiju

Vojnomedicinska akademija, Beograd

Kratak sadràj: Cilj istraìvanja je da se utvrdi klini~ki zna~aj citokina faktora nekroze tumora-alfa (TNF-alfa), interleukina (IL)-12, IL-15 i IL-18 u proceni aktivnosti reumatoidnog artritisa (RA). Pore|enjem koncentraci-ja kod 30 bolesnika sa visoko, 14 sa umereno i 20 bolesnika sa blago aktivnim RA utv|eno je da bolesnici savisokim stepenom aktivnosti oboljenja imaju zna~ajno ve}e (p<0,01; p<0,05) koncentracije ispitivanih citokina ireumatoidnog faktora u krvi i sinovijskoj te~nosti kao i C-reaktivnog proteina u serumu u odnosu na bolesnike saumereno i blago aktivnim oboljenjem. Zaklju~ili smo da koncentracije citokina uti~u na teìnu RA i mogu biti dobripokazatelji stepena op{te aktivnosti RA. Rad moè pruìti doprinos razja{njenju nedovoljno poznatih stavova opatogenetskoj ulozi i zna~aju odre|ivanja IL-18, IL-15, IL-12 i TNF-alfa u aktivnoj bolesti.

Klju~ne re~i: citokini, artritis, reumatoidni faktor, C-reaktivni protein

Received: February 25, 2004

Accepted: April 7, 2004


Introduction

A large group of workers exposed to mercuryvapours may be found in the chlor alkali industry,where chlorine and caustic soda are produced usingthe electrolysis of brine in mercury cells. A typical mer-cury cell, 10’20 m2, may contain up to 3 tons of mer-cury, and there are often about 100 mercury cells at aplant. Although the process is closed, leakage of mer-cury occurs as a result of technical faults, and alsoduring repairs and maintenance. The workers of theseplants are exposed to mercury mainly in the form ofvapour but to some degree also as the dust of mercu-ry salts (1).

It is relatively easy to diagnose mercurialism withan acute or subacute course by such symptoms asmetallic taste in the mouth, excessive salivation, gin-givitis, stomatitis, diarrhoea and nephritis. CNS symp-toms include nervousness, timidity and uncontrolledfierceness. It is much more difficult to detect the initialsymptoms of intoxication showing gradual, yet deep-ening changes in various metabolic processes. At thebeginning, they consist of the elimination of themicroelements necessary for life (based on competi-tive interactions) such as Fe, Ca and Zn (2). A recentreview of world literature collected by Moszczynsky (3)irrefutable shows the strong, negative influence ofmercury on the immune system.

The aim of this investigation was to determinate(on easily accessible material such as the blood ofworkers exposed to mercury vapours) changes con-cerning the basic haematological measurements.

UC 577,1; 61 ISSN 0354-3447


THE BASIC HAEMATOLOGICAL MEASUREMENTS IN PERIPHERAL BLOODFROM WORKERS EXPOSED TO MERCURY VAPOURS

Radmila Maksimovi}1, Ljuba Mandi}2, Slavica Spasi}3

1Medical Centre, Kru{evac2Faculty of Chemistry, University of Belgrade, Belgrade3Faculty of Pharmacy, University of Belgrade, Belgrade

Summary: In the present study was assessed the influence of occupational exposure to mercury vapourson the basic haematological parameters (erythrocyte, leukocyte and platelet count, haemoglobin concentration,haematocrit, MCV, MCH and MCHC). Studies were carried out on 138 workers involved in the production of chlo-rine using the mercuric electrolysis method (divided into three groups: permanently, periodically and earlierexposed to mercury vapours), as well as on 38 healthy workers. The shift time - weighted averages for mercurywas determined in the workplace air before research; mean value was significantly over maximum tolerated dose.The mercury content in the blood and urine of exposed workers was determined by atomic absorption spec-trophotometry. In all three groups 95th percentile values of mercury in blood and urine are significantly over MTD.Peripheral blood cell parameters were determined using an automatic cell counter. In the group exposed to mer-cury vapours, was found a statistically significant increase of erythrocyte count with a concomitant decrease inMCV. The mean values of haemoglobin concentration, MCHC and platelet count were higher in the group ofworkers exposed to mercury vapours, but the difference was not statistically significant. There were no significantdifferences in haematocrit, MCH and leukocytes between the studied groups. Our results indicate that long-termand permanent exposure to mercury vapours induces changes in the important haematological parameters of theperipheral blood.

Key words: mercury, haematological parameters


Radmila Maksimovi}Rasadnik I H4/29, 37000 Kru{evace-mail: [email protected]


Subjects and Methods

Subjects

The study has been performed on 138 workersinvolved in the production of chlorine using the mer-curic electrolysis method, which were divided intothree groups:

Group I, GI ’ workers (N = 33, average age 36.4± 7.21 yrs) permanently exposed to mercury vapoursfor 8.4 ± 4.7 yrs;

Group II, GII ’ workers (N = 87, average age33.9 ± 7.60) periodically exposed to mercury vapoursfor 7.7 ± 5.3 yrs;

Group III, GIII ’ workers (N = 18, average age42.3 ± 5.8 yrs) earlier (minimum 5 years ago) exposedto mercury vapours for 10.3 ± 4.9 yrs.

Control group, CG ’ (N = 38, average age 31.8± 9.4 yrs) consisted of healthy workers, not exposedto mercury vapours.

Methods

The metallic mercury concentrations in work-place air were determined using Analyser of mercuryvapour »Jerome instrument corporation S« . The mer-cury content in the blood and urine of workers wasdetermined by atomic absorption spectrophotometer»Perkin-Elmer«. Blood samples were obtained fromfasting subjects together with blood samples for deter-mination complete blood counts. The eight haemato-logical parameters were determined using haemato-logical analyser »Coulter counter«.

The results were subjected to statistical analysisby analysis of variance and Student-t test.

Results

Mercury in blood and urine

The air for the determination of the mercury con-centrations was taken in different areas of the work-place on morning and during work time. The shift time’ weighted averages determined for mercury in the

workplace air before research were 0.045 ± 0.072mg/m3 (0.006 to 0.293 mg/m3); mean value was sig-nificantly over maximum tolerated dose, MTD (0.01mg/m3).

Maximum tolerated dose for mercury in blood is0.175 mmol/L and in urine 0.1 mmol/L. Workers ingroup permanently exposed to mercury vapours havemean value of mercury in blood close to MTD, butmean in urine are significantly over MTD (Table I).Mean values in blood in two other groups are bellowMTD, mean value in urine in group periodicallyexposed is slightly higher than MTD and in group ear-lier exposed is bellow MTD. In all three groups 95th

percentile values of mercury in blood and urine aresignificantly over MTD.

Mercury concentration in blood and urine ofworkers significantly correlated with mercury concen-tration in workplace air: blood: r = 0.415, y = 0.0094+ 0.382x; urine: r = 0.474, y = 0.069 + 0.354x.

Haematological parameters

There were no significant differences in haema-tocrit, MCH and leukocytes between the studiedgroups (Table II).

Erythrocytes count was significantly higher in thegroup permanently exposed to mercury vapours, 4.74× 1012/L vs. 4.47 × 1012/L in the control group (t =2.09, p = 0.04). The other two groups of workers werenot statistically different from the control group(Figure 1).

There was substantial drop in the mean corpus-cular volume (MCV) value from 102.0 fL in the controlgroup to 97.4 fL in the group permanently exposed tomercury vapours (t = 2.044, p = 0.044). The valuesin the group of workers periodically exposed to mer-cury vapours were slightly higher than the values in theother groups with extremely high 95th percentile value(Figure 2).

The mean values of haemoglobin concentration,MCHC and platelet count were higher in the group ofworkers permanently exposed to mercury vapours, butthe difference was not statistically significant.

Table I Mercury concentrations in blood and urine of control group and workers exposed to mercury vapours

Group

CGGIGIIGIII

mean±SD0.009 ± 0.00800.172 ± 0.1230.102 ± 0.0870.087 ± 0.093

5th’95th P0’0.022

0.055’0.4570’0.2370’0.202

mean± SD0.015 ± 0.0330.379 ± 0.3880.103 ± 0.0980.045 ± 0.053

5th’95th P0’0.078

0.069 ’1.0170.006’0.2800.006 ’0.132

Blood (mmol/L) Urine (mmol/L)


Discussion

Elemental mercury has been detected in theurine of workers exposed to mercury vapour. Theoccurrence of trace amounts of elemental mercury isconsidered to be due to reduction of mercuric ion bycertain types microorganisms present in the urine anddirect urinary excretion following glomerular filtrationof elemental mercury persisting in the blood. Butdeterminations of the inorganic species of mercury inurine samples can provide information on both recentexposure and the body borden resulting from chronic

exposure to mercury vapour (4). Our results supportthis assertion. Almost all workers have had increasevalues of mercury in urine.

Data collected for all studied groups, i.e. groupsexposed to mercury vapours and control subjects aregenerally contained within normal range. However,when we compared group of workers permanentlyexposed to mercury vapours with control subjects, themean value of erythrocyte count was significantlyhigher and mean value of MCV was significantly lowerin the group permanently exposed to mercury. The

Table II The values of haematological parameters in the studied groups

Group

CGGIGIIGIII

Group

CGGIGIIGIII

Group

CGGIGIIGIII

Group

CGGIGIIGIII

mean ± SD141.6 ± 15.86148.0 ± 12.05144.7 ± 11.98145.0 ± 15.79

mean ± SD0.45 ± 0.0450.46 ± 0.0290.46 ± 0.0290.46 ± 0.042

mean ± SD31.8 ± 1.4931.3 ± 2.4532.1 ± 3.7431.2 ± 2.90

mean ± SD7.50 ± 1.967.58 ± 1.577.52 ± 1.987.60 ± 2.00

5th ’ 95th P111.9 ’ 160.1124.0 ’ 160.0125.7 ’ 160.0125.6 ’ 160.0

5th ’ 95th P0.39 ’ 0.500.40 ’ 0.490.41 ’ 0.500.39 ’ 0.50

5th’95th P28.4 ’ 35.428.0 ’ 36.027.6 ’ 37.827.7 ’ 35.4

5th ’ 95th P4.8 ’ 11.14.8 ’ 9.85.0 ’ 10.35.0 ’ 10.9

mean ± SD4.47 ± 0.614.74 ± 0.464.54 ± 0.494.68 ± 0.60

mean± SD102.0 ± 9.9197.4 ± 9.27

103.0 ± 11.99100.2 ± 9.76

mean ± SD313.2 ± 22.07323.0 ± 22.38311.9 ± 23.14311.6 ± 18.33

mean ± SD219.4 ± 33.0240.3 ± 43.5236.6 ± 42.4234.3 ± 39.9

5th ’ 95th P3.58 ’ 5.403.97 ’ 5.503.84 ’ 5.373.63 ’ 5.41

5th ’ 95th P88.9 ’ 116.785.7 ’ 112.389.3 ’ 122.287.2 ’ 114.4

5th ’ 95th P275.5 ’ 350.0293.1 ’ 351.2278.9 ’ 354.5281.4 ’ 340.4

5th ’ 95th P169.7 ’ 281.0171.8 ’ 300.0166.3 ’ 296.8178.5 ’ 290.0

Haemoglobin (g/L)

Haematocrit (L/L) MCV (fL)

MCH (pg)

Leukocytes (×109)

MCHC (g/L)

Platelet (×109)

Erythrocytes count (×1012)

Figure 1. Mean value and 5th and 95th percentile valuesof erythrocyte count in control group and in groups of workers exposed to mercury vapours; * p < 0.05

*

CG GI GII GIII

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

Eryt

hroc

yte

coun

t, ×

1012

/L

Figure 2. Mean value and 5th and 95th percentile values of MCV in control group and in groups of workers

exposed to mercury vapours; * p < 0.05

*

CG GI GII GIII

140

130

120

110

100

90

80

70

MC

V, fL


quantitative health status results obtained after treat-ment with toxic compounds or heavy metals wherestudied parameters fall below the physiological valuesare relatively easy to explain. It is not easy to commentresults obtained in the present study. One possibleexplanation is that concerning differences in the levelof physical exercise, which is higher in the group per-manently exposed to mercury than the control groupconsisting of not physically hardworking people. It isknown that physical exercise (6’8 h) increases loss ofwater from the body, which entails a »thickening« ofthe peripheral blood (5, 6). The blood »thickening« isaccompanied by increased water reapsorption in therenal tubules and increased oxygen consumption bythe renal tissue, resulting in local hypoxia whichincreases the erythropoietin synthesis. That effect isrevealed by the increased number of erythrocytes (7).

The decrease of MCV in the group permanentlyexposed to mercury vapour may result from the dimin-ished iron incorporation into erythrocytic cell lineage,as indicated by increased values of serum ferritin,transferrin and TIBC (8, 9). However, in further studieson cases of mercury intoxication, the level of Cu aswell as cerulloplasmin activity in the serum should betaken into account. Copper deficiency is frequently thecause of decrease in MCV and MCHC (10).

Our results indicate that long-term and perma-nent exposure to mercury vapours induces changes inthe important haematological parameters of the peri-pheral blood, but explanation that phenomenonrequires further, broader research.

VREDNOSTI OSNOVNIH HEMATOLO[KIH PARAMETARA U PERIFERNOJ KRVI RADNIKA IZLO@ENIH UTICAJU PARA @IVE

Radmila Maksimovi}1, Ljuba Mandi}2, Slavica Spasi}3

1Medicinski centar, Kru{evac2Hemijski fakultet, Univerzitet u Beogradu, Beograd

3Farmaceutski fakultet, Univerzitet u Beogradu, Beograd

Kratak sadràj: U radu je ispitivan uticaj izloènosti parama ìve iz radne sredine na osnovne hematolo{keparametre (broj eritrocita, leukocita i trombocita, koncentraciju hemoglobina, hematokrit, MCV, MCH i MCHC).U studiju je uklju~eno 138 radnika koji rade u proizvodnji hlora procesom elektrolize (podeljeni su u tri grupe: stal-no izloèni, povremeno izloèni i ranije izloèni uticajima para ̀ ive), kao 38 zdravih radnika. Pre studije su odre|enekoncentracije ìve u vazduhu radne sredine; srednje vrednosti su bile znatno iznad maksimalne doze tolerancije(MTD). Koncentracije ìve u krvi i urinu radnika koji su izloèni ìvi odre|ena je atomskom apsorpcionom spek-trometrijom. U sve tri grupe je vrednost 95-tog percentila bila znatno iznad MTD. Hematolo{ki parametri suodre|eni uz pomo} automatskog broja~a. U grupi koja je stalno izloèna parama ìve, dobijeno je zna~ajno po-ve}anje srednje vrednosti broja eritrocita, kao i zna~ajno smanjenje vrednosti MCV. Srednje vrednosti koncen-tracije hemoglobina, MCHC i broja trombocita u grupi radnika koji su stalno izloèni uticaju ìve bile su vi}e negou kontrolnoj grupi, ali ta razlika nije statisti~ki zna~ajna. Izme|u posmatranih grupa nije bilo zna~ajne razlike uvrednostima hematokrita, MCH i broja leukocita. Dobijeni rezultati pokazuju da dugotrajna i stalna izloènost para-ma ìve moè da dovede do promena u vrednostima va`nih hematolo{kih parametara.

Klju~ne re~i: ìva, hematolo{ki parametri


References

1. Menke R, Wallis G. Detection of mercury in air in thepresence of chlorine and water vapour. Am Ind Hyg AssJ 1980; 41: 120’4.

2. Goyer RA. Toxic and essential metal interaction. AnnRev Nat 1997; 17: 37’50.

3. Moszczynski P. Immunological disorders in menexposed to metallic mercury vapour. A review. CenterEur J Public Health 1998; 7: 10’4.

4. Yoshida M. Relation of mercury exposure to elementalmercury levels in the urine and blood. Scand J WorkEnviron Health 1985; 11: 33’7.

5. Szygula Z. Erythrocytic system under the influence ofphysical exercise and training. Sport Med 1990; 10:181’97.

6. Neuhaus D, Gaehtgens P. Haemorheology and long-term exercise. Spot Med 1994; 18: 10’21.

7. Schmidt W, Massen N, Tegtbur U, Braumann KM.Changes in plasma volume and red cell formation aftermarathon competition. Eur J Appl Physiol 1989; 57:490’8.

8. Zolla L, Lupidi G, Bellelli A, Amiconi G. Effect of mer-curic ions on human erythrocytes. Biochim BiophysActa Biomembr 1997; 1328: 273’80.

9. Strom S, Johnson RI, Uyeki EM. Mercury toxicity tohemopoietic and tumor colony-forming cells and itsreversal by selenium in vitro. Toxicol Appl Pharmacol1979; 49: 431’6.

10. Marek K, Zajac-Nedza M, Rola E, Wocka-Marek T,Langauer-Lewowicka H, Witecki K. Examination ofhealth effects after exposure to metallic mercury vapoursin workers engaged in production of chlorine and aceticaldehyde. I. Evaluation of general health status. Med Pr1995; 46: 101’9.




Uvod

Kalkuloza urinarnog trakta je veoma ~esto obo-ljenje sa prose~nom prevalencijom od 4’10% (1) igodi{njom incidencijom od 0,5% u zemljama Evrope(2). Od ove bolesti nije po{te|eno nijedno geografskopodru~je, etni~ka grupa niti jedno ìvotno doba. Klini-~ki je najmanifestnija izme|u tre}e i {este decenijeìvota. U osnovi stvaranja kamena je proces kristali-zacije u prezasi}enom urinu. On nastaje kao rezultatporeme}ene ravnoteè izme|u promotera (kalcijum,okslat, mokra}na kiselina i cistin) ~iji je stepen eks-krecije u urinu pove}an i inhibitora (Mg, citrat, piro-fosfat i glikozaminoglikani) ~iji je nivo ili inhibitornipotencijal u urinu snièn. Udruèna promena pHvrednosti i zapremine urina dovode do povoljnih uslo-va za stvaranje novih kristala, agregata kristala i veli-kih kristalnih ~estica koje u kasnijem toku prerastajuu vidljiv konkrement (3). Rezultati in vitro istraìvanja

(4, 5) su potvr|eni i u klini~kim studijama, koje supokazale zna~ajno sniène vrednosti inhibitora krista-lizacije u urinu osoba sa kalkulozom u pore|enju sazdravima (6’9). Prisustvo infekcije izazvane mikroor-ganizmima koji razlaù ureu, poreme}aji u morfologi-ji urinarnog trakta (stenoze, dilatacije, o{te}enjaurotela) i protoka urina (staza, refluks) takodje delujukao faktori rizika za stvaranje kamena ili pojavu nje-govog recidiva.

U proteklih nekoliko decenija na~injen je zna~-ajan napredak u le~enju ove bolesti primenom manjeinvazivnih metoda ’ ekstra i intrakorporalne dezinte-gracije kamena. Me|utim, rizik od pojave recidivaostaje i dalje visok, 50’100% u nele~enih i 10’15% ule~enih bolesnika (10). Novostvoreni kamen u urinar-nom traktu zna~ajno oteàva klini~ku sliku i evolucijubolesti jer pove}ava mogu}nost razvoja arterijske hi-pertenzije, a u 20% bolesnika i razvoj hroni~ne bubre-`ne slabosti (11). Visok procenat recidiva, pored toga{to je terapijski problem, ukazuje i na nepotpuno utvr-|enu etiologiju bolesti kao i na neadekvatnu primenumera prevencije. Zbog navedenog, pravilna klini~kaevaluacija pacijenata je od neprocenjivog zna~aja.

UC 577,1; 61 ISSN 0354-3447

Jugoslov Med Biohem 23: 387–392, 2004 Stru~ni radProfessional paper

NOVI PROTOKOL ZA LABORATORIJSKO ISPITIVANJE PACIJENATA SA KALKULOZOM URINARNOG TRAKTA

Dragica Milenkovi}1, Aleksandar Vuksanovi}1, Nata{a Lali}2, Sanja Simi}-Ogrizovi}1, Violeta Dopsaj2

1Institut za urologiju i nefrologiju,2Institut za medicinsku biohemiju, Klini~ki centar Srbije, Beograd

Kratak sadràj: Kalkuloza urinarnog trakta je bolest koja je jo{ uvek zna~ajan socio-medicinski problemzbog visoke stope recidiva (50’100% u nele~enih pacijenata), kao i mogu}nosti pojave hroni~ne bubre`ne sla-bosti (u 20% pacijenata). Iz tih razloga je pravilna klini~ka evaluacija pacijenata od neprocenjivog zna~aja. U ciljuefikasnijeg i racionalnijeg pristupa u utvr|ivanju mogu}ih uzroka nastanka kamena u urinarnom traktu i izboranajbolje terapije i mera prevencije va`no je sprovo|enje laboratorijskih ispitivanja prema inoviranom Protokolukoji je usagla{en sa preporukama Evropskog udruènja urologa (EAU) iz 2003. god. U zavisnosti od hemijskogsastava kamena, klini~ke kategorije bolesti kao i specifi~nih faktora rizika, program ispitivanja moè biti: mi-nimalan (analiza prvog jutarnjeg uzorka urina i urinokulture; odre|ivanje kalcijuma, albumina, kreatinina imokra}ne kiseline u serumu i analiza sastava kamena) i pro{iren (kvantifikacija svih relevantnih parametara zanastanak kamena i procena bubre`ne funkcije u serumu i 24-oro ~asovnom urinu (ispitivanjem dva uzastopnauzorka urina). Diferencijalna dijagnoza utvr|enog metaboli~kog poreme}aja, zasniva se na odre|ivanju parat-hormona u serumu, cAMP u urinu i testova optere}enja kalcijumom, amonijum hloridom i [13C2] oksalatom.

Klju~ne re~i: kalkuloza urinarnog trakta, metaboli~ki poreme}aj, protokol laboratorijskog ispitivanja, tes-tovi optere}enja

Adresa autora:

Doc Dr Dragica Milenkovi}Institut za urologiju i nefrologijuKlini~ki centar Srbije, Resavska 51, 11000 Beograd


U cilju {to efikasnijeg i racionalnijeg pristupa uutvr|ivanju mogu}ih uzroka nastanka urinarnog ka-mena i/ili pojave njegovog recidiva, va`no je, pre sve-ga, utvrditi kojoj klini~koj kategoriji pripada pacijent.Na osnovu poznatog hemijskog sastava kamena kaoi odre|enih parametara koji ukazuju na teìnu bolestiOdbor za zdravstvenu za{titu Evropskog udruènjaurologa (EAU) je 2003. god. preporu~io klini~ku kat-egorizaciju pacijenata sa kalkulozom, koja omogu}a-va ciljanu primenu dijagnosti~kih protokola i adekvat-niju terapiju (12, l3), (v. Tabelu I).

Postoji vi{e parametara koji se javljaju kao fak-tori rizika za pojavu recidiva. Na osnovu stepena nji-hovog prisustva moè se predvideti teìna bolesti ipojava recidivne kalkuloze. Koriste}i analizu speci-fi~nih faktora rizika pacijent se moè pravilnije svrsta-ti u odre|enu kategoriju bolesti (Tabela II).

Cilj ovog rada je da prikaè novi Protokol za la-boratorijsko ispitivanje pacijenata sa kamenom uri-narnog trakta u nas koji je usagla{en sa preporukamaEvropskog udruènja urologa iz 2003. godine, kao ida se ukaè na korist od njegovog uvodjenja kaostandardnog postupka u klini~ku praksu.

Protokol laboratorijskih ispitivanja

Ispitivanje pacijenata sa kalkulozom urinarnogtrakta sprovodi se u ambulantnim i/ili bolni~kim us-lovima (kada ne postoji mogu}nost adekvatnog tran-sporta 24h uzoraka urina do laboratorije, kao i u slu-~ajevima kada se radi o pro{irenom programu ispi-tivanja).

Optimalno vreme za zapo~injanje ispitivanja jenajmanje mesec dana od prestanka bubre`ne kolikeili posle uklanjanja kamena. Protokol se ne nastavlja iu slu~ajevima kada pregled jutarnjeg uzorka urina po-kaè hematuriju ili piuriju.

U zavisnosti od hemijskog sastava kamena, kli-ni~ke kategorije bolesti kao i prisustva specifi~nih fak-tora rizika, program ispitivanja moè biti minimalan ipro{iren.

Minimalan program ispitivanja obuhvata:

1. Pregled prvog jutarnjeg uzorka urina

’ pH, eritrociti, leukociti, nitriti (test trakama)’ Odre|ivanje specifi~ne teìne (test traka/uro-

metar) ’ Pregled sedimenta urina (kristalurija)

2. Urinokultura

Tabela I Klini~ke kategorije kalkuloze urinarnog trakta

KAMEN KOJI NE SADR@I KALCIJUM

Infekcijom izazvan kamen (Infection stone) INF*

Kamen mokra}ne kiseline i njenih soli (Uric acid/sodium, ammonim urate stone) UR

Cistinski kamen (Cystine stone) CY

KALCIJUMSKI KAMEN

Prva pojava kamena bez rezidualnih fragmenata So

(First time stone former without residual stone or stone fragments)

Prva pojava kamena sa rezidualnim fragmentima Sres

(First time stone former with residual stone or fragments)

Recidivna kalkuloza umerene teìne bez rezidualnih fragmenata Rm-o

(Recurrent stone former with mild disease without residual stone)

Recidivna kalkuloza umerene teìne sa prisutnim fragmentima Rm-res

(Recurrent stone former with mild disease witht residual stone)

Recidivna kalkuloza te{kog stepena sa ili bez rezidualnih fragmenata Rs

(Recurrent stone former with severe disease with or without residual stone)

Kalkuloza+specifi~ni faktori rizika Risk(Stone forming patient with specific risk factor)

* Me|unarodne skra}enice prema preporukama EAU-a

Tabela II Specifi~ni faktori rizika za pojavu kamena urinarnog trakta

Po~etak bolesti pre 25. godine ìvotaPrisustvo fosfata u hemijskom sastavu kamenJedan funkcionalan bubregUdruène bolesti: hiperparatireoidizam, hipertireoza,renalna tubularna acidoza, Crohn-ova bolest, jejuno-ilealni by-pass, stanja malapsorpcije, sarkoidozaUno{enje visokih doza: kalcijuma, vitamina D, C, sulfonamida, triamterena i Indinavir-a.Morfolo{ke promene urinarnih puteva pra}ene zastojem mokra}e ili veziko-ureteralnim refluksom(uro|ene ili ste~ene).


3. Pregled seruma: kalcijum, albumin, kreatinin, mo-kra}na kiselina

4. Analiza kamena

Ovaj program ispitivanja se sprovodi kod svihkategorija bolesnika sa kalkulozom urinarnog trakta,a na osnovu analize parametara indikuju se dalja ispi-tivanja. Vrednosti pH urina ve}e od 5,8 ukazuju namogu}nost postojanja renalne tubularne acidoze avrednosti pH preko 7,0 na urinarnu infekciju. Pozi-tivan nalaz na test trakama za leukocite i nitrite ukazu-je tako|e na prisustvo infekcije. Ukoliko pregled sedi-menta urina pokaè pove}an broj eritrocita, mogu}aje mehani~ka lezija mukoze urinarnih puteva usledprisustva kamena, infekcije ili glomerulonefritisa. Po-ve}an broj LE (vi{e od 5) u sedimentu urina tako|eukazuje na infekciju. Vrednosti kalcijuma i mokra}nekiseline u serumu iznad gornjih granica referentnihvrednosti postavljaju sumnju na hiperparatireoidizami hiperurikemiju i zahtevaju dalja metaboli~ka ispiti-vanja (Tabela III).

Analiza kamena se radi posle njegove spontaneeliminacije ili posle primenjene terapije ’ dezintegra-cije ili operacije. Prema preporukama Odbora zazdravstvenu za{titu EAU, referentne metode za ispiti-vanje sastava kamena su infracrvena spektroskopijaili difrakcija x-zracima.

Pro{iren program ispitivanja

Ovaj se program sprovodi kod pacijenata kojipripadaju klini~kim kategorijama komplikovane kal-kuloze (S-res,R m-res,R-s i Risk, Tabela I).

Pored svih analiza navedenih u minimalnomprogramu ispitivanja, neophodan je i pregled dvauzorka 24h urina koji se sakupljaju pri uobi~ajenomreìmu ishrane i unosu te~nosti i uzorci se ~uvaju nahladnom mestu (+4 °C).

UZORAK I ’ sakuplja se bez konzervansa ili uzprethodni dodatak 10 mL 5% rastvora timola u izo-propanolu (za svaku bocu od 2,0 L)

Odre|uje se: 1. zapremina urina2. urea, kreatinin, albumin, mokra}na

kiselina, kalijum, fosfat, magnezijumi citrat

UZORAK II: sakuplja se uz prethodni dodatak15’30 mL 6 mol/L rastvora HCL (za svaku bocu od2,0 L)

Odre|uje se: 1. zapremina urina

2. kalcijum i oksalat

Zaki{eljavanje urina je neophodno da bi se spre-~ilo taloènje soli kalcijuma (u formi oksalata i fosfa-ta) kao i oksidacija askorbinske kiseline u oksalat.Urea, kreatinin, mokra}na kiselina, K, Ca, i fosfat userumu odre|uju se na dan dono{enja prvog 24h

uzorka urina, ~ime je omogu}eno odredjivanje kliren-sa kreatinina. Pored navedenih parametara, koji suobavezni, odre|uju se Na i Cl u serumu i u urinuprvog uzorka koji uz vrednosti uree, fosfata i K ukazu-ju na navike u ishrani.

Odre|ivanje navedenih parametara u urinu jemogu}e i iz uzoraka koji se sakupljaju u kra}im vre-menskim intervalima (4 ili 6 sati). U tom slu~aju sevrednosti prikazuju na gram kreatinina a ne na ukup-nu diurezu.

Na osnovu seta parametara dobijenih analizom24 h uzoraka urina mogu}e je izra~unati indeksezasi}enosti urina kalcijum oksalatom i kalcijum fos-fatom (14) kao i prediktivni indeks rizika ’ PRI (15).Vrednosti ovih indeksa daju uvid u stepen poreme-}ene ravnoteè izme|u promotera i inhibitora krista-lizacije. Tako|e predvi|aju mogu}nost pojave recidi-va, {to je od zna~aja za klini~ko pra}enje pacijenatasa kalkulozom.

Kada vrednosti ispitivanih parametara odstupa-ju od referentnih, ispitivanje se ponavlja. Poznato jeda na ekskreciju supstanci od zna~aja za stvaranjeurinarnog konkrementa moè da uti~e i na~in ishranebilo da prikrije, smanji ili pove}a postoje}i metabo-li~ki poreme}aj. Iz tih razloga, protokol pro{irenogispitivanja se ponavlja posle sprovo|enja standardnedijete (2) u trajanju od tri dana, kada se u kontrolnim,bolni~kim uslovima postiè stabilizacija metabolizma.Pore|enjem vrednosti analiziranih parametara iz uzo-raka urina I i II pre i posle dijete, mogu}e je otkriti oneporeme}aje u ekskreciji, na koje uti~e na~in ishrane.

Ukoliko se ispitivani parametri u urinu vrate nanormalne vrednosti posle standardne dijete uzrok po-

Tabela III Grani~ne vrednosti parametara u urinu i serumu

Parametri

pH (dnevni profil)

Specifi~nateìna

Zapremina

Kalcijum

Fosfat

Mokra}na kiselina

Magnezijum

Citrat

Oksalat

Cistin

Kreatinin

24h urin

< 5,8 ili >6,8

>1,010 g/cm3

< 2,0 L

>5,0 mmol/dU

>35 mmol/dU

>4,0 mmol/dU

<3,0 mmol/dU

<2,5 mmol/dU

>0,5 mmol/dU

>0,8 mmol/dU

7,0 ’13,0 mmol/L (`)

13,0 ’18,0 mmol/L (m)

Serum

2,0’2,5 mmol/L

0,81’1,29 mmol/L

119’380 mmol/L

25’100 mmol/L


reme}aja su nepravilnosti u ishrani. Odràvanje vred-nosti urinarnih parametara izvan referentnih granicaukazuje na metaboli~ki poreme}aj i zahteva sprovo-|enje testova optere}enja kalcijumom, amonijumhloridom ili test apsorpcije [13C2] oksalata.

Test optere}enja kalcijumom

Test optere}enja kalcijumom modifikovan pre-ma Pak-u (17) izvodi se na slede}i na~in: I dan: dijeta bez mle~nih proizvoda

18h ’ ve~era20h unos 300 mL vode osiroma{ene kalciju-mom23h unos 300 mL vode osiroma{ene kalciju-mom

II dan: 07h ’ uriniranje, zatim unos 600 mL vodeosiroma{ene kalcijumom07’09h prvi period sakupljanja urina (vrednostiposle gladovanja)09h ’ doru~ak (1 sendvi~, puter, dèm, 2 {oljevo}nog ~aja + 1 tbl od 1000 mg kalcijuma11h ’ unos 300 mL vode osiroma{ene kalciju-mom09’13h drugi period sakupljanja urina (vredno-sti posle optere}enja)

U uzorcima urina I i II odredjuje se koncentraci-ja kalcijuma (mmol/L) i kreatinina (mmol/L) i izra~u-nava se indeks Ca/Cr.

Testom optere}enja kalcijumom omogu}ava serazlikovanje tri tipa hiperkalciurije i to:

1. Apsorptivna hiperkalciurija koja nastaje kao po-sledica pove}anog stepena apsorpcije kalcijuma izintestinalnog trakta. Ona moè biti nezavisna odunete koli~ine kalcijuma (tip I) i zavisna, kada sepojavljuje samo pri pove}anom unosu kalcijuma(tip II).

2. Renalna hiperkalciurija koja je rezultat gubitkakalcijuma na nivou distalnih tubula usled poreme-}aja u njegovoj reapsorpciji. Tada se, zbog pove}a-

nog gubitka kalcijuma urinom javlja sekundarni hi-perparatireoidizam.

3. Resorptivna hiperkalciurija koja je posledica pri-marnog hiperparatireoidizma koji se karakteri{ezna~ajnom resorpcijom kalcijuma iz ko{tanog tkivai njegovom pove}anom apsorpcijom iz intestinal-nog trakta. Odre|ivanje koncentracije PTH u krvi icAMP u urinu olak{ava diferencijalnu dijagnozu hi-perkalciurije.

U 20% pacijenata i pored sprovedenog Proto-kola laboratorijskih ispitivanja uzrok hiperkalciurijeostaje neotkriven (idiopatska hiperkalciurija).

Test optere}enja amonijum hloridom

Indikacija za sprovo|enje ovog testa je sumnjana postojanje renalne tubularne acidoze, tj. stanja ne-adekvatne sekrecije vodonikovih jona na nivou distal-nih tubula. Sumnja na postojanje ovog oblika meta-boli~ke acidoze postoji u slu~ajevima kada se pHvrednost urina ne sniàvaju ispod 5,8 u toku nekolikouzastopnih odre|ivanja dnevnog profila pH (18).

Test se izvodi ambulantno, a pH vrednost urinase odredjuje isklju~ivo pH-metrom, neposredno posakupljanju svakog uzorka urina.

Test se izvodi na slede}i na~in:

Ukoliko se pH vrednosti urina snize do 5,4 is-klju~uje se postojanje renalne tubularne acidoze, aukoliko nisu ispod 5,4 da bi se dokazao tip renalnetubularne acidoze treba uraditi gasne analize krvi.Niske vrednosti bikarbonata (ispod 22 mmol/L) i pHkrvi (ispod 7,35) ukazuju na kompletnu, dok normal-ne vrednosti ukazuju na inkompletnu renalnu tubu-larnu acidozu (v. Tabelu V).

Test apsorpcije [ 13C2] oksalata

Hiperoksalurija je vaàn faktor rizika u nastankukalcijum oksalatne kalkuloze. Nastaje kao posledicanaslednih poreme}aja u metabolizmu oksalne kise-line (endogena ili primarna hiperoksalurija) ili usledpove}anog unosa oksalne kiseline hranom, kao izbog pove}anog stepena apsorpcije pri razli~itim pa-

08h: doru~ak + 0,1g NH4Cl/kg tt + 150 mLvo}nog ~aja

09h: sakupljanje I uzorka urina i unos 150 mLvo}nog ~aja

10h: sakupljanje II uzorka urina i unos 150 mLvo}nog ~aja

11h: sakupljanje III uzorka urina i unos 150 mLvo}nog ~aja

12h: sakupljanje IV uzorka urina i unos 150 mLvo}nog ~aja

13h: sakupljanje V uzorka urina

Tabela IV Indeks Ca/Cr

Normalno Apsorptivna hiperkalciurijaRenalna/resorptivnahiperkalciurijaRenalna hiperkalciurija:cAMP pove}anResorptivna hiperkalciurija:pove}an PTH

Preoptere}enja do 0,337do 0,337 ≥ 0,338

Posleoptere}enja

do 0,563 ≥ 0,564≥ 0,564

tolo{kim stanjima u intestinalnom traktu (egzogena ilisekundarna hiperoksalurija).

Test apsorpcije [13C2] oksalata se upravo i izvo-di kod pacijenata sa dokazanom hiperoksalurijom ucilju razlikovanja osnovnog uzroka nastanka poreme-}aja i pravilnog izbora na~ina le~enja. Test se spro-vodi u hospitalnim uslovima, pri unosu 2400 mLte~nosti na dan i posle 2-dnevne standardne dijete(19), na slede}i na~in:

I dan 08’20h: sakupljanje urina (+30 mL 2,5 mol/LHCl)

20’08h: sakupljanje urina (+30 mL 2,5 mol/LHCl)

II dan 08h: unos 33,8 mg [13C2] oksalne kiseline uformi natrijumove soli u kapsuli od 50 mg

09h: doru~ak08’14h: sakupljanje urina (+15 mL 2,5 mol/L

HCl)14h: ru~ak



Nivo ekskretovanog oksalata obeleènog radio-izotopom ugljenika u 24h uzorcima urina sakupljanihI i II dana odre|uje se gasnom hromatografijom. Raz-lika u nivoima ve}a od 10% smatra se zna~ajnom.

Zna~aj protokola

Primena novog Protokola obezbe|uje racional-nost i efikasnost u klini~koj evaluaciji pacijenata sakalkulozom urinarnog trakta zbog sasvim preciznihindikacija za izvo|enje testova laboratorijskih ispiti-vanja za svaku klini~ku kategoriju bolesti. Kontrolisa-nost uslova pri skupljanju uzoraka materijala za ana-lizu, sprovo|enje standardnih dijeta i adekvatno ~u-vanje uzoraka urina pove}avaju pouzdanost rezultata.Po{tovanje algoritma ispitivanja omogu}ava da se nanajbrì na~in utvrdi postoje}i metaboli~ki poreme}aj,odnosno otkrije egzogeni faktor (gre{ke u ishrani ilipatolo{ka intestinalna apsorpcja materija odgovornihza nastanak kamena). Utvr|ivanje uzroka bolestiobezbe|uje da se u svakom konkretnom slu~aju od-redi najadekvatnija terapija, odnosno da se primenemere profilakse u cilju spre~avanja pojave recidivabolesti.

Izra~unavanje odre|enih indeksa rizika, na os-novu parametara dobijenih primenom ovog Proto-kola, omogu}ava precizniju procenu teìne bolesti,predvi|anje njenog toka kao i pra}enje efekata pri-menjene medikamentozne terapije.


Tabela V Diferencijalna dijagnoza renalne tubularne acidoze

Biohemijski parametripH krvi Bikarbonati plazmeKalijum Hloridi Ca i fosfor u urinu Citrat u urinu

Kompletna

snièn snièni snièn povi{eni povi{eni snièn

Inkompletna

normalan normalni normalan normalninormalni snièn

UPDATE ANALYTIC PROGRAM FOR LABORATORY INVESTIGATION IN URINARY TRACT CALCULOSIS

Dragica Milenkovi}1, Aleksandar Vuksanovi}1, Nata{a Lali}2, Sanja Simi}-Ogrizovi}1, Violeta Dopsaj2

1Institute of Urology and Nephrology 2Institute of Medical Biochemistry, Clinical Centre of Serbia, Belgrade

Summary: Urinary tract calculosis due to it's high recurrence rate (50’100% in untreated patients) andpossibility to cause chronic renal failure in 20% stil represents a great social and medical problem. From thataspect, an adequate clinical evaluation in stone forming patients is of paramount importance. An effective andrational attitude in detecting possible causes of stone formation demands the adequate analytic program in bio-chemical investigations. Presented program is adjusted to the guidelines on urolithiasis, recommended byBoard EAU (European Association of Urologists) Healthcare Office, for year 2003. Regarding to known che-mical stone composition, category of stone former and presence of an specific risk factor, analytic programcould be minimal (fasting morning spot urine sample analysis, urineculture and blood analysis (calcium, albu-mine, creatinine and urate and stone analysis) and extended (quantification of all parameters in serum and intwo consequtive 24h urine collections, relevant for stone formation and evaluation of renal function). In order toidentify metabolic disorders, an quantification of parathormone in serum, cAMP in urine and absorption/loadingtests with calcium, NH4Cl and [12C2] oxalate are recommended.

Key words: urinary tract calculosis, metabolic disorder, analytic program, absorption/loading tests

Literatura

1. Gambaro G, Reis-Santos JM, Rao N. Nephrolithiasis:Why doesn't our learning progress. Eur Urol 2004; 45:547’56.

2. Hesse A, Tiselius HG, Jahnen A. Urinary stones: Diag-nosis, treatment and prevention of recurrence, 2nd

revised and enlarged ed. Basel: Karger 2002; 8.

3. Fleisch H. Role of inhibitors and promoters of crystalnucccleation, growth and aggregation in the formationof calcium stone. In: Wikham J E A and Buck AP eds.Renal tract stones, Edinbourgh, London, Melbourneand New York, Churchill Livingstone 1990; 295’306.

4. Fleisch H. Inhibitors and promoters of stone formation.Kidney Int 1978; 13: 361’71.

5. Lieske JC, Deganello S, Toback FG. Cell-crystal inter-actions and kidney stone formation. Nephron 1999; 81(suppl. 1): 8’17.

6. Hesse A, Wurzel H, Vahlensieck W. The excretion ofglycosaminoglicans in the urine of calcium stone pati-ents and healthy persons. Urol Int 1986; 41: 81.

7. Milenkovi} D. Procena nivoa magnezijuma, pirofosfata,citrata i glikozaminoglikana u urinu kao faktora rizika unastanku kalkuloze. Doktorska disertacija, Medicinskifakultet, Beograd, 1991.

8. Milenkovi} D, Petroni} V, Hadì \oki} J, Mi}i} S, Tuli}C, Lali} N. Urinary levels of glycosaminoglicans in stoneforming subjects. Br J Urol 1997; 80 (suppl. 2): 323.

9. Greishar A, Nakagawa Y, Coe F. Influence of urine pHand citrate concentration on the upper limit of meta-stability for calcium phosphate. J Urol 2003; 169: 867’70.

10. Strohmaer WL. Course of calcium stone disease with-out treatment. What can we expect? Eur Urol 2000; 37:339’44.

11. Marangella M, Bruno A, Vitalo C, Cosseddu D, TricerriA, Linari F. The occurence of chronic renal failure incalcium nephrolithiasis. In: Vahlesieck W, Gasser G,Schoneich G Eds.: Urolithiasis, Amstredam ExcerptaMedica 1990; 15 ’8.

12. EAU guidelines ed 3, Arnhem: Drukkerij 2003; 19’20.

13. Tiselius HG. Etiology and investigation of stone dis-ease. Eur Urol 1998; 33/1 (Curric Urol 2.1): 1’7.

14. Tiselius HG. Solutiom chemistry and suppersaturationin kidney stones: Medical and surgical menagement.Edds Coe E, Flavus MJ, Pak CYC, Parks JH, PremingerGM. Philadelphia, Lipkot Raven Publishers, 1996; 33’64.

15. Milenkovi} SD, Petroni} V, Lali} N, Hadì \oki} J, Mi}i}S, Tuli} C, Kozomara M. Modified predictive risk indexfor urinary calculosis. In. Kidney stones ed Borghi L,Micchi T, Briganti A, Schinchi T, Novarini A. Proce-edings of the 8 th European symposium on urolithiasis,Parma, Editoriale Bios 1999; 397’ 9.

16. Tiselius HG. Factors influencing the course of calciumstone disease. Eur Urol 1996; 363’70.

17. Pak CYC, Kaplan RA, Bone H, Townsend J, Woters O.A simple test for the diagnosis of absorptive, resorptiveand renal hypercalciuria. New Engl J Med 1975; 292:497’500.

18. Chafe L, Gaul MH. First morning urine pH in the diag-nosis on renal tubular acidosis with nephrolithiasis. ClinNephrol 1994; 41: 159’162.

19. Von Unruh GE, Langer MA, Paar DW, Hesse A. Massspectrophotometric-selected ion monitoring assay foran oxalate absorption test applying (12C2) oxalate. JChromatogr 1998; 716: 343’9.




Jugoslov Med Biohem 2004.; 23 (4) 393

Uvod

Somatostatin je tetradekapeptid, prvo izolovan izhipotalamusa ovce 1973. godine, a kasnije je otkrivenu gastrointestinalnom traktu, pankreasu, centralnom iperifernom nervnom sistemu i tireoidnoj `lezdi (1).Poznato je da izaziva inhibiciju ve}eg broja drugih hor-mona, kao i inhibiciju sekrecije i motiliteta u gastroin-testinalnom traktu. Eksperimentalne studije ukazujuda se somatostatin metaboli{e u jetri i bubrezima i do-kazana je degradacija somatostatina u izolovanim he-patocitima (2, 3). Ispitivanja u ljudi za sada daju kon-tradiktorne rezultate.

U ve}ini patolo{kih stanja kao {to su oboljenjaèluca, tankog i debelog creva dolazi do odstupanjapojedinih gastrointestinalnih peptida, pa i somatosta-tin (4). Objavljene studije ukazuju na hipoteti~ki zna~ajsomatostatina u patofiziologiji gastritisa, inflamatornihoboljenja creva i kolorektalnog kancera s jedne strane,i mogu}nosti primene u terapijske svrhe (5’7). Ciljovog rada je bio da se razjasni mogu}a uloga jetre umetabolizmu somatostatina merenjem njegovih vred-nosti u serumu bolesnika sa razli~itim stepenom o{te-

}enja jetre, utvrdi veza izme|u razvoja intestinalne me-taplazije i vrednosti somatostatina, ispita vrednost so-matostatina, u bolesnika sa ulceroznim kolitisom iKronovom bole{}u u odnosu na fazu i lokalizaciju bo-lesti i eventualna odstupanja vrednosti somatostatinau bolesnika sa kolorektalnom neoplazijom.

Materijal i metode

Ispitano je ukupno 50 (35 mu{kog pola i 15ènskog pola) bolesnika sa histolo{ki potvr|enomalkoholnom cirozom jetre, prose~ne starosti 49 godi-na. Dobijeni rezultati posmatrani su sa dva aspekta: uodnosu na prisustvo ciroze i u odnosu na stepen he-patalne insuficijencije. Izvr{ena je podela prema kli-ni~kim kriterijumima na kompenzovani i dekompenzo-vani oblik ciroze.

Sa oboljenjem perniciozne anemije i atrofi~noggastritisa sa ahlorhidrijom bilo je ukupno 15 bolesnika,prose~ne starosti 59 godina. Dijagnoza perniciozneanemije postavljena je na osnovu megaloblastne kos-tne srì i pozitivnog Schilling testa, a atrofi~nog gastri-tisa gastroskopski uz histopatolo{ki nalaz.

Endoskopskim ispitivanjem uz histopatolo{kuanalizu postavljena je dijagnoza ulceroznog kolitisa uukupno 31 bolesnika, od ~ega 17 u akutnoj fazi bo-lesti, a 14 u remisiji. Tako|e je pra}eno i 32 bolesnikasa M. Crohn razli~ite lokalizacije (21 na tankom, 5 na

UC 577,1; 61 ISSN 0354-3447

Jugoslov Med Biohem 22: 393 – 396, 2003 Stru~ni radProfessional paper

Adresa autora:

Prof. dr Nada Kosti}KBC Dr Dragi{a Mi{ovi}Heroja Milana Tepi}a br.1, BeogradTel: 367’20’25

SOMATOSTATIN U OBOLJENJIMA GASTROINTESTINALNOG TRAKTA

Nada Kosti}1, Branislava Brki}1, Zorica âparevi}1, Verica Milo{evi}2

1Klini~ko-bolni~ki centar »Dr Dragi{a Mi{ovi} - Dedinje«, Beograd2Institut za biolo{ka istraìvanja »Sini{a Stankovi}«, Beograd

Kratak sadràj: Somatostatin u serumu odre|en je RIA metodom, kod 50 pacijenata sa cirozom jetre, 15sa pernicioznom anemijom i atrofi~nim gastritisom, 31 sa inflamatornim oboljenjem creva, 32 sa kolorektalnimtumorima i 40 kontrolnih osoba. Kod pacijenata sa cirozom jetre na|ene su zna~ajno vi{e vrednosti somatostati-na u odnosu na kontrolnu grupu (p<0,01). Pacijenti sa pernicioznom anemijom i ulceroznim kolitisom u akutnojfazi bolesti imali su zna~ajno niè vrednosti somatostatina (p<0,005). Kod pacijenta sa kolorektalnim tumorimasomatostatin je bio zna~ajno niì nego u kontrolnoj grupi (p<0,01). Dobijeni rezultati pokazuju da je jetra uklju-~ena u metabolizam somatostatina. Atrofi~ni gastritis, ulcerozni kolitis i M. Crohn, kao i kolorektalni tumori bili suudruèni sa zna~ajnim promenama u nivou somatostatina {to sugeri{e mogu}u patofiziolo{ku i terapeutsku ulogusomatostatina u ovim oboljenjima.

Klju~ne re~i: somatostatin, ciroza jetre, atrofi~ni gastritis, ulcerozni kolitis, M. Crohn, kolorektalni tumori

debelom crevu i 6 istovremeno na tankom i debelomcrevu).

Mogu}a veza izme|u somatostatina i kolorektal-ne neoplazije ispitivana je na 42 bolesnika, prose~nestarosti 60 godina. Dijagnoza je postavljena kolonos-kopski uz histopatolo{ku verifikaciju i to u 16 adeno-matozni polip, u 8 vilozni adenom i u 18 adenokarci-nom.

Svih 138 bolesnika kao i 40 kontrolnih osoba biloje hospitalizovano na Klinici za internu medicinu idetaljno ispitano (kompletne biohemijske analize i krvnaslika, rendgenska ispitivanja, ultrazvu~na dijagnostika iendoskopski pregledi uz histopatolo{ke analize).Izuzimaju}i grupu sa alkoholnom cirozom jetre, svimostalim bolesnicima isklju~ena su druga oboljenja, presvega bubre`na i hepatalna insuficijencija.

Svi ispitanici bili su bez medikamentne terapije iujutru na{te uzimana je krv za odre|ivanje bazalnihvrednosti somatostatina. Analiza je vr{ena RAI meto-dom uz kori{}enje laboratorijskog, eksperimentalnogtest reagensa (dobijenog iz Laboratorije HammersmithHospital u Londonu). Referentne vrednosti za somato-statin bile su 17’150 pmol/L.

U statisti~koj obradi kori{}eni su Studentov t testi druge uobi~ajene statisti~ke metode.

Rezultati

Alkoholna ciroza jetre

Bazalne vrednosti somatostatina u serumu bilesu zna~ajno vi{e kod svih bolesnika sa alkoholnomcirozom jetre u odnosu na kontrolnu grupu (p<0,01)(Tabela I). Vrednosti somatostatina u grupi bolesni-ka sa dekompenzovanim oblikom ciroze bile su zna-~ajno vi{e u odnosu na kompenzovani oblik (p<0,05)(Tabela I).

Perniciozna anemija i atrofi~ni gastritis

Svi bolesnici sa pernicioznom anemijom imali suhroni~ni atrofi~ni gastritis, a u 7 bolesnika histolo{ki jepotvr|ena i intestinalna metaplazija. Nivoi somatostati-na u serumu bili su zna~ajno niì u bolesnika sa perni-cioznom anemijom i hroni~nim atrofi~nim gastritisomu odnosu na kontrolnu grupu (p<0,01). Sve vrednostiu ispitivanih bolesnika bile su ispod normalnih vred-nosti za somatostatin. Kod bolesnika sa intestinalnommetaplazijom na|ene su zna~ajno niè vrednosti uodnosu na bolesnike sa samo hroni~nim atrofi~nimgastritisom (p<0,01) (Tabela II).

Ulcerozni kolitis i Kronova bolest

Ispitivanjem su utvr|ene zna~ajno vi{e vrednostisomatostatina u obe grupe bolesnika (105,5 ± 21,4pmol/L) u odnosu na kontrolnu grupu (50,3 ± 10,2pmol/L) (p<0,01). U odnosu na fazu bolesti vrednostibazalnog somatostatina bile u zna~ajno niè u akutnojfazi u odnosu na remisiju (p<0,005). U odnosu nalokalizaciju Kronove bolesti vrednosti somatostatinabile su zna~ajno vi{e u grupi sa promenama na tan-kom crevu (p<0,001) (Tabela III). Sve ispitivanevrednosti somatostatina bile su unutar referentnihvrednosti za somatostatin.


N20305040

Ispitivane grupeKompenzovana cirozaDekompenzovana cirozaSve ciroze jetreKontrolna grupa

Somatostatin, pmol/L44,0 ± 9,8 X

68,6 ± 20,1XX

50,7 ± 30,3**28,0 ± 10,1

Tabela I Nivoi somatostatina u plazmi bolesnika sa alkoholnom cirozom jetre

** p < 0,01 nivo statisti~ke zna~ajnosti razlika u odnosu na kontrolnu grupuX p < 0,05 nivo statisti~ke zna~ajnosti razlika bolesnika sa dekompenzo-

vanom cirozom jetre u odnosu na grupu sa kompenzovanomXX p < 0,01 nivo statisti~ke zna~ajnosti razlika bolesnika sa dekompenzo-

vanom cirozom jetre u odnosu na kontrolnu grupu

N158

7

40

Grupe bolesnikaPerniciozna anemijaSamo atrofi~ni gastritisAtrofi~ni gastritis i interstinalna metaplazijaKontrolna grupa

Somatostatin, pmol/L9,4 ± 10,6*

13,6 ± 12,1

3,2 ± 2,3**

49,5 ± 20,1

Tabela II Somatostatin u serumu bolesnika sa pernicioznom anemijom

* zna~ajna razlika (p<0,01) za bolesnike sa pernicioznom anemijom uodnosu na kontrolnu grupu

** zna~ajna razlika (p < 0,01) podgrupa perniciozne anemije sa i bez inesti-nalne metaplazije

N311714322156

40

Ispitivane grupeUlcerozni kolitis’ u akutnoj fazi’ u remisijiKronova bolest’ tanko crevo’ debelo crevo’ obaKontrolna grupa

Somatostatin, pmol/L

8,1 ± 4,4*39,9 ± 14,7

44,1 ± 15,2**28,7 ± 13,244,7 ± 2,919,5 ± 20,1

Tabela III Somatostatin u serumu bolesnika sa ulceroznim kolitisom i M. Crohn

* p < 0,005** p < 0,001

Kolorektalni tumori

U grupi bolesnika sa kolorektalnim adenokarci-nomom i viloznim adenomom vrednosti somatostati-na bile su zna~ajno niè u odnosu na kontrolnu grupu(p<0,01), ali unutar normalnih vrednosti.

Diskusija

Do sada saop{teni kontradiktorni rezultati u lite-raturi (3, 8) ovo ispitivanje ~ine aktuelnim. Rezultatipovi{enih bazalnih vrednosti somatostatina u skladusu sa pretpostavkom da se somatostatin metaboli{e ujetri, {to ne isklju~uje njegovu ulogu kao neurotrans-mitera i lokalnog endokrinog regulatora pa i mogu}-nosti da se metaboli{e na mestima gde se i osloba|a.Pove}anje somatostatina u cirozi jetre, a posebno de-kompenzovane, mogu}e je objasniti uticajem intra-hepati~ne holestaze, portalne hipertenzije i postoja-njem intra i ekstrahepati~nih {antova (10). Navedenerezultate treba posmatrati i u svetlu upotrebe somato-statina u farmakolo{kom tretmanu portalne hiperten-zije (11).

Poznato je prisustvo hipergastrinemije kod bo-lesnika sa pernicioznom anemijom i hroni~nim atro-fi~nim gastritisom, {to se obja{njava sekundarno po-ve}anim osloba|anjem gastrina zbog ahlorhidrije,uzrokovane upravo atrofi~nim gastritisom, a ne isklju-~uje se ni uloga imunolo{kih mehanizama (postojanjeautoantitela na gastrinske receptore) (14). Niske vred-nosti somatostatina mogu se objasniti pre pove}animodnosom G/D }elija {to je i dokazano, a {to sugeri{eda su niske vrednosti pre posledice ahlorhidrije, nego

tipi~nog efekta hipergastrinemije, jer je poznato dase somatostatin osloba|a pri niskim antralnim pH(15’17).

Nalaz zna~ajno niìh vrednosti somatostatina uakutnoj fazi bolesti je u saglasnosti sa ispitivanom li-teraturom (18). Tako|e ve}e vrednosti somatostatinau bolesnika sa intestinalnom lokalizacijom odraàvajumesto osloba|anja somatostatina uzrokovano najve-rovatnije nekroti~nim promenama. S druge strane odzna~aja je razmatranje upotrebe somatostatina u bo-lesnika sa te{kim oblicima kolitisa (18, 19).

Poslednjih godina otkriveno je prisustvo recepto-ra za somatostatin i druge peptide kod eksperimen-talnog kancera kolona (20). Tako|e potvr|ena je imu-noreaktivnost }elija serotonina, enteroglukagona, pep-tida YY i dr. (21, 22). Niè vrednosti somatostatina uzpovi{ene vrednosti gastrina, IGF-1 i drugih peptidauklapaju se u patofiziolo{ki koncept rasta kolorektalnihtumora, gde endokrini sistem o~igledno ima odre|enuulogu. S obzirom na navedene promene moglo bi serazmi{ljati o upotrebi neuroendokrinih peptida, pa isomatostatina u dijagnostici i terapiji kolorektalnogkancera (23).

Iz svega izloènog moè se zaklju~iti da poredpoznatog op{teg inhibitornog dejstva somatostatinana sekreciju neuroendokrinih tumora, kao i na sekre-ciju i motilitet gastrointestinalnog trakta, somatostatino~igledno ima ulogu i u patofiziologiji gastritisa, infla-matornih oboljenja creva, kolorektalnog kancera. Ovoje od posebnog zna~aja kada se razmatraju mogu}-nosti upotrebe somatostatina u terapijske svrhe.


SOMATOSTATIN LEVELS IN GASTROINTESTINAL DISEASE


1Clinical Hospital Centre »Dr Dragi{a Mi{ovi}« »Dedinje«, Belgrade2Institute for Biological Research »Sini{a Stankovi}«, Belgrade

Summary: Serum levels of somatostatin were determined by RIA method in 50 patients with liver cirrhosis,15 with pernicious anemia and atrophic gastritis, 31 with inflamatory bowel disease, 32 with colorectal tumorsand in 40 control persons. In patients with liver cirrhosis somatostatin levels were significantly higher than in con-trol group (p<0.01). Patients with pernicious anemia and ulcerative colitis in acute phase of the disease had sig-nificantly lower levels of somatostatin (p<0.01) (p<0.005). In patients with colorectal tumors somatostatin weresignificantly lower then in control group (p<0.01). Our results show that the liver is involved in somatostatinmetabolism. Atrophic gastritis, ulcerative colitis and M.Crohn, so as colorectal neoplasia were associated with sig-nificantly changes in somatostatin levels which suggest the potential pathophysiologic and terapeutic role ofsomatostatin in those disease.

Key words: somatostatin, liver cirrhosis, atrophic gastritis, ulcerative colitis, M. Crohn, colorectal tumors

Literatura

1. Gerich JE, Paton GS. Somatostatin. Med Clin North Am1978; 62, 375’ 83.

2. Conlon JM, Whittaker J, Hammond V, Alberti KGM.Metabolism of somatostatin and its analogues by theliver. Biochim Biophys Acta 1981; 677: 234’ 2.

3. Sacks H, Cass TL. Clearance of imunoreactive somato-statin by rat liver. J Clin Invest 1981; 67: 419’29.

4. Kosti} N. Regulatorni peptidi (patofiziologija i klini~kizna~aj). Zavod za ud`benike i nastavna sredstva, Beo-grad, 1999.

5. Di Lorenzo C, Lucanto C, Flores AF, Idries S, HymanPE. Effect of sequential erythromycin and octreotide onantroduodenal manometry. J Pediatr Gastroenterol Nutr1999; 29 (3): 293’6.

6. Eliakim R, Fan QX, Babyatsky MW. Chronic nicotineadministration differentially alters jejunal and colonicinflammation in interleukin-10 deficient mice. Eur JGastroenterol Hepatol 2002; 14 (6): 607’14.

7. Sadij-Ouatas Z, Lasfer M, Julien S, Feldmann G, Rayl-Desmars F. Doxorubicin and octreotide induce a 40 kDabreakdown product of p53 in human hepatoma andtumoral colon cell lines. Biochem J 2002; 15: 364 (Pt 3):881’5.

8. Sheppard M, Shapiro B, Pimstone B, Kronhein S. Me-tabolic clearance and plasma half disappearance time ofexogenous somatostatin in man. Clin Endocrinol Me-tabol 1979; 48: 50’6.

9. Kelback H, Tronier B, Bahnsen M, Munkgard S. Fastingplasma somatostatin in alcoholic liver disease. Scand JClin Lab Invest 1983; 43: 597’ 601.

10. Munkgaard S, Kelbeek H, Tronier B. Elevated plassomatostatin in cirrhosis of the liver. N Engl J Med 1981;301: 1429’30.

11. Gonzales-Abraldes J, Bosch J, Garcia-Pagan JC. Phar-macological tretment of portal hypertension. Curr OpinInvestig Drugs 2001; 2 (10): 1407’13.

12. Matrella E, Valatas V, Notas G, Roumpaki H, Xidakis C,Hadzidakis A, et. al. Bolus somatostatin but not octreo-tide reduces hepatic sinusoidal pressure by a NOin-dependent mechanism in chronic liver disease. AlimentPharmacol Ther 2001; 15 (6): 857’ 64.

13. Zhang HB, Wong BC, Zhou XM, Guo XG, Zhao SJ, WangJH, et. al. Effects of somatostatin, octreotide and pitre-ssin plus nitroglycerine on systemic and portal hae-modynamics in the control of acute vericeal bleeding. IntJ Clin Pract 2002; 56 (6): 447’51.

14. Aizupurua HJ, Ungar B, Toch BH. Autoantibody to thegastrin receptor in pernicious anemia. N²Engl J Med1985; 313: 479’ 83.

15. Ligumsky M, Wengrower D, Karmeli F, Rachmilewitz D.Somatostatin release by human gastric mucosa. ScandJ Gastroenterol 1988; 23: 687’90.

16. Arnold R, Hulst MV, Neuhof CH, Schwarting H, BekerHD, Creutzfeld W. Antral gastrin-producting G-cells andsomatostatin producing ’ D-cells in different states ofgastric acid secretion. Gut 1982; 23: 285’91.

17. Alonso Falcon F, Codoceo Alquinta R, Polanco Allue I,Aquado Gil A, Fontan Casariego G. Study of gastro-intestinal polypeptides controlling gastric acid secretionin patirnts with primary antibody deficiency. Rev EspEnferm Dig 1999 1; 91 (1): 54’ 60.

18. van Bergeijk JD, Wilson JH, Nielsen OH, von Triptiz C,Karvonen AL, Lygren I, et. al. Octreotide in patients withactive ulcerative colitis with high dose corticosteroids.Eur J Gastroenterol Hepatol 2002; 14 (3): 243’8.

19. Su X, Burton MB, Gebhart GF. Effect of octreotide onresponses to colorectal distension in the rat. Gut 2001;48 (5): 676’82.

20. Szepeshazi K, Schally AV, Halmos G, Armatis P, HebertF, Sun B., et. al. Targeted cytotoxic somatostatin ana-logue AN-238 inhibits somatostatin receptor-positiveexperimental colon cancers independently of their p53status. Cancer Res 2002 1; 62 (3): 781’8.

21. Sitohy B, El-Salhy M. Colonic endocrine cells in rats withchemically induced colon carcinoma. Histol Histopathol2001; 16 (3): 833’8.

22. Cox HM, Tough IR, Zandvliet DW, Hollidey ND. Con-stitutive neuropeptide Y Y(4) receptor expression in hu-man colonic adenocarcinoma cell lines. Br J Pharmacol2001; 132 (1): 345’3.

23. Smith-Jones PM, Bischof C, Leimer M, Gludovacz D,Angelberger P, Pangerl T, et. al. DOTA-lanreotide: anovel somatostatin analog for tumor diagnosis and the-rapy. Endocrinology. 1999; 140 (11): 5136’ 48.




Jugoslov Med Biohem 2004.; 23 (4) 393

Uvod

Somatostatin je tetradekapeptid, prvo izolovan izhipotalamusa ovce 1973. godine, a kasnije je otkrivenu gastrointestinalnom traktu, pankreasu, centralnom iperifernom nervnom sistemu i tireoidnoj `lezdi (1).Poznato je da izaziva inhibiciju ve}eg broja drugih hor-mona, kao i inhibiciju sekrecije i motiliteta u gastroin-testinalnom traktu. Eksperimentalne studije ukazujuda se somatostatin metaboli{e u jetri i bubrezima i do-kazana je degradacija somatostatina u izolovanim he-patocitima (2, 3). Ispitivanja u ljudi za sada daju kon-tradiktorne rezultate.

U ve}ini patolo{kih stanja kao {to su oboljenjaèluca, tankog i debelog creva dolazi do odstupanjapojedinih gastrointestinalnih peptida, pa i somatosta-tin (4). Objavljene studije ukazuju na hipoteti~ki zna~ajsomatostatina u patofiziologiji gastritisa, inflamatornihoboljenja creva i kolorektalnog kancera s jedne strane,i mogu}nosti primene u terapijske svrhe (5’7). Ciljovog rada je bio da se razjasni mogu}a uloga jetre umetabolizmu somatostatina merenjem njegovih vred-nosti u serumu bolesnika sa razli~itim stepenom o{te-

}enja jetre, utvrdi veza izme|u razvoja intestinalne me-taplazije i vrednosti somatostatina, ispita vrednost so-matostatina, u bolesnika sa ulceroznim kolitisom iKronovom bole{}u u odnosu na fazu i lokalizaciju bo-lesti i eventualna odstupanja vrednosti somatostatinau bolesnika sa kolorektalnom neoplazijom.

Materijal i metode

Ispitano je ukupno 50 (35 mu{kog pola i 15ènskog pola) bolesnika sa histolo{ki potvr|enomalkoholnom cirozom jetre, prose~ne starosti 49 godi-na. Dobijeni rezultati posmatrani su sa dva aspekta: uodnosu na prisustvo ciroze i u odnosu na stepen he-patalne insuficijencije. Izvr{ena je podela prema kli-ni~kim kriterijumima na kompenzovani i dekompenzo-vani oblik ciroze.

Sa oboljenjem perniciozne anemije i atrofi~noggastritisa sa ahlorhidrijom bilo je ukupno 15 bolesnika,prose~ne starosti 59 godina. Dijagnoza perniciozneanemije postavljena je na osnovu megaloblastne kos-tne srì i pozitivnog Schilling testa, a atrofi~nog gastri-tisa gastroskopski uz histopatolo{ki nalaz.

Endoskopskim ispitivanjem uz histopatolo{kuanalizu postavljena je dijagnoza ulceroznog kolitisa uukupno 31 bolesnika, od ~ega 17 u akutnoj fazi bo-lesti, a 14 u remisiji. Tako|e je pra}eno i 32 bolesnikasa M. Crohn razli~ite lokalizacije (21 na tankom, 5 na

UC 577,1; 61 ISSN 0354-3447

Jugoslov Med Biohem 22: 393 – 396, 2003 Stru~ni radProfessional paper

Adresa autora:

Prof. dr Nada Kosti}KBC Dr Dragi{a Mi{ovi}Heroja Milana Tepi}a br.1, BeogradTel: 367’20’25

SOMATOSTATIN U OBOLJENJIMA GASTROINTESTINALNOG TRAKTA


1Klini~ko-bolni~ki centar »Dr Dragi{a Mi{ovi} - Dedinje«, Beograd2Institut za biolo{ka istraìvanja »Sini{a Stankovi}«, Beograd

Kratak sadràj: Somatostatin u serumu odre|en je RIA metodom, kod 50 pacijenata sa cirozom jetre, 15sa pernicioznom anemijom i atrofi~nim gastritisom, 31 sa inflamatornim oboljenjem creva, 32 sa kolorektalnimtumorima i 40 kontrolnih osoba. Kod pacijenata sa cirozom jetre na|ene su zna~ajno vi{e vrednosti somatostati-na u odnosu na kontrolnu grupu (p<0,01). Pacijenti sa pernicioznom anemijom i ulceroznim kolitisom u akutnojfazi bolesti imali su zna~ajno niè vrednosti somatostatina (p<0,005). Kod pacijenta sa kolorektalnim tumorimasomatostatin je bio zna~ajno niì nego u kontrolnoj grupi (p<0,01). Dobijeni rezultati pokazuju da je jetra uklju-~ena u metabolizam somatostatina. Atrofi~ni gastritis, ulcerozni kolitis i M. Crohn, kao i kolorektalni tumori bili suudruèni sa zna~ajnim promenama u nivou somatostatina {to sugeri{e mogu}u patofiziolo{ku i terapeutsku ulogusomatostatina u ovim oboljenjima.

Klju~ne re~i: somatostatin, ciroza jetre, atrofi~ni gastritis, ulcerozni kolitis, M. Crohn, kolorektalni tumori

debelom crevu i 6 istovremeno na tankom i debelomcrevu).

Mogu}a veza izme|u somatostatina i kolorektal-ne neoplazije ispitivana je na 42 bolesnika, prose~nestarosti 60 godina. Dijagnoza je postavljena kolonos-kopski uz histopatolo{ku verifikaciju i to u 16 adeno-matozni polip, u 8 vilozni adenom i u 18 adenokarci-nom.

Svih 138 bolesnika kao i 40 kontrolnih osoba biloje hospitalizovano na Klinici za internu medicinu idetaljno ispitano (kompletne biohemijske analize i krvnaslika, rendgenska ispitivanja, ultrazvu~na dijagnostika iendoskopski pregledi uz histopatolo{ke analize).Izuzimaju}i grupu sa alkoholnom cirozom jetre, svimostalim bolesnicima isklju~ena su druga oboljenja, presvega bubre`na i hepatalna insuficijencija.

Svi ispitanici bili su bez medikamentne terapije iujutru na{te uzimana je krv za odre|ivanje bazalnihvrednosti somatostatina. Analiza je vr{ena RAI meto-dom uz kori{}enje laboratorijskog, eksperimentalnogtest reagensa (dobijenog iz Laboratorije HammersmithHospital u Londonu). Referentne vrednosti za somato-statin bile su 17’150 pmol/L.

U statisti~koj obradi kori{}eni su Studentov t testi druge uobi~ajene statisti~ke metode.

Rezultati

Alkoholna ciroza jetre

Bazalne vrednosti somatostatina u serumu bilesu zna~ajno vi{e kod svih bolesnika sa alkoholnomcirozom jetre u odnosu na kontrolnu grupu (p<0,01)(Tabela I). Vrednosti somatostatina u grupi bolesni-ka sa dekompenzovanim oblikom ciroze bile su zna-~ajno vi{e u odnosu na kompenzovani oblik (p<0,05)(Tabela I).

Perniciozna anemija i atrofi~ni gastritis

Svi bolesnici sa pernicioznom anemijom imali suhroni~ni atrofi~ni gastritis, a u 7 bolesnika histolo{ki jepotvr|ena i intestinalna metaplazija. Nivoi somatostati-na u serumu bili su zna~ajno niì u bolesnika sa perni-cioznom anemijom i hroni~nim atrofi~nim gastritisomu odnosu na kontrolnu grupu (p<0,01). Sve vrednostiu ispitivanih bolesnika bile su ispod normalnih vred-nosti za somatostatin. Kod bolesnika sa intestinalnommetaplazijom na|ene su zna~ajno niè vrednosti uodnosu na bolesnike sa samo hroni~nim atrofi~nimgastritisom (p<0,01) (Tabela II).

Ulcerozni kolitis i Kronova bolest

Ispitivanjem su utvr|ene zna~ajno vi{e vrednostisomatostatina u obe grupe bolesnika (105,5 ± 21,4pmol/L) u odnosu na kontrolnu grupu (50,3 ± 10,2pmol/L) (p<0,01). U odnosu na fazu bolesti vrednostibazalnog somatostatina bile u zna~ajno niè u akutnojfazi u odnosu na remisiju (p<0,005). U odnosu nalokalizaciju Kronove bolesti vrednosti somatostatinabile su zna~ajno vi{e u grupi sa promenama na tan-kom crevu (p<0,001) (Tabela III). Sve ispitivanevrednosti somatostatina bile su unutar referentnihvrednosti za somatostatin.


N20305040

Ispitivane grupeKompenzovana cirozaDekompenzovana cirozaSve ciroze jetreKontrolna grupa

Somatostatin, pmol/L44,0 ± 9,8 X

68,6 ± 20,1XX

50,7 ± 30,3**28,0 ± 10,1

Tabela I Nivoi somatostatina u plazmi bolesnika sa alkoholnom cirozom jetre

** p < 0,01 nivo statisti~ke zna~ajnosti razlika u odnosu na kontrolnu grupuX p < 0,05 nivo statisti~ke zna~ajnosti razlika bolesnika sa dekompenzo-

vanom cirozom jetre u odnosu na grupu sa kompenzovanomXX p < 0,01 nivo statisti~ke zna~ajnosti razlika bolesnika sa dekompenzo-

vanom cirozom jetre u odnosu na kontrolnu grupu

N158

7

40

Grupe bolesnikaPerniciozna anemijaSamo atrofi~ni gastritisAtrofi~ni gastritis i interstinalna metaplazijaKontrolna grupa

Somatostatin, pmol/L9,4 ± 10,6*

13,6 ± 12,1

3,2 ± 2,3**

49,5 ± 20,1

Tabela II Somatostatin u serumu bolesnika sa pernicioznom anemijom

* zna~ajna razlika (p<0,01) za bolesnike sa pernicioznom anemijom uodnosu na kontrolnu grupu

** zna~ajna razlika (p < 0,01) podgrupa perniciozne anemije sa i bez inesti-nalne metaplazije

N311714322156

40

Ispitivane grupeUlcerozni kolitis’ u akutnoj fazi’ u remisijiKronova bolest’ tanko crevo’ debelo crevo’ obaKontrolna grupa

Somatostatin, pmol/L

8,1 ± 4,4*39,9 ± 14,7

44,1 ± 15,2**28,7 ± 13,244,7 ± 2,919,5 ± 20,1

Tabela III Somatostatin u serumu bolesnika sa ulceroznim kolitisom i M. Crohn

* p < 0,005** p < 0,001

Kolorektalni tumori

U grupi bolesnika sa kolorektalnim adenokarci-nomom i viloznim adenomom vrednosti somatostati-na bile su zna~ajno niè u odnosu na kontrolnu grupu(p<0,01), ali unutar normalnih vrednosti.

Diskusija

Do sada saop{teni kontradiktorni rezultati u lite-raturi (3, 8) ovo ispitivanje ~ine aktuelnim. Rezultatipovi{enih bazalnih vrednosti somatostatina u skladusu sa pretpostavkom da se somatostatin metaboli{e ujetri, {to ne isklju~uje njegovu ulogu kao neurotrans-mitera i lokalnog endokrinog regulatora pa i mogu}-nosti da se metaboli{e na mestima gde se i osloba|a.Pove}anje somatostatina u cirozi jetre, a posebno de-kompenzovane, mogu}e je objasniti uticajem intra-hepati~ne holestaze, portalne hipertenzije i postoja-njem intra i ekstrahepati~nih {antova (10). Navedenerezultate treba posmatrati i u svetlu upotrebe somato-statina u farmakolo{kom tretmanu portalne hiperten-zije (11).

Poznato je prisustvo hipergastrinemije kod bo-lesnika sa pernicioznom anemijom i hroni~nim atro-fi~nim gastritisom, {to se obja{njava sekundarno po-ve}anim osloba|anjem gastrina zbog ahlorhidrije,uzrokovane upravo atrofi~nim gastritisom, a ne isklju-~uje se ni uloga imunolo{kih mehanizama (postojanjeautoantitela na gastrinske receptore) (14). Niske vred-nosti somatostatina mogu se objasniti pre pove}animodnosom G/D }elija {to je i dokazano, a {to sugeri{eda su niske vrednosti pre posledice ahlorhidrije, nego

tipi~nog efekta hipergastrinemije, jer je poznato dase somatostatin osloba|a pri niskim antralnim pH(15’17).

Nalaz zna~ajno niìh vrednosti somatostatina uakutnoj fazi bolesti je u saglasnosti sa ispitivanom li-teraturom (18). Tako|e ve}e vrednosti somatostatinau bolesnika sa intestinalnom lokalizacijom odraàvajumesto osloba|anja somatostatina uzrokovano najve-rovatnije nekroti~nim promenama. S druge strane odzna~aja je razmatranje upotrebe somatostatina u bo-lesnika sa te{kim oblicima kolitisa (18, 19).

Poslednjih godina otkriveno je prisustvo recepto-ra za somatostatin i druge peptide kod eksperimen-talnog kancera kolona (20). Tako|e potvr|ena je imu-noreaktivnost }elija serotonina, enteroglukagona, pep-tida YY i dr. (21, 22). Niè vrednosti somatostatina uzpovi{ene vrednosti gastrina, IGF-1 i drugih peptidauklapaju se u patofiziolo{ki koncept rasta kolorektalnihtumora, gde endokrini sistem o~igledno ima odre|enuulogu. S obzirom na navedene promene moglo bi serazmi{ljati o upotrebi neuroendokrinih peptida, pa isomatostatina u dijagnostici i terapiji kolorektalnogkancera (23).

Iz svega izloènog moè se zaklju~iti da poredpoznatog op{teg inhibitornog dejstva somatostatinana sekreciju neuroendokrinih tumora, kao i na sekre-ciju i motilitet gastrointestinalnog trakta, somatostatino~igledno ima ulogu i u patofiziologiji gastritisa, infla-matornih oboljenja creva, kolorektalnog kancera. Ovoje od posebnog zna~aja kada se razmatraju mogu}-nosti upotrebe somatostatina u terapijske svrhe.


SOMATOSTATIN LEVELS IN GASTROINTESTINAL DISEASE


1Clinical Hospital Centre »Dr Dragi{a Mi{ovi}« »Dedinje«, Belgrade2Institute for Biological Research »Sini{a Stankovi}«, Belgrade

Summary: Serum levels of somatostatin were determined by RIA method in 50 patients with liver cirrhosis,15 with pernicious anemia and atrophic gastritis, 31 with inflamatory bowel disease, 32 with colorectal tumorsand in 40 control persons. In patients with liver cirrhosis somatostatin levels were significantly higher than in con-trol group (p<0.01). Patients with pernicious anemia and ulcerative colitis in acute phase of the disease had sig-nificantly lower levels of somatostatin (p<0.01) (p<0.005). In patients with colorectal tumors somatostatin weresignificantly lower then in control group (p<0.01). Our results show that the liver is involved in somatostatinmetabolism. Atrophic gastritis, ulcerative colitis and M.Crohn, so as colorectal neoplasia were associated with sig-nificantly changes in somatostatin levels which suggest the potential pathophysiologic and terapeutic role ofsomatostatin in those disease.

Key words: somatostatin, liver cirrhosis, atrophic gastritis, ulcerative colitis, M. Crohn, colorectal tumors

Literatura

1. Gerich JE, Paton GS. Somatostatin. Med Clin North Am1978; 62, 375’ 83.

2. Conlon JM, Whittaker J, Hammond V, Alberti KGM.Metabolism of somatostatin and its analogues by theliver. Biochim Biophys Acta 1981; 677: 234’ 2.

3. Sacks H, Cass TL. Clearance of imunoreactive somato-statin by rat liver. J Clin Invest 1981; 67: 419’29.

4. Kosti} N. Regulatorni peptidi (patofiziologija i klini~kizna~aj). Zavod za ud`benike i nastavna sredstva, Beo-grad, 1999.

5. Di Lorenzo C, Lucanto C, Flores AF, Idries S, HymanPE. Effect of sequential erythromycin and octreotide onantroduodenal manometry. J Pediatr Gastroenterol Nutr1999; 29 (3): 293’6.

6. Eliakim R, Fan QX, Babyatsky MW. Chronic nicotineadministration differentially alters jejunal and colonicinflammation in interleukin-10 deficient mice. Eur JGastroenterol Hepatol 2002; 14 (6): 607’14.

7. Sadij-Ouatas Z, Lasfer M, Julien S, Feldmann G, Rayl-Desmars F. Doxorubicin and octreotide induce a 40 kDabreakdown product of p53 in human hepatoma andtumoral colon cell lines. Biochem J 2002; 15: 364 (Pt 3):881’5.

8. Sheppard M, Shapiro B, Pimstone B, Kronhein S. Me-tabolic clearance and plasma half disappearance time ofexogenous somatostatin in man. Clin Endocrinol Me-tabol 1979; 48: 50’6.

9. Kelback H, Tronier B, Bahnsen M, Munkgard S. Fastingplasma somatostatin in alcoholic liver disease. Scand JClin Lab Invest 1983; 43: 597’ 601.

10. Munkgaard S, Kelbeek H, Tronier B. Elevated plassomatostatin in cirrhosis of the liver. N Engl J Med 1981;301: 1429’30.

11. Gonzales-Abraldes J, Bosch J, Garcia-Pagan JC. Phar-macological tretment of portal hypertension. Curr OpinInvestig Drugs 2001; 2 (10): 1407’13.

12. Matrella E, Valatas V, Notas G, Roumpaki H, Xidakis C,Hadzidakis A, et. al. Bolus somatostatin but not octreo-tide reduces hepatic sinusoidal pressure by a NOin-dependent mechanism in chronic liver disease. AlimentPharmacol Ther 2001; 15 (6): 857’ 64.

13. Zhang HB, Wong BC, Zhou XM, Guo XG, Zhao SJ, WangJH, et. al. Effects of somatostatin, octreotide and pitre-ssin plus nitroglycerine on systemic and portal hae-modynamics in the control of acute vericeal bleeding. IntJ Clin Pract 2002; 56 (6): 447’51.

14. Aizupurua HJ, Ungar B, Toch BH. Autoantibody to thegastrin receptor in pernicious anemia. N²Engl J Med1985; 313: 479’ 83.

15. Ligumsky M, Wengrower D, Karmeli F, Rachmilewitz D.Somatostatin release by human gastric mucosa. ScandJ Gastroenterol 1988; 23: 687’90.

16. Arnold R, Hulst MV, Neuhof CH, Schwarting H, BekerHD, Creutzfeld W. Antral gastrin-producting G-cells andsomatostatin producing ’ D-cells in different states ofgastric acid secretion. Gut 1982; 23: 285’91.

17. Alonso Falcon F, Codoceo Alquinta R, Polanco Allue I,Aquado Gil A, Fontan Casariego G. Study of gastro-intestinal polypeptides controlling gastric acid secretionin patirnts with primary antibody deficiency. Rev EspEnferm Dig 1999 1; 91 (1): 54’ 60.

18. van Bergeijk JD, Wilson JH, Nielsen OH, von Triptiz C,Karvonen AL, Lygren I, et. al. Octreotide in patients withactive ulcerative colitis with high dose corticosteroids.Eur J Gastroenterol Hepatol 2002; 14 (3): 243’8.

19. Su X, Burton MB, Gebhart GF. Effect of octreotide onresponses to colorectal distension in the rat. Gut 2001;48 (5): 676’82.

20. Szepeshazi K, Schally AV, Halmos G, Armatis P, HebertF, Sun B., et. al. Targeted cytotoxic somatostatin ana-logue AN-238 inhibits somatostatin receptor-positiveexperimental colon cancers independently of their p53status. Cancer Res 2002 1; 62 (3): 781’8.

21. Sitohy B, El-Salhy M. Colonic endocrine cells in rats withchemically induced colon carcinoma. Histol Histopathol2001; 16 (3): 833’8.

22. Cox HM, Tough IR, Zandvliet DW, Hollidey ND. Con-stitutive neuropeptide Y Y(4) receptor expression in hu-man colonic adenocarcinoma cell lines. Br J Pharmacol2001; 132 (1): 345’3.

23. Smith-Jones PM, Bischof C, Leimer M, Gludovacz D,Angelberger P, Pangerl T, et. al. DOTA-lanreotide: anovel somatostatin analog for tumor diagnosis and the-rapy. Endocrinology. 1999; 140 (11): 5136’ 48.





• 8 ’12 maj 2005, Glasgow

16th IFCC-FESCC European Congress of Clinical Chemistryand Laboratory Medicine

Internet: www.glasgow2005.org

• 24’29 jul 2005, Orlando, FL, USA

19th International Congress of ClinicalChemistry and Laboratory Medicine (IFCC Congress)

57th National Meeting of the American Association of Clinical Chemistry (AACC)

Informacije: Secr.: AACC Customer Service, 2101 LStreet NW, Suite 202, Washington, DC20037’1526, USA

Tel: + (1) 202 587 0717; Fax: + (1) 202 833 4576;E-mail: custserv¤aacc.orgInternet: www.aacc.org

Jugoslov Med Biohem 23: 397, 2004 Obave{tenjaTechnical reports

UC 577,1; 61 ISSN 0354-3447

SASTANCI U OBLASTI KLINI^KE HEMIJE

DRU[TVO MEDICINSKIH BIOHEMIÂRA SRBIJE I CRNE GORE

Farmaceutski fakultet Vojvode Stepe 45011 221 Beograd

Pristupnicu popuniti {tampanim slovima, i zajedno sa kopijom petog primerka dostaviti na navedenu adresu!

P R I S T U P N I C AU ^LANSTVO DMBSCG

Ime i prezime:

Adresa:

Telefon:

Datum:

Kategorija ~lanstva:

REDOVNO ^LANSTVO

PRIDRU@ENO ^LANSTVO

STUDENTI I PENZIONERI ’

Potpis:

^lanarina se upla}uje za teku}u godinu na teku}i ra~un DMBSCG broj 255-0006390101000-02, Privredna bankaBeograd a.d., Beograd i obezbe|uje prijem ~asopisa »Jugoslovenska medicinska biohemija« kao i svih obave{tenjao aktivnostima Dru{tva medicinskih biohemi~ara Srbije i Crne Gore.

1 200,00 din

15 000,00 din

BESPLATNO



JUGOSLOVENSKA MEDICINSKA BIOHEMIJAje ~lan

Dru{tva biomedicinskih ~asopisa i biltena SR Jugoslavije

Dru{tvo biomedicinskih ~asopisa i biltena SR Jugoslavije

Yugoslav Society of Biomedical Journals & Bulletins

Adresa:

Dru{tvo biomedicinskih ~asopisa i biltena SR Jugoslavije,Medicinski fakultet, Zavod za farmakologiju, Pregradak 380,

21 000 Novi Sad, Srbija i Crna Gora. Telefon: (021) 22’ 172; Faks: (021) 615’771

Mailing Address:

Yugoslav Society of Biomedical Journals & Bulletins,Medical School, Department of Pharmacology, P.O.B. 380,

21 000 Novi Sad, Serbia and Montenegro. Phone: 381’ 21’ 22 172; Fax: 381’ 21’ 615 771

UPUTSTVO AUTORIMA

»Jugoslovenska medicinska biohemija« objavljuje ori-ginalne nau~ne i stru~ne radove iz oblasti klini~kehemije, medicinske biohemije i drugih srodnih disci-plina u kojima se primenjuju hemija, biohemija, mo-lekularna biologija i imunohemija za izu~avanje nor-malnih i patolo{kih procesa u humanom organizmu.Na predlog recenzenata radovi se klasifikuju u sle-de}e stalne kategorije ~asopisa: a) li~ni stav, b) pre-gledni ~lanci, c) originalni nau~ni radovi, d) stru~niradovi, e) predhodna saop{tenja, i f) izlaganja sanau~nih skupova. âsopis objavljuje obave{tenja,prikaze knjiga, izve{taje o radu DMBSCG, IFCC-a idrugih srodnih organizacija, pisma uredni{tvu, kao iinforma-cije o novinama u oblasti proizvodnje reage-nasa i in-strumenata iz oblasti klini~ke hemije.

Svi radovi se {alju Uredni{tvu Jugoslovenske medi-cinske biohemije, Zavod za medicinsku biohemiju,Farmaceutski fakultet, Vojvode Stepe 450, 11221, Beo-grad, Fah 146.

Prispele radove Redakcijski odbor {alje dvojici re-cenzenata anonimno. Rukopisi koji ne zadovoljavajuutvr|ene kriterijume vrati}e se autorima na preradu,ako tako predloè recenzenti.

Prvi otisak ~lanka se {alje autoru radi ispravke.Rukopisi se ne vra}aju.

1. Tekst radaîtav rad, uklju~uju}i sve priloge treba poslati u dup-likatu i to original i odgovaraju}u fotokopiju rada.Original mora da sadrì sve priloge izra|ene nana~in opisan pod 2. Rad treba da bude otkucan sadvostrukim proredom, tako da na jednoj stranici budenajvi{e 30 redova; s leve strane treba ostaviti beli rub{irok oko 4 cm. Pregledni ~lanci i originalni radovimogu da imaju do 15 kucanih strana, a ostali doosam strana sa svim prilozima. Radovi moraju da ima-ju slede}i sadràj:

1.1. Naslov rada treba da bude jasan i {to kra}i,otkucan na posebnom listu papira s imenima autorakoji se navode prvo punim imenom, zatim prezime-nom. Ispod toga treba navesti ta~an naziv ustanoveu kojoj je rad ura|en. Na dnu stranice treba napisatiadresu autora sa kojim }e se vr{iti korespodencija.

1.2. Na posebnoj stranici se prilaè kratak sadràj,koji ne sme da bude duì od 120 re~i. Ne sme bitiopisan, ve} mora da sadrì sve bitne ~injenice izneteu radu: kratak i precizan prikaz problema, metode ra-da, bitne rezultate i osnovne zaklju~ke.

1.3. Kratak sadràj na engleskom jeziku se tako|eprilaè na posebnoj stranici papira. Treba da sadrì inaslov rada na engleskom jeziku.

1.4. Klju~ne re~i se navode ispod teksta kratkogsadràja bilo da je na srpskom ili engleskom jeziku.Treba navesti od 2–5 klju~nih re~i koje su zna~ajneza brzu identifikaciju i klasifikaciju sadràja rada.

1.5. Uvod rada treba pisati jasnim jezikom uz na-vo|enje su{tine problema i svrhe istraìvanja. U uvo-du se navode poznati radovi koji su u vezi sa opi-sanom problematikom.

1.6. U eksperimentalnom delu treba detaljno opi-sati kori{}ene materijale i matode. Ako su primenjenemetode poznate u literaturi nije ih potrebno detaljnoopisivati, ve} navesti izvorni literaturni podatak. Ako seu radu prikazuje nova metoda ili modifikacija po-znate metode potrebno ih je detaljno opisivati. Ako jeu radu primenjena statisti~ka analiza rezultata potre-bno je navesti koje su metode primenjene.

1.7. Rezultate treba jasno i precizno prikazati.Zna~ajnost rezultata treba obraditi statisti~ki. Rezulta-te treba izraàvati u skladu sa me|unarodnim mernimsistemom (SI).

1.8. U diskusiji treba interpretirati dobijene rezultatei njihovo pore|enje sa postoje}im rezultatima prema na-vedenoj literaturi. Na osnovu ovako upore|enih rezulta-ta treba iskazati zaklju~ke do kojih se u radu do{lo.

1.9. U celini rad treba pisati u tre}em licu i izbe-gavati pasivne glagolske oblike.

2. Tabele i slike

Rad treba da sadrì razuman broj slika i tabela.2.1. Tabele se pi{u na posebnom listu papira.

Ozna~avaju se rimskim brojevima. U zaglavlju tabeletreba napisati kratak informativan opis (legendu). Utabelama ne treba koristiti skra}enice, osim uobi~aje-no usvojenih prema nomenklaturi i SI sistemu. Utekstu treba nazna~iti mesto gde dolazi odgovaraju}atabela.

2.2. Ilustracije (slike, crteì) se ozna~avaju arap-skim brojevima prema redosledu kako se u tekstupojavljuju (u tekstu treba nazna~iti mesto ilustracije).Na posebnom listu papira redom treba navesti svelegende (opise) priloènih ilustracija.Prilaù se crno-bele fotografije na sjajnom, kvalitet-nom papiru. Na pole|ini slike treba mekom olovkomnapisati broj slike i naslov rada i ime autora. Crteì seizra|uju tu{em na paus papiru i prilaù se originalu.Slova i znakovi moraju da budu jasni, jednake veli-~ine i odgovaraju}ih proporcija za {tampu. Na vrhucrteà treba napisati ime prvog autora i pomo}ustrelice ozna~iti vrh slike ili crteà.

3. Podaci o literaturi

Popis literature se pi{e na posebnom papiru premaredosledu javljanja u tekstu.Literatura se u tekstu ozna~ava arapskim brojevima uzagradi, prema redosledu pojavljivanja. U popisu ci-tirane literature podatke pore|ati po redosledu pokojem se prvi put pojavljuje u tekstu. Za naslove ~a-sopisa koristiti skra}enice prema Index Medicusu (Listof Journals Indexed). Jugoslovenski ~asopisi koji se neindeksiraju u ovoj publikaciji skra}uju se na osnovuListe skra}enih naslova jugoslovenskih serijskih publi-kacija. Vankuverska pravila precizno odre|uju redo-sled podataka i znake interpunkcije kojima se oniodvajaju, kako je u nastavku dato u pojedinim pri-merima. Navode se svi autori; ukoliko ih je preko {est,navesti prvih {est i dodati »et. al.«.


Primeri:

^lanci u ~asopisima

• Standardni ~lanakGoate AM, Haynes AR, Owen MJ, Farral M James LA, Lai LY,et al. Predisposing locus for Alzheimer's disease on chro-mosome 21. Lancet 1989; 1: 352–5.

• Organizacija kao autorThe Royal Marsden Hospital Bone-marrow TransplantationTeam. Failure of bone-marrow graft without preconditi-oning in posthepatitis marrow aplasia. Lancet 1977; 2:742–4.

• Nisu navedena imena autoraCoffee drinking and cancer of the pancreas (editorial).BMJ 1981; 283: 628.

• Volumen sa suplementomMagni F, Rossoni G, Berti F. BN-52021 protects guinea pigfrom heart anaphylaxis. Pharmacol Res Commun 1988; 20Suppl 5: 75 –8.

• Sveska sa suplementomGardos G, Cole JO, Haskell D, Marby D, Pame SS, Moore P.The natural history of tardive dyskinesia. J Clin Psycho-pharmacol 1988; 8 (4 Suppl): 31S–37S.

Knjige i druge monografije

• Jedan ili vi{e autoraEisen HN. Immunology: an introduction to molecular andcellular principles of the immune response. 5th ed. NewYork: Harper and Row, 1974: 406.

• Urednik(ci) kao autorDanset J, Colombani J, eds. Histocompatibility testing 1972.Copenhagen: Munksgaard, 1973: 12– 8.

• Poglavlje u knjiziWeinstein L, Shwartz MN. Pathologic properties of inva-ding microorganisms. In: Soderman WA Jr, Soderman WA.eds. Pathologic physiology: mechanisms of disease. Phi-ladelphia: Saunders, 1974: 457–72.

• Rad u zborniku radovaHarley NH. Comparing radon daughter dosimetric and riskmodels. In: Gammage RB. Knye SV. eds. Indoor air and hu-man health. Proceedings of the Seventh Life Sciences Sym-posium: 1984 Oct 29-31; Knoxville (TN). Chelsea (MI): Lewis.1985: 69–78.

• Disertacije i tezeCairns RB. Infrared spectroscopis studies of solid oxygen.Dissertation. Berkeley, California; University of California,1965.

UURREEDDNNII[[TTVVOO


INSTRUCTIONS TO AUTHORS

»Jugoslovenska medicinska biohemija« is the journalpublishing scientific and specialized articles on all as-pects of clinical chemistry, medical biochemistry andrelated scientific disciplines where chemistry, bioche-mistry, molecular biology and immunochemistry aredealing with the study of normal and pathologic pro-cesses in human beings. All manuscripts are reviewedand after final decision are classified in the followingcategories: a) personal view, b) review articles, c) ori-ginal papers, d) professional papers, e) preliminaryreports, and f) review of scientific meetings. There arealso different reports and news, book reviews, reportson the activity of the Society of Medical Biochemistry of Serbia and Montenegro, IFCC and other related orga-nizations, letters to the editor, and information aboutinnovations, new reagents and instruments in the fieldof clinical chemistry.

All manuscripts should be addressed to:The Editor, »Jugoslovenska medicinska biohemija«,

Department of Medical Biochemistry, University Schoolof Pharmacy, Vojvode Stepe 450, 11 221 Belgrade, P.O.Box 146, Serbia and Montenegro.

All manuscripts will be reviewed by two anonymousreviewers. Manuscripts which do not satisfy the pro-posed criteria will be returned to the author for adap-tation to reviewers suggestions. Final decision for pu-blishing will be made by the Editorial Board.

The author will receive first proofs for correction.Manuscripts are not returned.

1. Manuscript Preparation

The complete manuscript, including enclosures,should be sent in two copies (original and a photo-copy). Enclosures to original copy should be prepa-red according to instructions given in section 2. Themanuscript should be typed or printed double-spa-ced (30 lines on a page), with a 4 cm left margin.Review articles and original papers should not exceed15 pages and other articles 8 pages, including allenclosures. The manuscript has to be arranged asfollows:

1.1. The title should be short and clear, and typedon the separate sheet, with full names of authors, fo-llowed by the name of institution. Exact postal ad-dress of the author to whom communications shouldbe sent is typed at the bottom.

1.2. A summary should be short and clear, typedon the separate sheet, not exceeding 120 words. Thesummary should point to the problem, methods, resultsand discussion.

1.3. A short summary in Serbian language shouldbe typed on the separate sheet, beginning with theSerbian title.

1.4. At the end of the Serbian and English sum-maries up to five key words should be written for in-dexing purposes.

1.5. Introduction should be clear, pointing to theessence of the problem and the purpose of thestudy. References related to the problem discussed inmanuscript should be cited.

1.6. The experimental part should include descrip-tion of material and methods used. If methods arewidely known, they should not be described, but onlyreferences indicated. If the article deals with a newmethod or modified method, full description shouldfollow. Methods used in statistical analyses should beindicated.

1.7. Results should be precise and clear, statisti-cally processed and expressed according to the In-ternational System of Units (SI).

1.8. Results should be discussed and compared toreference results. Conclusions should be drawn on thebasis of these comparisons.

1.9. The manuscript should be written in the thirdperson and passive tense avoided.

2. Tables and figures

The number of tables and figures should be rati-onal.

2.1. Tables are typed on separate sheets, with thetable number in roman numerals, and title centeredabove the table and explanatory note below thetable. Abbreviations are not used in the tables, ex-cept the accepted nomenclature and SI system.Place of tables in the text should be indicated.

2.2. Figures and graphs are submitted on sheetsseparate from the text, with the figures and graphsnumber in arabic numerals, and place in the textindicated. Figure and graph legends are typed onsheets separate from the text.

Black and white photographs of good quality aresubmitted. The first author’s name, figure number andtop location are indicated on the back of each illustra-tion.

Original drawings, made in India ink on tracingpaper, are submitted. Letter symbols and signs shouldbe bright, of equal size and appropriate printing pro-portions. The first author’s name, drawing number andtop location are indicated on the back of illustration.

3. References

References are typed on sheets separate from thetext and follow the text. References are identified inthe text by arabic numerals in parentheses, and num-bered consecutively in the order in which they arementioned in the text. If one reference is cited seve-ral times in the text, the same number is indicated inparentheses.

Citations such as »personal communication«, »un-published data« or »in press« are not accepted. Ab-breviations of journals conform to those used in IndexMedicus (List of Journals Indexed). After the volume,first and last page of the cited article are indicated,



followed by the year of publication. Vancouver rulesprecisely determine the order of data and punctua-tion marks as given in examples. List all authors if six orless; otherwise list first six and add »et al.«.

Periodicals:

• Example for articles in journalsGoate AM, Haynes AR, Owen MJ, Farral M James LA, LaiLY, et al. Predisposing locus for Alzheimer’s disease onchromosome 21. Lancet 1989; 1: 352– 5.

• Example for institution as authorThe Royal Marsden Hospital Bone-marrow TransplantationTeam. Failure of bone-marrow graft without preconditi-oning in posthepatitis marrow aplasia. Lancet 1977; 2:742–4.

• Example for articles without authors' namesCoffee drinking and cancer of the pancreas (editorial).BMJ 1981; 283: 628.

• Example for volume with supplementMagni F, Rossoni G, Berti F. BN-52021 protects guinea pigfrom heart anaphylaxis. Pharmacol Res Commun 1988;20 Suppl 5: 75– 8.

• Example for number with supplementGardos G, Cole JO, Haskell D, Marby D, Pame SS, Moore P.

The natural history of tardive dyskinesia. J Clin Psycho-pharmacol 1988; 8 (4 Suppl): 31S –37S.

Books and monographs

• Example for one or more authorsEisen HN. Immunology: an introduction to molecular andcellular principles of the immune response. 5th ed. NewYork: Harper and Row, 1974: 406.

• Example for editor(s) as author(s)Danset J, Colombani J, eds. Histocompatibility testing 1972.Copenhagen: Munksgaard, 1973: 12– 8.

• Example for chapter in a bookWeinstein L, Shwartz MN. Pathologic properties of inva-ding microorganisms. In: Soderman WA Jr, Soderman WA.eds. Pathologic physiology: mechanisms of disease. Phi-ladelphia: Saunders, 1974: 457–72.

• Example for articles in the proceedingsHarley NH. Comparing radon daughter dosimetric and riskmodels. In: Gammage RB. Knye SV. eds. Indoor air andhuman health. Proceedings of the Seventh Life SciencesSymposium: 1984 Oct 29– 31; Knoxville (TN). Chelsea (MI):Lewis. 1985: 69–78.

• Example for dissertation and thesisCairns RB. Infrared spectroscopic studies of solid oxygen.Dissertation. Berkeley, California; University of California, 1965.

EEDDIITTOORRIIAALL BBOOAARRDD

Documents

UC 577,1; 61 ISSN 03543447 JUGOSLOVENSKA ......JUGOSLOVENSKA MEDICINSKA BIOHEMIJA YUGOSLAV MEDICAL BIOCHEMISTRY Adresa uredni{tva Jugoslovenska medicinska biohemija, Dru{tvo medicinskih