nau^ni odbor - dmbs · 2009-06-03 · medicinski biohemi~ari su u svom domenu rada pod-vrgnuti...

NAU^NI ODBOR

PredsednikVidosava \or|evi}

PotpredsednikJovan Kavari}

^lanoviNada Majki}-SinghGordana Bjelakovi}Jasmina Mimi}-Oka

Branislava Brki}Svetlana Ignjatovi}

Danica Pribilovi}-Popovi}Mirka Ili}

ORGANIZACIONI ODBOR

PredsednikNada Majki}-Singh

Potpredsednik Olivera Jankovi}

Sekretar Tatjana Vodnik

^lanoviAleksandra Po{ti}-Grujin

Dejan Krivokapi}Vera Cvetkovi}Borislav Nal~i}Janko Pejovi}

LOKALNI ORGANIZACIONI ODBOR

Ivana Stojanovi}Tatjana Jevtovi}

Tatjana Cvetkovi}Vladan ]osi}

Lilika Zvezdanovi}Slavica Kundali}

ADRESA ORGANIZACIONOG ODBORA

Institut za medicinsku biohemijuKLINI^KI CENTAR SRBIJE

Vi{egradska 26, 11000 Beograd

Tel/Fax: 011 361‡56‡31Telefoni: 011‡361‡7777, lok. 36-56 i 33-62

(Aleksandra Po{ti}-Grujin i tehni~ki sekretar DMBJ Cica Lazarevi})

86 Jugoslov. Med. Biohem. 2002.; 21 (2)

ULOGA LABORATORIJSKE MEDICINE ZASNOVANE NA DOKAZIMA U ZDRAVSTVENOJ SLU@BI

N. Majki}-Singh

Institut za medicinsku biohemiju, Klini~ki centar Srbije, Beograd

Laboratorijska medicine zasnovana na dokazi-ma (eng. evidence-based laboratory medicine,EBLM) koristi najbolje dokaze koji se dobijaju u vidurezultata laboratorijskih testova za dono{enje odlukeo zbrinjavanju svakog pojedina~nog pacijenta. Ovajpristup je mogu} jedino na osnovu objedinjavanjalaboratorijskih i klini~kih iskustava o na~inu le~enjakoja proizilaze iz sistematskih istraìvanja u ovimoblastima. To zna~i da se definicija EBLM zasniva nadva elementa: iskustvu i dokazima koji poti~u iz sis-tematskih istraìvanja. Mada izraz medicina zasno-vana na dokazima (eng. evidence-based medicine,EBM) poti~e sa kanadskog Mc Master Univerziteta odgrupe koju je predvodio Dr Garad Guyatt, mnogi po-laù pravo na kreiranje ovog izraza i njegove su{tine.Shodno ovome, brojni su faktori koji su poslednjih 30godina uticali na formiranje mi{ljenja i pokreta poz-natog pod imenom Evidence-Based Medicine i skra-}enicom EBM. Jedan od faktora koji uti~u na formi-ranje navedene filozofije je da se svaki lekar susre}esa brojnim medicinskim informacijama koje poti~u izrazli~itih izvora, drugi faktor je globalni fenomenpove}avanja tro{kova le~enja, a tre}i je da su danaspacijenti i sami dovoljno edukovani da zahtevajunajbolju dijagnostiku i le~enje. To zna~i da se medi-cina koja se zasniva na dokazima mora oslanjatina sve tri napred navedene pretpostavke. Protokolikoji se pripremaju za le~enje obolelih moraju uzeti uobzir efikasnost i efektivnost zdravstvene slu`be. Dabi se ovo ostvarilo laboratorijski stru~njaci morajupa`ljivo odabirati laboratorijske testove, pomo}ukojih }e se dobiti najvi{e klini~kih informacija. Prematome, laboratorijska medicina zasnovana na do-kazima ima za cilj da podràva klini~ku dijagnozu izbrinjavanje oboljenja primenom novih znanja kojadovode do standardne procedure koja }e najboljeomogu}iti laboratorijsko ispitivanje. U laboratorijskojmedicini broj testova stalno se pove}ava; uvode senovi testovi, me|utim stari testovi se retko skidaju saprograma. S obzirom da su sredstva zdravstvenih

Jugoslov. Med. Biohem. 2002.; 21 (2) 99

THE ROLE OF EVIDENCE-BASED LABORATORY MEDICINE

IN HEALTH SERVICE

N. Majki}-Singh

Institute of Medical Biochemistry, Clinical Centre of Serbia, Belgrade

Evidence-based laboratory medicine (EBLM) isthe use of the current best evidence of the utility of la-boratory tests in making decisions about the care ofindividual patients. This practice means integratinglaboratory and clinical experience with the last avai-lable external evidence from systematic research. Itmeans that the definition of EBLM focuses on twokey elements: experience and evidence from syste-matic research. Although the term evidence-basedmedicine (EBM) was created in Canada at Mc MasterUniversity by a group led by Dr Gard Guyatt, there arevarious claims as to the origin of its practice.Regardless of its origins, many factors have cometogether over the past 30 years to drive the move-ment to EBM. One factor is those individual physi-cians, faced with numerous medical informations;the second factor is the global phenomenon ofincreasing health care costs and third is that patientswho have generaly more education, want the best indiagnostics and therapies. It means that evidence-based medicine has been driven by the need to copewith information overload, by cost-control, and bypublic impatient for the best in diagnostics and treat-ment. Clinical guidelines care maps, and outcomemeasures are quality improvement tools for theappropriateness, efficiency, and effectiveness ofhealth services. Laboratory professionals must directmore effort to demonstrating the impact of laborato-ry tests on a greater variety of clinical outcomes.Evidence-based laboratory medicine aims to adviseclinical diagnosis and management of diseasethrough systematic researching and disseminatinggeneralisible new knowledge that meets the standardof critical review on clinically effective practice of lab-oratory investigations. In laboratory medicine, theuse of tests increases; new tests are constantly intro-duced, but »old« tests are seldom removed from therepertoire. This, together with limited public funds forthe health care, should underline the challenge forlaboratory professionals to provide evidence for the

UC 577,1; 61 ISSN 0354-3447

Jugoslov. Med. Biohem. 21: 99–104, 2002 Predavanja

Lectures


fondova ograni~ena, name}e se potreba izbora onihtestova koji }e pruìti najvi{e dijagnosti~kih informa-cija. Dijagnostika se mora izvoditi u laboratoriji kojaje osigurala dobru laboratorijsku praksu u pre-anali-ti~koj, post-analiti~koj i analiti~koj fazi laboratorijskogdijagnosti~kog procesa. Shodno navedenom ovo pre-davanje }e se baviti izborom, metodologijom i faza-ma laboratorijske medicine zasnovane na dokazi-ma.

NOVE TEHNOLOGIJE U KLINI^KOJ HEMIJI

L. M. Lorens, R. I. Mekonel, J. V. Lamont, S. P. Ficdèrald

Kompanija Randox Laboratories, Crumlin, Co Antrim, Severna Irska

Upotreba novih tehnologija dovodi do razvoja no-vih proizvoda za dijagnozu oboljenja. Najva`niji faktorkoji uti~e na razvoj novih proizvoda iz oblasti in vitrodijagnostike je cena. Zdravstvene organizacije poste-peno su smanjile budète za laboratorijske testove,zbog ~ega su i klini~ke laboratorije prinu|ene daumanje tro{kove. Osim {to je neophodno da im seponude razumne cene, klini~ke laboratorije zahtevajutestove koji su laki za upotrebu, brzo daju rezultate,ta~ni su i precizni do najmanjeg detalja. Takvi zahte-vi su podelili trì{te in vitro dijagnostike na dva sek-tora ’ automatizacija i testiranje uz postelju pacijenta.Pokazalo se da je najefikasniji na~in da se zadovoljenavedene potrebe, potpuno razvijen, automatizovanisistem koji moè da obuhvati celokupni proces; odpunjenja uzorka do krajnjih analiza i dijagnoza. Teh-nologije koje su zaslu`ne za novine u automatizacijisu progres u oblasi elektronskog interfejsa, robotika,nanotehnologija, mikrofabrikacija, sistemi detekcije ive{ta~ke neuralne mreè. Osim novina u sistemima,tako|e se pojavljuje i ~itav niz novih testova. Oni surezultat napredovanja u oblasti genomike, pro-teomike i od nedavno, teranostike. Deo industrije invitro dijagnostike koja nije uklju~ena u visoku auto-matizaciju, koristi se sve vi{e u oblasti testiranja uzpostelju pacijenta. Tehnologije koje u~estvuju u na-pretku ove oblasti su imunohromatografija, mikroele-ktronika i neinvazivne tehnologije. Ova prezentacija}e omogu}iti uvid u nove tehnologije iz oblasti invitro dijagnostike, s posebnim akcentom na restruk-turiranje klini~kih laboratorija uz pomo} automatiza-cije i testiranja uz postelju pacijenta.

utility of different tests. This practice means integra-ting laboratory and clinical experience with the bestavailable external evidence from systematic research.Therefore, it is important that advice given by labora-tory medicine professionals are sound and based onevidence in the pre-analytical, analytical, and post-analytical phases of the diagnostic process. The lec-ture provides an insight into the rationale, methodo-logy and the phases of the EBLM.

NEW TECHNOLOGIES IN CLINICALY CHEMISTRY

L. M. Lawrens, R. I. McConnell, J. V. Lamont, S. P. FizGerald

Randox Laboratories Ltd., 55 Diamond Road,Crumlin, Co Antrim BT29 4QY, Northern Ireland

The application of new technologies is leading tonew products for the diagnosis of human disease.The most significant factor influencing the develop-ment of new in vitro diagnostic (IVD) products iscost. Healthcare organizations have gradually redu-ced the amounts they are willing to pay for laborato-ry tests and this has put pressure on clinical labora-tories to reduce their charges. In addition to reducedcosts, clinical laboratories require tests that are easyto use, give rapid results and do not compromise onaccuracy or reliability. Such cost-containment pres-sures have divided the IVD market into two dominat-ing sectors’automation and point-of-care. The deve-lopment of totally automated systems that can han-dle everything from sample loading to final analysisand diagnosis has been one of the most effectiveways of meeting these challenges. Technologieswhich have driven developments in automation inclu-de advances in electronic interfaces, robotics, nan-otechnology, microfabrication, detection systemsand artificial neural networks. In addition to the sys-tems themselves, a wider range of tests are alsoemerging. These are being driven by advances in ge-nomics, proteomics and, more recently, theranos-tics. The proportion of the IVD industry not engagedin high-volume automation is becoming increasinglydevoted to moving testing as close as possible to thepoint-of-care. Techologies underpinning these deve-lopments include immunochromatography, micro-electronics and noninvasive technologies. This pres-entation will provide an overview of new techologiesemerging in the field of IVD and will outline the sig-nificant implications that automation and point-of--care testing will have on the inevitable restructuringof clinical laboratories.

KVALITET ANALITI^KIH ODRE\IVANJA U MEDICINSKOJ BIOHEMIJI: MERENJE NESIGURNOSTI

S. Ignjatovi}

Institut za medicinsku biohemiju, Klini~ki centar Srbije i Farmaceutski fakultet,

Univerzitet u Beogradu

Veliki broj medicinskih odluka zasniva se na rezul-tatima analiti~kih odre|ivanja u klini~ko-biohemijskimlaboratorijama. Iz ovih razloga je neophodno da seima uvid u kvalitet dobijenih rezultata. Jedan od os-novnih uslova za klini~ko-biohemijske laboratorijepredstavlja implementacija mera kontrole kvalitetarada kako bi se omogu}ilo dobijanje rezultata zado-voljavaju}eg kvaliteta. U ove mere spadaju: kori{}enjeproverenih analiti~kih metoda odre|ivanja, primenata~no definisanog postupka unutra{nje kontrolekvaliteta rada, u~e{}e u programima spolja{nje kont-role kvaliteta rada, akreditacija zasnovana na stan-dardima ISO 17025, kao i utvr|ivanje »traceability«rezultata odre|ivanja. Kao posledica ovih zahteva,medicinski biohemi~ari su u svom domenu rada pod-vrgnuti stalnom pritisku da demonstriraju kvalitet svo-jih rezultatata, kao i spremnost u pru`anju podatakao njihovoj pouzdanosti. To uklju~uje o~ekivani stepenslaganja rezultata sa drugim rezultatima i ne zavisi odprimenjene analiti~ke metode. Odre|ivanje nesig-urnosti (engl. uncertainty,) smatra se korisnom me-rom ovog slaganja. Nesigurnost odre|ivanja uklju-~uje vi{e komponenti. U praksi, nesigurnost rezultatapoti~e od nedovoljne definisanosti uslova odre|ivan-ja, uzorkovanja, uticaja vezanih za »matriks«, interfer-encije, uslova u kojima se vr{i odre|ivanje, nesig-urnosti opreme kojom se meri zapremina ili masa,referentnih vrednosti, aproksimacija i pretpostavkikoje su inkorporirane u metodu i proceduru odre|i-vanja, kao i slu~ajnih varijacija. Mogu}nost primeneanaliti~kih metoda za rutinska odre|ivanja procenjujuse postupkom »validacije«. Pomo}u ovih prou~avan-ja dobijaju se podaci o sveukupnim karakteristikamapojedina~nih faktora koji uti~u na odre|ivanje. Tako-|e, podaci mogu da se primene pri proceni nesigur-nosti rezultata koji su dobijeni kori{}enjem rutinskihmetoda odre|ivanja. U principu, merenje nesigurnos-ti je jednostavno i uklju~uje slede}e etape: specifika-ciju koncentracije analita (ono {to se odre|uje, engl.measurand), identifikaciju izvora nesigurnosti, me-renje komponenata nesigurnosti i izra~unavanje uku-pne nesigurnosti. U radu }e biti iznet postupak izvo-|enja svih gore navedenih etapa u merenju nesigur-nosti, kao i mogu}nost pojednostavljenja cele proce-dure u zavisnosti od raspolo`ivih podataka o kombi-novanim uticajima razli~itog porekla.

QUALITY OF ANALYTICAL MEASUREMENTSIN MEDICAL BIOCHEMISTRY: QUANTIFYING UNCERTAINTY

S. Ignjatovi}

Institute of Medical Biochemistry, Clinical Centre of Serbia and School of Pharmacy,

University of Belgrade, Belgrade, Yugoslavia

Many important decisions are based on the resultsof analytical measurements in medical biochemistry.Whenever decisions are based on analytical results, itis important to have some indication of the quality ofthe results. Now it is a formal requirement for clinicallaboratories to introduce quality assurance measuresto ensure that they are capable of and are providingdata of the required quality. Such measures include:the use of validated methods of analysis; the use ofdefined internal quality control procedures; participa-tion in external quality schemes; accreditation basedon ISO 17025, and establishing tracebility of the re-sults of the measurements. As a consequence of the-se requirements, medical biochemists are, for theirpart, coming under increasing preassure to demon-strate the quality of their results, and in particular todemonstrate their fitness for purpose by giving ameasure of the confidence that can be placed on theresult. This is expected to include the degree to whicha result would be expected to agree with other results,normally irrespective of the analytical methods used.One useful measure of this is measurement uncer-tainty. Uncertainty of measurement comprises, ingeneral, many components. In practice the uncer-tainty of the result may arise from many possiblesources, including examples such as incomplete de-finition, sampling, matrix effects and interferences,environmental conditions, uncertainties of massesand volumetric equipment, reference values, approx-imations and assumptions incorporated in the meas-urement method and procedure and random varia-tion. The fitness for purpose of analytical methodsapplied for routine testing is most commonly asse-ssed through method validation studies. Such studiesproduce data on overall performance an on individualinfluence factors which can be applied to the estima-tion of uncertainty associated with the results of themethod in normal use. Uncertainty estimation is sim-ple in principle. The steps involved are: specificationmesurand, identification uncertainty sources, quan-tification uncertainty components and calculationcombinied uncertainty. The aim of this study will beto provide guidance on the execution of all the stepslisted above and to show how the procedure may besimplified depending on the information that is avail-able about the combined effect of a number ofsources.


UPRAVLJANJE ZA[TITOM RADNE I @IVOTNE SREDINE U ZDRAVSTEVNIM

LABORATORIJAMA

M. Ili}, A. Po{ti}-Grujin, N. Majki}-Singh


Upravljanje za{titom ìvotne i radne sredine u lab-oratorijama moè se ostvariti samo efikasnom kon-trolom svih {tetnosti koje postoje u laboratorijama ikoje se mogu ispoljiti bilo kada. Za{tita otpo~injesamim prepoznavanjem {tetnosti (hazarda) a ostvaru-je se razumnim pristupom, dobrim pona{anjemosoblja, pravilnim radnim procesom u svim labora-torijskim celinama i stalnim sprovo|enjem dobre lab-oratorijske prakse i tehnika pri laboratorijskom radu.U ve}ini slu~ajeva neèljeni doga|aji i nesre}e pri lab-oratorijskom radu doga|aju se zbog nepa`nje i lo{ihradnih uslova. Klini~ke laboratorije uglavnom manip-uli{u sa devet klasa opasnih materija, a prema kriter-ijumima Ujedinjenih nacija to su: 1) eksplozivne ma-terije, 2) komprimovani gasovi, 3) zapaljive te~nosti,4) zapaljive ~vrste materije, 5) oksidacione materije,6) toksi~ne materije, 7) radioaktivne materije, 8) ko-rozivne materije i 9) ostalo, u {ta se ubrajaju i infek-tivni agensi. S obzirom na brojne {tetnosti, u ostvari-vanju za{tite osoblja klini~kih laboratorija veoma jezna~ajno pravilno obeleàvanje istih i upozoravanjelaboratorijskog osoblja. Odgovornost za sprovo|enjemera sigurnosti leì prvenstveno na rukovodiocu la-boratorije, odnosno poslodavcu, ali i na svim zaposle-nim. Sprovo|enje mera za{tite u laboratoriji otpo~injeod pisanih uputstava sa kojima treba da budu upoz-nati svi laboratorijski izvr{ioci. To zna~i da svaka kli-ni~ko-biohemijska laboratorija mora da ima zvani~anprogram za{tite. Osim toga poslodavac je duàn daobezbedi pouzdane laboratorijske radne procedure,stalni nadzor nad radom zaposlenih, informacije o na-~inu za{tite, obuku, li~nu za{titnu opremu i medicin-sku za{titu. Institut za medicinsku biohemju Klini~kogcentra Srbije kao vode}a institucija za oblast klini~ko-biohemijske dijagnosti~ke laboratorijske prakse serti-fikovao je sistem menadèmenta kvalitetom premastandardu ISO 9001:2001 i akreditovao sve svoje lab-oratorije prema standardu ISO 17025. Poslovnikomo kvalitetu i kroz pojedina dokumenta za sprovo|enjesistema kvaliteta predvi|eno je obezbe|ivanje odgo-varaju}ih uslova radne sredine za obavljanje laborato-rijskih ispitivanja i za{tite laboratorijskih radnika. Do-kumenta koja se odnose na upravljanje za{titom rad-ne i ìvotne sredine u Institutu ovde }e biti diskuto-vana.

PROTECTION MANAGEMENT OF WORKING ENVIRONMENT IN HEALTH

LABORATORIES

M. Ili}, A. Po{ti}-Grujin, N. Majki}-Singh

Institute for Medical Biochemistry, Clinical Center of Serbia, Belgrade

Protection management of working environmentin the laboratories may be implemented only throughefficient control of all laboratory hazards, which maybe manifested at any time. The protection involvesfirst of all the recognition of the hazards and is imple-mented by reasonable approach, good behaviour ofthe personnel, adequate work process in all laborato-ries and continuing application of good laboratorypractice and techniques in the laboratory work. In themajority of cases the laboratory hazards and acci-dents result from negligence and bad working condi-tions. Clinical laboratories, according to the UnitedNations criteria, chiefly manipulate with the followingnine classes of hazardous materials: 1) explosive ma-terials, 2) compressed gases, 3) combustible fluids,4) combustible solid materials 5) oxidants, 6) toxicmaterials, 7) radioactive materials, 8) corrosive mate-rials and 9) others, which include ineffective agents.Adequate labeling warning of these numerous haz-ards is very important for the protection of the per-sonnel in the clinical laboratories. The responsibilityfor the implementation of safety measures lies prima-rily on the head of the laboratory i.e. employer butalso on the entire personnel. The first step in theimplementation of protection measures in the labora-tory is the instruction manual with which all laborato-ry workers must be acquainted. This means that eachclinical-biochemical laboratory must have an officialprotection programme. Besides, it is upon the emplo-yer to provide reliable laboratory procedures, con-stant supervision over the work of the employees,information on the manner of protection, training,protective equipment and medical protection for thepersonnel. The Institute for Medical Biochemistry ofthe Clinical Center of Serbia, as a leading institutionfor the field of clinical-biochemical diagnostic labora-tory practice, has certified the quality managementsystem according to ISO 9001: 2001 and accreditedall its laboratories according to ISO 17025. Throughquality control manual and through some documentsfor the implementation of quality system, the ade-quate working and environmental conditions, for car-rying out laboratory tests and for the protection oflaboratory workers, are provided. The documents re-lating to the protection management of working envi-ronment are discussed here.


ZAHTEVI ZA KOMPETENTNOST LABORATORIJA ZA ISPITIVANJE

I NJIHOVA PRIMENA

T. Ne{i}, R. Bo{kovi}

Bonex inènjering, Beograd

Dokazivanje tehni~ke kompetentnosti, neutralnos-ti, nepristrasnosti i nezavisnosti laboratorija za ispiti-vanje vaàn je segment ukupnog procesa za{tite gra-|ana od proizvoda i usluga neodgovaraju}eg kvalite-ta, jer se time ja~a poverenje u objektivnost labora-torija i ta~nost rezultata ispitivanja. Ovo dokazivanjesprovodi se kao deo postupka akreditacije koji se vodipred nadle`nim organom. Akreditacija je zvani~nipostupak u kojem organizacija za akreditaciju, nakonsprovedenog ocenjivanja, potvr|uje da je laboratorijakompetentna za obavljanje ispitivanja u definisanomobimu akreditacije. Poslovi akreditacije u na{oj zemljipovereni su Jugoslovenskom akreditacionom telu(JUAT). Laboratorija za ispitivanje koja èli da budeakreditovana treba da uskladi sistem rada premazahtevima standarda JUS ISO IEC 17025. Ovaj stan-dard usvojen je i objavljen u na{oj zemlji jo{ marta2001. godine. Me|utim, prema zvani~nim podacimaJUAT-a, do kraja 2001. godine tek jedna laboratorijaza ispitivanje akreditovana je prema zahtevima stan-darda. Ostale laboratorije akreditovane su prema ra-nije vaè}em JUS ISO IEC Uputstvu 25 i/ili standarduJUS EN 45001 (21 regularnih re{enja o akreditaciji i56 privremenih re{enja). Podaci pokazuju da se labo-ratorije za ispitivanje u na{oj zemlji sporo prestruk-turiraju na ispunjavanje novih zahteva. Bez obzira kojisu razlozi za ovakvo stanje kod nas, treba se okrenutibudu}nosti i obezbe|enju dovoljnog broja laboratori-ja koje imaju potvr|enu kompetentnost za vr{enjedefinisanih ispitivanja i to upravo prema zahtevimastandarda JUS ISO IEC 17025. Cilj ovog rada je daprikaè op{te zahteve koje laboratorije za ispitivanjetreba da ispune, kako bi mogle potvrditi svoju kom-petentnost. Standard JUS ISO IEC 17025 propisujeop{te zahteve koji su formulisani tako da vaè za svekategorije laboratorija za ispitivanje i laboratorija zaba`darenje. Zahtevi su svrstani u dve osnovne grupe:zahtevi koji se odnose na menad`ment i tehni~kizahtevi. Zahtevi koji se odnose na menad`ment ti~use organizacionih, upravlja~kih i komercijalnih aspe-kata rada laboratorije, uklju~uju}i i zahteve za sistemkvaliteta. Moè se re}i da se ova grupa zahteva pri-menjuje na vrlo sli~an na~in u svim laboratorijama zaispitivanja, jer se u njima razmatraju elementi, zajed-ni~ki za sve poslovne sisteme. Ono, na {ta svakapojedina~na laboratorija za ispitivanje treba da po-sveti posebnu pa`nju, su tehni~ki zahtevi. Za definisa-nu oblast i obim ispitivanja, laboratorija mora obezbe-diti i dokazati:

’ da su zaposleni kompetentni i osposobljeni za vr{e-nje ispitivanja i drugih poslova u laboratoriji na kojesu raspore|eni,


GENERAL REQUIREMENTS FOR THE COMPETENCE OF TESTING

LABORATORIES AND THEIR IMPLEMENTATION

T. Ne{i}, R. Bo{kovi}

Bonex engineering, Belgrade

Demonstration of technical competence, impar-tiality and independence of testing laboratories is animportant part of the process of consumer protectionagainst nonconforming products or services. Itstrengthens the confidence in objectivity of laborato-ries and exactness of test results. Demonstration ofcompetence is an integral part of accreditationprocess. Accreditation is the official recognition, per-formed by an authorized organization hat a laborato-ry is competent to perform testing activities withindefined scope of accreditation. Organization, perfor-ming accreditation in our country is called Yugoslavaccreditation body. Any testing laboratory, seekingaccreditation, should align its internal organizationand system of work with the requirements of thestandard JUS ISO IEC 17025, which has been adopt-ed and issued in our country in the March, 2001.Unfortunately, official data of the Yugoslav accredita-tion body show that till December, 2001. only onetesting laboratory was accredited according to therequirements of this standard. Other testing labora-tories were accredited according to canceled JUSISO IEC Guide 25 and/or standard JUS EN 45001(21 regular certificate of accreditation and 56 tempo-rary certificates). These data show that testing labo-ratories in our country very slowly move toward newrequirements. Whatever the reasons for this situa-tion, we recommend that testing laboratories shouldanticipate the future trends and take timely measuresto demonstrate their technical competence accord-ing to the standard JUS ISO IEC 17025. The objec-tives of the paper is to explain general requirementsthat testing laboratories should fulfill in order to con-firm their competence in the certain field of test-ing.The requirements of the standard are separatedin two main groups: management requirements andtechnical requirements. Management requirementsrelate to organizational, managerial, administrativeand commercial aspects of work, including require-ments for the quality system. These requirementscan be implemented in the similar way in testing lab-oratories, since they consider elements, common forall business systems. A laboratory should pay specialattention to the fulfillment of technical requirements.For the defined field and scope of testing, laboratoryshould assure and demonstrate the following:

’ competence of personnel ’ for testing activities andother activities they are assigned,

’ adequacy of the equipment, ’ adequacy of environmental conditions regarding

people, samples, equipment and testing methods,

’ da je oprema pogodna za obavljanje definisanihispitivanja,

’ da su uslovi radne sredine odgovaraju}i za ljude,uzorke, opremu i metode koje se primenjuju,

’ da se primenjuju adekvatne metode ispitivanja ~ijaje validnost potvr|ena,

’ da postupak s uzorcima obezbe|uje validnost rezul-tata ispitivanja,

’ da se primenjuju mere kojima se obezbe|uje pove-renje u rezultate ispitivanja i

’ da se primenjuje definisan postupak izdavanja izve-{taja o ispitivanju.

Pored pomenutog standarda u svakoj oblasti ispi-tivanja primenjuje se i regulativa koja se odnosi na tuoblast: zakoni, pravilnici, principi dobre laboratorijskeprakse, tehni~ki standardi itd. Svaka laboratorija tre-ba da utvrdi sopstvene ciljeve i potrebe, kao i zahteveokru`enja i va`e}e regulative koju mora u svom raduispunjavati. Ukoliko je me|u tim zahtevima i ciljevimai zahtev da laboratorija za ispitivanje bude akreditova-na, potrebno je usmeriti snage ka ispunjenju ovihzahteva i to na sistemati~an na~in i uz potpunu koor-dinaciju svih aspekata poslovanja laboratorije.

’ adequacy of testing methods whose validity hasbeen confirmed,

’ adequacy of procedures with test samples whichprovide confidence in the test results,

’ use of procedures and measures that provide con-fidence in the testing results,

’ adequacy of the procedure for issuing test report.

In addition to the a.m. standard, within each testarea exist other applicable legislation, e.g. law, prin-ciples of good laboratory practice, technical stan-dards etc. Each laboratory should establish its owngoals and needs taking into account the require-ments of business environment and legislative thathas to be implemented. If requirements for accredi-tation are part of these requirement, testing laborato-ry should focus towards fulfillment of these require-ments too.

PATOFIZIOLOGIJA CEREBRALNE ISHEMIJE

Mehanizmi uklju~eni u neuronalna o{te}enja

T. Úzben

Medicinski fakultet Akdeniz, Katedra za biohemiju, Antalija, Turska

Cerebralna ishemija je osnovni uzrok morbiditeta imortaliteta. Mnoge reakcije koje se dogode u toku iza vreme cerebralne ishemije dobro su poznate, aliipak ne poznaju se toliko dobro da bi se mogli u pot-punosti razjasniti mehanizmi o{te}enja mozga. Tro-{enje energije je osnovna tema svake diskusije o pa-tofiziologiji ishemijskih povreda. Danas su u centrupa`nje slede}e osobine ishemijskog i postishemijskogmozga: razvoj acidoze, formiranje otoka, prekomernostvaranje kalcijuma, ekscitotoksi~nost glutamata,formiranje slobodnih radikala i prekomerno stvaranjeazot oksida. Ovi i drugi manje poznati procesi dopri-nose stvaranju nepovratnog o{te}enja. Mozak uogromnoj meri zavisi od protoka krvi, odnosno stal-nog dotoka kiseonika i glukoze. Nakon samo neko-liko minuta od napada ishemije, potrebe za energi-jom su ve}e od mogu}nosti mozga da anaerobnosinteti{e ATP. Tro{enje energije ima ogroman zna~aju genezi kasnijih povreda, jer se laktat i nepuferovanijoni vodonika akumuliraju u tkivu. Prekid rada svihmehanizama zavisnih od energije, uklju~uju}i i jonskepumpe, dovodi do opadanja membransko-jonskoggradijenta, otvaranja selektivnih i neselektivnih jon-skih kanala i izjedna~avanja ve}ine intracelularnih iekstracelularnih jona (anoksi~na depolarizacija).Usled celularne akumulacije jona formira se citotok-si~an edem. Gubitak ATP brzo dovodi do velikogpriliva i otpu{tanja kalcijuma iz intarcelularnih proso-tra. Ekstracelularne koncentracije glutamata primet-no su pove}ane u tkivu ishemijskog mozga, {to jeposledica i pove}anog otpu{tanja aminokiselina izneurona i njegovog smanjenog unosa u glija }elije ineurone. Glutamat koji je oslobo|en iz depolarizo-vanih presinapti~kih krajeva aktivira nekoliko post-sinapti~kih receptor/kanal kompleksa, koji su nazvaniprema odgovaraju}im agonistima (kviskalat, kainat iNMDA-prioritetni receptor). Jedini koji propu{ta jonekalcijuma je N-metil-D-asparat (NMDA) receptor/ka-nal kompleks. Smatra se da intracelularni vi{ak Ca2+

tokom ishemije ostavlja nekoliko posledica, uklju~u-ju}i aktiviranje fosfolipaze A2, prekid oksidativne fos-forilacije, promenu funkcije receptora, osloba|anje


PATHOPHYSIOLOGY OF CEREBRAL ISCHEMIA

Mechanisms Involved In Neuronal Damage

T. Úzben

Akdeniz University School of Medicine, Department of Biochemistry, 07070, Antalya, Turkey

Cerebral ischemia represents a major cause ofmorbidity and mortality. Many events occuring du-ring and after cerebral ischemia are well known, butthey are not known enough to fully elucidate themechanisms of brain damage. Central to any discus-sion of the pathophysiology of ischemic lesions isenergy depletion. At present, the following features ofthe ischemic and postischemic brain are the focus ofinterest: development of acidosis, edema formation,calcium overload, glutamate excitotoxicity, free radi-cal formation and nitric oxide overproduction. Theseevents, with other less known ones, contribute alto-gether to the occurence of irreversible damage. Thebrain is critically dependent on its blood flow for acontinuous supply of oxygen and glucose. Withinminutes of the onset of ischemia, energy demandsexceed the brain's capacity to synthesize ATP anaer-obically. Energy depletion has fundamental impor-tance in the genesis of subsequent injurious events.Lactate and unbuffered hydrogen ions accumulate intissue. Failure of all energy dependent mechanisms,including ion pumps, leads to the deterioration ofmembrane ion gradients, opening of selective andunselective ion channels and equilibration of mostintracellular and extracellular ions (anoxic depolariza-tion). Cellular accumulation of ions cause formationof cytotoxic edema. Loss of ATP rapidly leads to amassive calcium influx and release of calcium fromintracellular compartments. Extracellular concentra-tions of glutamate are markedly elevated in ischemicbrain tissue as a concequence of both enhancedrelease of the amino acid from neurons and itsimpaired uptake into glia and neurons. Glutamatereleased from depolarized presynaptic endings acti-vates several post-synaptic receptor /channel com-plexes which are named according to the preferredagonist (the quisqualate, kainate and NMDA-prefer-ring receptor). Of these, the N-methyl-D-aspartate(NMDA) receptor/channel complex is permeable tocalcium ions. Intracellular Ca2+ overload duringischemia is thought to have several deleterious con-sequences including activation of phospholipase A2,

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Opening Lecture

toksi~nih ekscitatornih aminokiselina (EAA), uvo|e-nje neurona u stanje hiperekcitabilnosti. Intracelularnivi{ak Ca2+ tako|e moè prouzrokovati lavinu proce-sa, na primer formiranje raznih vrsta reaktivnog kiseo-nika. Usled povrede mo`danih }elija otpu{taju se jonigvo`|a, koji mogu da stimuli{u reakcije slobodnihradikala. Pored toga, u sivoj i beloj masi CNSa nalazise visoka koncentracija askorbinske kiseline. Me{a-vine askorbat/gvo`|a i askorbat/bakra generi{u slo-bodne radikale. Jo{ jedan izvor kiseoni~nih slobodnihradikala je intramitohondrijalni lanac prenosa elek-trona. Slobodni radikali koji se stvaraju u mitohondri-jama tako|e mogu da izazovu ta~kaste mutacije,umreàvanje DNK i prekid u lancu DNK u mitohon-drijalnim genima. O{te}enje mitohondrijalnog geno-ma dovodi do oslabljene respiracije, dalje pove}ava-ju}i mogu}nost proizvodnje radikala kiseonika. Osimtoga, mozak ima slabu aktivnost katalaze i samo ma-lu koli~inu superoksida dizmutaze i glutation peroksi-daze. Azot monoksid (NO) nedavno se pojavio kaovaàn posrednik }elijskih i molekularnih procesa iuti~e na patofiziologiju cerebralne ishemije. Pove}a-nje Ca2+ aktivira enzim azot monoksid sintetazu(NOS; EC 1.14.13.39), koji dalje katalizuje sintezuazot monoksida iz gvanidin azota L-arginina i moleku-larnog kiseonika. NO se proizvodi u neuronima, glija}elijama i vaskularnom endotelijumu u centralnomnervnom sistemu (CNS). Efekti su razli~iti u zavisnos-ti od porekla. NO je posrednik koji ima i neurotok-si~ne i neuromodulatorske efekte. Neuronalni NO jepredloèn kao neurotoksi~ni agens koji uti~e na NMDAtoksi~nost i pove}anu akutnu ishemijsku povredu.On je uzrok citotoksi~nosti tokom formiranja gvo`|e--NO kompleksa sa nekoliko enzima, uklju~uju}i tran-sportni lanac mitohondrijalnog elektrona, oksidacijuprotein sulfhidrila i DNK nitraciju. NO moè da uti~ena odumiranje }elije kroz formiranje jakih oksidantnihperoksinitrata (ONOO’). ONOO’¯se raspada nahidroksilni slobodni radikal (OH’.) i na nitrogen diok-sid radikal (NO2) koji je snaàn aktivator lipidne per-oksidacije. S druge strane, vaskularni NO, kao snaànvazodilator i inhibitor agregacije trombocita, moèdelovati povoljno u ranim fazama fokalne cerebralneishemije, olak{avaju}i dotok kolateralne krvi do teri-torije zahva}ene ishemijom. Prvobitna uverenja da in-hibitori sinteze NO mogu da se suprotstave tok-si~nosti glutamata u }elijskoj kulturi, dovela su do te-meljnih ispitivanja uloge NO u patofiziologiji cere-bralne ishemijske povrede u organizmu. Ipak, studijeo efikasnosti inhibitora NO sintaze u modelima fokal-ne cerebralne ishemije navele su razli~ite pronalaske,od onih drasti~nih neuroprotekcija do pogor{anja is-hemijske povrede. Osnovni razlog koji podupire nekeod postoje}ih kontroverzi je upotreba neselektivnihNOS inhibitora. Da bi se smanjila ishemijska povreda,trebalo bi da se razviju selektivni inhibitori za neuro-nalne izoforme.

Klju~ne re~i: cerebralna ishemija, glutamat, azotoksid.


uncoupling of oxidative phosphorylation, alteration ofreceptor function, toxic excitatory amino acid (EAA)release, precipitating neurons in a state of hyperex-itability. Intracellular Ca2+ overload can also set off acascade of events which may lead to the formation ofreactive oxygen species. Injury to brain cells mayrelease iron ions that can stimulate free radical reac-tions. In addition, there is a high concentration ofascorbic acid in the gray and white matter of theCNS. Ascorbate/iron and ascorbate/copper mixturesgenerate free radicals. One more source of oxygenfree radicals is the intramitochondrial electron trans-fer chain. Free radicals produced in mitochondriacould also have the ability to cause point mutations,DNA cross link and DNA strand breaks in mitochon-drial genes. Damage to the mitochondrial genomeresults in impaired respiration, further increasing thepossibility of oxygen radical production. In addition,the brain is poor in catalase activity and has onlymoderate amounts of superoxide dismutase and glu-tathione peroxidase. Nitrogen monoxide (NO) hasrecently emerged as an important mediator of cellu-lar and molecular events which impacts the patho-physiology of cerebral ischemia. An increase in intra-cellular Ca2+ activates the enzyme NOsynthase (NOS;EC 1.14.13.39) which catalyzes the synthesis of NOfrom the guanido nitrogen of L-arginine and mo-lecular oxygen. NO is produced in neurons, glia cellsand vascular endothelium in central nervous system(CNS). Depending on their origin, its effects are va-ried. NO is a mediator having both neurotoxic andneuromodulator effects. Neuronal NO was proposedas the neurotoxic agent mediating NMDA toxicity andincreasing acute ischemic damage. It causes cyto-toxicity through formation of iron-NO complexes withseveral enzymes including mitochondrial electrontransport chain, oxidation of protein sulfhydryls andDNA nitration. NO may mediate cell death alsothrough formation of the potent oxidant peroxynitrite(ONOO’). ONOO’ decomposes to the hydroxyl freeradical (OH’.) and to the radical nitrogen dioxide(NO2) which is a potent activator of lipid peroxidation.On the other hand, vascular NO as a potent vasodila-tor and an inhibitor of platelet aggregation, may bebeneficial in the early stages of focal cerebral ische-mia. It may facilitate collateral blood flow to theischemic territory. The original observation that inhi-bitors of NO synthesis can antagonize glutamate tox-icity in cell culture has led to extensive investigationof the role of NO in the pathophysiology of cerebralischemic injury in vivo. However, studies of the effi-cacy of NO synthase inhibitors in models of focalcerebral ischemia have generated widely disparatefindings, ranging from dramatic neuroprotection toexacerbation of ischemic damage. The main reasonthat underlies some of the existing controversies isthe use of non-selective NOS inhibitors. To diminishthe ischemic injury, selective inhibitors for neuronalisoform should be developed.

Key word: cerebral ischemia, glutamate, nitricoxide.

OKSIDATIVNI STRES I KANCER

S. Kakari

Onkolo{ka bolnica Sveti Sava Mystra 10, Voula 16673, Atina, Gr~ka

Razvoj i postojanje aerobnih organizama u pris-ustvu kiseonika povezano je sa generisanjem »reak-tivnih« kiseoni~nih (ROS) i azotnih (RNS) vrsta, kojisu, {to je paradoks, odgovorni za oksidativna o{te-}enja biolo{kih makromolekula (DNK, proteini, lipidiitd.). Ovi procesi su uklju~eni u inicijaciju progresijeoboljenja kao {to su arteroskleroza, ishemija-reper-fuzija povreda, inflamacija, traumatska paraplegija ikancer. U ovom radu bi}e re~i o ulozi slobodnihraidikala (F.R.) i ROS/RNS u karcinogenezi i {tetnostioksidativnog stresa (O.S.), poreme}aja u antioksida-tivnom odbrambenom sistemu u razvoju i progresijikancera. Oksidativni stres je rezultat poreme}ajaredukcionih i oksidativnih procesa u }eliji. U prook-sidativnim stanjima }elije, intra}elijski ROS/RNS supovi{eni, zbog povi{ene proizvodnje ili nemogu}nostida se neutrali{u. Kao posledica, oksidativni stresmogu da prouzrokuju mnogobrojne razli~ite grupefaktora, kao {to su radijacija, hiperbari~ni kiseonik,ksenobioti~ni metaboliti, tip Fenton metal joni (Fe2+,Cu2+), modulatori citohroma P-450, nedostatakhvata~a slobodnih radikala i helatori metalnih jona ineadekvatne aktivnosti antioksidantnog enzima i ne-enzimskih antioksidanata. Ako se ima u vidu va`nosto{te}enja DNK u karcinogenezi, kompleksnom pro-cesu, o~igledno je da je bilo koji faktor koji mo`e damodifikuje hemiju DNK, potencijalni karcinogen.Postoje tri osnovne odlike karcinogeneze:1. mogu}nost da se proizvodi permanentna struktu-

ralna povreda DNK, kao {to su mutacije osnovnogpara, ubacivanje, brisanje i sekvencionirano umno-`avanje,

2. mogu}nost da se aktiviraju citoplazmi~ni i nuk-learni signali transdukcionih putanja, i

3. mogu}nost da se modulira aktivnost streasogenihgena i proteina da bi se regulisali efektivni geni kojisu povezani sa rastom }elije, njenom diferencijaci-jom i odumiranjem.Bitno je da slobodni radikali i ROS/RNS mogu da

izazovu sve ove promene.Razvoj neoplasti~nih }elija iz normalnih }elija

uklju~uje tri glavna koraka: inicijaciju, promociju iprogresiju. Inicijacija je nepovratna specifi~na prome-


OXIDATIVE STRESS AND CANCER

S. Kakari

St. Sava Anticancer Hospoital10 Mystra St., Voula 16673, Athens, Greece

The development and existence of aerobic orga-nisms in the presence of oxygen is associated withthe generation of »reactive« species of oxygen (ROS)and nitrogen (RNS) which, paradoxically, are respon-sible for oxidative damage to biologic macromole-cules (DNA, proteins, lipids, etc.). These processesare involved in the initiation or progression of dis-eases like atherosclerosis, ischaemia-reperfusioninjury, inflammation, traumatic paraplegia and can-cer. This discussion focuses on the role of of free rad-icals (F.R.) and ROS/RNS in carcinogenesis, and thedetrimental consequences of Oxidative Stress (O.S.)and of disturbances in the antioxidative defense sys-tem in the development and progression of cancer.

Oxidative stress is the result of disturbances in theredox homeostasis of cells. In prooxidant states ofthe cell intracellular ROS/RNS are elevated, obvious-ly because of overproduction or inability for their neu-tralization. Consequently, oxidative stress may beproduced by many different groups of environmentalfactors such as radiation, hyperbaric oxygen, xenobi-otic metabolites, Fenton-type metal ions (Fe2+,Cu2+), modulators of cytochrome P-450, lack of freeradical scaverngers and of metal chelators, and frominadequate antioxidant enzyme activities and non-enzymic antioxidants. In view of the importance ofDNA damage in carcinogenesis, a complex process,it is obvious that any factor able to modify DNAchemistry is a potential carcinogen. There are threecritical properties of carcinogens:1. The capacity to produce permanent structural

DNA lesions such as base-pair mutations, inser-tions, deletions, and sequence amplification.

2. The ability to activate cytoplasmic and nuclear sig-nal transduction pathways, and

3. The capacity to modulate the activity of stressgenes and stress proteins to regulate effector ge-nes related to cell growth, differentiation anddeath.Importantly, free radicals and ROS/RNS have the

properties to cause all of these changes. The development of neoplastic cells from normal

cells involves three major steps: initiation, promotionand progression. Initiation is an irreversible specific

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Plenary Lecture

na u DNK ciljne }elije (npr. agensi iz neposredne oko-line, kao {to je duvanski dim, mogu da indukujuonkogeno stanje). Promocija uklju~uje reverzibilnustimulaciju ekspanzije zahva}enih }elija ili reverzibilnupromenu stanja gena u toj }eliji ili progenu. Nekiagensi, potencijalni potpomaga~i, ne mogu sami dapobude kancer, ali mogu da pove}aju razvijanje kan-cera tako {to pobu|uju iniciraju}e agense. Kompletnikarcinogeni mogu da budu i inicijatori i promoteri.Primeri kompletnih karcinogena uklju~uju onkogenuDNK i RNA viruse, joniziranje i ultraljubi~astu radijaci-ju i razne policikli~ne hidrokarbonate. ROS/RNS suizgleda uglavnom uklju~eni u promotivnu fazu kar-cinogeneze, iako neki ROS mogu da budu slabi kom-pletni karcinogeni. Suprotno tome, neki antioksidan-ti su anti-promoteri i anti-karcinogeni. Progresija,tre}a faza karcinogeneze, karakteri{e razvoj ane-ploidne i cionalne varijacije tumora. To dovodi doinvazije tumora i metastaze. êsto se de{ava da tkivotretirano karcinogenima pokazuje pove}an nivo 8-hidroksideoksiguanozina (8-OhdG), koji je markeroksidativnog o{te}enja DNK. To navodi na zaklju~akda je oksidativni stres uklju~en u karcinogenezu,nezavisno od karcinogena koji je inicirao ovaj proces.Oksidativni stres prouzrokuje celularni na vi{e na~ina,putem o{te}enja DNK, koje moè biti direktno, npr.putem hidrokslinih radikala (OH.) koji su generisani unukleusu (preko Fenton reakcije) ili indirektno putemvezivanja produkata lipidne peroksidacije, kao {to sualdehidi, tj. 4-HNE (4-hidroksinonenal) sa bazomDNK, zbog nesposobnosti polimeraze i oporavka enz-ima DNK i zbog pucanja DNK usled nukleaze, koju jeaktiviralo pove}anje intracelularnog slobodnog Ca2+,koji je uzrokovan oksidativnim stresom. Veliki brojtumora pojavljuje se u kasnijim godinama, nakonkompleksne interakcije genetskih faktora i faktora izneposredne okoline, kao {to su na~in ìvota (pu{enje,preterano uzimanje alkohola, itd.), ishrana, infektivnimikroorganizmi i radijacija. Duvan je najve}i uzro~nikkancera i nekoliko drugih bolesti zabeleènih u svetu.U tre}ini slu~ajeva duvan je uzro~nik kancera, a u~etvrtini je uzro~nik sr~anih oboljenja, usled ~egasvake godine u Sjedinjenim Ameri~kim Dràvamaranu smrt pogodi 400000 osoba. Pu{enje je glavnifaktor rizika kancera plu}a, usta larinksa, bubrega,mokra}ne be{ike, esofagusa, stomaka i pankreasa.Svaki udahnuti dim formira vi{e od milijardu oksir-adikala. Kad se uporedi sa udahnutim, izdahnuti dimsadrì 10 puta vi{e ONOO’, koja je najotrovnija cito-toksi~na reaktivna vrsta i predstavlja opasnost zapasivnog pu{a~a. Nedavna studija u Kanadi potvrdilaje 6 prethodnih ispitivanja na populaciji koja su poka-zala da pasivno pu{enje predstavlja potencijalni rizikne samo za pojavu kancera plu}a, ve} i kanceradojke. Pu{enje moè da aktivira sinergiju sa drugimpotencijalnim karcinogenima koji su povezani sa na-~inom ìvota, kao {to je preterano konzumiranje alko-hola i kafe, kao i slaba ishrana. Drugi po redu faktorpojave kancera je, posle pu{enja, preterano uzimanjealkohola.


alteration in the DNA of the target cell (e.g. environ-mental agents ’ as in tobacco smoke ’ may induceoncogene expression). Promotion involves thereversible stimulation of the expansion of the initiatedcell or the reversible change of gene expression inthat cell or its progeny. Some agents, capable pro-moters, are unable to induce cancer on their own,but can enhance cancer development by initiatingagents. Other compounds are complete carcinogensin that they are both initiators and promoters.Examples of complete carcinogens include onco-genic DNA and RNA viruses, ionizing and ultravioletradiation and various polycyclic hydrocarbons.ROS/RNS appear to be involved primarily in the pro-motion phase of carcinogenesis, although someROS may also be weak complete carcinogens.Conversely, some antioxidants are anti-promotersand anti-carcinogens. Progression, the third phase ofcarcinogenesis, is characterized by the developmentof aneuploidy and cional variation in the tumor.These, in turn, lead to tumor invasion and metasta-sis. Frequently, tissues treated with carcinogensexhibit increased levels of 8-hydroxydeoxyguanosine(8-OhdG) ’ a marker of oxidative DNA damage ’suggesting that oxidative stress is involved in car-cinogenesis, independent of the carcinogen that ini-tiated this process. Oxidative stress causes cellular bymultiple ways such as by DNA damage which may bedirect, for example by hydroxyl radicals (OH) gener-ated in the nucleus (by Fenton reaction) or indirectby binding of lipid peroxidation products such asaldehydes i.e. 4-HNE (4-hydroxynonenal) with DNAbases, from inability of polymerases and of theenzymes of DNA repair, and from DNA breakdown bynucleases activated by the intracellular free Ca2+

increase, caused by oxidative stress. The greatmajority of tumors appear in adulthood following acomplex interaction of genetic and environmentalfactors such as: lifestyle (e.g. smoking, excessivedrinking etc.), diet, infectious microorganisms andradiation. Tobacco abuse is the most important sin-gle cause of cancer and several other diseases in theworld, contributing to one third of cancer cases andone fourth of coronary heart disease, and about 400,000 premature deaths each year in the United States.Smoking is a major risk factor for cancer of the lung,mouth, larynx, kidney, urinary bladder, esophagus,stomach and pancreas. Cigarette smoke results inthe formation of more than a billion oxyradicals ineach puff. Compared to the inhaled, the exhaled to-bacco smoke (side-stream) smoke contains 10 timesmore ONOO’, the most cytotoxic reactive species,and is of great concern to passive smokers. RecentCanadian study confirmed six previous populationstudies, which showed that passive smoking, in addi-tion to lung cancer, is a potential risk factor for breastcancer in women. Furthermore, smoking may actsynergistically with other potential carcinogens rela-ted to lifestyle such as alcohol and coffee over-con-sumption in addition to poor diet among others.Alcohol over-consumption is second in importanceonly to smoking as a cause of cancer.


HIPERHOMOCISTEINEMIJA I KARDIOVASKULARNI POREME]AJI

A. Tzontcheva

Medicinski fakultet i Institut za klini~ku laboratoriju i klini~ku imunologiju »Aleksandar bolnica«,

Sofija, Bugarska

Homocistein (Hcy) je normalni metabolit esenci-jalne aminokiseline metionina, za koji vlada intereso-vanje poslednjih nekoliko godina. Istra`iva~i su otkrilida homocistein direktno o{te}uje krvne sudove i izra-zito je povezan sa sr~anim udarima, {logom i perifer-nim vaskularnim oboljenjima. Homocistein se vazujeza LDL pri ~emu se modifikuje njegova struktura tipaoksidacije ili glikozilacije. Epidemiolo{ka izu~avanjaukazuju da su pove}ane koncentracije homocisteinau plazmi nezavisni faktor rizika za kardiovaskularnaoboljenja (KVO) i osetljiv marker funkcije kobalaminai folata (Fol). Hcy posreduje endotelnu }elijsku tok-si~nost pove}avanjem }elijske gustine i stvaranja ko-lagena. Folna kiselina zna~ajno sni`ava koncentracijehomocisteina u plazmi. Izrazita inverzna korelacija jena|ena izme|u ukupnog homocisteina i serumskogfolata. Graham sa sar. (1997) je u velikoj multicentri~-noj evropskoj studiji koja je uklu~ivala 750 slu~ajeva i800 kontrola na{ao da je pove}ani tHcy isto takoizrazit nezavisni faktor rizika za KVO oboljenja kao ipu{enje i hiperlipidemija. Uo~eno je da je odnos iz-me|u nivoa Hcy i vaskularnih oboljenja postepen.Pove}anje homocisteina za svakih 5 mmol/L u plazmije pra}eno u~estalo{}u koronarnog oboljenja od 1,6do 1,8 puta (CJ Boushley i sar., 1995). Srednje doumerena hiperhomocisteinemija je na|ena kodvaskularnih oboljenja pra}enih polimorfizmom meti-lentetrahidrofolat reduktaze (MTHFR) (P. Frosst i sar.,1995). Na{ cilj je bio da se ispita odnos izme|u ho-moicisteina u plazmi i infarkta mokarda pore|enjemsamog uticaja HCy i kombinacije visokog Hcy i vita-min B12 i doprinosa polimorfizma MTHFR ovom od-nosu.

HYPERHOMOCYSTEINEMIA AND CARDIO-VASCULAR FAILURE

A. Tzontcheva

Medical University Sofia, Chair of Clinical Laboratoryand Clinical Immunology »Alexander Hospital«,

Sofia, Bulgaria

Homocysteine (Hcy) ’ a normal metabolite of theessential amino acid methionine, came into the lime-light several years ago. Researchers discivered thathomocysteine can directly damage blood vessels andis strongly associate with heart attacks, strokes andperipheral vascular disease. Homocysteine attachesto LDL and modifies its structure, much like oxidationand glycosylation do. Accumulating findings fromepidemiological studies provide a compelling case forconsidering that increased plasma homocysteinelevel is an independent risk factor for cardiovasculardisease (CVD) and a sensitive marker of cobalamineand folate (Fol) function. Hcy mediates endothelialcell toxicity by increasing both cell toxicity by increas-ing both cell density and collagen production. Folicacid effectively lowers homocysteine concentration inplasma. A strong inverse correlation was foundbetween total homocysteine and serum folate.Graham et. al. reported rhat increased risk factor forCVD on the basis of a large multicenter Europeanstudy involving 750 cases and 800 controls (1997).The relationship between Hcy levels abd vascular dis-ease is considered to be graded. Each 5 mmol/Lincrease in fasting plasma Hcy are associated with an1.6 to 1.8-fold increase in incidence in fasting plasmaHcy are associated with an 1.6 to 1.8-fold increase inincidence of coronary disease (CJ Bousshley et al.,1995). Mild-to-moderate hyperhomocysteinemiaseen in vascular disease associates with methylenete-trahydrofolate reductase (MTHFR) polymorphism (P.Frosst et al., 1995). Our objectives were to estimatethe relationship between fasting plasma Hcy andmyocardial Infarction (MI), comparing the influenceof Hcy alone with those of combination of high Hcyand low Fol and Vit. B12 and to examine the contri-bution of MTHFR polymorphism to that relationship.

UC 577,1; 61 ISSN 0354-3447

Jugoslov. Med. Biohem. 21: 93, 2002 Prenarno predavanje

Plenary Lecture


CLINICAL USEFULNESS OF SERUM CYSTATIN C IN PATIENTS WITH VARIOUS KIDNEY DISEASES

T. Gruev, S. Domazetovska, K. Stefanovski, K. âkalarovski

Institut of Clinical Chemistry, Clinic of NephrologyClinical Centre, Skopje, Macedonia

Cystatin C (cystein proteinase inhibitor) is a lowweight protein which s is produced constantly by allnucleated cells indenpedently of j different pathologi-cal conditions and eliminated from the blood byglomerular filtration. Cystatin C has been proposedas an alternative marker of GFR , which meets manyof the criteria of a good GFR marker. Serum CystatinC closely reflects the glomerutar filtration rates in pa-tients with different kidney diseases and in patientsafter kidney transplantation. The patients (120) withdifferent diseases (diabetic nephropathy, chronic di-seases) and 38 after transplantation was investigated.The concentration of Cystatin C and beta 2 microglo-bulin was determined using the DAKO test. Creatini-ne clearence estimated by the Cockcroft-Gaultformula was) used as a indicator of GFR. Patientswith chronic diseases were divided into subgroupsaccording to GFR(mL/mm). In all studied groups(glomerulonephritis, GN with nephrotic proteinuria,pyelonephrites and transplantation) was differences(except glomerilonephritis) in correlation coefficientsbetween Ccr and Cystain C and Ccr and beta 2microglobulin. However, serum Cystatin C is a bettermarker of GFR than beta 2 Microglobulin in patientswith nephrotic proteinuria (p=0.620, p=0.410).

KLINI^KA PRIMENA CISTATINA C KOD PACIJENATA SA RAZLIÎTIM

BUBRE@NIM OBOLJENJIMA

T. Gruev, S. Domazetovska, K. Stefanovski, K. âkalarovski

Institut za klini~ku biohemiju, Klinika za nefrologijuKlini~ki centar, Skopje, Makedonija

Cistatin C (cistatin proteazni inhibitor) je proteinsa niskom molekulskom teìnom, koji se stvara kon-stantno u svim nuklearnim }elojama nezavisno od ra-zli~itih patolo{kih stanja, a elimini{e se preko krvi glo-merularnom filtracijom. Isti je predloèn kako alter-nativni marker za GFR, a poslednja ispitivanjapokazuju da je dobar marker za GFR. Cistatin C sereflektira sa glomerularnom filtracijom kod pacijena-ta sa razli~itim bubre`nim oboljenjima i kod pacijena-ta sa izvr{enom bubre`nom transplantacijom. Ispiti-vano je 120 pacijenata sa dijabeti~nom nefropatijomi hroni~nim oboljenjima i 38 pacijenata sa izvr{enombubre`nom transplantacijom. Koncentracija CistatinaC i beta-2 mikroglobulina je odre|ivana imunotur-bidimetriskim metodom sa testovima DAKO. Odre-|ivanje kreatin klirensa vr{eno je prema formuli Coc-roft-Gaut kao indikator za GFR. Pacijenti sa hroni~-nim oboljenjima su podeljeni na subgrupe premaGFR (mL/min). Kod svih ispitivanih grupa (glomeru-lonefritis, glomerulonefritis sa nefrotskom proteinuti-jom, pielonefriti i transplantacija) dobili smo razli~Itekoeficijente korelacije izme|u kreatin klirensa i cis-tatina C i kreatin klirensa i beta-2 mikroglobulina.Rezultati pokazuju da je Cistatin C bolji marker zaGFR u odnosu na beta-2 mikroglobulin kod pacije-nata sa nefrotskom proteinurijom.

UC 577,1; 61 ISSN 0354-3447


Plenary Lecture


UC 577,1; 61 ISSN 0354-3447


Plenary Lecture

IMUNSKI SISTEM I PROMENE U OKSIDATIVNOM METABOLIZMU

B. Kamenov, H. Dimitrijevi}, S. Pljaski}, T. Zaharov

De~ja klinika, Klini~ki centar Ni{

Postoji mnogo elemenata koji povezuju aktivacijuimunskog sistema i oksidativni metabolizam. Klini~kielement ove veze su: anemija u hroni~nim bolestima,nishodna regulacija oksidativnog metabolizma peri-fernih fagocita u toku hroni~nih inflamacija, nishodnaregulacija oksidatnog metabolizma i disfunkcijaimunskog sistema u sindromu hroni~nog umora,uklanjanje gvo`|a u toku hroni~nih inflamacija, sin-drom hladnih ruku i nogu, simptomatska glavoboljau hroni~nim inflamatornim bolestima, bronhoopstru-kcija u toku inflamacije zbog infekcije ili alergije. Ana-liza klini~kih manifestacija kod 250-ro dece s hroni~-nom disfunkcijom imunskog sistema pokazuje da sunaj~e{}e klini~ke manifestacije vezane za hemopoet-ski sistem (anemija 78,8%, sideropenija 64,8%, mo-nocitoza 49,2%, virocitosa 31,6%, trombocitoza11,6%, leukopenija 5.6%, trombocitopenija 2.8%),limfadenopatija 62%, sindrom hroni~nog umora36,5%, vaskulitis 28,8%, glavobolja 21,6%, sviranje ugrudima 14,4%, Raynold-ov sindrom 8,4%. Antitelaspecifi~na za CMV na|ena su kod 92,4%, HSV u51,2%, EBV u 23,6%. Makrofagi dobijaju najve}i deogvo`|a eritrofagocitozom. Kada su makrofagi ktivi-rani oni pove}avaju sintezu feritina, {to je pod kon-trolom citokinske mre`e, i {to dovodi do deponovanjagvo`|a, umesto njegovog osloba|anja. Citokini po-put TNF-beta, IL-1 beta, IL-6, IFN-gama inhibirajustvaranje Epo i proliferaciju BFU-E i CFU-E. Sli~neefekte pokazuje sni`enje IL-10 {to je ~esto u hroni-~nim inflamacijama. Citokini i drugi medijatori akti-vacije imunskog sistema pokazuju efekte na nivouendotela dovode}i do vazokonstrikcije ili bronhoop-strukcije bronhijalnog sistema. Aktivacija imunskogsistema dovodi do izmena u oksidativnom metaboliz-mu na nivou metabolizma gvo`|a, stvaranja hemo-globina i eritrocita, snabdevanja tkiva krvlju ili venti-lacije {to su komponente imunoregulacije i patoge-neze bolesti, ali i zna~ajni elementi prognoze i le~enja.

IMMUNE SYSTEM AND OXIDATIVE METABOLISM DOWN REGULATION

B. Kamenov, H. Dimitrijevi}, S. Pljaski}, T. Zaharov

Pediatric Clinic, Clinical Centre Ni{, Yugoslavia

There are many elements connecting the immunesystem activation to oxidative metabolism. Clinicalelements of this connections are: anemia in chronicdiseases, down regulation of the oxidative burst of theperipheral phagocytes during chronic inflammation,oxidative metabolism down regulation and immunesystem dysfunctions in chronic fatigue syndrome, ironwithdrawal during chronic inflammations, syndromeof cold legs and hands or symptomatic headache,bronchobostruction during inflammatation becauseof infection or allergy. Analysis of the clinical manifes-tation in 250 children with chronic immune dysfunc-tions has shown that the most common manifesta-tions are related to the haemopoietic system (anemia78,8%, sideropenia 64,8%, monocytosis 49,2%, viro-cytosis 31,6%, thrombocytosis 11,6%, leucopenia5.6%, thrombocytopenia 2.8%), lymphadenopathy62%, chronic fatigue syndrome 36,5%, vasculitis28,8%, headache 21,6%, wheezing 14,4%, raynoldsyndrome 8,4%. Antibodies specific for CMV werepresent in 92,4%, HSV in 51,2%, EBV in 23,6%. Ma-crophages acquire most of their iron by erythropha-gocytosis. When macrophages are activated they in-crease the synthesis of feritin which is under control ofthe cytokine network leading to iron storage insteadiron release. Cytokines like TNF-beta, IL-1 beta, IL-6,IFN gamma inhibit Epo production, and proliferationof BFU-E, CFU-E. Similar effects have low levels of IL-10 which is common in chronic inflammations. Cyto-kines and other mediators of immune system activa-tion have effects on the level of the endothelium lea-ding to vasoconstriction, or bronchial system to bron-choconstriction. Immune system activation leads tooxidative metabolism modulation on the level of ironmetabolism, hemoglobin and red blood cell produc-tion, blood supply or ventilation, which are part of theimmunoreguation and pathogenesis of the disease,but my be important elements for prognosis and treat-ment.

BIOHEMIJSKA OSNOVAPATOGENEZE DEPRESIJE

V. R. Paunovi}

Institut za psihijatriju, Klini~ki centar Srbije, Beograd

Biohemijski patogenetski supstrat depresije ~inepreme}aji serotonergi~ke, noradrenergi~ke i delimi~-no dopaminergi~ke neurotransmisije. Kako sistemineurotransmisije funkcioni{u u stalnoj me|usobnojkomunikaciji, ~itav niz modulatomih interakcija dovo-di do poreme}aja u drugim sistemima neurotrans-misije/neuromodulacije. Primarni biohemijski disba-lans javlja se u serotonergi~kim strukturama. Sma-njena je koncentracija L-hidroksitriptofana i serotoni-na u plazmi. Smanjena produkcija serotonina dovodido promena funkcionalnosti serotoninskih presinap-ti~kih 5-HT1 (promena senzitivnosti) i postsinapti~kih5-HT2 (hipersenzibilizacija) receptora. Smanjena pro-dukcija i obrt serotonina potvr|uju se smanjenjemkoncentracija 5-hidroksindolsir}etne kiseline (5-HIAA) u cerebrospinalnoj te~nosti. Promene koncen-tracije serotonina u trombocitima tako|e ukazuju naporeme}aj centralnih serotonergi~kih mehanizama,kao i pove}anje broja trombocitnih 5-HT2 receptora islabije vezivanje imipramina i paroksetina za trombo-cite. Snièna funkcija serotonergi~kog sistema dovo-di do hipersenzibilizacije postsinapti~kih beta-2 adre-noceptora, {to ukazuje na zna~ajne interakcije sero-tonergi~kog i noradrenergi~kog sistema u procesimaorganizacije afektiviteta. Disfunkcionalnost noradre-nergi~kog sistema je druga bitna komponenta pato-genetskog procesa. Produkcija i obrt noradrenalina upo~etku patolo{kih doga|anja mogu biti snièni,nepromenjeni ili pove}ani. Sniènje noradrenergi~ketransmisije moè biti uslovljeno nedostatkom nora-drenalina ili promenom osetljivosti noradrenergi~kihreceptora (ushodna regulacija i hipersenzibilizacijaalfa-adrenergi~kih presinapti~kih auto- i heterorecep-tora). Smanjene koncentracije 3-metoksi-4-hidroksi-fenilglikola (MHPG) u cerebrospinalnoj te~nosti i urinu,ukazuje na snièn obrt noradrenalina. Presinapti~kadisfunkcija indukuje i promene na subcelularnompostsinapti~kom nivou: u bipolarnom poreme}aju na-|ena je hiperaktivnost G-proteina, promene aktivnostisistema fosfoinozitida i poja~ano osloba|anje intrace-lularnog kalcijuma. Niz drugih pokazatelja (smanjeni


BIOCHEMICAL BASIS OF DEPRESSION

V. R. Paunovi}

Institute of Psychiatry, Clinical Centre of Serbia, Belgrade

Dysfunctional central serotonergic, noradrenergicand, at lesser degree, dopaininergic mechanisms arethe core biochemical factors of pathogenesis of de-pression. Continuous interactions of different sys-tems of neurotransmission generate, as a result ofdysfunctional monoaminergic systems, changes inother systems of neurotransmission/neuromodula-tion. The primary biochemical disturbance appearsin serotonergic structures. Reduced plasmatic con-centrations of L-hydroxytryptophan and serotonin arefound in depressed subjects. Lower rates of synthesisand lack of serotonin induce functional changes inpresynaptic 5-HT1 (altered sensitivity) and postsy-naptic 5-HT2 (upergulation) serotonergic receptors.Lowered concentrations of 5-hydroxyindolacetic acidin CSF reflect diminished turnover and serotonergictransmission. The changes of platelet serotonin pointto altered central serotonergic mechanisms, as wellas increased number of platelet 5-HT2 receptors andweakened thrombocyte binding of imipramine andparoxetine. The lowered serotonergic transmissionleads to hypersensitization of postsynaptic beta-2adrenoceptors, revealing important interactionsbetween serotonergic and noradrenergic systems inorganization of affectivity. Dysfunction of noradrener-gic central system is the second core component ofpathogenesis of depression. Noradrenaline biosyn-thesis and turnover may be augmented, diminished,or even unchanged at the beginning of depressivedisorder. In most of depressive subentities, dysfunc-tion is based on lowered noradrenergic transmission,due to the lack of noradrenaline or to altered sensi-tivity of noradrenergic receptors (upregulation of alp-ha-2 presynaptic auto- and heteroreceptors). Lowe-red CSF and urinary concentrations of 3-methoxy-4-hydroxy-phenylglycol (MHPG) reveal noradrenergicdysfunction in depression. Presynaptic disturbancesinduce changes on the postsynaptic subcellular level:hyperactivity of G-protein and phosphoinositide sys-tem with augmented intracellular calcium release isfound in bipolar depression. Other functional sys-

UC 577,1; 61 ISSN 0354-3447


Lectures

odgovori na stimulaciju hormona rasta, oslabljen od-govor TRH na stimulaciju TSH, izostajanje supresijeosloba|anja kortizola posle aplikacije deksameta-zona, slab odgovor ACTH, kortizola i prolaktina na sti-mulaciju fenfluraminom) ukazuje na poreme}aj nor-adrenergi~kih i serotonergi~kih regulatomih meha-nizama u depresiji. Podaci dobijeni vizualizacijommozga (SPECT, PET) otkrivaju smanjenje potro{njeglikoze u frontalnim regijama u kojima se vr{i sin-teti~ka obrada informacija. U toku su studije neuro-tropnih faktora (BDNF), stimulisanih serotonergi~kimi noradrenergi~kim mehanizmima, ~ija je aktivacijasubcelularno posredovana CREB proteinom.

ULOGA PATOLO[KOG TAU PROTEINA U NEURODEGENERACIJI

G. Oci}

Institut za neurologiju KCS, Beograd

Depoziti neurofilamenata sa patolo{kim oblicimatau proteina nalaze se kod brojnih degenerativnihoboljenja mozga, uklju~uju}i pored hereditarnih tau-patija, Alchajmerovu bolest, progresivnu supranuk-learnu paralizu i kortikobazalnu degeneraciju.Hereditarne taupatije obuhvataju frontotemporalnedemencije i me{ovite sindrome kao {to su fron-totemporalna demencija sa parkinsonizmom, dezin-hibicija ’ demencija ’ parkinsonizam ’ amiotrofijakompleks, palidopantonigralna degeneracija i{izofreniji sli~no pona{anje sa degeneracijomamigdala. Tau protein se u mozgu odraslih nalazi u 6izomorfnih oblika generisanih pojedina~nim genomna 17. hromozomu putem alternativnog cepanjaiRNK. Oni se me|usobno razlikuju prema sadr`aju 3(tau-3, -3S, -3L) ili 4 (tau-4, -4S, -4L) za mikrotubulevezuju}ih domena u blizini C-terminala. KodAlchajmerove bolesti ovi oblici tau proteina nalaze seu hiperfosforilisanom stanju zahvaljuju}i povi{enojaktivnosti protein kinaza ili sni`enoj aktivnosti proteinfosfataza. Hiperfosforilisani tau protein ulazi u sastavparova helikalnih filamenata intracelularno lociranihneurofibrilarnih klubadi kod Alchajmerove bolesti.Kod raznih oblika hereditarnih taupatija do danas jeopisano oko 20 mutacija na 17. hromozomu egzon-skog i intronskog tipa, koje dovode do promeneodnosa tau-3 i tau-4 izomorfa u korist poslednjeg.Pojava brojnih, uvijenih filamenata sa~injenihprete`no od tau-4 izomorfa uzrokovana je navedenimmutacijama. Patolo{ki oblici tau proteina ~ine jedanod klju~nih mehanizama neurodegeneracije sa krajn-jim ishodom izumiranja neurona.


tems may be affected in depression due to the alte-red neurotransmision/neuromodulation. Blunted res-ponses at the level of HPA, thyroid axes, and to thegrowth hormone stimulation, diminished response ofACTH, cortisol and prolactin to serotonin agonist,fenfluramine, are in some way, markers of depressivedisorder. New visualization techniques (SPECT, PET)reveal metabolic changes in frontal lobes (syntheticinformation processing), that clinically correlate withdepressive mood and cognition. Studies of neu-rotropic factors (BDNF), stimulated by serotonergicand noradrenergic mechanisms, and activated byCREB protein, are in progress.

THE ROLE OF PATHOLOGICAL TAU PROTEINS IN NEURODEGENERATION

G. Oci}

Institute for Neurology, CCS, Belgrade

Neurofilament deposits accompanied with patho-logical forms of tau protein are found in a number ofbrain degenerative diseases, including hereditarytauopathies, Alzheimer's disease, progressivesupranuclear paralysis and corticobasal degenera-tion. Hereditary tauopathies encompass frontotem-poral dementias and mixed syndromes such as fron-totemporal dementia associated with parkinsonism ,disinhibition ’ dementia ’ parkinsonism ’ amiotro-phy complex, pallidopantonigral degeneration andschizophrenia like behaviour with amygdala degener-ation. In adult brain tau protein is expressed in 6 iso-morphic forms generated by single gene located at17 chromosome by alternative cleavage of mRNA.They differ according to the content 3 (tau-3, -3S, ’3L) or 4 (tau-4, -4S, -4L) of microtubuli bound C-ter-minal domain. In Alzheimer's disease these tau pro-tein forms are hyperphosphorylated due to increasedprotein kinase activities or decreased protein phos-phatase activities. Hyperphosphorylated tau proteinis the structural component of intracellular neurofib-rillar glome hellicoidal filament pairs in Alzheimer’sdisease. Up to now about 20 mutations at 17 chro-mosome (exonic or intronic type), leading to thechange of tau-3 and tau-4 isomorph relation, aredescribed in different types of hereditary tauopathies.Appearance of numerous convoluted filaments most-ly composed of tau-4 isomorph is induced by men-tioned mutations. Pathological forms of tau proteinrepresent one of the key mechanisms of degenera-tion leading to death of neurons.

BIOHEMIJSKE OSNOVE ANKSIOZNOSTI

S. Miljkovi}, S. To{i}-Golubovi}

Medicinski fakultet, Ni{

Anksioznost je ubikvitarna pojava koja proìmasveukupno ljudsko ìvljenje ’ normalno i patolo{ko. Usklopu normalne emocionalne reaktivnosti ona aktivi-ra budnost, selektivnu pa`nju i procesiranje informa-cija, deluju}i kao op{ti alarmni signal. U patolo{kimstanjima ovaj signal gubi autenti~nost, t.j. aktivira se iu prilikama koje ne predstavljaju opasnost po indi-viduu. Bez obzira na brojne kontroverze o mestuanksioznosti u razli~itim klini~kim entitetima i mogu-}im komorbidnim manifestacijama (na pr. sa depre-sijom), anksioznost je najzastupljenija u stanjima stra-ha: generalizovanim anksioznim poreme}ajima, pa-ni~nim poreme}ajima i fobijama. Autori u saop{tenjurazmatraju biohemijske promene u okviru neuro-transmisije i neuromodulacije koje leè u osnovinavedenih tipova reakcije straha. Pouzdana nau~nasaznanja iz ove oblasti predstavljaju osnovu zaprou~avanje kompleksnijih stanja anksioznosti isolidne implikacije za suptilnije terapijske zahvate. Po-sebno se razmatraju uloge GABA-ergi~nog, nora-drenergi~nog i serotonergi~nog sistema. Isti~e sezna~ajna uloga GABA-ergi~kih inhibitornih procesa(direktnih i indirektnih) preko modulacije svih ascen-dentnih sistema koji u~estvuju u bihejvioralnoj orga-nizaciji stanja straha. Zna~aj Locus coeruleus-a inorepinefrina u noradrenergi~koj transmisiji potvr|enje eksperimentalno na ìvotinjama, dok u klinici ovipodaci sluè kao osnova u terapiji anksioznosti ne-benzodiazepinskim agensima. Najzad, razmatra sekontroverzna uloga serotonergi~ke transmisije(anksiogena ili anksioliti~ka?), uloga parcijalnih ago-nista u adaptaciji 5-HT1 receptora i mogu}a relacijasa stanjima depresije. Osim generalnog pristupa,isti~u se biohemijske specifi~nosti pojedinih klini~kihentiteta stanja straha, koje predstavljaju osnovu zauspe{nu primenu psihofarmakoterapije ovih stanja.

THE BIOCHEMICAL BASIS OF ANXIOSITY

S. Miljkovi}, S. To{i}-Golubovi}

Medical Faculty, Ni{, Yugoslavia

Anxiosity is ubiquitous phenomenon pervadingthe entire human living ’ normal and pathological. Innormal emotional reactivity, it activates awakeness,selective concern, information processing, acting asgeneral alarm signal. In pathological states this signalloses authenticity, being activated in situations thatare not dangerous for the person. Aside from numer-ous controversies about the site of anxiosity in differ-ent clinical entities and possible comorbid manifesta-tions (for example depression), anxiosity is mostlypresent in the states of fear: generalised anxious dis-orders, panic disorders and fobias. In this report theauthors discuss the biochemical changes in neuro-transmission and neuromodulation, underlying thementioned types of fear reaction. Relevant scientificknowledge in this area represents the basis for inves-tigation of more complex states of anxiosity and solidimplications on more subtle therapeutic approaches.The roles of GABA-ergic, noradrenergic and seroton-ergic systems are discussed separately. The impor-tant role of GABA-ergic inhibitory processes (directand indirect) via modulation of all ascendant systemsinvolved in behavioural organisation of fear state isemphasized. The importance of Locus coeruleus andnorepinephrine in noradrenergic transmission isexperimentally confirmed on animals, while clinicallythese data are basic support in nonbenzodiazepinedrug therapy of anxiosity. Finally, the controverse roleof serotonergic transmission (anxiogenic or anxiolytic?), the role of partial agonists in 5-HT1 receptor adap-tation and possible relation to depression states arealso of concern. Apart of general approach, the bio-chemical specificities of some clinical entities of fearstates, representing the basis of these states psy-chopharmacotherapy, are emphasized.


BOLESTI TRINUKLEOTIDNIH PONOVAKA:PATOFIZIOLO[KI MEHANIZMI

I KLINI^KI ZNA^AJ

V. S. Kosti}

Institut za neurologiju, Klini~ki centar Srbije, Beograd

Na kraju 20. veka identifikovano je 14 bolesti kojenastaju kao posledica ekspanzija nestabilnih trinuk-leotidnih ponovaka, ustanovljavaju}i ovaj nekada je-dinstveni mutacioni mehanizam kao osnovu sve ve-}eg broja bolesti centralnog nervnog sistema. Bolestitrinukleotidnih ponovaka mogu se zavisno od lokaci-je samih trinukleotidnih ponovaka, grubo grupisati udve celine: (a) bolesti koje uklju~uju nekodiraju}e po-novke (npr. Friedreichova ataksija, miotoni~ka distro-fija, spinocerebelarne ataksije ’ SCA tip 8 i 12) i (b)bolesti u kojima se ponovci nalaze unutar kodiraju}ihregiona. Ova druga grupa bolesti je uzrokovana eg-zonskim ekspanzijama CAG ponovaka, koje se potomtransliraju u duge poliglutaminske nizove (otuda ibolesti glutaminskih ponovaka). U ovu grupu jedin-stvenih oboljenja spadaju brojne SCA, spinobulbarnami{i}na atrofija, Huntingtonova bolest i dentatoru-bropalidoluizijalna atrofija. Ovaj tip mutacija je odklju~ne va`nosti za razumevanje patofiziolo{kih meh-anizama u razvoju razli~itih bolesti, a posebno ulogestrukturno izmenjenih proteina (npr. hantingtin iliataksini sa produ`enim poliglutaminskim nizovima)tokom procesa njihove degradacije proteasomsko-ubikvitinskim mehanizmima. Uz to, ovaj tip mutacijaomogu}io je i razumevanje ~itavog niza klini~kih fe-nomena, tipa anticipacije i razli~itih brzina progresijepatolo{kog procesa. Najzad, identifikacija patofizio-lo{kih mehanizama otvara vrata i za nove terapijskestrategije u, ina~e rezistentnim, neurodegenerativnimprocesima.

TRINUCLEOTIDE REPEAT DISEASES: PATHOPHYSIOLOGICAL MECHANISMS

AND CLINICAL SIGNIFICANCE

V. S. Kosti}

Institute for Neurology, Clinical Center of Serbia,Belgrade, FR Yugoslavia

At the end of the twentieh century 14 diseases thatresulted from expansions of unstable trinucleotiderepeats were identified. The formerly unique mutationmechanism has been recognized as the basis ofincreasing number of diseases of the central nervoussystem. The trinucleotide repeat diseases may beroughly divided into two groups, according to loca-tion of the repeats themselves: (1) diseases that affectthe non-coding repeats (e.g. Friedrich's ataxia,myotonic dystrophy, spinocerebellar ataxias [SCA]type 8 and 12) and (b) diseases where the repeats arein the coding regions. This latter group of diseases iscaused by exonic expansions of CAG repeats that aresubsequently translated into long ploygluytaminetracts SCAs, spinobulbar muscular atrophy, Huntin-gton's disease, and dentatorubropallidoluysian atro-phy. This type is mutations is crucially important forunderstanding of pathophysiological mechanisms indevelopment of miscellaneous diseases, particularlythe role of structurally altered proteins (e.g., hunting-tin or ataxins with prolonged polyglutamine tracts) du-ring the process of their degradation by proteosomal-ubiquitine mechanisms. Besides, this type of muta-tions has made it possible for us to understand aseries of clinical phenomena, such as anticipation anddifferent rates of progression of pathological process-es. Finally, identification of pathophysiological mech-anisms opens the door to new therapeutic strategiesin otherwise resistant neurodegenerative processes.


NEUROBIOLOGIJA CEREBROSPINALNETE^NOSTI: FAZNOST U RAZVOJU

ISHEMI^NOG NEURONALNOG O[TE]ENJA

M. @ivkovi}

Klinika za neurologiju, Klini~ki centar, Ni{

Cerebrospinalna te~nost suptilno reflektuje pro-mene metaboli~kog statusa kod ishemije mozga. Ciljovog istraìvanja bio je da se evaluiraju neurohemi-jske promene u ranoj postishemiji, sa idejom da sedefini{e vremenski interval (terapijski prozor) u komeaplikacija medikamenata povoljnije uti~e na metabo-li~ke promene u ishemiji mozga. Sadràj energetskihmetabolita (glikorahija, laktat, piruvat) i cAMP ispiti-vani su u likvoru 65 bolesnika, a biogenih amina (nor-adrenalin, dopamin, serotonin, 5-HIAA) kod 85 obo-lelih pacijenata. Kao kontrolna grupa u ispitivanjeuklju~eno je 10 pacijenata bez znakova akutnog cere-brovaskularnog oboljenja. Uzorci likvora su zamrza-vani i nakon toga ispitivani enzimskim fluorometrij-skim i radiometrijskim tehnikama. Unutar prvih 6 satiod po~etka bolesti je uo~eno zna~ajno pove}anje sle-de}ih parametara: glikorahije (230%), laktata (1300%),piruvata (270%), cAMP-a (600%), noradrenalina(478%), dopamina (303%), serotonina (173%), 5-HIAA(205%), 5-HIAA/5HT (118%). Rezultati istraìvanja uka-zuju da je rani postishemi~ni period (prvih 6’12 sati odpo~etka bolesti) faza burnih metaboli~kih zbivanja, {tomoè biti svojevrstan terapijski prozor adekvatnogtretmana.

BIOCHEMISTRY OF CEREBROSPINALFLUID: PHASIC RECOGNITION

OF ISCHEMIC DAMAGE

M. @ivkovi}

Department of Neurology, Faculty of Medicine, Ni{

Cerebrospinal fluid (CSF) reflects brain extracellularfluid composition, thus being valuable in assessingmetabolic state of the tissue in various conditions. Inthe present investigation, we have assessed the tem-poral patern of the biochemical changes in the CSF ofpatients with stroke; i.e 85 patients were classified intotwo groups according to the time that had elapsedafter the onset of the stroke and prior the administra-tion of therapy. Control lumbar CSF samples wereimmediately frozen and analyzed using the appropriateenzymatic, fluorometric and radiometric techniques.Within first 6 hours the following biochemical parame-ters were found increased: glucose (230%), lactate(1300%), piruvate (270%), cyclic AMP (600%), nora-drenaline (478%), dopamine (303%), serotonine(173%), 5-HIAA (205%), 5-HIAA/5HT (118%). Ourresults suggest that there is an »active phase« from theonset of the stroke to the 6’12th hour afterwards; dur-ing this interval dynamic changes are found. Thisphases are documented by means of the therapyadministrated before or after 12 hours folowing theonset of the stroke.


UC 577,1; 61 ISSN 0354-3447


Lectures

PROSPEKTIVNA STUDIJA O STATUSUHOMOCISTEINA KOD KARAKTERISTI^NIH

GRUPA PACIJENATA

D. Mirkovi}, S. Ignjatovi}, N. Majki}-Singh


Vrlo va`no polje istraìvanja u svetu, kada je u pi-tanju rizik za nastanak koronarne bolesti s jedne, od-nosno rizik za nastanak karakteristi~nih tromboti~kihpromena vaskularnog sistema s druge strane, jeutvr|ivanje karakteristi~nih vrednosti homocisteine-mije. Evidentna je i genetska predispozicija pojedinihgrupa pacijenata za nastanak hierhomocisteinemije.Po{to ovakve vrste podataka nema za na{u popula-ciju, ispitivan je nivo ukupnog homocisteina kod trikarakteristi~ne grupe pacijenata: hroni~nih bolesnikasa anginom pektoris (AP), pacijenata sa preleànimakutnim infarktom miokarda (AIM) i grupe pacijenatasa bubre`nom insuficijencijom na stalnoj hemodijal-izi. Grupa pacijenata sa simptomima AP imala je 77osoba, a broj pacijenata sa preleànim AIM, kojima jekrv uzimana maksimalno 48 ~asova nakon mani-festacije AIM, imala je 148 pacijenata. U pore|enju sakontrolnom grupom od 25 osoba na|eno je statis-ti~ki zna~ajno pove}anje koncentracija ukupnog ho-mocisteina, uz primetno izraènije pove}anje homo-cisteinemije za grupu pacijenata sa preleànim AIM.Ovo ukazuje na pove}anu sintezu homocisteina utoku i eventualno pre nastanka lezija sr~anog mi{i}a,odnosno potvr|uje pozitivnu korelaciju izme|u po-ve}anih vrednosti ukupnog homocisteina i pove}anjarizika za nastanak koronarne bolesti uklju~uju}i i AIM.Kod nezavisne grupe od 117 hroni~nih bolesnika nastalnoj hemodijalizi na|eno je statisti~ki najizraènjepove}anje vrednosti ukupnog homocisteina u odno-su na kontrolnu grupu. Ovakav podatak moè sedovesti u vezu sa izraènom toksi~nim delovanjemhomocisteina na intimu krvnih sudova. Mehanizamnastanka izraène hiperhomocisteinemije kod grupepacijenata na hemodijalizi za sada je nepoznat, a utvr-|ivanje mehanizma istog moè imati veliki zna~aj usagledavanju problema kvalitetnog saniranja pome-nutih pacijenata.

PROSPECTIVE STUDY ABOUT HOMOCYSTEINE STATUS

IN ORDERED PATIENT GROUPS

D. Mirkovi}, S. Ignjatovi}, N. Majki}-Singh


One of the very important field of investigation,when we put question about coronary risk factor, andrisk for trombotic changes in vascular system, res-pectively, is homocysteine levels. Genetic predisposi-tion for hyperhomocysteinemia in typical patient gro-ups, are also evident. There are no data like those forour population, then we have investigated whole ho-mocysteine levels in three typical patient groups:chronic patient, with angina pectoris (AP), patientwho went through acute miocardial infarction (AMI),and patients with kidney insufficiency, on permanenthemodialysis. Patient group with AP symtoms had 77persons, while number of patients in group with AMI,whose blood was collected maximum 48 hours afterattack, had 148 persons. In comparsion with controlgroup, which was consisted of 25 healthy persons,statistical significant elevation for whole homocys-teine levels was detected, for both groups, withmarked elevation for (AMI) group. This data appearon increased synthesis of homocysteine duringattack, and before rise of coronary muscule lesion,confirmated positive correlation among elevatedwhole homocysteine levels and increased risk for co-ronary diseases, including AIM. By independent groupwith 117 patients on permanent hemodialysis, mostsignificant homocysteine elevation has been detectedagainst control group. This fact can be bind with toxichomocysteine action on intima. Mechanism for hiper-homocysteinemy in this patient group is not clear andits determination can give important results in qulita-tive treatmant of these patients.


UTRO[AK MANOZA VEZUJU]EG PROTEINAU SERUMU I SINOVIJALNOJ TE^NOSTIKOD BOLESNIKA SA REUMATOIDNIM

ARTRITISOM

Z. V. Miju{kovi}1, I. Zgradi}3, M. ôli}2, R. Nikolajevi}1, G. Radunovi}4, Lj. Rackov3,

Z. An|elkovi}3, M. Popovi}3

1Institut za medicinsku biohemiju 2Institut za medicinska istraìvanja

3Klinika za reumatologiju i klini~ku imunologiju,Vojnomedicinska akademija

4Institut za reumatologiju, Beograd

Izmenjen proces glikozilacije kod bolesnika sareumatoidnim artritisom (RA) manifestuje se defici-tom u sadràju galaktoze unutar glikanskog lanca naFc fragmentu IgG, a G0IgG je dominatna forma. Ta-kve strukturne promene mogu da promovi{u noveepitope i autoreaktivnost ili da olak{aju stvaranjekompleksa agregiranih G0IgG, koji aktiviraju »pse-udoklasi~ni« put aktivacije komplementa, posred-stvom manoza vezuju}eg proteina (MbP). Da bi seispitivala povezanost ovih fenomena sa pomenutomaktivacijom komplementa kod RA, odre|ena je kon-centracija MbP u 59 seruma i sinovijalnih te~nost(ST), imunonefelometrijskom metodom (DADE Be-hring). Bolesnici su razvrstani u 2 grupe: 24 sa ume-renom (UA) i 35 sa visokom aktivno{}u (VA) RA pre-ma klini~ki utvr|enim kriterijumima. Kontrolnu grupu(KG) ~inilo je od 15 bolesnika sa lezijama meniskusa.Prose~ne vrednosti ± SD za MbP u serumu uznosilesu za KG 2,1 ± 1,0; 2,6 ± 1,5 za UA i 3,3 ± 1,9 u grupisa VA, dok su vrednosti u ST bile 1,1 ± 0,8; 1,0 ± 0,9;2,9 ± 1,7 mg/L. Koncentracije MbP bile su unutarfiziol{kih granica ili povi{ene u serumu a u ST su bileniè za oko 40’55% u odnosu na serum. Statisti~kazna~ajnost je zabeleèna u koncentracijama MbP userumu i ST izme|u grupa sa VA i UA RA, a samo userumu izme|u VA i KG (p<0,05). Nije konstatovano~ekivan utro{ak MbP u serumu i ST, te rezultati ovogispitivanja niti opovrgavaju ustanovljenu ulogu MbP uaktivaciji komplementa, niti idu u prilog ovakve hi-poteze.

MANNOSE-BINDING PROTEIN CONSUMPTION IN SERUM

AND SYNOVIAL FLUID OF RHEUMATOIDARTHRITIS PATIENTS

Z. V. Miju{kovi}1, I. Zgradi}3, M. ôli}2, R. Nikolajevi}1, G. Radunovi}4, Lj. Rackov3,

Z. An|elkovi}3, M. Popovi}3

1Institute for Medical Biochemistry2Institute for Medical Research

3Clinic for Rheumatology and Clinical Immunology,Military Medical Academy

4Institute for Rheumatology, Belgrade, Yugoslavia

Altered glycosylation at rheumatoid arthritis (RA)patients was demonstrated by deficiency in galactosecontent of the N-linked carbohydrate in IgG Fc mo-lecule, while G0IgG form is dominating. Such struc-tural change might expose new epytopes that couldelicit autoreactivity or, enhance complex formationcontaining aggregated G0IgG and complement»pseudoclassical« activation via mannose-bindingprotein (MbP) attachment. In order to prove connec-tion between RA and mentioned anticomplementaryactivity, 59 RA patients sera and synovial fluid (SF)were assayed for the level of MbP by the latex-enhan-ced immunonephelometry (DADE-Behring) method.Patients were divided in two groups: 24 with moder-ate (MA) and 35 with severe activity (SA), accordingto the established clinical criteria. Control group (GG)was consisted of 15 patients suffering from meniscuslesion. Mean ± SD sera MbP values (mg/L) were forCG 2.1 ± 1.0; 2.6 ± 1.5 in MA and 3.3 ± 1.9 in SAgroup and 1.1 ± 0.8; 1.0 ± 0.9; 2.9 ± 1.7 in SF, res-pectively. MbP serum concentrations were either nor-mal or elevated and SF concentration representedabout 40’55% of serum values. Statistical signifi-cance was detected in sera and SF MbP values in SAversus. MA groups and only in sera in VA versus. CG(p<0.05). However, undetectable MbP consumptionin both fluids from this examination, do not refute theproposed role of MbP-mediated complement activa-tion, but provide no support for that hypothesis.


BIOHEMIJSKA DIJAGNOSTIKA HEMATOLO[KIH BOLESTI

DE^IJEG UZRASTA

G. Bjelakovi}, G. Kosti}, M. Ili}, T. Jevtovi}

Institut za biohemiju, Medicinski fakultet i De~ija interna klinika, Klini~ki centar, Ni{

Hematolo{ke bolesti u de~ijem uzrastu su veoma~este. Analiza hematolo{kog statusa kao i standard-nih biohemijskih analiza krvi, koje predstavljaju odrazfunkcije hematopoeznog sistema, od vitalnog zna~ajasu u otkrivanju i prepoznavanju oboljenja. Sva hema-tolo{ka oboljenja javljaju se bilo zbog poreme}ajafunkcije hematopoeznog sistema na nivou produkci-je, stepena vitalnosti krvnih }elija i pre`ivljavanja u cir-kulaciji {to ima odraza na hemogram, kao i biohe-mijske karakteristike krvne plazme i seruma. Osnovnihematolo{ki pokazatelji postojanja krvnih oboljenja(osim poreme}aja koagulacije krvi) su: ukupan brojkrvnih }elija (eritrocita, leukocita sa utvr|ivanjem leu-kocitarne formule, ukupan broj trombocita), koncen-tracija hemoglobina, vrednost hematokrita, brzina se-dimentacije eritrocita, osmotska rezistencija eritroci-ta. Od standardnih biohemijskih analiza kod ovih bo-lesti odre|uje se vrednost ukupnog bilirubina uz po-sebno sagledavanje vrednosti indirektnog bilirubina,nivo gvo`|a u serumu, transferina (TIBC), serumskogbakra, ceruloplazmina. Elektroforeza proteina i he-moglobina tako|e se koriste. Enzimolo{ka ispitivanja,osim povremenog merenja ukupne aktivnosti laktatdehidrogenaze, retko se primenjuju. Osnovna idejaovog prikaza je sagledavanje ve}ih biohemijskih mo-gu}nosti u rasvetljavanju prirode hematolo{kih pore-me}aja (osim poreme}aja hemostaze), prvenstvenokroz {iri dijapazon enzimolo{kih analiza, specifi~nihenzima u krvnim }elijama {to ima direktni odraz naaktivnost enzima u serumu; ispitivanje aktivnosti enz-ima vezanih za razgradnju glukoze glikolizom (LDHsa izoenzimima, aldolaze A, piruvat kinaze, alkalnefosfataze, kisele fosfataze), enzima vezanih za pen-tozni put razgradnje glukoze i metabolizam glutationa(glukozo-6-fosfat dehidrogenaze, glutation sintetaze,glutation reduktaze, glutation peroksidaze), enzimevezane za metabolizam purina i pirimidina (5-nuk-leotidaze, AMP-dezaminaze, adenozin dezaminaze,citidin dezaminaze i enzime vezane za metabolizampoliamina i RNK (arginaza, poliamino oksidaza i alka-


BIOCHEMICAL INVESTIGATION OF HEMATOLOGICAL DISEASES

IN CHILHOOD

G. Bjelakovi}, G. Kosti}, M. Ili}, T. Jevtovi}

Institute of Biochemistry, Faculty of Medicine,Pediatric Clinic, Clinical Center, Ni{

Hematological diseases in childhood occur veryfrequently. Analysis of hematological status as well asstandard biochemical analysis of blood, which arethe result of hematopoetic system function, is ofgreat importance in discovering and recognition ofthese diseases. All hematological diseases appear asa result of disturbance of hematopoetic system func-tion at the level of the production and viability inblood stream; this disturbance has reflection on thehemogram and on the biochemical analysis of bloodplasma and serum. The basic hematological param-eters of blood diseases (except disturbance of bloodcoagulation) are: total blood count cells (erythro-cytes, leukocytes with differential leukocytes formu-las, trombocytes) hemoglobin concentration, theamount of hematocrit (PCV, packed cell volume),erythrocytes sedimentation rate, erythrocytes osmot-ic fragility. Among the standard biochemical analysisin these diseases total bilirubin with the calculationfraction of indirect bilirubin, the amount of serumiron and transferine (TIBC), serum copper and ceru-loplazmin are determined. The protein and hemoglo-bin electrophoresis are in use as well. The measure-ment of lactate dehydrogenase activity infrequentlyanother enzymological investigations are rare in use.Enzymological researches, measurement of lactatedehydrogenase activity, are rarely in use. The basicidea of this study is evaluation of another biochemi-cal possibility with the aim to understand the natureof hematological disturbance (beside the hemostasedisturbances), by the use the great specter of enzymeanalysis, especially the enzymes which are present indifferent blood cell and with the direct reflection onthe serum enzymes activities such as: the investiga-tion of enzymes of glycolysis (LDH with isoenzymes,aldolase A, pyruvate kinase, alkaline and acid phos-phatase), enzymes of pentose phosphates pathwayand glutathione metabolism (glucose-6-phosphatdehydrogenase, glutathione synthetase, glutathionereductase, glutathione peroxidase), enzymes of puri-

UC 577,1; 61 ISSN 0354-3447


Lectures

lne ribonukleaze). Merenje sadràja vitamina C u krvi,dijagnostikovanje hipovitaminoze piridoksina krozmerenja aktivnosti transaminaza, pre i posle in vitrosuplementacije sa piridoksal-fosfatom, elektroforet-sko ispitivanje formimino-glutaminske kiseline u uri-nu (Figlu) u cilju rasvetljavanja hipovitaminoze folnekiseline, kao i merenje koli~ine metilmaloni~ne aci-demije u cilju diagnostikovanja hipo ili avitaminozevitamina B12 tako|e predstavljaju specifi~ne biohemij-ske analize primenljive u okviru biohemijske dijagnos-tike. Zbog va`nosti hemoglobina u regulaciji pH krvi,ispitivanje acidobaznog statusa kod bolesti koje zah-vataju eritropoezu moglo bi da bude od koristi u sa-gledavanju stepena poreme}aja intermedijernog me-tabolizma. Dosada{nji rad na ispitivanju pomenutihenzima i ostalih biohemijskih parametara u raznimhematolo{kim oboljenjima, uro|enim ili ste~enim,ukazuju na mogu}nost i potrebu izvo|enja pomenu-tih biohemijskih analiza u savremeno opremljenim la-boratorijskim uslovima.

IZBOR PARAMETARA HEMOSTAZE ZA PRA]ENJE NORMALNE

I KOMPLIKOVANE TRUDNO]E

T. Vodnik, S. Ignjatovi}, N. Majki}-Singh


Koncentracije proteina plazme uklju~enih u proceskoagulacije menjaju se tokom normalne trudno}e {toremeti ravnoteù koja postoji izme|u prokoagu-lantnog i antikoagulantnog sistema. Ove promeneobuhvataju pove}anje aktivnosti faktora koagulacije,pove}ano stvaranje fibrina i supresiju fibrinolize. Naovaj na~in se fiziolo{kim mehanizmima smanjuje rizikod gubitka krvi u trudno}i, ali se pove}ava rizik odpojave tromboze. Usled tromboze placentarnih krvnihsudova moè se javiti placentarna insuficijencija kojadovodi do pojave ponavljaju}ih abortusa, zastoja urastu fetusa, eklampsije, intrauterine smrti ploda i pre-vremenog poro|aja. Poslednjih godina istraìvanjapokazuju da su mogu}i uzroci ovih pojava promenekoje se de{avaju unutar protein C koagulantnog sis-tema. Aktivirani protein C (APC) svoje antitromboti~kodelovanje ispoljava proteoliti~kom razgradnjom fakto-ra V i VIII. Mutacija na genu za faktor V (tzv. Factor VLeiden mutacija) stvara mutirani faktor V koji ispoljavanormalnu prokoagulantnu aktivnost ali je manjeosetljiv na APC. Ove promene vode stabilizaciji kom-pleksa protrombinaze, stvara se vi{e trombina kojiaktivira jo{ ve}e koli~ine proteina C, ali rezistentnog,


ne and pyrimidine metabolism (5-nucleotidase, AMP-deaminaze, adenosine desaminase, cytidine deami-nase and enzymes which participate in metabolism ofpolyamines and RNA (arginase, polyamine oxidaseand alkaline ribonuclease). The measurement of vita-min C amount, activity of transaminases before andafter supplementation with pyridoxal phosphate inorder to prove the hypovitaminose of pyridoxine, thedetermination of formiminoglutamic acid (Figlu) bymethods of electrophoresis in order to prove hypovi-taminoseof folic acid and the determination of met-hylmalonic acidemie with the intention to prove thehypo or avitaminose of vitamin B12 represent the pos-sibilities in biochemical diagnostics of hematologicaldiseases. As a result of the importance of hemoglo-bin in the blood pH regulation, the investigation ofacid-base status may give some greater possibility inlaboratory diagnostic methods. Our work in the paston the determination of the mentioned enzymes acti-vities and other biochemical parameters in differenthematological diseases, inherited or acquired, showthe need and practical possibility for application of allthese methods in modern equpped laboratories.

CHOICE OF HAEMOSTASIS PARAMETERSFOR MONITORING OF NORMAL AND COMPLICATED PREGNANCY

T. Vodnik, S. Ignjatovi}, N. Majki}-Singh

Institute of Medical Biochemistry, Clinical Center of Serbia, Belgrade

The concentrations of plasma proteins involved inthe process of coagulation are changed during thenormal pregnancy, disturbing the balance betweenprocoagulant and anticoagulant system. Thesechanges include the increased activity of coagulationfactors, the increased fibrin production and suppres-sion of fibrinolysis. In this way, the risk of loosingblood during pregnancy is reduced by physiologicalmechanisms, but the hazard of developing thrombo-sis is increased. Placental insufficiency may occurdue to thrombosis of placental blood vessels, leadingto repeated miscarriages, retardation of foetal growth,eclampsia, intrauterine foetal death and preterm birth.The causes of these incidents are the changes occur-ring within protein C coagulation system. ActivatedProtein C inhibits blood coagulation by factor Va andVIIIa proteolytic degradation. The mutation in genefor factor V (so-called Factor V Leiden mutation),where the arginine 506 is replaced by glutamine, pro-duces the mutated factor V having normal procoag-ulant activity but it is less responsive to APC. Thecondition characterized by poor anticoagulant respo-nse to APC is called the APC resistance. The aim of

~ime se postiè stanje hiperkoagulabilnosti. Stanjekoje karakteri{e slab antikoagulantni odgovor na APCuzrokovan mutacijom na genu za faktor V naziva seAPC rezistencija. Cilj ovog rada je bio da se ispita stan-je koagulantnog, antikoagulantnog i fibrinoliti~kog sis-tema kod trudnica sa normalnom i komplikovanomtrudno}om razli~ite gestacijske starosti. Tako|e ispi-tana je i funkcionalnost protein C antikoagulantnogsistema u trudno}i razli~itih gestacijskih starosti i APCrezistencija. Za procenu stanja hemostaznog sistemaodre|ivani su slede}i parametri: protrombinsko vre-me, parc. protrombinsko vreme, fibrinogen, fibrin mo-nomer, D-dimer, antitrombin III, TAT kompleks, plaz-minogen, PAI, faktor XIII, protein C, protein S, proteinC Global i aktivirani protein C. Pokazano je da su fi-brinogen, fibrin monomer, TAT kompleks i PAI kaoparametri hemostaze dobri markeri veli~ine hiperkoa-gulabilnog stanja kod trudnica. Vrednosti za PT, aPTT,D-dimer, faktor XIII i antitrombin III u ovom ispitivanjuostale su uglavnom u granicama normalnih vrednostiili su se neznatno menjale u poodmakloj trudno}i.Vrednosti proteina S kod ve}ine trudnica bile su sni-ène, dok su za protein C bile u granicama normalnihvrednosti. Globalna aktivnost protein C antikoagulan-tnog puta i odnos sa prokoagulantnim sistemom po-kazana je kvantitativnim merenjem kroz ProC Globaltest. Odre|ivanje aktiviranog proteina C kroz test APCsenzitivnosti koristi se kao pomo} u identifikaciji oso-ba sa APC rezistencijom odnosno sa pove}anim rizi-kom od tromboze. U ovom radu statisti~ki zna~ajnoniè vrednosti PC-NR i APC-NR dobijene su u sva trivremenska perioda kod trudnica sa ponavljaju}impoba~ajima i trudnica sa hipertenzijom u odnosu natrudnice sa normalnim tokom trudno}e. Na taj na~inje u ovom radu pokazano da je antikoagulantna aktiv-nost aktiviranog proteina C zna~ajno smanjena u trud-no}i, naro~ito u trudno}i pra}enoj komplikacijama.Dijagnosti~ka vrednost parametara hemostaze kaomarkera tromboti~kih promena kod trudnica ispitanaje ROC analizom. Sveukupnim ispitivanjem je potvr-|eno da su PC-NR i APC-NR pokazali zadovoljavaju}udijagnosti~ku ta~nost kao markeri tromboti~kih prom-ena u trudno}i. U ovom radu je pokazano da su PC-NR i APC-NR dobri markeri tromboti~kih promena uplacentarnim krvnim sudovima ta~nije dobri pokaza-telji razvoja rezistencije na aktivirani protein C u trud-no}i. Odre|ivanje ovih parametara kao i odre|ivanjefibrinogena, fibrin monomera, TAT kompleksa i PAIvrlo je korisno za otkrivanje i pra}enje trudnica satrombozom. Zbog velike uloge koju ovi parametri ima-ju u periodu trudno}e, postoje brojne preporuke zanjihovo uvo|enje u rutinsko odre|ivanje. Na taj na~inbi se pravovremeno utvrdilo odsustvo ili prisustvo jed-nog iz niza faktora rizika koji prati stanje trudno}e.


this study was to test the condition of coagulant, anti-coagulant and fibrinolytic system in normal and com-plicated pregnancy as well as the function of proteinC anticoagulant system in pregnancy of various ges-tation periods and APC resistance. The following pa-rameters were measured for the evaluation of hae-mostasis system: prothrombin time, partial pro-thrombin time, fibrinogen, fibrin monomer, D-dimer,antithrombin III, TAT complex, plasminogen, PAI,factor XIII, protein C, protein S, protein C Global andactivated protein C. The testing was performed inpregnant women with normal and complicated preg-nancy of various gestation periods. A special groupconsisted of pregnant women having two or moreconsecutive miscarriages. It was shown that fibrino-gen, fibrin monomer, TAT complex and PAI, asparameters of haemostasis, were good markers forthe extent of hypercoagulable condition in pregnantwomen. PT, aPTT, D-dimer, protein C and antithrom-bin III values stayed within normal range. Values ofprotein S were decreased in pregnancy. Pro C Globaltest is a coagulation test for the determination of theanticoagulatory capacity of the protein C system inhuman plasma. The determination of the APC sensi-tivity is thus used to identify persons with an elevatedthromboembolic risk. In this study, significantly lowervalues of PC-NR and APC-NR were obtained in allthree time periods in pregnant women with hyperten-sion and repeated miscarriages in comparison tohealthy controls, what verified that anticoagulantactivity of activated protein C was significantly redu-ced during pregnancy, and especially in pregnancyassociated with complications. The diagnostic valueof haemostasis parameters as markers of thromboticchanges in pregnant women was tested by ROC ana-lysis. According to the obtained results, PC-NR andAPC-NR showed satisfactory diagnostic accuracy asmarkers of thrombotic changes in pregnant women,more precisely, they were found to be good indica-tors of resistance to activated protein C in pregnan-cy. Therfore it is very useful to introduce these pa-rameters in daily work. Thereby it would be affirmabsence or presence pregnancy risk factors in time.

LEPTIN: FIZIOLO[KA ULOGA I DIJAGNOSTI^KI ZNAÂJ

B. Brki}, G. Lazovi}, V. Milo{evi}

KBC »Dr Dragi{a Mi{ovi}«, Ginekolo{ka klinika,Klini~ki centar Srbije, Institut za biolo{ka istraìvanja

»Sini{a Stankovi}«, Beograd

Leptin je proteinski hormon od 167 aminokiseli-na, molekulske mase 16 kDa, koga prete`no stvarajuadipociti. Tercijarna struktura leptina sli~na je cito-kinima i laktogenim hormonima. Leptin cirkuli{e ukrvi u koncentraciji koja je proporcionalna koli~inimasnog tkiva, odnosno indeksu telesne mase. Leptincirkuli{e u biolo{kim te~nostima kao slobodan pro-tein, i u obliku vezuju}e solubilne izoforme za njegovreceptor. Leptin se sekretuje pulsatilno i ima cirkadi-jalni ritam, sa no}nim pikom izme|u 01.00h i 02.00h.Poznato je da leptin i odgovaraju}i receptor, pred-stavljaju integralne komponente fiziolo{kog signalnogsistema regulisanja energetskih depoa i energetskogbalansa. Leptin se stvara u adipoznom tkivu, placen-ti, stomaku i popre~no prugastim misi}ima. Leptin jemo}an regulator unosa hrane sposoban je da zna~aj-no smanji ekspresiju najmo}nijeg peptida gladi Y(NPY) za koga je dokazano da je fiziolo{ki transdjuserapetita u mozgu. Dejstvo na suprimiranje unosahrane ostvaruje preko mRNK u hipotalamusu, kojikontroli{e apetit i termogenezu. Koncentracija leptinau serumu kog zdravih osoba u pozitivnoj je korelacijisa sadràjem masnog tkiva i telesnim masenimindeksom (BMI). Ona je vi{a kod debelih nego kodmr{avih osoba. Koncentracija leptina u serumu zdra-vih èna je 12,1 ± 7,6 ng/mL, prema BMI 21,9 ± 0,2kg/m2 i procentu masnog tkiva 41,3 ± 1,6 %, a u zdra-vih mu{karaca nivo leptina iznosi 3,6 ± 1,6 ng/mL,BMI 23.0 ± 0,8 kg/m2, procenat masnog tkiva 27,5 ±1,2%. Uo~ava se da ène imaju vi{e nivoe leptina, kaoi procenat masnog tkiva u odnosu na BMI, negomu{karci. Ova razlika proisti~e iz razli~ite distribucijesubkutanog adipoznog tkiva kod ène i mu{karca {touslovljava razli~it stepen sinteze leptina. Osnovnauloga leptina je da informi{e CNS o vi{ku ili manjkuenergetskih depoa i aktivno u~estvuje u energetskoj ineuroendokrinolo{koj adapataciji na te promene. Po-red uloge u regulisanju telesne teìne leptin u~estvu-je i u regulisanje reproduktivnog sistema. Tako leptin


PLEPTIN: THE ROLE IN HUMAN PHYSIOLOGY AND DIAGNOSTIC

SIGNIFICANCE

B. Brki}, G. Lazovi}, V. Milo{evi}

KBC »Dr Dragi{a Mi{ovi}«, Clinical Centre of Serbia,Institute of Biological Research »Sini{a Stankovi}«,

Belgrade

Leptin is a 16 kDa protein hormone producedpredominantly by adipocytes. Leptin, Tertiary struc-ture of leptin, resembles that of cytokines and lacto-genic hormones. Leptin circulates in the blood atconcentrations paralleling the amount of fat reserves.In biological fluids leptin circulates both as a free pro-tein and in a form that is bound to the soluble iso-form of its receptor. Leptin secretion is pulsatile andshows a circadian rhythm, with a nocturnal risereaching its peak between 01.00h and 02.00h. Leptinand its receptor, are now known to be integral com-ponents of a physiological signalling system that reg-ulate fuel stores and energy balance. Constitutive lep-tin expression has been demonstrated only in adi-pose tissue, placenta, stomach and sceletal muscle.Leptin has been reported to be a potent regulator offood intake. There is evidence indicating that at leastsome of the effects of leptin occur through receptor-mediated regulation of the hypothalamic proteinneuropeptide Y (NPY). Leptin mRNA expression inthe hypothalamus is suppressed by fasting, suggest-ing a role of brain leptin in the central regulationappetite. Serum leptin concentrations in healthy indi-viduals positively correlate with the body fat contentand body mass index. They are higher in obese thanin lean subjects. Serum concentration of leptin inhealthy non-obese women is 12.1 ± 7.6 ng/mL, inaccordance with BMI 21.9 ± 0.2 kg/m2 and fat tissuepercent 41.3 ± 1.6%, while in healthy man leptin levelis 3.6 ± 1.6 ng/mL, BMI 23.0 ± 0.8 and fat tissue per-cent 27.5 ± 1.2%. It is noticeable that the womenhave higher leptin level as well as fat tissue percent inaccordance BMI than the man. The distinction isresult of different subcutane adipose tissue distribu-tion in women and men causing different leptin syn-thesis level. Beside its role in body weight regulation,leptin also stimulates the reproductive axis and takesan active role in enters puberty. In adult patients,homozygous missense mutations of the gene encod-

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Lectures

aktivno u~estvuje i u zapo~injanju puberteta. U odra-slih osoba, homozigota nastanak mutacija na genukoji kodira leptinski receptor dovodi do izraènog ra-nog nastanka gojaznosti, smetnje u razvoju puberte-ta, primarne amenoreje kod èna i klini~kih karakter-istika hipogonadizma u mu{karca. Neki radovi ukazu-ju da leptin u~estvuje kao metaboli~ki kofaktor upatogenezi hiperandrogenizma u bolesnica sa policis-ti~nim jajnicima. Veza leptina i trudno}e je naro~itozna~ajna. Leptin ima tendenciju porasta u tre}em tri-mestru trudno}e, {to korelira sa ukupnim porastommasnog tkiva kod trudnice. Tako|e uti~e na veli~inuCNS, na hematopoetske }elije, kao i na funkciju bu-brega.

ENZIMI METABOLIZMA PURINA U SERUMUBOLESNIKA SA HIPERLIPIDEMIJAMA

S. Kundali}, G. Koci}, V. ]osi}, T. Jevtovi}-Stoimenov, V. B. \or|evi}

Centar za medicinsku biohemiju, Klini~ki centar Ni{;Biohemijski institut, Medicinski fakultet, Ni{

Metabolizam purina zna~ajan je za normalnofunkcionisanje endotelnih }elija, a enzimi katabolizmapurina zauzimaju posebno mesto u o{te}enom endo-telu ateroskleroti~nih sudova hiperlipidemija. Kataliti-~ka aktivnost 5'-nukleotidaze (5'-NT), adenozin deza-minaze (ADA), ksantin dehidrogenaze (XD) i ksantinoksidaze (XO) ispitivana je kod tri grupe bolesnika sahiperlipidemijama: grupa bolesnika sa hiperlipopro-teinemijom, grupa bolesnika sa dijabetom i grupa sahipertenzijom. Prema vrednostima lipidnih para-metara: holesterol (HOL), triacilgliceroli (TG), HDL-holesterol (HDL-c) i LDL-holesterol (LDL-c), bolesni-ci su svrstavani u tipove hiperlipoproteinemija po Fre-driksonu, a podgrupe tipova formirane su na osnovuvrednosti hiperholesterolemije i hipertrigliceridemije.Naj~e{}e zastupljeni tipovi hiperlipoproteinemija usve tri grupe bili su IIa, IIb i tip IV. Enzimi metaboliz-ma purina bili su statisti~ki zna~ajno izmenjeni u seru-mu bolesnika sve tri grupe. Aktivnost 5'-NT statisti~kije zna~ajno rasla u serumu bolesnika sa hipertenzi-jom (p<0,001), kao i u podgrupi 2b tipa IIb HLP-a itipu IV HLP-a (p<0,001 i p<0,01). Registrovana jestatisti~ki zna~ajna pozitivna korelacija izme|u aktiv-nosti 5'-NT i aterogenih lipidnih parametara (p<0,001), a negativna korelacija sa HDL-c (p<0,05).Zabeleèn je statisti~ki zna~ajan porast aktivnostiADA u grupi bolesnika sa dijabetom, kao i tipovimaIIa, IIb i IV HLP-a (p<0,001). Postoji statisti~ki zna-~ajna pozitivna korelacija izme|u aktivnosti ADA iglukoze (p<0,001), kao i izme|u ADA i aterogenih li-pidnih parametara. Zna~ajna negativna korelacija jezabeleèna izme|u aktivnosti ADA i protektivnogHDL-c (p<0,001). Aktivnost XO je pokazivala zna-


ing the leptin receptor determine severe early-onsetobesity, failure to enter puberty, primary amenorhoeain female and clinical features of hypogonadism inmales. Some articles suggest that leptin takes part inhyperandrogenism pathogenesis as a metabolic co-factor in female patients with polycystic ovaries. A re-lation between leptin and pregnancy is of great signi-ficance. Leptin has increasing tendency in the thirdmonth of pregnancy that correlates with total incre-ase of the fat tissue in pregnant women. Leptin alsoexerts influence on CNS value, on hematopoethiccells and on the kidney function.

PURINE METABOLISM ENZYMES IN HYPERLIPIDEMIC PATIENTS

S. Kundali}, G. Koci}, V. ]osi}, T. Jevtovi}-Stoimenov, V. B. \or|evi}

Centre of Medical Biochemistry, Clinical Centre, Ni{Institute of Biochemistry,

University School of Medicine, Ni{

Since purine metabolism is important forendothelial cells normal function, purine catabolismenzymes have a special place in damaged endotheli-um of atherosclerotic blood vessels in hyperlipide-mias. The activities of 5-nucleotidase (5'-NT), adeno-sine deaminase (ADA), xanthine dehydrogenase (XD)and xanthine oxidase (XO) were investigated in threegroups of hyperlipidemic patients: patients with hy-perlipoproteinemia, diabetic patients and hyperten-sion group. According to the values of lipid parame-ters: cholesterol (CHOL), triacylglycerols (TG), HDL-cholesterol (HDL-c) and LDL-cholesterol (LDL-c),the patients were allocated into hyperlipoproteinemictypes by Fredrikson, while type subgroups wereformed based on the values of hypercholesterolemiaand hypertriglyceridemia. The most frequent types ofhyperlipoproteinemias in all three groups were IIa, IIband type IV. The enzymes of purine metabolism sho-wed statistically significant changes in serum of allthree groups patients. 5'-NT activity was siginificant-ly increased in serum of patients with hypertension(p<0.001), as well as in subgroup 2b (HLP type IIb)and HLP type IV (p<0.001; p<0.01, respectively).We found statistically significant positive correlationbetween 5'-NT activity and atherogenic lipid parame-ters (p<0.001) and negative correlation with HDL-c(p<0,05). Statistically significant increase of ADAactivity was noted in diabetic patients, as well as inHLP types IIa, IIb and IV (p<0.001). There is statis-tically siginificant positive correlation between ADAactivity and glucose value (p<0.001), as well as ADAand atherogenic lipid parameters. Significant nega-tive correlation was found between ADA activity and

~ajan porast u serumu sve tri grupe pacijenata (p<0,001) u odnosu na kontrolnu grupu, kao i u svimtipovima HLP-a, a zabeleèna je i zna~ajna pozitivnakorelacija izme|u XO i aterogenih lipida, a zna~ajnanegativna korelacija izme|u XO i HDL-c. Statisti~kizna~ajno sniènje aktivnosti XD registrovano je u hi-perlipidemijama sve tri grupe bolesnika (p<0,001),kao i u svim tipovima HLP-a. Zapaèna je zna~ajnanegativna korelacija izme|u aktivnosti XD i ateroge-nih lipida, a zna~ajna pozitivna korelacija sa protek-tivnim HDL-om. Krajnji produkt metabolizma purina i»scavanger« slobodnih kiseoni~nih radikala, mokra}-na kiselina, bila je statisti~ki zna~ajno pove}ana u hi-perlipidemijama. Dobijeni rezultati sugeri{u mogu}-nost odre|ivanja enzima metabolizma purina kao mar-kera o{te}enja endotela i eventualne terapijske korek-cije u prakti~nom radu.

PATOFIZIOLOGIJA I KLINI^KI ZNAÂJATEROGENOG LIPOPROTEINSKOG

FENOTIPA I MALIH GUSTIH LDL ÊSTICA

M. \eri}

Zavod za laboratorijsku medicinu, Klini~ki centar, Novi Sad

Brojne epidemiolo{ke studije potvrdile su pove-zanost izme|u serumskih triglicerida i prevremeneateroskleroze, ali metaboli~ka osnova ove udruènos-ti nije dobila zadovoljavaju}e obja{njenje. Uspostav-ljena je »vladavina« holesterolske hipoteze, da bi,nedavno, bio definisan holesterolski paradoks ’ se-rumska koncentracija holesterola nije dobar diskrim-inacioni parametar za procenu rizika koronarnesr~ane bolesti. Postalo je o~igledno da aterogenostLDL lipoproteina kod ve}ine osoba proizilazi iz nji-hove male veli~ine, ve}e gustine i pove}anog broja~estica, a ne iz njihovog doprinosa serumskom holes-terolu. Subfrakcije malih gustih LDL (LDL-III) imajuniì afinitet za LDL receptore, duè se zadràvaju ucirkulaciji, imaju vi{i afinitet za oksidaciju i vezivanjeza proteoglikane arterijskog zida. Predominacija LDL-III (fenotip B) poti~e od defekta u metabolizmu tri-gliceridima bogatih lipoproteina i posledica je pojaverecipro~nog prenosa lipida tokom delipidacionekaskade, uz u~e{}e holesterol estar transfer proteina,lipoproteinske i hepati~ne lipaze, i, uz centralnu uloguHDL ~estica. Karakteristi~no premo{}avanje izme|uklasa lipoproteina predodre|enih da na periferiju do-premaju trigliceride i ~estica koje prenose holesterol,dovodi do uspostavljanja tzv. aterogenog lipoprotein-skog fenotipa (hipertrigliceridemija, pove}anje LDL-III, apolipoproteina B i VLDL-triglicerida, a sniènjeHDL-holesterola i apolipoproteina A).


protective HDL-c (p<0.001). XO activity showed sig-nificant increase in serum of all three groups patients(p<0.001) compared to control group and all HLPtypes. There is also a significant positive correlationbetween XO and atherogenic lipids and significantnegative correlation between XO and HDL-c. Sta-tistically significant decrease of XD activity was regis-tered in hyperlipidemias in all three examined groups(p< 0.001), as well as in all HLP types. Significantnegative correlation between XD activity and athero-genic lipids and significant positive correlation withprotective HDL were also found. The end product ofpurine metabolism and scavenger of free radicals,acidum uricum, was significantly increased in hyper-lipidemias. The obtained results suggest a possibilityof purine metabolism enzymes estimation, using themas markers of endothelial damage, but also a poten-tial of therapeutic correction in practice.

PATHOPHYSIOLOGY AND CLINICAL SIGNIFICANCE OF ATHEROGENIC

LIPOPROTEIN PHENOTYPE AND SMALLDENSE LDL PARTICLES

M. \eri}

Department of Laboratory Medicine, Clinical Center, Novi Sad

Many epidemiological studies have confirmed theassociation of the serum triglyceride level and prema-ture atherosclerosis, but the metabolic basis of thisassociation has not been so far fully explained. Thecholesterol hypothesis has prevailed, and recently thecholesterol paradox has been defined ’ the serumcholesterol level is not a reliable discriminatory para-mater for the risk evaluation of coronary heart disease.It is obvious that LDL lipoprotein atherogenicity inmost patients occurs due to their small size, higherdensity and enlarged number of particles, but not dueto their contribution to total cholesterol. The subfrac-tions of small dense LDL (LDL-III) have lower affinityfor the LDL receptors, persist longer in the circulation,have higher affinity for oxidation and arterial wall pro-teoglicans binding. The predominance of LDL-III (phe-notype B) results from the defect in the metabolism oftriglyceride rich lipoproteins and is a concequence ofthe reciprocal lipid transfer throughout the delipidationcascade, with the action of cholesterol ester transferproteins, lipoprotein and hepatic lipase, and with thedominant role of HDL particles. The characteristicconnection between the lipoprotein classes pre-desposed to cary triglycerides to the perifery and theparticles which transfer cholesterol, induces the estab-lishment of the so-called atherogenic lipoprotein phe-notype (hypertriglyceridemia, increase in LDL-III level,apolipoproteine B and VLDL-triglyceride, and decrea-se in HDL-cholesterol and apolipoprotein A).

UTICAJ AZOT MONOKSIDAI PEROKSINITRITA NA METABOLIZAM

NUKLEOTIDA: PRO ILI ANTIAPOPTOTI^NIEFEKTI?

G. Koci}, T. Jevtovi}, D. Pavlovi}, R. Koci}, V. \or|evi}, G. Bjelakovi}

Institut za biohemiju i Klinika za endokrinologiju,Medicinski fakultet, Ni{

Apoptoza je forma }elijske smrti ~iji je fiziolo{kizna~aj odràvanje homeostaze tkiva u uslovima delo-vanja razli~itih toksi~nih, inflamtornih stimulusa, kao ihemoterapeutika u terapiji kancera. S obzirom na toda apoptoti~ne }elije podleù fagocitozi unutar tkiva,razgradnja DNK na niskomolekularne fragmente imaza{titnu ulogu u spre~avanju eventualnog ulaska ve-}ih fragmenata u obli`nje }elije i poreme}aja njihovoggenoma. Do sada je skrenuta pa`nja na vi{e tipovarazli~itih endonukleaza koje bi bile zna~ajne u inter-nukleozomalnoj degradaciji genomske DNK u tokuapoptoze, premda je najve}i zna~aj pridavan Mg2+-zavisnoj alkalnoj DNazi. Drugi rezultati ukazuju nazna~aj i kisele DNaze II, koja je aktivna usled acidi-fikacije i lizozomalne aktivacije. Vi{e tipova kaspaza jeuklju~eno u apoptozu ali je nedavno pokazano dakodiraju}i deo i DNaze I i DNaze II sadrì mesto kojeje prepoznatljivo za razgradnju od strane kaspaza, {tone isklju~uje u~e{}e i drugih takozvanih »fragmenta-cionih faktora« koji bi ubrzali digestiju DNK. Azotmonoksid (NO) je vi{estruko aktivan molekul koji imadihotomnu regulatornu ulogu. Kompleksnost njegov-ih biolo{kih efekata proisti~e iz inreakcije s razli~itimbiomolekulima, kao {to su reaktivni kiseonikoviradikali, metalni joni i proteini. îni se da redoks sis-tem }elije ima odlu~uju}i zna~aj u formiranju per-oksinitrita i mogu}eg delovanja NO u pravcu multip-likacije }elije i njenog preìvljavanja. Pored ovih direk-tnih i indirektnih inreakcija, efekat NO i peroksinitritamoè biti moduliran i dozno-zavisan, a izgleda dazna~ajno uti~e i tip }elija. Visoke koncentracije sigur-no dovode do apoptoze ili nekroze. Dugotrajno izla-ganje, u toku hroni~ne inflamacije moè biti predis-poniraju}i faktor u pravcu tumorogeneze kao posle-dice o{te}enja DNK, poreme}aja programa }elijskesmrti i signalne aktivacije proliferacije. Cilj ove studijebio je pra}enje uloge enzima koji u~estvuju u raz-gradnji nukleotida u recipro~noj kontroli apoptoze i


THE INFLUENCE OF NITRIC OXIDEAND PEROXYNITRITE ON NUCLEOTIDE

METABOLISM: PRO OR ANTIAAPOPTOTICEFFECTS?

G. Koci}, T. Jevtovi}, D. Pavlovi}, R. Koci}, V. \or|evi}, G. Bjelakovi}

Institute of Biochemistry and Clinic for Endocrinology, Faculty of Medicine, Ni{

Apoptosis is a form of cell death utilized physio-logically to maintain tissue homeostasis, as well as inresponse to various toxic, inflamatory stimuli andcancer chemotherapeutic agents. Because appoptot-ic cells usually undergo phagocytosis by their neigh-bours within tissues, the cleavage of DNA to low-mol-lecular weight material may serve as a protective fun-ction limiting the probability of transfer of genes in apotentially active site from dying cells to the nuclei oftheir viable neighbors. Many different endonucleaseshave been proposed as candidates responsible forthe internucleosomal cleavage of the genomic DNAobserved during apoptosis, but it was mainly attrib-uted to Mg2+-dependent alcaline DNase I. Otherreports have also implicated the importance of acidDNase II as a result of intracellular acidification andlysosomal enzyme activation. Several caspases areincluded in apoptosis and it was recently demon-strated that coding sequence of both DNase I andDNase II contains a potential caspase-cleavage site,but the presence of other »fragmentation factors«capable of triggering DNA digestion is also observed.Nitric oxide (NO) represents a multi faceted moleculewith a dichotomous regulatory role. The complexityof its biological effects is a consequence of itsnumerous potential interactions with other mole-cules, such as reactive oxygen species, metal ionsand proteins. The redox state of the cells appears tobe a crucial parameter for the formation of peroxyni-trite and action of NO on cell multiplication and sur-vival. Beside direct or indirect interactions, the effectof NO and peroxynitrite can be also modulated in adose-dependent manner and seems to be cell-spe-cific. Higher concentration can induce apoptosis ornecrosis. But long-term exposure in certain condi-tions like chronic inflamatory states may predisposecells to tumorogenesis through DNA damage, alte-ration of programmed cell death or activation of pro-

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Lectures

proliferacije u normalnoj i regeneri{u}oj jetri pacova utoku simultanog davanja NO ili peroksinitrita. In vivoi in vitro efekat evaluiran je merenjem aktivnostiDNaze I, DNaze II, alkalne RNaze, 5'-nukleotidaze,adenozin dezaminaze i ksantin oksidaze. Dobijenirezultati ukazuju na doznu zavisnost efekata, pri ~emuje velika raznolikost rezultata dobijena u in vitro studi-jama, premda svi ispitivani enzimi imaju tendencijusni`avanja aktivnosti. Suprotno tome, in vivo rezultatipokazuju zna~ajan porast aktivnosti, dok su ve}edoze ispoljavale letalni efekat.

HORMONSKI ZAVISNI TUMORI

M. Medi}-Stojanoska

Klinika za endokrinologiju, dijabetes i bolesti metabolizma, Institut za interne bolesti,

Klini~ki centar, Novi Sad

Steroidni i polipeptidni hormoni stimuli{u rastve}eg broja razli~itih tkiva deluju}i direktno ili indirek-tno, preko faktora rasta. Ovi hormoni uti~u na eks-presiju i aktivnost nekih gena koji kontroli{u pro-gramiranu }elijsku smrt ili apoptozu. U karcinogenezi,ako su celularni mehanizmi postoje}eg autonomnogtkivnog rasta posledica uticaja hormona, nastaju hor-monski zavisni tumori. Poznato je da su hormonskizavisni tumori: karcinomi dojke, endometrijuma iprostate, kao i dobro diferentovani karcinomi {titaste`lezde (papilarni, folikularni i me{oviti, papilarno-folikularni). Estrogen i progesteron imaju ulogu u raz-voju karcinoma dojke i endometrijuma, androgeni ukarcinomu prostate, a TSH u karcinomu {titaste `le-zde. Dva su glavna, ali ne i isklju~iva, mehanizma zaobja{njenje estrogenom-indukovane neoplazme. Prviuklju~uje receptorom posredovane aktivnosti, a drugise odnosi na direktna genotoksi~na dejstva. Ulogaprogesterona u nastanku karcinoma dojke i dalje jekontroverzna, nasuprot njegovoj protektivnoj ulozi unastanku karcinoma dojke. Androgeni, sli~no estro-genima, stimuli{u razvoj karcinoma prostate. Me|u-tim, i estrogeni i androgeni mogu delovati indirektnopreko faktora rasta na autokrini ili parakrini na~in. Unastanku ovih karcinoma zna~ajnu ulogu ima i inter-akcija epitelnih struktura s okolnom stromom. Kar-cinom nastaje kao rezultat akumulacije mutacija ge-na koji reguli{u }elijsku proliferaciju. Geni zna~ajni zarazvoj karcinoma reguli{u razli~ite faze }elijskog cik-lusa, rezistenciju na apoptozu i odgovor na signalekoji diktiraju }elijsku diferencijaciju. U naslednom


liferative signaling pathway. The aim of this study wasto evaluate the possible role of nucleotide-metaboly-zing enzymes in reciprocal control of apoptosis andproliferation in normal and regenerating rat liver dur-ing simultaneous exposure to either NO or peroxyni-trite. In vivo and in vitro effect was evaluated bymeasuring the activity of DNase I, DNase II, alkalineRNase, 5-nucleotidase, adenosine deaminase andxanthine oxidase. Observed effect point out the im-portance of proposed doses in evaluating the effects,where a broad difference in enzyme activity wasobserved during in vitro experimental study, but allinvestigated enzymes tended to show significant de-crease of the activity. In vivo results, contrary sho-wed significant increase, while higher doses of pero-xynitrite were lethal.

HORMONE RESPONSIVE TUMORS

M. Medi}-Stojanoska

Clinic of Endocrinology, Diabetes and MetabolicDiseases, Institute of Internal Medicine, Clinical

Centre, Novi Sad, Yugoslavia

Steroid and polypeptide hormones stimulate thegrowth of a large variety tissues acting either directlyor indirectly, through growth factors. These hormo-nes have impact on the expression and activity ofseveral genes controlling programmed cell death orapoptosis. In carcinogenesis, if the cellular mecha-nisms underlying autonomous tissue growth arelinked to the promoting effects of hormones, the re-sult may be hormone-responsive tumors. It is knownthat breast, endometrial and prostate cancer, well-differentiated thyroid cancers (i.e. papillary, follicularand mixed papillary-follicular tumors) are hormone--responsive neoplasms. Estrogen and progesteronehave the role in development of breast and endome-trial carcinoma, androgens in prostatic cancer andTSH in thyroid cancers. There are two general butnot exclusive possible mechanisms for estrogen-in-duced neoplasia. The first one involves receptor-me-diated promoting effects and the second a direct ge-notoxic action. The role of progesterone in develop-ment of breast cancer still remains controversial,opposite to his protective role in development of en-dometrial carcinoma. Androgenes similar to estro-gens stimulate development of prostatic cancer. Butestrogens and androgens may act indirectly throughgrowth factors in autocrine or paracrine fashion. Theinteraction between surrounding extracellular matrixand epithelial structure may have important role inthe development of these tumors. Cancer resultsfrom the accumulation of mutation in genes that re-gulate cellular proliferation. Genes important for the

obliku karcinoma dojke odgovorne su mutacije natumorskim supresorskim genima BRCA1 i BRCA2.Mutacije na BRCA1 genu nalaze se i kod porodi~nogoblika karcinoma prostate. Mutacije TSH receptora,kao i mutacije na sva tri Ras gena dokazane su koddobro diferentovanog karcinoma {titaste `lezde. Ina-ktivacija tumorskog supresorskog gena p53 odgovor-na je za nastanak brojnih humanih karcinoma, pa ihormonski zavisnih. U sporadi~nom obliku karcino-ma, pored navedenih mutacija, mogu biti prisutne imnoge druge mutacije. Brojni su faktori rizika zarazvoj hormonski zavisnih tumora uklju~uju}i ìvotnudob, na~in ìvota, dijetske navike i drugi. U radu }ebiti prikazana klini~ka slika, dijagnostika i savremeniterapijski stavovi hormonski zavisnih tumora.

MOLEKULARNE I BIOHEMIJSKE KARAKTERISTIKE APOPTOZE U TOKU KARCINOGENEZE

V. V. Balti}

Institut za onkologiju, Sremska Kamenica

Apoptoza je evolutivno konzerviran i genetski od-re|en visoko regulisan, sloèn aktivni i energetski za-vistan mehanizam }elijske samodestrukcije koji sluìza odràvanje homeostaze u vi{e}elijskim organizmi-ma. Mnogobrojni fiziolo{ki i patolo{ki signali (faktorirasta, hipoksija, toplota, radijacija, hemioterapija,gubitak athezije i dr.), imaju sposobnost da pokrenuapoptoti~ki program i da moduliraju signale urazli~itim i kontinuiranim putevima. Neki signali smrtiu }eliji deluju preko mehanizama (Fos, TNFR), drugipromovi{u smrt (BAX) a tre}i blokiraju promociju}elijske smrti (BCL-2). Posle stimulacije za }elijskusmrt, signal se prenose i amplifikuje, preko proteoli-ti~kog cepanja caspaza kroz: mitohondrijalni/citohromC-put (BCL−2→Apf-1→caspaza-9→caspaza 3), ne-mitohondrijalni put preko vezivanja za TNFR (Fas,TRAIL receptori) →caspaza 8→caspaza 3 kao i prekodirektnog cepanja, i aktivacije caspaza preko citoli-ti~kih T-}elijskih produkata. Familija BCL-2 proteinaposle signala smrti u apoptoti~koj kaskadi moè dadoprinese inhibiranju apoptoze. U apoptozi proapop-toti~ki molekuli BID, BAD i BAX se modifikuju itranslociraju. Na primer, BAD molekuli reaguju nametaboli~ki stres, BID mogu da aktiviraju caspazu 8.Apoptoti~ki put biohemijski komunicira sa }elijskomproliferacijom. Onkoproteini c-myc, i adenovirus E1Aindukuju proliferaciju i apoptozu preko Rb proteina,transkripcionog korepresora p300 i dr. Tako|e, i gu-


development of cancer regulate diverse phase of thecell cycle, resistance to apoptosis and the responseto signals that direct cellular differentiation. Mutati-ons in one germ-line allele of tumor-suppressor ge-nes BRCA1 and BRCA2 are responsible for cases offamilial brest cancer. Mutations on BRCA1 gen arepresent in familial prostatic cancer also. TSH recep-tor mutations, as mutation of all of three Ras geneshave been shown in well-differentiated thyroid carci-noma. Inactivation of tumor-suppressor gen p53 isresponsible for development of many kinds of hu-man cancers and hormone-responsible tumors too.Besides these mutations, in the case of sporadic tu-mor, many others mutations may be present. Nou-merous of risk factors are responsible for develop-ment of hormone-responsible tumors including age,lifestyle, diet and others. Clinical fetures, diagnosisand current terapeutical approach of hormone-res-ponsible tumors have been shown in this lecture.

MOLECULAR AND BIOCHEMICAL CHARACTERISTICS OF APOPTOSIS

DURING CARCINOGENESIS

V. V. Balti}

Institute of Oncology, Sremska Kamenica

Apoptosis is evolutionary conserved, geneticallydefined, highly regulated, complex, active and ener-gy dependent mechanism of cell self-destruction thatserves to maintain homeostasis in multicellular orga-nisms. Numerous physiological and pathologic sig-nals (growth factors, hypoxia, heat, radiation, che-motherapy, loss of adhesion and others) are capableto trigger the program of apoptosis and to modulatethe signals in different and continuous pathways.Some cell death signals act through mechanisms(Fas, TNFR), other promote cell death (BAX), whilethere are those, such as BCL-2, that block cell deathpromotion. After stimulation, death-inducing signalsare transmitted and amplified by means of proteolyt-ic cleavage of caspase through mitochondrial/cyto-chrome c-pathway (BCL-2→Apf-1→caspase-9→cas-pase 3), nonmitochondrial pathway by binding toTNFR (Fas, TRAIL receptors) caspase 8→caspase 3,and also through direct cleavage and caspase activa-tion via cytolytic T cell products. The family of BCL-2 proteins, after death-inducing signal in apoptoticcascade can contribute to the inhibition of apoptosis.Pro-apoptotic molecules BID, BAD and BAX are mo-dified and translocated. For example, BAD moleculesrespond to metabolic stress, while BID moleculesmay activate caspase 8. Apoptotic pathway commu-nicates biochemically with cell proliferation. Onco-proteins c-myc and adenovirus E1A induce prolifera-


tion and apoptosis via Rb proteins, transcriptive core-pressor, etc. The loss of the function of p53 tumorsuppressor gene due to DNA damage by hypoxia oroncogene activation, lead to the cessation of cellcycle or to apoptosis. Mutations of protooncogeneand tumor suppressor gene are responsible for thedisruption of homeostasis between proliferation andapoptosis. Mutations of p53 gene induce the positiveregulation of apoptosis thus causing continuous andexcessive proliferation. Apoptosis may also be indu-ced by the expression of tumor suppressor gene viadifferent mechanisms of signal network. In differentphases of carcinogenesis and by means of differentmechanisms the program of apoptotic death is per-formed. Multicenter clinical trials using antisense oli-gonucleotide BCL-2 (G3139) or in the combinationwith conventional chemotherapy in case of non-Hod-king's lymphomas, or in combination with paclitaxelin case of prostatic carcinoma show that there is apossible way for the treatment of these tumors.

bitak funkcije p53 supresornog gena tumora, zbogDNK o{te}enja, hipoksijom kao i aktivacija onkoge-na, dovodi do zaustavljanja }elijskog ciklusa ili apop-toze. Mutacije protoonkogena i gena supresora tu-mora odgovorne su za naru{avanje homeostaze iz-me|u proliferacije i apoptoze. Mutacije p53 gena us-lovljavaju pozitivnu regulaciju apoptoze {to prouzro-kuje kontinuiranu prekomernu proliferaciju. Tako|e iekspresija supresornih gena tumora, uslovljava prekorazli~itih mehanizama signalnih sistema proces apop-toze. U razli~itim fazama karcinogeneze preko razli-~itih mehanizama ostvaruje se program apoptoti~kesmrti. Multicentri~na klini~ka ispitivanja sa antisensoligonukleotid BCL-2 (G3139) i u kombinaciji sa kon-vencionalnom hemioterapijom u Non-Hodgkin B-}e-lijskim limfomima ili u kombinaciji sa paclitaxelom ukarcinomu prostate, pokazuje da postoji mogu}nostza le~enje ovih tumora.

EVIDENCE® - PRVI U SVETU TEHNOLO[KISISTEM PROTEINSKOG BIOÎPA

L. M. Lorens, R. I. Mekonel, J. V. Lamont, S. P. Ficdèrald

Kompanija Randox Laboratories, Crumlin, Antrim, Severna Irska

Jedan od osnovnih problema sa sada{njim dijag-nosti~kim testovima je nemogu}nost da se izmeriopseg analita u jednom jedinom testu i nepreciznostdijagnoze, u smislu ograni~enih informacija. Da bi seprevazi{ao ovaj problem, kompanija Randox Labora-tories Ltd. prva je u{la u razvoj tehnolo{kog dijagnos-ti~kog sistema proteinskog bio~ipa. On je zasnovan nanizu testova koji }e iz jednog uzorka istovremeno mer-iti nekoliko najva`nijih biohemijskih markera. Panelitestova su na jednom mestu, tako da mere klju~nemarkere za odre|eno stanje bolesti. Trenutno su dos-tupni paneli koji uklju~uju lekove koji se zloupotreb-ljavaju, parametre za bolesti srca, hormone fertiliteta,tiroidne hormone, alergense i antibiotike, a u razvojusu i ostali paneli. Da bi omogu}io automatsko testi-ranje, Randox je nedavno prvi u svetu izbacio na trì{teanalizator bio~ipa ’ Evidence®. Ovaj sistem moè daanalizira 80 uzoraka na sat, s 20 testova u odre|enomnizu, {to daje broj od 3 600 testova na sat. Zbog togaEvidence® imunoanalizator ima najuspe{niji u~inak odsvih imunoanalizatora tog tipa u svetu. Druge pred-nosti su jo{ multianalit format koji omogu}ava istovre-meno pra}enje vi{e parametara. Odnosi izme|u poje-dina~nih markera koji se mere omogu}uju protein-skom bio~ipu da pokazuje visoku prediktivnu vrednosts obzirom na stanje bolesti. Koli~ina uzorka po testutako|e je smanjena kada se uporedi sa pojedina~nimtestovima. Na primer 10 mL uzorka na bio~ipu kojisadrì 10 testova ekvivalentno je 1 mL uzorka po testu.Ovo je posebno va`no kod uzoraka ~ija je koli~inaograni~ena, tj. vrlo mala. Dana{nji dijagnosti~ki testovizahtevaju 5’100 mL uzorka za svaki test. Tehnologijaproteinskog bio~ipa bi}e va`na osnova za budu}a di-jagnosti~ka testiranja. Ne samo da se danas koristi zauobi~ajene dijagnosti~ke testove, ve} se lako moè pri-lagoditi novim testovima po{to su identifikovani novimarkeri. U tom smislu, novi testovi se stalno dodajupostoje}im dijagnosti~kim panelima, a veliki broj novihdijagnosti~kih panela se trenutno razvija. Detaljnija ana-liza tehnologije proteinskog bio~ipa i Evidence® sledina workshop-u.

EVIDENCE® THE WORLD'S FIRST PROTEINBIOCHIP ARRAY TECHNOLOGY SYSTEM

L. M. Lawrence, R. I. McConnell, J. V. Lamont, S. P. FitzGerald

Randox Laboratories Ltd., 55 Diamond Road, Crumlin, Co Antrim BT20 4QY, Northern Ireland

One of the main problems with current diagnostictests is the inability to measure a range of analyses ina single test and the inaccuracy of diagnosis that isinherent with such limited information. To overcomethis problem Randox Laboratories Ltd. has pioneeredthe development of the world's first protein biochiparray diagnostic system. This is based on an array oftests that will measure a number of key biochemicalmarkers simultaneously from a single patient sample.Panels of tests (arrays) are compiled so that theymeasure the key markers for a particular disease state,panels that are currently available include drugs ofabuse, cardiac disease, fertility hormones, thyroid hor-mones, allergens and antibiotic drug residues with anumber of other panels in the development stege. Toenable automated testing Randox has recently laun-ched the world's first biochip array (analyzer ’ Eviden-ce®. This system has ihs capability to analyse 80 pa-tients samples per hour. With 20 tests on a particulararray this gives si throughput of 3.600 tests per hourmaking Evidence® the world’s highest throughputimunoassay analyzer. Other advantages of the systeminclude the milti-analyte format enabling the clinicianto monitor many parameters at once. Relationshipsbetween the individual markers can be measured giv-ing the potential for protein biochip arrays to have ahigh predictive value with respect to disease state. Thesample volume per test is also dramatically reducedwhen compared to carrying out individual tests. Forexample 10 mL sample on a biochip containing 10tests is the equivalent of 1 mL sample per test. This isparticularly important ih samples where the volumecan be very small. Current diagnostic tests require5’100 mL sample for each test. Protein biochip tech-nology will be an important platform for future diag-nostic testing. Not only is it currently being used for themore conventional diagnostic tests but it can easilyaccommodate new and innovative tests as new mark-ers are identified. In this respect new tests are conti-nually being added to existing diagnostic panels and anumber of new diagnostic panels are currently underdevelopment. Full details of protein biochip array tech-nology and Evidence® will be presented at this work-shop.


UC 577,1; 61 ISSN 0354-3447

Jugoslov. Med. Biohem. 21: 137, 2002 Workshop 1

Technofarm ’Randox

VISOK NIVO TEHNOLOGIJENEOPHODNOST U 21. VEKU

Dimension® RXL nova tehnologija omogu}ava jednostavan, brz i ekonomi~an

proces rada u klini~koj laboratoriji

O. Jankovi}

Interlabexim, Beograd

Aparat Dimension® RxL je jedan sistem sa mnogomogu}nosti. Zahvaljuju}i jedinstvenoj modularnojtehnologiji, multi senzor modulu (IMT Quik LYTE),heterogenom modulu za imuno analize (HM) i poseb-nom sistemu rukovanja reagensima (RMS), konfigu-racija modula na aparatu Dimension® RxL mo`e dase prilagodi potrebama svake laboratorije. Dimen-sion® RxL je vi{e nego rutinski analizator za klini~kuhemiju, jer se sa HM modulom mogu raditi visokoosetljivi testovi za TSH, FT4, sr~ani troponin I, mio-globin, HCG, PSA i pra}enje terapije odre|enih leko-va. Modul IMT Quik LYTE omogu}ava odre|ivanjeNa+, K+, Cl’ za samo 48 sekundi, a mogu}e je ugra-diti i elektrodu za TCO2. Poseban sistem za reagenseRMS omogu}ava skladi{tenje i programiranje pripre-me radnog reagensa unapred za nekoliko dana. Ka-sete sa reagensima (FlexTM) automatski se ubacuju iuklanjaju bez prekida procesa rada. Reagens u apa-ratu je stabilan do 30 dana. Stabilnost kalibracijeiznosi izme|u dva i tri meseca. U zavisnosti od iza-branih modula, mogu}e je uraditi i do 750 testova zajedan sat. Aparat je jednostavan za rukovanje, prepo~etka rada automatski proverava nivoe reagenasa iuzorka i signalizira na monitoru ako ne{to nije u redu.Sistem je bezbedan jer se kivete proizvode u samomaparatu i nakon zavr{ene analize automatski zatvara-ju. Ovim procesom za{ti}en je kadar koji radi u labo-ratoriji, a tako|e izoluje se i potencijalno opasnibiolo{ki otpad. Potro{nja vode za jedan sat rada apa-rata iznosi svega 3,5 litara. Dnevno odr`avanje apara-ta je minimalno. Sve prethodno nabrojane karak-teristike omogu}avaju jednostavan, brz, pouzdan iekonomi~an proces rada, ali ono {to je najva`nije ta~-ne i precizne rezultate.

HIGH LEVEL OF TECHNOLOGYA MUST FOR 21ST CENTURY

Dimension® RXL new technology enablessimple, quick economical and reliable

workflow in clinical laboratory

O. Jankovi}

Interlabexim, Belgrade

The instrument Dimension® RxL is one systemwith many capabilities. The unique modular technol-ogy, multi sensor module (IMT Quik LYTE), hetero-geneous immunoassay module (HM) and speciallydesigned Reagent Inventory Management System(RMS), makes Dimension® RxL easy to configure,according to your laboratory needs. Dimension® RxLis more than a routine analyzer for clinical chemistry,since HM comprises high sensitive tests for TSH,FT4, CK-MB mass, cardiac Troponin I, MYO, HCG,PSA and therapeutic drug monitoring. IMT QuikLYTEtm module ensures that NA+, K+ and Cl’ resultsare available within 48 seconds. An optional TCO2-electrode is available. System for reagents RMS mod-ule enables storage, programming of the preparationof the reagent a few days in advance. ReagentCassettes FlexTM are automatically loaded andunloaded without routine run interruption. Reagenton-board stability is 30 days. Calibration stability isbetween 2 and 3 months. The performed throughputis up to 750 tests per hour, dependant on the mod-ules chosen. The instrument is simple to handle andbefore the operation it checks sample and reagentslevels, signalizing eventual inefficiency on the moni-tor. The system is safe since the cuvettes are madeon board and are automatically sealed. This guaran-tees biohazardous waste is isolated for safe disposal,protecting laboratory staff and saving time. Waterconsumption for one hour of routine workflow is only3,5 liters. Daily maintenance of the instrument is min-imal. All the above-mentioned characteristics enablesimple, quick, reliable end economical workflow, andthe most important, the accurate and precise results.


UC 577,1; 61 ISSN 0354-3447

Jugoslov. Med. Biohem. 21: 138, 2002 Workshop 2

Interlabexim ’ Dade Behring


F65

PROMENE PRIRODNIH ANTIKOAGULANASA KOD BOLESNIKA SA AKUTNIM INFARKTOM MIOKARDA

LEÊNIH TKIVNIM PLAZMINOGEN AKTIVATOROM

S. Mandi}-Radi}, S. Obradovi}, V. Subota, R. Nikolajevi}, B. Gligi}

Institut za medicinsku biohemiju, Klinika za urgentnu internu medicinu,Vojnomedicinska akademija, Beograd

Akutni infarkt miokarda (AIM) je najteà mani-festacija ishemijske bolesti srca, a naj~e{}e je posled-ica rupture plaka i intrakoronarne tromboze. Ove pro-cese prati aktivacija koagulacionog sistema i trom-bocita. Na bazi toga je razvijena odgovaraju}a anti-tromboti~na terapija, ~iji je cilj blokada aktivnostitrombocita, zaustavljanje hiperkoagulabilnosti krvi irazgradnja nastalog tromba. Cilj ovog rada bio je dase procene promene aktivnosti prirodnih antikoagu-lanasa kod bolesnika sa AIM koji su le~eni fibrinoli-ti~kom (tkivni plazminogen aktivator, tPA) uz antikoa-gulantnu terapiju (heparin). Odre|ivani su protein C(PC) i antitrombin III (ATIII) kod 10 zdravih osoba(kontrola) i kod 30 bolesnika sa AIM, potvr|enim pokriterijumima WHO. Krv je uzimana po prijemu boles-nika, (najvi{e 12 sati od pojave bola), a zatim 90 mi-nuta, 24 i 72 sata posle terapije. Primenjeni su sta-ndardni funkcionalni testovi firme Dade Behring.Aktivacija koagulacionog sistema i trombogeneza supotvr|ene povi{enim vrednostima fibrin monomera(p< 0,001) i D-dimera (p< 0,01). Nije na|ena sta-tisti~ki zna~ajna razlika izmedju aktivnosti PC i ATIIIkontrolne grupe i po~etnih vrednosti kod bolesnika.Snaàn skok aktivnosti PC (p< 0,001) nadjen je u 90minutu po davanju fibrinolitika. Aktivnost PC posle 24sata se izrazito smanjila, tako da je bila zna~ajno niài od po~etne (p< 0,01), dok se dalje odràvala bezpromena. Vrednosti ATIII su se smanjivale tokom te-rapije, i zna~ajno se razlikuju u svim ta~kama od po-lazne vrednosti (p< 0,01). Visoke vrednosti PC posle

FSLOBODNE TEME

FREE COMMUNICATIONS

F65

CHANGES OF NATURAL ANTICOAGULANTS IN PATIENTS

WITH ACUTE MYOCARDIAL INFARCTION RECEIVING TISSUE PLASMINOGEN

ACTIVATOR

S. Mandi}-Radi}, S. Obradovi}, V. Subota, R. Nikolajevi}, B. Gligi}

Institute of Medical Biochemistry, Department of Urgent Medicine,

Military Medical Academy, Belgrade

Acute myocardial infarction (AMI) is the most se-vere manifestation of an ischaemic heart disease,and it is predominantly due to plaque rupture andintracoronary thrombosis. Those processes are fol-lowed by the activation of a coagulation system andplatelets. The blockade of platelet activation, stop-page of blood hypercoagulability and destruction ofpresent thrombus are the goals of the correspondingantithrombotic therapy. The aim of the study was toevaluate the importance of natural anticoagulantchanges in patients with AMI who were treated withboth the fibrinolytic (tissue plasminogen activator,tPA) and anticoagulant therapies (heparin). Protein C(PC) and antithrombin III (ATIII) were determined in10 healthy individuals (control) and 30 patients withAIM, confirmed by WHO criteria. Blood was takenafter patients' admission to hospital (no longer than12 hours from initial feeling the pain), and than 90minutes, 24 hours and 72 hours after the therapy.The standard functional Dade Behring assays wereused. The activation of the coagulation system andthe thrombogenesis were confirmed by the increas-ing fibrin monomer (p< 0,001) and D-dimer values(p< 0,01). No statistically significant changes in PCand ATIII activities were found between the controlgroup and the baseline values in patients. The inten-sive rise in PC activities (p< 0,001) was found in 90thminutes after receiving tPA. After 24 hours PC activi-ty was remarkably decreased, moreover, it was lowerthan the baseline values (p< 0,01). Thereafter, itsactivity remained unchanged. The values of ATIII we-


tPA poti~u od uticaja fibrinolitika. Skok je zna~ajan zainaktivaciju prokoagulanata, aktiviranih faktora V iVIII, kao i inhibiciju PAI-1, koja ozna~ava profibrinoli-ti~ko dejstvo PC. Sniènje vrednosti ATIII poti~e odnjegove potro{nje u reakciji sa heparinom. Dobijenirezultati ukazuju na zna~ajnu ulogu antikoagulantnih~inilaca tokom terapije t-PA u bolesnika sa AIM.

F66

UTICAJ ALUMINOSILIKATA KAO DODATKA HRANI NA DELOVANJE

CIJANOBAKTERIJSKIH TOKSINA

G. Grubor-Laj{i}, R. Kova~evi}, Z. Svir}ev, N. Andri}, B. Stani}, S. Zori}, B. Stan~evi}

Institut za biologiju, PMF, Novi Sad

Cijanobakterije (modrozelene alge) proizvode {i-rok spektar sekundarnih metabolita, koji mogu bitivrlo toksi~ni, kao {to su mikrocistini i nodularini. Ka-rakteristi~ni za odre|ene rodove cijanobakterija, mi-krocistini dospevaju u vodenu sredinu (»cvetanje« vo-de). Prisustvo ovih toksina u ìvotnoj sredini je takopotencijalna opasnost ne samo za akvati~ne orga-nizme, ve} za ekosistem u celini. Mikrocistini su he-patotoksini. Poznato je da inhibiraju protein fosfatazu1 (PP1) i protein fosfatazu 2A (PP2A). Brojni radoviukazuju i na ulogu ovih cikli~nih heptapeptida kaopromotora kancera. U ovom radu ispitivan je uticajprirodnih zeolita i glina (preparat ATN), koji se doda-ju hrani, na delovanje mikrocistina. Pacovi hranjenihranom sa ovim dodacima bili su izloèni delovanjumikrocistina iz prirodnog izvora (jezerska voda uvreme »cvetanja«) i ekstrakta laboratorisjke kultureMicrocystis (PCC7806). Pra}ena je aktivnost gluta-tion S-transferaze (GST) u homogenatu jetre pacova,jer je poznato da aktivno{}u ovog enzima mikrocisti-ni formiraju konjugat sa glutationom, {to vodi dopada u aktivnosti samog GST. Toksi~nost mikrocisti-na kao hepatotoksina potvr|ena je i histolo{komanalizom jetre pacova. Pokazalo se da prisustvo ATN-a u hrani za pacove kod eksperimentalne grupe, uodnosu na kontrolnu, elimini{e {tetno delovanje mi-krocistina.

re decreased during the therapy and significant diffe-rences from baseline values there existed in eachobserved period (p< 0,01). The high PC values aftertPA are due to the fibrinolytic process influence. Therise is important for both procoagulants inactivation,such as the activated factors V and VIII, and for PAI-1inhibition, which means the profibrinolytic PC action.Decreasing of ATIII values is due to their consump-tion in reaction with heparin. The obtained results in-dicate the important role of anticoagulant factors du-ring tPA therapy in AMI patients.

F66

IMPACT OF ALUMOSILICATES AS ADDITIVES UPON

CYANOBACTERIAL TOXIN EFFECTS

G. Grubor-Laj{i}, R. Kova~evi}, Z. Svir}ev, N. Andri}, B. Stani}, S. Zori}, B. Stan~evi}

Institute for Biology, Faculty of Science andMathematics, Novi Sad

Cyanobacteria (blue-green algae) produces awide spectrum of secondary heavily toxic metabolitessuch as microcystins and nodularins. The former,being a characteristic of certain cyanobacterial gene-ra, contaminate water during cyanobacterial »bloom«therefore threatening not only the aquatic fauna, butalso an ecosystem in general. Microcystins are potentand highly specific hepatotoxins whose toxicity isbased on inhibition of type-1 (PP1) and type-2A (PP2A)protein phosphatase. A great number of papers pointout the role of this cyclic heptapeptides as potent tu-mor promoter. The purpose of this study was toinvestigate the impact of natural zeolites and clay(ATN) upon microcystins. Natural microcystins (lakewater at blooming) and extract of laboratory Micro-cystis culture (PCC7806) effects were tested withrats fed with ATN enriched food. Glutathione S-trans-ferase (GST) activity in rat liver homogenate was fol-lowed to show the correspondence between our re-sults and those telling us that the activity of this enzy-me affects the microcystin-glutathione conjugationtherefore causing GST activity drop. Microcystin tox-icity was confirmed also by the hystological survey ofrat liver. ATN-enriched rat food in the experimentalgroup eliminated toxic microcystin effect when com-pared with the control.


F67

EOZINOFILIJA KOD OBOLELIH OD TRIHINELOZE

Lj. Vorgi}, Lj. Ba~vanski, M. Frenc, Z. Papi}

Klini~ko biohemijska laboratorija, Op{ta bolnica »\or|e Joanovi}«, Zrenjanin

Trihineloza (trihinoza) je bolest izazvana nema-todom Trichinella spiralis. Dijagnoza se postavlja naosnovu tipi~ne klini~ke slike, izraène eozinofilije i epi-demiolo{kih podataka. Tok bolesti prolazi kroz 3 fazei to intestinalnu, generalizovanu i rekonvalescentnu.Intestinalna faza je kratka, nastaje posle inkubacije od2’3 dana, nakon konzumiranja zaraènog mesa. Is-poljava se mu~ninom, povra}anjem, gr~evima i bo-lom u trbuhu. Drugu generalizovanu fazu, karakteri{upovi{ena temperatura, edemi lica, naro~ito perior-bitalnog dela i gornjih kapaka, bolovi u mi{i}ima iizrazita eozinofilija. U ovom radu prikazane su vred-nosti eozinofilnih granulocita i ukupnih leukocita kodpacijenata koji su imali izraène simptome generali-zovane faze, a le~eni su na infektivnom odeljenju iliambulantno, tokom epidemije trihineloze. Od 150pacijenata, 94% imalo je eozinofiliju u prvoj nedelji odpojave simptoma. U drugoj nedelji od pojave simp-toma eozinofilija je bila jo{ izraènija, i javljala se kod98% pacijenata. Prose~an apsolutni broj eozinofila uprvoj nedelji iznosio je 2 764 u mm3, a u drugoj ne-delji 4 834 u mm3. Kod 8 pacijenata broj eozinofilabio je iznad 10 000 u mm3. Relativni broj eozinofilakretao se od 10’70%. Broj {tapi}astih granulocita,bio je zna~ajno pove}an, a broj eozinofilnih granulo-cita odre|ivan je i u rekonvalescentnoj fazi. Dobijenipodaci odgovaraju onima koji se mogu na}i i u litera-turi. Leukociti su odre|ivani na broja~u krvnih eleme-nata Coulter MD II serija, a eozinofilni granulociti ana-lizom krvnog razmaza, obojenog po Pappenheim-u.

F68

PROTEKTIVNI EFEKTI METIONINA I VITAMINA B12 NA FUNKCIJU JETRE

U HOLESTAZI

T. Jevtovi}, G. Bjelakovi}, G. Koci}, G. G. Bjelakovi}, D. Sokolovi}, T. Cvetkovi}

Institut za biohemiju, Medicinski fakultet, Ni{

Ekstrahepati~na holestaza izazvana opstrukcijomù~nih kanala dovodi do ozbiljnih o{te}enja jetre. Uosnovi ovih o{te}enja su strukturne i funkcionalnepromene membrane hepatocita. Posledica toga jerazvoj niza biohemijskih i funkcionalnih poreme}aja:

F67

EOSINOPHILIA IN TRICHINELLOSIS PATIENTS

Lj. Vorgi}, Lj. Ba~vanski, M. Frenc, Z. Papi}

Laboratory of Clinical Biochemistry, General Hospital »\or|e Joanovi}«, Zrenjanin

Trichinellosis (trichinella) is a disease caused by ne-matode Trichinella spiralis. Diagnosis is made accor-ding to typical clinical picture of impressive eosinophi-lia and epidemiology datas. There are 3 phases in thecourse of disease: intestinal, generalized and conva-lescent. Intestinal phase is short, it starts after shortincubation of 2’3 days after consumption of conta-gious meat. Typical symptoms are characterised bynausea, vomit, convulsions and abdominal pain. Thesecond, generalized, phase is characterized by hightemperature, facial edema, especially periorbital partand upper eyelid, muscle pain and an impressive eo-sinophilia. In this work we showed eosinophile granu-locytes and total leukocytes in patients with empha-sized symptomes of generalized phase who were trea-ted at the Infective departement of the hospital or out-patiently, during trichinellosis epidemic. From the totalof 150 patients, 94% had eosinophilia at the first weekafter symptomes appeared. During the second weekof getting symptomes eosinophilia was even moreephasized at even 98% of patients. The average abso-lute number of eosinophile at the first week was 2 746in mm3, at the second week it was 4 834 in mm3. In 8patients the number of eosinophile was more than 10000 in mm3. Relative number of eosinophile was bet-ween 10’70%. The number of bands is significantly in-creased. Number of eosinophile granulocytes was alsodeterminated in convalescent phase too. The obtainedresults correlate with datas in literature. Leukocyteswere determinated by using the counter of blood ele-ments Coulter MD II serie, and eosinophile granulo-cytes by analyzing peripheral blood smear, coloredaccording to Pappenheim.

F68

PROTECTIVE EFFECTS OF METHIONINEAND VITAMIN B12 ON LIVER FUNCTION

IN CHOLESTASIS

T. Jevtovi}, G. Bjelakovi}, G. Koci}, G. G. Bjelakovi}, D. Sokolovi}, T. Cvetkovi}

Institute of Biochemistry, School of Medicine, Ni{, Yugoslavia

Serum proteins and uric acid measurements areuseful parameters in assessing hepatic function inliver diseases. In cholestatic liver diseases, liver tissueis hypoxic and anaerobic metabolism predominates.


hipoksija, smanjena sinteza ATP i razvoj energetskekrize u }elijama sa pojavom sekundarne hiperurike-mije kao i poreme}aj sintetske funkcije jetre i pojavadisproteinemije. Dosada{nja istraìvanja su pokazalada S-adenozil-metionin (SAM) ima zna~ajnu ulogu ureakcijama transmetilacije i transulfuracije koje suva`ne u odràvanju stabilnosti i fluiditeta membranehepatocita. S obzirom na to da vitamin B12 u~estvu-je kao koenzim u resintezi metionina iz homocisteina,cilj ovog rada bio je da se ispitaju efekti metionina ivitamina B12 na nivo serumskih proteina i mokra}nekiseline kod holestatskih pacova. U eksperimentu sukori{}eni Wister pacovi mu{kog pola. @ivotinje supodeljene u 6 grupa. I-kontrola; II-»sham« operisani;III-pacovi s podvezanim ù~nim kanalom; IV operi-sani pacovi tretirani vitaminom B12 (100 g/kg t.m. i.p.dnevno tokom 7 dana; V-operisani pacovi tretirani saobe supstance i VI-operisani pacovi tretirani metio-ninom (50 mg/kg t.m.i.p. dnevno tokom 7 dana). @i-votinje su `rtvovane dekapitacijom nakon 7 dana tre-tiranja. Serumski proteini (TP) i mokra}na kiselina(MK) bili su statisti~ki zna~ajno pove}ani kod pacovasa holestazom (71,2 ± 8,3 vs. kontrola 43,22 ± 3,07g/L; 65,92 ± 8,6 vs. kontrola 17,85 ± 8,6 mmol/L,p<0,001). Kod operisanih ìvotinja tretiranih metion-inom koncentracije ukupnih proteina i mokra}ne ki-seline bile su zna~ajno smanjene u odnosu na ne tre-tirane ìvotinje (69,74% za TP i 54,45% za MK); sa vi-taminom B12 (64,04 % TP; 53,32% MK) i kod paco-va tretiranih sa oba agensa (58,5% TP; 48,1% MK).

F69

HEPATO-PROTEKTIVNI EFEKAT METIONINAI VITAMINA B12 U EKSPERIMENTALNOJ

HOLESTAZI

D. Sokolovi}, G. Bjelakovi}, G. Koci}, T. Jevtovi}, D. Pavlovi}, B. \in|i}


S-adenozil metionin (SAM) nosilac je tiol grupe sadokazanim terapijskim efektima u holestazi i toksi~-nom o{te}enju hepatocita. Protektivni efakat SAM-aogleda se u spre~avanju o{te}enja mitohondrija, {tose postiè zahvaljuju}i smanjenju oksidativnog stresau mitohondrijama i popravljanjem ishemijom izazva-nih poreme}aja enargetskog metabolizma hepatoci-ta. Cilj rada bio je da se ispita hepato-protektivnavrednost vitamina B12 i metionina u eksperimental-nom modelu holestaze kod mi{eva. U eksperimentu

Decreased availability of oxygen is accompanied withdecreased ability to synthetise ATP and accumulationof ADP and AMP in cells. This accumulated adeninenucleotide is degraded at more rapid rate to severalpurine metabolites, including uric acid. Recent stu-dies have established the clinical efficacy of S-ade-nosil-L-methionine in the treatment of cholestasisassociated with hepatic diseases. SAM has funda-mental role in transmethylation and transsulphura-tion reactions that are necessary for the maintenanceof membrane fluidity. Since vitamin B12 is importantas coenzyme in methionine synthesis from homocis-teine, the aim of this study was to investigate theeffects of vitamin B12 and methionine on total proteinand uric acid level in sera of cholestatic rats. MaleWister rats were allocated to six groups: I-control; II-sham operated; III-rats with common bile duct li-gation; IV-cholestatic rats treated with vitamin B12 (100g/kg b.w.i.p. daily during 7 days; V-cholestatic ratstreated with both agents and VI cholestatic rats treat-ed with methionine (50 mg/kg b.w.i.p. daily during 7days). The animals were killed after seven days treat-ment. Serum protein (TP) and uric acid (UA) level weresignificantly increased in cholestatic rats (71.2 ± 8.3vs. control 43.22 ± 3.07 g/L; 65.92 ± 8.6 vs. control17.85 ± 8.6 mmol/L, p<0.001). In cholestatic animalstreated with methionine level of proteins and uric acidshowed significant depletion (69.74% for TP and54.45% for UA); with vitamin B12 (64.04 % TP; 53.32%UA) and in animals treated with both agents (58.5%TP; 48.1% UA). Since methionine and vitamin B12together have better influence on liver function incholestatic rats then agents given alone, combinationof these agents must be investigated in the future andapplied in human cholestatis therapy.

F69

PROTECTIVE-EFFECT OF METHIONINEAND VITAMIN B12 ON HEPATOCITES IN EXPERIMENTAL CHOLESTASIS

D. Sokolovi}, G. Bjelakovi}, G. Koci}, T. Jevtovi}, D. Pavlovi}, B. \in|i}

Institute of Biochemistry, Medical Faculty in Ni{, Yugoslavia

S-adenosylmethionine (SAM) is a thiol-containingcompound with known therapeutic effects oncholestasis and hepatotoxicity. SAM protects againstmitochondrial injury, which prevents mitochondrialoxidant stress and improves ischemia-induced hepat-ic energy metabolism. The aim of this study was toinvestigate the protective capacity of vitamin B12 andmethionin on hepatocites in experimental cholestasisin rats. Wistar rats were divided into six equal experi-mental groups: I’control (sham operated); II’animals


su kori{}eni mi{evi Wistar soja, podeljeni u {est eks-perimentalnih grupa: I’kontrola (la`no operisani);II’grupa tretirana metioninom ili vitaminom B12;III’mi{evi sa podvezanim d. choledocusom; IV’mi{e-vi sa holestazom dnevno tretirani vitaminom B12 (100g/kg telesne mase. i.p. tokom 7 dana); V’mi{evi saholestazom tretirani sa oba agensa; VI’mi{evi saholestazom dnevno tretirani metioninom (50 mg/kgtelesne mase i.p. tokom 7 dana). @ivotinje su `rtvo-vane nakon 7 dana od otpo~injanja tretmana. Sred-nje vrednosti ukupnog i direktnog bilirubina u seru-mu bile su zna~ajno pove}ane u grupama sa induko-vanom holestazom u odnosu na kontrolu (0,24 “0,064 vs. 0,004 “ 0,0001 mmol/L, za ukupni i 0,167“ 0,047 vs. 0,01 “ 0,002 mmol/L, za direktni;p<0,01). Nije na|ena zna~ajnija razlika u vrednosti-ma ukupnog i direktnog bilirubina kod mi{eva iz IIIgrupe u pore|enju sa IV, V i VI grupom. Jedino jedirektni bilirubin pokazao blago sniènje vrednostikod ìvotinja sa holestazom tretiranih vitaminom B12u pore|enju sa onima bez protektivnog tretmana aprisutnom holestazom (0,167 “ 0,047 vs. 0,132 “0,022 mmol/L; p<0,1). Serumski nivoi alanin amino-transferaze-ALT zna~ajno su niì u grupi bez holesta-ze tretiranoj vitaminom B12 u pore|enju sa kontrol-nom grupom (28,9 “ 3,35 vs. 36,32 “ 2,19 U/L,p<0,01). Sli~no sniènje nije na|eno u grupi bez ho-lestaze tretiranoj metioninom (32,56 “ 3,38 vs.36,32 “ 2,19 U/L). Sve grupe sa holestazom imalesu vi{e vrednosti ALT-a (III’150,8 “ 45,3; IV’72,48“ 34,65; V’84,2 “ 21,14 i VI’78,3 “ 13,2 U/L) upore|enju sa kontrolom (p<0,01). Protektivni tret-man vitaminom B12, metioninom i njihovom kombi-nacijom (IV, V i VI grupa) zna~ajno sniàva vrednostiALT-a u odnosu na ìvotinje sa holestazom bez pro-tektivnog tretmana’III grupa (p<0,01). Metionin i vi-tamin B12 mogu spre~iti o{te}enje mitohondrija he-patocita uzrokovano eksperimentalnom holestazom ibiti zna~ajno sredstvo u terapiji holestaze.

F70

UTICAJ INTRAVENSKOG DAVANJA GVO@\ANA ANTIOKSIDANTNI STATUS PLAZME

I ERITROCITA

M. Plje{a1, K. Ille1, A. Radojevi}-Savi}1, N. Dimkovi}2, T. Simi}1, @. Davi~evi}1,

J. Mimi}-Oka1

1Institut za biohemiju2Institut za bubre`ne bolesti Zvezdara,

Medicinski fakultet, Beograd

Eritropoetin i odgovaraju}a primena gvo`|a va`nisu vidovi le~enja anemije kod bolesnika s hroni~nominsuficijencijom bubrega. Me|utim, postavlja se pita-nje da li intravenska nadoknada gvo`|a vodi preza-si}enju� transferina i veoma visokim vrednostima gvo-

treated with methionin or B12; III’rats with commonbile duct ligation; IV’cholestatic rats treated with vita-min B12 (100 g/kg b.w. i.p. daily during 7 days);V’cholestatic rats treated with both agents andVI’cholestatic rats treated with methionine (50mg/kg b.w. i.p. daily during 7 days). The animals we-re killed after seven days of initial treatment. Averagelevels of total and direct bilirubin were significantlyhigher in cholestatic rats compared with control(0.24 “ 0.064 vs. 0.004 “ 0.0001 mmol/L, for totaland 0.167 “ 0.047 vs. 0.01 “ 0.002 mmol/L, fordirect; p<0.01). There was non-significant differenceof average levels of total and direct bilirubin in ratsfrom III group compared with IV, V and VI group.Only direct bilirubin showed slightly decreasing incholestatic rats treated with vitamin B12 compared tocholestatic rats without protective treatment (0.167“ 0.047 vs. 0.132 “ 0.022 mmol/L; p<0.1). Serumlevels of alanine aminotransferase-ALT showed sig-nificant decrease in non-cholestatic group treatedwith B12 compared to control (28.9 “ 3.35 vs. 36.32“ 2.19 U/L, p<0.01). Similar decreasing was not ob-served in non-cholestatic rats treated with methionin(32.56 “ 3.38 vs. 36.32 “ 2.19 U/L). All cholestaticrats had higher ALT (III’150.8 “ 45.3; IV’72.48 “34.65; V’84.2 “ 21.14 and VI’78.3 “ 13.2 U/L)compared with control (p<0.01). Protective treat-ment with B12, methionin and both agents (IV, V andVI group) significantly decrease ALT in cholestaticrats compared with rats in III group (p<0.01). Methi-onine and vitamin B12 could prevent hepatocite mito-chondrial injury induced by experimental cholestati-sis, and could be important in cholestatic therapy.

F70

EFFECT OF INTRAVENOUS IRON THERAPYON PLASMA AND RED BLOOD CELLS

ANTIOXIDANT STATUS

M. Plje{a1, K. Ille1, A. Radojevi}-Savi}1, N. Dimkovi}2, T. Simi}1, @. Davi~evi}1,

J. Mimi}-Oka1

1Institute of Biochemistry2Institute for Renal Diseases Zvezdara,

School of Medicine Belgrade, Yugoslavia

Erythropoietin and appropriate iron administra-tion are important aspects of managing the anaemiain end-stage renal disease (ESRD) patients. However,concerns have been raised about parenteral ironsupplementation leading to an »over saturation« of


`|a, {to mo`e da pogor{a ve} postoje}i oksidativnistres kod ovih pacijenata. Poznato je da intravenskaprimena gvo`|a u pojedina~nim dozama pogoršavaoksidativni stres kod pacijenata na hemodijalizi (HD).S druge strane, uticaj ponavljane intravenske prime-ne gvo`|a je manje prou~en. Zbog toga su u ovojstudiji markeri oksidativnog stresa (reaktivni karbonil-ni derivati-CRD, tiol grupe-SH) i aktivnosti antioksi-dantnih enzima superoksid dizmutaze, (SOD), gluta-tion peroksidaze, (GPX) i katalaze, (CAT) odre|ivani uplazmi i eritrocitima kod 10 pacijenata kojima je da-vano gvo`|e u dozi od 625 mg (feroglukonat, Fer-rlecit 62,5 mg) u toku 10 uzastopnih hemodijaliza.Krv je uzimana pre prve i poslednje doze gvo`|a. Re-zultati (srednja vrednost ± SD) dobijeni u plazmi suslede}i:

Markeri oksidativnog stresa

Aktivnosti antioksidantnih enzima

Sli~ni rezultati dobijeni su i u eritrocitima (rezultatinisu prikazani). Nadoknada gvo`|a popravlja hema-tolo{ke parametre kod bolesnika na hemodijalizi.Korekcija anemije nije pra}ena zna~ajnom razlikom uprocenjivanim markerima oksidativnog stresa. Na os-novu ovih podataka mo`e se zaklju~iti da terapijagvo`|em u ukupnoj dozi od 625 mg ne pogor{avaoksidativni stres kod pacijenata na HD.

F71

ODRE\IVANJE INTERAKCIJA IZME\U DOPAMINSKOG

D1 RECEPTORA I G-PROTEINA

S. \uranovi}1,2, V. [o{ki}1, J. Predi}1

1Hemijski fakultet, Univerzitet u Beogradu2Institut za bolesti djece KC Crne Gore

Dopaminski receptori pripadaju velikoj familiji re-ceptora, sa sedam transmembranskih domena, kojiinteraguju sa G proteinima (7TM receptori). Mjesta na

transferrin and excessively high iron levels, whichmay aggravate oxidative stress already present inthese patients. It is known that i.v. iron application ina single dose increases oxidative stress in haemodial-ysis (HD) patients. However, the effects of repeatedi.v. iron supplementation are less studied. Therefore,in the present study, markers of oxidative stress (car-bonyl reactive derivatives, CRD, thiol groups, SH),and antioxidant enzyme activities superoxide dismu-tase (SOD), glutathione peroxidase (GPX) and cata-lase (CAT) were determined in plasma and red bloodcells (RBC) of ten pts given a total iron dose of 625mg (ferrogluconat, Ferrlecit, 62.5 mg) during 10consecutive HD. Blood samples were taken beforethe first and the last dose of iron. The results (mean± SD) obtained in plasma were:

Markers of oxidative stress

Antioxidant enzyme activities

The similar results have also been found in RBC (datanot shown). Iron supplementation improves haema-tological parameters in HD pts. The correction ofanaemia was not followed by significant differencesin evaluated markers of oxidative stress. Based onthis data it can be concluded that iron therapy in totaldose of 625 mg does not aggravate oxidative stressin HD pts.

F71

DETERMINATION OF INTERACTIONSBETWEEN DOPAMINE

D1 RECEPTOR AND G-PROTEINS

S. \uranovi}1,2, V. [o{ki}1, J. Predi}1

1Faculty of Chemistry, University of Belgrade2The Institute for Children's Diseases in Podgorica,

KC Crna Gora, Yugoslavia

Dopamine receptors belong to the large family of se-ven-transmembrane domain G-protein-coupled re-ceptors (7TM receptors). Regions of human dopa-

pre Fe

1,05 ± 0,220,58 ± 0,21

posle Fe

1,14 ± 0,150,48 ± 0,20

CRD, mmol/g protSH, mmol/L

before Fe

1.05 ± 0.220.58 ± 0.21

after Fe

1.14 ± 0.150.48 ± 0.20

CRD, mmol/g protSH, mmol/L

pre Fe

96 ± 28190 ± 31

36 ± 14

posle Fe

91 ± 17211 ± 37

43 ± 16

SOD, U/LGPX, U/LCAT, kU/L

before Fe

96 ± 28190 ± 31

36 ± 14

after Fe

91 ± 17211 ± 37

43 ± 16

SOD, U/LGPX, U/LCAT, kU/L


dopaminskim receptorima koja u~estvuju u interakci-jama sa G proteinima nisu u potpunosti definisana.Smatra se da su poreme}aji u dopaminskim recep-torima kao i u mehanizmu prenosa signala prekoovih receptora odgovorni za nastanak odre|enihbolesti nervnog sistema. U ovoj studiji opisana jeupotreba jednostavne in vitro metode za odre|ivan-je interakcija fragmenata CPL4 hD1 receptora sarazli~itim Ga subjedinicama. U ovu svrhu su frag-menti CPL4 hD1 klonirani u pGEX-2T plazmid ieksprimirani u E. coli BL21 kao fuzioni proteini saglutation S-transferazom (MF CPL4-GST i VF CPL4-GST). Fuzioni proteini pre~i{}eni su afinitetnom hro-matografijom na Glutathion Sepharose-i CL4B. Gasubjedinice eksprimirane su u E. coli JM109 (Gas) iE. coli BL21 DE3 (Gao i Gai1), i pre~i{}ene kao »His-tag« proteini na afinitetnom matriksu Ni-NTA Sepha-rose-i. Postojanje interakcija detektovano je na 12%PAA gelu SDS-PAG elektroforezom, i kolorimetrijskiodre|ivanjem aktivnosti glutation S-transferaze. Ovakolorimetrijska metoda omogu}ava kvantifikacijuinterakcija. Utvr|eno je da je CPL4 D1 receptora~ovjeka odgovorna za interakcije ovog receptora saGas i Gao subjedinicama. Tako|e je utvr|eno da sustrukturni motivi iz MF CPL4 hD1 receptora odgo-vorni za interakciju sa Gas subjedinicom, a strukturnimotivi iz VF CPL4 hD1 receptora za interakciju saGao subjedinicom.

F72

U^ESTALOST POJAVE HIPOSURFAKTOZEKOD PRIJEVREMENO RO\ENE DJECE

P. Radovi}1, M. Batri}evi}2

1Institut za bolesti djece KC Crne Gore, Podgorica 2Privatna biohemijska laboratorija »Dr Tomanovi}«,

Podgorica

Hiposurfaktoza plu}a novoro|an~eta je nedosta-tak plu}nog surfaktanta zbog nezrelosti plu}a novo-ro|en~eta. Glavni uzrok nedostatka surfaktanta jenezrelost plu}a zbog skra}ene gestacije novoro|en-~eta. Bolest se karakteri{te tahipnejom, dispnejom icijanozom sa po~etkom u prvim satima do najkasni-je 24 sata `ivota, a zavr{ava se ili letalno ili potpunimizlje~enjem u toku nekoliko dana. Metaboli~ka aci-doza je jedna od klju~nih karika u patogenetskomlancu hiposurfaktoze plu}a kod novoro|an~eta. Sho-dno tome u pra}enju acido-baznog statusa o~ekujese pozitivan bilans H+ jona u ekstracelularnim pros-torima, smanjenje koncentracije bikarbonata u plaz-mi, smanjenje pCO2, smanjenje pH. U Institutu zadje~ije bolesti KBC Podgorica, na Odeljenju za neo-natologiju lije~eno je u periodu izme|u 1. 1. 1998. i

mine receptor of D1 subtype that are involved in Gprotein-coupling are not well known. It is consideredthat disorders in dopamine receptors as well as inmechanisms of transduction of signals through thesereceptors are responsible for developing of particulardiseases of nerve system. A simple in vitro methodhas been used for estimating the interactions bet-ween various Ga subunits and fragments of CPL4hD1 receptor. For this purpose, fragments of CPL4hD1 were cloned in pGEX-2T vector and expressed inE. coli BL21 as a fusion proteins with glutathion S-transferase (MF CPL4-GST and VF CPL4-GST). Fu-sion proteins were purified by affinity chromatogra-phy on Glutathione-Sepharose CL4B. Ga subunitswere expressed (Gas in E. coli JM109; Gao and Gai1in E. coli BL21 DE3), and purified on affinity matrixNi-NTA Sepharose. Interactions were detected on12% polyacrilamide gels by SDS PAG electrophoresisand by a colorimetric assay for glutathione S-trans-ferase. Colorimetric assay can be used for quantifica-tion of the interactions. It was shown that CPL4 ofhuman D1 receptor is involved in the coupling of thisreceptor to Gas and Gao subunits. It was also shownthat structural motives from MF CPL4 hD1 receptorare needed for interaction with Gas subunit, andstructural motives from VF CPL4 hD1 receptor areneeded for interaction with Gao subunit.

F72

INCIDENCE OF HYPOSURFACTOSA WITH PREMATURE BABIES

P. Radovi}1, M. Batri}evi}2

1The Institute for Children's Diseases in Podgorica, KC Crna Gora, Yugoslavia

2Private Biochemistry Laboratory »Dr Tomanovi}«,Podgorica, Crna Gora, Yugoslavia

Hyposurfactosa of an infant lung is caused by lackof lung surfactant due to immaturity of a newborninfant lung. The main cause of lack of surfactant isimmaturity of lungs due to shortened gestation of anewborn infant. The disease is characterised by tac-hypnoea, dyspnoea, cyanosis, that starts in the firsthours until at least 24 hours of life and finishes eitherlethally or is completely cured in the course of sever-al days. Metabolic acidosis is one of key links in path-ogenic chain of hyposurfactosa of lungs with new-born infants. Following acid-base status, positive ba-lance of H+ extra cellular areas, decrease of: concen-tration of bicarbonate in plasma, pCO2, and pH areexpected. At the Institute for Children's Diseases inPodgorica (KBC Podgorica), at the Department of


1. 01. 1999. godine 63 djece ro|enih prije vremena.Od tog vremena 54. djece (85,7%) lije~eno je od hi-posurfaktoze plu}a. Za potpunost dijagnoze kori{}enje acidobazni status, pri ~emu se iz kapilarne krvi, uzi-manjem uobi~ajenim postupkom i mjerenjem nagasnom analizatoru AVL 990 elektohemijskom meto-dom upotpunila dijagnostika datog oboljenja i potvr-di zaklju~ak koji slijedi: ispitivanja ukazuju da se inci-denca hiposurfaktoze kod prijevremeno ro|ene djecene razlikuje od literarnih podataka.

F73

UTICAJ NATRIJUMOVE SOLI MONOKETOHOLNE KISELINE

NA VREDNOSTI GLUKOZE U KRVI ZDRAVIH I DIJABETI^NIH PACOVA

S. Koji}1, M. \eri}1, M. Mikov2, A. Ra{kovi}3, K. Kuhajda4, S. Kevre{an5

1Zavod za laboratorijsku medicinu, Klini~ki centar Novi Sad,

2Zavod za farmakologiju, toksikologiju i klini~ku farmakologiju, Medicinski fakultet, Novi Sad,

3Zavod za farmakologiju, Medicinski fakultet, Podgorica4Institut za hemiju, PMF Novi Sad5Poljoprivredni fakultet, Novi Sad

Dok su prve hemijske transformacije ù~nih kiseli-na izvo|ene da bi se odredila njihova struktura, danasse posebna pa`nja poklanja izu~avanju farmakolo{kihefekata sintetisanih derivata ù~nih kiselina. Dokazanje njihov uticaj na pobolj{anje farmakokinetskih i far-makodinamskih osobina pojedinih lekova (pre svegainsulina i oralnih hipoglikemika), a sve je vi{e intere-sovanja i za njihova direktna farmakolo{ka delovanja.Ovo ispitivanje izvedeno je da bi se ustanovili direktniefekti natrijumove soli monoketoholne kiseline navrednosti glukoze u krvi zdravih i dijabeti~nih pacova.Ispitivan je uticaj jednokratne i sedmodnevne per-oralne, supkutane i intravenske primene kiseline (2mg/kg TT) na vrednosti glukoze u krvi, kod zdravih(n=50) i dijabeti~nih (n=50) pacova mu{kog pola,soja »Wistar«, hranjenih standardizovanom ishranomza laboratorijske ìvotinje. Diabetes mellitus induko-van je hemijskim putem, intraperitonealnom aplikaci-jom aloksana (100 mg/kg TT), a vrednosti glukoze ukrvi odre|ivane su u uzorku krvi iz repne vene, po-mo}u test traka, na aparatu Accutrend Glucose mini,Boehringer Mannheim. Pore|ene su vrednosti gluko-ze u krvi nakon 3 sata (akutni tretman) i 7 dana (hro-ni~ni tretman) od aplikacije ispitivane ù~ne soli, uodnosu na vrednosti pre aplikacije. Najve}i hipogli-kemijski efekat u grupi dijabeti~nih pacova, i akutnim

Neonatology, 63 premature babies were treated inthe period from Jan. 1 1998 to Jan. 1 1999. Sincethat time 54 infants suffering from hyposurfactosahave been treated (85.7%). To make diagnosis pre-cise, acid-base status was used, taking capillaryblood in usual way and measuring it on the gas ana-lyzer AVL 990 by electrochemical method confirmingthe diagnosis of hyposurfactosa of lungs. The con-clusion is: our research indicates that incidence ofhyposurfactosa with premature babies does not differfrom data found in medical literature.

F73

THE INFLUENCE OF SODIUM MONOKETOCHOLATE ON LEVEL

OF BLOOD GLUCOSE IN HEALTHY AND DIABETIC RATS

S. Koji}1, M. \eri}1, M. Mikov2, A. Ra{kovi}3, K. Kuhajda4, S. Kevre{an5

1Institute for Laboratory Medicine, Clinical Center Novi Sad

2Institute for Pharmacology, Toxicology and ClinicalPharmacology, Medical School Novi Sad

3Institute for Pharmacology, Medical School Podgorica4Institute for Chemistry, PMF Novi Sad

5Agricultural Faculty, Novi Sad

In the past, chemical transformations of bile acidswere done to determine their structure. Nowadays,particular attention is paid to pharmacological effectsof the synthetic derivatives of bile acids. It has beenproven that they improve pharmacokinetic and phar-macodynamic features of certain medications (insu-lin and anti-diabetic agents, above all) and there isincreasing interest concerning their direct pharmaco-logical effects. The aim of this investigation was todetermine the direct effects of sodium monoketo-cholate on blood glucose levels in healthy and dia-betic rats. We investigated the impact of applicationof the salt on blood glucose levels of healthy (n=50)and diabetic (n=50) male »Wistar« rats fed withstandardized laboratory animal food. We comparedthe effects of one dose and a seven day peroral, sub-cutaneous and intravenous treatment (2 mg/kg bodyweight). Diabetes mellitus was induced chemically,by intraperitoneal application of Aloxane (100 mg/kgbody weight), and blood glucose levels were deter-mined from the blood of the tail vein using the teststrips (Accutrend Glucose Mini, Boehringer Mann-heim). We compared the blood glucose levels afterthree hours (acute treatment) and seven days (chron-ic treatment) after application of the salt, with the lev-els before the treatment. The largest hypoglycemic


i hroni~nim tretmanom, ostvaren je intravenskom(’16,46% i ’18,83%), a zatim supkutanom (’11,98%i ’12,37%) i peroralnom (’5,41% i ’6,82%) prime-nom, iako ni jedno sniènje nije dostiglo statisti~kuzna~ajnost. U grupi zdravih pacova, suprotno o~eki-vanjima, ustanovljen je porast vrednosti glukoze ukrvi nakon svih na~ina aplikacije ù~ne soli, bez obzi-ra na duìnu trajanja tretmana, ali ovaj porast nije biostatisti~ki zna~ajan. Prisutan hipoglikemijski efekat ugrupi dijabeti~nih pacova, iako bez statisti~ke zna~aj-nosti, mogao bi biti od zna~aja, te rezultati zahtevajukriti~ku proveru na ve}em uzorku radi ispitivanja mo-gu}nosti {ire terapijske primene.

F74

INSULINU SLI^NI FAKTORI RASTA KOD OSOBA INFICIRANIH BAKTERIJAMA

HELICOBACTER PYLORI ILI FRANCISELLA TULARENSIS

I. Bari~evi}1, O. Nedi}1, J. A. Nikoli}1, S. Marjanovi}1, E. Ristanovi}2, B. Lako2

1Institut za primenu nuklearne energije-INEP, Zemun2Vojnomedicinska akademija,

Institut za mikrobiologiju, Beograd

Insulinu sli~ni faktori rasta I i II (IGF-I i -II), njihovivezuju}i proteini (IGFBP) i IGFBP-proteaze (BP-Pr)imaju zna~ajnu ulogu u }elijskom rastu i metaboliz-mu. Bakterijske infekcije, pored primarnog imunogodgovora, ~esto izazivaju sekundarne posledice nanivou drugih fiziolo{kih sistema u organizmu. Ciljovog rada bio je da se ispitaju promene IGF/IGFBPsistema kod osoba inficiranih bakterijama Helicoba-cter pylori ili Francisella tularensis, koje su izabranekao model sistemi. Helicobacter pylori kolonizujeepitelni sloj zida èluca i proksimalni duodenum, amoè izazvati gastritis, stvaranje èluda~nog i duode-nalnog ~ira, kao i adenokarcinom. Francisella tula-rensis je uzro~nik tularemije, u organizam prodirekroz (ne)o{te}enu koù, disajne i gastrointestinalneorgane, a naj~e{}i simptomi su lokalna infekcija i lim-foadenopatija sa groznicom. Koncentracije IGF-I iIGF-II u serumu odre|ivane su radioimunolo{kimtestovima (RIA, INEP-Zemun) uz 125I obeleène li-gande. IGFBP u serumu okarakterisani su autoradio-grafski, nakon SDS-PAGE i elektrotransfera. Refe-rentne vrednosti za IGF-I su 9’45 nmol/L, a za IGF-II50’90 nmol/L. Rezultati su pokazali da su koncen-tracije IGF-I i IGF-II u cirkulaciji uglavnom bliske don-joj granici referentnog opsega kod inficiranih pacije-nata u odnosu na kontrolnu grupu. Prose~ne vred-

effect in the group of diabetic rats was achieved byintravenous (’16.46% and ’18.83%), then subcuta-neous (’11.98% and ’12.37%) and peroral (’5.41%and ’6.82%) therapy even though none of theseeffects were statistically significant. In the group ofhealthy rats, unexpectedly, we established the incre-ase in blood glucose levels after all three kinds ofapplication of bile salt, regardless of the length of thetreatment, but this increase was not statistically sig-nificant. The hypoglycemic effect that was observedin diabetic rats, even though not statistically signifi-cant, could be important, therefore the results re-quire critical verification using a larger sample to in-vestigate a possibility of therapeutic application.

F74

INSULIN-LIKE GROWTH FACTORS IN SUBJECTSINFECTED WITH THE BACTERIA HELICOBACTER PYLORI

OR FRANCISELLA TULARENSIS

I. Bari~evi}1, O. Nedi}1, J. A. Nikoli}1, S. Marjanovi}1, E. Ristanovi}2, B. Lako2

1Institute for the Application of Nuclear Energy-INEP,Belgrade-Zemun

2Military Medical Academy, Institute of Microbiology, Belgrade

Insulin-like growth factors I and II (IGF-I and -II),their binding proteins (IGFBP) and IGFBP proteases(BP-Pr) have important roles in cell growth andmetabolism. Besides the primary immune response,bacterial infections frequently cause secondary reac-tions in other physiological systems in the organism.The main purpose of this work was to examinechanges in the IGF/IGFBP system in subject infectedwith the bacteria Helicobacter pylori or Francisellatularensis, chosen as models. Helicobacter pyloricolonizes the epithelial layer of the gastric mucosaand proximal duodenum and is a predominant causeof chronic gastritis, gastric and duodenal ulcers andgastric adenocarcinoma. Francisella tularensis is themain agent in tularemia, passing through (non)dam-aged skin, respiratory and gastrointestinal organsinto the organism, causing local infection and lym-phoadenopathy with fever. The concentrations ofIGF-I and IGF-II in serum were determined byradioimmunoassay (RIA, INEP-Zemun) with ligandslabeled with 125I. IGFBP in serum were determinedby autoradiography, after SDS-PAGE and electro-transfer. Reference values were 9’45 nmol/L for IGF-I and 50’90 nmol/L for IGF-II. The results showedthat IGF-I and IGF-II concentrations were generally inthe lower reference range in the circulation of infect-


nosti za IGF-I (’x ± SD) kod osoba inficiranih sa H.pylori i F. tularensis bile su: 10,9 ± 3,05, odnosno16,5 ± 9,25 nmol/L. Za IGF-II dobijene su vrednosti:55,0 ± 15,94, odnosno 61,5 ± 15,86 nmol/L. Auto-radiografska ispitivanja pokazala su pove}anje pris-ustva IGFBP mase 34 kDa (IGFBP-2) kod 60% ispi-tanika sa H. pylori i 30% sa F. tularensis uz sma-njeno prisustvo proteinske trake IGFBP-3 (45/40kDa) kod ve}ine inficiranih. Dobijeni rezultati ukazujuda je infekcija sa H. pylori u ve}oj meri uticala na IGF/IGFBP sistem u serumu nego infekcija sa F. tularen-sis.

F75

PROBLEM INTERFERENCIJE SALICILNEKISELINE SA NEKIM PARAMETRIMA

U SVE@EM URINU KOD ZDRAVIH OSOBA

S. \. Jovanovi}1, D. Pap2

1Institut za zdravstvenu za{titu dece i omladine, Novi Sad

Medicinski fakultet, Novi Sad2Zdravstveni centar, Sremska Mitrovica

Problem interferencije salicilne kiseline u sveèmurinu pra}en je kod 20 zdravih osoba pri jednokratnojoralnoj dozi od 0,5 g. Ispitivani su slede}i parametri:natrijum (metoda ISE), kalijum (metoda ISE), hloridi(titraciona metoda sa ìvinim nitratom), fosfati (meto-da sa amonijum-molibdatom), ukupni kalcijum (kom-pleksometrijska metoda), urea (sa ureazom po Bert-holetu) i kreatinin (Jaffeeova metoda sa alkalnim pi-kratom) kao naj~e{}e ispitivani parametri bubre`nefunkcije. Salicilati su odre|eni metodom po Trinderu.Odre|ene su koncentracije gore navedenih parame-tara u momentu peroralnog davanja salicilata i poslel, 2, 3 i 4 sata od aplikacije. Vrednosti natrijuma, hlo-rida, ukupnog kalcijuma i fosfata pokazuju skoro rav-nomeran pad u odnosu na po~etnu vrednost sve doposlednjeg (~etvrtog) sata kada dolazi do manjegporasta. Kalijum i urea rastu u prvom satu posle ~egase sniàvaju u drugom i tre}em satu i ponovo raste u~etvrtom. Kreatinin neravnomerno raste (sa sniàvan-jem u drugom satu). Promene kod natrijuma (t=3,36), hlorida (t= 2,03), ukupnog kalcijuma (t=5,12) i fosfata (t= 10,29) za t > 1,99 i p > 0,05 sta-tisti~ki su zna~ajne dok kod uree i kreatinina (t od0,57 i 0,45) nema statisti~ke zna~ajnosti. Ispitivanjain vitro direktnim delovanjem salicilne kiseline u kon-centracijama od 10, 50, 100 i 300 mmol/L nisu po-tvrdila zna~ajnije promene ispitivanih parametara.Evidentno je da kod ispitivanih elektrolita postoji uti-

ed subjects compared with those in a control groupof healthy individuals. The mean values for IGF-I (’x ±SD) in subjects infected with H. pylori and F.tularensis were 10.9 ± 3.05 and 16.5 ± 9.25 nmol/L,respectively. The values obtained for IGF-II were 55.0± 15.94 and 61.5 ± 15.86 nmol/L, respectively.Autoradiography showed increased intensity of the34 kDa IGFBP band (IGFBP-2) in 60% of the exam-ined cases of H. pylori and in 30% with F.tularensis,while the IGFBP-3 protein bands were fainter (45/40kDa) in most examined persons. The obtained resultsshowed that H. pylori infection had a greater influ-ence on the IGF/IGFBP system in serum than infec-tion with F. tularensis.

F75

THE PROBLEM OF INTERFERENCE OF SALICYLIC ACID IN DETERMINATIONOF SOME PARAMETERS IN FRESH URINE

SAMPLES IN ADULT INDIVIDUALS

S. \. Jovanovi}1, D. Pap2

1Department of Laboratory, Clinic od Pediatrics,

Faculty of Medicine, Novi Sad2Medical Center, Sremska Mitrovica, Yugoslavia

The problem of interference of salicylic acid is fol-lowed in fresh urine samples of 20 adult persons afteroral application of 0.5 g of the drug. The followingparameters were investigated: sodium and potassion(method of ISE), chloride (method of titrimetric titra-tion with Hg-nitrate), phosphate (method with am-monium-molybdate), total calcium (complexometricmethod) followed by BUN (urease by Bertholet) andcreatinine (Jaffee method with alcaline picrate) as themost frequent parameters of kidney function.Salicylates were determinated according to Trinder.The concentrations were determined in the momentof per oral application of salicylic acid and 1, 2, 3 and4 hours after. The values of sodium, chloride, totalcalcium and phosphate show nearly linear decreasecompared to the initial value up to the last (forth)hour when an increase was measured. In potassiumand similar in BUN there is an increase in the firsthour followed by the decrease in 2nd, 3rd and 4th

hour. Creatinine demonstrates irregular decrease.The changes of sodium (t= 3.36), chloride (t= 2.03),total calcium (t= 5.12) and phosphate (t= 10.29) fort > 1.99 and p > 0.05 are statistically significantwhile BUN (t= 0.57) and creatinine (t= 0.45) did notpresent significant changes. Investigations in vitro bydirect adding of salicylic acid in concentration of 10,50, 100 and 300 mmol/L did not produce significantchanges in concentrations of investigated parame-


caj salicilne kiseline i u malim dozama ovog leka teda o ovome treba da se vodi ra~una pri interpretacijirezultata. Efekat nastaje najverovatnije usled direk-tnog dejstva na proksimalne }elije tubula bubrega.

F76

NIVO UKUPNOG MAGNEZIJUMA I KALCIJUMA U SERUMU BOLESNIKA

SA DIJABETES MELITUSOM

D. Miljkovi}1, N. Kosti}2

1Dom zdravlja Varvarin2Zdravstveni centar Kru{evac

Razli~ite studije su ukazale na pove}anu u~esta-lost nedostatka magnezijuma kod bolesnika s dijabe-tes melitusom i postojanje veze izme|u hipomag-nezemije i hiperglikemije. Magnezijum ima antago-nisti~ko svojstvo u odnosu na kalcijum i mo`e seozna~iti kao fiziolo{ki antagonist kalcijuma. Cilj radaje bio da se odrede koncentracije ukupnog serum-skog magnezijuma i kalcijuma kod bolesnika s dija-betes melitusom i uporede sa nivoom ukupnog se-rumskog magnezijuma i kalcijuma kontrolne grupezdravih osoba. Ispitivanje je obuhvatilo 166 dijabe-ti~ara, oba pola, 97 (58,4%) `ena i 69 (41,6%) mu{-karaca, starosne dobi od 18 do 71 godine. Kontrolnugrupu je ~inilo 30 zdravih odraslih osoba, oba pola.Uzorci krvi uzimani su nakon dvanaesto~asovnog gla-dovanja a posle izdvajanja seruma, serum je ~uvan na’20 °C do trenutka rada. Analize su ra|ene na vi{e-kanalnom selektivnom autoanalizatoru Targa 3000,primenom gotovih komercijalnih testova. Magnezijumje odre|ivan kolorimetrijski sa ksilidil plavim, firmeDialab, sa referentnim vrednostima 0,8’1,1 mmol/L.Odre|ivanje kalcijuma zasniva se na kolorimetrijskojmetodi firme Dialab, sa referentnim vrednostima2,24’2,74 mmol/L. Prose~na vrednost ukupnog se-rumskog magnezijuma kod bolesnika sa dijabetesmelitusom iznosila je −x = 0,848 ± 0,17 mmol/L, aprose~na vrednost ukupnog serumskog kalcijuma −x= 2,61 ± 0,18 mmol/L. Prose~na vrednost ukupnogserumskog magnezijuma kod ispitanika kontrolnegrupe iznosila je −x = 0,83 ± 0,11 mmol/L,a kalciju-ma −x = 2,58 ± 0,17 mmol/L. Nije bilo statisti~ki zna-~ajne razlike u koncentracijama serumskog magnezi-juma izme|u dijabeti~ara i kontrolne grupe (t = 1,25p > 0,05) i koncentracijama serumskog kalcijumadijabeti~ara i kontrolne grupe (t = 0,86 > p 0,05).Hipomagnezemija je na|ena kod 63 (46,6%) bolesni-ka sa dijabetes melitusom, a u kontrolnoj grupi kod

ters. It could be concluded the investigated elec-trolytes are influenced by salicylic acid even in slightdoses, thus one should pay close attention wheninterpreting results. The effect is most probable theconsequence of direct influence of salilcylic acid onproximal tubular cells in kidney.

F76

THE LEVEL OF TOTAL SERUM MAGNESIUM AND CALCIUM IN THE SERUM OF PATIENTSWITH DIABETES MELLITUS

D. Miljkovi}1, N. Kosti}2

1Health Center Varvarin2Health Center Kru{evac

Various studies have shown that there is a mag-nesium deficit in patients with diabetes mellitus andthe correlation between hypomagnesia and hyperglycemia. The characteristic of magnesium is that itis physiological antagonist with calcium. The aim ofthe presented study was to determine concentrationsof the total serum magnesium and calcium in pa-tients with diabetes mellitus and to compare it withthe level of total serum magnesium and calcium inhealthy persons’ control group. The research invol-ved 166 diabetes patients of both sexes, 97 (58.4 %)of them were female and 69 (41.6 %) were male. Theage was between 18’71 years. The control groupwas consisted of 30 healthy adults of both sexes.Blood samples were taken upon the 12-hour-starva-tion and separation of serum. Serum was stored at’20 °C. Analysis were performed on multi-channel se-lective auto-analyzer Targa 3000. Ready-to-use com-mercial tests were used. The method used to deter-mine magnesium was colorimetric Xylidyl Blue, DialabCompany, with referent values 0.8’1.1 mmol/L. Cal-cium was determined on colorimetric method, DialabCompany, with referent values 2.24’2.74 mmol/L.Mean value of total serum magnesium in patientswith diabetes mellitus was −x=0.848 ± 0.17 mmol/L.Mean value of total serum calcium was −x=2.61 ±0.18 mmol/L. Mean value of total serum magnesiumin those from control group was −x=0.83 ± 0.11mmol/L, and of total serum calcium was −x=2.58 ±0.17 mmol/L. Statistically significant differences inserum magnesium concentrations between patientswith diabetes and control group (t = 1.25 p> 0.05)and serum calcium concentrations between patientswith diabetes and control group (t = 0.86 p> 0.05)were not observed. Hypomagnesia was found in 63(46.6 %) patients with diabetes mellitus, and in con-trol group in 14 (46.6 %) persons. In statistical terms


14 (46,6%) osoba {to statisti~ki ne predstavlja zna-~ajnu razliku (p > 0,05). Postoji visoka negativna ko-relacija izme|u nivoa serumskog magnezijuma i kal-cijuma kod bolesnika sa dijabetes melitusom (r =’ 0,526, p>0,01). Prose~ne vrednosti serumskogmagnezijuma i kalcijuma kod bolesnika sa dijabetesmelitusom nisu izlazile izvan okvira normalnih vred-nosti. Visoka negativna korelacija na|ena je izme|univoa serumskog magnezijuma i kalcijuma koja po-tvr|uje o~uvanost fiziolo{kog antagonizma ovih ele-menata i kod dijabeti~ara.

F77

PARAMETRI INTRAAMNIONSKE INFEKCIJE KOD TRUDNICA U TERMINU

B. Jovovi}1, B. Nati}1, N. Le~i}2, D. Jano{evi}2, J. Jovanovi}1

1Klini~ko-biohemijska laboratorija, Vojna bolnica, Ni{

2Klinika za aku{erstvo i ginekologiju, Klini~ki centar, Ni{

Amnionska te~nost ~esto se koristi za procenubiohemijskog statusa amnionske {upljine. Koncen-tracija glukoze odre|ene u amnionskoj te~nosti moèda posluì za brzu dijagnostiku intraamnionske infek-cije jer je katabolizam glukoze, aktiviran neutrofilima,primarni mehanizam ovog procesa. Neutrofili infici-rane amnionske te~nosti koriste glukozu dva do triputa vi{e nego neutrofili amnionske te~nosti neinfici-ranih porodilja. U trudno}i sa intraamnionskom in-fekcijom smanjeno je osloba|anje transferina iz spe-cifi~nih granula leukocita uz zaklju~ak da u ovom slu-~aju transferin ne ispoljava sna`nu antibakterijskuaktivnost. Cilj je bio da se proceni koncentracija tran-sferina odre|enog u amnionskoj te~nosti 26 porodi-lja. Porodilje su podeljene u dve grupe ra~unaju}ikoncentraciju glukoze od 0,077 mmol/L kao gra-ni~nu vrednost intraamnionske infekcije. Vrednostiglukoze odre|ene su na automatskom analizatoruAxon, a koncentracija transferina sa Dipro-ovim tes-tom imunoturbiodometrijskom metodom. Rezultatipokazuju da je koncentracija transferina zna~ajno ni-à (p<0,001) u ne inficiranoj u odnosu na vrednosttransferina u inficiranoj amnionskoj te~nosti. Niì nivoglukoze i pove}an nivo transferina u amnionskoj te~-nosti dokaz je prisustva infraamnionske infekcije kodporodilja.

this is not a significant difference (p> 0. 05). There isa high negative correlation between levels of serummagnesium and calcium in patients with diabetesmellitus (r = ’0.526 p>0.01). Mean values of serummagnesium and calcium in patients with diabetesmellitus were not out of normal values' ranges. Highnegative correlation between levels of serum magne-sium and calcium was found. It confirms the fact thatthe physiological antagonism of these elements ispreserved even in patients with diabetes.

F77

THE PARAMETERS OF INTRAAMNIOTICINFECTION AT DELIVERY

B. Jovovi}1, B. Nati}1, N. Le~i}2, D. Jano{evi}2, J. Jovanovi}1

1Clinical-Biochemical Laboratory, Military Hospital, Ni{, Yugoslavia

2Clinic for Obstetric and Ginecology, Clinical Center, Ni{, Yugoslavia

Amniotic fluid is very often used to assess themicrobiological state of the amniotic cavity. The glu-cose concentration of amniotic fluid has predictivevalue for the rapid diagnosis of intraamniotic infec-tion. Evidence hase been reported suggesting thatglucose catabolism by activated neutrophyls is theprimary mechanism involved in these processes.Neutrophyls obtained from infected amniotic fluidconsume glucose at a rate two or three times higherthan neutrophyls from non-infected patients. Thetransferrin is reduced from specific granular leuko-cytes in infected amniotic fluid at delivery. Then,transferin does not show strong anti-bacterium acti-vity. The main topic of this study was to evaluate theconcentration of transferin in amniotic fluid obtainedfrom 26 patients at delivery. The patients were divid-ed into two groups considering the concentracion ofglucose of 0,077 mmol/L. Glucose was estimatedroutinely on standard Axon analyser. The transferrinconcentration was determinated by immunoturbidi-metric assay Dipro diagnostic. The significantly hig-her transferrin level (p<0.001) was detected in amni-otic fluid of infected in comparison to non-infectedpatients. Lower glucose level and increase transferrinlevel is evidence of presence of intraamniotic infec-tion at delivery.


F78

ZNA^AJ DIFERENCIJALNE DIJAGNOSTIKE U ODRE\IVANJU

KONCENTRACIJE LAKTATA U LIKVORUKOD PACIJENATA S BAKTERIJSKIM

I VIRUSNIM MENINGITISOM

N. Maksi}1, S. Nikoli}2

1Institut za medicinsku biohemijii, Klini~ki centar Srbije, Beograd

2Institut za infektivne i tropske bolesti, Klini~ki centar Srbije, Beograd

Koncentracija laktata u likvoru odre|ivana je kodpacijenata sa purulentnim (15), tuberkuloznim (6) ivirusnim meningitisom (25). Kontrolnu grupu ~iniloje deset pacijenata sa meningealnim sindromom ~ijije citobiohemijski nalaz likvora bio normalan. Kodsvih pacijenata sa purulentnim meningitisom vred-nosti koncentracije laktata u likvoru bile su povi{ene(>6,2 mmol/L). Najvi{e vrednosti laktata (>15mmol/L) odre|ene su kod pacijenata sa razvojemrespiratorne insuficijencije. Pore|enjem vrednostikoncentracija laktata dobijenih kod pacijenata sapurulentnim meningitisom i ostalih grupa dobijena jestatisti~ki zna~ajna razlika (p< 0,01). Koncentracijelaktata bile su u korelaciji sa vrednostima brojaleukocita (r = 0,78, p<0,01) i stepenu zastupljenostipolimorfonukleara u likvoru (r = 0,80, p<0,01). Po-ve}ane koncentracije laktata (> 2,95 mmol/L) dobi-jene su kod svih pacijenta sa tuberkuloznim meningi-tisom, a i razlika izme|u ovih vrednosti i vrednostidobijenih kod pacijenata sa virusnim meningitisom imeningealnim sindromom bila je statisti~ki zna~ajna(p< 0,01). Koncentracija laktata u likvoru pacijenatasa virusnim meningitisom i meningealnim sindromomnije se statisti~ki zna~ajno razlikovala (p >0,05).

F79

KORELACIJA DVOVALENTNIH JONA SA KREATININOM U SERUMU

BOLESNIKA NA DIJALIZI

N. Kosti}1, B. Gagi}2, S. \or|evi}-Cvetkovi}1

1Biohemijska laboratorija, Zdravstveni centar, Kru{evac

2Apotekarska ustanova, Krusevac

S napredovanjem hroni~ne bubre`ne insuficijen-cije (HBI) nastaju ireverzibilna o{te}enja funkcional-nih jedinica nefrona i definitivni gubitak odre|enihfunkcija bubrega, na primer ekskretorne. Usled toga

F78

DIFFERENTIAL DIAGNOSTIC SIGNIFICANCE IN DETERMINATION

LIQUOR LACTATE IN PATIENTS WITH BACTERIAL AND VIRAL MENINGITIS

N. Maksi}1, S. Nikoli}2

1Institut of Medical Biochemistry, Clinical Center of Serbia, Belgrade

2Institut of infective and tropic diseases, Clinical Center of Serbia, Belgrade

Liquor lactate concentration was determined inpatients with purulent (15), tuberculous (6) and viralmeningitis (25). Ten patients with meningeal syn-drom with normal cytobiochemical liquor analysiscomprised the control group. In all patients withpurulent meningitis lactate values were increased(>6,2 mmol/L) and the highest values (>15 mmol/L)were determined in patients with concurrent develop-ment of respiratory insufficiency. Statistically ana-lysed liquor lactates values in patient with purulentmeningitis were significantly higher when comparedto other examined groups of patients (p< 0,01). Thelactate values correlated with total leukocyte count (r=0,78, p<0,01) and liquor polimorphonuclears rate(r=0,80, p<0,01). Increased lactate values (> 2,95mmol/L) were also found in all patients with tubercu-lous meningitis and the difference between these val-ues and values obtained in patients with viral menin-gitis and meningeal syndrom were statistically signif-icant (p< 0,01). There was no difference betweenliquor lactate of patients with viral meningitis and tho-se with meningeal syndrom (p >0,05).

F79

CORRELATION BETWEEN TWO-VALENCEIONS AND CREATININ IN SERUM OF HAEMODIALYZED PATIENTS

N. Kosti}1, B. Gagi}2, S. \or|evi}-Cvetkovi}1

1Biochemistry Laboratory, Health Center, Kru{evac

2Pharmaceutical Institution, Kru{evac

Kidney loses some functions: for example excre-tion, when chronic kidney insufficient (CKI) progres-sion. Disordered metabolism of two-valence ions,calcium and phosphate, in serum is consequence of


nastaje poreme}aj metabolizma dvovalentnih jona userumu, kalcijuma i fosfora. Ispitivano je 95 osobaoba pola. Osobe s normalnom funkcijom bubregapredstavljale su kontrolnu grupu (N=20), korelativnepo polu i starosti s grupom bolesnika (N=75) sa hro-ni~nom bubre`nom insuficijencijom u terminalnojfazi koji se le~e ponovljenim hemodijalizama na Inter-nom odeljenju bolnice u Kru{evcu. Ispitanici su pre-ma vrednostima kreatinina u serumu, razvrstani u trigrupe (grupa D1 kreatinin<1000 mmol/L; D2 od 1001’ 1500 mmol/L; D3>1501 mmol/L). Cilj je bio da seispita da li se sa porastom vrednosti kreatinina me-njaju koncentracije dvovalentnih jona (kalcijuma ifosfora) u serumu bolesnika na dijalizi. Odre|ivana jei aktivnost alkalne fosfataze koja zajedno sa ovim jo-nima odra`ava stanje ko{tanog metabolizma. Svi pa-rametri ispitivani su gotovim komercijalnim testovimafirmi: Bio Systems (urea kineti~ki), Elitech (kreatininipo Jaffe-u i kalcijum kolorimetrijski), Dialab (aktiv-nost alkaline fosfataze, IFCC), a fosfor vlastitim rea-gensima na biohemijskom analizatoru Targa 3000.Statisti~kom obradom uo~ava se zna~ajno vi{i t zasve ispitivane parametre na nivou p<0,01. S poras-tom kreatinina fosfor pokazuje trend porasta ali upo-re|uju}i vrednosti izme|u grupa bolesnika statisti~kazna~ajnost postoji izme|u grupa D1 i D3 (t=3,2;p<0,01), D2 i D3 (t=3,08; p<0,01). Hipokalcemijaje, u odnosu na KG, prisutna u svim grupama na ni-vou zna~ajnosti p<0,01, uravnote`ena je sa tenden-cijom pada kod bolesnika sa HBI (D1=2,07 mmol/L;D2=2,03 mmol/L, D3=1,99 mmol/L), a u negativnojkorelaciji je sa kreatininom u serumu. Alkalna fosfa-taza pokazuje statisticku zna~ajnost u odnosu na KG(t=0,01; p<0.01). Izme|u grupa zna~ajna je samokod D1 i D2 pa se zaklju~uje da ne prati porast kre-atinina. Hipokalcemija, a posebno hiperfosfatemija,znak su definitivnog iscrpljenja nefrona i smatraju sejednim od osnovnih patofiziolo{kih mehanizama.

F80

UTICAJ INTRAVENTRIKULARNO INJEKTOVANOG KOLHICINA

NA APOPTOZU ]ELIJA U HIPOKAMPALNOM GIRUSU PACOVA

B. Milojkovi}1, M. Rajkovi}1, A. Isakovi}2,T. Kravi}2, V. Bumbasirevi}1, B. \uri~i~1

1Institut za biohemiju2Institut za histologiju i embriologiju, Medicinski

fakultet, Beograd

Kolhicin je agens koji se vezuje za za tubulin iinhibira agregaciju mikrotubula. Direktna aplikacijaovog agensa u razli~ite regione mozga izaziva neuro-toksi~ne efekte. U ovoj studiji je ispitivan efekat intra-

that. Thus, we have examined 95 persons, both se-xes. Control group (N=20) was compared with grouppatients (N=75) with CKI in terminal phase who wereon therapy. There were three groups of patients com-pared by creatinin in serum (group D1<1000 mmol/L;D2 1001’1500 mmol/L, D3>1501 mmol/L). The aimof the study was to find if increase of creatinin affectsconcentration of Ca and PO4 ions in serum of pa-tients with CKI. Activity of AP was determinated,which with Ca and PO4 reflected bone metabolism.All parameters were determinated by finished factorytests: Bio Systems (urea), Elitech (creatinin, Ca),Dialab (AP). Statistical analyses show higher t for allparameters for p<0,01. Creatinine tends to increasewhen phosphate increases. There were statisticallysignificant differences between D1 and D3 group(t=3.2; p<0.01), D2 and D3 (t=3.03, P<0.01).Hipocalcemia was present in all groups in compa-rison to control group, it had tendency to decreasein patients with CKI (D1=2.07 mmol/L; D2=2.03mmol/L and D3=1.99 mmol/L) and it was in nega-tive correlation with creatinine in serum. AP show sta-tistical significance in comparison to control group(t=0.01; P<0.01). It was statistically significant cor-relation between D1 and D2 group which means thatactivity of AP did not follow increase of creatinine. Hi-pocalcemia and hiperphosphatemia were sign of fa-tigue of nephrone. They are one of the basic patophi-siological mechanism.

F80

THE EFFECT OF INTRAVENTRICULARYINJECTED COLCHICINE ON APOPTOSIS

OF CELLS IN THE HYPOCAMPAL GYRUS OF RATS

B. Milojkovi}1, M. Rajkovi}1, A. Isakovi}2,T. Kravi}2, V. Bumbasirevi}1, B. \uri~i~1

1Institute of Biochemistry2Institute for Histology and Embryology, University School of Medicine, Belgrade

Colchicine, a chemical that binds to tubulin andinhibits microtubule assembly, directly applicatedinto various brain regions has neurotoxic effect. Inthis study we have investigated the effect of intraven-


ventrikulamo injektovanog kolhicina na }elije hipo-kampalnog girusa. Wistar pacovi su anestezirani hlo-ral hidratom. Kolhicin je injektovan uz pomo} stere-taksi~kog rama u levu bo~nu komoru (2 mmol/L).Kontrolnim ìvotinjama je injektovana identi~nazapremina ve{ta~ke cerebrospinalne te~nosti. @ivoti-nje su posle 48h anestezirane hloral hidratom, per-fundovane kroz ascedentnu aortu 0.5% NaN02 azatim je tkivo fiksirano 4% paraformaldehidom ili2,5% glutaraldehidom. Mozak je brzo izdvajan iz lo-banjske duplje i suspendovan u fiksativ (paraformal-dehid ili glutaraldehid). Tkivo je zatim obra|ivano ikalupljeno u parafin (za svetlosnu mikroskopiju) iliEPON (za elektronsku mikroskopiju). Tip }elijskesmrti indukovane kolhicinom definisane detektova-njem jednolan~anih prekida DNK, kori{}enjem TU-NEL tehnike. Veliki broj }elija hipokampusa pokazaoje je TUNEL pozitivnu reakciju 48h nakon jedno-kratnog intraventrikulamog injektovanja kolhicina.Bojenjem hipokampalnih ise~aka hematoksilin-eozi-nom pokazane su morfolo{ke promene karakter-isti~ne za apoptozu. Elektronsko-mikroskopska ana-liza ultratankih ise~aka tako|e je detektovala pro-mene tipi~ne za apoptozu u }elijama hipokampusa iu }elijama ependima komora. Apoptoza nije zapa-èna u ise~cima mozga ìvotinja iz kontrolne grupe,osim u zoni oko injekcione povrede. Ova studija jepokazala da intraventrikulamo injektovan kolhicin in-dukuje apoptozu u }elijama hipokampusa.

F81

DIJAGNOSTI^KA VREDNOST SRÂNIH MARKERA U AKUTNOM

KORONARNOM SINDROMU

R. Kova~evi}1, N. Majki}-Singh2, S. Ignjatovi}2, R. Obrenovi}2, M. Golubovi}2, I. Obradovi}2,

V. \ur|evi}-Obradovi}2, M. Dajak2,G. Kartaljevi}2, B. @ugi}2

1Dedinje Institut za kardiovaskularne bolesti, Beograd2Institut za medicinsku biohemiju,

Klini~ki centar Srbije, Beograd

Biohemijski markeri o{te}enja miokarda imajuzna~ajnu ulogu u dijagnostikovanju akutnog korona-rnog sindroma, posebno kod bolesnika sa nespeci-fi~nim klini~kim i EKG nalazom. U uzorcima krvi uze-tih kod 20 bolesnika sa akutnim infarktom miokarda(AIM) i 6 bolesnika sa nestabilnom anginom pectorisna prijemu i 4, 8, 16, 24, 48 i 72 sata posle prijemau koronarnu jedinicu odre|ivani su troponin T (cTnT)na ES300 analizatoru (Boehringer Mannheim), mio-globin (Mi) na Behring Elisa-Procesoru II, ukupna CKi CKMB na Synchronu CX 5 analizatoru (Backman) imasena koncentracija CKMB (CKMBm) na IMX anal-

tricular colchicine on cells of hippocampal gyrus.Wistar rats were anaesthetised with Chloral hydrateand stereotoxically injected with colchicin into the leftlateral ventricule (2 mmol/L) 48h before sacrifice.Control animals were injected with the same volumeof ACSF. For histochemistry, rats were anaestetisedwith chloral hydrate, perfused through the ascendingaorta with 0.5% NaNO2, followed by fixation with 4%paraphormaldehyde or 2.5% glutaraldehyde. Thebrains were rapidly dissected and immersed in thesame fixative. The tissue was later processed andembedded in parafin for light microscopy and EPONfor electron microscopy. We have also characterizedthe mode of neuronal cell death, induced by col-chicine by detecting single DNA strand breaks usingthe TUNEL technique. Many hippocampal cells, 48hafter a single inraventricular injection of colchicineexhibited a positive TUNEL reaction. Hematoxillin-eosin staining of hippocampal sections reveals fea-tures fitting morphological criteria for apoptosis.Electron microscopy analysis of ultra thin sectionsalso reveals features of apoptosis in these cells. Apo-ptotic morphology was also observed in the ependy-mal cells. No apoptotic cells were observed in brainsections obtained from control rats, except in thearea around the place of injection. Our study showsthat intraventriculary injected colchicine inducedapoptosis of cells in hippocampus.

F81

DIAGNOSTIC ACCURACY OF CARDIACMARKERS IN ACUTE

CORONARY SYNDROME

R. Kova~evi}1, N. Majki}-Singh2, S. Ignjatovi}2, R. Obrenovi}2, M. Golubovi}2, I. Obradovi}2,

V. \ur|evi}-Obradovi}2, M. Dajak2,G. Kartaljevi}2, B. @ugi}2

1Dedinje Institute of Cardiovascular Diseases, Belgrade2Institute of Medical Biochemistry, Clinical Centre of Serbia, Belgrade

Biochemical markers of myocardial injury have animportant role in the diagnosis of acute coronary syn-drome particular in patients with unspecific clinicalsymptoms and ECG findings. Serum samples from20 patients with acute myocardial infarction (AIM)and 6 patients with unstable angina were obtained onadmission and 4, 8, 16, 24, 48 and 72 hours afteradmission to the coronary care unit. We determinedtroponin T (cTnT) on ES300 analyzer (BoehringerMannheim), myoglobin (My) on Behring Elisa-Pro-cesor II, total CK i CKMB on Synchronu CX 5 analy-zer (Backman) and CKMB mass (CKMBm) on IMX

izatoru (Abbott). U istraìvanje su uklju~eni samo onibolesnici koji su se javili u bolnicu 1’ 6 sati od pojavebola u grudima. Uzimani su uzorci krvi i u kontrolnojgrupi od 12 bolesnika sa ortopedskim operacijama.Za procenu dijagnosti~ke vrednosti ispitivanih para-metara primenjena je ROC analiza. Bolesnici sa AIM:Izra~unate povr{ine ispod krivih ROC AUC za vre-menski interval 4 sata posle prijema iznose: za cTnT =0,9879; CKMBm = 0,9111; CKMB = 0,9278; za Mi= 0,8139; za CK = 0,6333. Za isti vremenski inter-val, pri »cuttof« vrednostima od 0,05 ng/mL za cTnT,16,3 ng/mL za CKMBm i 16 U/L za CKMB na|ene suodli~ne dijagnosti~ke efikasnosti (0,96 za cTnT, 0,93za CKMBm i 0,89 za CKMB), osetljivosti (0,93 zacTnT, 0,93 za CKMBm i 0,93 CKMB), specifi~nosti(1,00 za cTnT, 0,92 za CKMBm i 0,83 za CKMB),pozitivne i negativne prediktivne vrednosti koje senalaze izme|u 0,87 i 1,00 pri dijagnostikovanju AIM.Bolesnici sa anginom pectoris: na prijemu su bilenormalne vrednosti za cTnT i CKMBm da bi porasleposle 16 sati od prijema. Sr~ani markeri cTnT,CKMBm i CKMB sa visokom dijagnosti~kom ta~-no{}u obezbe|uju ranu identifikaciju i potvrdu AIM.cTnT potvr|uje ili isklju~uje dijagnozu akutnog ko-ronarnog sindroma 16 sati posle prijema.

F82

REFERENTNI INTERVALI ZA JONIZOVANIMAGNEZIJUM I DRUGE SERUMSKE

KATJONE MERENI SA AVL988-4JONOANALIZATOROM

B. Radosavljevi}1, N. Majki}-Singh2

1Institut hemija u medicini, Medicinski fakultet,

Univerzitet u Beogradu, 2Institut za medicinsku biohemiju, Klini~ki Centar Srbije, Beograd

Sve ve}i klini~ki zna~aj odre|ivanja koncentracijejonizovanog magnezijuma (iMg), jedine fiziolo{kiaktivne forme, u {irokom spektru patofiziolo{kih sta-nja, nalaè da referentne vrednosti budu dobro defi-nisane. Primena komercijalnih analizatora zasnovanihna jon-selektivnim elektrodama je jedina pouzdanametoda za rutinsko klini~ko odre|ivanje iMg u krvi.Prikazano istraìvanje je realizovano sa jonoanaliza-torom AVL988-4 (AVL List GmbH) koji koristi visokopre~i{}enu jonoforu ETH 7025 selektivnu za iMg. Userumu se koncentracije iMg i jonizovanog kalcijumasmanjuju sa pove}anjem pH zbog njihove konkuren-cije sa vodoni~nim jonima za vezivna mesta na albu-minu i drugim proteinima. Ispitivanje je izvedeno sa163 zdrava dobrovoljca (113 mu{karaca, 50 èna)starosti od 18 do 77 godina. Krv je sakupljena anaer-

analyzer (Abbott). This study included only patientswho arrived in hospital within 1’6 hours after theonset of chest pain. Serum samples were obtained incontrol group of 12 patients who underwent ortho-pedic surgery. To access diagnostic accuracy ofstudied markers we used ROC analysis. Patients withAIM: calculated areas under curves ROC AUC 4hours after admission were: for cTnT=0.9879; forCKMBm=0.9111; for CKMB=0.9278; for Mi=0.8139; for CK=0.6333. For the same time intervalat cutoff values of 0.05 ng/mL for cTnT, 16.3 ng/mLfor CKMBm and 16 U/L for CKMB we found excellentdiagnostic efficacy (0,96 for cTnT, 0,93 for CKMBm,0.89 for CKMB), sensitivity (0.93 for cTnT, 0,93 forCKMBm, 0.93 for CKMB), specificity (1.00 for cTnT,0.92 for CKMBm, 0.83 for CKMB), positive predictiveand negative predictive values ranged from 0.87 and1.00 in diagnosis of AIM. Patients with unstable angi-na: on admission there were normal values also forCKMBm and cTnT but their values raised after 16hours. Cardiac markers cTnT, CKMBm and CKMBwith high diagnostic accuracy confirm early identifi-cation of AIM. cTnT confirm or exclude the diagnosisof acute coronary syndrome 16 hours after admi-ssion.

F82

REFERENCE INTERVALS FOR IONIZEDMAGNESIUM AND OTHER SERUM

CATIONS MEASURED WITH AVL988-4 IONANALYZER

B. Radosavljevi}1, N. Majki}-Singh2

1Institute of Chemistry in Medicine, School of

Medicine, University of Belgrade, Institute of Medical Biochemistry,

2Clinical Centre of Serbia, Belgrade, Yugoslavia

Growing clinical importance of determination ofconcentration of ionized magnesium (iMg), the onlyphysiologically active form, in a broad spectrum ofpathophysiological conditions, prescribes for the ref-erence values to be well defined. The application ofcommercial analyzers based on ion-selective elec-trodes is the only reliable method for routine clinicaldetermination of iMg in blood. The presented studywas realised with the ionanalyzer AVL988-4 (AVL ListGmbH) which used a highly purified ETH 7025 iono-phore that is selective for iMg. In serum iMg and ion-ized calcium concentrations decrease with increasedpH because of their competition with hydrogen ionsfor binding sites on albumin and other proteins. Theexamination was performed on 163 healthy volun-teers (113 males, 50 females) aged ranging from 18


obno u plasti~ne »Terumo« vakutejnere sa koagula-cionim aktivatorom za serum. {to pre su odre|ivanekoncentracije iMg kao i koncentracije kalcijumovih(iCa), natrijumovih (iNa) i vodoni~nih jona (pH vred-nost). Dobijene veli~ine za referentne intervale odre-|ene neparametarskom statisti~kom metodom kao iza srednje vrednosti su slede}e (izra`eno u mmol/L):iMg (0,67’0,87, 0,76), iCa (1,10’1,36, 1,25), iCa(korigovano na pH 7.4) (1.15’1.35, 1.26), iNa(136,40’ 145,28, 141,28) i pH (7,34’7,59, 7,41).Kori{}eni analizator se pokazao kao pouzdani ure|ajza pra}enje raznih fiziolo{kih stanja. Ne postoji jo{nijedna preporu~ena standardizovana procedura od-re|ivanja iMg kao ni precizno definisana povezanostkoncentracije iMg i koncentracija jona vodonika i pro-teina. Tako|e, nije dovoljno ispitano postojanje inter-ferencija. Svaki od tri danas postoje}a tipa analizato-ra kao i brojni tipovi vakutejnera daju razli~ite vred-nosti za iMg ispitivanog uzorka. Literaturni izvori oiMg iznena|uju raznoliko{}u citiranih srednjih vred-nosti, referentnih intervala i njihovih {irina. Zbog sve-ga iznesenog prikazani rezultati predstavljaju samoprvi poku{aj u ustanovljenju referentnih vrednosti na-{e populacije. Me|utim, izvesni publikovani podaci,zatim ~injenica da uo~ene niske koncentracije iMg userumima sa visokim pH vrednostima nisu matema-ti~ki korigovane i najposle neadekvatna ishrana na-{eg stanovni{tva, uzeto sve zajedno, sugeri{u u`i re-ferentni opseg i ne{to vi{e koncentracije referentnihgranica za iMg u odnosu na predstavljene vrednosti.

F83

UKUPNI ANTIOKSIDANTNI KAPACITET PRE I POSLE HEMODIJALIZE U ODNOSU NA KRVNU SLIKU

S. Pan~evska1, B. Dejanova2, P. Dejanov3, V. Maleska2

1Klini~ka biohemija2Institut za fiziologiju

3Klini~ka nefrologija, Medicinski fakultet, Skopje, Makedonija

Cilj ove studije bio je odre|ivanje ukupnog antiok-sidativnog kapaciteta (TAC) u plazmi u odnosu nahemokoncentraciju pacijenata posle hemodijalize(HD). Ispitano je 36 pacijenata podvrgnutih regu-larnoj HD. Oni nisu bili pu{a~i i niko nije primaoantioksidanse. Pacijenti su bili dijalizovani 3 putanedeljno, 4’5 ~asova. Uzimani su uzorci krvi pre i poHD. Nivo TAC-a je odre|ivan testom firme RANDOX(Krumlin, Vel. Britanija), a broj eritrocita, nivo he-moglobina i hematokrita uobi~ajenim metodama.Pre HD, nivo TAC-a je imao zna~ajno vi{u vrednost,

to 77 years old. Blood was collected anaerobically byplastic »Terumo« vacutainer blood collection tubeswith clot activator for serum. As soon as possible iMgconcentrations as well as concentrations of calcium(iCa), sodium (iNa) and hydrogen ions (pH value) we-re determined in serum. The obtained values for thereference intervals determined by nonparametric sta-tistic method and for mean values were as follows(expressed in mmol/L): iMg (0.67-0.87, 0.76), iCa(1.10 ’1.36, 1.25), iCa (corrected at pH 7.4) (1.15’1.35, 1.26), iNa (136.40 ’145.28, 141.28) and pH(7.34’7.59, 7.41). The utilised analyzer proved to bea reliably device in monitoring of various physiologi-cal states. There is still no any recommended stan-dardized procedure for iMg determination as well asaccurately defined relationship between iMg concen-tration and concentrations of hydrogen ions and pro-teins. Also, the existence of interferences is insuffi-ciently investigated. Each of three today existingtypes of analyzers, like numerous types of vacuta-iners, gives dissimilar values for iMg of the examinedsample. Finally, literature sources about iMg surprisewith diversity of cited mean values, reference intervalsand their broadnesses. These results therefore presentonly the first attempt to establish the reference valuesof our population. However, some published data,secondly, the fact that noticed low iMg concentrationsin serums of high pH values were not mathematicallycorrected and, in the end, nonadequate diet of ourinhabitants, taken together, suggest the narrower ref-erence range and some higher concentrations of re-ference limits for iMg than those presented here.

F83

TOTAL ANTIOXIDANT CAPACITY BEFORE AND AFTER HEMODIALYSIS

RELATED TO BLOOD COUNT

S. Panchevska1, B. Dejanova2, P. Dejanov3, V. Maleska2

1Clinical Biochemistry Unit, 2Institute of Physiology,

3Clinic of Nephrology, Medical Faculty, Skopje, Macedonia

The aim of the study was to determine whetherthe hemoconcentration in patients after hemodialysis(HD) session influence on plasma total antioxidativecapacity (TAC) level. A number of 36 patients under-going regular HD were examined. None of themsmoked or received any antioxidants. The patientswere dialyzed 3 times per week for 4’5 hours. Theblood samples were withdrawn before and after HD.TAC level was determined by using the combinationassay kit from RANDOX (Crumlin, Great Britain) andthe ordinary tests were performed for red blood cell



count, hemoglobin and hematocrit level. Before theHD session TAC showed significantly higher level1.49±0.1 mmol/L compared to the level after the HD,1.29±0.1 mmol/L (p<0.001). Lower red blood cellcount was found before the HD, 3.2±0.6×1012/L com-pared to the count 3.88±0.9x1012/L and so were thelevel of hemoglobine, 10.0±1.9 g/L v.s. 11.8± 27.9 g/Land hematocrit, 27.95±5.8% v.s. 33.0±8.1%. Due tohemoconcentration, expected TAC level was 1.49mmol/L (related to red blood cell count after HD);expected TAC level was 1.75 mmol/L (related to hemo-globin level after HD); expected TAC level was 1.76mmol/L (related to hematocrit level after HD). Theobtained results show that significantly decreased TAClevel after HD session was more expressed whencompared with red blood cell count, hemoglobin andhematocrit level determined after HD. These findingsmay contribute, beside TAC level, also for otherparameters correction, obtained after HD session,due to existing hemoconcentration.

F84

DETERMINATION OF URINARYMETANEPHRIN, VANILLYMANDELIC ACID AND 5-HIAA IN EXCLUDING

PHEOCHROMOCYTOMA

S. Panchevska, S. Subeska-Stratrova, V. Arsova, T. Gruev

Institute of Biochemistry, Clinic of Endocrinology and Metabolic Disorders Medical Faculty,

Skopje, Macedonia

Confirming or excluding pheochromocytoma insuspected hypertensive patients may be difficult. Thepurpose of this stady was to identify a test, for betterexcluding of pheochromocytoma. The 24-hour uri-nary metanephrine and vanillymandelic acid valueswere determined in a groupe of 68 women and 28men with clinically excluded pheochromocytomaand normal urinary catecholamine values. The speci-ficity of both tests was determined. The specificity ofthe urinary metanephrine was 97 % with women and90 % with men that means 3 % false positive resultswith women and 10 % in men. The specificity of vanil-lymandelic acid was 91 % with women and 85 % withmen that means 9 % false positive test results withwomen and 15 % with men. Higher percentage offalse positive results in men was discovered probablyas a result of a higher exposition to stressful events.These results suggest that urinary metanephrine isan accurate test of the peripheral sympathetic activi-ty and could be a useful tool in excluding pheochro-mocytoma better than vanillymandelic acid. Deter-

1,49±0.1 mmol/L u pore|enju sa vrednostima posleHD, 1,29±0,1 mmol/L (p< 0,001). Pre HD dobijen jeniì broj eritrocita, 3,2± 0,6x1012/L u odnosu na nji-hov broj po HD, 3,88± 0.9x1012/L kao i nivo hemo-globina, 100±19 g/L v.s. 118±27 g/L i hematokrita,27,95±5,8 % v.s. 33,0±8,1 %. Zbog hemokoncen-tracije, o~ekivani nivo TAC-a bio je 1,49 mmol/L (uodnosu broja eritrocita po HD); o~ekivani nivo TAC-abio je 1,75 mmol/l (u odnosu nivoa hemoglobina poHD); o~ekivani nivo TAC-a bio je 1,76 mmol/l (uodnosu hematokritske vrednosti po HD). Dobijenirezultati pokazuju da postoji zna~ajno nià TAS vred-nost po HD koja je izraènija u pore|enju sa brojemeritrocita, nivoom hemoglobina i hematokrita odre|i-vanih po HD. Ova ispitivanja mogu doprineti, osim zanivo TAC-a, i za korekciju drugih parametara, dobi-venih posle HD, zbog postoje}e hemokoncentracije.

F84

ODRE\IVANJE URINARNOG METANEFRINA, VANILMANDELI^NE

KISELINE I 5-HIDROKSI-INDOL SIR]ETNEKISELINE (5-HIAA) U ISKLJUÎVANU

FEOHROMACITOMA

S. Pan~evska, S. [ubeska-Stratova, V. Arsova, T. Gruev

Institut za klini~ku biohemiju, Kliniku za endokrinologiju i bolesti metabolizma,

Medicinski fakultet, Skopje, Makedonija

Potvr|ivanje ili isklju~ivanje feohromacitoma kodsuspektnih hipertenzivnih pacijenata moè biti te{ko.Cilj ove studije bio je da se utvrdi koji je test bolji zaisklju~ivanje feohromacitoma. Vrednosti 24-~asovnogurinarnog metanefrina i vanilmandeli~ne kiseline bilisu odre|eni pri ispitivanju grupa od 68 èna i 28mu{karaca sa klini~ki isklju~enim feohromacitom inormalnim vrednostima urinarnih kateholamina.Specifi~nost oba testa bila je odre|ena. Specifi~nosturinarnog metanefrina iznosila je 97% kod èna i 90%kod mu{karca, {to predstavlja 3% la`no pozitivnihrezultata kod ène i 10% kod mu{karaca. Specifi~-nost vanilmandeli~ne kiseline bio je 91% kod èna i85% kod mu{karaca, {to ~ini 9% la`no pozitivnih re-zultata kod ène i 15% kod mu{karaca. Ve}i procenatla`no pozitivnih rezultata bio je utvr|en kod mu{kara-ca, verovatno kao rezultat vi{e izloènosti stresnimsituacijama. Ovi rezultati potvr|uju da je urinarni me-tanefrin sigurniji test za aktivnost simpatikusa i moèda bude korisnije sredstvo u isklju~ivanju feohroma-citoma od vanilmandeli~ne kiseline. Pri odre|ivanju

mination of vanillymandelic acid is not necessarywhen performing urinary metanephrine becouse ofits lower specificity in exclusion of pheochromocy-toma. 5-hydroxyindolacetic acid (5-HIAA) levels weredetermined whit 68 women and 28 men with clinical-ly excluded pheochromocytoma. The levels of exa-mined parameter 5-hydroxyindolacetic acid (5-HIAA)didn't show signaficant correlation.


metanefrina nije potrebno odre|ivati vanilmande-li~nu kiselinu, zbog njegove ni`e specifi~nosti u isklju-~ivanju feohromacitoma. Vrednosti 5-hidroksi-indolsir}etne kiseline, bili su odre|eni kod 68 `ena i 28mu{karaca sa klini~ki isklju~enim feohromacito-mom. Vrednosti ispitanog parametra, 5-HIAA, nije po-kazao zna~ajnu korelaciju i povezanost.

F85

TISSUE TYPE PLASMINOGEN ACTIVATOR AND PLASMINOGEN ACTIVATOR

INHIBITOR TYPE1 IN WOMEN DURINGMENOPAUSE

P. Kandikjan, S. Petrovska, B. Dejanova, D. Kocmanovski

Institute of Physiology, Medical Faculty, Skopje, R. Macedonia

Cardiovascular diseases, especially coronary heartdisease and cerebrovascular disease, are the leadingcauses of death, as well as morbidity and disability inmiddle-aged and older women. Hemostatic factors,such as high levels of plasma fibrinogen, plasminogenactivator inhibitor type 1 (PAI-1) and tissue type plas-minogen activator (t-PA) are associated with throm-boembolic complications in menopausal women. Theaim of this study was to determine the plasma levelsof t-PA and PAI-1,and the concentration of follicle-stimulating hormone (FSH) and estradiol (E2) in thedifferent phases of the reproductive life of women.We examined 58 women divided in 3 groups accord-ing some criteria (regularity of menstrual cycle andFSH serum concentration): control group (n=22) ofwomen with regular menstrual cycle; premenopausalwomen (n=15) with iregular menstrual cycle and se-rum concentration of FSH< 25 mIU/mL; menopau-sal women (n=21) at least 12 months after amenor-rhoea, with serum concentration of FSH>25 mIU/mL. It was determined the FSH and E2 serum con-centration as well as the t-PA and PAI-1 in 3 groupsof women. Our results show statistical significantdifference (p< 0.01) in t-PA and PAI-1 plasma level(p<0.001), between control group and the group ofpremenopausal and menopausal women. There isno statistical significance in the t-PA and PAI-1 plas-ma level between premenopausal and menopausalwomen. These data suggest the significant increaseof fibrinolytic inhibitors (PAI-1) in premenopausal andmenopausal women, that could be an important riskfactor of thromboembolic complications (deep ve-nous thrombosis, cerebrovascular ubsults, etc.) inthis most vulnerable period of women's life.

E57

UOÂVANJE GENOMSKE NESTABILNOSTIU ORALNIM LEUKOPLAKIJAMA AP-PCR-om

N. Dedovi}1, D. Trifunovi}1, N. Tani}2, T. Drami}anin1, B. Dimitrijevi}1

1Institut za nuklearne nauke »Vin~a«, Beograd2Institut za biolo{ka istraìvanja »Sini{a Stankovi}«,

Beograd

Po definiciji Svetske zdravstvene organizacije(SZO), leukoplakija (LP) je beli~asta lezija na sluzokoìusne duplje koja se po klini~kim i histopatolo{kim kri-terijumima ne moè svrstati ni u jednu drugu bolest.LP se dele u tri kategorije koje se razlikuju po stepenumaligne alteracije. Stepen njihove maligne alteracijeje relativno nizak i kre}e se od 0,2’17%, pri ~emunaj~e{}e transformi{u u planocelularni karcinom koji~ini 90% ukupnog broja oralnih karcinoma. înjenicada LP mogu maligno da alteriraju navela je mnogeistraìva~e da ih svrstaju u prekancerozne lezije. Zbogtoga je analizirana genomska nestabilnost DNK izolo-vane iz LP kao i prisustvo onkogenih mutacija u dvanaj~e{}e izmenjena gena u humanim tumorima ’p53 antionkogena i H-ras onkogena. Genomska ne-stabilnost analizirana je DNK profilisanjem (DNA fin-gerprint) normalnog i promenjenog tkiva metodomAP-PCR. DNK fingerprint je sofisticirana metoda kojaomogu}ava otkrivanje kvantitativnih (amplifikacija ilidelecija DNK sekvenci) i kvalitativnih (mutacije) razli-ka izme|u vrlo srodnih genoma (normalno ’ izmen-jeno tkivo). Pore|enjem DNK profila analiziranih uzo-raka, genomska nestabilnost otkrivena je kod 8 od 9ispitanika (89%). S obzirom da vi{e od 50% oralnihkarcinoma ima razvijene lokalne ili udaljene metas-taze u trenutku dijagnostikovanja, uo~avanje prome-na na molekularnom nivou (utvr|ivanje stepenagenomske nestabilnosti), posebno u prekanceroza-ma, bitan je faktor u prognozi ovih glaveno-vratnihtumora.

E57

DETECTION OF GENOMIC INSTABILITY IN ORAL LEUKOPLAKIA BY AP-PCR

N. Dedovi}1, D. Trifunovi}1, N. Tani}2, T. Drami}anin1, B. Dimitrijevi}1

1Institute of Nuclear Sciences »Vin~a«, Belgrade2Institute for Biological Research »Sini{a Stankovi}«,

Belgrade

Leukoplakia (LP), as defined by the WHO, is whitelesion of oral mucosa which can not be clinically andpathologically classified as any other disease. LPcould be divided into three categories that differ bypropensity for malignant alteration. Frequency ofmalignant alteration is relatively low in the range of0.2’17%. The most frequent being squamous cellcarcinoma comprising 90% of oral malignancies. Thefact LP have the propensity for malignant alterationinspired research to classify them as premalignantlesion. Along this line, we initiated genomic instabili-ty in DNA from LP and two most frequently mutatedcancer genes ’ p53 and RAS. Genomic instability (GI)was determined by DNA profiling (fingerprinting) ofnormal and paired tumor tissue by the AP-PCRapproach. This method is known to be superior toconventional microsatellite mapping since it providesscanning of anonymous regions of the genome pro-viding both qualitative and semi-quantitative dataabout chromosomal gains, losses and point muta-tions. Such DNA profiling revealed GI in 89% of thecases. Having in mind that more than 50% of oralcarcinomas display metastatic disease upon identify-ing, detection of changes on molecular level (dete-ction of genome instability level) especially in prema-lignant period, is important issue when prognosingoral carcinomas.


EDIJAGNOSTIKOVANJE MALIGNIH STANJA

DIAGNOSTICS OF MALIGNANT DISORDERS

E58

INAKTIVACIJA P16 I P53 ANTIONKOGENA U PLANOCELULARNIM

KARCINOMIMA USNE

T. Drami}anin1, N. Dedovi}1, M. Dragi}1, D. Trifunovi}1, N. Tani}2, B. Dimitrijevi}1,

K. Krtolica1


Beograd

Osnovna epigenetska modifikacija kod sisara jestemetilacija citozina u promotorskom regionu koja voditranskripcionoj inaktivaciji datog gena. Inaktivacijaodre|enih gena hipermetilacijom, smatra se jednim odmehanizama vezanih za nastanak kancera. Hiperme-tilacija promotora na p16 genu dovodi do promena u}elijskom ciklusu i do nekontrolisanog }elijskog rasta.Podaci koji se mogu na}i u literaturi ukazuju da velikibroj (oko 30%) tumora glaveno-vratne regije imametilovana CpG ostrva u promotoru p16 antionkoge-na. Metilacioni status ovog gena kod planocelularnihtumora usne duplje utvr|en je metodom metilaciono-specifi~nog PCR-a (MSP). MSP se zasniva na ampli-fikaciji natrijum-bisulfitom modifikovane DNK specijal-no kreiranim parovima prajmera. Jedan par prajmerakomplementaran je targetu na kojem su nemetilovanicitozini modifikovani u uracil, dok je drugi par speci-fi~an za isti segment DNK sa citozinima koji ukoliko sumetilovani ne podle`u hemijskoj modifikaciji. MSPanalizom modifikovane DNK, koja je izolovana iz 12tumorskih uzoraka, u tri slu~aja (25%) na|ena je meti-lacija promotorskog regiona p16 gena. Istovremeno,za iste uzorke ura|ena je i PCR-SSCP analiza »vru}ih«egzona (E5, E7, E8) p53 tumor supresor gena. Ta~ka-ste mutacije detektovane su kod 3 analizirana uzorka(25%). U radu }e biti diskutovana uloga ovih gena(p16, p53) u regulaciji }elijskog ciklusa.

E59

PCR-SSCP U OTKRIVANJU NASLEDNIHOBLIKA KANCERA: LI-FRAUMENI SINDROM

PRIKAZ SLU^AJA

D. Trifunovi}1, N. Tani}2, N. Dedovi}1, T. Drami}anin1, B. Dimitrijevi}1


Beograd

Kancer je ste~eno geneti~ko oboljenje somatske}elije. Mutacije u germinalnim }elijama javljaju se uretkim slu~ajevima naslednih sindroma kao {to su:


E59

INACTIVATION OF p16 AND p53 TUMORSUPPRESSOR GENES IN ORAL

SQUAMOUSE CELL CARCINOMA

T. Drami}anin1, N. Dedovi}1, M. Dragi}1, D. Trifunovi}1, N. Tani}2, B. Dimitrijevi}1,

K. Krtolica1


Belgrade

The main epigenetic modification in mammals isthe methylation of cytosine nucleotide residue in pro-moter region which causes gene inactivation. Geneinactivation by hypermethylation is considered to beone of the mechanisms in cancerogenesis. Hyper-methylation of p16 promoter causes abnormal cellcycling and uncontrolled cell growth. According tothe literature, about 30% of head and neck cancershave methylated CpG islands in promoter region ofp16 tumor suppressor gene. We determined methy-lation status of p16 in oral squamous cell carcinomaby methylation specific PCR (MSP). MSP is based onamplification of bisulfite modified DNA by speciallycreated pairs of primers. One pair of primers is spe-cific for unmethylated DNA where cytosines are con-verted to uracil, while the other pair of primers iscomplementary to the same target DNA with methy-lated cytosines which do not undergo chemical mod-ification. We analyzed modified DNA of 12 samplesby MSP. p16 promoter was methylated in three cases(25%). At the same time, PCR-SSCP of »hot exons«(E5, E7, E8) for p53 tumor suppressor gene wereanalyzed. Four, out of 12 analyzed samples (30%)had point mutations. We will discuss the role of p16and p53 in regulation of cell cycle.

E59

PCR-SSCP IN DETECTION OF INHERITEDFORMS OF CANCER: LI-FRAUMENI

SYNDROME CASE REPORT

D. Trifunovi}1, N. Tani}2, N. Dedovi}1, T. Drami}anin1, B. Dimitrijevi}1


Belgrade

Cancer is acquired genetic disorder of somaticcell. Mutations in germ cell lines occurs only in rarecases of inherited syndromes such as: retinoblas-


retinoblastom, medularni tiroidni karcinom, Li-Frau-meni sindrom, itd. PCR-SSCP (polimorfizam konfor-macije jednolan~anih fragmenata) tehnikom analizi-ran je p53 tumorski supresor gen u tkivu pacijenta saprethodno dijagnostikovanim primarnim tumorombutne kosti i sekundarnim tumorom na plu}ima. Uoba slu~aja histopatolo{ka analiza je ukazala na sar-kom (haemangiopericytoma malignum). Kao nega-tivnu kontrolu u PCR-SSCP analizi kori{}ena je DNKizolovana iz krvi pacijenta i DNK izolovana iz krvizdrave osobe. Testom je dokazano prisustvo mutaci-je u 7. egzonu p53 gena u DNK izolovanoj iz ise~kaprimarnog tumora. Mutaciju u istom egzonu p53 ge-na detektovali smo i u DNK iz krvi pacijenta, {to do-kazuje da je re~ o naslednom sindromu. U analizu jenaknadno uvr{tena i DNK iz krvi oba roditelja. Rezul-tati su pokazali mutaciju u 7. egzonu i kod majke pa-cijenta. Iako vrlo jednostavna, PCR-SSCP metodaomogu}uje otkrivanje retkih naslednih oblika kance-ra, kao {to je u ovom slu~aju Li-Fraumeni sindrom.Prednost ove metode ogleda se i u otkrivanju ta~-kastih mutacija koje se ~esto javljaju kod naslednihsindroma, a koje se ne mogu otkriti klasi~nim histo-patolo{kim niti citogeneti~kim analizama.

E60

ULOGA g-GLUTAMIL TRANSPEPTIDAZE I ALKALNE FOSFATAZE

U PREOPERATIVNOJ PROCENI POSTOJANJA METASTAZA

KARCINOMA BUBRE@NOG PARENHIMA

S. Cimbaljevi}1, D. Dragi~evi}2, T. Simi}1, J. Mimi}-Oka1

1Institut za biohemiju, Medicinski fakultet, Beograd2Institut za urologiju i nefrologiju,

Klni~ki centar Srbije, Beograd

U ovom radu je ispitivan zna~aj odre|ivanja aktiv-nosti g-glutamil transpeptidaze (GGT) i alkalne fosfa-taze (AF) u preoperativnoj proceni postojanja metas-taza kod bolesnika sa karcinomom bubre`nog paren-hima. Aktivnost GGT i AF je odre|ivana u serumu 22bolesnika sa klini~ki dijagnostikovanim metastazamakarcinoma bubre`nog parenhima (grupa I). Rezultatikoji su dobijeni u ovoj grupi analizirani su prema me-stu postojanja metastaza. Rezultati odre|ivanja aktiv-nost GGT i AF kod bolesnika sa metastazama upore-|eni su sa enzimskim aktivnostima u grupi 23 boles-nika sa klini~ki lokalizovanim karcinomom bubre`nogparenhima (grupa II). U grupi bolesnika sa metas-tazama (grupa I), pove}anje aktivnosti oba enzima za-pa`eno je kod 7 bolesnika. Sedam pacijenata sa me-tastazama imalo je samo pove}anu aktivnost AF.Izolovano pove}anje aktivnosti GGT zabele`eno jekod 2 bolesnika sa metastazama. Dva bolesnika sa

toma, medullary thyroid cancer, Li-Fraumeni syn-drome, etc. We used PCR-SSCP to analyze p53 tu-mor suppressor gene in tissue of a patient previouslydiagnosed with primary sarcoma and secondary lungcarcinoma. Negative control was blood of the patientand of healthy person. Our analysis showed mutationin exon 7 of p53 gene in primary tumor and theblood of the patient. The same mutation was find inthe blood of a parent (mother). This results suggest-ed that this was the case of inherited syndrome (Li-Fraumeni syndrome). PCR-SSCP, although very sim-ple method, gives us the possibility to detect evenrare cases of inherited cancers such as Li-Fraumeni.The main advantage of this method is detection ofpoint mutations which are common for inherited syn-dromes and could not be detected by histopatho-logic or cytogenetic analyses.

E60

THE ROLE OF γ-GLUTAMYL TRANSPEPTIDASE AND ALKALINE

PHOSPHATASE IN THE PREOPERATIVEMETASTATIC EVALUATION

OF RENAL CELL CARCINOMA

S. Cimbaljevi}1, D. Dragi~evi}2, T. Simi}1, J. Mimi}-Oka1

1Institute of Biochemistry, School of Medicine2Institute of Urology, Clinical Centre of Serbia,

Belgrade, Yugoslavia

We evaluated g-glutamyl transpeptidase (GGT)and alkaline phosphatase (AP) activities as indicatorsof metastatic disease in patients with renal cell carci-noma. The activities of GGT and AP in sera of 22patients with clinically diagnosed metastatic renal cellcarcinoma (Group I) were determined and analysedwith respect to the site of metastases. These resultswere then compared to GGT and AP activities in 23patients with clinically localized renal cell carcinoma(Group II). Among patients with metastases (GroupI), an increase in both GGT and AP activities wasobserved in 7 patients. Seven patients with metas-tases had increased AP activity, only. Increased GGTactivity only, was found in 2 patients. Two of the pa-tients with clinically suspected metastases in lympha-tic nodes and 4 patients with clinically proven metas-tases did not exhibit an increase in neither GGT nor


klini~ki suspektnim metastazama u limfnim `lezdamai 4 bolesnika sa klini~ki dokazanim metastazama nisuimali pove}anu aktivnost ni GGT niti AF. Kod boles-nika sa klini~ki lokalizovanim karcinomom parenhi-ma bubrega (grupa II) istovremeno pove}anje aktiv-nosti GGT i AF uo~eno je kod 4 bolesnika, dok jejedan pacijent imao samo pove}anu aktivnost AF. Zadva od ovih bolesnika kasnije je potvr|eno da imajumetastaze karcinoma bubre`nog parenhima, a pre-ostala tri su imala tumore izuzetno velike mase.Aktivnost GGT pove}ana je u visokom procentu bo-lesnika sa karcinomom bubre`nog parenhima sa me-tastazama. Aktivnost GGT i AP je u opsegu normal-nih vrednosti kod ve}ine bolesnika sa lokalizovanimkarcinomom bubrega. Odre|ivanje aktivnosti GGTtreba, zajedno sa odre|ivanjem aktivnosti AF, uklju-~iti u protokol za preoperativnu procenu postojanjametastaza kod bolesnika sa karcinomima bubre`nogparenhima.

E61

TIROKALCITONIN I KONCENTRACIJEUKUPNOG KALCIJUMA I FOSFATA

U JEDNOM SLUÂJU MEDULARNOGTIROIDNOG KARCINOMA

V. Radonji}1, R. Milunovi}2, P. Radovi}2

1Dom zdravlja Kotor2Klini~ki centar Podgorica

Tirokalcitonin je hormon sintetisan u parafoliku-larnim }elijama u tiroidnoj `lezdi (C }elijama). Nje-gova aktivnost se zasniva na redukciji serumskog kal-cijuma preko bubrega i ko{tanog mineralnog meta-bolizma. Kod medularnog karcinoma tiroidee posto-ji hiperplazija C }elija i porast koncentracija ovog hor-mona. U ovom radu ispitan je uticaj visokih koncen-tracija tirokalcitonina na koncentracije ukupnogkalcijuma (Ca2+) i fosfata (PO4

3’) u serumu i urinukod pacijentkinje obolele od medularnog karcinomatiroidee. Pacijentkinji je dijagnostikovan medularnikarcinom tiroidee u 29. godini ìvota i ona je pra}enatokom 5 godina. Ura|ena je totalna tireotomija i 4operacije u recedivima, a sada je na supstitucionojterapiji sa LT-4. Koncentracija tirokalcitonina odre-|ena je dvopoloàjnim radioimunotestom (CIS-biointernational), ukupni kalcijum kolorimetrijskom me-todom, fosfati UV metodom i alkalna fosfataza (AP,EC 3.1.3.1.) kineti~ki na 30 °C (DEA-pufer) RANDOXreagensima. Koncentracije tirokalcitonina su varirale;posle hiru{ke operacije bile su niè, a pre operacijeveoma visoke (od 219 do 2800 pg/mL). Koncentra-cija ukupnog kalcijuma bila je konstantno nià, pro-se~no 1,86 mmol/L i izlu~ivanje je snièno prose~no

AP enzyme activities. In patients with clinically docu-mented localized renal cell carcinoma (Group II) theactivities of both GGT and AP were increased in 4patients, while AP activity only, was elevated in onepatient. Two of these patients were later confirmed tohave metastatic tumor, while other three had extremetumor masses. GGT was increased in a large percentof patients with metastatic renal cell carcinoma, andit was normal in the majority of patients with localizedrenal cell carcinoma. We conclude that GGT as wellas the AP should be included in the preoperativeevaluation of patients with renal cell carcinoma.

E61

THYROCALCITONIN AND CONCENTRATIONS OF TOTAL

CALCIUM AND PHOSPHATE IN ONE CASEOF MEDULLARY THYROID CARCINOMA

V. Radonji}1, R. Milunovi}2, P. Radovi}2

1Health Centre of Kotor2Clinical Centre of Podgorica

Thyrocalcitonin (TC) is hormone synthesized bythe parafollicular cells of the thyroid gland (C cells).Its activity is based on reduction of the plasma calci-um by the kidneys and by bone turnover. Themedullary thyroid carcinoma is characterized byhyperplasia of C cells and rise of basal concentra-tions of this hormone. We investigated the influenceof high concentrations TC on the total calcium(Ca2+) and inorganic phosphorous (PO4

3’) value inplasma and urine at the patient that had become illof medullary thyroid carcinoma. Patient was diag-nosed medullary thyroid carcinoma at 29 years ofage, and she has been followed for the period of 5years. She was done total thyroidectomy and foursurgeries after recedives. She was undergone repla-cement therapy with LT-4. Thyrocalcitonin concen-trations were determinated using two sites imunora-diometric assay (CIS bio international). Calcium wasassayed by colorimetric method, inorganic phospho-rous UV method and activities of total alkaline pho-shase (AP, EC 3.1.3.1) by kinetic method at 30 °C(DEA-buffer) by RANDOX reagent. TC concentra-tions varies, they are lower after surgery and they arevery high before surgery (of 219 to 2800 pg/mL).


2,96 mmol u dnevnom urinu. Ukupna koncentracijafosfata u serumu bila je prose~no 1,41 mmol/L, izlu-~ivanje u urinu 35,72 mmol u dnevnom urinu. Aktiv-nost ukupne AP bila je veoma snièna (51,33 U/L).Bolja korelacija je prona|ena izme|u koncentracijatirokalcitonina i koncentracija izlu~enog kalcijuma(r=0,79), kao i izlu~enih fosfata (r=0,92). Serumskekoncentracije Ca2+ i PO4

3’ su konstantne, verovatnozahvaljuju}i homeostatskim mehanizmima u organiz-mu. Snièna aktivnost AP ide u prilog hipotezi da CTdirektno inhibira osteoklaste i ko{tani promet.

E62

TUMORSKI MARKERI I ADENOZIN DEAMINAZA U SERUMIMA PACIJENATA SA PLEURALNIM IZLJEVIMA MALIGNE

I TUBERKULOZNE ETIOLOGIJE

J. Nikoli}1, M. Vu~i}2

1Zavod za biohemiju, Medicinski fakultet Univerziteta u Banjoj Luci

2Klinika za plu}ne bolest, Klini~ki centar, Banja Luka

Nedavno smo pokazali da odre|ivanje karcinoem-brionalnog antigena (CEA ), karbohidratnog antigena19-9 (CA 19-9) i aktivnosti adenozin deaminaze (ade-nozin aminohidrolaza, EC 3.5.4.4; ADA) u pleuralnimizljevima moè da doprinese u diferecijalnoj dijagnos-tici pleuralnih izljeva tuberkulozne i maligne etiologi-je. Cilj ovog rada je bio da se provjeri da li kod istihpacijenata navedeni biohemijski markeri odre|eni userumima imaju isti dijagnosti~ki zna~aj. Ispitivanje jeobuhvatilo 30 bolesnika sa izljevom maligne etiologi-je i 30 bolesnika sa tuberkuloznim izljevom. Tumorskiobiljeìva~i (markeri) odre|ivani su imunoradiometri-jskim metodama, a kataliti~ka aktivnost ADA-e spek-trofotometrijskom metodom. Koncentracija AFP-a userumima dvije grupe ispitanika nije se zna~ajno raz-likovala, dok su koncentracije CEA-a i CA 19-9 sta-tisti~ki zna~ajno bile povi{ene (p<0,001) kod pacije-nata s malignim bolestima plu}a (kako primarnim,tako i metastatskim tumorima) u odnosu na pacijen-te s tuberkulozom. Nasuprot tome, kataliti~ka aktiv-nost serumske ADA-e bila je zna~ajno ve}a (p<0,01)kod pacijenata s tuberkuloznim izljevima. Rezultatiukazuju da se u diferencijalnoj dijagnostici pleuralnihizljeva maligne i tuberkulozne etiologije moè koristi-ti odre|ivanje koncentracije CEA i CA 19-9 u serumu,kao i aktivnosti serumske ADA-e.

Concentrations of total Ca2+ were constant by lower,median 1.86 mmol/L and secreting was reduced,median 2.96 mmol per day. Concentrations of PO4

3’in plasma are median 1.41 mmol/L, secreted per daymedian 35.72 mmol in urine. Activities of total APwas very decreased (51.33 U/L). Better correlationwas found between TC values and values secretedCa2+ (r=0.79) and PO4

3’ (r=0.92). Values of plasmaCa2+ and PO4

3’ are kept constant in organism, pro-bably due to the mechanism of homeostasis. Thedecriase activities of total AP support the hypothesis,that TC direct by reduces the osteoclasts cells andbone turnover.

E62

TUMOUR MARKERS AND ADENOSINEDEAMINASE IN THE SERA OF PATIENTS

WITH HYDROTORAX OF MALIGNANT AND TUBERCULOUS ETIOLOGY

J. Nikoli}1, M. Vu~i}2

1Institute of Biochemistry, Faculty of Medicine,University of Banja Luka

2Clinic for Lung Diseases, Clinical Centre, Banja Luka

We have shown recently that determination ofcarcinoembryonic antigen (CEA), carbohydrate anti-gen 19-9 (CA 19-9) and adenosine deaminase(adenosine aminohydrolase, EC 3.5.4.4; ADA) activi-ty in pleural effusions can contribute greatly to differ-ential diagnostics of pleural effusions of tuberculousand malignant etiology. The aim of the present studywas to find out whether the above listed biochemicalparameters of the same patients determined in theblood sera could be of the same diagnostic value.Thirty patients with hydrotorax of malignant etiologyand 30 with tuberculous hydrotorax were examined.Tumor markers were determined by immunoradio-metric assays and catalytic ADA activity was estimat-ed spectrophotometrically. Concentration of AFP inthe sera of these two groups of patients was not sig-nificantly different, but the concentrations of bothCEA and CA 19-9 were significantly higher (p<0.001)in the patients with malignant lung diseases (both inprimary tumors and metastases) in comparison withthose suffering from tuberculosis. Opposite to that,catalytic activity of serum ADA was significantly high-er in the latter group (p<0.01). These results suggestthat determination of serum concentrations of CEAand CA 19-9, as well as activity of serum ADA canhelp in differential diagnostics of both pleural effu-sions of malignant and tuberculous etiology.

E63

SERUMSKI TUMORSKI MARKERI CEA I CA 15-3 KOD PACIJENATA

S KARCINOMOM DOJKE

G. Prtenjak1, V. Balti}1, Lj. Miljkovi}1, J. Pe{i}1, V. Ristovski2

1Institut za onkologiju Sremska Kamenica2Institut za kardiovaskularne bolesti, Sremska Kamenica

U pra}enju toka bolesti kod pacijenata s karci-nomom dojke koristi se nekoliko tumorskih markeraod kojih su CEA i CA 15-3 verovatno najvi{e prou-~avani. Cilj ovoga rada bio je da prikaè ulogu CEA iCA 15-3 u postoperativnom toku pacijenata, odnosdobijenih rezultata u zavisnosti od starosti pacijenata,prisustva metastaza u limfnim `lezdama i stadijumaklini~ke progresije bolesti po UICC (TNM klasifikaci-ja). Analizirani rezultati odre|ivani su u serumima 400èna koje su operisane od karcinoma dojke na Insti-tutu za onkologiju u Sremskoj Kamenici. Kao kontrol-na grupa kori{}eni su serumi 50 zdravih èna. Analizesu ura|ene pomo}u IMx MEIA (Abbott) testova i IMxanalizatora. Referentna vrednost za CEA je do 3 ng/mL,a za CA 15’3 do 28 U/mL. Vrednosti oba tumorskamarkera dobijena u serumima zdravih èna nalaze seu okviru referentnih vrednosti i zna~ajno su niì uodnosu na vrednosti pacijenata s karcinomom dojke(p<0,005). Rezultati pokazuju da su vrednosti CEA iCA 15-3 zna~ajno vi{e u odnosu na stadijume bolesti(p<0,005). Nije na|ena statisti~ki zna~ajna korelacijaCEA i CA 15-3 u odnosu na grupe èna u pre i post-menopauzalnom periodu, kao ni kod grupa pacijena-ta s pozitivnim i negativnim aksilarnim limfnim `lez-dama. Na osnovu dobijenih rezultata moè se zaklju-~iti da su CEA i CA 15-3 va`ni indikatori pacijentovogstanja i dobri tumorski markeri u pra}enju progresijebolesti kod pacijenata s karcinomom dojke.

E64

MOLEKULSKE VARIJANTE PARAPROTEINA KOD BOLESNIKA SA MALIGNIM MONOKLONSKIM

GAMAPATIJAMA

T. Risti}1, N. Milo{evi}-Jov~i}2, V. Ili}2, D. ]iri}2, S. Petrovi}2

1Centar za medicinsku biohemiju, Klini~ki centar, Ni{2Institut za medicinska istraìvanja, Beograd

Paraproteini su imunoglobulini koje sinteti{u}elije B }elijske linije koje su podlegle neoplasti~nojtransformaciji. Otkrivanje paraproteina u serumu i/ili


E63

SERUM TUMOUR MARKERS CEA AND CA 15-3 INPATIENTS

WITH BREAST CANCER

G. Prtenjak1, V. Balti}1, Lj. Miljkovi}1, J. Pe{i}1, V. Ristovski2

1Institute of Oncology Sremska Kamenica2Institute of Cardiovscular Diseases, Sremska Kamenica

Several tumour markers are used to monitorbreast cancer patients and CEA and CA 15-3 areprobably the most studied. The aim of this study wasto present the role of CEA and CA 15-3 in postoper-ative course of our patients and to show correlationof obtained results and patient’s age, presence ofmetastases to the axillary lymph nodes, stage of theclinical progression by UICC (TNM classification).The analyzed results were determined in sera of 400breast cancer patients, surgically treated at the Insti-tute of Oncology Sremska Kamenica. Sera from 50healthy women were used as control group. Theanalyses were done with IMx MEIA tests (Abbott) andIMx analyzer. The CEA limit value of the normal rangewas 3 ng/mL, and CA 15-3 was 28 U/mL. The valuesof both tumour markers found in the serum of healthywomen remained within normal value and significant-ly lower comparing to the value at patients with breastcancer (p<0.005). Further analysis of the results sho-wed that the values CEA and CA 15-3 are significant-ly higher comparing to the stage of clinical progres-sion (p<0.005). No significant correlation was foundcomparing CEA and CA 15-3 values in women beforeand postmenopausal, and patients with metastases inlymph nodes and group without metastases. On thebasis of the results of this study it may be concludedthat CEA and CA 15-3 are important indicators ofpatients status and good tumour markers in the fol-low-up of breast cancer progression.

E64

MOLECULAR VARIANTS OF PARAPROTEINS IN PATIENTS WITH MALIGNANT MONOCLONAL

GAMMAPATHIES

T. Risti}1, N. Milo{evi}-Jov~i}2, V. Ili}2, D. ]iri}2, S. Petrovi}2

1Center of Medical Biochemistry, Clinical Center, Ni{2Institute for Medical Research, Belgrade

Paraproteins are immunoglobulins that are pro-duced by a single neoplastic clone of B cells. Detec-tion, quantitative and qualitative analysis of parapro-


urinu bolesnika sa malignim monoklonskim gama-patijama (MMG), njihova kvantifikacija i imunohemi-jska identifikacija izotipa te{kih i tipa lakih lanaca unjihovim molekulima va`an su korak u postavljanjudijagnoze, pra}enju toka bolesti i efekta terapije iprognozi odre|ene MMG. Ovaj rad prikazuje rezultateispitivanja 52 bolesnika sa MMG i pokazuje kolika jeprocentualna zastupljenost (najmanje 2,1%, najvi{e56,5% od ukupnih serumskih proteina) i koncentraci-ja paraproteina u serumu (opseg: 1,42 g/L’92,7 g/L)i u kojoj meri se ona odra`ava na koncentraciju ukup-nih serumskih proteina, kakva je zastupljenost struk-turno kompletnih (80,95%) i nekompletnih (19,05%)imunoglobulinskih molekula me|u paraproteinima,kao i zastupljenost pojedinih imunoglobulinskih klasa(62,7% IgG, 15,6% IgA, 21,56% IgM), tipova lakihlanaca (60,78% kapa, 39,22% lambda tipa), IgG pod-klasa (77,7% IgG1) i njihovih alotipova (svi IgG1 subili G1m(f) alotipa) i bi(tri)klonskih kombinacija (23%),kakva je elektroforetska pokretljivost (90% beta2,gama1, gama2) i rastvorljivost paraproteina (5,78%krio- i 13,4% euglobulini) i kolika je i kakva je para-proteinurija. Rezultati su posebno zna~ajni sa sta-novi{ta molekulskih kurioziteta koji su otkriveni ovimispitivanjima.

teins in serum and/or urine are essential in makingthe diagnosis, following the disease progression andmonitoring the treatment response in patients withmalignant monoclonal gammapathies (MMG). Thisstudy deals with the analysis of investigation of 52patients with MMG. It shows: serum level of parapro-teins can be very low as well as extremely high (ran-ged 1.42 g/L’92.7 g/L); 80.95% of paraproteins werein the form of structurally complete, intact moleculesand 19.05% were free light chains; 62.7% of para-proteins with complete structure belonged to IgG,15.6% to IgA and 21.6% to IgM class and had kappa(60.8%) or lambda (39.22%) light chain types;among IgG paraproteins the most frequent werethose of IgG1 subclass with G1m(f) allotype; 23% ofparaproteins comprised the bi(tri)clonal combina-tions; 90% of paraproteins were situated in beta2,gamma1 or gamma2 electrophoretic area; 19.18%of paraproteins had unusual solubility (cryo-, euglob-ulins); urinary paraproteins were found to be kappaor lambda light chains and in some cases the frag-ments of heavy chains. A number of molecular curio-sities among paraproteins were revealed during thestudy.

D49

UTICAJ SUPSTITUCIONE TERAPIJE NA KONCENTRACIJE KALCIJUMA,

FOSFATA, MAGNEZIJUMA I AKTIVNOSTALKALNE FOSFATAZE KOD DECE

SA HIPOTIROIDIZMOM

V. Radonji}1, Z. Jeli}-Ivanovi}3, V. Kalimanovska3, P. Radovi}2, R. Milunovi}2

1Dom zdravlja Kotor2Institut za de~ije bolesti KC Podgorica

3Farmaceutski fakultet, Beograd

Tiroidni hormoni su veoma va`ni za razvoj ske-letnog sistema. Pretpostavlja se da je njihovo dejstvoposledica stimulacije, sekrecije i dejstva hormonarasta. Koncentracije kalcijuma, fosfata i magnezijumau tesnoj vezi su sa remodeliranjem kosti, dok aktiv-nost alkalne fosfataze (AP) moè da bude pokazateljosteoblasti~ne aktivnosti. Cilj ovog rada bio je ispiti-vanje uticaja supstitucione terapije na koncentracijeCa2+, PO4

3’, Mg2+ kao i aktivnost AP i eventualni po-reme}aj u rastu ove dece tokom detinjstva. Za po-re|enje je kori{}ena kontrolna grupa (n=100) zdravedece uzrasta od mesec dana do 18 godina, podelje-na u 5 starosnih podgrupa. Grupa dece obolele odhipotiroidizma na supstitucionoj terapiji sa LT-4(n=56) bila je iste starosne dobi. Da bi se proceniouticaj supstitucije na vrednosti ispitivanih parametaraupore|ene su vrednosti srednjih koncentracija,Studentovim t-testom grupe obolele dece sa supsti-tucijom i kontrolne grupe. Neparametarskim x2 tes-tom ispitana je mera slaganja izme|u distribucijafrekvenci, odnosno nivoa i kvaliteta razlika koncen-tracija ispitivanih parametara izme|u kontrolne grupei grupe dece posle duèg primanja supstitucione te-rapije. Ca2+ i Mg2+ odre|eni su kolorimetrijskim tes-tom, PO4

3’ UV metodom, a AP kineti~ki na 30 °C(DEA-pufer) RANDOX testovima. Aktivnost AP je niàu svim grupama, ali sa statisti~kom zna~ajno{}u ugrupama od 1,1’5 god. (p<0,05) i 10,1’15 god.(p<0,05) u odnosu na kontrolnu grupu. Distribucija


DHORMONIHORMONS

D49

INFLUENCE OF THE REPLACEMENT THERAPY ON CONCENTRATIONS

OF CALCIUM, PHOSPHATE, MAGNESIUMAND ACTIVITIES IN CHILDREN

WITH HYPOTIROIDISM


1Health Centre of Kotor2Institute of Infant’s Disease, Clinical Centre of Podgorica

3Pharmaceutical Faculty, Belgrade

The thyroid hormones are very important for de-velopment of skeleton system. Their effect are resultsof stimulating secretion and influence of growth hor-mone. Concentration of Ca2+, PO4

3’ and Mg2+ areclosely related with bone turnover, and activities alka-line phosphatase (AP, EC 3.1.3.1) could indicateosteoblastic activity. We have previously aimed theinfluence of the replacement therapy on concentra-tions of Ca2+, PO4

3’, Mg2+, and activities of AP andpossible disturbance in growth of these children dur-ing chilhood. The control group of healthy infants(N=100) within 1 month to 18 years of age was sep-arated in 5 age subgroup of age, and was taken forcomparison. The group of ill infants patient withhypothyroidism under the replacement therapy withLT-4, (N=56) were classified into the same groups,as the healthy group. In order to evaluate the influ-ence of the replacement therapy on values of investi-gated parameters, the values of median cocentra-tions of the parameters were compared between dis-eased children group and the control group, by useof Student t-test. Using X2 test the correlation extentbetween frequency distribution were investigated,distinctions of concentrations evaluated parametersbetween the control group and the group of infantsafter longterm replacement therapy. Ca2+ and Mg2+

were determined by the colorimetric method, PO43’

UV method, and AP by kinetic method at 30 °C(DEA-buffer) by RANDOX reagent. Activities of AP is

frekvenci niìh, srednjih i vi{ih vrednosti statisti~ki jezna~ajno izmenjena u svim starosnim grupama(p<0,05) u pore|enju sa distribucijom u kontrolnojgrupi. Koncentracije Ca2+ i PO4

3’ posle supstitucioneterapije nisu statisti~ki izmenjene (p>0,05), dok jedistribucija frekvenci niìh, srednjih i vi{ih vrednostiizmenjena u dve starije starosne grupe 10,1’15 god.(p<0,001) i 15,1’18 god. (p<0,001). Za Mg2+ sta-tisti~ki zna~ajno vi{e vrednosti na|ene su u uzrastu1,1’5 god. (p<0,02) i 10,1’15 god. (p<0,001) kadase o~ekuje najintezivniji metabolizam kostiju. Distri-bucija niìh, srednjih i vi{ih vrednosti zna~ajno se raz-likuje kod dece na supstituciji u odnosu na distribuci-ju u kontrolnoj grupi. Moè se zaklju~iti da je koddece sa hipotiroidizmom na supstituciji sa LT-4 ko{-tani metabolizam i rast vremenski pomeren ka stari-jem uzrastu.

D50

KORELACIJA IZME\U SLOBODNOG TIROKSINA

I ODNOSA T4/TBG U DECE SA HIPOTIROIDIZMOM:

UTICAJ SUPSTITUCIJE


1Dom zdravlja Kotor2Institut za de~ije bolesti KC Podgorica

3Farmaceutski fakultet, Beograd

Odre|ivanju indeksa tiroksin/tiroksin vezni globu-lin (T4/TBG) daje se veliki zna~aj, a posebno imaprednost u slu~aju izmene koncentracija TBG, jeromogu}ava rasvetljavanje problemati~nih slu~ajevatireoidne disfunkcije. Ispitanici u ovoj studiji bila suzdrava deca (n=100) uzrasta od mesec dana do 18godina i deca obolela od hipotireoidizma na supstitu-cionoj terapiji L-T4 (n=56) iste starosne dobi. Ispitanje i poseban serumski uzorak (n=20) dobijen iz pup-~ane vrpce zdravo ro|ene dece. Koncentracije hor-monskih parametara slobodnog tiroksina (fT4), ukup-nog tiroksina (T4), tiroid veznog globulina (TBG)odre|ene su upotrebom DELFIA fluoroimunotesta.Prose~an odnos T4/TBG u kontrolnoj grupi je izno-sio 0,22, a u grupi dece na supstituciji 0,33 sa zna-~ajnom statisti~kom razlikom (p<0,001). Koeficijentkorelacije u kontrolnoj grupi izme|u fT4 i T4/TBGje r=0,41 sa jedna~inom zavisnosti yx=0,131+


lower in all groups, but with statistical significance ingroups 1.1’5 years (p<0.05) and 10.1’15 years(p<0.05) compared to the control group. The fre-quency distribution of lower, middle and higher val-ues is statistical significantly changed in all goups ofage (p<0.05) in comparison with the control groupdistribution. Concentrations of Ca2+ and PO4

3’ afterthe replacement therapy weren't statistically signifi-cantly changed (p>0.05), and the distribution fre-quency of lower, middle and higher values were chan-ged in two older group of age 10.1’15 years (p<0.001) and 15.1’18 years (p<0.001) for both elec-trolit. We got statisticaly significant higher values forMg2+ at 1.1’5 years (p<0.02) and 10.1’15 years(p<0.001) when it is expected the most intensivebone metabolism. Distribution of lower, middle andhigher values are significantly different at the childrenwith replacement in comparison with the controlgroup. We conclude that the bone metabolism at thechildren with hypotiroidism on replacement with LT-4and their growth is temporally moved to the older age.

D50

CORRELATION BETWEEN THE FREE THYROXIN AND THE THYROXIN/

THYROXIN-BINDING GLOBULIN RATIO IN CHILDREN WITH HYPOTHYROIDISM:

REPLACEMENT INFLUENCE


1Health Centre of Kotor2Institute of Infant's Disease, Clinical Centre of Podgorica

3Pharmaceutical Faculty

Determination of thyroxin/thyroxin-binding globu-lin (T4/TBG) has the great importance, which is pri-ority, especially in the cases of TBG concentrationschanges, because it enables elucidation of problem-atic cases thyroid disfunction. Serum samples fromN=100 euthyroid children (1 month to 18 years ofage) were examined, and children with hypothyroi-dism with replacement therapy L-T4, N=56 of thesame age. The sample taken from the healthy borninfants umbilical cord (N=20) constitutes specialgroup. Concentrations of hormones parameters, freethyroxin (fT4), thyroxin (T4) and thyroxin-binding glo-bulin (TBG) were mesured using DELFIA reagents.Average ratio T4/TBG was 0.22 in the control group,and in the group of children with reaplecement ther-apy was 0.33 (p less than 0.001). Coefficient correla-tion in the control group between fT4 and T4/TBGwas r=0.41 with dependence equation yx=0.131+


0,0075x, {to odgovara kriterijumu srednje zavisnostiizme|u ove dve promenljive. Koeficijent korelacije ugrupi dece na supstituciji iznosio je r=0,84 sa jedna-~inom zavisnosti y=0,176+0,01030x, {to odgovarakriterijumu visoke korelacije. Bolji korelacioni odnosu grupi dece na supstituciji u odnosu na kontrolnugrupu verovatno je uzrokovan ~injenicom da u kon-trolnoj grupi deca u mla|oj starosnoj dobi imaju vi{ekoncentracije TBG, a u grupi dece na supstituciji ovopove}anje je suprimirano vi{im koncentracijama T4.Zadovoljavaju}a korelacija izme|u ove dve promen-ljive dokazuje da iz jedne promenljive moèmo izra-~unati drugu s potpunom dijagnosti~kom upotreblji-vo{}u, {to daleko pojeftinjuje dijagnosti~ki postupak.

D51

ACTH ]ELIJE HIPOFIZE MU@JAKA PACOVA

POSLE TRETMANA OKTREOTIDOM

V. Milo{evi}1, B. Brki}2, B. Filipovi}1, S. Velkovski3, V. Star~evi}3

1Institut za biolo{ka istraìvanja2KBC £Dr Dragi{a Mi{ovi}«

3Institut za fiziologiju, Medicinski fakultet, Beograd

Ispitivani su efekti intracerebroventrikularno (i.c.v.)ubrizganog analoga somatostatina, oktreotida, na rastadrenokortikalnih (ACTH) }elija adenohipofize mu`ja-ka Wistar pacova. Sve eksperimentalne ìvotinje prim-ile su tri doze po 1 mg Oktreotida rastvorenog u 10 mLfiziolo{kog rastvora, svaki drugi dan. Kontrole su treti-rane na isti na~in fiziolo{kim rastvorom. Pacovi su`rtvovani petog dana po poslednjoj primljenoj dozioktreotida. ACTH }elije bojene su imunocitohemijskiPAP metodom. Stereolo{kom analizom pokazano jeda mu`jaci pacova i.c.v. tretirani oktreotidom, poka-zuju statisti~ki zna~ajno smanjenje (p<0,05) svih ispi-tivanih morfometrijskih parametra u odnosu na odgo-varaju}u kontrolu. Zapremina }elija smanjena je(p<0,05) za 44%, jedara za 18%, a volumenska gusti-na za 31% u pore|enju sa odgovaraju}om kontrolom.Na osnovu dobijenih rezultata moè se zaklju~iti dacentralno aplikovan oktreotid deluje inhibitorno na rastACTH }elija adenohipofize mu`jaka pacova.

0.00075x, which corresponds to the criterion of themiddle dependence between of these two variables.Corelation coefficient in the group of children withreplacement therapy was r=0.84 with dependenceequation y=0.176+0.01030x, which corresponds tothe criterion of very high correlation. Beter correlationratio in the group of children with replacement thera-py compared to the control group was probablycaused by the fact that children of the younger grouphave higher concentrations TBG to 5 years, and thisgroup is neutralized by higher concentrations of T4 inthe group of children with replacement therapy. Satis-factory correlation between these two variables provesthat we can estimate one variable from the secondone with complete diagnostic usage, which means thediagnostic procedure becomes a lot cheaper.

D51

ACTH CELLS IN THE PITUITARY OF MALE RATS AFTER TREATMENT

WITH OCTREOTIDE

V. Milo{evi}1, B. Brki}2, B. Filipovi}1, S. Velkovski3, V. Star~evi}3

1Institute for Biological Research2Clinical Centre »Dr Dragi{a Mi{ovi}«

3Institute of Physiology, School of Medicine,University of Belgrade

The effects of the intracerebroventricular (i.c.v.)application of somatostatin analogue, Octreotide, ongrowth pituitary adrenocorticotropes (ACTH cells)were examined in adult male Wistar rats. The animalswere subjected to i.c.v. administration of three 1 mgdoses Octreotide dissolved in 10 mL saline every se-cond day. Controls were treated in the same way withthe same volume of saline only. Five days after thelast injection, the animals were sacrificed. ACTH-pro-ducing cells were studied using the peroxidase-antiperoxidase (PAP) immunohistochemical proce-dure. Stereological analyses showed that i.c.v. treat-ment males with Octreotide significantly decreased(p<0.05) all morphometric parameters in compari-son with corresponding control animals. The volumeof ACTH cells and their nuclei were decreased by44% and by 18% respectively compared to the con-trols. The volume of density was also significantdecreased (p<0.05) by 31% in comparison with cor-responding controls. These findings suggest thatcentrally administered somatostatin analogue Octre-otide reduced growth of ACTH cells in pituitary ofmale rats.


D52

KARAKTERISTIKE RADIOIMUNOLO[KOG TESTA ZA

ODRE\IVANJE KONCENTRACIJE TBG-a(RIA hTBG, INEP)

I. Petrovi}, S. Savin, D. Cveji}

Institut za primenu nuklearne energije - INEP, Zemun

Tiroksin-vezuju}i globulin (TBG) glavni je trans-portni protein tireoidnih hormona u cirkulaciji. Direkt-no odre|ivanje koncentracije ovog proteina je va`nojer se ona menja u odre|enim klini~kim stanjima, {tose odraàva na nivoe ukupnih a donekle i slobodnihtireoidnih hormona. U INEP-ovoj laboratoriji formiranje radioimunolo{ki test za odre|ivanje koncentracijeTBG-a i ispitane su njegove karakteristike. Osetljivosttesta je veoma visoka i iznosi 2,0 mg TBG/L. Velikareproducibilnost ovog testa ogleda se u niskim koefi-cijentima varijacije (< 5%) za izra~unate preciznostiodre|ivanja koncentracije TBG-a u tri uzorka serumakako u seriji tako i izme|u serija. Nespecifi~no vezi-vanje obeleènog antigena veoma je nisko i ne prelazi2% ukupnog vezivanja. Linearnost testa pokazana jeu opsegu koncentracija standardne krive (0’80 mg/L)pore|enjem koncentracija o~ekivanih i izmerenihvrednosti TBG-a u razblaènjima seruma (r = 0.999).Pokazano je da pove}ano prisustvo lipida u serumuuti~e na ta~nost merenja koncentracije TBG-a (r =0.767). Ispitivanje stabilnosti RIA hTBG testa ufunkciji vremena, odre|ivanjem koncentracije TBG-au razli~itim uzorcima seruma, pokazalo je da ovaj testmoè da se koristi u roku od 32 dana od momentajodovanja TBG-a (F<F 0,05

tab, F7,320,05=2.31). Ta~nost

testa je ispitana kori{}enjem me|unarodnih kontrolaza standardizaciju Lyphochek Immunoassay Controls(Bio-Rad). Pore|enjem vrednosti koncentracija TBG-a, koje su odre|ene u nepoznatim uzorcima seruma(n = 25) sa RIA hTBG testom i Kodak Amerlite TBGAssay, na|en je zna~ajan stepen korelacije (r = 0.84).Dobijeni rezultati ukazuju da je RIA hTBG precizna,osetljiva i pouzdana metoda za odre|ivanje koncen-tracije TBG-a u humanom serumu.

D52

THE PROPERTIES OF A RADIOIMMUNOASSAY

FOR THE QUANTITATION OF TBG (RIA hTBG, INEP - ZEMUN)

I. Petrovi}, S. Savin, D. Cveji}

Institute for the Application of Nuclear Energy - INEP, Zemun

Thyroxine-binding globulin (TBG) is the majortransport protein for thyroid hormones in the circula-tion. Direct determination of its concentration isimportant because changes occur in certain clinicalconditions which affect total and, to some degree,free thyroid hormone concentrations. A radioimmu-noassay for the quantitation of TBG was preparedand characterised. The sensitivity is favourable (2.0mg TBG/L). The highly satisfactory precision of theassay is reflected in the low coefficients of variation(less than 5%) found for both intra- and inter-assayprecision at three concentrations. Non-specific bind-ing of the labelled antigen is very low (less than 2% ofthe total binding). Linearity of the assay within therange of the standard curve (0 ’80 mg/L) was shownby comparing the expected and measured concen-trations of TBG in serial dilutions of sera (r = 0.999).TBG could not be measured accurately in lipemicsera (r = 0.767). Reproducibility was checked by de-termining the concentration of TBG in samples ofsera over a period of 32 days from the day of antigeniodination and (F<F0,05

tab , F7,320,05=2.31). The assay

was standardised for accuracy using LyphochekImmunoassay Controls (Bio-Rad). A significant cor-relation was obtained between RIA hTBG and theKodak Amerlite TBG Assay (r = 0.84; n = 25). Theseresults show that RIA hTBG is a sensitive, precise andreliable method for determination of TBG in humansera.


D53

VREDNOSTI T4/TBG INDEKSA U SERUMUZDRAVIH I OSOBA SA POREME]AJIMA

FUNKCIJE [TITASTE @LEZDE: KORELACIJA SA KONCENTRACIJOM FT4

I. Petrovi}1, S. Savin1, S. Spasi}2, D. Cveji}1,N. Paunkovi}3, J. Paunkovi}3

1Institut za primenu nuklearne energije - INEP, Zemun

2Institut za medicinsku biohemiju, Farmaceutski fakultet, Univerzitet u Beogradu

3Slu`ba za nuklearnu medicinu, Medicinski centar Zaje~ar

Za evaluaciju funkcionalnog stanja tireoideedanas se, pored odre|ivanja koncentracija ukupnihtireoidnih hormona (T4 i T3), odre|uje i slobodna,fiziolo{ki aktivna frakcija. Pored direktnog odre|ivan-ja koncentracije slobodnih hormona (FT4 i FT3), zaprocenu slobodne frakcije koristi se i T4/TBG indeks,koji se izra~unava iz koncentracija ukupnog T4 i TBG-a. U ovoj studiji odre|ene su koncentracije TBG-a,ukupnog T4, FT4 i izra~unat je T4/TBG indeks kodzdravih, eutireoidnih osoba (n = 102; 45 mu{karacai 57 èna) kao i kod pacijenata sa poreme}ajimafunkcije {titaste `lezde: hipotireozom (n = 33) i hiper-tireozom (n = 66). Odre|eni su opsezi referentnihvrednosti za svaki parametar. Dobijene vrednostiT4/TBG indeksa kod zdravih su u opsegu od 2,1 do4,4. Pokazano je da su koncentracije TBG-a kod ènane{to vi{e, ali da se ne razlikuju zna~ajno (p > 0,05)u odnosu na mu{ku populaciju zdravih. Koncentra-cije TBG-a u hipotireozi su zna~ajno vi{e (p < 0,05)dok su kod hipertireoidnih osoba ne{to niè, ali se nerazlikuju zna~ajno od eutireoidnih vrednosti (p >0,05). Vrednosti T4/TBG indeksa sniène su u hipo-tireozi (0,5’2,2), a povi{ene u hipertireozi (4,7’13,1).Pore|enjem vrednosti T4/TBG indeksa i koncen-tracije FT4 kod svih ispitanika dobijen je zna~ajanstepen korelacije (r = 0,93). Utvr|ene su referentnevrednosti za T4/TBG indeks kod eutireoidnih osoba iosoba sa hipo i hiperfunkcijom {titaste `lezde. Prak-ti~na provera primenljivosti T4/TBG indeksa u ispiti-vanju funkcije {titaste `lezde pokazana je korelacijomsa FT4.

D53

THE T4/TBG RATIO IN HEALTHY SUBJECTSAND IN PATIENTS WITH DISORDERS

OF THYROID FUNCTION: CORRELATION WITH FT4 CONCENTRATIONS

I. Petrovi}1, S. Savin1, S. Spasi}2, D. Cveji}1,N. Paunkovi}3, J. Paunkovi}3

1Institute for the Application of Nuclear Energy - INEP, Zemun

2Institute for Medical Biochemistry, Faculty of Pharmacy, University of Belgrade

3Nuclear Medicine, Medical Center Zaje~ar

Nowadays, besides the determination of total thy-roid hormone (T4 and T3) concentrations, it is impor-tant to measure the proportion of free, physiological-ly active, hormone for the proper evaluation of thy-roid function. The proportion of free hormone can beestimated by direct measurement of free hormoneconcentrations (FT4 and FT3) and by the T4/TBGratio which is calculated from the concentrations oftotal T4 and TBG. This study concerns the determi-nation of TBG, total and free T4 and T4/ TBG ratio insera from healthy, euthyroid persons (n = 102; 45males and 57 females) as well as sera obtained frompatients with thyroid function disorders: hypothyrosis(n = 33) and hyperthyrosis (n = 66). The referencerange for each parameter was determined. The val-ues obtained for T4/ TBG ratio in healthy subjectsvaried from 2.1 to 4.4. It was shown that TBG con-centrations in females were slightly higher than thosein males but the elevation was not statistically signi-ficant (p > 0.05). Significantly higher levels of TBGwere found in hypothyrosis (p < 0.05), while theywere slightly but not significantly lower in hyperthyro-sis (p > 0.05) compared with euthyroid subjects. Thevalues for T4/TBG ratio were lower in hypothyrosis(0.5’2.2) and elevated in hyperthyrosis (4.7’13.1). Asignificant correlation was obtained between the val-ues of T4/ TBG ratio and FT4 in all examined subjects(r = 0.93). The reference ranges of T4/TBG ratio ineuthyroid subjects and in patients with hypothyrosisand hyperthyrosis were determined. The correlationof T4/ TBG ratio with FT4 showed the practical use-fulness of this ratio in assessment of thyroid function.

D54

DIJAGNOSTI^KA OSETLJIVOST, SPECIFI^NOST I PREDIKTIVNOST

ODRE\IVANJA UKUPNOG T4, T4/TBG INDEKSA I FT4 U EVALUACIJI

TIREOIDNOG STATUSA

I. Petrovi}1, S. Spasi}2, S. Savin1, D. Cveji}1,N. Paunkovi}3, J. Paunkovi}3

1Institut za primenu nuklearne energije - INEP, Zemun2Institut za medicinsku biohemiju,

Farmaceutski fakultet, Univerzitet u Beogradu3Slu`ba za nuklearnu medicinu,

Medicinski centar Zaje~ar

Odre|ivanje koncentracije ukupnog T4 u serumupredstavlja jednu od osnovnih laboratorijskih analizaza klini~ku dijagnostiku poreme}aja funkcije {titaste`lezde. êsto se de{ava da ova analiza nije dovoljnapa se dopunjuje odre|ivanjem slobodne frakcije tireo-idnih hormona: FT4 i/ili T4/TBG indeksa. Za klini~kupraksu je veoma va`no da se utvrdi kakva je osetlji-vost, specifi~nost i prediktivnost odre|ivanja ovih pa-rametara za dijagnozu hipotireoze i hipertireoze. Uku-pni T4, T4/TBG indeks i FT4 odre|eni su kod pacije-nata sa hipotireozom (n = 33), hipertireozom (n =66) kao i u grupi zdravih, eutireoidnih osoba (n =102). Pokazano je da za dijagnozu hipotireoze najve-}u osetljivost ima FT4 (97%). Osetljivost odre|ivanjaT4/TBG indeksa je visoka (85%) ali ipak zna~ajno niàod FT4 (t > ttab), dok je najmanje osetljiv parametarukupni T4 (64%). Specifi~nosti T4/TBG indeksa i FT4vrlo su visoke (96% odnosno 99%), dok je kod ukup-nog T4 zna~ajno nià (46%). Najve}u pozitivnu predik-tivnu vrednost (PPV) ima FT4 (97%), zatim T4/TBGindeks (88%) a najniù ukupni T4 (28%). Pore|enjemnegativnih prediktivnih vrednosti (NPV) pokazano jeda nema zna~ajne razlike izme|u FT4 (99%) i T4/TBGindeksa (95%), dok je vrednost zna~ajno nià zaukupni T4 (80%) (t > ttab). Za dijagnozu hipertireozepokazano je da sva tri parametra imaju maksimalnuosetljivost, specifi~nost i prediktivnost (99% ili 100%).Utvr|eno je da T4/TBG indeks i FT4 imaju veomadobre karakteristike, mada se prednost FT4 ogleda uve}oj osetljivosti i PPV pa je zato bolji parametar zadijagnozu hipotireoze, dok odre|ivanje ukupnog T4nije analiza izbora. Za dijagnostiku hipertireoze svakiod ova tri parametra moè da se koristi sa podjed-nakom sigurno{}u.

D54

DIAGNOSTIC SENSITIVITY, SPECIFICITYAND PREDICTIVE VALUE OF TOTAL T4,

T4/TBG RATIO AND FT4 IN THE EVALUATION OF THYROID STATUS

I. Petrovi}1, S. Spasi}2, S. Savin1, D. Cveji}1,N. Paunkovi}3, J. Paunkovi}3

1Institute for the Application of Nuclear Energy - INEP, Zemun

2Institute for Medical Biochemistry, Faculty of Pharmacy, University of Belgrade

3Nuclear Medicine, Medical Center Zaje~ar

The determination of serum total T4 concentra-tion is one of the fundamental laboratory analyses forclinical diagnosis of thyroid function disorders.However, very often it is not sufficient because theproportion of free hormone should be known bydetermining FT4 and/or the T4/TBG ratio. For clinicalpractice it is very important to establish the sensitivi-ty, specificity and predictive value of these tests in thediagnosis of hypothyrosis and hyperthyrosis. Total T4,T4/TBG ratio and FT4 were determined in patientswith hypothyrosis (n = 33), hyperthyrosis (n = 66)and in healthy, euthyroid subjects (n = 102). For thediagnosis of hypothyrosis the highest sensitivity wasfound for FT4 (97%). The sensitivity of T4/TBG ratiowas also high (85%) but significantly less than for FT4(t >ttab), while the least sensitive parameter was totalT4 (64%). The specificities of T4/TBG ratio and FT4were very high (96% i.e. 99%), while it was signifi-cantly lower for total T4 (46%). The highest positivepredictive value (PPV) was given by FT4 (97%), thenT4/TBG ratio (88%) and lastly total T4 (28%). It wasshown that the negative predictive values (NPV) werenot significantly different between FT4 (99%) andT4/TBG ratio (95%), but total T4 had a significantlylower NPV (80%) (t >ttab). All three parameters werefound to have excellent sensitivity, specificity and pre-dictive values (99% or 100%) for the diagnosis ofhyperthyrosis. Thus, both T4/TBG ratio and FT4 hadvery good characteristics, but FT4 is the better test forthe diagnosis of hypothyrosis because of the morefavourable sensitivity and PPV, whereas determina-tion of total T4 was of little value. Each of these threeparameters could be used with equal certainty in thediagnosis of hyperthyrosis.


D55

ABNORMALNOSTI TIREOIDNIH HORMONA KOD PACIJENATA

SA BUBRE@NOM INSUFICIJENCIJOM

S. \or|evi}-Cvetkovi}, S. Milojevi}

Biohemijska laboratorija, Urolo{ko odeljenje Zdravstveni centar Kru{evac

Endokrini poreme}aji kod pacijenata na dijalizipredmet su mnogih istraìvanja. Interpretacija dobi-jenih rezultata ~esto nije jednostavna jer je endokrinisistem kod pacijenata na dijalizi poreme}en na supti-lan i kompleksan na~in. Klini~ke manifestacije ovihendokrinih poreme}aja razli~ite su u u~estalosti iteìni a neki koji su pra}eni zna~ajnim biohemijskimabnormalnostima nemaju poseban klini~ki zna~aj.Cilj ovog rada bio je da se prati metabolizam tireoid-nih hormona kod pacijenata sa bubre`nom insufici-jencijom. Ispitano je 40 bolesnika na hemodijalizi,prose~ne starosne dobi 51 godina (20’68), koji sedijaliziraju u proseku 56 meseci, tri puta nedeljno po5 ~asova, pomo}u vi{e tipova dijalizatora. U okviru ti-reoidnog statusa odre|ivan je: tiroksin (T4), trijod-tironin (T3) i tireostimuliraju}i hormon (TSH) imuno-hemijskim postupkom (ELISA) Human-ovim testovi-ma. T4 je izraàvan u nmol/L, T3 u ng/dl, a TSH ummol/L. Rezultati pokazuju da su kod pacijenata nadijalizi koncentracije T4 bile normalne ili sniène (p>0,05), dok su vrednosti T3 bile zna~ajno sniène(p<0,05) u odnosu na kontrolnu grupu. Koncentra-cije TSH su bile normalne u odnosu na referentnevrednosti. Bolesti bubrega koje su pra}ene poreme-}ajem u metabolizmu proteina, pra}ene su i sma-njenjem sinteze i koncentracije TBG. Zato se sinteza ikoncentracija tiroksina u krvi smanjuje, ali samo uizraènim slu~ajevima. I konverzija T4 u T3 moè bitismanjena pa se i koncentracija T3 smanjuje. Klini~ki,i pored smanjenih vrednosti T4 i T3, ve}ina pacijena-ta na dijalizi je eutireoti~na sa normalnim vrednosti-ma TSH. Dodavanje tireoidnih hormona u cilju ko-rekcije niskih vrednosti tireoidnih hormona u serumu,ne treba sprovoditi jer mogu nastati zna~ajni kata-boli~ki poreme}aji.

D56

STRESOGENI UTICAJ RADNE SREDINE NA NIVO HORMONA [TITNE @LEZDE

S. Stoilkovi}, M. Popovi}

Zdravstveni centar Vranje

Veliki broj pacijenata koji dolaze iz pogona Yum-ko imaju pove}ani nivo hormona {titne `lezde. To jebio razlog da se ispita kod zaposlenih da li postoji


D55

THYROID HORMONES ABNORMALITIES AT PATIENTS WITH RENAL INSUFFICIENCY

S. \or|evi}-Cvetkovi}, S. Milojevi}

Biochemical Laboratory, Department of UrologyHealth Care Center of Kru{evac

Endocrine disorders at patients on dialysis are sub-ject of many researches. Interpretation of obtainedresults is not always easy, as the endocrine system atpatients on dialysis is disordered in a sensitive andcomplex way. Clinical manifestation of these endo-crine disorders is different per frequency and gravity,and some of them, that have been followed by signifi-cant biochemical abnormalities do not have a specialclinical importance. The aim of this study is to deter-mine the thyroid hormones metabolism at patientswith renal insufficiency. It has been tested 40 patientson hemodialysis, average of 51 years (20’68), dialyzedmostly 56 months, three times a week per 5 hours,using a different dialysators. Within thyroid status ithas been determined as following: thyroxine (T4),triiodothyronine (T3) and thyrotropin (TSH) by meansof immunochemical procedure (ELISA) of Human'stests. T4 is expressed in nmol/L, T3 in ng/dl, and TSHis expressed in mmol/L. The results show that concen-tration of T4 at patient on dialysis is normal or lower(p>0.05), but values of T3 are significantly lower(p<0.05) referring to the control group. The concen-tration of TSH is normal regarding to the referring val-ues. Kidney diseases followed by disorders in metabo-lism of albumen, are also followed by reducing of syn-thesis and concentration of TBG. For that reason syn-thesis and concentration of thyroxine in blood isreduced, but only at distinguished cases. The conver-sion of T4 in T3 can be also reduced so that the con-centration of T3 is reduced, too. Clinically, besidesreduced values of T4 and T3, most patients on dialysisare euthyreotic with normal TSH values. Adding thy-roid hormones in order to correct lower values of thy-roid hormones in serum should not be done, becausesignificant catabolic disorders can develop.

D56

EFFECT OF WORKING ENVIRONMENTSTRESS ON THE LEVEL OF THYROID

HORMONS

S. Stoilkovi}, M. Popovi}

Health centre, Vranje

Huge number of patients with increased level ofthyroid hormones come from factory Yumco for aprofessional help, which was the reason to examine if


stresogeni uticaj buke na {titnu `lezdu i njenu aktiv-nost. Cilj je bio da se utvrde {tetni efekti na zaposlenezavisno od duìne njihove izloènosti buci. Ispitivanisu pacijenti koji do tada nisu evidentirani (n=125), akontrolnu grupu su ~inili radnici Yumko-a koji nerade u uslovima buke (n=75). Tako|e, ispitivan je istepen o{te}enja u zavisnosti od duìne radnog staà:grupa radnika podeljena je na: I grupa ’ 38 radnikasa staòm do 10 godina; II grupa ’ 47 radnika sastaòm do 20 godina i III grupa ’ 38 radnika sa sta-òm preko 20 godina. Nivo hormona T3 i TSH ispiti-van je MEIA metodom, a T4 FPIA metodom na apa-ratu ABBOTT IMx. U prvoj grupi srednje vrednostiiznosile su za T4=134,8 nmol/L, u drugoj T4=128,4nmol/L, a u tre}oj T4=141,0 nmol/L. Srednja vred-nost T4 za kontrolnu grupu iznosila je T4=120,6nmol/L. Za T3 srednje vrednosti bile su u prvoj grupiT3=2,15 nmol/L, u drugoj T3=1,98 nmol/L i u tre}oj2,28 nmol/L. Srednja vrednost za T3 u kontrolnojgrupi iznosila je T3=1,90 nmol/L. Srednje vrednostiza TSH iznosile su u prvoj grupi TSH=3,7 mIJ/L, udrugoj TSH=3,32 mIJ/L. Rezultati su statisti~ki obra-|eni i nije na|ena statisti~ki zna~ajna korelacija u~es-talosti povi}enih vrednosti ni u jednoj od ovih grupau odnosu na kontrolnu grupu. Zna~i, s obzirom nanepostojanje ispitivane povezanosti, name}e se po-treba traènja drugih razloga za ve}u u~estalost obo-lelih.

there is a negative effect of stressful noise on thyroidgland and its activity, while a patient is engaged atwork. The aim of the study was to evaluate effects ofprofesional damage on the organism of exposedworkers, especially on thyroid gland, in dependenceon duration of exposure to this efects. We examined200 patients, 125 were exposed workers and 75 werecontrol subjects. We were interested in level of da-mage depending on duration of exposure, thus weewamined three groups of patients: I group ’ 38workers who have been exposed for ten years; IIgroup ’ 47 workers who have been exposed for 20years; III group ’ 38 workwrs who have been exposedfor more than 20 years. We studied the level of thy-roid hormons T3 and TSH with MEIA method, T4 withFPIA method with ABBOTT IMx. Results are statisti-caly presented. In I group middle value is forT4=134.8 nmol/L, middle value for II group forT4=128.4 nmol/L and in III group T4=141.0 nmol/L.Middle value for T4 in control group is 120.6 nmol/L.Middle value for T3 in I group is T3=2.28 nmol/L.Middle value for T3 in control group is T3=1.90nmol/L. Middle value for TSH in I group is TSH=3.7mIJ/L. Middle value in II group is TSH=3.32 mIJ/L andin III group is TSH=2.98 mIJ/L. Middle value for con-trol group is TSH=3.30 mIJ/L. There are no statisti-cally significant in any of the groups with respect tothe control group. We must examine other reasonsfor such a huge frequency of diseased, becouse thereis no studied connection.

C39

POVEZANOST TE@INE ISHEMIJSKEBOLESTI SRCA SA KONCENTRACIJOM

LIPOPROTEINA (a)

S. Vujani}, D. Din~i}, R. Nikolajevi}

Instut za medicinsku biohemiju, Klinika za urgentnu internu

medicinu, Vojnomedicinska akademija, Beograd

Lipoprotein (a) ’ Lp(a) pored LDL ~estice pred-stavlja najaterogeniju lipoproteinsku frakciju u krvi injegove visoke koncentracije kombinovane sa dru-gim faktorima rizika jako pove}avaju rizik za nastanakishemijske bolesti srca (IBS). Cilj ovog istraìvanja bioje da se utvrdi da li postoji povezanost teìne IBS sakoncentracijom Lp(a). U na{e istraìvanje bilo jeuklju~eno 166 ispitanika oba pola u dobi od 40 do 74godine koji su bili podeljeni u 3 osnovne grupe: kon-trolna grupa (K0) zdravih (n=59, neinvazivnim meto-dama isklju~eno postojanje IBS), kontrolna grupa(K1) bolesnih (n=34, bez zna~ajnih stenoza krvnihsudova srca), eksperimentalna grupa (E) bolesnih(n=73, sa stenozama krvnih sudova srca >50%); ovagrupa podeljena je na 3 podgrupe: E1-jednosudov-nom, E2-dvosudovnom, E3-trisudovnom koronar-nom bole{}u. Koncentracija Lp(a) je odre|ivana usveèm izolovanom materijalu-serumu, nefelometrij-skom metodom na aparatu BN100 firme Dade Be-hring. Dobijeni rezultati pokazuju da je koncentracijaLp(a) (log. koncentracija Lp(a)) bila statisti~ki zna-~ajno pove}ana u E-grupi bolesnih u odnosu na K0 iK1 grupu i u E3-podgrupi u odnosu na E1 i E2 pod-grupu. Nema statisti~ki zna~ajne razlike u koncen-traciji Lp(a) izme|u K0 i K1 grupe i izme|u E1 i E2podgrupe. Na osnovu dobijenih rezultata moè se za-klju~iti da je u ovim istraìvanjima dobijena poveza-nost koncentracije Lp(a) sa teìnom IBS, odnosnobrojem obolelih krvnih sudova.

C39

CORRELATION OF THE ISCHAEMIC HEARTDISEASE SERIOUSNESS

WITH LIPOPROTEIN (a)

S. Vujani}, D. Din~i}, R. Nikolajevi}

Intitute of Medical Biochemistry, Clinic for Urgent Internal MedicineMilitary Medical Academy, Belgrade

Lipoprotein (a) ’ Lp(a), in addition to the LDL par-ticle, represents the most atherogenic lipoproteinfraction in blood and its high concentrations com-bined with other risk factors significantly increase arisk of the ischaemic heart disease (IHD). A target ofthis study was to determine if there were a correlationbetween the IHD seriousness and Lp(a) concentra-tions. This study included 166 male and female indi-viduals from 40 to 74 years of age who were dividedinto three major groups: control group (K0) of heal-thy individuals, n=59 (presence of IHD was excludedby using non-invasive methods), control group (K1)of sick individuals, n=34 (without significant stenosisof heart blood vessels), experimental group (E) of sickindividuals, n=73 (with stenosis of heart blood ves-sels > 50%) and it was divided into three sub-groups:E1 ’ one-vascular, E2 ’ two-vascular, E3 ’ three-vas-cular coronary heart disease. Lp(a) concentrationswere determined in a newly isolated material ’ serumby a nephelometric method using a Dade BehringBN100 analyser. Results obtained indicated thatLp(a) concentrations /log.Lp(a) concentractions/ we-re statistically significantly increased in the E-group ofsick individuals in relation to the K0 and K1 groups,and in the E3-sub-group in relation to the E1 and E2sub-groups. There was no statistical evidence of asignificant difference in Lp(a) concentrations betweenthe K0 and K1 groups and the E1 and E2 sub-groups. On the basis of the obtained results, we canconclude that our study has found the correlation ofLp(a) concentrations with the IHD seriousness, i.e.the number of blood vessels affected by the disease.


CLIPIDI, LIPOPROTEINI I APOLIPOPROTEINI

LIPIDS, LIPOPREOTEINS AND APOLIPOPROTEINS


C40

EFEKAT SIMVASTATINA NA LIPIDNI STATUS

J. Vasi}, N. Bogavac-Stanojevi}, V. Spasojevi}-Kalimanovska, S. Spasi}, I. ]e}ez

Institut za medicinsku biohemiju, Farmaceutski fakultet, Beograd

Primena simvastatina, inhibitora regulatornog en-zima HMG-CoA-reduktaze u biosintezi holesterola, odizuzetnog je zna~aja za sniàvanje visokih vrednostiholesterola i LDL-holesterola. U okviru ovog radaobra|eni su rezultati multicentri~ne studije ’ Zocor®

Monitored Treatment Program koju je sprovela firmaMSD u na{oj zemlji. Pacijenti sa dislipidemijom iklini~kim simptomima koronarne bolesti (132 paci-jenta, 58 mu{karaca i 74 ène) tretirani su sa simvas-tatinom u dozi od 10 i 20 mg. Efekat statina na lipid-ni status ispitivan je odre|ivanjem ukupnog holes-terola (uH), HDL-holesterola (HDL-H), LDL-holes-terola (LDL-H) i triglicerida (TG) pre po~etka terapijei posle 4, 8 i 12 nedelja. Ispitivanja su pokazala zna-~ajno sniènje vrednosti LDL-H od bazalnih 5,21 ±1,40 mmol/L do 3,15 ± 1,04 mmol/L (p<0,001) po-sle 3 meseca. Procentualne promene lipidnih para-metara posle prvog, drugog i tre}eg meseca u odno-su na bazalne vrednosti najve}e su za LDL-H i iznose:’22,41%, ’33,20% i ’37,67%. Tako|e je dobijenozna~ajno sniènje od 7,71 ± 1,47 mmol/L do 5,54 ±1,18 mmol/L posle 3 meseca terapije (p<0,001), aprocentualno sniènje vrednosti po mesecima bilo je:-32,03%, -25,40 i -27,10%. Simvastatin deluje i nasniènje TG (3,29 ± 2,44 mmol/L do 2,14 ± 1,24mmol/L, p<0,001), procentualna promena bila jemanja u odnosu na holesterol (’15,75%, ’21,45% i’25,76%). Za HDL-H dobijen je porast vrednosti odpo~etnih 1,00 ± 0,26 mmol/L do 1,2 ± 0,25 mmol/L(p<0,001). Procentualni porast HDL-H po mesecimaiznosio je: 9,57%, 20,39% i 21,41 % i statisti~ki sezna~ajno razlikovao za tre}i mesec (p<0,05). Efekatsimvastatina ispitivan je i na osnovu promene rizi~nihvrednosti lipidnih parametara kod ispitivanih pacije-nata. Pre primene terapije 89,4% pacijenata imalo jerizi~ne vrednosti uH (>6,3 mmol/L), a posle 3 mese-ca terapije samo 23,9%. Na po~etku ispitivanja 79,4%pacijenata imalo je rizi~ne vrednosti LDL-H (>4,1mmol/L), da bi se kasnije taj broj pacijenata smanjiona 15,8%. Utvr|eno je da simvastatin ima zna~ajanefekat na sniàvanje LDL-H, a umeren na porastHDL-H, {to omogu}ava primenu ovog leka u pri-marnoj i sekundarnoj prevenciji kardiovaskularnihoboljenja.

C40

EFFECT OF SIMVASTATIN ON LIPID PARAMETERS

J. Vasi}, N. Bogavac-Stanojevi}, V. Spasojevi}-Kalimanovska, S. Spasi}, I. ]e}ez

Institute of Medical Biochemistry, University School of Pharmacy, Belgrade

Treatment with simvastatin, which inhibits HMG-CoA reductase ’ the rate limiting enzyme step in cho-lesterol biosynthesis ’ has become the most suc-cessful drug treatment in lowering total plasma cho-lesterol and LDL-cholesterol concentrations. Wepresent the results of the multicentric study-Zocor®

Monitored Treatment Program (MSD) for our coun-try. Patients with dyslipidemia and clinical symptomsfor coronary artery disease (132 patients, 58 malesand 74 females) were treated with simvastatin atdoses of either 10 or 20 mg. Effect of simvastatin onlipid status was evaluated by determination of totalcholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C) and triglycerides (TG) at thebeginning and after 4, 8 and 12 weeks of treatment.Simvastatin therapy results in lower LDL-C levelsfrom 5.21 ± 1.40 mmol/L to 3.15 ± 1.04 mmol/L(p<0.001) after 3 months. Percent change of lipidparameters after one, two or three months from thebaseline were the greatest for LDL-C: ’22.41%,’33.20% and ’37.67%. Total cholesterol was alsoreduced from 7.71 ± 1.47 mmol/L to 5.54 ± 1.18mmol/L (p<0.001) and percent change was:’32.03%, ’25.40% and ’27.10%. Similar resultswere obtained for TG from 3.29 ± 2.44 mmol/L to2.14 ± 1.24 mmol/L after three months of treatment(p<0.001), but percent change was lower than forcholesterol (’15.75%, ’21.45% and ’25.76%). Incontrast to other lipid parameters, HDL-C raised aftertreatment with simvastatin from 1.00 ± 0.26 mmol/Lto 1.20 ± 0.25 mmol/L (p<0.001). Percent change inHDL-C was: 9.57%, 20.39% and 21.41% and thesechange was statistically significant (p<0.05). Thereducing effect of simvastatin on TC and LDL-C wasanalyzed in view of the percent of subjects with high-risk values of cholesterol. Before treatment most ofthe patients (89.4%) were with high-risk values for TC(>6.3 mmol/L) and after 3 months the percent wasonly 23.9%. Similar results were obtained for LDL-C.With high-risk values (>4.1 mmo/L) were 79.4% ofpatients and after treatment with simvastatin 15.8%.The hypolimidemic effect of simvastatin is principallybased on reduction of LDL-C and that is major con-cern in both primary and secondary prevention of co-ronary heart disease.

C41

FAKTORI RIZIKA ZA ATEROSKLEROZU KOD PACIJENATA S ESENCIJALNOM

HIPERTENZIJOM

J. Ili}1, J. Kotur-Stevuljevi}2, N. Bogavac-Stanojevi}2,

V. Spasojevi}-Kalimanovska2

1Klini~ko-biohemijska laboratorija, Zdravstveni centar Sveti Luka, Smederevo

2Institut za medicinsku biohemiju, Farmaceutski fakultet, Beograd

Hipertenzija je jedan od klju~nih faktora rizika zarazvoj ateroskleroze pa i kardiovaskularnih bolesti.Pored pu{enja, gojaznosti i dislipidemije hipertenzijaspada u varijabilne faktore rizika na koje se moè de-lovati preventivno i tako umanjiti rizik od aterosklero-ze. S tim ciljem ispitivan je i aterogeni rizik kod paci-jenata s esencijalnom hipertenzijom odre|ivanjemukupnog holesterola (uH), HDL-holesterola (HDL-H),LDL-holesterola (LDL-H) i triglicerida (TG) i izra~una-vanjem odnosa: uH/HDL-H i LDL-H/HDL-H. Pore-me}aji lipidnog statusa kod pacijenata s esencijal-nom hipertenzijom ispitivani su u grupi od 185 osoba(82 ène i 103 mu{karca) i pore|enjem sa kontrol-nom grupom od 97 osoba (61 èna i 36 mu{karaca)bez hipertenzije. Lipidni parametri u serumu odre-|ivani su standarnim biohemijskim metodama. Onisu upore|eni kod normotenzivnih i hipertenzivnihosoba i u zavisnosti od pu{enja i BMI. Pore|enjemlipidnih parametara pacijenata s hipertenzijom uodnosu na kontrolnu grupu dobijena je statisti~ki zna-~ajna razlika za sve parametre (p<0,001, Student’ovt-test). Pacijenti sa hipertenzijom imali su vi{e vred-nosti u odnosu na kontrolu: uH (6,24 ± 2,32 vs 5,44± 2,09 mmol/L), LDL-H (4,30 ± 2,07 vs 3,70 ± 1,81mmol/L), TG (2,68 ± 4,35 vs 1,43 ± 1,62 mmol/L),odnos uH/HDL-H (6,72 ± 4,85 vs 4,72 ± 2,07) i LDL-H/HDL-H (4,63 ± 3,41 vs 3,19 ± 1,82), a niè vred-nosti HDL-H (1,01 ± 0,58 vs 1,20 ± 0,54 mmol/L).Lipidni parametri za pacijente i kontrolnu grupu ispi-tivani su posebno za mu{karce i ène i dobijeni susli~ni rezultati. Ispitivanja su pokazala i statisti~kizna~ajnu razliku izme|u pacijenata i kontrole za svelipidne parametre kod pu{a~a (p<0,001) i to za obapola. Pacijenti sa hipertenzijom i BMI od 25 do 30kg/m2 statisti~ki se zna~ajno razlikuju u odnosu naodgovaraju}u kontrolnu grupu za TG, uH/HDL-H,LDL-H/HDL-H (p<0,001), za LDL-H (p<0,01) i zauH i HDL-H (p<0,05). Dobijeni rezultati ukazuju nazna~ajan poreme}aj u metabolizmu lipida kod pacije-nata s hipertenzijom i na interakciju drugih faktorarizika za aterosklerozu, a pre svega na uticaj pu{enja.

C41

ATHEROGENIC RISK FACTORS IN PATIENTS WITH ESSENTIAL

HYPERTENSION

J. Ili}1, J. Kotur-Stevuljevi}2, N. Bogavac-Stanojevi}2,

V. Spasojevi}-Kalimanovska2

1Clinical Chemistry Laboratory, Svetog Luke, Health Centre, Smederevo

2Institute of Medical Biochemistry, University School of Pharmacy, Belgrade

Objective of the present study was to test thehypothesis that hypertension, smoking, obesity anddyslipidemia are major variable factors of atheroscle-rosis disease. The atherogenic risk for hypertensivepatients was assessed by measuring total cholesterol(TC), triglycerides (TG), HDL-cholesterol (HDL-C)and LDL-cholesterol (LDL-C) and calculating theratio: TC/HDL-C and LDL-C/HDL-C. The examinedgroup consisted of 185 hypertensive patients (82females and 103 males) and a control group of nor-motensive individuals 97 (61 females and 36 males)matched by age and sex. Serum lipid parameterswere examined by standard biochemical methods.Levels were also compared in normotensive andhypertensive individuals according to smoking andBMI. All lipid parameters in hypertensive patientswere statistically different compared with the controlgroup (p<0.001, Student t-test). Hypertensive pa-tients compared with normotensive had higher valuesof TC (6.24 ± 2.32 vs 5.44 ± 2.09 mmol/L), LDL-C(4.30 ± 2.07 vs 3.70 ± 1.81 mmol/L), TG (2.68 ± 4.35vs 1.43 ± 1.62 mmol/L), ratio TC/HDL-C (6.72 ± 4.85vs 4.72 ± 2.07) and LDL-C/HDL-C (4.63 ± 3.41 vs3.19 ± 1.82) and lower values of HDL-C (1.01 ± 0.58vs 1.20 ± 0.54 mmol/L). Similar results were obtainedseparately for women and men. Lipid levels in smok-ing normotensive and hypertensive groups were sig-nificantly different (p<0.001) for both sex. Lipid dis-turbance in hypertensive patients with BMI (25’30kg/m2) was statistically significant difference com-pared with control group for TG, TC/HDL-C, LDL-C/HDL-C (p<0.001), for LDL-C (p<0.01) and for TC,HDL-C (p<0.05). We conclude that disturbances ofplasma lipid profile in hypertensive patients may bemainly due to an interaction with smoking and withan additional effect of other risk factors.



C42

ME\UODNOS LIPIDNIH PARAMETARA I INDEKSA TELESNE MASE

KOD [KOLSKE DECE

M. Babi}, D. Pap, A. Antonijevi}

Zdravstveni centar Uìce, Dom zdravlja Poèga,Zdravstveni centar S. Mitrovica

Ukupni holesterol (TC), HDL-holesterol (HDL-C),LDL-holesterol (LDL-C) i trigliceridi (TG), odre|eni suu serumima 68 u~enika starosti 14 godina iz Poègei Sremske Mitrovice. Utvr|eni su odnosi TC, HDL-C,LDL-C i TG u zavisnosti od porasta indeksa telesnemase (BMI). Ispitanici su podeljeni u tri grupe u zavis-nosti od BMI (Grupa 1: BMI< 25 kg/m2; grupa 2: BMI25’28 kg/m2; grupa 3: BMI > 28 kg/m2. KoncetracijeTC i TG odre|ene su enzimskim metodama. HDLholesterol odre|en je enzimskom metodom u super-natantu koji je dobijen posle precipitacije drugih li-poproteina sa fosfovolframovom kiselinom i magne-zijum hloridom. LDL-C dobijen je ra~unskim putempomo}u Friedewaldove jedna~ine. Rezultati su sta-tisti~ki obra|eni i grafi~ki prikazani. Rezultati lipidnihparametara (srednja vrednost “ 2 SD) u odgovara-ju}im grupama prema indeksu telesne mase iznosilisu: za holesterol (grupa 1: 4,25 “ 1,22 mmol/L; gru-pa 2: 5,96 “ 2,02 mmol/L; grupa 3: 6,23 “ 2,52mmol/L); za LDL-holesterol (grupa 1: 3,20 “ 1,80mmol/L; grupa 2: 4,14 “ 2,20 mmol/L; grupa 3: 4,34“ 2,96 mmol/L); za HDL-holesterol (grupa 1: 1,51 “0,64 mmol/L; grupa 2: 1,49 “ 0,70 mmol/L; grupa3: 1,48 “ 0,72 mmol/L); i za trigliceride (grupa 1:0,90 “ 0,88 mmol/L; grupa 2: 1,90 “ 1,44 mmol/L;grupa 3: 2,56 “ 1,82 mmol/L). Rezultati ispitivanjapokazali su da izme|u BMI i TC, LDL-C i TG postojistatisti~ki zna~ajna razlika (p < 0,05), dok ista nijeutvr|ena izme|u BMI i HDL-C (p > 0,05). Sa poras-tom BMI rasle su i vrednosti ukupnog holesterola,LDL-holesterola i triglicerida. Pove}ane vrednosti na-vedenih aterogenih lipida predstavljaju pove}an kar-diovaskularni rizik kod dece starosti 14 godina.

C42

CORRELATION OF LIPIDS PARAMETERSAND BODY MASS INDEX IN SCHOOL CHILDREN

M. Babi}, D. Pap, A. Antonijevi},

Medicale Centre Uìce, Helth Centre Poèga, Medical Centre Sremska Mitrovica

The total cholesterol (TC), high density lipopro-tein cholesterol (HDL-C), low density cholesterol(LDL-C) and triglycerides (TG) level were determinedin serum samples of 68 children from Poèga andSremska Mitrovica, aged 14 years. The relationsbetween TC, HDL-C, LDL-C and TG, and increase inbody mass index (BMI) were examined. Participantswere divided into three groups according to BMI(Group 1: BMI< 25 kg/m2; group 2: BMI 25’28kg/m2; group 3: BMI > 28 kg/m2. Concentrations ofTC i TG were determinated by enzymatic colorimet-ric method, as well as HDL-C in supernatant afterpercipitation of other lipoproteins with addition ofphosphotungsten acid and MgCl2. LDL-C was calcu-lated using the Friedewald formula. The results werestored in a computer, processed statistically and pre-sented graphically. The overall fasting values (mean“ 2 SD) in the defined groups according to BMIwere: for cholesterol (group 1: 4,25 “ 1,22 mmol/L;group 2: 5,96 “ 2,02 mmol/L; group 3: 6,23 “ 2,52mmol/L); for LDL-cholesterol (group 1: 3,20 “ 1,80mmol/L; group 2: 4,14 “ 2,20 mmol/L; group 3:4,34 “ 2,96 mmol/L); for HDL-cholesterol (group 1:1,51 “ 0,64 mmol/L; group 2: 1,49 “ 0,70 mmol/L;group 3: 1,48 “ 0,72 mmol/L); and for triglycerides(group 1: 0,90 “ 0,88 mmol/L; group 2: 1,90 “ 1,44mmol/L; group 3: 2,56 “ 1,82 mmol/L). A statisti-cally significant dependance between BMI and TC,LDL-C and TG was established (p < 0,05), while theinterrelation between BMI and HDL-C was not signif-icant (p > 0,05). The mean values for cholesterol,lLDL-cholesterol and triglycerides were increased incorrelation with increased BMI. The concetrations oftotal cholesterol, LDL-cholesterol and triglyceridesmay be a sign of the present cardiovascular risk inchildren aged 14 years.


C43

APOLIPOPROTEIN B U PROCENIATEROGENOG RIZIKA LIPIDSKOG

POREKLA KOD OSOBA S GRANI^NOPOVI[ENIM NIVOIMA UKUPNOG HOLESTEROLA I TRIGLICERIDA

O. Brklja~, Lj. Lepanovi}, M. \eri}

Zavod za laboratorijsku medicinu, Klini~ki centar Novi Sad, Novi Sad

Smatra se da je apolipoprotein B (apo) pouzdani-ji parametar za procenu aterogenog rizika nego LDL-H (H=holesterol), budu}i da je koncentracija apo Bekvivalentna broju aterogenih lipoproteinskih ~estica,dok sadràj H u jednoj partikuli moè zna~ajno vari-rati. Ova ~injenica bila bi od posebnog klini~kogzna~aja kod osoba s poèljnim vrednostima LDL-H(do 3,5 mmol/L), uz grani~no povi{ene nivoe uku-pnog H (5,2’6,5 mmol/L) i triglicerida (TG) (1,7’2,3mmol/L). U ovoj studiji izvr{ena je procena zna~ajaodre|ivanja apo B kod osoba s poèljnim nivoimaLDL-H, 100 s normotrigliceridemijom i grani~no po-vi{enim ukupnim H i 37 s istovremenim grani~nimpovi{enjem ukupnog H i TG. Kontrolnu grupu ~iniloje 50 normolipidemi~nih osoba. Parametri su odre|-ivani standardnim biohemijskim metodama. Rezultatipokazuju da je apo B (g/L) zna~ajno povi{en i kodosoba s grani~nim povi{enjem ukupnog H (0,82 ±0,14) i kod osoba s grani~nim povi{enjem ukupnogH i TG (0,89 ± 0,11). U obe grupe registrovano je izna~ajno povi{enje Lp(a) lipoproteina (g/L) (0,19 ±0,16; 0,21 ± 0,20). Dobijeni rezultati ukazuju da jeapo B pouzdaniji parametar za procenu aterogenogrizika kod osoba s grani~no povi{enim nivoima ukup-nog H i TG, nego LDL-H, ~iji bi porast mogao, tako-|e, da uputi na istovremeno povi{enje nivoa ateroge-nog lipoproteina Lp(a).

C44

FOSFOLIPIDSKI SASTAV SUBCELULARNIH ORGANELA

K. Bajin Kati}, Z. Kova~evi}

Institut za biohemiju, Medicinski fakultet, Novi Sad

Cilj rada bio je da se izvr{i analiza fosfolipidnogprofila pojedinih subcelularnih organela jetre pacova.Organele su izolovane metodom diferencijalnog cen-trifugiranja, a pojedine frakcije fosfolipida analiziranesu pomo}u originalne metode dvodimenzionalne hro-matografije na tankom sloju i odre|ivanjem fosfora.

C43

APOLIPROTEIN B IN ESTIMATION OF ATHEROGENIC RISK LIPIDAL

ORIGIN IN PERSONS WITH BORDERLINEHIGH CONCENTRATIONS OF TOTAL CHOLESTEROL AND TRIGLYCERIDES

O. Brklja~, Lj. Lep{anovi}, M. \eri}

Institute for Laboratory MedicineClinical Center Novi Sad, Novi Sad

Opinion that apoliprotein B (apo) is more reliableparameter for estimation atherogenic risk than LDL-H (H=cholesterol), as the concentration of apo B isequivalent to the number of atherogenic lipoproteinparticles, while contents of H in one particle may varysignificantly. This fact could be of special clinicalimportance for the persons with desirable concentra-tions of LDL-H (under 3.5 mmol/l) and borderlinehigh levels of H (5.2’6.5 mmol/L) and triglycerides(TG) (1.7’2.3 mmol/L). In this study estimation ofapo B determination importance is done in personswith desirable levels of LDL-H, 100 normotriglyceri-demic with borderline high levels of total H and 37with simultaneous borderline high levels of total Hand TG. Control groupe consisted 50 normolipidem-ic persons. Parameters were measured by standardbiochemical methods. Results show that apo B (g/L)is significantly increased as well in persons with bor-derline high levels of total H (0.82 ± 0.14) and in per-sons with borderline high levels of total H and TG(0.89 ± 0.11). In both groups significant increase ofLP (a) lipoprotein (g/L) (0.19 ± 0.16; 0.21 ± 0.20) reg-istrated. Our results speak in favor of apo B as morereliable parameter for estimation of atherogenic riskfor the persons with borderline high levels of total Hand TG than LDL-H, which increase may as well referto simultaneous increase of atherogenic lipoproteinLp (a) levels.

C44

PHOSPHOLIPID COMPOSITION OF SUBCELLULAR ORGANELLES

K. Bajin Kati}, Z. Kovacevi~

Institute of Biochemistry, Medical Faculty Novi Sad

The aim of the study has been to analyze theprofile of phospholipides of some subcellular orga-nelles in a rat liver. Organelles were isolated by thedifferential centrifugation method and the fractions ofphospholipides were analyzed by the method of two-dimensional chromatography on a thin layer and by


phosphorus determination. The analysis of a phos-pholipid core fraction has shown that the highest per-centage belongs to phosphatidylcholine (40%) andphosphatidylethanolamine (30%). The highest per-centage of cardiolipin has been found in mitochondr-ial fraction which is in accordance with the fact thatthis phospholipid is located exclusively in the innermembrane of the organelles. Lysosomal fraction ischaracterized by substantial reduction in cardiolipinand, compared with other fractions, high percentageof phosphatidylinositol. In microsomal fraction thehighest percentage of lysolecithin has been recordedwhich is probably the result of increased activity ofphospholipase A. The obtained differences in phos-pholipid composition of membranes of subcellularorganelles indicate that they differ in their physical,chemical and biological characteristics. The resultssupport the difference in biological features of thecellular membranes such as enzyme profile and activ-ity, transport processes and the mechanisms of ener-gy transduction and transformations.

C45

OXYDATIVE CELL DEMAGE; ACUTE ISHEMIC PROCESS

AND ITS MODEL

R. Mihajlovi}1, M. Jovanovi}2, S. Vidakovi}1, A. Jelenkovi}2, A. Jovi~i}3

1Institute for Rehabilitation, Belgrade 2Institute for Medicial Research, and

3Clinic for Neurology, Military Medical Academy,Belgrade

Ishemia is shape of hipoxy a which shows in vivowhen blood flow is interupted in specific regions(heart, brain). Interupted of tissue supplying with oxy-gen is reason of patological changes. After ishemiacell becomes specially sensitive on reoxygenation,which cause oxydative damage during reperfusion.Partial oxygen pressure, in that moment, is increasedin comparation with ishemic tissue parts which givespossibilities of moving whole seria of step reactionswhich results with creation of toxical metabolites. Ongreat number of models ishemia heart and brain ofanimals had shown that become in creation of lipidperoxides, respectively to damage and destruction ofcell membrane. The aim of our project is to show thatstage of tissue hipoxy a cause with spending time ofinquires in hipobaric room is reason for moving ofpatophysiological changes same or similar as instage of heart and brain ishemia. Range of lipid per-oxide (LP) is measured in blood plasma of pilots(n=12) before (control) and 20 min, after half an

Analiza fosfolipidne jedarne frakcije pokazuje danajve}i procenat pripada fosfatidilholinu (40%) i fos-fatidiletanolaminu (30%). Najve}i procenat kardioli-pina na|en je u mitohondrijalnoj frakciji {to je uskladu sa ~injenicom da ovaj fosfolipid ulazi isklju~ivou sastav unutra{nje membrane organela. Lizozomal-nu frakciju karakteri{e izrazito smanjenje kardiolipinai vi{a koncentracija fosfatidilinozitola nego u svimostalim frakcijama. U mikrozomalnoj frakciji zabele-èn je najve}i procenat lizolecitina {to je verovatnorezultat poja~ane aktivnosti fosfolipaze A. Dobijenerazlike u fosfolipidskom sastavu membrana pojedinihsubcelularnih organela ukazuju da se one razikuju posvojim fizi~ko-hemijskim i biolo{kim karakteristika-ma. Rezultati obja{njavaju razliku u biolo{kim osobi-nama pojedinih celularnih membrana kao {to su pro-fil i aktivnost enzima, transportni procesi i mehanizmiprenosa i transformacije energije.

C45

OKSIDATIVNO O[TE]ENJE ]ELIJE; AKUTNI ISHEMI^NI DOGA\AJ

I NJEGOV MODEL

R. Mihajlovi}1, M. Jovanovi}2, S. Vidakovi}1, A. Jelenkovi}2, A. Jovi~i}3

1Insitut za rehabilitaciju 2Institut za medicinska istraìvanja VMA3Klinika za neurologiju VMA, Beograd

Ishemija je oblik hipoksije koji, in vivo, nastajekada je prekinut dotok krvi u odre|eni region (srce,mozak). Prekid snabdevanja tkiva kiseonikom uzrokje nastalih patolo{kih promena. Posle ishemije }elijapostaje posebno osetljiva na reoksigenaciju, {to imaza posledicu nastanak oksidativnog o{te}enja u tokureperfuzije. Parcijalni pritisak kiseonika, u ovom mo-mentu, znatno je pove}an u odnosu na ishemi~nedelove tkiva {to otvara mogu}nost pokretanja ~itaveserije lan~anih reakija koje za rezultat imaju stvaranjetoksi~nih metabolita. Na velikom broju modela ishe-mije srca i mozga, na ìvotinjama, pokazano je da do-lazi do stvaranja lipidnih peroksida, odnosno o{te}e-nja i destrukcije }elijskih membrana. Cilj ovog radabio je da se pokaè da stanje hipoksije tkiva, izazvanoboravkom ispitanika u hipobari~noj komori dovodido pokretanja patofoziolo{kih promena sli~nih/istihkao i u stanjima ishemije mozga i srca. Meren je nivolipidnih peroksida (LP) u plazmi pilota (n=12) pre(kontrola) i 20 min posle polu~asovnog boravka u hi-pobari~noj komori. Drugu grupu sa~injavali su paci-


jenti (n=45) sa ishemijom srca. Kontrolna grupa sas-tojala se od 23 dobrovoljna davaoca krvi. Slede}aispitivana grupa (n=45) bili su bolesnici sa ishemijommozga. Kontrola su bili pacijenti (n=20) sa drugimneurolo{kim oboljenjima, bez medikamentozne ter-apije. Pacijenti sa ishemijom srca i mozga kao i ispi-tanici koji su boravili u hipobari~noj komori pokazujusli~an porast nivoa lipidnih peroksida u plazmi uodnosu na kontrolne grupe. Najve}i porast nivoa LPimali su oboleli od ishemije srca (198%), zatim odinfarkta mozga (178%) i na kraju u plazmi ispitanihpilota (164%). Ovakav trend rasta LP, kao indikatorao{te}enja }elijske membrane moè da se objasni~injenicom da kod ishemije srca lipidni peroksididirektno dospevaju u cirkulaciju, bez prolaska hema-to-encefalne barijere, {to je slu~aj sa ishemijommozga. Tako|e, na primeru ispitanih pilota pokazanoje da ishemija dovodi do oksidativne povrede tkiva in-dukovane hipoksijom, pri ~emu je stepen povredepove}an prelaskom iz stanja hipoksije u stanje nor-moksije tkiva (ali u relativnom odnosu hiperoksije).

C46

INTENZITET LIPIDNE PEROKSIDACIJE KOD ZDRAVIH OSOBA I INSULIN

ZAVISNIH DIJABETIÂRA

A. Kozi}1, M. Popovi}2, K. \akovi}3, B. Mari}4

1Dom zdravlja Dr Milorad Mika Pavlovi}, In|ija2Institut za hemiju, PMF Novi Sad

3Zavod za farmakologiju i toksikologiju, Medicinski fakultet, Novi Sad4AD Jugoinspekt, Novi Sad

Peroksidacija lipida je oksidativna degradacijanezasi}enih masnih kiselina, koje ulaze u sastav lipi-da. Ona ima zna~ajnu ulogu u patogenezi mnogihbolesti kao {to su Alchajmerova bolest, dijabetes,Parkinsonova bolest i dr. Cilj ovog rada bio je da seodredi intenzitet peroksidacije lipida (LPx), kod zdrav-ih ljudi oba pola i dijabeti~ara (insulin zavisnih) sta-rosti izme|u 20 i 75 godina i korelacija ovih para-metara u odnosu na starost i dijabetes, u specifi~nimekolo{kim uslovima In|ije. Ovi rezultati su bitni zbogpore|enja sa svetskim rezultatima ura|enim u sli~-nim ili razli~itim ekolo{kim uslovima. Izloèna ispiti-vanja obuhvatila su 200 zdravih ljudi, starosti od 20do 75 godina i 80 ljudi obolelih od diabetes mellitus-a (tip I) starosti izme|u 20 i 60 godina. Zdravstvenostanje ispitanika proveravano je odre|ivanjem rutin-skih laboratorijskih parametara (sedimentacija, krvnaslika, glukoza, urea i transaminaze). Svaka grupaobuhvatila je dvadeset ispitanika, sa pribli`no istim

hour spent time in hiperbaric room. The secondgroup consists of heart ishemia patients (n=45).Control group consists of 23 voluntary blood givers.Next inquire group (n=45) were patients with brainishemia. Control are patients (n=20) with other neu-rological desesase without medicament therapy.Patients with heart and brain ishemia and patientswho were in hipobaric room show similar increase ofrange of lipid peroxide in blood plasma in compari-son with control groups. The highest increase ofrange LP is shown on patients with heart ishemia(198%), after them come patients with brain attack(178%) and at the end are inquired pilots (164%).This increased trend of LP as indicator of damagecell membrane can be explained with fact that heartishemia lipid peroxides are directly come to blood cir-culation without passing hemato-encephal barier,which is the case with brain ishemia. Also, on theexample on iquired pilots we can say that ishemiacome to oxydative tissue damages induces withhipoxy and then the level of damage is increased withcrossing from stage of hipoxy a to stage of normoxytissue (but in relative relation hiperoxy).

C46

INTENSITY OF LIPID PEROXIDATION IN HEALTHY SUBJECTS

AND INSULIN-DEPENDENT DIABETICS

A. Kozi}1, M. Popovi}2, K. \akovi}3, B. Mari}4

1Health Centre Dr Milorad Mika Pavlovic, In|ija2Faculty of Sciences, Institute of Chemistry, Novi Sad

3Faculty of Medicine, Institute of Pharmacology and Toxicology, Novi Sad4AD Jugoinspekt, Novi Sad

Lipid peroxidation (LPx) represents an oxidativedegradation of unsaturated fatty acids, the basiccomponents of lipid molecules, and it has a veryimportant role in the aerobic cell pathology. Thisphenomenon is to a great extent involved in thepathogenesis of a number of diseases: Alzheimer dis-ease, diabetes, Parkinson disease, etc. The objectiveof this study was to determine the intensity of LPx inhealthy subjects and insulin-dependent diabetics,aged between 20 and 75 and correlate these param-eters to the age and diabetes in the specific ecologi-cal environment of Indjija. The intensity of LPx, bothin plasma and hemolysate of healthy subjects, in-creases with age. The LPx values obtained for healthyfemale subjects, measured in both plasma and he-molysate, show an increase with age, and are some-what lower compared with the corresponding valuesobserved with the healthy male. On comparing ourresults with those from the literature we can see that


brojem mu{karaca i èna. Intenzitet LPx odre|en jemetodom po Buege i Austu. Na|eno je da se inten-zitet peroksidacije lipida kod zdravih mu{kih osobapove}ava sa starenjem i u plazmi i u hemolizatu.Intenzitet LPx kod zdravih èna pove}ava se sastarenjem i u plazmi i u hemolizatu i ne{to je niì uodnosu na LPx kod zdravih mi{karaca. Pore|enjemdobijenih podataka sa podacima u literaturi, utvr|e-no je da je intenzitet lipidne peroksidacije vi{i i u pla-zmi i u hemolizatu kod zdravih osoba oba pola. Inten-zitet lipidne peroksidacije pove}ava se kod dijabeti-~ara oba pola sa staro{}u (i u hemolizatu i u plazmi),i to je pove}anje ve}e u odnosu na zdrave ispitanikeiste starosne grupe. Pore|enjem vrednosti odre|iva-nja LPx kod dijabeti~ara sa podacima iz literature do-bijeno je bolje slaganje nego u slu~aju pore|enjavrednosti zdravih osoba sa podacima u literaturi.

C47

PROMENE NIVOA LIPIDNE PEROKSIDACIJE U KRVNOJ PLAZMI

PACOVA TRETIRANIH 5-FLUOROURACILOM I SELENOM

O. Jozanov-Stankov1, M. Demajo1, I. \uji}2

1Institut za nuklearne nauke »Vin~a«, Beograd2IHTM ’ Centar za hemiju, Beograd

Ve}ina danas kori{}enih citostatika u terapiji ma-lignih oboljenja, ostvaruje svoje efekte bilo prekopodsticanja ili direktnim stvaranjem slobodnihradikala (SR), najvi{e veoma reaktivnih oblika kiseoni-ka, sa ciljem da zaustave produkciju malignog tkiva.Nespecifi~nost ovih reakcija, me|utim, poga|a po-red malignog i zdravo tkivo {to izaziva dodatno anga-òvanje antioksidativnog odbrambenog sistema uorganizmu, kao i promene parametara koji govore oprisustvu oksidativnog stresa. Cilj ovih istraìvanja bioje da se na zdravom eksperimentalnom pacovu ispi-ta koliko davanje dosta kori{}enog citostatika 5-fluo-rouracila (FU) uti~e na stepen peroksidacije lipida(LP) u krvi kao jednog od parametara negativnihefekata pove}ane produkcije SR. Slede}i zadatak bioje da se vidi da li pove}ano uno{enje mikroelementaselena (Se), kao va`nog u~esnika antioksidativneodbrane, uti~e na nivo LP u krvnoj plazmi usled tret-mana sa FU-om. @ivotinje su dobijale organski vezanselen (Se) u vodi za pi}e (u dozi koja odgovara oko100 mg/dan) mesec dana pre tretmana FU. FU jeaplikovan i.p. u 5 jednakih dnevnih doza (ukupnakoli~ina odgovara jednom uobi~ajenom terapijskomciklusu za ~oveka). Pacovi su `rtvovani osmog danaod po~etka davanja FU i u izdvojenoj krvnoj plazmiodre|ivan nivo LP, mere}i koncentraciju tzv. pro-dukata reakcije sa tiobarbiturnom kiselinom (TBK-P).Rezultati eksperimenta pokazali su da aplikovanje FU

our results, both for plasma and hemolysate, are re-gularly higher in healthy subjects of both sexes.Intensity of LPx in both plasma and hemolysate ofdiabetic patients increases with age and this increaseis higher compared with that observed with healthysubjects of the same age. The LPx values obtainedfor the diabetic patients correlate better with thosefound in the literature than the values obtained forhealthy subjects with the corresponding literaturedata.

C47

CHANGES IN THE LEVELS OF LIPID PEROXIDATION IN BLOOD PLASMA

OF RATS TREATED WITH 5-FLUOROURACIL AND SELENIUM

O. Jozanov-Stankov1, M. Demajo1, I. \uji}2

1Institute of Nuclear Sciences »Vin~a«, Belgrade2Institute of Chemistry, Technology and Metallurgy

’ Center of Chemistry, Belgrade, Yugoslavia

Most of the cytostatics used today in the therapyof malignant diseases express their effects eitherthrough inducing or by the direct production of freeradicals (SR), mostly very reactive oxygen specieswith the purpose to eliminate the production of ma-lignant tissue. As these reactions are non-specific,beside the malignant tissue, healthy tissue is alsoaffected, causing additional engagement of the anti-oxidant defense system in the organism as well asinducing changes in parameters which indicate pres-ence of oxidative stress. The purpose of this researchwas to examine how much the currently used cyto-static 5-fluorouracil (FU) injected into healthy experi-mental rats, influences the blood level of lipid perox-idation (LP), as one of the parameters demonstratingthe negative effects of increased SR production. Thenext task was to determine if an increased intake ofthe microelement selenium (Se), as an importantpart of the antioxidative defense system, has an effecton the level of LP in blood plasma after treatmentwith FU. The animals received organically bound Sein drinking water (dose which corresponds to about100 mg/day) for a month before treatment with FU.The cytostatic was injected i.p. in 5 equal daily doses(the total amount corresponds to one standard hu-man therapeutical cycle). The rats were sacrificed 8days after the start of FU injection and blood plasmalevels of LP were measured as the amount of the


izaziva zna~ajno pove}anje LP nivoa u plazmi eksper-imentalnog pacova. Pove}an unos Se snizio je bazal-ni nivo LP u plazmi a tako|e je redukovao porast LPpri tretmanu FU-om, {to ukazuje da bi dodavanje Sepri terapiji ovim citostatikom moglo biti od zna~aja uklini~koj praksi.

C48

DIJAGNOSTI^KI ZNAÂJ PRODUKATALIPIDNE PEROKSIDACIJE

U RAZLIKOVANJU PLEURALNIH IZLIVA

J. Lali}1, I. \or|evi}2, S. Stojiljkovi}1, L. Zvezdanovi}1, B. Blagojevi}2

1Centar za medicinsku biohemiju, Klini~ki centar, Ni{2Klinika za plu}ne bolesti i tuberkulozu,

Klini~ki centar, Ni{

Biohemijske analize pleuralne te~nosti prvi su koraku razlikovanju pleuralnih izliva (PI) na eksudate (E) itransudate (T). Mada se Light-ovi kriterijumi naj~e{}ekoriste za razdvajanje E od T, oni su manje ta~ni uidentifikaciji transudata. Zbog toga se, da bi se pobolj-{ala ta~nost dijagnoze, koriste novi parametri. Poznatoje da slobodni radikali kiseonika (SRK) prouzrokujuo{te}enja u mnogim biolo{kim tkivima. SRK ispolja-vaju svoj citotoksi~ni efekat uzrokuju}i lipidnu peroksi-daciju za koju se smatra da je odgovorna za eksudaci-ju te~nosti u pleuralni prostor. Shodno tome, cilj ovograda bio je odre|ivanje malondialdehida (MDA), kaokrajnjeg produkta lipidne peroksidacije u pleralnoj te~-nosti (MDAp) i serumu (MDAs) i odnosa MDAp/s; kao ipore|enje dobijenih rezultata s drugim dobro utvr|e-nim kriterijumima. Analizirano je 30 pacijenata sa PIkoji su Light-ovim kriterijumima bili klasifikovani kaoE(20) i T(10). MDA je odre|ivan spektrofotometrij-skom metodom sa TBA. Dobijeni rezultati su pokazalizna~ajno pove}anje nivoa MDAs kod obe grupe pacije-nata: E (7,27 ± 1,29 mmol/L), T (7,75 ± 0,89 mmol/L),u pore|enju sa kontrolnom grupom (5,10±0,87 mmol/L), p<0,001. Nivo MDAp u E (5,16 ± 1,28 mmol/L)bio je tako|e zna~ajno pove}an u odnosu na T (2,67 ±0,61 mmol/L) p<0,001. Sli~no je i odnos MDAp/s biozna~ajno ve}i u E (0,72 ± 0,17 mmol/L) nego u T (0,34± 0,05 mmol/L), p<0,001. Koriste}i grani~nu vrednostod (0,4) za MDAp/s odnos (senzitivnost 95% i speci-fi~nost 90%) uspe{no su odvojeni E od T. Na osnovuovih rezultata moè da se zaklju~i da odre|ivanje pro-dukata lipidne peroksidacije moè biti korisno u dijag-nozi-diferentovanju pleuralnih izliva na E i T, {to je odzna~aja za pravilno le~enje pacijenata sa PI.

thiobarbituric acid reactive substances (TBARS). Theexperimental results showed that treatment with FUinduces a significant increase of LP levels in the plas-ma of experimental rats. The increased intake of Selowered the basal level of plasma LP and it alsorestricted the increase of LP after treatment with FU,indicating that the supplementation of Se duringtherapy with this cytostatic may be of significance inclinical practice.

C48

DIAGNOSTIC SIGNIFICANCE OF LIPIDPEROXIDATION PRODUCTS

IN DIFFERENTIATION OF PLEURAL EFFUSIONS

J. Lali}1, I. \or|evi}2, S. Stojiljkovi}1, L. Zvezdanovi}1, B. Blagojevi}2

1Centre of Medical Biochemistry, Clinical Centre, Ni{2Clinic for Lung Diseases and Tuberculosis,

Clinical Centre, Ni{

Biochemical analyses of pleural fluid are the firststep in differentiation of pleural effusions (PE) intoexudates (E) and transudates (T). Although Light'scriteria have been used most commonly for the sep-aration of E from T, they are less accurate in identi-fying transudates. Therefore, new parameters havebeen used to improve the accuracy of diagnosis.Free oxygen radicals (FOR) are known to producedamage in many bilogical tissues. FOR exert theircytotoxic effect by causing lipid peroxidation which isbelived to be responsible for the exudation of fluidinto the pleural space. Based on this idea, the aim ofthis study was the determination of malondialdehyde(MDA), as the final product of lipid peroxidation inpleural fluid (MDAp) and in serum (MDAs) and pleu-ral fluid to serum MDA ratio (MDAp/s); and to com-pare our results with other well estabilished criteria.We analysed 30 patients with PE who were classifiedas E(20) and T(10) by Light's criteria. MDA wasmeasured by spectrophotometric method with TBA.Our results showed significant increase of MDAs levelin both groups of patients: E (7.27 ± 1.29 mmol/L)and T (7.75 ± 0.89 mmol/L) in comparison with con-trol group (5.10 ± 0.87 mmol/L) p<0.001. MDAplevel in E (5.16 ± 1.28 mmol/L) was also significantlyincreased than in T (2.67 ± 0.61 mmol/L), p<0.001.Similary, MDAp/s ratio was significantly higher in E(0.72 ± 0.17 mmol/L) than in T (0.34 ± 0.05 mmol/L)p<0.001. Using a cut-off value (0.4) for MDAp/s ratio(sensitivity 95% and sprecificity 90%) effectively sepa-rated E from T. Based on these results we can con-clude that determination of lipid peroxidation prod-ucts may be useful in diagnosis-differentiation ofpleural effusions into E and T. It is of significance forthe correct treatment of patients with PE.

B23

PRO-ANTIOKSIDATIVNI STATUS U EKSPERIMENTALNOJ

UNILATERALNOJ NEFREKTOMIJI

T. Cvetkovi}, V. B. \or|evi}, D. Pavlovi}, G. Koci}, P. Vlahovi}


Uklanjanje jednog bubrega dovodi do kompenza-tornog pove}anja bubre`ne mase preostalog bubrega{to se karakteri{e pove}anjem stepena glomerularnefiltracije (40’70%) i tubularne reapsorpcije. Unilateral-na nefrektomija (UN) tako|e je pra}ena pove}anompotro{njom kiseonika i produkcijom slobodnih radi-kala u preostalom bubregu. Cilj ove studije bio je daispita promene pro-antioksidativnog statusa tokomunilateralne nefrektomije. Unilateralna nefrektomijaizvedena je na mu{kim pacovima Wistar soja (n=6)nakon podvezivanja krvnih sudova i uretera. Kontrol-nu grupu ~inilo je 6 la`no operisanih ìvotinja. Nakon48 sati pacovi su `rtvovani i va|ena je heparinizovanakrv iz abdominalne aorte. Bubrezi su brzo izva|eni,dekapsulirani i homogenizovani (10% homogenat) naledu. U plazmi je odre|ivana koncentracija uree ikreatinina, a koncentracija malondialdehida (MDA),glutationa (GSH) kao i aktivnost glutation-S-transfe-raze (GSH-ST) i katalaze u plazmi i tkivu bubrega.Urea (9,27 “ 0,69 mmol/L) i kreatinin (79,60 “11,16 mmol/L) bili su statisti~ki zna~ajno pove}ani uUN grupi (p< 0,001) u pore|enju s kontrolnom gru-pom (6,41 “ 0,78 urea; 46,25 “ 4,41 kreatinin). U tojgrupi zna~ajno je pove}ana koncentracija MDA u pla-zmi (13,50 “ 1,27 mmol/L; prema kontroli 9,94 “1,16; p<0,001), a nepromenjena u bubregu. Koncen-tracija GSH u krvi UN grupe (14,79 “ 1,28 mmol/g Hb;prema kontroli 10,58 “ 1,15; p<0,05) kao i aktivnostGSH-ST u plazmi (19,99 “ 5,09 mmol/L/min; premakontroli 13,57 “ 3,01; p<0,05) bila je pove}ana, asnièna u tkivu bubrega. Aktivnost katalaze u plazmi ieritrocitima bila je nepromenjena u pore|enju sa kon-trolnom grupom dok je u bubregu snièna (8,95 “3,28 mmol/mg proteina) u odnosu na kontrolnu gru-


BENZIMI

ENZYMES

B23

PRO-ANTIOXIDATIVE STATUS IN EXPERIMENTAL

UNILATERAL NEFRECTOMY

T. Cvetkovi}, V. B. \or|evi}, D. Pavlovi}, G. Koci}, P. Vlahovi}

Institute of Biochemistry, Medical Faculty, Ni{

The removal of left kidney leads to the compen-satory renal mass elevation characterized by increasein GFR (40’70%) and tubular reapsorption. This uni-lateral nephrectomy (UN) is also followed by intensiveoxygen consumption and elevated free radicals pro-duction of the remnant kidney. The present studywas designed to explore a change in pro-antioxidativestatus during experimental unilateral nephrectomy.Unilateral nephrectomy was done on 6 male Wistarrats (180’200 g) after blood vessels and ureteral li-gation. The control group consisted of 6 sham-ope-rated animals. After forty-eight hours the rats weresacrificed and heparinized blood was collected fromthe abdominal aorta. Kidney was quickly removed,decapsulated and homogenized (10% homogenate)on ice. The plasma urea and creatinine concentra-tion were determined. The concentration of malondi-aldehyde (MDA), content of glutathione (GSH), activ-ity of glutathione-S-transferase (GSH-ST) and cata-lase were determined in the plasma and kidney. Theurea (9.27 “ 0.69 mmol/L) and creatinine (79.60 “11.16 mmol/L) in the UN group were elevated(p<0.001) in comparison to control group (6.41 “0.78 urea; 46.25 “ 4.41 creatinine). In this groupMDA concentration was also significantly increasedin the plasma (13.50 “ 1.27 mmol/L; vs control 9.94“ 1.16; p<0.001), remaining unchanged in kidney.The concentration of GSH in blood of UN group(14.79 “ 1.28 mmol/g Hb vs control 10.58 “ 1.15;p<0.05) as well as the activity of GSH-ST in plasma(19.99 “ 5.09 mmol/L/min; vs control 13.57 “ 3.01;p<0.05) were increased, and decreased in kidney(p<0.05). Activity of catalase was significantly decre-ased in kidney of UN group (8.95 “ 3.28 mmol/mg

pu (8,95 “ 3,28 mmol/mg proteina p<0,05). Iako jebubreg izloèn oksidativnom stresu, antioksidativnifaktori uspe{no uklanjaju slobodne radikale i tako ga{tite. Dobijeni rezultati pokazuju da je period od 48sati suvi{e kratak za sintezu novih proteina (katalaza,GSH i GSH-ST) koji su uklju~eni u sistem antioksida-tivne odbrane bubrega.

B24

ANTIOKSIDANTNI ZA[TITNI SISTEMMIOKARDA POSLE OPERACIJE

NA OTVORENOM SRCU

V. Ristovski, Lj. Ristovski, V. [}eki}, V. Mujovi}, N. Radovanovi}

Univerzitetska klinika za kardiovaskularnuhirurgiju, Novi Sad

Precizan klini~ki zna~aj slobodnih kiseoni~kihradikala posle operacije na otvorenom srcu jo{ uveknije rasvetljen. Smatra se da se slobodni kiseoni~kiradikali stvaraju odmah po operaciji {to do sada nijepotvrdjeno. Superoksid dizmutaza (SOD, EC1.15.1.1) jedan je od najva`nijih enzima antioksidant-nog sistema. SOD katalizuje pretvaranje superoksidradikala u vodonik peroksid koji se dalje metaboli{edo kiseonika i vode. Zato se ubraja u najva`nije za{tit-nike od dejstva slobodnih kiseoni~kih radikala. U ovojstudiji odre|ivane su aktivnosti SOD u eritrocitima ibiohumoralni status u plazmi kod 60 pacijenata posleoperacije na otvorenom srcu pre i 1/2, 6, 24, 48, 72sata posle operacije. U isto vreme odredjivani su idrugi markeri o{te}enja srca (CK, CK-MB, LDH, AST)kao i ukupni proteini, mokra}na kiselina, bilirubin i dr.SOD je odre|ivan Randox-ovim testom na automat-skom analizatoru Cobax Fara II (Roche) na 37 °C, aCK, CK-MB, ukupni proteini, mokra}na kiselina, bili-rubin i dr. standardnim biohemijskim metodama.SOD je izraèna u U/g Hb. Rezultati pokazuju da jeaktivnost SOD bila zna~ajno nià 1/2 sata posle ope-racije na otvorenom srcu (p<0,05) kao i da su CK,CK-MB, LDH, AST zna~ajno vi{i posle operacije naotvorenom srcu (p<0,05) u odnosu na kontrolnugrupu. U ispitivanim vremenskim intervalima zabele-èno je smanjenje ukupnih proteina. Dobijeni rezultatiukazuju na sniènje aktivnosti SOD, verovatno zboghiperprodukcije slobodnih kiseoni~kih radikala. Po-vi{ena produkcija slobodnih kiseoni~kih radikala ide uprilog hipotezi koja uklju~uje slobodne kiseoni~ke ra-dikale u patofiziolo{ke mehanizme reperfuzije.


protein) compared to the control (15.57 “ 3.06;p<0.005). Despite kidney was exposed to oxidativestress, antioxidative factors successfully removed freeradicals. These results indicate that in the kidney 48hperiod is too short for the new synthesis of proteins(catalase, GSH and GSH-ST) involved in antioxidativeprocesses.

B24

ANTIOXIDANT DEFENSE SYSTEM IN MYOCARDIUM AFTER OPEN

HEART SURGERY

V. Ristovski, Lj. Ristovski, V. [}eki}, V. Mujovi}, N. Radovanovi}

University Clinic of Cardiovascular Surgery, Novi Sad, Yugoslavia

The precise clinical importance of free oxygenradicals after open heart surgery has not yet beenelucidated. It is belived that free oxygen radicals areproduced as early as during the operations; howeverthis has not yet been confirmed. Superoxide dismu-tase ( SOD, EC 1.15.1.1) is one of the most impor-tant enzymes in the antioxidant system. SOD cataly-ses the transformation of superoxide radicals intohydrogen peroxide which is furter metabolized to oxy-gen and water. Therefore, it is the most importantprotector against the effects of these radicals. Thestudy concerns the determination of SOD activity inerythrocytes and biohumoral status of plasmaobtained from 60 patients after open heart surgery,before and 1/2, 6, 24, 48, 72 hours after operation.At the same time other markers of heart disease (CK,CK-MB, LDH, AST) and total protein, uric acid, biliru-bin, ect. SOD activities were examined using Randoxreagent and automated Cobas Fara II analyser(ROCHE) at 37°C and CK, CK-MB, total protein, uricacid, bilirubin, using standard biochemical methods.SOD was expressed in U/g Hb. Results showed thatSOD activities were significantly lower 1/2 hour afteropen heart surgery (p<0.05) and significantly higherlevels of CK, CK-MB, LDH, AST (p<0.05) comparingwith controls. During the monitoring decreased totalprotein levels were observed. The obtained resultsindicate the reduction in SOD activity, probably be-cause of hyperproduction of free oxygen radicals.The higher production of free radicals supports thehypothesis which includes these radicals in thepathophysiological mehanism of reperfusion.


B25

ANTIOKSIDANTNI KAPACITET LIMFOCITABOLESNIKA U TERMINALNOM STADIJUMUHRONI^NE BUBRE@NE INSUFICIJENCIJE

I HEMODIJALIZIRANIH BOLESNIKA

@. Davi~evi}, T. Simi}, K. Ille, A. Savi}-Radojevi}, M. Plje{a, J. Mimi}-Oka

Institut za biohemiju, Medicinski fakultet, Beograd

Bolesnici u terminalnom stadijumu hroni~ne bu-bre`ne insuficijencije (HBI) izloèni su oksidativnomstresu, koji ima ulogu i u patogenezi i progresiji uremij-skih komplikacija. Imunske }elije posebno su osetljivena oksidativni stres zbog visokog sadràja polinezasi-}enih masnih kiselina i povi{ene produkcije kiseoni~-nih slobodnih radikala. Poreme}aji funkcije imunskogsistema bolesnika sa HBI verovatno su povezani saoksidantno/antioksidantnim disbalansom u imunskim}elijama. Cilj ovog istraìvanja bio je odre|ivanje enzim-skog antioksidantnog kapaciteta limfocita bolesnika sterminalnom bubre`nom insuficijencijom i hemodijal-iziranih bolesnika. Odre|ivana je aktivnost antioksi-dantnih enzima superoksid dizmutaze (SOD), glutationperoksidaze (GPX), katalaze (CAT), glutation S-trans-feraze (GST) i glutation reduktaze (GR) u limfocitimanedijaliziranih pacijenata u terminalnom stadijumu HBI(20 pacijenata) i hemodijaliziranih pacijenata (12 paci-jenata). Dobijeni rezultati pokazuju da se aktivnost anti-oksidantnih enzima pove}ava sa progresijom hroni~nebubre`ne insuficijencije, a najzna~ajnije pove}anje je ulimfocitima bolesnika u terminalnom stadijumu HBI(Ccr manji od 21 mL/min). Promene u aktivnosti anti-oksidantnih enzima u limfocitima mogu biti adaptivnimehanizam za }elije usled hiperprodukcije slobodnihradikala u HBI. Porast aktivnosti antioksidantnih enzi-ma uo~en kod nedijaliziranih bolesnika delimi~no jesmanjen kod bolesnika le~enih hemodijalizom.

B26

ZNAÂJ ODRE\IVANJA b-NAG I g-GT U URINU DIJABETIÂRA

SA RAZLIÎTIM STEPENOM MIKROALBUMINURIJE

D. Stevanovi}1, R. Obrenovi}2, N. Majki}-Singh2, V. Jovanovi}1

1Biohemijska laboratorija, Zdravstveni centar Zaje~ar2Institut za medicinsku biohemiju,


Merenje enzimurije u nefrologiji je koristan neinva-zivan test za procenu anatomskog integriteta tubulo-intersticijuma. Cilj rada bio je ispitivanje uticaja mi-kroalbuminurije na anatomski integritet tubula u ko-

B25

LYMPHOCYTE ANTIOXIDANT CAPACITY IN END STAGE RENAL DISEASE AND HEMODIALYSIS PATIENTS

@. Davi~evi}, T. Simi}, K. Ille, A. Savi}-Radojevi}, M. Plje{a, J. Mimi}-Oka

Institute of Biochemistry, Medical Faculty, Belgrade

Chronic renal failure (CRF) patients in end stagerenal disease (ESRD) are more exposed to oxidativestress, a state which also play a role in pathogenesisand progression of uremic complications. Immunecells are particularly sensitive to oxidative stress be-cause of high percentage of polyunsaturated fattyacids and a higher production of reactive oxygenspecies. Thus, the impairement in immune system ofCRF pts is probably related to an oxidant/antioxidantimbalance in immune cells. In this study activity ofantioxidant enzymes (superoxide dismutase (SOD),glutathione peroxidase (GPX), catalase (CAT), glu-tathione S-transferase (GST) and glutathione reduc-tase (GR) were studied in lymphocytes of non-dial-ysed patients in ESRD (20 pts) and of patients onregular hemodialysis (HD) treatment (12 pts). Theresults obtained show that antioxidant enzyme activi-ties increase with progression of chronic renal insuf-ficiency, and the more pronounced augmentationwere found in lymphocytes from the ESRD patients(with Ccr less than 21 mL/min). These changes couldbe an adaptive mechanism for the cells due to ahyperproduction of free radicals in CRF. The increaseof antioxidant activity seen in ND pts were partly abol-ished in patients undergoing HD.

B26

THE IMPORTANCE OF DETERMINING URINARY β-NAG AND γ-GT IN TYPE 2

DIABETIC PATIENTS WITH DIFFERENTMICROALBUMINURIA DEGREE

D. Stevanovi}1, R. Obrenovi}2, N. Majki}-Singh2, V. Jovanovi}1

1Biochemical Laboratory, Health Centre Zaje~ar2Institute of Medical Biochemistry, Clinical Centre of Serbia, Belgrade

Measuring of enzymuria in nephrology representuseful, non-invasive test for evaluation of anatomicintegrity of tubulo-interstitium. The investigationswere carried out with the aim to determine the influ-

relaciji sa kataliti~kom aktivno{}u lizozomalnog enzi-ma N-acetil-b-D-glukozaminidaze (b-NAG) i enzima~etkastog pokrova tubula g-glutamil transferaze (g-GT) u urinu. Aktivnosti enzima izraène su na mmolurinarnog kreatinina. Kori{}en je 24h urin, ~ime suizbegnute varijacije ekskrecije u toku dana. Ispitivanisu bolesnici oboleli od diabetes mellitusa tipa II.Pacijenti su podeljeni u 3 grupe: I grupa, pacijenti samikroalbuminurijom manjom od 0,01 g/24h; IIgrupa, sa mikroalbuminurijom od 0,01 do 0,3 g/24h,III grupa, sa mikroalbuminurijom od 0,3 do 3 g/24h.Za statisti~ku obradu podataka kori{}en je Mann-Witney-ev neparametarski test i neparametarskaanaliza varijanse po Kruskall-Wallisu. Rezultatipokazuju da je porast aktivnosti b-NAG u urinu dija-beti~ara u III grupi bila statisti~ki zna~ajna u odnosuna bolesnike I (0,45 “ 0,23) i II (0,85 “ 0,39) grupe.Bolesnici II grupe imali su statisti~ki zna~ajno ve}uaktivnost b-NAG u odnosu na bolesnike I grupe.Porast aktivnosti g-GT bio je, statisti~ki, zna~ajno ve}ikod bolesnika III grupe (3,43 “ 1,16) samo u odno-su na bolesnike I (1,52 “ 1,19) grupe. Na osnovunavedenih rezultata zaklju~eno je da je b-NAG oset-ljiviji parametar nego g-GT za procenu uticaja mi-kroalbuminurije na anatomski integritet tubula i pro-cenu stepena o{te}enja tubula.

B27

UTICAJ pH NA ODRE\IVANJE AKTIVNOSTI

N-ACETIL-b-D-GLUKOZAMINIDAZE

J. A}imovi}, B. Boj~evski, V. Jovanovi}, Lj. Mandi}

Hemijski fakultet, Beograd

Odre|ivanje aktivnosti N-acetil-b-D-glukozami-nidaze (EC 3.2.1.30; NAG) u urinu pokazalo je daNAG moè biti veoma koristan pokazatelj renalniho{te}enja do kojih dolazi u razli~itim patolo{kim stan-jima i pod dejstvom razli~itih toksi~nih supstanci. Priodre|ivanju aktivnosti urinarnog NAG-a kod ze~evakoji su bili izloèni dejstvu te{kih metala na|ene suvrednosti niè od o~ekivanih za renalna o{te}enja.Urini su imali boju od ùte do mrke. U urinima mrkeboje pH vrednost je bila ve}a od 8, {to je dovelo dotoga da se u ovom radu ispita zavisnost aktivnostiNAG-a od pH. Aktivnost NAG-a odre|ivana je sa 2-metoksi-4-(2-nitrovinil)-fenil-N-acetil-b-D-glukoza-minidom kao supstratom. U slabo-kiseloj sredini (pHoko 6) ukupna aktinost urinarnog NAG-a je stabilna,


ence of microalbuminuria on anatomic integrity oftubules in correlation with catalytic activity of lysoso-mal enzyme N-acetyl-beta-D-glucosaminidase (b-NAG) as well as catalytic activity of enzyme g-glu-tamyl transpherase (g-GT) in urine. The activities ofenzymes are represented in mmol of urinary creati-nine. We used 24h urine, whereby daily variation ofexcretion were avoided. In this study, patients withtype 2 diabetes mellitus were examined. Patientswere divided in 3 groups: I group, patients withmicroalbuminuria less than 0.01 g/24h; II group, withmicroalbuminuria from 0.01 to 0.3 g/24h and IIIgroup, with microalbuminuria from 0.3 to 3 g/24h.For statistical analysis, Mann-Witney non-parametartest and non-parametar analysis of variance accord-ing to Kruskall-Wallis were used. Results showed thatb-NAG activity in urine of diabetic patients from IIIgroup (1.91 “ 1.44) was significantly higher com-pared with b-NAG activity in urine of patients from I(0.45 “ 0.23) and II group (0.85 “ 0.39). In patientsfrom II group b-NAG activity (0.85 “ 0.39) increasedstatistically significant compared with b-NAG activityin patients from I group (0.45 “ 0.23). Activity of g-GT significantly increased in patients from III group(3.43 “ 1.16) only compared with patients from Igroup (1.52 “ 1.19). Based on reported results, weconcluded that b-NAG represent more sensitive para-metar than g-GT for evaluation of the influence ofmicroalbuminuria on anatomic integrity of tubulesand for evaluation of tubule damage degree.

B27

THE INFLUENCE OF pH ON THE N-ACETYL-b-D-GLUCOSAMINIDASE

ACTIVITY DETERMINATION

J. A}imovi}, B. Boj~evski, V. Jovanovi}, Lj. Mandi}

Faculty of Chemistry, Belgrade

It was shown that activity of urinary N-acetyl-b-D-glucosaminidase (EC 3.2.1.30; NAG) may be usefulparameter of renal damage in some pathologicalstates as well as under toxicological substances ac-tion. In rabbits exposed to heavy metals urinary NAGactivities were smaller than expected for renal inju-ries. The color of urines was yellow to dark. pH ofdark urines was higher than 8. To clear these findingsthe influence of pH on NAG activity was investigatedin this paper. For determination of NAG activity 2-methoxy-4-(2-nitrovinyl)-phenyl-N-acetyl-b-D-glu-cosaminide as substrate was used. Under mild acidiccondition (about pH 6), there was no significant lossof total urinary NAG activity. In contrast, under alka-line condition (pH > 8) the total enzymatic activity of


dok se u alkalnoj sredini (pH > 8) ona izrazito sma-njuje. Da bi se sagledao uzrok ovog smanjenja aktiv-nosti ispitani, su izoenzimski profili NAG-a u slabo-ki-seloj i baznoj sredini (pri razli~itim pH vrednostima).Za razdvajanje izoenzimskih oblika primenjena je jo-noizmenjiva~ka hromatorgafija na DEAE celulozi.Analiza profila je pokazala je da se aktivnost A oblikaNAG-a u baznoj sredini zna~ajno smanjuje (inakti-vacija A oblika). Budu}i da je u normalnom urinu (pH5’7) udeo aktivnosti A oblika u ukupnoj aktivnostiNAG-a znatno ve}i (84,64 ± 2,75 %) od udela B obli-ka (8,62 ± 2,19 %), odre|ivanje ukupne aktivnostiNAG-a u baznoj sredini daje pogre{ne rezultate. Sto-ga u slu~aju baznih urina renalna o{te}enja treba pro-cenjivati na osnovu aktivnosti B oblika.

B28

AKTIVNOST SUPEROKSID DIZMUTAZE U PLAZMI I ERITROCITIMA PACIJENATA

OBOLELIH OD [IZOFRENIJE

N. D. Petronijevi}1, D. V. Mi}i}1, B. \uri~i}1, D. Marinkovi}2,

V. R. Paunovi}2

1Institut za biohemiju, Medicinski fakultet, 2Institut za psihijatriju,


Poreme}aj antioksidantne odbrane i pove}anjelipidne peroksidacije na|eno je u pacijenata obolelihod {izofrenije. Mogu}nost da o{te}ena antioksidant-na odbrana i izraèno peroksidativno o{te}enje mogubiti integralni deo patofiziolo{kog procesa {izofrenogoboljenja intenzivno se razmatra. Cilj ove studije jebio da se odredi aktivnost superoksid dizmutaze(SOD, EC 1.15.1.1.) u plazmi i eritrocitima: 1’pacije-nata sa prvom epizodom psihoze (n=20) pre prime-ne terapije i zatim posle 7, 14 i 21. dana tretmana;2’hroni~nih le~enih pacijenata (n=52) u fazi egza-cerbacije. Svi pacijenti su razvrstani u grupe premaCrow-u, kao Tip-I i Tip-II {izofrenije (SCH). Aktivnostsuperoksid dizmutaze je merena spektrofotometrijskina osnovu autooksidacije epinefrina. Dobijeni rezul-tati pokazali su da je u grupi pacijenata sa prvom epi-zodom psihoze, pre le~enja, aktivnost SOD u eritroc-itima i plazmi bila zna~ajno snièna (p<0,05) u pore-|enju sa kontrolom, samo kod pacijenata sa Tipom-I SCH. Posle prve tri nedelje tretmana, aktivnost SODu plazmi i eritrocitima bila je u okviru kontrolnih vred-nosti u obe ispitivane grupe. Kod hroni~nih pacijena-ta koji su dobijali neuroleptike u periodu duèm odjedne godine, zapaèno je statisti~ki zna~ajno pove-

NAG was rapidly lost. To understand the cause ofNAG activity decrease, the isonzyme profiles in mildacidic and alkaline conditions (under different pH val-ues) were investigated. For separation of isoenzymeforms the ion-exchange chromatography on DEAEcellulose was applied. The analysis of profiles showedthat activity of NAG isoenzyme A under alkaline con-dition significantly decreases (it seems to be inacti-vated). As isoenzyme A is a major form in normalurine (pH 5’7) which contribution in total activity(84.64 ± 2.75 %) is higher than contribution of isoen-zyme B (8.62 ± 2.19 %), the results obtained for totalNAG activity under alkaline condition may be wrong.Thus our results suggest that for alkaline urine, NAGisoenzyme B should be measured for detection renaldamage.

B28

SUPEROXIODE DISMUTASE ACTIVITY IN PLASMA AND EYTHROCYTES

OF PATIENTS SUFFERING FROM SCHIZOPHRENIA

N. D. Petronijevi}1, D. V. Mi}i}1, B. \uri~i}1, D. Marinkovi}2,

V. R. Paunovi}2

1Institute of Biochemistry, Faculty of Medicine, 2Institute of Psychiatry, Clinical Center of Serbia,

Belgrade, Yugoslavia

Impaired antioxidant defense and increased lipidperoxidation has been reported in schizophrenic pa-tients. The possibility that an impaired antioxidantdefense and increased peroxidative injury might beintegral to the schizophrenic disease process is underintensive consideration. The aim of the present studywas to assess activity of superoxide dismutase (SOD,EC 1.15.1.1.) in plasma and erythrocytes of: 1’pa-tients with first episode of psychosis (n=20) beforemedication and then after 7, 14 and 21. days of treat-ment; 2’chronic medicated schizophrenic patients(n=52) in the phase of exacerbation. All patients we-re selected in groups with Crow's Type-I or Type-II ofschizophrenia (SCH). Superoxide dismutase activitywas measured by spectrophotometric assay basedon epinephrine autoxidation. Our results revealedthat in the group of patients with first episode of psy-chosis, before medication, SOD activities in erythro-cytes and plasma were significantly decreased(p<0.05) comparing to controls, only in patients withType-I of SCH. After first 21 days of treatment theactivities of SOD in plasma and erythrocytes were inthe range of control values in both investigatedgroups. In chronic patients that have received neu-


}anje (p<0,05) aktivnosti SOD i u plazmi i u eritroci-tima pacijenata sa oba tipa SCH. Moè se zaklju~itida smanjenje aktivnosti SOD kod pacijenata saprvom epizodom SCH Tipa I, pre le~enja, predstavljapromenu koja je najverovatnije karakteristika samogprocesa bolesti. Pove}anje aktivnosti SOD koje pratiprolongirani tretman antipsihoticima moglo bi bitiposledica pove}anog oksidativnog tonusa uzrokova-nog pove}anim metabolizmom kateholamina ili sa-mog leka.

B29

UTICAJ SALICILNE KISELINE NA AKTIVNOST ALFA-AMILAZE, BETA-GLUKURONIDAZE I NAG U SVE@IM UZORCIMA URINA

S. \. Jovanovi}

Odeljenje laboratorije, Pedijatrijska klinika,Medicinski fakultet, Novi Sad

Cilj rada bio je da se ispita uticaj niskih doza sali-cilne kiseline (aspirin od 0,5 g na dan) na 3 glikozid-hidrolaze izlu~enih u sveìm uzorcima urina. Ispitiva-nja su izvedena kod 15 zdravih odraslih dobrovoljaca.Odre|ivani su slede}i enzimi: alfa-amilaza (EC3.2.1.1), beta-glukuronidaza (EC 3.2.1.31) i NAG (EC3.2.1.30). Prvi enzim je vaàn za funkciju pankreasa,a druga dva za ispitivanje bubre`nih tubula. In vitroispitivanjima na|eno je neznatno sniènje aktivnostibeta-glukuronidaze (beta-glu) i N-acetil-beta-D-glu-kuronidaze (NAG) kao enzima proksimalnih tubula,dok se alfa-amilaza nije menjala. Aspirin je davan di-rektno u sveè uzorke urina (kumuliran uzorak) ukoncentracijama 10, 50, 100 i 300 mmol/L. Uo~enoje nelinearno sniènje NAG (od 4,09 do 2,18 U/L,srednje vrednosti) i skoro linearan pad beta-glu (od55,3 do 23,0/L, srednje vrednosti). Alfa-amilazapokazuje sli~an trend (od 277 ka 198 U/L u tre}emsatu ~emu sledi porast aktivnosti u ~etvrtom satu(246 U/L). U isto vreme koncentracija salicilata rasteu prvom i drugom satu posle ingestije, a zatim sledisniènje u tre}em i ~etvrtom satu. Sniènje enzimskeaktivnosti moè da se tuma~i kao direktno dejstvosalicilata na proksimalne }elije tubula. Nema sumnjeda male doze salicilata interferiraju pri odre|ivanjuenzimske aktivnosti.

roleptics during period longer than one year, statisti-cally significant increase (p<0.05) in SOD activitywas noticed in plasma and erythrocytes of patientswith both types of SCH. We can conclude that thedecrease of SOD activity noticed in first episode TypeI patients before medication represents the changewhich is likely to be characteristic of the diseaseprocess itself. Elevation of SOD activity after pro-longed treatment with antipsychotics might be theconsequence of increased oxidative tonus caused byincreased catecholamine or drug turnover.

B29

INFLUNCE OF SALICILYC ACID ON THE ACTIVITY OF ALPHA-AMYLASE,

BETA-GLUCURONIDASEAND NAG IN FRESH URINE SAMPLES

S. \. Jovanovi}

Department of Laboratory, Clinic of Pediatrics, Faculty of Medicine Novi Sad, Yugoslavia

The aim of the study was to investigate the influ-ence of low doses of salicylic acid (aspirin, 0.5 g/day)in 15 healthy adult volonteers on 3 glycosid hydro-lases excreted in fresh urine samples. The enzymeswere as following: alpha-amylase (EC 3.2.1.1), beta-glucuronidase (EC 3.2.1.31) and NAG (EC 3.2.1.30).The first enzyme is important for pancreas examina-tions and the two others for investgations of kidneytubules. Investigations in vitro gave slight fall of theactivities of beta-glucuronidase (beta-glu) and (N-acetyl-beta-D-glucuronidase NAG) as proximal tubu-lar enzymes, while alpha-amylase showed almost nochanges. Aspi-rin was added directly to fresh urinesamples in concentrations of 10, 50 100 and 300mmol/L. Investigations in vivo (0.5 g/day) were per-formed in urine from zero to four hours after applica-tion. Non linear fall of NAG (from 4.09 to 2.l8 U/L,mean values), nearly linear fall of beta-glu (from 55.3to 23.0 U/L, mean values) were marked. Alpha-amy-lase presented similar trend (from 277 to 198 U/L) inthe third hour followed by increase of the activity inthe fourth hour (246 U/L). In the same time salicylicacid showed an increase in the first and the secondhour after ingestion followed by the decrease in thethird and the fourth hour. The depression of enzymeactivities could be seen as an direct action of salicylicacid on proximal tubular cells. There is no doubt theproblem in interference (influence) is present in theenzyme determination using even low doses of sali-cylic acid.


B30

PRA]ENJE AKTIVNOSTI GLUKOZA-6-FOSFAT DEHIDROGENAZE I METHEMOGLOBINA KOD RADNIKA

IZLO@ENIH PARAMA ORGANSKIH RASTVARAÂ

V. Kneèvi}, D. Milosavljevi}, O. Kneèvi}

Slu`ba za laboratorijsku dijagnostiku, Dom zdravlja u Kragujevcu,

Medicinski fakultet, Kragujevac

Biotransformacijom benzena i njegovih derivatastvaraju se reaktivni intermedijeri koji produkuju slo-bodne radikale. ]elije su za{ti}ene od o{te}enja iza-zvanih slobodnim radikalima razli~itim endogenimmehanizmima sa antioksidativnom ulogom. Jednaod zna~ajnih manifestacija oksidativnog stresa je pro-mena aktivnosti glukoza-6-fosfat dehidrogenaze. Glu-koza-6-fosfat dehidrogenaza (G-6-PDH, EC 1.1.1.49.)je klju~ni enzim u pentozo-fosfatnom ciklusu kojim sepreko NADPH obezbe|uje redukovani glutation kaoglavni neenzimski antioksidant. U ovoj studiji su odre-|ivane aktivnosti G-6-PDH u eritrocitima i koncen-tracije methemoglobina (Met-Hb) kod 30 radnika izlo-ènih parama organskih rastvara~a. G-6-PDH je od-re|ivan Randox-ovim testom na jednokanalnom-flek-sibilnom automatskom analizatoru Minilab na 37 ‰C, aMet-Hb je odre|ivan posebnom biohemijskom meto-dom na spektrofotometru Emerald III, koriste}i karak-teristi~an vrh apsorbancije na 630 nm, a zatim sman-jenje apsorbancije proporcionalno koncentraciji met-hemoglobina posle dodavanja cijanida. G-6-PDH jeizraàvan u U/g Hb, a Met-Hb % ukupnog Hb. Rezul-tati pokazuju da je aktivnost G-6-PDH zna~ajno niàkod izloènih ovim organskim rastvara~ima (p<0,05).Procenat methemoglobina zna~ajno je vi{i kod izlo-ènih benzenu i njegovim derivatima (p<0,05) u od-nosu na kontrolnu grupu. Rezultati ove studije uka-zuju na sniène vrednosti G-6-PDH verovatno zbogdominacije prooksidanasa, odnosno stvorenog oksi-dativnog stresa. Tako|e, prisustvo slobodnih radikalauti~e na gvo`|e da gubi elektron pri ~emu nastajeferimethemoglobin i superoksid anjon radikal. Odre-|ivanje ovih parametara u hemolizatu moè da ukaèna toksi~nost benzena i njegovih derivata sa niskimkoncentracijama (niìm od MDK), pre klini~ke eks-presije.

B30

MONITORING GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITY,

AND MET-Hb WITHIN THE GROUP OF WORKERS PREVIOUSLY EXPOSED TO ORGANIC DISSOLVENT VAPOURS

V. Kneèvi}, D. Milosavljevi}, O. Kneèvi}

Department of Laboratory Diagnostics, Health Center in Kragujevac,

The University of Medical Sciences, Kragujevac

Biotransformation of benzene, as well as bio-transformation of benzene derivaties, forms reactiveintermediers, that create free radicals. Various endo-gene mechanisms with antioxidant functions, protectcells from damages caused by free radicals. One ofthe significant demontrations of oxidating stress ischange in activity of glucose-6-phosphate dehydro-genase (G-6-PDH). G-6-PDH is the key enzyme inpentose-phosphate cycle, that through NADPH, pro-vides reduced glutation as main non enzyme antioxi-dating agent. This study is about determination ofthe activity of G-6-PDH in human red blood cells andthe concentration of Met-Hb in red cells within thepopulation of 30 workers previously exposed to vapo-urs of organic dissolvants. G-6-PDH has been deter-mined by Randox test on one chanel, flexible, auto-matic analyser Minillab at 37 ‰C. Met-Hb has beendetermined by special biochemical method on spec-trophotometer »EMERALDIII«, applying characteristicabsorbtion peak on 630 nm, and then decrease ofabsorbtion proportional to Met-Hb concentrationgained after addition of cyanide. G-6-PDH is beingexpressed in U/g Hb, and Met-Hb is being expressedin % of total Hb. The results show that G-6-PDH acti-vity is significantly lower within the group of exposedto organic dissolvant vapours (p< 0.05). In compari-son with the control group, the percentage of Met-Hbis significantly higher within the group of exposed tobenzene and benzene derivates (p<0.05). The resultsof this study indicate the decreased values of G-6-PDH due to peroxidant domination i.e. created oxi-dating stress. The presence of free radicals also caus-es Fe loss of electron, while creating ferimethemo-globin and superoxide anion radical. Determinationof these parameters in haemolysate, can point out tothe toxic influence of benzene and it's derivatives withtheir decreased concentrations (lower than MDK),even before it's clinical expression.

B31

KATABOLIZAM POLIAMINA U JETRI U ISHEMIJSKO-REPERFUZIONOM

O[TE]ENJU BUBREGA

I. Stojanovi}1, T. Cvetkovi}1, P. Vlahovi}2, G. Bjelakovi}1, T. Jevtovi}1,

D. Sokolovi}1, J. Ba{i}1

1Institut za biohemiju, Medicinski fakultet, Univerzitet u Ni{u

2Klinika za nefrologiju, Klini~ki centar, Ni{

U cilju ispitivanja eventualnog uticaja ishemijsko-reperfuzionog o{te}enja bubrega na metabolizam po-liamina, ispitivana je aktivnost diamino- i poliaminoksidaze u jetri pacova podvrgnutih ishemiji, a zatimreperfuziji bubrega, kao i ìvotinja tretiranih kverceti-nom (20 mg/kg telesne mase) pre ishemijsko-reper-fuzionog o{te}enja. Srednja vrednost aktivnosti poli-amin oksidaze u jetri pacova s ishemijsko-reperfuzio-nim o{te}enjem bubrega iznosi 1,41 “ 0,19 jed./mgproteina, {to je statisti~ki zna~ajan pad u odnosu navrednost u jetri ìvotinja kontrolne grupe (1,93 “ 0,11).Pod uticajem kvercetina aktivnost poliamin oksidaze setako|e zna~ajno smanjuje (1,46 “ 0,11) u odnosu nakontrolnu grupu, ali nema statisti~ki zna~ajnih prome-na u odnosu na aktivnost enzima izmerenu u grupi saishemijsko-reperfuzionim o{te}enjem. Aktivnost dia-mino oksidaze u jetri pacova podvrgnutih ishemijsko-reperfuzionom o{te}enju (1,14 “ 0,09) statisti~ki jezna~ajno nià od aktivnosti u kontrolnoj grupi (1,52 “0,18). Pretretman kvercetinom dovodi do jo{ izrazitijegsniènja aktivnosti ovog enzima (1,005 “ 0,06) uodnosu na kontrolne vrednosti, sa stastisti~komzna~ajno{}u i u odnosu na vrednost u grupi ìvotinja sishemijsko-reperfuzionim o{te}enjem. Smanjeni kata-bolizam poliamina u jetri pacova kao odgovor na ishe-mijsko-reperfuziono o{te}enje bubrega verovatno uka-zuje na te`nju za odrànjem visokog intracelularnogsadràja putrescina u cilju ispoljavanja njegovih protek-tivnih efekata u parenhimu jetre.

B32

AKTIVNOSTI SUPEROKSID DIZMUTAZE,KATALAZE I PEROKSIDAZE KOD ZDRAVIHLJUDI I INSULIN ZAVISNIH DIJABETIÂRA

A. Kozi}, M. Popovi}, V. Iveti}, S. Gavrilovi}

1Dom zdravlja Dr Milorad Mika Pavlovi}, In|ija2PMF, Institut za hemiju, Novi Sad

3Zavod za fiziologiju, Medicinski fakultet, Novi Sad4Institut za hirurgiju, Medicinski fakultet, Novi Sad

Oksidativni stres ima klju~nu ulogu u brojnim bio-lo{kim fenomenima kao {to su diferencijacija, trans-dukcija signala, starenje i u patogenezi mnogih boles-ti (karcinom, dijabetes, ateroskleroza i dr.). Kod dija-


B31

LIVER POLYAMINE CATABOLISM IN RENAL ISCHEMIA-REPERFUSION

IN RATS

I. Stojanovi}1, T. Cvetkovi}1, P. Vlahovi}2, G. Bjelakovi}1, T. Jevtovi}1,

D. Sokolovi}1, J. Ba{i}1

1Institute of Biochemistry, Medical Faculty, University of Ni{

2Clinic for Nephrology, Clinical Center, Ni{

Diamine and polyamine oxidase activities in liverof rats underwent renal ischemic-reperfusion dama-ge as well as of animals pretreated by quercetine (20mg/kg b.w.) in the same experimental model, werestudied in order to examine eventual influence ofischemic-reperfusion injury on liver polyamine catab-olism. Polyamine oxidase activity was significantlydecreased (1.41 “ 0.19 U/mg proteins) in liver of ratswith renal ischemia-reperfusion compared to thecontrol values (1.93 “ 0.11). Under the influence ofquercetine the activity of polyamine oxidase signifi-cantly decreased as well (1.46 “ 0.11), but withoutstatistically significant changes related to the activitymeasured in the group of rats with ischemia-reperfu-sion. Diamine oxidase activity in liver of rats under-went ischemia-reperfusion was significantly lower(1.14 “ 0.09) in comparison to the control value(1.52 “ 0.18). Quercetine pretreatment caused morepronounced decrease of activity (1.005 “ 0.06),which was statistically significant compared to con-trol as well as ischemia-reperfusion group of animals.Reduced polyamine catabolism in liver followingrenal ischemia-reperfusion injury could point out aneffort of maintaing high liver putrescine pool taking inaccount its protective role in liver.

B32

ACTIVITY OF SUPEROXIDE DISMUTASE,CATALASE AND PEROXIDASE

IN HEALTHY PERSONS AND INSULINEDEPENDENT DIABETICS

A. Kozi}, M. Popovi}, V. Iveti}, S. Gavrilovi}

1The health center dr Milorad Mika Pavlovi}, In|ija2Faculty of Sciences, Institute of Chemistry, Novi Sad3Faculty of Medicine, Institute of Phisiology, Novi Sad

4Faculty of Medicine, Institute of Surgery, Novi Sad

Oxidative stress plays very important role in a lar-ge number of biological phenomena such as diffe-rentiation, transformation, transduction of signals,


beti~ara prisustvo hiperglikemije dovodi do pove}aneprodukcije reaktivnih kiseoni~nih ~estica, kroz auto-oksidaciju glukoze i neenzimsku glikaciju. Smanjenjeaktivnosti antioksidativnog odbrambenog mehaniz-ma, pove}ava osetljivost dijabeti~ara na oksidativnao{te}enja. U ovom radu odre|ivane su aktivnosti en-zimskih antioksidativnih sistema (SOD, CAT i Px), uspecifi~nim ekolo{kim uslovima In|ije kod zdravihljudi i kod dijabeti~ara. Aktivnost SOD kod zdravihosoba ima relativno visoke vrednosti do 50 godinastarosti, a zatim opada. CAT pokazuje maksimalnuaktivnost u periodu 30’40 godina kod zdravih oso-ba, a zatim se aktivnost CAT smanjuje. Aktivnost Px(za razliku od prethodnih parametara) ima veomaniske vrednosti kod zdravih osoba do 40 godina, a za-tim njena aktivnost intenzivno raste. Aktivnost SOD iCAT daleko je ve}a kod dijabeti~ara u odnosu nazdrave ispitanike iste starosne grupe, ali se ove aktiv-nosti smanjuju sa starenjem. Ovi rezultati su bitnizbog pore|enja s rezultatima sli~nih istra`ivanja kojasu vr{ena u svetu u odgovaraju}im ekolo{kim uslovi-ma, sli~nim na{im i razli~itim od na{ih.

B33

ENZIMI ANTIOKSIDATIVNE ZA[TITE U KRVI I LIKVORU BOLESNIKA SA BAKTERIJSKIM I VIRUSNIM

MENINGITISOM

L. Zvezdanovi}1, S. Stani{i}2, V. ]osi}1, J. Lali}1, V. B. \or|evi}1

1Centar za medicinsku biohemiju2Klinika za infektivne bolesti,

Klini~ki centar Ni{

Bakterijski i virusni meningitis pra}eni su zna~aj-nom produkcijom slobodnih radikala koji su odgo-vorni za o{te}enja nastala u inflamatornom odgov-oru. U uslovima jakog oksidativnog stresa anga`ovanje antioksidativni sistem koga ~ine enzimi: superoksiddizmutaza (SOD), glutation peroksidaza (GSH-Px),katalaza (CAT) kao i drugi elementi endogene za{tite.U ovoj studiji je odre|ivana aktivnost SOD, GSH-Px iCAT u krvi i likvoru 19 bolesnika sa bakterijskim (BM)i 11 bolesnika sa virusnim meningitisom (VM). Kon-trolnu grupu ~inilo je 12 ispitanika le~enih pod dijag-nozom »meningizam«. SOD i GSH-Px odre|ivani suRandox-ovim testovima na automatskom analizatoruSynchron CX-5. Aktivnost katalaze u plazmi odre|i-vana je spektrofotometrijskom metodom Koroljuka isaradnika a u eritrocitima kineti~ko-spektrofotometri-

aging, and also in pathogenesis of some diseases(cancer, diabetes, atherosclerosis, etc.). Among dia-betics, increased production of reactive oxygenspecies is result of presence of hyperglycemia, occu-rred throughout autooxidation of glucose and non-ensymatic glycation. Decreasing antioxidative protec-tion mechanisms increase sensitivity of diabetics tooxidative damages. In this study, the activity of anti-oxidative enzymatic systems (SOD, CAT and Px) weredetermined among healthy people and diabetics inspecific ecological environment of In|ija. SODamong people, within age under 50 years of ageshows relatively high activity values, and then thisactivity decrease. CAT shows maximum activity dur-ing period of 30’40 years of age among healthy peo-ple, and then the CAT activity decrease. The activi-ty of Px (difference from the previous parameters) hasrather low values among healthy people under 40years of age, and then Px activity increase. SOD andCAT activity are significantly increased within diabe-tics compared to healthy people of same age, butthese activities show the tendency to decrease withaging. These results are important to compare withsimilar researches worldwide, done in specific eco-logical enviroments.

B33

ANTIOXIDATIVE PROTECTION ENZYMES IN BLOOD AND CEREBROSPINAL FLUID

OF THE PATIENTS WITH BACTERIAL AND VIRAL MENINGITIS

L. Zvezdanovi}1, S. Stani{i}2, V. ]osi}1, J. Lali}1, V. B. \or|evi}1

1Centre for Medical Biochemistry2Clinic for Infectious Diseases,

Clinical Centre Ni{

Bacterial and viral meningitis are associated withsignificant production of free radicals, responsible forthe inflammatory response damage. In the condi-tions of intense oxidative stress antioxidative systemis engaged comprising the following enzymes: super-oxyde dismutase (SOD), glutathion peroxidase(GSH-Px), catalase (CAT) as well as other endoge-nous protection elements. In this study we measuredthe activity of SOD, GSH-Px and CAT in blood andcerebrospinal fluid of 19 patients with bacterial (BM)and 11 with viral meningitis (VM). Twelve examineestreated under the diagnosis of »meningism« com-prised the control group. SOD and GSH-Px weredetermined with Randox-tests on automatic analyzerSYNCHRON CX-5. Plasma catalase activity was de-termined with spectrophotometric method by Koro-


ljuk et al., and erythrocyte CAT activity with kinetic-spectrophotometric method by Beutler. In thepatients with BM and VM there was a significant ele-vation of SOD activity in erythrocytes (p<0.001) whi-le GSH-Px activity was not significantly altered com-pared to controls. Marked changes in the cell ele-ments content in the cerebrospinal fluid of the stu-died patients were not associated with SOD activitychanges, but at the first diagnostic punction a mar-kedly increased GSH-Px activity in BM (p<0.001) andVM (p<0.05) was observed. In the conditions of in-creased H2O2 activity CAT was increased in erythro-cytes (p<0.01) and plasma (p<0.05) of BM patients.The observed increase of activity of antioxidative pro-tection enzymes probably represents a compensatoryresponse to increased oxygen free radical produc-tion. These findings suggest the possibility of ration-al application of antagonistic »coctails« with SODand CAT which may reduce deleterious effects ofreactive oxygen metabolites released from polymor-phonuclear leucocytes.

B34

INHIBITORY EFFECTS OF METFORMIN ON XANTHINE OXIDASE ACTIVITY

V. ]osi}, S. Kundali}, L. Zvezdanovi}, V. B. \or|evi}

Center of Medical Biochemistry, Clinical Centre Ni{, Yugoslavia

The biguanide metformin is the second mostcommonly prescribed oral antidiabetic agent in Euro-pe, where it is taken as monotherapy in 40% of thepatients. Although metformin has been used exten-sively the effects of this drug are not completelyunderstood. Previous studies have shown that met-formin reduces blood glucose levels predominantlyby decreasing gluconeogenesis. Also, metformin de-creases fasting plasma insulin concentration and en-hances peripheral tissue sensitivity to insulin. Recentstudy showed that metformin has been demonstra-ted as a direct inhibiting agent for glycation and/orglycoxidation. In this study the effects of metforminon plasma xanthine oxidase (XO, EC 1.1.3.22) acti-vity were investigated in 46 patients with type 2 dia-betes mellitus as well as in vitro study. The activity ofXO was assayed by the spectrophotometric methodof Kalckar with slight modification. We noted signifi-cant decrease in xanthine oxidase activity (p<0.001)during monotherapy with metformin. Also, our invitro studies showed that therapeutic as well as moretimes higher metformin doses inhibited significantlyXO activity (p<0.001). Simultaneously, a significant

jskom metodom Beutler-a. Kod bolesnika sa BM i VMpostoji zna~ajan porast aktivnosti SOD u eritrocitima(p<0,001), dok aktivnost GSH-Px u eritrocitima nijestatisti~ki zna~ajno promenjena u odnosu na kontrol-nu grupu. Uo~ljive promene u sadràju }elijskih ele-menata u likvoru ispitivanih bolesnika nisu pra}enepromenama aktivnosti SOD, ali je ve} pri prvoj dijag-nosti~koj punkciji dobijena zna~ajno pove}anaaktivnost GSH-Px u bakterijskom (p<0,001) i virus-nom meningitisu (p<0,05). U uslovima pove}aneprodukcije H2O2 aktivnost CAT pove}ana je u eritro-citima (p<0,01) i plazmi (p<0,05) bolesnika sa bak-terijskim meningitisom. Zabeleèn porast aktivnostienzima antioksidativne za{tite verovatno predstavljakompenzatorni odgovor na pove}anu produkciju slo-bodnih radikala kiseonika. Ova saznanja upu}uju namogu}nost racionalne primene antagonisti~kih»koktela« sa SOD i CAT koji mogu redukovati {tetneefekte reaktivnih metabolita kiseonika oslobo|enih izpolimorfonuklearnih leukocita.

B34

INHIBITORNI EFEKAT METFORMINA NA AKTIVNOST KSANTIN OKSIDAZE

V. ]osi}, S. Kundali}, L. Zvezdanovi}, V. B. \or|evi}

Centar za medicinsku biohemiju, Klini~ki centar Ni{

Bigvanidin metformin je drugi naj~e{}e prepisi-vani oralni antidijabetik u Evropi, koji se koristi umonoterapijske svrhe kod 40% pacijenata. Iako semetformin kao antidijabetik ~esto koristi, pun efekatovog leka jo{ nije u potpunosti razja{njen. Prethodnestudije su pokazale da metformin smanjuje nivoglukoze u krvi uglavnom suprimiranjem procesaglukoneogeneze. Tako|e, metformin smanjuje kon-centraciju insulina u plazmi i poja~ava osetljivost per-ifernih tkiva prema insulinu. Novije studije pokazujuda metformin direktno inhibira procese glikozilacije.U ovom radu prou~avan je efekat metformina naaktivnost ksantin oksidaze (XO, EC 1.1.3.22) kod 46pacijenata sa tipom 2 dijabetes melitusa, kao i u invitro uslovima. Aktivnost XO odre|ivana je modifiko-vanom spekrofotometrijskom metodom po Kalckar-u. Na|eno je statisti~ki zna~ajno smanjenje aktivnos-ti XO (p<0,001) za vreme monoterapije metformi-nom. Tako|e, rezultati dobijeni u in vitro eksperi-mentu pokazuju da terapijske, kao i mnogo vi{e do-ze metformina, zna~ajno inhibiraju aktivnost XO(p<0,001). Istovremeno je zabeleèna statisti~ki zna-~ajna korelacija izme|u doze leka i inhibicije XO


(p<0,001). Dakle, jedna od mogu}nosti je da met-formin direktno uti~e na aktivnost XO, {to moè bitidodatni korisni efekat ovog leka. Dobijeni rezultati in-direktno sugeri{u da metformin moè da smanji tok-si~na o{te}enja tkiva upravo preko inhibicije XO.

B35

PROMENA ENZIMSKE AKTIVNOSTI KOD PACIJENATA SA TRIHINELOZOM

Lj. Ba~vanski, Lj. Vorgi}, M. Frenc, Z. Papi}

Klini~ko biohemijska laboratorija, Op{ta bolnica »\or|e Joanovi}«, Zrenjanin

Trihineloza je parazitska infekcija izazvana nema-todom Trichinella spiralis. Oboljenje odlikuju 3 fazei to intestinalna, invazivna i rekonvalescentna. Posleintestinalnog stadijuma, larve putem krvi migriraju dopopre~no-prugaste muskulature, gde se inkapsulira-ju. Direktna trauma mi{i}nih }elija dovodi do pove}a-nog osloba|anja citoplazmatskih enzima i do prome-ne enzimskog profila u serumu. Prema klasifikacijineuromuskularnih oboljenja, trihineloza pripada gru-pi inflamatornih miopatija. U ovom radu kod pacije-nata sa klini~kom slikom trihineloze i eozinofilijompra}ene su aktivnosti AST, ALT, CK i LDH tokominvazivne faze. Aktivnosti navedenih enzima odre|enesu na automatskom analizatoru Synchron CX4(Beckman) na 37 °C, pomo}u komercijalnih testovafirmi Randox i Biosystems. Na|en je umereni porastsvih ispitivanih enzima, sa maksimalnim vrednostimaizme|u 3. i 5. nedelje od po~etka infekcije. Cilj radabio je da se ispita uloga navedenih enzima kao po-kazatelja parazitne infekcije.

B36

Na,K-ATPaza ERITROCITA KAO MOGU]I MARKER U DIJAGNOSTICI

IDIOPATSKE HIPERTENZIJE

T. Stojanovi}

Institut za biohemiju, Medicinski fakultet, Kragujevac

Pokazano je da merenje aktivnosti Na,K-ATPaze umembranama eritrocita moè biti potencijalni markerrazli~itih oboljenja; jedno od njih je i idiopatska

correlation was observed between doses of metfor-min and XO activity (p<0.001). So, one more possi-bility is that metformin may directly influence theactivity of XO and that may be additional benefitialeffect of this medicament. Our results indirectly sug-gest that metformin can reduce toxic tissue damagethrough the inhibition on XO activity.

B35

CHANGE OF ENZYME ACTIVITIES IN PATIENTS WITH TRICHINELLOSIS

Lj. Ba~vanski, Lj. Vorgi}, M. Frenc, Z. Papi}

Laboratory of Clinical Biochemistry, General Hospital »\or|e Joanovi}«, Zrenjanin

Trichinellosis is a parazite infection aquired by ne-matode Trichinella spiralis. Disease is distinguishedby 3 phases: intestinal, inavasive and convalescent.After intestinal stadium larvaes migrate via blood tostriate musculature where they incapsulate. Directtrauma of muscular cells leads to increased exemp-tion of cytoplasmic enzymes, and to changes of en-zyme profile in serum. According to classification ne-uromuscular diseases trichinellosis belongs to thegroup of inflammatory miopathy. In this study we havebeen monitoring the activity of AST, ALT, CK andLDH during invasive phase in patients with clinicaldiagnosis of trichinella and eosinophilia. The level ofthe observed parameters was defined using automat-ic analyzer Synchron CX4 (Beckman) at 37 °C withcommercial tests of Randox and Biosystems compa-nies. The obtained results showed moderate rise of allexamined enzymes. Maximal values were found bet-ween the third and the fifth week after infection. Thegoal of our work was to define the significiance ofenzymes as an indicator of parazite infection.

B36

THE ERYTHROCYTE Na,K-ATPase AS A MARKER INDIAGNOSIS

OF IDIOPATHIC HYPERTENSION

T. Stojanovi}

Institute of Biochemistry, School of Medicine,University of Kragujevac

Measuring of the Na,K-ATPase activity in the ery-throcyte membranes has been introduced as a po-tential marker for several diseases; one of those is


hipertenzija. U grupi od 20 pacijenata sa idiopatskomhipertenzijom (15 èna i 5 mu{karaca, starosti 28’58godina) i bez ikakve terapije, izmerena je aktivnostNa,K-ATPaze u membranama eritrocita. Dobijeni re-zultati iznose 2,41 ± 0,22 Pi/mg proteina/min {to jezna~ajno nià aktivnost u pore|enju sa kontrolnomgrupom zdravih ispitanika (3,78 ± 0,16 nmol Pi/mgproteina/min). Prethodno odre|ene referentne aktiv-nosti Na,K-ATPaze u membranama eritrocita iznosilesu 3,43’4,17 Pi/mg proteina/min bez zna~ajne razlikeizme|u èna i mu{karaca. Moè se pretpostaviti daendogena jedinjenja u cirkulaciji ispitanika sli~na di-goksinu ili uabainu mogu biti uzrok inhibicije ili dis-funkcije aktivnosti Na,K-ATPaze. Za ova jedinjenja se,ina~e, pretpostavlja da su jedan od uzroka u patoge-nezi idiopatske hipertenzije. Na osnovu dobijenih rezul-tata moè se zaklju~iti da merenje aktivnosti Na,K-ATPaze u membranama eritrocita moè biti koristanmarker u laboratorijskoj dijagnostici idiopatske hiper-tenzije.

B37

VREDNOSTI ANTIOKSIDATIVNIH ENZIMA I AZOT-MONOKSIDA KOD DECESA O[TE]ENJEM ENDOTELA

T. Stankovi}, V. \or|evi}, B. Kamenov, V. ]osi}, L. Zvezdanovi}

De~ja interna klinika, Centar za medicinsku biohemiju, Klini~ki centar, Ni{

U stanjima izmenjene imunolo{ke reaktivnostiorganizma remete se regulatorni mehanizami istvaraju uslovi za o{te}enje }elija i tkiva. Sinteza reak-tivnih metabolita kiseonika i azot-monoksida kojaprati imunolo{ke procese moè pokrenuti oksida-tivno o{te}enje endotelnih }elija i/ili indukovati nji-hovu apoptozu. U ovom radu pra}ena je aktivnostantioksidativnih enzima (superoksid dizmutaza, kata-laza, glutation peroksidaza) i koncentracija azot-mo-noksida kod 40 dece sa o{te}enjem endotela krvnihsudova. Uporedo su pra}eni i osnovni hematolo{kiparametri, kao i vrednosti oksidativne sposobnosti fa-gocita preko NBT-testa. Aktivnosti superoksid dizmu-taze i glutation peroksidaze odre|ivane su komerci-jalnim Randox-ovim testovima, a katalaze metodompo Beutler-u. Koncentracija azot-monoksida u plazmiodre|ivana je fotometrijski testom firme CaymanChemical. Analiza anamnesti~kih parametara, prisus-tvo monocitoze i sniènje stimulisane oksidativnesposobnosti fagocita ukazuju na dugotrajnu disregu-laciju imunskog sistema kod svih pacijenata. Aktiv-nost superoksid dizmutaze bila je zna~ajno nià(906,11 ± 177,46 U/gHb; p<0,01), a katalaze zna~aj-no vi{a (12,37 ± 2,43 U/gHb×104; p<0,01) u odno-

idiopathic hypertension. In the group of 20 patientswith idiopathic hypertension (15 females and 5 ma-les, aged 28 to 58) and without any medications theNa,K-ATPase activity in the erythrocyte membraneswere significantly lower as compared to the controlgroup of healthy volunteers (2.41 ± 0.22 and 3.78 ±0.16 nmol Pi/mg protein/min, respectively). The refe-rent value for healthy adults obtained by the sameprocedure were 3.43’4.17 nmol Pi/mg protein/min(SD 0.37; CV 9.8%) without difference between malesand females. Endogenous circulating digoxin- orouabain-like substances could be a reason of theenzyme inhibition, or dysfunction of Na,K-AT-Paseactivity may be involved in the pathogeneses of theidiopathic hypertension. According to these results,the measuring of Na,K-ATPase activity could bepotentially a useful routine marker in laboratory diag-nosis of idiopathic hypertension.

B37

VALUES OF ANTIOXIDATIVE ENZYMES AND NITRIC OXIDEIN CHILDREN WITH ENDOTHELIAL DAMAGE

T. Stankovi}, V. \or|evi}, B. Kamenov, V. ]osi}, L. Zvezdanovi}

Pediatric Clinic, Center of Medical Biochemistry,Clinical Center, Ni{

In state of changed immunological reactivity dis-turbed regulatory mechanisms make conditions forcell or tissue injury. Synthesis of reactive oxygenintermediates and nitric oxide which follows theimmune processes may contribute to oxidative injuryof endothelial cells and/or induce their apoptosis.This study examine the activity of antioxidativeenzymes (superoxide dismutase, catalase, glu-tathione peroxidase) and concentration of nitric oxidein 40 children with endothelial cell damage. Thebasic hematological parameters and values of fago-cytic oxidative capacity (NBT-test) were also exam-ined. The activity of superoxide dismutase and glu-tathione peroxidase were determined by Randoxcommercial tests, while the activity of catalase wasdetermined using Beutler's method. Plasma nitricoxide concentration was measured by CaymanChemical's photometric test. Analysis of anamnesticdata, registered monocytosis and decrease of stimu-lated fagocytic oxidative capacity suggested chronicdisregulation of immune system in all patients.Superoxide dismutase activity was significantlydecreased (906.11 ± 177.46 U/gHb; p<0.01), cata-lase activity was significantly increased (12.37 ± 2.43


su na dobijene vrednosti u kontrolnoj grupi dece(1125,78 ± 114,08 U/gHb, odnosno 9,30 ± 0,95U/gHb×104), dok aktivnost glutation peroksidaze nepokazuje zna~ajne razlike. Koncentracije azot-mo-noksida kao bitnog regulatornog molekula u limiti-ranju proinflamatornih de{avanja pokazuju varijacijeod zna~ajno vi{ih (72,18 ± 20,79 mmol/L) kod decesa manifestacijama po tipu eritematoznih, makulo-papuloznih i papuloznih promena, do zna~ajno ni`ih(33,08 ± 3,56 mmol/L) kod makuloznih promena, dokse u odnosu na kontrolnu grupu (42,14 ± 3,95 mmol/L)kod promena koje se manifestuju petehijama i pur-purom vrednosti zna~ajno ne razlikuju. Dobijenirezultati koji se odnose na aktivnost antioksidativnihenzima indirektno ukazuju da kod dece sa izmenje-nom imunolo{kom reaktivno{}u postoji povi{enaprodukcija reaktivnih metabolita kiseonika {to dopri-nosi o{te}enju endotela, dok disbalans u sintezi azot-monoksida direktno uti~e na stepen tog o{te}enja.

B38

INHIBITORNO DEJSTVO UREMI^KIHTOKSINA NA AKTIVNOST ENZIMA

N. Kosti}1, D. Miljkovi}2

1Biohemijska laboratorija, Op{ta bolnica, Kru{evac2Dom zdravlja, Varvarin

Naj~e{}i na~in le~enja hroni~ne bubre`ne insufici-jencije (HBI) u terminalnoj fazi je dijaliza. Proceshemodijalize obezbe|uje smanjenje uremijskih toksinaakumuliranih u krvi. Ove nedovoljno definisane sup-stance su uzrok mnogih komplikacija a mogu dovestii do fatalnog ishoda bolesnika u uremiji Svojom tok-si~no{}u uti~u na enzime, smanjuju}i njihovu aktiv-nost. Cilj rada bio je da se ispita uticaj hemodijalize naenzime odre|ivanjem nivoa enzima u serumu bolesni-ka sa HBI pre hemodijalize i pore|enjem istih posletretmana. Ispitivana je i korelacija enzima pacijenatana dijalizi sa grupom zdravih osoba (KG). Odre|ivanaje aktivnost enzima: alanin-aminotransferaze (ALT),alkaline fosfataze (AP), gama glutamiltransferaze(GGT) i holinesteraze (ChE). Ispitivanje je obuhvatilo33 osobe (N=33) oba pola sa HBI koje su se nalazilena hroni~nom programu hemodijalize 3 puta nedeljnopo 5 sati. Zna~ajno je vi{i t u serumu pre dijalize a AP(p<0,005), GGT (p<0,001) i ChE (p<0,01) u odno-su na KG, a posle dijalize pokazuje statisti~ki zna~aj-nost za: ALT (P<0,05), AP (P<0,05) i za GGT(p<0,001). Upore|uju}i aktivnost enzima pre i poslehemodijalize dobijene srednje vrednosti posle dijalizeimaju trend opadanja, {to se mo`e smatrati posledi-com inhibitornog uticaja uremijskih toksina akumuli-ranih u uremiji. Statisti~kom obradom tih vrednostinije dokazana statisti~ka zna~ajnost (p>0,05 kod svihenzima t je manji od teorijskog).

U/gHb×104; p<0.01) according to values in controlgroup of children (1125.78 ± 114.08 U/gHb, or 9.30± 0.95 U/gHb×104), while glutathione peroxidaseactivity did not show significant changes. The con-centration of nitric oxide as important immunoregu-latory molecule involved in restriction of proinflama-tory events showed variations from significantlyincreased (72.18 ± 20.79 mmol/L) in children witherythematic, papulous, and maculo-papulous chan-ges to significantly decreased (33.08 ± 3.56 mmol/L)in children with maculae according to values in con-trol group (42.14 ± 3.95 mmol/L), while children withpetechial and purpural manifestation did not showsignificant changes. The obtained results related toantioxidative enzyme activities indirectly indicate thehigher production of reactive oxygen intermediates inchildren with disturbed immunological reactivitywhich contributes to endothelial cell damage, whilethe disbalance in synthesis of nitric oxide directlyinfluence the extent of endothelial cells damage.

B38

INHIBITORY EFFECTS OF UREMIC TOXINS ON ENZYME ACTIVITY

N. Kosti}1, D. Miljkovi}2

1Biochemistry Laboratory - Health Center, Kru{evac2Health Center - Varvarin

Haemodialysis is the proper manner of therapy inchronic kidney insufficiency (CHI). This therapy ena-bles decrease of uremic toxins accumulated in theblood. This, ever now, unknown supstances, causedmany complications and it may be fatal. Decrease ofenzyme activity is also consequence of their toxicity.The aim in our work was to find enzyme activity (ALT,AP, GGT and ChE) in serum of patients with CKIbefore and after haemodialysis. The study has beenperformed on group patients (N=33) both sexes,with CKI who have been on chronic programme dial-ysis thrice per week about five hours, and controlgroup (N=20). Statistical significant increase (t) ofactivity were found before dialysis for AP (p<0.05),GGT (p<0.01) and ChE (p<0.01) in comparison tocontrol group. After dialysis statistical significancewere found for ALT (p<0.05), AP (p<0.05) and GGT(p<0.01). Enzyme activity after haemodialysis incomparison to activity before haemodialysis showeddecrease of activity, but that decrease is not statisti-cally significant.

A1

KLINI^KA VREDNOST BIOHEMIJSKIH PARAMETARA

U DIFERENCIJACIJI PLEURALNIH IZLIVA,EKSUDATA/TRANSUDATA

B. Pra`i}, V. Cvetkovi}, B. Cvejin, A. Vukeli}, M. Kr~o, V. Kuruc

Zavod za klini~ku biohemiju, Zavod za patologiju, Zavod za mikrobiologiju,

Klini~ka odeljenja,Institut za plu}ne bolesti, Sremska Kamenica

Osnova svakog dijagnostikovanja pleuralnih izlivazasniva se na razlikovanju pleuralnih eksudata i tran-sudata. Bazi~ni algoritam je sa~injen od parametarakoji su postavljeni prema Light-u. Mada, u nekimgrani~nim slu~ajevima, taj kriterijum nije dovoljan inije pouzdan. Otuda je da bi se dijagnostikovanjepleuralnih izliva u~inilo validnijim i svrsishodnijim,pove}an broj biohemijskih parametara, odre|ivanjemholesterola u izlivu i serumu. Cilj ovog rada bio je dase u na{im uslovima sagleda dijagnosti~ka validnosttih parametara. Merena je koncentracija holesterola iLDH u izlivu i serumu, testovima firme Dialab, aukupni proteini odre|ivani su biuretskom metodom.Ispitano je 419 izliva i seruma kod pacijenata saplu}nim bolestima razli~ite etiologije i ne-plu}nog po-rekla (kardijalni, abdominalni). Izlivi su grupisani u 11grupa prema citolo{koj dijagnozi ’ limfocitni tip izliva(76), makrofagni (26), makrofagno-limfocitni tip izliva(30), purulentni (62), maligni (72), eozinofilni tip izliva(11), limfocitno-makrofagni (76), tip izliva ’ me{ani}elijski sastav, tip izliva ’ ostalo, hemoragi~ni tip izlivai specifi~ni tip izliva sa bakteriolo{kom dijagnozom.Dobijene vrednosti za osetljivost po grupama kretalesu se izme|u 30 i 100%, a za specifi~nost izme|u 67i 80%. U literaturi, vrednosti za ove parametre surazli~ite. U tabeli su date srednje vrednosti dijagnos-ti~ke validnosti svih grupa po ispitanim biohemijskimparametrima.


AMETODE U KLINI^KOJ HEMIJI

METHODS IN CLINICAL CHEMISTRY

A1

BIOCHEMICAL PARAMETERS IN THE DIFFERENTIAL DIAGNOSTICS

OF PLEURAL EFFUSIONS, EXUDATES/ TRANSUDATES: DIAGNOSTIC EVALUATION

B. Pra`i}, V. Cvetkovi}, B. Cvejin, A. Vukeli}, M. Kr~o, V. Kuruc

Biochemistry, Pathology, Microbiology and Clinical Departments,

Institute for Pulmonary Diseases, Sremska Kamenica

The differential diagnostics of any pleural effusionis essentially based upon differentiating between apleural exudate and transudate. The basic algorithmincludes the parameters selected by Light, but insome border-line cases it, however, appears to beinsufficient and unreliable. To improve the efficacyand reliability of diagnosing pleural effusions, wehave increased the number of biochemical parame-ters to be determined, including cholesterol levels inboth the effusion and the serum. The objective of thisstudy is to perform a diagnostic evaluation of theseparameters. Cholesterol and LDH concentrations inboth the effusion and serum were measured byDialab tests, while total proteins were determined bythe biuret method. The study included 419 effusionsand serums from the patients with diverse etiology oftheir pulmonary disorder. The effusions were sub-classified into 11 types, depending on their cytologi-cal diagnosis: lymphocytic effusions (76), macropha-geal (26), macrophage-lymphocytic (30), purulent(62), malignant (72), eosinophilic (11), lymphocyte-macrophageal (76), effusions with a mixed cellularcontent, hemorrhagic and specific effusion type witha bacteriologic diagnosis and teffusions of non-pul-monary origin (cardiac, abdominal). The parametersmeasured within these effusion subtypes range from30% to 100% in sensitivity and from 67% to 80% inspecificity. The literature also reports a great diversi-ty in this respect. The table below reviews the diag-nostic validity of all examined biochemical parame-ters in all effusion subgroups.

Dobijena ujedna~ena klini~ka vrednost laboratorij-skih odre|ivanja navedenih biohemijskih parametara,ukazuje da se sa istom pouzdano{}u moè koristitiodre|ivanje holesterola u izlivu i serumu i njihov od-nos, kao jednostavna dopuna Light-ovog kriterijumaza diferencijanlo dijagnostikovanje pleuralnih izliva.

A2KARCINOEMBRIONALNI ANTIGEN

U HUMANOJ SALIVI

S. Golubovi}

Institut za primenu nuklearne energije - INEP, Zemun

Karcinoembrionalni antigen (CEA) jedan je od naj-vi{e kori{}enih tumorskih markera. Odre|ivanje kon-centracije CEA u serumu vaàn je parametar za prog-nozu i pra}enje terapije kod pacijenata s kolorektal-nim karcinomom. CEA pripada imunoglobulin (Ig)superfamiliji proteina ~iji ~lanovi u~estvuju u procesi-ma }elijske adhezije. U nekoliko sprovedenih istraì-vanja pokazana su homo- i heteroadhezivna svojstvaCEA i strukturno veoma homologih molekula CEAfamilije, ali njihova uloga u zdravom i patolo{kom tki-vu nije razja{njena. Imunohemijskim metodama mo-lekuli CEA familije detektovani su u salivi i salivarnim`lezdama. Velika prednost pri ispitivanju salive, u od-nosu na druge telesne te~nosti, njeno je jednostavnoi neinvazivno uzorkovanje. U ovom radu ispitana jekoncentracija CEA u salivi i serumu klini~ki zdravihosoba (n = 10). Uzorkovana je cela humana saliva,bez ikakve stimulacije. Od iste osobe uzimani su uzor-ci salive tri puta dnevno u dva uzastopna dana. Salivaje centrifugirana, supernatant dijalizovan i kori{}en zaispitivanje. Paralelno je, ujutro do 11 sati, uzimanavenska krv istih osoba i za analiziranje je upotreblja-van serum. Koncentracija CEA u salivi i serumu odre-|ena je imunoradiometrijskim testom (IRMA CEA,INEP). Dobijeni rezultati pokazali su da su serumskinivoi CEA u svim ispitanim uzorcima bili niì od 5mg/L. Opseg koncentracija izmerenih u odgovaraju-


The evaluated biochemical parameters, measu-red in our samples by laboratory techniques, exhibitan almost even clinical value, leading to a conclusionthat determination of the cholesterol levels should beused for the differential diagnosis of pleural effusionsas a complementary but equally reliable parameter toLight's diagnostic criteria.

A2

CARCINOEMBRYONIC ANTIGEN IN HUMAN SALIVA

S. Golubovi}

Institute for the Application of Nuclear Energy – INEP, Zemun

Carcinoembryonic antigen (CEA) is one of themost widely used tumor marker. The measurementof CEA serum concentration is an important para-metar in predicting prognosis and monitoring thera-py in patients with colorectal carcinoma. CEA be-longs to the immunoglobulin (Ig) superfamily, whichmembers are involved in the events of cell adhesion.Several studies revealed that CEA and highly relatedmolecules of the CEA family are homo- and hetero-adhesion molecules, but still their function in healthand disease have not been elucidated. The CEA fam-ily members have been detected in saliva and salivaryglands by immunochemical methods. An importantadvantage of saliva as a diagnostic fluid, in relation toother body fluids, is its simple and non-invasive col-lection. In this study the concentration of CEA in sali-va and serum from clinically healthy individuals (n=10) was investigated. Human whole saliva was col-lected by spitting, without any stimulation three timesper day, during two days. Saliva was cleared by cen-trifugal force and the supernatant was dialyzed andused for investigation. An appropriate amount ofblood is taken from the same persons till 11 a.m. andthe serum was used for testing. CEA concentration insaliva and serum was determined by an immunora-diometric assay (IRMA CEA, INEP). The obtainedresults showed that serum levels of CEA were below

Parametar

LDH PP/S LDHP/S PROTPROT PHOL PP/S HOL

Cut-off

≥ 3,3 mkat/L≥ 0,6≥ 0,5

≥ 30 g/L≥ 1,5 mmol/L

≥ 0,3

Osetlji-vost(%)888990908889

Specifi-~nost(%)808080808080

Pv(+)(%)

858383828483

Pv(’)(%)

758183847980

Ef(%)

838484848384

Parameter

LDH PFPF/S LDHPF/S PROTPROT PFCHOL PFPF/S CHOL

Cut-off

≥ 3.3 mkat/L≥ 0.6≥ 0.5

≥ 30 g/L≥ 1.5 mmol/L

≥ 0.3

Sens.(%)

888990908889

Spec.(%)

808080808080

Pv(+)(%)

858383828483

Pv(’)(%)

758183847980

Ef(%)

838484848384


5 mg/L in all examined samples. The CEA levels inthe corresponding salivary samples were higher, ran-ging from 67’1200 mg/L. Such findings indicatedthat salivary CEA is localy produced, without eventu-ally contribution of serum exudate to determined va-lues. A circadian rhythm was demonstrated for saliva-ry CEA, although CEA levels were very variable. Iden-tical daily variations of salivary CEA concentration,reaching the maximum value around 7 a.m. were de-tected for all ten investigated healthy persons. Theimportance of rhytm characteristics of tumor mark-ers is still unknown. The circadian rhytm of salivaryCEA and CEA rhytm alteration, deserves furtherinvestigation and await aplication. It could be correla-ted with pathological changes and useful for recogni-zing early disease.

A3

TOTAL PROTEIN LEVELS IN URINE AND CEREBROSPINAL

FLUID BY DYE-BINDING METHODS

S. Stankovi}, E. ôlak, N. Aleksi}-Markovi}, N. Majki}-Singh


Determination of total protein in urine and cere-brovascular fluid is valuble in the diagnosis of renaland central nervous system disorders respectively.Urinary protein elevations are commonly seen in stre-nuous exercise, fever and hypothermia, nephrosis anddiabetic nephropathy and urinary tract infections.Determination of total protein in cerebrovascular fluidaids in the diagnosis of such conditions as meningitis,CNS tumours and cerebral haemorrhage. The choiceof method for quantitation of urinary and cerebro-spinal fluid (CSF) total protein is not easy. For routineuse total protein in urine and CSF were determined bydye-binding methods with Coomasie Briliant Blue G-250. Our aim was to adapt and evaluate the methodusing Randox test (Randox Laboratories Ltd., UnitedKingdom). This test is based on principle that Pyro-gallol red complexes with proteins in an acid environ-ment containing molybdate ions, and the resultingblue coloured complex absorbs maximally at 600 nm.Colorimetric determination applied on biochemicalanalyzers could simplify determination; so it was adap-ted to centrifugal analyzer IL Monarch 2000 (Instru-mentation Laboratory, Milan, Italy). Imprecision with-in-assay was evaluated with replicate measurement oftotal protein concentration using urine controls atthree protein levels. The coefficients of variation were

}im salivarnim uzorcima iznosio je 67’1200 mg/L, {toukazuje da se CEA u salivi lokalno produkuje i da nepoti~e od eventualnog serumskog eksudata. Pokaza-no je da postoji pravilnost u cirkadijalnom ritmu, a neu izmerenim nivoima, salivarnog CEA. Istovetnednevne promene koncentracija CEA u salivi, sa mak-simalnom vredno{}u dobijenom u 7 h ujutro, detek-tovane su kod svih ispitanih zdravih osoba. Zna~ajritmi~kih promena koncentracija tumorskih markeranedovoljno je istraèn. Dnevni ritam izlu~enog sali-varnog CEA, i njegova promena, zasluùje dalje ispi-tivanje radi mogu}e korelacije s patolo{kim prome-nama i primene u ranoj dijagnozi oboljenja.

A3

ODRE\IVANJE UKUPNIH PROTEINA U URINU I LIKVORU

METODOM VEZIVANJA BOJE

S. Stankovi}, E. ôlak, N. Aleksi}-Markovi}, N. Majki}-Singh


Odre|ivanje koncentracije ukupnih proteina u uri-nu i likvoru je va`no za dijagnozu oboljenja bubrega icentralnog nervnog sistema. Proteini u urinu se po-ve}avaju usled: intenzivnog ve`banja, groznice i hipo-termije, nefroze i dijabeti~ne nefropatije i kod infekcijaurinarnog trakta. Odre|ivanje proteina u likvoru va`noje za dijagnozu meningitisa, tumora CNS-a i cere-bralne hemoragije. Odre|ivanje ukupnih proteina uurinu znatno je komplikovanije od odre|ivanja u se-rumu. Za odre|ivanje ~esto se koriste metode koje sezasnivaju na vezivanju boje za proteine npr. CoomasieBriliant Blue G-250. U ovom radu je primenjena i ispi-tana metoda koja se zasniva na vezivanju boje Pyro-gallol crveno za proteine u testu firme Randox (RandoxLaboratories Ltd., United Kingdom). Ova metoda sezasniva na osobini Pyrogallol crvenoj da gradi komple-kse sa proteinima u kiseloj sredini u prisustvu molib-data, pri ~emu nastaje plavo obojeni kompleks ~ija seapsorbancija meri na 600 nm. S obzirom da odre-|ivanje na biohemijskim analizatorima u mnogomepojednostavljuje tok analize, kolorimetrijski test je pri-lago|en za odre|ivanje na centrifugalnom analizatoruIL Monarch 2000 (Instrumentation Laboratory, Milan,Italy). Nepreciznost u seriji ispitana je ponovnim odre-|ivanjem koncentracije ukupnih proteina u tri kontrolerazli~itih koncentracija. Dobijeni su koeficijenti varijaci-je 1,72%, 2,43%, i 3,11% pri koncentracijama ukup-


nih proteina od 0,35 g/L, 1g/L i 5 g/L, redom. Sli~nikoeficijenti varijacije dobijeni su pri ispitivanju nepre-ciznosti iz dana u dan (2,63%, 3,17%, 4,01%). Metodaje linearna do 5 g/L. Ispitana je i korelacija izme|u me-toda sa CBB G250 and Pyrogallol red i dobijen je ko-eficijent korelacije r=0,958 i jedna~ina prave za uku-pne proteine u urinu yx=’0,170+1,395x, Sy,x=1,335g/L (N=150), a za proteine u likvoru r=0,992, yx =0,066 + 0,901x, Sy,x=0,141g/L) (N=50). S obziromna dobijene rezultate ova metoda je adekvatna zame-na za metodu sa CBB G250 i moè da ima prakti~anzna~aj.

A4

SLOBODNE AMINOKISELINE PLAZME I URINA KOD DECE

SA POREME]AJEM FUNKCIJE CENTRALNOG NERVNOG SISTEMA

M. Milo{evi}-To{i}, J. Borota, J. Stoj~evi}

Zavod za biohemiju, Klini~ki centar, Novi Sad

Ovim radom èleli smo da utvrdimo vezu izme|uporeme}aja funkcije centralnog nervnog sistema(CNS) kod dece sa poreme}ajem sadràja slobodnihaminokiselina u plazmi i u urinu. Na uzorku od 52deteta sa dijagnozama: psihomotorna retardacija(RPM) (27 pacijenata), epilepsija (18 pacijenata) i kon-vulzije (7 pacijenata) odre|ivane su slobodne amino-kiseline u serumu i u 24h urinu jonoizmenjiva~komhromatografijom na HPLC aminokiselinskom analize-ru Beckman System 6300 uz kori{}enje litijumskihpufera i kolone od 10 cm. Pra}ene su 44 ninhidrinpozitivne supstance. Od ukupnog broja pacijenata sadijagnozom RPM kod 9 pacijenata ili 33,33% sve anali-zirane aminokiseline nalazile su se u granicama refer-entnih vrednosti. Generalizovana hiper-aminoacidemi-ja na|ena je kod 7 pacijenta ili 25,92%, dok su povi-{ene koncentracije taurina kao i lizina i glutaminskekiseline u plazmi bile zastupljene kod 3 odnosno 2 pa-cijenta. U urinu ovih pacijenata naj~e{}e je bio povi{enhidroksiprolin bilo sporadi~no ili u sklopu generalizo-vane hiperaminocidurije. Kod pacijenata sa epilepsi-jom u 77% slu~ajeva na|en je nalaz u granicama re-ferentnih vrednosti za uzrast deteta. Samo u po jed-nom slu~aju na|ena je povi{ena koncentracija GABAu plazmi i hidroksiprolina, BABA i serina u urinu. Kodpacijenata sa epilepsijom samo u jednom slu~aju suna|ene povi{ene koncentracije metionina, lizina iarginina dok su se kod svih ostalih pacijenata sve ana-lizirane aminokiseline u plazmi i u urinu nalazile u gra-

1.72%, 2.43%, and 3.11% for concentration of totalprotein of 0.35 g/L, 1g/L, 5 g/L, respectively. The sim-ilar results were obtained for the between-day impreci-sion (2.63%, 3.17%, 4.01%). This method is linear upto 5 g/L. The comparison of results between methodswith CBB G250 and Pyrogallol red yielded the follow-ing regression parameters for total protein determi-nation in urine (yx=’ 0.170+1.395 x, r=0.958, Sy,x=1.335 g/L) (N=150) and in CSF (yx=0.066 +0,901x,r=0.992, Sy,x=0.141 g/L) (N=50). On the basis ofthese findings method with Pyrogallol red offers anadequate alternative to the method with CBB G-250and could have a practical importance.

A4

FREE AMINO ACIDS IN THE BLOOD AND URINE OF CHILDREN

WITH FUNCTIONAL DISURBANCES OF THE CENTRAL NERVOUS SYSTEM

M. Milo{evi}-To{i}, J. Borota, J. Stoj~evi}

Department of Biochemistry, Clinical Center, Novi Sad

The aim of this paper is to determine the connec-tion between some functional disturbances of theCNS in children and changes in the amino acids con-tent in plasma and urine. 27 cases with psychomotorretardation (PMR), 18 with epilepsy and 7 with con-vulsions were examined in regard to the content offree amino acids in plasma and 24h urine, with ionex-change chromatography using HPLC AminoacidAnalyzer Beckman System 6300 with lithium buffersand 10 cm colomn. 44 ninhydrin positive substanceswere analyzed. Out of 27 examined cases with PMRnormal finding was confirmed in the plasma and/orurine of 9 pattients or 33.33%. General hyper-aminoacidemia was found in 7 patients or 25.92 %,while higher concentrations of taurine, lyzine and glu-tamic acid were confirmed in 2 and 3 cases respec-tively. The most frequent finding was higher content ofhydroxyproline in urine either sporadically or as a partof general hyperaminoaciduria. In cases with epilepsyin 77% normal findings in regard to the age of patientwere confirmed. Higher concentrations of GABA inplasma and hydroxyproline, BABA and serine in urinewere found in sporadic cases. Higher concentrationsof methionine, lyzine and arginine were found only inone case of epilepsy out of all cases with normal val-ues of amino acids. It is well known that some func-tional disturbances of the CNS can be associated withgenetic disorders of aminoacids metabolism, but inour examined cases typical picture, in regard to the


nicama referentnih vrednosti. Iako neki poreme}ajifunkcije CNS mogu biti udruèni sa genetskim pore-me}ajem metabolizma aminokiselina na analiziranomuzorku dece nije na|ena tipi~na slika u sadràju ami-nokiselina koja bi ukazivala na ovaj poreme}aj, nitikonzistentno ponavljanje promena sadràja aminokise-lina za odre|enu dijagnozu poreme}aja funkcije CNS.

A5

CISTATIN C KAO NOVI MARKER ZA PROCENU JAÎNE

GLOMERULSKE FILTRACIJE

R. Obrenovi}1, E. Jak{i}2, S. Beatovi}2, D. Petrovi}3, B. Stojimirovi}4, N. Majki}-Singh1

1Institut za medicinsku biohemiju, Klini~ki centar Srbije,

2Institut za nuklearnu medicinu, Klini~ki centar Srbije,

3Klinika za urologiju i nefrologiju, KBC »Kragujevac«,

4Institut za urologiju i nefrologiju, Klini~ki centar Srbije

U klini~koj nefrologiji merenje ja~ine glomerulskefiltracije (GFR) je potreban i koristan postupak za pro-cenu indeksa bubre`ne funkcije. Za indirektnu pro-cenu stepena glomerulske filtracije u klini~koj praksikoriste se: koncentracija kreatinina u serumu, klirenskreatinina, klirens inulina (zlatni standard), klirens raz-li~itih radionukleidnih markera (99mTc-DTPA) kao iproteini plazme male molekulske mase (a1-mikroglo-bulin (protein HC), b2-mikroglobulin, retinol-vezuju}iprotein (RBP). Najnoviji me|u ovim pokazateljima jecistatin C. Koncentracija cistatina C u serumu uglav-nom zavisi od ja~ine glomerulske filtracije. Zbog togaje cystatin C u serumu dobar unutra{nji (endogeni)parametar za procenu stepena ja~ine glomerulske fil-tracije (GFR). Cilj ovog rada bio je da se ispitapovezanost izme|u 99mTc-DTPA klirensa, recipro~nevrednosti koncentracije cistatina C u serumu (1/cis-tatin C) i recipro~ne vrednosti koncentracije kreatini-na u serumu (1/kreatinin). Ispitivanje je obuhvatilo 61osobu oba pola (37 èna i 24 mu{karaca), prose~nestarosti 44,76 ± 12,93 godina koji su imali normalnudo umereno o{te}enu bubre`nu funkciju. Cystatin Cu serumu odre|en je PENIA metodom (Particle-En-hanced Nephelometric Immuno-Assay), firme Be-hring; srednja vrednost iznosila je 0,88 ± 0,21 mg/L.Srednja vrednost za kreatinin u serumu bila je 86,06± 18,14 mmol/L. Dinamskom scintigrafijom bubregaodre|ivan je klirens 99mTc-DTPA i dobijena je sredn-ja vrednost 98,52 ± 23,08 mL/min. Ispitivanjempovezanosti izme|u 99mTc-DTPA klirensa i recipro-

content of amino acids, was not established to con-firm genetic disorder, or consistant repetition of chan-ges of amino acid characteristic for diagnose of func-tional disturbances of the CNS.

A5

CYSTATIN C NEW MARKERFOR ASSESSMENT OF GLOMERULAR

FILTRATION RATE

R. Obrenovi}1, E. Jak{i}2, S. Beatovi}2, D. Petrovi}3, B. Stojimirovi}4, N. Majki}-Singh1

1Institute for Medical Biochemistry, Clinical Center of Serbia

2Institute of Nuclear Medicine, Clinical Center of Serbia

3Clinic of Urology and Nephrology, Clinical-Medical Center »Kragujevac«4Institute of Urology and Nephrology,

Clinical Center of Serbia

In clinical nephrology, the measurement of glo-merular filtration rate (GFR) is necessary and helpfulprocedure to estimate the index of renal function. Forindirect determination of glomerular filtration rate inclinical practice, the following parameters are used:creatinine concentration in serum, creatinine clear-ance, inulin clearance (»golden standard«), clear-ance of various radionuclide markers (99mTc-DTPA)as well as plasma proteins of small molecular mass(a1-microglobulin (protein HC), b2-microglobulin,retinol-binding protein (RBP). Among these indica-tors, the most recent is Cystatin C. Cystatin C con-centration in serum depends mainly upon theglomerular filtration rate. Accordingly, serum cystatinC is favorable internal (endogenous) parameter formeasurement of glomerular filtration rate (GFR). Theobject of this study was to examine the association of99mTc-DTPA clearance, reciprocal value of cystatin Cconcentration in serum (1/cystatin C) and reciprocalvalue of serum creatinine concentration (1/creati-nine). The study included 61 individuals of both sexes(37 females and 24 males), mean-age of 44.76 ±12.93 years, who had normal to moderately impairedrenal function. Cystatin C in serum was determinedby PENIA method (Particle-Enhanced NephelometricImmuno-Assay), Behring, and the mean value was0.88 ± 0.21 mg/L. Mean value of serum creatininewas 86.06 ± 18.14 mmol/L. Dynamic scintigraphy ofkidneys was used to determine 99mTc-DTPA clear-


~ne vrednosti koncentracije cistatina C u serumu(1/cistatin C) i recipro~ne vrednosti koncentracijekreatinina u serumu (1/kreatinin) utvr|eno je daizme|u 99mTc-DTPA klirensa i 1/cistatina C postojinajbolja korelacija (P<0,01). Koncentracija kreatini-na u serumu nije idealan parametar za procenu ja-~ine glomerulske filtracije (P<0,05). Cistatin C u se-rumu je bolji parametar u odnosu na kreatinin u seru-mu, za procenu stepena ja~ine glomerulske filtracije.

A6

OCENA METODE ANALIZATORA KODAK EKTACHEM 250

ZA ODRE\IVANJE AKTIVNOSTI ALANIN AMINOTRANSFERAZE

S. Veselinovi}1, V. Keserovi}1, Z. Jeli}-Ivanovi}2, D. Vu~i}3, M. Sekulovi}4

1Zdravstveni centar »Dr Laza K. Lazarevi}«, [abac2Farmaceutski fakultet, Beograd

3Dom zdravlja »Dr Milutin Ivkovi}«, Beograd4Dom zdravlja »Vo`dovac«, Beograd

Metoda »suve hemije« primenjena na analizatoruKodak Ektachem 250 za odre|ivanje aktivnosti alaninaminotransferaze koja se zasniva na kori{}enju L-ala-nina i natrijum a-ketoglutarata kao supstrata uz do-datak piridoksal-5-fosfata ispitivana je i pore|ena sadrugim metodama »vla`ne hemije« koje ne koriste pi-ridoksal-5-fosfat u reakcionom puferu. Dobijeni su za-dovoljavaju}i rezultati za nepreciznost. Vrednosti ko-eficijenata varijacije za preciznost u seriji iznose 0,5%do 5,3%, dok su koeficijenti varijacije za preciznost izdana u dan iznosili 3,1% do 8,7%. Utvr|eno je da jeKodak Ektachem metoda za odre|ivanje aktivnostiALT ta~na; vrednosti dobijene analizom kontrolnogseruma sa normalnom i povi{enom aktivno{}u ALTnisu se zna~ajno razlikovale od pravih vrednosti(p>0,05). Aktivnost ALT odre|ivana je u 50 uzorakaseruma sa analizatorom Kodak Ektachem i pore|enasa odgovaraju}im rezultatima dobijenim sa drugimanalizatorima. Statisti~kom obradom rezultata dobi-jene su slede}e korelacije: Ektachem prema RA-XT(Technicon): y=’9,092+1,443x; r=0,991; Ektachemu odnosu na Monarch (International Laboratory):y=’12,830+1,578x; r=0,996; Ektachem u odnosuna Hitachi 704: y=1,285+1,525x; r=0.996.Regresione jedna~ine kao i odgovaraju}i Bland-Altma-novi dijagrami razlika pokazali su da su vrednosti zaALT na analizatoru Kodak Ektachem bile vi{e od vred-nosti odre|ivanih na analizatorima Technicon RA-XT,

ance, and the mean value of 98.52 ± 23.08 mL/minwas obtained. The analysis of association of 99mTc-DTPA clearance and reciprocal value of cystatin Cconcentration in serum (1/cystatin C) and reciprocalvalue of creatinine concentration in serum (1/creati-nine) verified the best correlation between 99mTc-DTPA clearance and 1/cystatin C (P<0.01). Crea-tinine concentration in serum is not an ideal param-eter to determine the glomerular filtration rate(P<0.05). Cystatin C in serum is better parameter inrelation to serum creatinine, to determine the glo-merular filtration rate.

A6

EVALUATION OF ALANINE AMINOTRANSFERASE ASSAY

ON THE KODAK EKTACHEM 250 ANALYSER

S. Veselinovi}1, V. Keserovi}1, Z. Jeli}-Ivanovi}2, D. Vu~i}3, M. Sekulovi}4

1Health Center »Dr Laza K. Lazarevi}«, [abac2Faculty of Pharmacy, Belgrade

3Health Center »Dr Milutin Ivkovi}«, Belgrade4Health Center »Vo`dovac«, Belgrade

A »dry chemistry« Kodak Ektachem 250 alanineaminotransferase (ALT) assay based on the use of L-alanine and sodium a-ketoglutarate as substrate withaddition of pyridoxal-5-phosphate was evaluated andcompared with other »wet chemistry« methods with-out addition of pyridoxal-5-phosphate. The impreci-sion study yielded satisfactory results: within-run CVsranged from 0.5% to 5.3%, whereas day to day CVswere in range from 3.1% to 8.7%. The Kodak Ekta-chem ALT assay was found to be accurate: the val-ues obtained in control sera with normal and elevat-ed ALT activities did not significantly differ from thecorresponding target values (p>0.05). The ALTactivities were measured in 50 serum samples byKodak Ektachem and compared with the correspon-ding results obtained by other analysers. The follow-ing correlations were found: Ektachem vs. RA-XT(Technicon): y=’9.092+1.443x; r=0.991; Ekta-chem vs. Monarch (International Laboratory):y=’12.830+1.578x; r=0.996; Ektachem vs. Hitachi704: y=1.285+ 1.525x; r=0.996. The regressionequations, as well as the corresponding Bland-Alt-man difference plots showed that Ektachem ALT va-lues were higher than those measured by TechniconRA-XT, Monarch or by Hitachi 704. The inter-instru-ment bias may be due to higher reference values ofKodak Ektachem method and the fact that ALT acti-


Monarch ili Hitachi 704. Dobijene razlike u vrednosti-ma za ALT na razli~itim analizatorima mogu biti rezul-tat vi{ih referentnih vrednosti metode Kodak Ektachemi ~injenice da je aktivnost ALT odre|ivana bez dodatkapiridoksal-5-fosfata u reakcionom puferu na ana-lizatorima Technicon RA-XT, Monarch i Hitachi 704.

A7

EVALUACIJA MEGX (MONOETILGLICINEKSILIDID) TESTA

M. Ili}1, S. Stankovi}1, P. Svorcan2, B. Dap~evi}2, B. Radevi}3, D. Meci}2,

N. Majki}-Singh1

1Institut za medicinsku biohemiju, Klini~ki centar Srbije, Beograd

2Institut za gastroenterohepatologiju, Klini~ko-bolni~ki centar Zvezdara, Beograd

3Institut za kardiovaskularne bolesti, Dedinje, Beograd

Monoetilglicineksilidid (MEGX) je primarni metabo-lit lidokaina. Nastaje oksidativnom N-deetilacijom uhepati~nim mikrozomima uz u~e{}e citohroma P-450.Zbog ekstenzivnog metabolizma lidokaina, pokazanoje da metabolizam lidokaina moè da posluì kaoindikator funkcije jetre. MEGX test je superioran uodnosu na druge konvencionalne testove, u procenikvaliteta donor organa za transplantaciju jetre, dokniske vrednosti MEGX-a kod donora zna~ajno ko-reliraju sa slabim graft preìvljavanjem kod recipienata.Tako|e ovaj test sluì za preoperativnu procenuhepati~ne funkcionalne rezerve i zna~ajno korelira sapostoperativnim tokom, a predstavlja i prognosti~kiindikator hroni~nih bolesti jetre. Tokom ispitivanja pa-cijentima je davan lidokain i.v. u bolusu 1 mg/kg te-lesne teìne, a 15 min nakon toga pacijentima je uzi-man uzorak krvi. Za odre|ivanje MEGX-a u serumukori{}en je test firme Abbot Laboratories (Abbot ParkIL 60 064). Test se zasniva na fluorescentno polariza-cionoj imuno-metodi (FPIA). Nepreciznost u seriji ispi-tana je odre|ivanjem koncentracije MEGX-a koriste}itri kontrole u kojima su koncentracije MEGX-a bile 50,100, 200 ng/mL. Dobijeni su koeficijenti varijacijemanji od 5%. Sli~ni koeficijenti varijacije dobijeni su ipri ispitivanju nepreciznosti iz dana u dan (2,58%,4,04%, 4,26%, redom za tri pomenute kontrole). Re-zultati ispitivanja linearnosti dali su srednji »recovery«97,24 ± 1,04%. Dobijeni rezultati pokazuju da su anal-iti~ke karakteristike testa takve da omogu}avaju pouz-dano i brzo odre|ivanje MEGX-a. Uvo|enjem u prak-su testa za odre|ivanje koncentracije MEGX-a, prvi putna na{im prostorima omogu}ena je preoperativnaprocena funkcionalnog stanja jetre kod pacijenata saindikacijama za resekciju jetre. Dobijeni rezultati dopri-nose predvi|anju hirur{ke intervencije.

vities were determined without pyridoxal-5-phos-phate in the assay buffer on the analysers TechniconRA-XT, Monarch and Hitachi 704.

A7

MEGX (MONOETHYLGLYCINEXYLIDE) ASSAY-EVALUATION OF METHOD

M. Ili}1, S. Stankovi}1, P. Svorcan2, B. Dap~evi}2, B. Radevi}3, D. Meci}2,

N. Majki}-Singh1

1Institute of Medical Biochemistry, Clinical Center of Serbia, Belgrade

2Department of Gastroenterohepatology, ZvezdaraUniversity Hospital, Belgrade

3Dedinje Institut of Cardiovascular Diseases, Belgrade

MEGX (monoethylglycinexylidede) is the primarymetabolite of lidocaine. MEGX is formed by oxidativeN-de-ethylation by the hepatic cytochrome P-450 sys-tem. Measurable blood concentrations of MEGX arefound in patients treated with lidocaine. Due to thehigh extraction ration and excessive metabolism oflidocaine, it has been demonstrated that the quantita-tive measurement of lidocaine metabolism can serveas a sensitive indicator of the liver function. MEGX testis advantageous in relation with other conventionaltests, for the assessment of donor's organs needed forliver transplantation. Thus, low MEGX values in donorssignificantly correlate with low graft survival in therecipients. Besides, this test serves for pre-operativeevaluation of the liver functional mass and is signifi-cantly correlated with post-operative course. Thereforeit represents a reliable prognostic indicator of chronicliver diseases. In this study, the patient received a sin-gle bolus dose of lidocaine (1mg/kg body mass we-ight) and blood samples were drawn 15 min after. Se-rum MEGX was determined by commercial kit (AbbotLaboratories, Abbot Park IL 60 064). This assay is ba-sed on fluorescence polarization immunoassay (FPIA).A within run precision was evaluated with replicate me-asurement of MEGX concentration using three com-mercial controls with different MEGX concentrations(50, 100, 200 ng/mL). The obtained coefficients of va-riation (CVs) were lower than 5%. With these three con-trols similar results were obtained for the between runprecision (2.58%, 4.04%, 4.26%, respectively). Averagerecovery was 97.24 ± 1.04%. On the basis of thesefindings, analytical characteristics of this commercialkit prove to be a reliable test for measuring MEGX con-centrations. The introduction of the test for determina-tion MEGX concentration in our clinical practice ena-bled for the first time the preoperative assessment ofthe liver functional state in patients indicative of theliver resection. The obtained results contribute to theforecast of surgical procedure.


A8

»SANDWICH« IMUNO-ODRE\IVANJE KONCENTRACIJE TROPONINA I

U SERUMU

S. Stankovi}, E. ôlak, N. Aleksi}-Markovi}, D. Vukosavljevi}, N. Majki}-Singh


Troponin je proteinski kompleks koji reguli{e kon-trakciju popre~no-prugastih mi{i}nih }elija i sastoji seiz tri komponente: troponina C, troponina I i troponi-na T. Troponin I javlja se u tri izoforme, od kojih sedve nalaze u skeletnim mi{i}ima i jedna u sr~anommi{i}u. Sr~ani troponin I se osloba|a u cirkulacijunakon akutnog infarkta miokarda (AMI). On se u krvipojavljuje 4’8 sati nakon po~etka sr~anog bola, do-stiè maksimalnu koncentraciju 12’16 ~asova i osta-je pove}an 5’9 dana nakon AMI, pa se zbog togaprimenjuje za rano dijagnostikovanje AMI. Cilj ovograda bio je da se ispitaju analiti~ke karakteristike ko-mercijalnog testa za odre|ivanje koncentracije sr~a-nog troponina I u serumu (Opus, Behring-Dade).Test se zasniva na principu sendvi~ odre|ivanja safluorescentno obeleènim sekundarnim antitelom.Ovim testom sr~ani troponin I se odre|uje u roku od15 minuta. Nepreciznost u seriji odre|ivana je ko-ri{}enjem tri razli~ite komercijalne kontrole (DADE®

TRU-Liquid Cardiac Control, MASTM, Level 1,2,3, Cat.No. B5930-1, B 5930-2, B5930-3) i merenjem sr~a-nog troponina I u svakoj 10 puta. Nepreciznost izdana u dan odre|ivana je merenjem koncentracijesr~anog troponina I u istim komercijalnim kontrolnimserumima u toku 7 dana. Linearnost metode ispiti-vana je razblaìvanjem seruma sa visokom vredno{}usr~anog troponina I sa uzorcima seruma u kojima sekoncentracija sr~anog troponina I nije mogla detek-tovati. Za nepreciznost u seriji za prvu kontrolu dobi-jen je Kv=3,72%, dok su koeficijenti varijacije za dru-ge dve kontrole iznosili 4,46%, 3,34%. Dobijeni koefi-cijenti varijacije za nepreciznost iz dana u dan iznosilisu 4,76%, 4,96%, 4,21%. Ispitivanjem linearnosti do-bijen je »recovery« izme|u 96’101%. Na osnovu do-bijenih rezultata moè se zaklju~iti da analiti~ke karak-teristike ovog komercijalnog testa omogu}avaju brzoi pouzdano odre|ivanje koncentracije sr~anog tro-ponina I.

A8

SANDWICH IMMUNOASSAY FOR DETERMINATION OF TROPONIN

I CONCENTRATION IN SERUM



Troponin is the contraction regulating proteincomplex of striated muscle which consists of threedistinct components: troponin C, troponin I and tro-ponin T. The troponin I subunit exists in three iso-forms, two in skeletal muscle and one in cardiacmuscle. Cardiac troponin I is released into blood afteracute myocardial infarction (AMI). It is measurable inserum within 4’ 8 hours following onset of chest pain,peak at 12’16 hours and remain elevated 5’9 daysfollowing an AMI; therefore, it can be used in earlydiagnosis of AMI. The aim of this study was to evalu-ate the analytical characteristics of a two-site or sand-wich fluorogenic enzyme linked imunoassay (Opus,Behring-Dade) for determination of cardiac troponinI concentration in serum. With this test serum cardiactroponin I concentration can be determined within 15min. Three commercial controls (DADE® TRU-LiquidCardiac Control, MASTM, Level 1,2,3, Cat. No.B5930-1, B 5930-2, B5930-3) with different cardiactroponin I levels were analyzed 10 times each to esti-mate the intra-assay precision. Inter-assay precisionwas determined by the evaluation of the same controlsover a period of 7 days. The linearity of dilution wastested using serum samples containing high cardiactroponin I concentrations. Serial dilutions were madewith a normal human sample that has previously beenevaluated and is known to be negative for troponin I.The obtained coefficient of variation for intra-assayprecision of the first control was CV=3.72%, while thecoefficients of variation for two other controls were4.46% and 3.34%, respectively. The obtained coeffi-cient of variation for inter-assay precision of three con-trols were: 4.76%, 4.96% and 4.21%. Linearity »recov-ery« was between 96’101%. Therefore, we concludethat analytical characteristic of this commercial kitprove to be a reliable test for measuring cardiac tro-ponin I concentrations.


A9

»SANDWICH« IMUNO-ODRE\IVANJE MASE CK-MB U SERUMU



Merenje aktivnosti serumskih enzima kao sr~anihmarkera dugo je sluìlo za evaluaciju kardiovaskular-nih oboljenja. Razvitkom imuno-odre|ivanja, omogu-}eno je odre|ivanje koncentracije enzima, nezavisnood toga da li je enzim aktivan ili ne. Prvi test za odre-|ivanje koncentracije CK-MB mass opisan je 1985.godine, a CK-MB mass danas predstavlja »zlatni stan-dard« me|u biohemijskim markerima za dijagnosti-kovanje infarkta miokarda. Odre|ivanje masene kon-centracije CK-MB ograni~eno je jer se ona pove}avatek nakon 4’6 h od pojave prvih simptoma i ne moèda se koristi za utvr|ivanje nestabilne angine pektoriskod pacijenata. Cilj ovog rada bio je da se ispitajuanaliti~ke karakteristike komercijalnog testa za odre-|ivanje masene koncentracije CK-MB u serumu (Opus,Behring-Dade). Test se zasniva na principu sendvi~imuno-odre|ivanja. Masena koncentracija CK-MB userumu moè se odrediti za 10 minuta. Nepreciznostmetode procenjena je analiziranjem tri komercijalnekontrole (DADE® TRU-Liquid Cardiac Control,MASTM, Level 1,2,3, Cat. No. B5930-1, B 5930-2,B5930-3) sa razli~itim masenim koncentracijamaCK-MB. Vrednosti koeficijenata varijacije za preciz-nost u seriji iznosile su 1,78% do 3,04%, dok su vred-nosti koeficijenata varijacije za preciznost iz dana udan bile u opsegu od 2,61% do 4,62%. Utvr|eno jeda je metoda za odre|ivanje masene koncentracijeCK-MB na Opus ta~na. Vrednosti dobijene analizomkontrolnog seruma sa normalnom i povi{enom ma-senom koncentracijom CK-MB u serumu nisu se zna-~ajno razlikovale od pravih vrednosti (P>0,05). Rez-ultati ispitivanja linearnosti dali su »recovery« izme|u92’101%. Dobijeni rezultati pokazuju da su analiti~kekarakteristike testa takve da omogu}avaju pouzdanoi brzo odre|ivanje masene koncentracije CK-MB userumu.

A9

SANDWICH IMMUNOASSAY FOR DETERMINATION OF CK-MB MASS

CONCENTRATION IN SERUM



The initial markers for cardiac diseases wereenzymes whose activity was measured to determinethe extent of cardiac damage. The development ofimmunoassay measurements enabled determinationof the enzyme concentration, which was not depend-ent on whether or not the enzyme was active. The firstmanual CK-MB mass assay was described in 1985and today CK-MB mass is still considered the »goldstandard« as a biochemical marker for acute myocar-dial infarction. The utility of CK-MB mass is limitedbecause blood concentrations do not increase until4’6 hours after symptom's onset, and CK-MB was»not effective« for identifying patients with unstableangina pectoris. The aim of this study was to evaluatethe analytical characteristics of a two-site or sandwichfluorogenic enzyme linked imunoassay (Opus, Be-hring-Dade) for determination of CK-MB mass con-centration in serum. With this test CK-MB mass se-rum concentration can be determined within 10 min.The imprecision was assessed by analyzing threecommercial controls (DADE® TRU-Liquid CardiacControl, MASTM, Level 1, 2, 3, Cat. No. B5930-1, B5930-2, B5930-3) with different CK-MB mass levels.Within-run coefficient of variation were in the rangefrom 1.78% to 3.04%, whereas day-to-day coeffici-ents of variation were in the range from 2.61% to4.62%. The Opus CK-MB mass assay was found tobe accurate: the values obtained in control sera withnormal and elevated CK-MB mass values did not sig-nificantly differ from the corresponding target values(P>0.05). Linearity »recovery« was between 92’101%.Therefore, we conclude that analytical characteristicof this commercial kit proves to be a reliable test formeasuring CK-MB mass concentrations.


A10

VREDNOSTI UREE I ACIDOBAZNIH PARAMETARA KOD PACIJENATA

NA HEMODIJALIZI

E. ôlak1, S. Stankovi}1, M. Radovi}2, N. Majki}-Singh1


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

Zbog smanjenog obima bubre`nog rada u hro-ni~noj bubre`noj insuficijenciji (HBI) ~esto dolazi doizraène metaboli~ke acidoze i patolo{kog kataboliz-ma pra}enog hipoksijom. Uzrok nastanka metabo-li~ke acidoze je smanjena tubularna sekrecija amoni-jaka, usled smanjene sinteze, uslovljene smanjenimbrojem bubre`nih kanali}a. Ispitivan je acidobaznistatus 74 pacijenata obolela od HBI, koji su na pro-gramu hemodijalize, od toga 36 mu{karaca starostiod 21’ 85 godina i 38 èna starosti od 30 ’78 godina.Pacijentima su odre|ivani slede}i parametri: pH,pCO2, pO2, HCO3

’, TCO2 i urea pre i posle hemodija-lize s ciljem da se procene vrednosti ureje i stepenstabilizacije acidobaznog statusa posle hemodijalize.Vrednosti pH, HCO3

’, TCO2 posle hemodijalize (pH7,428 ± 0,060; HCO3 25,41 ± 3,44, TCO2 26,57 ±3,56) pokazale su statisti~ki zna~ajan porast(P<0,001) u odnosu na vrednosti pre hemodijalize(pH 7,350 ± 0,050; HCO3

’ 20,88 ± 2,92, TCO2 22,03± 3,00), dok su vrednosti uree posle hemodijalize bilestatisti~ki zna~ajno manje (P<0,001) u odnosu naone pre hemodijalize. U grupama mu{karaca i èna,vrednosti pH, HCO3

’, TCO2 posle hemodijalize bilesu statisti~ki zna~ajno ve}e (P<0,001) u odnosu navrednosti pre hemodijalize, dok su vrednosti ureeposle hemodijalize bile statisti~ki zna~ajno manje(P<0,001) u pore|enju sa onima pre hemodijalize.Ovim je pokazano da kod pacijenata sa HBI oba polapostoji pozitivan uticaj hemodijalize na kompenzo-vanje nastale metaboli~ke acidoze.

A10

THE ANALYSIS OF ACID-BASE BALANCEAND UREA VALUES

IN HAEMODIALYZED PATIENTS



2Institute of Urology and Nephrology,Clinical Center of Serbia, Belgrade

Due to reduced scope of renal function in chron-ic renal failure (CRF), it is not rare that it comes tomarked metabolic acidosis and pathologic catabo-lism associated with hypoxia. The cause of metabolicacidosis is diminished tubular secretion of ammonia,due to reduced synthesis, stipulated by lower numberof renal canaliculi. Acid-base balance and values ofurea were analyzed in 74 patients suffering from CRF,who were on haemodialysis programme, out of whom36 were males aged 21’ 85 and 38 were femalesaged 30’78 years. The following parameters weremeasured in these patients: pH, pCO2, pO2, HCO3

’,TCO2, urea before and after haemodialysis with aview to evaluate the degree of stabilization of acid-base balance and urea values after haemodialysis.The values of pH, HCO3

’, TCO2 after haemodialysis(pH 7.428 ± 0.060; HCO3

’ 25.41 ± 3.44, TCO2 26.57± 3.56) were significantly increased (P<0.001) inrelation to values before haemodialysis (pH 7.350 ±0.050; HCO3

’ 20.88 ± 2.92, TCO2 22.03 ± 3.00) andvalues of urea after haemodialysis were significantlydesreased (P<0.001) in relation to values of ureabefore haemodialysis. In male and female groups, thevalues of pH, HCO3

’, TCO2 after haemodialysis weresignificantly higher (P<0.001) and the values of ureasignificantly lower (P<0.001) in comparison to thosebefore haemodialysis. These data suggested that the-re was a positive effect of haemodialysis in patientsof both sexes to the compensation of the existingmetabolic acidosis and high levels of urea in CRFpatients.


A11

UTICAJ ETIOLOGIJE HRONI^NEBUBRE@NE INSUFICIJENCIJE NA VREDNOSTI ACIDOBAZNIH

PARAMETARA, UREE, KREATININA, KALCIJUMA I FOSFATA

KOD HEMODIJALIZIRANIH PACIJENATA



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

Hroni~na bubre`na insuficijencija (HBI) javlja seusled smanjenog broja bubre`nih kanali}a koji pro-gresivno propadaju. Ova bolest je pra}ena prome-nom acidobaznog statusa pacijenata. Cilj ispitivanjaje bio da se utvrdi da li postoji povezanost izme|u eti-ologije nastanka HBI i stepena acidobaznog poreme-}aja, kao i izme|u etiologije nastanka HBI i vrednostiuree, kreatinina, kalcijuma i neorganskog fosfata.Ispitivan je acidobazni status 74 pacijenta obolela odHBI, koji su na programu hemodijalize, od toga 36mu{karaca starosti od 21’ 85 godina i 38 èna sta-rosti od 30’78 godina. U zavisnosti od osnove bolestikoja je kao posledicu imala HBI, pacijenti su pode-ljeni u pet grupa: pacijenti sa tubulsrnom intersticijal-nom nefrozom-TIN (18 pacijenata), policisti~nom bo-le{}u bubrega-ADPKD (13 pacijenata), glomerulone-fritisom (GN) (22 pacijenta), hipertenzijom i nefroan-giosklerozom (HTA-Nascl) (9 pacijenata) i peta grupakoju su ~inili pacijenti ~ija osnovna bolest nije utvr|e-na-ESRD (12 pacijenata). Pacijentima su odre|ivanislede}i parametri: pH, pCO2, pO2, HCO3

’, TCO2,urea, kreatinin, kalcijum i neorganski fosfat pre i po-sle hemodijalize s cijem da se utvrdi uticaj osnovnogoboljenja na vrednost ovih parametara. Utvr|eno jeda ne postoji statisti~ki zna~ajna razlika u vrednosti-ma odre|ivanih parametara (P>0,05) u odnosu naosnovno oboljenje, ni pre, ni posle dijalize, kako kodmu{karaca, tako i kod èna. Utvr|ena je statisti~kizna~ajna razlika unutar svih pet grupa bolesti u vred-nostima pH, HCO3

’, TCO2, uree i kreatinina poslehemodijalize (P<0,05). Tako|e je pokazano da sevrednosti pH, HCO3

’ i TCO2 statisti~ki zna~ajno naj-vi{e pove}avaju nakon hemodijalize u grupi pacijena-ta sa TIN-om, a zatim kod pacijenata sa policisti~nombole{}u bubrega, dok se vrednosti uree i kreatininanajvi{e sniàvaju posle hemodijalize kod pacijenata saHTA-Nascl, a zatim kod pacijenata sa TIN-om.

A11

THE EFFECT OF ETIOLOGY OF CHRONIC RENAL FAILURE

ON ACID-BASE PARAMETER, UREA, CREATININE, CALCIUM AND INORGANIC

PHOSPHORUS VALUES IN HAEMODIALYZED PATIENTS



2Institute of Urology and Nephrology, Clinical Center of Serbia, Belgrade

Chronical renal failure (CRF) occurs due toreduced number of renal canaliculi deteriorating pro-gressively. This disease is associated with the changeof patient's acid-balance. The object of the study wasto define the correlation between the etiology of CRFand the degree of acid-base balance disorder andbetween etiology of CRF and values of urea, creati-nine, calcium and inorganic phosphorus. Acid-basebalance was tested in 74 patients suffering from CRF,being on haemodialysis program, out of whom 36were males aged 21’ 85 and 38 females aged 30 ’78years. In relation to underlying disease resulting inCRF, the patients were divided into five groups: I-tubular interstitial nephrosis-TIN (18 patients), II-poly-cystic kidney disease-ADPKD (13 patients), III-glomerulonephritis-GN (22 patients), IV-hypertensionand nephroangio-sclerosis (HTA-Nascl) (9 patients),and V-consisting of patients whose underlying dis-ease was not diagnosed-ERSD (12 patients). The fol-lowing parameters were measured in our patients:pH, pCO2, pO2, HCO3

’, TCO2, urea, creatinine, calci-um and inorganic phosphorus before and afterhaemodialysis with a view to determine the effect ofunderlying disease to the value of these parameters.It was concluded that there was no statistically signif-icant difference of values of measured parameters(P>0.05) in relation to underlying disease eitherbefore or after haemodialysis, both in males andfemales. Statistically significant difference was foundin pH, HCO3

’, TCO2, urea and creatinine values, with-in all five disease groups, in patients before and afterhaemodialysis (P<0.05). Likewise, it was shown thatpH, HCO3

’ and TCO2 values were significantly incre-ased to the maximum after haemodialysis in patientswith TIN, and urea and creatinine values significantlydecreased after haemodialysis, the most in patientswith HTA-Nascl and TIN.

A12

KOMPARATIVNO ODRE\IVANJE BILIRUBINA U SERUMU METODOM

JENDRASSIK-GROF PRIMENOM DVA AKCELERATORA

E. ôlak, S. Stankovi}, D. Pejak


Naj~e{}e kori{}ena metoda za odre|ivanje bilirubi-na zasniva se na diazo reakciji koju je prvi opisao Erlichjo{ 1883 a koja je po~ela da se primenjuje od 1916.godine. Danas postoje brojne modifikacije ove meto-de za odre|ivanje bilirubina koje se me|usobno razli-kuju, uglavnom, po akceleratorima. Kao akceleratorinaj~e{}e se koriste kofein-benzoat, metanol, Brij 35,urea-benzoat, dimetilsulfoksid itd. U ovom radu su na120 uzoraka seruma, metodom sa diazo reagensompri ~emu su kao akceleratori kori{}eni kofein-natrijumbenzoat i dimetilsulfoksid, odre|ivani ukupan i konju-govani bilirubin. Cilj rada bio je da se utvrdi da li pos-toji korelacija izme|u dva pomenuta postupka, kao i dase odredi s kojim akceleratorom se moè posti}i ve}aosetljivost i preciznost. Pore|enjem rezultata ove dvemetode dobijen je koeficijent korelacije za ukupanbilirubin r=0,993 i jedna~ina prave yx = ’3,523 +1,210x (Sy,x = 7,884 mmol/L), a za konjugovani biliru-bin r=0,989 i jedna~ina prave yx = ’2,596 +1,426x(Sy,x = 7,078 mmol/L). Metoda za odre|ivanje bilirubi-na u kojoj se kao akcelerator koristi dimetilsulfoksid,s obzirom na dobijene rezultate, predstavlja adekvatnuzamenu za metodu za odre|ivanje bilirubina sa kofein-benzoatom kao akceleratorom.

A13

MODIFIKACIJA STERNHEIMER-OVOGBOJENJA U ANALIZI SEDIMENTA URINA

M. Milivojevi}, N. Lali}


Ispitivanje urina je jedna od najstarijih tehnika kojase danas koristi u laboratorijskoj praksi. Mikroskop-ska analiza sedimenta urina naj~e{}e podrazumevaispitivanje nativnog neobojenog preparata svetlosnimmikroskopom. Ova tradicionalna metoda diferenci-jacije sedimenta je i najteà zbog malog kontrastaizmedu elemenata i pozadine; naro~ito je te{ko uo~itihijaline cilindre, sluzi razne vrste }elija. Tehnike boje-


A12

COMPARATIVE DETERMINATION OFBILIRUBIN IN SERUM USING JENDRASSIK-GROF METHOD:

COMPARISON OF TWO DIFFERENT ACCELERATORS

E. ôlak, S. Stankovi}, D. Pejak


The most widely used methods for bilirubin meas-urement are those based on the diazo reaction,which was first described in 1883 by Ehrlich and thenapplied in 1916 by van den Bergh and Miller. Today,numerous variations of these procedure have beendeveloped. All of them utilize one of a variety of acce-lerators such as: caffeine-benzoate, methanol, Brij35, urea-benzoate, dimetylsulfoxide etc. The totaland conjugated bilirubin have been determined in120 human serum by diazo reaction using coffeine-benzoate and dimetylsulfoxide as accelerators. Theaim of this work was to compare these two accelera-tors in order to find what kind of correlation existsbetween these two procedures and also to define theaccelerator which is responsible for better precisionand sensitivity of the method. The comparison ofresults between these two methods yielded the fol-lowing regression parameters: for total bilirubin yx =’3.523 + 1.210x (r = 0.993, Sy,x = 7.884 mmol/L)and for conjugated bilirubin yx = ’2.596 + 1.426x (r= 0.989, Sy,x = 7.078 mmol/L). On the basis of thesefindings, method of determination of bilirubin withdimetylsulfoxide as accelerator offers an adequatealternative to the other mentioned method with caf-feine-benzoate as accelerator.

A13

MODIFICATION OF STERNHEIMER'SSTAINING IN URINE SEDIMENT ANALYSIS

M. Milivojevi}, N. Lali}


Urine analysis is one of the oldest techniques stillused in laboratory practice. Microscopic analysis ofthe urinary sediment most frequently includes exam-ination of the plain, unstained preparation usingbrightfield microscop. This traditional method of sed-iment differentiation is the most difficult one due tothe low contrast between the elements and back-ground; it is particularly difficult to distinguish hyaline


nja pomaù u vizuelizaciji razli~itih elemenata u sedi-mentu urina. Papaniuolau je standardno bojenje zadiferncijaciju elemenata sedimenta urina, koje zbogsloènog postupka i duìne trajanja nije primenljivo usvakodnevnom radu. Zato je u na{oj laboratoriji pri-menjeno Sternheimer-ovo citogenetsko bojenje. Ovaoriginalna metoda, koja je prvi put publikovana uJAMA, (1975; 231: 826) modifikovana je i prilago-|ena na{im uslovima. Primenom ove tehnike bojen-ja jasno se boje }elije sa jedrom: citoplazma ovih}elija je svetlocrvena sa tamno crvenim granulama,dok je jedro tamnoljubi~asto obojeno. U ovako obo-jenim preparatima mogu}e je diferencirati poli-morfonuklearne }elije (PMN) i mononuklearne }elije(MN). Ova metoda se primenjuje za identifikaciju iz-menjenih leukocita nazvanih Sternheimer-Malbinove}elije (glitter }elije) koje su ve}e od leukocita a kodkojih je svetloplavo obojena citoplazma i jasno seuo~ava Brawnovo kretanje. Kvalitet ovako dobijenihpreparata je odli~an, metoda je brza i jednostavna aza razliku od originalne metode ostaje deo sedimen-ta neodvojen za druga alternativna ispitivanja. Ovametoda se primenjuje u rutinskom radu od maja2001. godine i do sada je na ovaj na~in obra|enonekoliko stotina uzoraka sedimenata urina.

A14

ME\ULABORATORIJSKA UPOREDNAODRE\IVANJA KONCENTRACIJE BIOLO[KI AKTIVNIH SUPSTANCI POMO]U RADIOIMUNOTESTOVA

Lj. Hajdukovi}-Dragojlovi}1, S. Golubovi}1, V. Resanovi}1, M. Joksimovi}2, M. Jankovi}1,

A. Gruden-Movsesijan1, I. Petrovi}1, S. Marjanovi}1, J. Krsmanovi}1, G. Kosti}1

1Institut za primenu nuklearne energije ’ INEP, Zemun2Vojnomedicinska akademija, VMA, Beograd

U oblasti radioimunodijagnostike u na{oj zemljido sada nije uspostavljena organizovana spolja{njakontrola rada u laboratorijama. Kako postoji potrebaza me|ulaboratorijskim uporednim ispitivanjima, od-re|ivane su koncentracija TSH, T3, T4, TBG, FSH,LH, PROG i PSA u humanim serumima (n=18’49)paralelno u laboratorijama INEP-a i Instituta za nuk-learnu medicinu, VMA. Za ova ispitivanja kori{}eni suradioimunotestovi proizvedeni u INEP-u. Statisti~kaobrada podataka izvr{ena je uz pomo} MicrosoftExcel programa, a zasnivala se na izra~unavanju t i rvrednosti (Student-t test ’ izra~unavanje vrednosti t izrazlike izme|u parova odre|ivanja). Vrednosti koefici-

casts, mucus and different cell types. Staining tech-niques contribute to visualization of different ele-ments in the urine sediment. Papanicolau is standardstrain differentation elements in the urine sedimentbut it is also time-consuming and not useful for rou-tine work. Therefore, Sternheimer's cytogenetic strai-ning was applied in our laboratory. This original met-hod, which was initially described in JAMA, (1975;231: 826) is modified and adjusted for our condi-tions. Application of the straining method enablesclear distinguishing of the nuclear cell color: the cyto-plasm of the cells is light red with dark red granules,while the nucleus is dark purple colored. The prepa-rations stained in this way enable differentition of thepolymorphonuclear cells (PMN) and mononuclearcells (MN). The method is used for identification ofthe altered leukocytes names Sternheimer/Malbin’scells (glitter cells) which are larger than leukocytes,their cytoplasm is light blue colored and Brown’smovoment is easily observable. Quality of the obta-ined preparations is excellent, the method is quickand simple and, as opposed to the original method,a part of the sediment remains unstained for otheralternative examinations. The method has been rou-tinely applied since May 2001 and few hundred urinesediment samples were defined so far in this way.

A14

INTERLABORATORY COMPARATIVE MEASUREMENTS OF BIOLOGICALLY

ACTIVE SUBSTANCES USING RADIOIMMUNOASSAYS

Lj. Hajdukovi}-Dragojlovi}1, S. Golubovi}1, V. Resanovi}1, M. Joksimovi}2, M. Jankovi}1,

A. Gruden-Movsesijan1, I. Petrovi}1, S. Marjanovi}1, J. Krsmanovi}1, G. Kosti}1

1Institute for the Application of Nuclear Energy’ INEP, Zemun

2Military Medical Academy ’ MMA, Belgrade

External quality assessment is not well establishedin the field of radioimmunodiagnostics in our coun-try. Since there is a need for this kind of study, INEPand the Institute for Nuclear Medicine performed pre-liminary comparative measurements of TSH, T3, T4,TBG, FSH, LH, PROG and PSA in human sera(n=18 ’49), using INEP radioimmuno-kits (RIA andIRMA). Statistical evaluation of the obtained resultswas performed using the Microsoft Excel Programand based on calculation of t and r values (paired twosample for means). The coefficients of correlation(ranging from 0.862 to 0.997) indicated a close rela-tion between the results obtained in two laboratories


jenata korelacije (r = 0,862 do r =0,997) ukazale suna dobru korelaciju izme|u rezultata dobijenih u dvelaboratorije kod svih ispitanih analita. Me|utim, sta-tisti~ki zna~ajne razlike na|ene su za sve ispitivaneanalite osim kod uporednih odre|ivanja FSH i TSH.Generalno, u laboratoriji VMA dobijane su vi{e vred-nosti u odnosu na laboratoriju INEP-a, {to potvr|ujepotrebu za reevaluacijom referentnih vrednosti u sva-koj laboratoriji. Ovo je u saglasnosti sa preporukomproizvo|a~a radioimunotestova da svaka laboratorijatreba da ustanovi svoj opseg referentnih vrednosti.Sprovedena me|ulaboratorijska ispitivanja predstavl-jaju prvi korak u pravcu uvo|enja spolja{nje kontrolekvaliteta s ciljem da ona obuhvati sve laboratorije ko-je koriste testove tipa RIA i IRMA.

A15

SMERNICE ZA PRIMENU SPECIFI^NIHPROTEINA U KLINI^KIM OBOLJENJIMA

D. Pap1, S. Bugarinovi}2, V. Cveji}3

1Klini~ko-biohemijska laboratorija, Zdravstveni centar Sremska Mitrovica

2Specijalna neuropsihijatrijska bolnica, Kovin3Institut za biohemiju, Medicinski fakultet, Beograd

Ljudsko telo sadr`i niz raznovrsnih proteina, odkojih su neki rastvorljivi u te~nostima unutar ili van}elija, a neki su nerastvorljivi i strukturni. U plazmi jeidentifikovano vi{e od 300 proteina, ali se oni veomarazlikuju po koncentraciji, pa }e u ovom ~lanku bitirazmatrani samo oni kojih ima najvi{e. U poslednjojdeceniji, znanje o njihovoj ekspresiji i }elijskim funkci-jama je zna~ajno pove}ano ~ime je, obezbe|enosnov za pove}anu klini~ku primenu ovih proteina.Pozitivni proteini akutne faze su grupa proteina ~ija sekoncentracija pove}ava za vreme inflamatornih pro-cesa hirur{kog, toplotnog, neoplasti~nog, infektivnogili nekog drugog porekla. Proteini od teku}eg klini~-kog interesa su C-reaktivni protein, a1-kiseli glikopro-tein, haptoglobin, a1-antitripsin, a1-antihimotripsin iceruloplazmin. U radu je dat prikaz savremenih saz-nanja o dijagnosti~kom i klini~kom aspektu proteinaakutne faze i drugim specifi~nim proteinima kao {tosu prealbumin, albumin, mikroalbumin, transferin,kapa i lambda lakim lancima, reumatoidnom faktoru,a2-makroglobulinu, b2-makroglobulinu, imunoglob-ulinima M, G, A, komponentama komplementa C3 iC4, antitrombinu III, apolipoproteinima (apoA1 iapoB), properdin faktoru B, lipoproteinu (a), alfa-fe-toproteinu, mijelinskom baznom proteinu (MBP) i S-100 proteinu. U ovom radu prikazane su mogu}nosti

for all the examined analytes. However, statisticallysignificant differences were found for all the analytesexcept for FSH and TSH. Generally, higher valueswere obtained in the laboratory of the Institute forNuclear Medicine compared with the INEP laborato-ry, which confirms the need for reevaluation of refe-rence values in each laboratory. It is in agreementwith the general suggestion of producers of radio-immuno kits, that each laboratory should establishits own range of reference values. These interlabora-tory comparative measurements represent the firststep towards the establishment of external qualityassessment aiming at encompasing all laboratorieswhich use radioimmuno kits.

A15

GUIDELINES FOR THE USE OF SPECIFICPROTEINS IN CLINICAL DISORDERS

D. Pap1, S. Bugarinovi}2, V. Cveji}3

1Clinical-Biochemical Laboratory, Medical Center Sremska Mitrovica

2Psychiatric Hospital, Kovin3Institute of Biochemistry,

School of Medicine, Belgrade

The human body contains countless differentproteins, some are soluble in fluids within or outsidethe cells, some are insoluble and structural. In plas-ma, more than 300 proteins have been indentified,but they differ greatly in concentration and only themost abundant will be considered in this study.During the last decade, knowledge about theirexpression and cellular functions has rapidly accu-mulated providing the basis for increase of clinicalapplication of these proteins. Positive acute phaseproteins are the group of proteins whose concentra-tion increases significantly during an inflammatoryprocess of surgical, thermal, neoplastic, infectious orany other origin. The proteins of current clinical inter-est are C-reactive protein (CRP), a1-acid glycopro-tein, haptoglobin, a1-antitrypsin, a1-antichymotryp-sin and ceruloplasmin. The article deals with reviewof up-to-date knowledge about diagnostic and clini-cal aspects of acute phase proteins and other specif-ic proteins as well as albumin, microalbumin, preal-bumin, transferrin, kappa and lambda light chain,rheumatoid factor, a2-macroglobulin, b2-microglo-bulin, immunoglobulins M, G, A, complement C3and C4, antitrombin III, apolipoproteins (apoA1 andapo B), properdin factor B, lipoprotein (a)-Lp(a),alpha-fetoprotein (AFP), myelin basic protein (MBP)


klini~ke primene ovih specifi~nih proteina i proteinatelesnih te~nosti sa smernicama za upotrebu ovihproteina u prognostici, dijagnostici i terapiji u razli~i-tim oboljenjima i patolo{kim stanjima.

A16

DIABETES MELLITUS: PRA]ENJE POMO]UFRUKTOZAMIN TESTA - GLIKOZILIRANOG

SERUMSKOG PROTEINA

D. Pap1, M. Babi}2, V. Cveji}3

1Klini~ko-biohemijska laboratorija, Zdravstveni centar Sremska Mitrovica

2Dom zdravlja Po`ega3Institut za biohemiju, Medicinski fakultet, Beograd

Diabetes mellitus (DM) kontroli{e se kratkoro~nimi dugoro~nim pra}enjem vrednosti glukoze. Kada suvrednosti glukoze pove}ane raste i koncentracija fruk-tozamina (FRU). Zato je odre|ivanje FRU va`no zakratkoro~nu i srednjoro~nu kontrolu glikemije u toku2’3 nedelje. Cilj ovog rada bio je ispitivanje dijagnos-ti~ke efikasnosti pra}enja regulisanosti DM primenomfruktozamin testa. Fruktozamin je odre|ivan kod 136zdravih osoba (kontrolna grupa) i 188 pacijenata obo-lelih od DM (eksperimentalna grupa). Eksperimen-talna grupa podeljena je u 4 grupe: M1 ’ 54 pacijentaobolela od DM ne-zavisnog od insulina le~enih dije-tom (NIDDM na dijeti); M2 ’ 68 NIDDM na terapijioralnim antidijabeticima; M3 ’ 32 NIDDM le~ena insu-linom; M4 ’ 34 pacijenta obolela od insulin zavisnogDM (IDDM). Pacijenti su bili oba pola, iste starosnedobi kao i kontrola grupa i ispitani su u toku tri nedel-je. FRU je odre|en NBT kolorimetrijskom metodom(test Menarini), a koncentracija glukoze standardnomenzimskom metodom (GOD-PAP metod). FRU i vred-nosti glukoze u serumu bile su zna~ajno vi{e (p<0,01) kod svih grupa pacijenata u pore|enju sa zdrav-im osobama za vreme perioda pra}enja DM. FRU jebio u zna~ajnoj korelaciji sa glikemijom u toku posled-nje 2 nedelje. U svim grupama dobijene su slede}evrednosti ispitanih parametara: M1 glukoza 7,36 ±1,39 mmol/L, a FRU od 258’320 mmol/L; M2 9,60 ±3,77 mmol/L; FRU 346’386 mmol/L; M3 12,25 ±3,62 mmol/L, FRU 447’509 mmol/L; M4 15,01 ±5,95 mmol/L; FRU 497’587mmol/L; kontrolna grupa5,05 ± 0,75 mmol/L; FRU 174’225 mmol/L. Istovre-menim odre|ivanjem oba parametra dobija se uvid ukontrolisanost bolesti i prepoznavanje skorije me-taboli~ke dekompenzacije. Dobijeni rezultati ukazujuda je odre|ivanje FRU srednjoro~an marker za pra}e-nje kontrolisanosti DM u odnosu na njihovu terapiju.

and S-100 protein. The present review summarizesthe possibilities of clinical application of cited speci-fic proteins and proteins of body fluids with guide-lines for the use of these proteins as prognostic, dia-gnostic and therapeutic tools in various disease con-ditions and disorders.

A16

DIABETES MELLITUS: MONITORING BY MEANS OF FRUCTOSAMINE - GLYCATED SERUM PROTEIN

D. Pap1, M. Babi}2, V. Cveji}3

1Clinical-Biochemical Laboratory, Medical Center Sremska Mitrovica

2Medical Center, Po`ega3Institute of Biochemistry, School of Medicine,

Belgrade

Diabetes mellitus (DM) can be assesed by the longterm monitoring and control of glucose levels as shortterm indicator. When blood glucose levels are abnor-mally elevated the concentration of fructosamine alsoincreases. The measurement of fructosamine (FRU) istherefore useful in monitoring short to mediumglycemic control in DM, over the past 2’3 weeks. Thepurpose of this study was to evaluate diagnostic effi-ciency for monitoring of DM by fructosamine assay.The studied subjects were the control group (136healthy subjects) and the experimental group (188 DMpatients). The experimental group divided in fourgroups: M1 ’ 54 non-insulin dependent DM patients(NIDDM) on diet; M2 ’ 68 NIDDM patients on oralantidiabetes therapy; M3 ’ 32 NIDDM patients oninsulin; M4 ’ 34 insulin dependent patients (IDDM).Patients were both sexes, age matched and monitor-ing in last 3 weeks. We performed FRU determina-tions (by NBT colorimetric method) ’ test kit Menarini.Glucose concentration was measured by GOD-PAPmethod (test kit Randox). FRU and glucose values inserum were significantly higher (p< 0.01) in all groupsof patients compared to the control group duringwhole period of monitoring of DM. FRU was signifi-cantly correlated with glycemia over the past 2 weeks.The results of examined parameters in all groups haveshown the following values: M1 for glucose 7.36 ±1.39 mmol/L; and FRU values ranged from 258’320mmol/L; M2 9.60 ± 3.77 mmol/L; FRU 346’386mmol/L; M3 12.25 ± 3.62 mmol/L; FRU 447’509mmol/L; M4 15.01 ± 5.95; FRU 497’587 mmol/L;control group 5.05 ± 0.75 mmol/L; FRU 174’225mmol/L. Simultaneous determination of both param-eters allows us to emphasize the recent metabolicdecompensation. The results suggest that fructosa-mine assay is useful medium-term marker to monitordiabetic patients in regard to their therapy.

A17

C-REAKTIVNI PROTEIN U KAPILARNOJ I VENSKOJ KRVI:

PORE\ENJE REZULTATA AGLUTINACIONOG SKRINING TESTA

S. Stojiljkovi}, S. Stojiljkovi}, J. Lali}

Centar za medicinsku biohemiju, Klini~ki centar, Ni{

C-reaktivni protein (CRP) je reaktant akutne fazebolesti. Zna~ajno se pove}ava kod niza patolo{kih po-reme}aja u organizmu. U {irokoj su primeni kvalita-tivne i semi-kvalitativne metode koje se zasnivaju naaglutinaciji CRP-a u serumu pacijenta i ~estica latek-sa prekrivenih specifi~nim antitelima za humani CRP.Vo|eni idejom da se do dijagnoze do|e br`e i jeftini-je, upore|eni su rezultati kvalitativnog aglutacionogtesta u serumu venske i kapilarne krvi istog pacijenta.Obra|ena su 43 pacijenta (17 CRP-pozitivnih i 26CRP-negativnih). Pra}eni su sedimentacija eritrocita ivrednosti fibrinogena plazme kao pokazatelji akutnogprocesa u organizmu. Dobijeno je apsolutno pokla-panje rezultata aglutinacionog skrining testa za CRP userumu venske i kapilarne krvi. Sedimentacija eritro-cita i fibrinogen u grupi CRP-pozitivnih pacijenata sta-tisti~ki su zna~ajno bili povi{eni (p < 0,001) u odno-su na grupu CRP negativnih pacijenata. Srednja vred-nost fibrinogena u grupi CRP-pozitivnih bila je 5,9 g/La u grupi CRP-negativnih 3,7 g/L. Srednja vrednostsedimentacije eritrocita u grupi CRP-pozitivnih iznosi-la je 41 arbitralnih jedinica, a u grupi CRP-negativnih17 arbitralnih jedinica. Pozitivni CRP aglutinacioniskrining-test je prema dobijenom vrlo siguran poka-zatelj prisustva akutnog procesa u organizmu. Mo-gu}nost da se on sa jednakom precizno{cu radi i ukapilarnom uzorku krvi doprinosi njegovoj br`oj i jef-tinijoj primeni.

A18

STANDARDIZACIJA KAO FAKTOR KONTROLE KVALITETA:

PRIKAZ IMUNORADIOMETRIJSKIH TESTOVA ZA ODRE\IVANJE

KONCENTRACIJE ALFA-FETOPROTEINA I PROLAKTINA

M. Jankovi}, S. Golubovi}

Institut za primenu nuklearne energije, INEP, Beograd

Standardizacija je deo laboratorijske kontrole kva-liteta. Cilj standardizacije metoda je da pobolj{a upo-redivost rezultata iz razli~itih laboratorija, pove}avaju-


A17

C-REACTIVE PROTEIN IN VENOUS AND IN CAPILLARY BLOOD:

THE COMPARISON OF AGGLUTINATIONSCREENING-TEST RESULTS

S. Stojiljkovi}, S. Stojiljkovi}, J. Lali}

Center of Medical Biochemistry, Clinical Centre of Ni{, Yugoslavia

C-reactive protein (CRP) is very important acute-phase reactant. For some pathological diseases CRPlevel is significantly increased. Qualitative and semi-qualitative methods based on agglutination of CRP inpatient serum and latex particles coated with specifichuman CRP antibodies are frequently used. Inattempt to get fast and inexpensive diagnose wecompared the results of qualitative agglutination testof venous and capillary blood of the same patient.Test had been made for 43 patients, 17 of thembeing CRP positive and 26 CRP negative. For thesame patients we measured sedimentation of ery-throcytes and the level of plasma-fibrinogen, as indi-cator of acute processes. We found out identicalresults for agglutination of CRP in venous as well asin capillary blood. The values of plasma fibrinogenand the sedimentation of erythrocytes in the CRP-positive group are significant higher (p<0.001) to theCRP-negative group. Mean value of plasma fibrino-gen in the CRP-positive group is 5.9 g/L and in theCRP-negative group is 3.7 g/L. Mean value of sedi-mentation of erythrocytes in the CRP-positive groupis 41 arbitary units, and in the CRP-negative group is17 arbitary units. In the conclusion we can say thatmethod for the direct detection of CRP is importantand precise marker to acute diseases. The fact thatthis method is showing as precise results in capillaryblood as in venous blood, contribute to its faster andinexpensive application.

A18

STANDARDISATION AS A QUALITY CONTROL FACTOR: OVERVIEW

OF IMMUNORADIOMETRIC TESTS FOR DETERMINATION

OF ALPHA-FETOPROTEIN AND PROLACTIN

M. Jankovi}, S. Golubovi}

Institute for the Application of Nuclear Energy, INEP, Belgrade

Standardisation is part of laboratory quality con-trol. The aim of methodological standardisation is toimprove the comparability of results from various lab-


}i njihovu ta~nost. Standardi sami po sebi ne garan-tuju uporedivost imuno-testova ali predstavljajuklju~nu komponentu. Proces u kome se esej kalibri-{e, sastoji se od nekoliko koraka u kojima se vrednostprimarnog standarda prenosi na sekundarne i terci-jarne kalibratore koji se koriste u finalnom testu. Touklju~uje odgovaraju}u selekciju standarda i metode,pufera i njihovih matriksa, kontrolu razblaìvanja, sta-tisti~ku evaluaciju i ukupnu kontrolu kvaliteta final-nog rezultata. Odràvanje kalibracije zahteva da sedeo ovog procesa ponavlja svaki put kada se uvodinova serija reagenasa i predstavlja osnovni faktorkontrole kvaliteta imunoradiometrijskih testova kojise proizvode u INEP-u. S tim u vezi, u ovom radu suprikazani rezultati kontrole kvaliteta testova za analizualfa-fetoproteina i prolaktina u humanom serumu.Koncentracije ovih antigena su odre|ivane imunora-diometrijskim testovima proizvo|a~a INEP (Zemun) iCIS bio international (Francuska), koji su standardi-zovani prema referentnim preparatima Svetske zdrav-stvene organizacije: 1IS 72/225 (alfa-fetoprotein) iIRP 84/500 (prolaktin). Statisti~ka obrada rezultatavr{ena je u Microsoft Excel programu izra~unavanjemvrednosti t, iz razlike izme|u parova kod paralelnihodre|ivanja. Na osnovu vrednosti koeficijenta kore-lacije (r=0.9897) za IRMA-AFP i ELSA2-AFP i koefi-cijenta korelacije (r=0.9722) za IRMA-PRL i RIA-gnostPRL, moè se zaklju~iti da su koncentracije alfa-feto-proteina i prolaktina izmerene pomo}u INEP-ovog iCIS-ovog kompleta u dobroj korelaciji.

A19

OPTIMIZACIJA KAPILARNE ELEKTROFOREZE ZA ODRE\IVANJE

MALIH MOLEKULA U PLAZMI

G. @uni}

Institut za medicinska istraìvanja, Vojnomedicinska akademija, Beograd

Kapilarna elektroforeza (KE) razlikuje se od svihdrugih vrsta elektroforeza ne samo zato {to se izvodi ukapilarama, ve} i zato {to je elektroosmoza glavna,mada ne i jedina, sila koja uti~e na pokretljivost kom-ponenti. Tako KE omogu}ava razdvajanje i velikih imalih molekula, a prednosti su odredjivanje malih ko-li~ina komponenata (10’18mola) iz malih uzoraka (nL)u kratkom vremenskom periodu. Me|utim, telesnete~nosti su kompleksne sme{e sa visokim sadràjemproteina i soli, koje modifikuju povr{inu kapilare {touti~e na uslove razdvajanja, vremena migracije kom-ponenata i osetljivosti detekcije pri KE komponenata ubiolo{kim te~nostima. Problemi su posebno izraèni

oratories by increasing the accuracy. Standards alo-ne do not guarantee comparability of immunoassays,but they represent a key component. Assay calibra-tion proceeds in several stages of transfer of the valueof the primary standard to secondary and tertiary cal-ibrators for use in the final analysis. This includes theproper selection of standards and assay methods,buffers and their matrices, control of dilution, statis-tical evaluation and overall control of the final results.Maintenance of calibration requires that part of thisprocess is repeated every time a new batch of rea-gents is introduced, and this is a basic part of thequality control of immunoradiometric tests, produ-ced by INEP. In this work we present the results ofsuch quality control of the tests for alpha-fetoproteinand prolactin in human sera. The concentrations ofthese antigens were determined immunoradiometri-cally (INEP, Zemun and CIS-bio international, Fran-ce), calibrated according to the World Health Orga-nization reference preparations: 1IS 72/225 (alpha-fetoprotein) and IRP 84/500 (prolactin). The resultsobtained were statistically evaluated with the aid ofMicrosoft Excel software, based on the calculation oft values (paired two sample for means). Coefficient ofcorrelation (r=0.9897) for IRMA-AFP and ELSA2-AFP and the coefficient of correlation (r=0.9722) forIRMA-PRL and RIA-gnost PRL indicated that the cor-responding concentrations of alpha-fetoprotein andprolactin, measured by the two different assays werein good correlation.

A19

OPTIMIZATION OF CAPILLARY ELECTROPHORESIS FOR

QUANTIFICATION OF SMALL MOLECULES IN PLASMA

G. @uni}

Institute for Medical Research, Military Medical Academy, Belgrade

Capillary electrophoresis (CE) is different fromother forms of electrophoresis not only in that it iscarried out within the confines of a narrow tube, butdue to electroosmosis as main driving force, amongthe others influencing mobilites of the compounds incapillary. Thus CE enables separation of both largeand small molecules and has many advantages suchas determination of small quantities (10’18mol) insmall sample volumes (nL) within a short period oftime. However, body fluids represent a complexmatrix with high levels of proteins and salts that canmodify the capillary surface influencing separatingconditions, migration times and sensitivity of their


pri analizi malih molekula (joni, peptidi, aminokiseline,itd.) u fiziolo{kim te~nostima, prvenstveno zbog sli~-nosti njihove elektroforetske pokretljivosti, velikog bro-ja razli~itih molekula i razli~itih nivoa njihovih fiziolo{-kih koncentracija. Zato rutinska primena KE u labora-torijama umnogome zavisi od re{enja ovih problema.Ovde su prikazane dve metode, razvijene u na{oj labo-ratoriji za kvantifikaciju nekih malih molekula u plazmipomo}u slobodne zonske KE uz merenje direktne iliinderektne apsorbancije. Koriste}i alkalni boratni pu-fer velike jonske ja~ine u kapilari (36,5 cm × 75 mm),pri potencijalu od 15 kV (inverzna polarnost), uz direk-tno merenje absorbancije na 214 nm razdvojeni sunitrati i nitriti za manje od 10 minuta. Metoda je po-godna za precizno i ta~no odredjivanje ovih anjona uplazmi i cerebrospinalnoj te~nosti bez prethodne de-proteinizacije. Imaju}i u vidu da se nivo njihove kon-centracije koristi kao pokazatelj stvaranja azot oksida,dobijena metoda je jednostavan i brz postupak za od-re|ivanje ovog multifunkcionalnog inter/intra }elijskogmedijatora. Nedavno smo razvili i 30-minutnu proce-duru, koja se zasniva na optimizaciji KE i detektovanjuindirektne apsorbancije, za razdvajanje i kvantifikaciju30 nederivatizovanih fiziolo{kih aminokiselina i pepti-da, koji se obi~no nalaze u biolo{kim te~nostima. Ele-ktroforeza se izvodi u kapilari (87 cm × 75 mm) pri po-tencijalu od 15 kV (normalna polarnost) uz p-amino-salicilnu kiselinu (pH 10,2) kao elektrolit. Ovaj postu-pak je jedna od prvih metoda KE za kvantifikacijuvelikog broja aminokiselina u biolo{kim te~nostima,koja se moè porediti sa jonoizmenjiva~kim ili HPLCmetodama, mada je manje precizna. U zaklju~ku bi semoglo re}i da }e kontinuirano prevazilaènje proble-ma vezanih za analizu biolo{kih te~nosti koriste}i KEbrzo omogu}iti uvo|enje ove nove tehnike u klini~kelaboratorije.

A20

ZNAÂJ PRIMENE SISTEMA KVALITETA

I INFORMACIONOG SISTEMA U KLINI^KO-BIOHEMIJSKOJ LABORATORIJI INSTITUTA

ZA KARDIOVASKULARNE BOLESTI – SREMSKA KAMENICA

V. Ristovski1, G. Prtenjak2

1Institut za kardiovaskularne bolesti, Sremska Kamenica

2Institut za onkologiju, Sremska Kamenica

Institut za kardiovaskularne bolesti u SremskojKamenici obuhvata Kliniku za kardiologiju i Kliniku zakardiovaskularnu hirurgiju. U okviru njih radi labora-torija za biohemiju koja mora pruàti visoko kvalitetne

detection during CE of the compounds in biofluids.There are particular problems in CE of small mole-cules (ions, peptides, amino acids, etc.) present inbiofluids, due to similarities in their electrophoreticmobility, their great number and different physiolog-ical concentrations. Routine use of CE in laboratoriesdepends mostly on resolving these problems. Hereare presented two methods, developed in our labora-tory for quantification of some small molecules inplasma employing free zone CE and direct or indirectabsorbance detection. Employing alkaline borate buf-fer with high ionic strength in uncoated capillary (36.5cm × 75 mm) at 15 kV (inverse polarity), with on-co-lumn UV detection at 214 nm, nitrite and nitrate wereseparated within less than 10 minutes. The methodis suitable for their precise and accurate quantifica-tions both in plasma and cerebrospinal fluid withoutdeproteinization. Considering their levels as com-monly used index of nitric oxide production, obtainedmethod represent simple and rapid procedure for themeasurement of this multifunctional inter/intra cellu-lar mediator. Also, recently we have developed 30minute single-run procedure, based on the optimiza-tion of CE and indirect absorbance detection capabi-lities, for the separation and quantification of 30underivatized amino acids and peptides, usually pre-sent in biological fluids. Electrophoresis was carriedout in a capillary (87 cm × 75 mm) at 15 kV potential(normal polarity) in running electrolyte (p-aminosali-cylic acid, pH 10.2). Obtained method represent oneof the first CE methods for free amino acids quantifi-cation in biofluids, comparable with ion-exchangedor HPLC procedure, although not as precise as theyare. In conclusion, continuous overcoming problemsassociated with analysis of biofluid by CE will bringthis new technuqie into clinical laboratories soon.

A20

THE IMPORTANCE OF THE IMPLEMENTATION OF MANAGMENTQUALITY SYSTEM AND INFORMATION

SYSTEM IN THE CLINICAL-BIOCHEMICALLABORATORY OF THE INSTITUTE OF CARDIOVASCULAR DISEASES

- SREMSKA KAMENICA

V. Ristovski1, G. Prtenjak2

1Institute of Cardiovascular Diseases, Sremska Kamenica

2Institute of Oncology, Sremska Kamenica

Institute of Cardiovascular Diseases in SremskaKamenica include Department of Cardiology and De-partment of Cardiovascular Surgery. Institut includeClinical-biochemical laboratory that must provide a


usluge davanjem ta~nih, preciznih i razumljivih poda-taka neophodnih pri le~enju kardiovaskularnih boles-nika. Potrebni testovi moraju odgovarati klini~komproblemu, analize moraju biti korektno i brzo izvede-ne uz ekonomsku opravdanost. Institut za kardiovas-kularne bolesti u Sremskoj Kamenici u protekloj2001. godini dobio je priznanje Biznis Partner {to gaje obavezalo da i u ovoj 2002. godini odr`i visok nivokvaliteta laboratorijskih usluga. Da bi to i ostvarilo,najva`nije je stalno sprovoditi unutra{nju i povreme-nu spolja{nju kontrolu kvaliteta. Ove kontrole morajuda obuhvate ne samo opremu ve} i stru~nost tehni~-kog i analiti~kog osoblja. Tehnike koje se veoma brzomenjaju i uvo|enje novih testova zahtevaju kontinui-ranu obuku i edukaciju tehni~kog i laboratorijskogosoblja. Obrada klini~kih rezultata u laboratoriji morabiti zasnovana na dva bitna zahteva: implementacijikvalitetne opreme pri organizovanju klini~ke labora-torije i ponovno razmatranje zadataka i poslova klini~-kog biohemi~ara. Uvo|enje sistema kvaliteta u labo-ratoriji i laboratorijskog informacionog sistema pred-stavlja budu}e izazove.

A21

PORE\ENJE FUNKCIONALNIH TESTOVA ZA ISPITIVANJE SISTEMA KOMPLEMENTA

U KLINI^KOJ PRAKSI

M. Mitrovi}1, B. Rodi}-Poli}1, N. Majki}-Singh2, G. Mitrovi}3

1Institut za transfuziju krvi RS2Institut za medicinsku biohemiju,

Klini~ki centar Srbije3Srbolek a.d.

Sistem komplementa ima veliku ulogu u pato-genezi odre|enih oboljenja. U ve}ini slu~ajeva kom-plement nije uzrok ve} samo jedan od faktora kojiu~estvuju u njihovom nastanku. U ovom radu upo-re|ivani su hemoliti~ki i esteroliti~ki testovi u dijag-nostici uro|enog i ste~enog angioneurotskog ede-ma, kao i hemoliti~ki i ELISA testovi u proceni stepe-na aktivacije komplementa u pra}enju toka bolesti.Odre|ivane su koncentracije komponenti komple-menta C3, C4, C1q, C1-inh, faktora B, kao i hemoli-ti~ka aktivnost klasi~nog i alternativnog puta aktivaci-je u uzorcima seruma i plazme pacijenata sa dijagno-zom uro|enog angioneurotskog edema i pacijenatakojima je transplantirana jetra. Hemoliti~ki testovianalizirani su nefelometrijski (Behring Laser-Nephe-lometer) i spektrofotometrijski (Bio System photome-ter BTS 310). Dobijeni rezultati pokazuju da funkcio-nalni testovi, sposobnost C1-inh da inhibira spontanu

high quality service by producing accurate, precise,relevant and comprehensive data that can be appliedto the medical of cardiovascular patients. The testsrequested must be appropriate to the clinical pro-blem, tests must be analytically correctly performedand economically justified. Institute of CardiovascularDiseases in Sremska Kamenica got reward »Bussi-nes Partner« last year. In the next period the task ofthe Institute will be to continue activities for highquality laboratorie service. In order to maintain a highquality level, an internal and an external periodical re-view of the quality system will be esential. This reviewmust cover the quality sistem, technical and analyticalcompetence. The very rapid changing techniquesand the introduction of new tests will require a con-tinuous training and education of laboratory staff.Management of clinical outcome in medical labora-tories must be based on two essential requirements:the implementation of a quality system in clinicallaboratory and a reconsideration of the tasks of aclinical biochemist. The implementation of manage-ment Quality System in laboratory and LaboratoryInformation Managment System is a challenge forthe future.

A21

THE COMPARISON OF FUNCTIONAL TESTSFOR EXAMINATION OF COMPLEMENT

SYSTEMS IN CLINICAL PRACTICE

M. Mitrovi}1, B. Rodi}-Poli}1, N. Majki}-Singh2, G. Mitrovi}3

1Blood Transfusion Institute of Serbia2Insistute of Medical Biochemystry,

Clinical Centre od Serbia3Srbolek AD

Complement system is very important in patho-genesis of certain diseases. At the most cases com-plement is not a cause, but just one of the factorswhich participate in their releasing. In this studyhemolytical and esterolitical tests are compared indiagnosis of hereditary and acquired angioneuroticedema, as far as hemolytic and ELISA tests in esti-mation of complement level of activation in followingdisease course. The concentrations of complementcomponents C3, C4, C1q, C1-inh, factor B havebeen determined, as far as hemolytical activity ofclassical and alternative way of activation in serumand plasma samples, in patients with diagnosis ofhereditary angioneurotical edema and patients whohave transplanted liver. Hemolytic tests were analy-zed by nephelometric method (Behring Laser-Nep-helometer) and by spectrophotometric method (BioSystem photometer BTS 310). These results show


aktivaciju komplementa, kao i njegova sposobnostinhibicije hidrolize sintetskog hromogenog supstrata,pouzdano detektuju poreme}aje u funkcionalnostiC1-inh. Rezultati ovog rada pokazuju da samoodre|ivanje koncentracija komponenti komplementanije pouzdan i dovoljan parametar kada se ima u vidufunkcionalno stanje sistema komplementa. Preporu-~uje se upotreba funkcionalnih testova u dijagnosticideficijencije sistema komplementa, jer se njima dobi-jaju najpouzdaniji rezultati.

A22

KAPACITET ELIMINACIJE GALAKTOZEU PROCENI FUNKCIONALNE

HEPATI^NE MASE

M. Ili}1, S. Stankovi}1, P. Svorcan2, B. Dap~evi}2, B. Radevi}3, D. Meci}2, A. Po{ti}-Grujin1,

N. Majki}-Singh1


2Institut za gastroenterohepatologiju, Klini~ko bolni~ki centar Zvezdara, Beograd

3Institut za kardiovaskularne bolesti, Dedinje, Beograd

Utvr|ivanje funkcionalne hepati~ke rezerve je jo{uvek kontroverzno. Hepati~na funkcija moè biti pro-cenjena na osnovu supstanci koje se selektivno meta-boli{u u jetri. Kvantitativni testovi za procenu funkcio-nalnog stanja jetre dele se na testove eliminacije i tes-tove zasnovane na detekciji odre|enog metabolitasupstance koja se metaboli{e u hepatocitima. Test eli-minacije galaktoze generalno reprezentuje funkcional-nu hepati~nu masu (metaboli~ki kapacitet), a elimina-cija galaktoze iz krvi zavisi od fosforilacije galaktokina-zom u hepatocitima. Ovaj test je dobar prognosti~kiindikator u nekim akutnim i hroni~nim bolestima jetre.Galaktoza je odre|ivana kod pacijenata sa operabilnimprimarnim malignim tumorom jetre, benignim tumo-rom jetre (hemangiomom), cisti~no izmenjenom je-trom, kao i u grupi pacijenata sa sekundarnim depozi-tima (metastazama) na jetri koje se mogu operativnoukloniti. D-galaktoza je davana pacijentima i.v. u bo-lusu 0,5 g/kg telesne teìne, a uzorci krvi uzimani sunakon 20, 25, 30, 35, 40 i 45 min. Koncentracija ga-laktoze odre|ivana je komercijalnim testom firmeRoche (Roche Diagnostics GmbH, Mannheim, Deut-schland). Dosada{nji rezultati pokazuju da je ovaj testpouzdan, reproducibilan i relativno lako izvodljiv, pa jestoga vaàn za rano prepoznavanje disfunkcije jetre,kao i za preoperativnu procenu funkcionalnog stanjajetre kod pacijenata sa indikacijama za resekcije jetre,a u smislu procene funkcionalnog stanja jetre.

that functional tests, capability of C1-inh to inhibitspontaneous activation of the complement and itscapability to inhibit hydrolyze of synthetic chromogensubstrate, are very reliable in distrurbance detectionin C1-inh functionality. The results of this study showthat determination of complement components con-centrations is not reliable and sufficient parameterconsidering functional status of complement system.Functional tests are recommended in deficiency ofcomplement system diagnostics because of theirmost reliable results.

A22

THE GALACTOSE ELIMINATION CAPACITY

IN FUNCTIONAL HEPATIC MASS

M. Ili}1, S. Stankovi}1, P. Svorcan2, B. Dap~evi}2, B. Radevi}3, D. Meci}2, A. Po{ti}-Grujin1,

N. Majki}-Singh1


2Department of Gastroenterohepatology, ZvezdaraUniversity Hospital, Belgrade

3Dedinje Institut of Cardiovascular Diseases,Belgrade, Yugoslavia

Assessment of functional hepatic mass is stillcontroversial. Hepatic function may be assessed onthe basis of substances which are selectively metabo-lized in the liver. There are two types of quantitativetests for the assessment of the liver functional state: 1)elimination tests and 2) tests based on the detection ofa certain substance metabolite, which is metabolizedin hepatocytes. Generally, the test of galactose elimi-nation represents functional hepatic mass (metaboliccapacity) whereas galactose elimination from theblood depends on phosphorylation by galactokinasein hepatocytes. This test is a good prognostic indica-tor in some acute and chronic liver diseases. Galacto-se was determined in patients with operable primarymalign liver tumor, benign liver tumor (hemangiom)cystically changed liver as well as in patients with se-condary deposits (metastasis) on the liver, which canbe surgically removed. Patients were administered witha single bolus dose of D-galactose (i.v. 0.5 g/kg. b.w.)and blood samples drawn after 20, 25, 30, 35 40 and45 min. Galactose concentration was determined bycommercial test (Roche Diagnostics GmbH, Mann-heim, Deutschland). The obtained results show thatthis test is reliable, reproducible and relatively easilyperformed. Thus, it represents an important parame-ter for the early recognition of the liver dysfunction aswell as for pre-operative assessment of the liver functi-onal state in patients with the indications for the liverresection.

nau^ni odbor - dmbs · 2009-06-03 · medicinski biohemi~ari su u svom domenu rada pod-vrgnuti...

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