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2nd FESCC SYMPOSIUMFOR BALKAN REGION

New Diagnostic Tools and Qualityin Laboratory Medicine

NOVI SAD, 17-18. October 2006.

Jugoslov Med Biohem 25: 417–425, 2006 2nd FESCC Symposium for Balkan RegionApstrakti – Abstracts

Jugoslov Med Biohem 2006; 25 (4): 417

VELIÎNA ÊSTICA KAO DIJAGNOSTI^KO SREDSTVO

V. Blaton KU-Leuven

Odeljenje klini~ke hemije Az StJan Av Rudderhove 10, 8000 Brugge (Be)Drugi FESCC Simpozijum za balkanski region,

18. oktobar, Novi Sad, Srbija

Prvi korak u separaciji i identifikaciji klasa serum-skih lipoproteina bilo je ultracentrifugiranje. Goffmani Lindgren su prvi razdvojili serumske lipoproteine uklase razli~itih gustina na osnovu gradijenata gustine.Njima je po{lo za rukom da okarakteri{u veli~inu ~e-stica i da ih dovedu u vezu sa rizikom za koronarno--sr~ane doga|aje. Morali smo do sada da ~ekamo dabismo bolje razumeli povezanost izme|u fizi~kih pa-rametara molekula i rizika za nastanak bolesti. Izazovza budu}nost je da se istraì uticaj fizi~kih parametara~estica na razvoj bolesti, da se prona|e metoda za di-jagnosti~ke mogu}nosti, i tako|e, da se istraè pute-vi za tretman. Epidemiolo{ke studije pokazale su poz-itivnu vezu izme|u koncentracija ukupnog holestero-la i mortaliteta usled koronarnih sr~anih bolesti(CHD). Ukupan holesterol ne predvi|a ta~no rizik zanastanak CHD kod mnogih pacijenata; odluka o tret-manu zasniva se na LDL-holesterolu, ali tako|e moèbiti potrebno uzeti u obzir heterogenost LDL-a. LDL~estice male gustine su vi{e aterogene od onih ve}egustine, dok flotacione LDL ~estice i oksidovani LDLtako|e pove}avaju aterogeni~nost. HeterogenostLDL okarakterisana je prema razli~itim fizi~kim osobi-nama LDL ~estica: veli~ini lipoproteina, flotacionojbrzini i gustini. Razmatraju se osobine nekih uobi-~ajenih metoda ispitivanja pomo}u kojih se obi~nokarakteri{u LDL podfrakcije. Gradijent gel elektro-foreza koja koristi uslove bez denaturacije obi~no seupotrebljava za karakterizaciju distribucije veli~ineLDL ~estica. Visokoefikasna gel-filtraciona hromato-grafija i nuklearna magnetna rezonantna (NMR) spek-troskopija od nedavno se primenjuju za odre|ivanjeveli~ine LDL ~estica. Nekoliko razli~itih metoda ultra-centrifugiranja na gradijentu gustine koje koristeanaliti~ke ultracentrifuge upotrebljavaju se za karak-terizaciju flotacione brzine LDL-a. Nekoliko metodakoje se zasnivaju na diskontinualnim gradijentimasoli koriste se za odre|ivanje LDL podklasa na osno-vu gustine. Ove razli~ite metode odre|ivanja pod-klasa LDL-a pokazuju visok stepen korelacije, uprkos

PARTICLE DIMENSION AS A DIAGNOSTIC TOOL

V. Blaton KU-Leuven

Department of Clinical Chemistry, Az StJan Av Rudderhove 10, 8000 Brugge (Be)2nd FESCC Symposium for the Balkan Region,

October 18, Novi Sad, Serbia

The first step in the separation and identificationof serum lipoprotein classes was ultracentrifugation.Goffman and Lindgren were the first to separate theserum lipoproteins in different density classes basedon density gradients. They were able to characterizethe particle sizes and to relate them to the risk of co-ronary heart events. We had to wait till now tounderstand more about the relationship between thephysical parameters of the molecules and the risk ofdisease. It is a challenge for the future to explore theinfluence of the physical parameters of a particle onthe development of a disease, to find the methods fordiagnostic possibilities and also to explore the waysfor treatment. Epidemiological studies have shown apositive relationship between total cholesterol con-centrations and mortality due to coronary heart di-sease. Total cholesterol does not accurately predictthe risk of CHD in many patients; the decision abouttreatment is based on LDL-cholesterol, but may alsoneed to take into account the LDL heterogeneity.Small dense LDL particles are more atherogenic thanlarge, buoyant LDL particles, and LDL-ox alsoincrease atherogenicity. LDL heterogeneity is cha-racterized according to the different physical proper-ties of LDL particles: lipoprotein size, flotation rateand density. Features of some common assays usedto characterize LDL subfractions are discussed. Gra-dient gel electrophoresis using no denaturing con-ditions is commonly used to characterize the LDLparticle size distribution. High-performance gel-fil-tration chromatography and nuclear magnetic re-sonance (NMR) spectroscopy have been recentlyapplied for the determination of LDL particle size. Se-veral different methods of density gradient ultracen-trifugation have been used to characterize LDL flota-tion rate using the analytical ultracentrifuge. Severalmethods based on discontinuous salt gradients areused to determine LDL subclasses based on density.These various methods of determining LDL subcla-sses show a high degree of correlation, despite thefact that they measure different physical properties of

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~injenici da mere razli~ite fizi~ke osobine LDL-a. Svemetode za merenje lipoproteina zahtevaju izvo|enjedve sekvencijalne operacije: separaciju lipoproteinada bi se njegov nivo izmerio zasebno od drugih, dokbi se kvantifikacija pojedinih va`nih pogodnosti po-stigla izbegavanjem separacionog koraka, naro~itokada se kvantifikuju lipoproteinske podklase. Kako supodklase lipoproteina veoma sli~ne jedna drugoj poveli~ini, gustini i sastavu, njihova separacija je teègreda veli~ine nego separacija glavnih lipoproteinskihklasa. Postoje va`ne prednosti kori{}enja spektro-skopske analize lipoproteinskih podklasa. Nova pro-cedura }e iskoristiti ono {to se ~ini prirodnim, ali segeneralno ne uvaàva ’ protonske NMR spektroskop-ske razlike ispoljene od strane lipoproteinskih ~esticarazli~ite veli~ine. Taj novi proces sada je uglavnompotpun. Kori{}enjem posve}enog intermedijarnogpolja (360 MHZ) NMR analizatora, rutinska kvantifi-kacija 15 razli~itih podklasa VLDL, LDL i HDL postièse za oko jedan minut. Kao {to se moè o~ekivati odmetode koja ne koristi reagense i zahteva minimalnumanipulaciju uzorka, ta~nost i preciznost su isto takodobre ili bolje nego one postignute sa najboljim alter-nativnim procedurama. Uz efikasnost NMR procesa,prvi put }e biti mogu}e sprovo|enje istraìva~kihstudija na velikim populacijama, kako bi se ispitaleveza izme|u podklasa i CHD doga|aja, kao i drugihbolesti, i procenio uticaj raznih reìma tretmana nanivoe podklasa i klini~ki ishod. Bitni koncepti osnov-ne NMR analize lipoproteinskih podklasa sasvim sujednostavni i ne zahtevaju veliko poznavanje u NMRspektroskopije. Kvantifikacija je postignuta u procesuod tri stupnja koji se sastoji od merenja NMR spektraplazme ili seruma, posle koga sledi kompjuterskadekonvolucija podataka spektra i izra~unavanje kon-centracija podklasa. Za stupanj merenja koji se izvo-di automatski na netretiranom uzorku plazme ili seru-ma (0,5 mL) potrebno je manje od jednog minuta.Stupnjevi dekonvolucije i izra~unavanja, koji se izvodena digitalizovanim podacima uz kori{}enje softvera zaspecijalizovane analize koji radi off-line na personal-nim kompjuterima, zahtevaju samo nekoliko sekun-di. Otkrivanje neprime}enog rizika je najva`niji za-datak. Klju~na uloga LDL-a u CVE i patogenezi CHDdobro je prihva}ena, kao i korisnost sniàvanja LDL-a kod pacijenata sa visokim rizikom. Ostaje spornoda li koristiti najbolje merenje za LDL da bi se identi-fikovali svi pojedinci koji }e imati korist od terapije.Mnoge studije pokazale su da su, pri postoje}em ni-vou LDL-a, pojedinci sa prete`no malim LDL ~es-ticama iskusili ve}i rizik nego oni sa LDL ~esticamave}e veli~ine. Trenutno nije razja{njeno da li su malimolekuli LDL-a su{tinski vi{e aterogeni nego oni ve}izato {to, pri datom nivou LDL-a, pojedinci sa malomveli~inom LDL ~estica imaju ve}i ukupan broj ~esti-ca. Broj ~estice LDL-a meren uz pomo} nuklearnemagnetne rezonance dosledno se pokazuje kaosnaàn, nezavistan prediktor CHD.

LDL. All methods of measuring lipoproteins requirethe performance of two sequential operations: sepa-ration of the lipoprotein to be measured from theothers, and quantification of several important bene-fits would be gained by avoiding the physical sepa-ration step, particularly when quantifying lipoproteinsubclasses. Because the subclasses of a lipoproteinare so closely similar to one another in size, density,and composition, their separation is an order of mag-nitude more difficult than the separation of major li-poprotein classes. There are major advantages inusing spectroscopic lipoprotein subclass analysis.The new procedure would exploit what appeared tobe natural, but remained generally unappreciated ’proton NMR spectroscopic differences exhibited bylipoprotein particles of different sizes. The new pro-cess is now largely complete. Using a dedicated inter-mediate-field (360 MHZ) NMR analyzer, routinequantification of 15 different subclasses of VLDL,LDL and HDL is achieved in about one minute. Asmight be expected from a method that uses noreagents and requires minimal sample manipulation,accuracy and precision are as good as, or betterthan, those achieved by the best alternativeprocedures. With the efficiency of the NMR process,it will be possible for the first time to conductresearch studies of large populations in order toexamine subclass associations with CHD events andother diseases, and to assess the impact of varioustreatment regimens on subclass levels and clinicaloutcomes. The basic concepts underlying NMR lipo-protein subclass analysis are quite simple and requireno background in NMR spectroscopy to be under-stood. Quantification is achieved in a three-stepprocess consisting of measurement of the plasma orserum NMR spectrum followed by computer de-convolution of the spectral data and calculation ofthe subclass concentrations. The measurement stepwhich is performed automatically on untreated pla-sma or serum specimens (’0.5 mL) takes less thanone minute. The deconvolution and calculation steps,which are performed on the digitized data using spe-cialized analysis software running off-line on a per-sonal computer, require just a few seconds. Disco-vering the unseen risk is the most important task.The key role of LDL in CVE and in the pathogenesisof CHD is well accepted, as is the benefit of loweringLDL in high risk patients. What remains controversialis whether we are using the best measure of LDL toidentify all individuals who would benefit from the-rapy. Many studies have shown that, at given level ofLDL-c, individuals with predominantly small LDLparticles experience greater risk than those with lar-ger-size LDL. It is not clear at the moment whethersmall LDL molecules are inherently more athero-genic than large ones because, at a given level ofLDL-c, individuals with small LDL particle size havemore LDL particles in total. Particle number of LDLmeasured by nuclear magnetic resonance hasconsistently been shown to be a strong, independentpredictor of CHD.

Jugoslov Med Biohem 2006; 25 (4) 419

PRIMENA TEHNIKE SELDI-TOF-MS U PROFILISANJU PROTEINA:

PREDNOSTI I ZAMKE

J. A. P. Bons1, W. K. W. H. Wodzig1, D. de Boer1, M. Drent2, M. P. van Dieijen-Visser1

1Odeljenje za klini~ku hemiju 2Respiratorna medicina,

Centar za kontrolu sarkoidoze,Univerzitetska bolnica »Maastricht«,

Maastricht, Holandija

Profilisanje proteina seruma tehnikom Surface-Enhanced Laser Desorption/Ionization Time-of-FlightMass Spectrometry (SELDI-TOF-MS) ~ini se kaoveoma va`no dijagnosti~ko sredstvo za ~itav nizbolesti. Senzitivnost i specifi~nost koje se posti`u ovomnovom tehnologijom ~esto su superiorne u odnosu narezultate postignute uz pomo} drugih biomarkera.Me|utim, njihova reproduktivnost i standardizacija jo{uvek su problemati~ne. Objasni}emo tehniku SELDI-TOF-MS i razmotriti neke va`ne aspekte u izu~avanjuproteoma, kako pre tako i posle analize, kao i postup-ke za kontrolu kvaliteta. U ovom pregledu tako|e supredstavljeni i na{i podaci o otkrivanju biomarkera zapostavljanje dijagnoze sarkoidoze.

PRIMENA 2D-HPLC SISTEMA ZA RAZDVAJANJE PROTEINA

I. Levreri1, L. Musante2, A. Petretto3, D. Cuccabitta3, G. Candiano2, G. Melioli1

1U.O. Laboratorio Centrale di Analisi2Laboratorio di Fisiopatologia dell’Uremia,

U.O. di Nefrologia3Laboratorio di Spettrometria di Massa,

Core facility,Instituto Scientifico Giannina Gaslini, Genova, Italy

The ProteomeLab™ PF 2D sistem za frakcionisanjeproteina je brz, poluautomatski 2D-HPLC aparat kojikoristi dve razli~ite metode za razdvajanje plazmaserumskih proteina: jonoizmenjiva~ku hromatografijukoja koristi {irok opseg pH u prvoj dimenziji i nepo-roznu reverzno-faznu hromatografiju u drugoj dimen-ziji. Samo zbog toga {to je ova metodologija nedavnopredstavljena u laboratorijama za proteomikane,malo se zna o karakteristikama PF 2D frakcionisanjaproteina u humanom serumu. Da bi se procenilaprimena sistema u okvirima klini~ke laboratorije,analizirane su osobine razdvajanja zasnovanog najonoizmenjiva~koj hromatografiji. Posle frakcionisa-nja proteina humanog seruma na linearnom gra-

APPLICATION OF SELDI-TOF-MS IN PROTEIN PROFILING: PROMISES AND PITFALLS

J. A. P. Bons1, W. K. W. H. Wodzig1, D. de Boer1, M. Drent2, M. P. van Dieijen-Visser1

1Departments of Clinical Chemistry 2Respiratory Medicine,

Sarcoidosis Management Centre, University Hospital Maastricht, Maastricht,

The Netherlands

Serum protein profiling by Surface-Enhanced La-ser Desorption/Ionization Time-of-Flight Mass Spec-trometry (SELDI-TOF-MS) appears to be an impor-tant diagnostic tool for a whole range of diseases.Sensitivities and specificities obtained with this newtechnology often seem superior to those obtainedwith current biomarkers. However, reproducibilityand standardization are still problematic. The presentreview explains the SELDI-TOF-MS technique anddiscusses some important aspects for proteomicsstudies, like pre- and post-analytical aspects andquality control procedures. Own data about biomar-ker discovery for the diagnosis of sarcoidosis are alsopresented in this review.

APPLICATION OF 2 D-HPLC SYSTEM FOR PLASMA PROTEIN SEPARATION

I. Levreri1, L. Musante2, A. Petretto3, D. Cuccabitta3, G. Candiano2, G. Melioli1

1U.O. Laboratorio Centrale di Analisi2Laboratorio di Fisiopatologia dell’Uremia,

U.O. di Nefrologia3Laboratorio di Spettrometria di Massa,

Core facility, Instituto Scientifico Giannina Gaslini, Genova, Italy

The ProteomeLab™ PF 2D protein fractionationsystem is a rapid, semi-automated, 2 D-HPLC instru-ment that uses two different methods to separate pla-sma serum proteins: ion-exchange chromatographyusing a wide range of pH in the first dimension andnon-porous reverse-phase chromatography in the se-cond dimension. Because this methodology has onlyvery recently been introduced in proteomic laborato-ries, little is known about the characteristics of PF 2Dfractionation of human serum proteins. To evaluatethe system’s application in a clinical laboratory set-ting, the characteristics of the ion-exchange chro-matography-based separation were analyzed. Follo-wing fractionation of human serum proteins on a li-

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dijentu pH (u opsegu od 8,5 do 4,0), svaka frakcija jesakupljena u hladnom modulu aparata. Razli~ite frak-cije iz prve dimenzije su zatim zajedno objedinjene istavljene na klasi~nu aparaturu za 2D gel elektrofo-rezu. Dobijene razli~ite ta~ke su zatim proverene pre-ko Swiss-Prot baze podataka. Ukupno 36 proteinahumanog seruma je identifikovano u razli~itim PF 2Dproizvedenim frakcijama. Pojedine va`ne osobine se-paracionog sistema su bile zapaène. Razli~ite elu-cione frakcije sadràle su razli~ite proteine {to poka-zuje pouzdanost sistema za frakcionisanje. Proteini sutako|e frakcionisani prema teoretskoj izoelektri~nojta~ki (pI). To je bilo u skladu sa dokazom da velikave}ina imunoglobulina, okarakterisana sa alkalnimpI, nije bila zadràna u koloni i bila je eluirana u neve-zanoj frakciji. Ovakav rezultat tako|e nagla{avaprakti~nu prednost: eluirane frakcije od pH 8 do pH4 sadràle su zapravo proteine seruma bez prisustvaimunoglobulina. Ovaj nalaz podràva neposrednuupotrebu PF 2D sistema u klini~kim procedurama,gde bi trebalo veliki broj proteina jasno identifikovatida bi se omogu}ila procena drugih manje prisutnih,ali potencijalno va`nih, tipova.

PROTEINSKI MIKROREDOVI U DIJAGNOZII ISTRA@IVANJUALERGIJSKIH OBOLJENJA

R. Hiller

Centre for Proteomic and Genomic Researchc/o Institute for Infectious Disease & Molecular

Medicine, University of Cape Town, Cape Town, South Africa

Mikroredovi proteina predstavljaju jedan odosnovnih postulata moderne proteomike, kako u ciljusistematizacije, tako i radi sistemske analize funkcija,interakcija i koli~ine proteina. Mogu}nost analiziranjauzoraka u minijaturnom obliku podstakla je naporeza razvijanje novih dijagnosti~kih pristupa u le~enjuraka, autoimunih oboljenja i alergija. U ovom radubi}e razmotrena najnovija dostignu}a u razvoju pro-teinskih mikroredova za profilisanje IgE antitela u di-jagnozi alergijskih oboljenja tipa 1.

near gradient of pH (ranging from 8.5 to 4.0), eachfraction was collected in a cool module of the instru-ment. Different fractions obtained from the first di-mension were then pooled together and loaded onclassic 2D gel electrophoresis instrumentation. Thedifferent spots obtained were then checked againstthe Swiss-Prot Database. A total of 36 human serumproteins were identified in different PF 2D-generatedfractions. Some important features of the separationsystem were observed. Different eluted fractions con-tained different proteins, thus demonstrating the reli-ability of the fractionation system. The proteins werealso fractionated according to the theoretical isoelec-tric point (pI). This was consistent with the evidencethat the vast majority of immunoglobulins, characte-rized by an alkaline pI, were not retained by the co-lumn and were eluted in the unbound fraction. Thisoutcome also underlies a practical advantage: fracti-ons eluted from pH 8 to pH 4 contained virtually im-munoglobulin-depleted serum proteins. This findingsupports an immediate use of the PF 2D system in aclinical setting, where abundant proteins should beclearly identified in order to enable evalutation of ot-her less abundant, but potentially relevant, species.

PROTEIN MICROARRAYS FOR DIAGNOSISAND RESEARCH OF ALLERGIC DISEASES

R. Hiller

Centre for Proteomic and Genomic Researchc/o Institute for Infectious Disease & Molecular

Medicine, University of Cape Town, Cape Town, South Africa

For both the systematic as well as for the system-wide analysis of protein function, interaction andabundance, protein microarrays represent one of thepillars underlying modern high-throughput Proteo-mics. The prospect of analysing samples in a highlyparallel while miniaturized fashion has fuelled effortsto develop novel diagnostic applications in the can-cer, autoimmune or allergy field. Here, I discuss re-cent advancements in the development of proteinmicroarrays for the profiling of IgE antibodies in thediagnosis of Type 1-related allergic diseases.


BIOMARKERI OBOLJENJA: PRISTUP ZASNOVAN NA DOKAZIMA

S. Ignjatovi}, N. Majki}-Singh

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

Univerziteta u Beogradu, Beograd, Srbija

Medicina zasnovana na dokazima (EBM) pri dono-{enju odluka o nezi pacijenata kombinuje individual-nu klini~ku ve{tinu sa najboljim raspoloìvim klini~-kim dokazima iz sistemati~nih istraìvanja. Klini~kave{tina odlikuje se ta~no{}u u proceni i sti~e se u~e-njem, klini~kim iskustvom i praksom. Klini~ki dokazidobijaju se iz klini~kih istraìvanja koja su usmerenana pacijenta, a koja ispituju ta~nost i preciznost dijag-nosti~kih testova i biomarkera, efikasnost i sigurnostterapeutskih postupaka i pouzdanost prognosti~kihindikatora. Kombinacija klini~ke ve{tine i dokumen-tovanih dokaza omogu}ava sigurniju, efikasniju i po-uzdaniju negu pacijenta. Vodi~i zasnovani na doka-zima naj~e{}e se koriste kao dodatne alatke pri do-no{enju medicinskih odluka. Formulisanje preporukazasnovanih na dokazima predstavlja vode}i princip upripremi vodi~i. U razvoju preporuka koje uklju~ujulaboratorijske testove i biomarkere treba primenitisistematsku i standardizovanu metodologiju koja jeprilago|ena laboratorijskoj medicini. Postoji velikibroj mogu}nosti za primenu i evaluaciju laborato-rijskih testova u dobrim klini~kim ispitivanjima. Jo{su ve}e mogu}nosti za uspostavljanje korelacijaizme|u razli~itih laboratorijskih postupaka i klini~kihnalaza, ishoda i dijagnoza, kao i za kori{}enje uzorakakoji su skladi{teni za ova ispitivanja. Era medicine za-snovane na dokazima zahteva od eksperata koji do-nose medicinske odluke prou~avanje nau~ne litera-ture kako bi se obezbedio visok kvalitet dokaza okorisnosti i ta~nosti dijagnosti~kih testova. Ovakvavrsta dokaza potrebnija je vi{e nego ikad zato {to listadijagnosti~kih testova raste eksponencijalno i u go-dinama koje dolaze na nju }e biti dodato jo{ vi{e bio-markera, proteomike i aplikacija profiliranja ekspre-sije gena.

BIOMARKERS OF DISEASES: AN EVIDENCE-BASED APPROACH

S. Ignjatovi}, N. Majki}-Singh

Institute of Medical Biochemistry, Clinical Center of Serbia and School of Pharmacy, University of Belgrade,

Belgrade, Serbia

Evidence-based medicine (EBM) combines indi-vidual clinical expertise with the best available clinicalevidence from systematic research in making deci-sions about the care of individual patients. Clinicalexpertise is the proficiency and judgment that indi-vidual clinicians acquire through knowledge, clinicalexperience, and practice. Clinical evidence comesfrom patient-centered clinical research which inves-tigates the accuracy and precision of diagnostic testsand biomarkers, the efficacy and safety of therapeu-tic regimes, and the reliability of prognostic indica-tors. The powerful combination of clinical expertiseand documented evidence results in safer, more effi-cacious and accurate care of the patient. Evidence-based guidelines are commonly used tools for sup-porting medical decisions. Formulating evidence-ba-sed recommendations has become a leading prin-ciple in guideline development. In laboratory medici-ne, guidelines provide recommendations on the useof a wide range of tests in detecting or predicting atarget condition, for staging and monitoring a di-sease, and for decisions to initiate, modify, or termi-nate treatments. Systematic, standardized, andexplicit methodology, adapted to laboratory medici-ne, should be followed when developing recommen-dations involving the use of laboratory tests andbiomarkers. There are many opportunities for theapplication and evaluation of laboratory tests in goodclinical trials. There are even greater opportunities forcorrelating various laboratory procedures with theclinical findings, outcomes and diagnoses, and usingthe stored samples collected for those studies. In thisera of evidence-based medicine, clinicians and otherdecision-makers turn to the scientific literature forhigh-quality evidence about the usefulness, preci-sion, and accuracy of diagnostic tests. Such evidenceis needed more than ever because the list of diag-nostic tests is growing exponentially, and even morebiomarkers, proteomics, and applications of geneexpression profiling will be added in the years tocome.

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B-TIP NATRIURETSKIH PEPTIDA KAOMO]NIH MARKERA U KARDIOLO[KIM

BOLESTIMA ’ ANALITI^KI I KLINI^KI ASPEKTI

A. Hammerer-Lercher1, B. Puschendorf1, J. Mair2

1Division of Clinical Biochemistry, Innsbruck Biocenter, Innsbruck Medical

University, Innsbruck, Austria2Department of Internal Medicine, Clinical Division of Cardiology,

Innsbruck Medical University, Innsbruck, Austria

Me|u svim natriuretskim peptidima i neurohormo-nima, za B-tip natriuretski peptid (BNP) i njegov N-ter-minalni prohormonski fragment (NT-proBNP) je po-kazano da su najbolji i najmo}niji markeri za identi-fikaciju pacijenata sa akutnom i hroni~nom sr~anominsuficijencijom (SI). Potpuno automatizovana odre|i-vanja BNP i NT-proBNP ahtevaju samo 15’20 minutaza dostizanje rezultata testa tako da je mogu}e »turn-around« vreme manje od 60 minuta, kao {to se zahte-va od strane vodi~a kardiolo{kih dru{tava. In vitro sta-bilnosti BNP i NT-proBNP su dovoljne za rutinskuupotrebu. Ve}ina komercijalno dostupnih testova, semako su sublicencirani, koriste razli~ita antitela. Ovo semoè objasniti da uop{teno, BNP i NT-proBNP odre|i-vanja sa razli~itim testovima pokazuju bliske korelacije,ali se ne slaù u apsolutnim vrednostima. Ova odre|i-vanja nisu dosada standardizovana i primena razli~itihkalibracionih materijala moè doprineti razli~itim rezul-tatima. Prema tome, referentni opsezi zavise od testakoji se koristi, i moraju biti odre|eni za svaki test po-sebno. Pove}anje vrednosti sa godinama mo`da je uvezi sa rastu}om frekvencijom subklini~ke renalne ilikardiolo{ke disfunkcije kod starijih osoba. Estrogenistimuli{u produkciju natriuretskih peptida kod èna, areferentni opsezi zavise od pola od adolescencije domenopauze. Odmah nakon ro|enja, nivoi BNP i NT-proBNP su znatno vi{i kod novoro|en~adi nego kodnjihovih majki. Visoka biolo{ka varijacija natriuretskihpeptida mora se uzeti u obzir pri interpretaciji serijerezultata BNP i NT-proBNP. Prema tome, samo znatnepromene BNP i NT-proBNP u toku pra}enja pacijentasu u vezi sa promenama klini~kog statusa SI. Zaklju~aksvih vode}ih studija je da su BNP i NT-proBNP kodpacijenata sa hroni~nom SI pre markeri za »isklju~e-nje« nego za »potvrdu« zbog ograni~enih kardiolo{kihspecifi~nosti. Pacijenti sa akutnom SI obi~no pokazujuvi{e nivoe BNP i NT-proBNP nego pacijenti sa hroni~-nom SI. Najve}a efikasnost testiranja BNP i NT-proBNPje pokazana kod pacijenata prisutnih na urgentnomodeljenju sa akutnom dispneom ili kod pacijenata sasimptomima koji ukazuju na hroni~nu SI. Mnoge studi-je ukazuju da kratkoro~na i dugoro~na prognoza kodSI moè biti procenjena odre|ivanje BNP i NT-proBNP.Ovi hormoni su nezavisni prediktori letalnog ishoda ilihospitalizacije zbog SI. Natriuretski peptidi su pove}anikod svih bolesti koje uti~u na kardijalnu ili renalnufunkciju i ravnoteù te~nosti. BNP i NT-proBNP su ta-

B-TYPE NATRIURETIC PEPTIDES AS POWERFUL MARKERS IN CARDIAC

DISEASES – ANALYTICAL AND CLINICAL ASPECTS

A. Hammerer-Lercher1, B. Puschendorf1, J. Mair2

1Division of Clinical Biochemistry, Innsbruck Biocenter, Innsbruck Medical

University, Innsbruck, Austria2Department of Internal Medicine, Clinical Division of Cardiology,

Innsbruck Medical University, Innsbruck, Austria

Among all natriuretic peptides and neurohormo-nes, B-type natriuretic peptide (BNP) and its N-termi-nal prohormone fragment (NT-proBNP) have beenshown to be the best and most powerful markers toidentify patients with acute and chronic heart failure(HF). The fully automated BNP and NT-proBNP assaysrequire only 15’20 min to achieve a test result so thata turn-around time of less than 60 min is possible, asrequested by the guidelines of the cardiological soci-eties. The in-vitro stabilities of BNP and NT-proBNPare sufficient for routine use. Most of the commercial-ly available assays, except if they are sublicensed, usedifferent antibodies. This may explain that in general,BNP and NT-proBNP assays show close correlations,but do not agree in absolute values. The assays havenot been standardized so far and the application of var-ious calibration materials may contribute to differentresults. Thus, reference ranges are dependent on theassay used, and reference ranges have to be deter-mined for each assay separately. The increasing valueswith age may be related to the increasing frequency ofsubclinical renal or cardiac dysfunction in the elderly.Estrogens stimulate the natriuretic peptide productionin females, and reference ranges depend on sex fromadolescence to menopause. Immediately after birth,BNP and NT-proBNP levels are substantially higher inneonates than in their mothers. The high biologicalvariation of natriuretic peptides must be consideredwhen interpreting serial BNP and NT-proBNP results.Therefore, only marked BNP or NT-proBNP changesduring follow-up are related to changes in the clinicalHF status. A conclusion of all major studies is that inpatients with chronic HF BNP and NT-proBNP arerather rule-out than rule-in markers because of limitedcardiac specificities. Patients with acute HF usuallyshow higher BNP and NT-proBNP levels than patientswith chronic HF. The greatest efficiency of BNP andNT-proBNP testing was demonstrated in patients pre-senting to the emergency department with acute dys-pnoea or in outpatients with symptoms suggestingchronic HF. Many studies indicate that short- and long-term prognosis in HF can be assessed by BNP or NT-proBNP determination. These hormones are inde-pendent predictors of death or HF hospitalizations.Natriuretic peptides are increased in all diseases affect-ing the cardiac or renal function and fluid balance.BNP and NT-proBNP are markers of cardiac dysfunc-


ko|e markeri kardijalne disfunkcije i kod pacijenata sarenalnom insuficijencijom, ali se tu moraju koristiti vi{evrednosti granica odlu~ivanja. Smanjene koncentracijeBNP i NT-proBNP kod gojaznih nisu potpuno razum-ljive i kontraverzni izve{taji su na|eni u literaturi. Uzaklju~ku, odre|ivanje BNP i NT-proBNP je mo}an testza isklju~enje SI. Osim toga, ovi markeri su korisnadopuna standardnim klini~kim ispitivanjima pacijenatasa suspektnom ventrikularnom disfunkcijom.

KOMPLEKSIRANI PSA I SERUM HER-2/neu:NOVI SERUMSKI MARKERI U LEÊNJU

RAKA PROSTATE I RAKA DOJKE

R. Neumann

European Scientific Affairs, Bayer Healthcare,Odeljenje za dijagnostiku, Leverkuzen, Nema~ka

Rak prostate je naj~e{}i oblik raka kod mu{karaca,a rak dojke naj~e{}e maligno oboljenje kod èna.Zahvaljuju}i napretku u razumevanju biologije tih kan-cera, le~enje i razvoj novih dijagnosti~kih sredstava kojise kre}u u pravcu »personalizovane medicine« trenu-tno su podvrgnuti klini~kom ispitivanju. Ovaj ~lanakopisuje unapre|enu ulogu jednog oblika antigena spe-cifi~nih za prostatu (prostate-specific antigen, PSA),takozvanog »kompleksiranog PSA« (cPSA), u otkriva-nju raka prostate, i uz to predstavlja pregled publikacijana temu klini~ke vrednosti novih testova koji otkrivajuizvan}elijski deo molekula HER-2 u serumu pacijentki-nja sa rakom dojke. Na osnovu navedenih publikacija,pokaza}e se da kompleksirani PSA ima manje ogra-ni~enu specifi~nost u odnosu na druge izoforme PSAkoji se trenutno uglavnom primenjuju u dijagnostiko-vanju raka prostate, kao i to da HER-2neu iz serumadaje informacije o ta~nom statusu HER-2, pruàju}i nataj na~in podatke klini~ki va`ne za potencijalno unapre-|ivanje le~enja pacijentkinja sa rakom dojke.

KLINI^KA VREDNOST BIOHEMIJSKIHMARKERA ZA REMODELIRANJE KOSTIJU

U ISPITIVANJU BOLESTI KO[TANOG METABOLIZMA

J. J. Stepan

Odeljenje za internu medicinu 3, Univerzitet»Charles«, Medicinski fakultet, ê{ka republika

Ko{tani markeri su veoma korisno dijagnosti~kosredstvo, mada je njihova klini~ka upotreba ogra-ni~ena specifi~nim tehni~kim i analiti~kim aspektima,kao i njihovom pre-analiti~kom varijabilno{}u. Ko{-

tion in patients with renal failure as well, but higherdecision limits have to be used. Decreased BNP andNT-proBNP concentrations in obesity are not fullyunderstood and controversial reports are found in theliterature. In summary, BNP or NT-proBNP determina-tion is a powerful test for ruling out HF. Furthermore,these markers are a useful addition to the standardclinical investigations of patients with suspected ven-tricular dysfunction.

COMPLEXED PSA AND SERUM HER-2/neu:NEW SERUM MARKERS FOR THE

MANAGEMENT OF PROSTATE AND BREAST CANCER

R. Neumann

European Scientific Affairs, Bayer Healthcare,Diagnostics Division, Leverkusen, Germany

Prostate cancer is the most common type of cancerin men, and breast cancer the most common mali-gnancy in women. Based on the improvements inunderstanding the biology of these cancers, treatmentand ongoing development of new diagnostic tools aim-ing towards »personalized medicine« are under clinicalevaluation. This article describes the improved value ofone of the immunodetectable forms of the prostate-specific antigen (PSA), the so-called »complexed PSA«(cPSA), in detecting prostate cancer, and also presentsan overview of publications on the clinical value of anew immunoassay detecting the extracellular part ofthe HER-2 molecule in serum of breast cancer patients.Based on the publications cited, it will become obviousthat complexed PSA improves the limitations of thespecificity of PSA isoforms currently mainly applied todiagnose prostate cancer, and that serum HER-2/neuoffers information about the actual HER-2 status thusadding information clinically relevant to the probabilityof optimizing therapy of breast cancer patients.

CLINICAL VALUE OF THE BIOCHEMICALMARKERS OF BONE REMODELING IN THE

ASSESSMENT OF BONE METABOLICDISEASES

J. J. Stepan

Department of Internal Medicine 3, Charles University, Faculty of Medicine,

Prague, Czech Republic

Bone markers have been useful research tools,with their clinical utility limited by their specific tec-hnical and analytical aspects and pre-analytical va-riability. Bone markers reflect different aspects of the

424

tani markeri, za razliku od mineralne gustine kosti,ukazuju na posebne aspekte kvaliteta kostiju, te sto-ga nude zasebnu i prognosti~ku perspektivu u ispi-tivanju promena mineralne gustine kosti i smanji-vanju rizika od fraktura. Smanjenje nivoa ko{tanihmarkera usko je povezano sa smanjenjem rizika odfrakture vratnih pr{ljenova usled kori{}enja raloksi-fena, rizedronata i alendronata. Posle anaboli~ke te-rapije teriparatidom, ubrzano formiranje ko{tanihmarkera je jasan pokazatelj reakcije gustine mineralaproteina. Postoje brojne mogu}nosti za kori{}enjeovih markera u kratkoro~nom pra}enju toka le~enjaosteoporoze, pored merenja mineralne ko{tane gu-stine, kako bi se otkrili pacijenti koji ne reaguju naterapiju. Upotreba ko{tanih markera u svakodnevnojklini~koj praksi zahteva standardizaciju merenja i pro-grama kontrole kvaliteta kako bi se smanjile varijacijeu podacima iz razli~itih laboratorija, definisali krite-rijumi za ubrzani metabolizam kostiju u pogledu re-ferentnih vrednosti, i postiglo preciznije odre|ivanjemarkera u pogledu geografskih oblasti i rasa, kao i urazli~itim klimatskim uslovima.

KVALITET UZORKA I NJEGOV UTICAJ NA KLINI^KO ODLUÎVANJE

D. Mirkovi}, M. Dajak, Z. [umarac, S. Ignjatovi}

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

Beograd, Srbija

Medicinsko tuma~enje klini~ko-laboratorijskihrezultata je komparativni proces odlu~ivanja u komese laboratorijski podaci pojedinca porede sa referent-nim vrednostima izvedenim na osnovu ispitivanja re-ferentne populacije. Ovakvo tuma~enje ~ini procesodlu~ivanja uslovno jednostavnim. Nedostatak zna-nja o kvalitetu uzorka i osobinama biolo{kog mate-rijala pravi raskorak izme|u laboratorije i lekara-kli-ni~ara. Odre|ivanje kontrolnog materijala je bitno zaanaliti~ki proces. Vaàn problem u ovom slu~aju jeodsustvo pravog uzorka i njegova promenljivost.Kontrola kvaliteta se obavlja obradom ve{ta~kog ma-terijala, uz nadu da }e isti verno odslikati karakteris-tike biolo{kog materijala pacijenta. U ovom radu bliè}emo se upoznati sa problemima vezanim za uzoraki njegov kvalitet. Kroz primere iz literature i sopstve-nog iskustva, govori}emo vi{e o:

’ lipemiji, turbiditetu, hemolizi i ikteri~nom biolo-{kom materijalu;

’ efektu uparivanja uzorka;’ kalibraciji;’ stabilnosti analita;’ pravilnom i pogre{nom kori{}enju referentnih

intervala;’ statisti~kom prikazivanju i pogre{noj interpre-

taciji rezultata;’ interferenciji lekova.

quality of bone than BMD and, therefore, may add anindependent, predictive value to the assessment ofchanges in bone mineral density and reductions inthe risk of fracture. The decrease in bone markerlevels is strongly related to the reduction in vertebralfracture risk through raloxifene, risedronate and alen-dronate. After anabolic therapy with teriparatide,early increases in bone formation markers are strongpredictors of BMD responses. There are potentialadvantages of using markers for monitoring anti-osteoporosis treatment in the short term, in additionto the bone mineral measurements, to identify non-responders or non-compliance. The transition of bio-chemical bone markers into everyday clinical practicerequires standardization of assays and quality controlprograms to reduce large inter-laboratory variationsof data, defining criteria of a high bone turnover interms of reference values, either young adult or age-matched, and better characterization of the markersacross geographic areas and races and under variousclinical conditions.

THE QUALITY OF THE SAMPLE AND ITSIMPACT ON CLINICAL DECISION MAKING

D. Mirkovi}, M. Dajak, Z. [umarac, S. Ignjatovi}

Institute of Medical Biochemistry, Clinical Centre of Serbia and Faculty

of Pharmacy, Belgrade, Serbia

The medical interpretation of clinical laboratorydata is a comparative decision-making process inwhich a laboratory test result for an individual is com-pared with a reference interval, derived from a re-ference population. This sentence seems to describean easy situation. Lack of knowledge about the quali-ty of the sample and the special issues of the biologi-cal material creates a gap between the laboratory andthe clinician. To measure quality control material isimportant for the analytical process. The major con-cern in this field is the absence of a real sample and itscommutability. We perform quality control with artifi-cial samples and hope they will successfully mimic realpatients’ material. In this paper we will move muchcloser to the issues concerning the samples and theirquality. With a lot of examples from literature and owninvestigation, we will learn more about:

’ lipemia, turbidity, hemolysis and icterus,’ volume displacement effect,’ calibration,’ stability of the analytes,’ use and misuse of reference intervals,’ statistical presentation and misleading interpre-

tation,’ special cases of drug interferences.Finally, we have to investigate better the sample

and the preanalytical process to provide reliable re-


Tako|e, potrebno je bolje prou~avanje uzorka ipreanaliti~kog postupka kako bi se obezbedila pouz-danost rezultata. Konsultacije su va`no sredstvo kojeotvara proces komunikacije, {to omogu}ava da labo-ratoriju prikaèmo klini~aru na nov na~in. Postojipotreba za prikazivanjem laboratorijskih rezultata nana~in koji svi njeni korisnici razumeju. Brzi i pouzdanirezultati odre|ivanja, kombinovani sa komentarom imogu}no{}u komunikacije, nude odgovaraju}i nivolaboratorijskog rada. Znanje i {irenje istog vodi kavi{em nivou laboratorijskog rada, u cilju pouzdanijegzbrinjavanja pacijenata.

ISO 15189 I PROGRAMI ZA PORE\ENJAIZME\U LABORATORIJA

J. Hejsek

Bio-Rad LaboratorijeNad ostrovem 7, Prag 147 00

ê{ka Republika

Novi standard ISO 15189, posebno osmi{ljen zaklini~ke laboratorije, izdat 2003. godine, svakoj akre-ditovanoj laboratoriji pruà jedinstvenu mogu}nostda na me|unarodnom nivou poredi svoje usluge sabilo kojom drugom akreditovanom laboratorijom usvetu. Isto to garantuju i multilateralni sporazumiizme|u akreditovanih organizacija {irom sveta. ISO15189 sadrì 23 Preduslova za kvalitetan sistem(Quality System Essentials, QSE) koje laboratorijamora da ispuni. Za kontrolu kvaliteta najva`niji jeQSE 5.6 ’ »Zagarantovan kvalitet u postupcimaistraìvanja«. Svi potrebni preduslovi iz te oblasti bi}enavedeni u prezentaciji. Prilikom akreditacije labora-torija, va`na uloga pridaje se programima za pore|e-nje izme|u laboratorija. Postoje dve vrste programaza me|ulaboratorijsko pore|enje: EQA, i pore|enjepoznatih rezultata laboratorije sa rezultatima srodnihlaboratorija, koji se mogu sprovoditi svakodnevno ilina mese~nom nivou. Tokom prezentacije razmo-tri}emo va`nost oba ova programa u odnosu na ISO15189. Kompanija Bio-Rad laboratorije trenutnonudi {irok izbor programa koji laboratorijama mogupomo}i u ispunjavanju preduslova potrebnih za ISO15189. Ima programa sa bazom na internetu koji selako koriste, kao i usavr{enijih verzija koje podràvajusistem Totalne gre{ke (Total Error) zasnovan na bio-lo{kim varijacijama. Najnoviji i najsavremeniji me|unjima primenjuje program Westgard EZ Rules, kojilaboratorijama moè pomo}i pri izboru najpogodni-jeg Westgard pravila, kao i velikog broja materijala zakontrolu kvaliteta za svaku analiziranu supstancu ’ uslu~aju da laboratorija unapred precizira zahtevanikvalitet.

sults. The consultation is an important tool whichopens a field of communication and will help us topresent the laboratory in a new way to the clinician.There is a strong need to show the value of a labora-tory that understands its customers. A rapid and reli-able test result combined with a comment or a con-sultation will add value to the work of the laborato-ries. Knowledge and sharing the knowledge will resultin high quality laboratory work, providing safer pa-tient care.

ISO 15189 AND INTERLABORATORY COMPARISON PROGRAMS

J. Hejsek

Bio-Rad LaboratoriesNad ostrovem 7, Praha 147 00

Czech Republic

In 2003, the new ISO 15189 standard especiallydeveloped for clinical laboratories was published. Itgives the accredited laboratory a unique option ofcomparing internationally its services with any otheraccredited laboratory in the world. This is also gua-ranteed by the multilateral agreements of accreditedorganisations throughout the world. ISO 15189 com-prises 23 Quality System Essentials which the labo-ratory has to comply with. For quality control, themost important one is QSE 5.6 ’ Assuring Qualityof Examination Procedures. All the essential require-ments of this chapter are mentioned in the presenta-tion. Very important role in the accreditation of a la-boratory is given to the interlaboratory comparisonprograms. There are two types of such comparisonprograms: EQA and comparison of lab’s knownresults with its peer group ’ which can be performedeither daily or on a monthly basis. Importance ofboth in relation to ISO 15189 is discussed in the pre-sentation. Bio-Rad laboratories currently offer a widerange of programs which can help laboratories tocomply with ISO 15189 requirements. There areeasy, internet-based programs and other, more so-phisticated versions which can also work with TotalError based on biological variation approach. Thenewest state-of-the-art program applies Westgard EZRules Program which can help laboratory to choosethe optimal Westgard Rules and number of qualitycontrol materials for each analyte ’ in case laborato-ry specifies the required quality.

XV KONGRESMED IC INSKEB IOHEM I JE I

L ABOR ATOR I JSKEMED IC INE

NOVI SAD, 17-21. oktobar 2006.

X V CONGRESSOF MED IC AL

B IOCHEM ISTRYAND L ABOR ATORY

MED IC INENOVI SAD, 17-21. October 2006.

P L E N A R N E S E KC I J E

P L E N A R Y S E S S I O N S

Sek c i j a 1G E N O M I P R O T E O M

Se s s i o n 1G E N O M A N D P R O T E O M


MODULATORI TARGET MESTA GENOMSKEI PROTEOMSKE REDOKS ]ELIJSKE SIGNALIZACIJE U KANCEROGENEZI: NOVE DIJAGNOSTI^KE I TERAPIJSKE

MOGU]NOSTI

D. Pavlovi}, G. Koci}, T. Jevtovi} Stoimenov

Institut za biohemiju,Medicinski fakultet Ni{, Srbija

U sloènom putu prenosa signala kroz }eliju delo-vanjem faktora spolja{nje sredine, pokre}e se trans-misija signala kroz specifi~nu komunikaciju, interakcijuprotein-protein (fosforilacija, defosforilacija, asocijaci-ja, disocijacija, oksidacija, glikacija, nitrozilacija, pro-teoliza) koja odre|uju biolo{ki odgovor }elije. Mnogiputevi prenosa signala kroz }eliju kao {to su JAK-STAT, MAP, PKC-kinazna kaskada, NF-kB put trans-dukcije signala, podleù down regulaciji posredovanojN-acetil-cisteinom i redukovanim glutationom, dokopadanje koncentracije GSH i prisutan redoks stresiniciraju njihovu aktivaciju. Aktivirani redoks signalniputevi su medijatori mitogenih efekata u kancero-genezi. Dve vrste gena, koji ina~e ~ine samo jedanmali deo genoma humane }elije, ostvaruju klju~nuulogu u kancerogenezi. Disfunkcija protoonkogena itumor supresor gena dovodi do poreme}aja u regu-laciji puteva prenosa signala koji kontroli{u }elijskiciklus, apoptozu, stabilnost genoma, diferencijaciju imorfogenezu. Promene u ovim va`nim fiziolo{kim pro-cesima odgovorne su ne samo za inicijaciju i promo-ciju maligne transformacije }elije ve} i za dalju pro-gresiju tumora. Slobodni radikali i njihovi oksidativnomodifikovani produkti dovode do aktiviranja kriti~nihsenzornih mesta u proteomu signalnih puteva kojikontroli{u }elijsku proliferaciju, apoptozu i promenu}elijskog fenotipa. Stoga je veoma va`no definisanjeterapijskih indikacija primene antioksidanata i drugihmodulatora target mesta redoks }elijske signalizacijegenomike i proteomike kao sloène mreè multifakto-rijalne onkogene kolaboracije u procesu kanceroge-neze.

REDOX CELL SIGNALING GENOMICS AND PROTEOMICS TARGET PLACE

MODULATORS IN CANCEROGENESIS: NEW DIAGNOSTIC AND THERAPEUTIC

POSSIBILITIES

D. Pavlovi}, G. Koci}, T. Jevtovi} Stoimenov

Institute of Biochemistry,Faculty of Medicine, Ni{, Serbia

Within the complex process of signal transductionthrough the cell under the influence of external fac-tors, signal transmission is initiated through a specif-ic communication, protein-protein interaction (phos-phorylation, dephosphorylation, association, dissoci-ation, oxidation, glycation, nitrosylation, proteolysis),determining the biologic response of the cell. Manyof the cellular signal transduction pathways, such asJAK-STAT, MAP, PKC-kinase cascade, NF-kB signaltransduction pathway, undergo down regulationmediated by N-acetyl-cystein and reduced glu-tathione, while the decline of GSH concentration andredox stress initiate their activation. Activated redoxsignaling pathways are the mediators of mitogeniceffects in cancerogenesis. Two types of genes, com-prising only a small part of the human cell genome,have a key role in cancerogenesis. Proto-oncogeneand tumor suppressor gene dysfunction induce dis-turbances in the regulation of signal transmissionpathways which control the cell cycle, apoptosis,genome stability, differentiation and morphogenesis.Alterations in these important physiologic processesare responsible not only for the initiation and promo-tion of malignant transformation of the cell, but forfurther tumor progression as well. Free radicals andtheir oxidatively modified products induce activationof critical sensory loci in the signal pathway proteo-ma, controling cellular proliferation, apoptosis andcellular phenotype alteration. Therefore, it is vital todefine the therapeutic indications for antioxidantsand other modulators of target places of redox cellsignaling genomics and proteomics as they are allpart of a complex network of multifactorial oncogenecollaboration in the process of cancerogenesis.

Jugoslov Med Biohem 25: 433’435, 2006 Plenarne sekcijePlenary sessions

434

ANTIOKSIDATIVNI BIOMARKERI I KANCEROGENEZA

S. B. Pajovi}, Z. S. Sai~i}, S. Peji}, J. Kasapovi}, V. Stojiljkovi}, D. T. Kanazir

Laboratorija za molekularnu biologiju i endokrinologiju, Institut za nuklearne nauke

»Vin~a«, Beograd, Srbija

Nastanak kancera odvija se u tri glavne faze (inici-jacija, promocija i progresija) koje uklju~uju oksida-tivni stres. Oksidativni stres defini{e se kao naru{a-vanje ravnoteè izme|u nivoa prooksidanata i antiok-sidanata, u pravcu pove}anja nivoa prooksidanata,{to za posledicu ima nastanak ireverzibilnih o{te}enja}elija. Da bismo procenili nivo oksidativnog stresa ukrvi pacijentkinja sa dijagnozom razli~itih formi trans-formacije uterusa, ispitivali smo nivo lipidne peroksi-dacije i aktivnost antioksidativnih enzima. Prikupljenisu uzorci krvi zdravih èna i pacijentkinja i u njima jeodre|ivana aktivnost enzima: superoksid dismutaze,katalaze, glutation peroksidaze, glutation reduktaze,kao i nivo lipidnih hidroperoksida. Rezultati pokazujuda promene ispitivanih parametara variraju u zavis-nosti od vrste enzima i dijagnoze. Me|utim, moè seuop{teno re}i da je zapaèno sniènje antioksidativneaktivnosti i pove}anje nivoa lipidne peroksidacije.Pored toga, nivo lipidnih hidroperoksida je negativnokorelisan sa aktivno{}u superoksid dismutaze i gluta-tion peroksidaze i pozitivno korelisan sa aktivno{}ukatalaze. Dobijeni rezultati tako|e pokazuju da jenaru{avanje antioksidativnog statusa u krvi izraènijekod pacijentkinja sa premalignim (hiperplazija) imalignim (adenokarcinom) lezijama, u pore|enju sapacijentkinjama kod kojih su benigne promeneuterusa dijagnostikovane kao polip ili miom.

ZNAÂJ »TISSUE MICROARRAY«TEHNIKE U DIJAGNOSTICI

I PROGNOSTICI NE-HODGINSKIH LIMFOMA, B ]ELIJSKOG POREKLA

G. Marjanovi}1, Lj. Veli~kovi}2, V. Marjanovi}3, L. Ma~ukanovi}-Golubovi}1, M. Milenovi}1

1Klinika za hematologiju i klini~ku imunologiju,Klini~ki centar ’ Ni{, Srbija

2Institut za patologiju, Klini~ki centar ’ Ni{, Srbija

3Klinika za de~iju hirurgiju i ortopediju, Klini~ki centar ’ Ni{, Srbija

Nova tehnologija »tissue microarray« (TMA) omo-gu}ava brzu i ekonomi~nu analizu stotine markera naistom setu uzoraka. Najzna~ajniji nedostatak TMAtehnologije je mala koli~ina tkiva koje se analizira, {toje nepogodno kod heterogenih tkiva kao {to su lim-

ANTIOXIDATIVE BIOMARKERS AND CANCEROGENESIS

S. B. Pajovi}, Z. S. Sai~i}, S. Peji}, J. Kasapovi}, V. Stojiljkovi}, D. T. Kanazir

Laboratory of Molecular Biology and Endocrinology, Vin~a Institute of Nuclear Sciences, Belgrade, Serbia

Cancer development includes three major steps,initiation, promotion and progression, in whichoxidative stress is involved. Oxidative stress is definedas an imbalance between the levels of prooxidantsand antioxidants in favour of the former and resultingin irreversible cell damage. We examined the lipidperoxidation levels and antioxidant enzyme activitiesin women diagnosed with different forms of uterinecell transformations in order to evaluate the extent ofoxidative stress in the blood of such patients. Bloodsamples of healthy subjects and gynecological pa-tients were collected and subjected to assays forsuperoxide dismutase, catalase, glutathione peroxi-dase, glutathione reductase and lipid hydropero-xides. The results show that alterations of the meas-ured parameters vary with the enzyme type and diag-nosis. However, both reduction in antioxidants andelevation of lipid peroxidation were observed in ge-neral. In addition, lipid hydroperoxide levels werenegatively correlated with superoxide dismutase andglutathione peroxidase activities, as well as positivelycorrelated with catalase activity. The obtained resultsalso show that perturbation of the antioxidant statusis more pronounced in blood of patients with prema-lignant (hyperplastic) and malignant (adenocarcino-ma) lesions, compared to those with benign uterinechanges such as polypus and myoma.

SIGNIFICANCE OF THE »TISSUE MICROARRAY« TECHNIQUE IN DIAGNOSIS AND PROGNOSIS OF

B NON HODGKIN’S LYMPHOMAS

G. Marjanovi}1, Lj. Veli~kovi}2, V. Marjanovi}3, L. Ma~ukanovi}-Golubovi}1, M. Milenovi}1

1Clinic of Hematology and Clinical Immunology,Clinical Center »Ni{«, Ni{, Serbia

2Institute of Pathology, Clinical Center »Ni{«, Ni{, Serbia

3Clinic of Child Surgery and Orthopedia, Clinical Center »Ni{«, Ni{, Serbia

The novel technology of tissue microarray (TMA)allows rapid and cost-effective analysis of hundredsof markers on the same set of specimens. Limitedamounts of tissue that could be analyzed and theproblem of tissue heterogeneity are the major draw-


fomi. Ovaj nedostatak je nadma{en velikim prednos-tima koje se ogledaju u homogenosti, efikasnosti iekonomi~nosti imunohistohemijske analize TMA uzo-raka. U Ne-Hodginskim limfomima TMA detekcijatranskripcionih faktora imunoglobulinskog genaOct1 i Oct2 i njihovog koaktivatora BOB1 je naro~itozna~ajna za razlikovanje T-}elijama bogatog B-}elij-skog limfoma od klasine-Hodgkinove bolesti, nodu-larno limfocitno predominantnog Hodgkinovog lim-foma i plazmablastnih limfoma. Predvi|anje pre`ivlja-vanja obolelih od pojedinih podtipova difuznog B lim-foma krupnih }elija upotrebom TMA bilo je kompati-bilno sa rezultatima cDNK »microarray« analiza.Detekcija p53 tumor supresorskog gena i Ki-67 pro-liferacionog antigena od velikog je prognosti~kogzna~aja za brojne podtipove ne-Hodginskih limfoma.Uprkos ~injenici da je ekspresija p53 i Ki-67 veomaheterogena, stepen poklapanja na TMA u odnosu naklasi~ne imunohistohemijske ise~ke iznosila je 90%za p53 i 92% za Ki-67 {to je dovoljno za svakodnevnirad. Nesumnjivo je da }e {iroka upotreba TMA posta-ti integralni deo svakodnevne klini~ke ali i istra`iva~keprakse u laboratorijama.

backs of the TMA technique for immunohistochemi-cal characterization of lymphomas. These problemsdo not outweigh the enormous advantages of TMA,namely the cost- and time-saving, and the mostlyhomogeneous results of immunohistochemistry. InNon Hodgkin's lymphomas (NHL), TMA detection ofOct1 and Oct2 immunoglobulin transcription factorsand their coactivator BOB1 showed particularly use-ful in distinguishing T-cell-rich B-cell lymphomasfrom classical Hodgkin's disease, nodular lympho-cyte predominant Hodgkin's lymphoma and plas-mablastic lymphomas. Outcome prediction for sub-types of diffuse large B-cell lymphomas, using a TMApanel with CD10, Bcl-6 and MUM1, was comparableto results of cDNA microarray analysis. Detection ofp53 tumor suppressor gene and Ki-67 proliferationantigen is important for the prognosis of many NHLsybtypes. In spite of their heterogeneity in expression,TMA showed 90 and 92% concordance rate, withconventional tissue sections for p53 and Ki-67 res-pectively, and that could be sufficient for routine pra-ctice. There is no doubt that the widespread use ofTMAs will become an integral part of daily practice inresearch and routine clinical laboratories.

Sek c i j a 2S L O B O D N E T E M E

Se s s i o n 2F R E E O R A L

P R E S E N TAT I O N


UMERENI HIPOTIROIDIZAM: KLINI^KI PROBLEM U ZDRAVOJ URBANOJ POPULACIJI INDIJE

V. Thakur

Odeljenje laboratorijske medicine, Bolnica i medicinski

istraìva~ki centar »Batra«, Nju Delhi, Indija

Blaìm oblikom hipotireoidizma naziva se klini~kostanje u kome dolazi do pove}anog lu~enja hormonakoji stimuli{u tireoideu, i to bez promena nivoa hor-mona koji ona lu~i. Takvo stanje se ~esto javlja kodosoba starijih od 50 godina, a ~e{}e je kod èna negokod mu{karaca. Uprkos ~injenici da moè izazvatiozbiljan hipotireoidizam, mi{ljenja o tome da li trebatragati za blaìm oblicima poreme}aja rada tireoideekod zdravog stanovni{tva bez izraènih simptoma surazli~ita. Ameri~ka Asocijacija za tireoideu podràvatakve preglede; radna grupa ameri~ke Slu`be za pre-ventivu, me|utim, takve preglede ne podràva. Iz ne-razvijenih zemalja se ne moè dobiti jasna slika jerraspolaù zanemarljivim brojem podataka iz teoblasti. Stoga je za sada nemogu}e osmisliti klini~keprincipe za uvo|enje kontrole rada tireoidee ili po~e-tak njenog le~enja. Postoje uverljivi dokazi da hipoti-reoidizam moè ubrzati koronarne arterijske bolesti.U ovoj studiji pregledali smo 678 zdravih ljudi bezsimptoma iz srednjeg i vi{eg ekonomskog staleà(526 mu{karaca i 152 ène) i otkrili da kod zna~ajnogbroja postoji blaì oblik hipotireoidizma (13%, n =88). Srednje vrednosti TSH bile su znatno ve}e u tojgrupi pacijenata nego kod pacijenata sa normalnimradom tireoidee (8,66 ± 4,39 IU/mL vs. 2,18 ± 0,93IU/mL, p<0,000). Koncentracije fT3 i fT4 bile su unormalnim granicama, iako je koncentracija fT4 bilaznatno nià nego kod pacijenata sa normalnimradom tireoidee (13,99 ± 2,87 pmol/L vs. 15,6 ±2,47 pmol/L, p<0,00). Na{li smo znatno ve}i brojTMT pozitivnih slu~ajeva kod grupe sa smanjenomfunkcijom tireoidee (p<0,00). Tokom 12 mesecipra}enja slu~ajeva pozitivnih na TMT otkrili smo da je55% pacijenata dobilo pravo koronarno arterijskooboljenje. Pacijenti sa smanjenim radom tireoideedobijali su blaè doze tiroksina. Terapeutski priru~niciu zapadnim zemljama preporu~uju le~enje pojedi-naca kod kojih je koli~ina TSH u serumu ve}a od10mIU/mL. Me|utim, nije sasvim izvesno da li }e ta-kav pristup biti uspe{an. Kad su u pitanju poreme}ajikoji nisu precizno definisani i kod kojih prednosti i

MILD HYPOTHYROIDISM: A CLINICAL PROBLEM IN HEALTHY

URBAN POPULATION OF INDIA

V. Thakur

Deptartment of Laboratory Medicine, Batra Hospital and Medical

Research Center, New Delhi, India

Mild hypothyroidism is a term used for describinga clinical condition where small elevations in thyroidstimulating hormone are seen without any change inthe thyroid hormone levels. The condition is quitecommon in elderly over the age of 50 and prevalentin women. Despite the fact that this condition canlead to overt hypothyroidism, controversy continueswhether asymptomatic healthy population should bescreened for mild thyroid dysfunction. AmericanThyroid Association supports the screening; howev-er, US Preventive Services task force does not sup-port screening. The picture is unclear from develo-ping countries, which only have scanty data availableon this subject. Thus, devising clinical guidelines inthis scenario for initiating hypothyroid screening orinitiating its treatment is impossible. There is com-pelling evidence that hypothyroidism could precipi-tate coronary artery disease. In a research study, wescreened 678 healthy asymptomatic individuals frommid to high socioeconomic strata (526 males and152 females) and revealed a substantial percentageof population having mild hypothyroidism (13%, n =88). The mean TSH levels were significantly higher inthis group of patients when compared with euthy-roids (8.66 ± 4.39 IU/mL vs. 2.18 ± 0.93 IU/mL,p<0.000). fT3 and fT4 concentrations were withinnormal limits, although the fT4 concentration wassignificantly lower than in euthyroids ( 13.99 ± 2.87pmol/L vs 15.6 ± 2.47 pmol/L, p<0.00). We found asignificantly higher number of TMT positive cases inthe hypothyroid group (p<0.00). In the 12 months offollow-up of TMT positive cases, we managed to findthat 55% of patients developed frank CAD. Thehypothyroid patients were treated with mild doses ofthyroxine. Therapeutic guidelines in the Westerncountries recommend treatment in individuals withserum TSH levels higher than 10 mIU/mL. But thereis gross clinical uncertainty if this approach will reallybenefit. In disorders for which the definition of thecondition is imprecise and the benefits and risks oftreatment are not clearly established, patient prefer-


440

mane le~enja nisu jasno utvr|ene, u dono{enju odlu-ke o le~enju presudnu ulogu ima stanje pacijenta.Sve u svemu, smatramo da treba nastaviti sa istra-ìvanjem metaboli~kih faktora, kao {to su visokosen-zitivni C reaktivni protein i homocistein, jer moguposluìti za rano predvi|anje koronarnih arterijskihbolesti kod ovog sindroma. Po~eli smo sa radom naHsCRP i homocisteinu, ali iz na{eg centra jo{ uveknema novih podataka.

CIRKULI[U]I sCD40 LIGAND U KARDIOVASKULARNIM BOLESTIMA

I INFLAMATORNIM STANJIMA

S. Stankovi}1, M. Ili}1, Z. Vasiljevi}2, B. Vujisi}-Te{i}2, N. Majki}-Singh1

1Institut za medicinsku biohemiju,Klini~ki centar Srbije, Beograd, Srbija 2Institut za kardiovaskularne bolesti, Klini~ki centar Srbije, Beograd, Srbija

CD40L, glikoprotein membrane tipa II, sastavljenod 261 amino-kiseline, ~lan familije faktora nekrozetumora (TNF), identifikovan je inicijalno na }elijamaimunolo{kog sistema (aktiviranim CD4+}elijama,mastocitima, bazofilima, eozinofilima i NK }elijama).CD40 je konstitutivno eksprimiran na B }elijama,makrofagama i dendriti~nim }elijama. CD40L i CD40su tako|e prisutni na endotelnim }elijama, glatkimmi{i}nim }elijama, monocitima i makrofagama. Vi{eod 95% cirkuli{u}eg CD40L nalazi se u trombociti-ma. CD40L i sCD40L imaju strukturne domene kojiomogu}avaju brojne funkcije: vezivanje za CD40 re-ceptor preko TNF homologog domena, vezivanje zaglikoprotein IIb/IIIa preko lizin-arginin-glutamat moti-va, izazivanje signalnih reakcija koje su posledica vezi-vanja CD40L za receptor i njegovih rastvorljivih pro-dukata cepanja. Funkcije trombocitnog CD40L po-sledica su interakcije ovih domena. Kada se ekspri-mira na povr{ini trombocita i izloì }elijama koje noseCD40, CD40L trombocita ima mogu}nost iniciranjarazli~itih inflamatornih odgovora, uklju~uju}i ekspre-siju adhezionih receptora, ekspresiju tkivnog faktora iosloba|anje hemokina. Cirkuli{u}i sCD40L ima pro-inflamatornu i protromboti~ku aktivnost. Pove}anekoncentracije sCD40L utvr|ene su u kardiovaskular-noj bolesti i brojnim inflamatornim stanjima, kao {tosu reumatoidni artritis, anemija srpastih }elija ili si-stemski eritemski lupus. Na{a iskustva sa odre|iva-njem sCD40L vezana su za njegovo odre|ivanje kodpacijenata sa akutnim koronarnim sindromom, mi-tralnom regurgitacijom i akutnim pankreatitisom.Iako dobijeni rezultati naizgled nemaju direktan uticajna klini~ku praksu, farmakolo{ko delovanje na inter-akciju CD40-CD40L dalo je zadovoljavaju}e rezultatekod razli~itih patolo{kih stanja.

ences play a critical role in treatment decisions. Ove-rall, we feel that metabolic factors like high sensitiveC reactive protein and homocystein should be furtherinvestigated as early predictors of coronary arterydisease in this syndrome. We have now started work-ing on HsCRP and homocystein, but no data is yetavailable from our center.

CIRCULATING sCD40 LIGAND IN CARDIOVASCULAR DISEASE

AND INFLAMMATORY CONDITIONS

S. Stankovi}1, M. Ili}1, Z. Vasiljevi}2, B. Vujisi}-Te{i}2, N. Majki}-Singh1

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

2Institute of Cardiovascular Diseases, Clinical Center of Serbia, Belgrade, Serbia

CD40L, 261-amino acid type II membrane glyco-protein, member of the tumor necrosis factor (TNF)family, was originally identified on the cells of theimmune system (activated CD4+ cells, mast cells,basophils, eosinophils, and natural killer cells). CD40is constitutively expressed on B cells, macrophagesand dendritic cells. CD40L and CD40 are also pres-ent on several cells of the vasculature, includingendothelial cells, smooth muscle cells, monocytesand macrophages. More than 95% of the circulatingCD40L exists in platelets. CD40L and sCD40L areknown to have structural domains that allow theseproteins to have multiple functions. First, the TNFhomology domain allows for binding to the receptor,CD40. Second, the lysine-arginine-glutamic acidmotif allows for binding to glycoprotein IIb/IIIa. Third,the trimeric structure of CD40L and its soluble clea-vage product allow for the induction of signalingreactions when bound to receptors. The functionalactivities of platelet CD40L are reflective of thesemultiple domains. When expressed on the surface ofplatelets and exposed to CD40-bearing vascularcells, platelet-associated CD40L is capable of initia-ting various inflammatory responses, includingexpression of inflammatory adhesion receptors,expression of the tissue factor, and release of chemo-kines. Soluble CD40L is also proinflammatory andhas prothrombotic activity. Although recent studieshave described increased levels of sCD40L in cardio-vascular diseases and a broad spectrum of inflam-matory conditions, such as rheumatoid arthritis, sick-le cell anemia or systemic lupus erythematosus, wewill present our experience with sCD40L in acutecoronary syndrome, mitral regurgitation and acutepancreatitis. Although our findings do not currentlyaffect clinical practice, pharmacological manipula-tion of the CD40-CD40L axis proved effective in va-rious pathological states.


PARAMETRI HEMOSTAZE U PACIJENATAKOD KOJIH JE INDIKOVANA KORONARNA

ENDARTEREKTOMIJA

V. Subota, S. Mandi}-Radi}, I. Petrovi}, \. Radak

Institut za medicinsku biohemiju VMA, Institut za KVB »Dedinje«,

Zavod za zdravstvenu za{titu radnika MUP,Beograd, Srbija

Dok se tradicionalni pristup predvi|anju arterijskeokluzije oslanja na faktore rizika koji su vezani za lipidei na~in `ivota, mnoge epidemiolo{ke studije potvr|ujuda visok rizik za aterosklerozu predstavljaju aktiviranafibrinoliza i koagulacija. Hroni~na inflamacija igrazna~ajnu ulogu u inicijaciji i progresiji ateroskleroze. Ciljna{eg rada bio je da se uporede promene parametarahemostaze u odnosu na povi{eni nivo C-reaktivnogproteina (CRP), kao markera sistemske inflamacije.Prou~avana grupa obuhvatila je 50 pacijenata podel-jenih u dve grupe od po 25 pacijenata, G1 (CRP 5mg/L) i G2 (CRP 5 mg/L). Nije bilo pacijenata saporeme}ajima hemostaze i perifernim vaskularnimbolestima. Ispitivani su parametri lipidnog statusa:trigliceridi, ukupni holesterol, HDL, LDL, Lp(a) iapolipoproteini A i B. Od parametara hemostaze odre-|ivani su: fibrinogen, protein C, koagulacioni faktor VII,D-dimer, 2-antiplazmin, plazminogen, inhibitor aktiva-tora plazminogena -1, Von Willebrand faktor, fibrinmonomer, agregacija trombocita sa ADP i epinefrinomkao agonistima, i broj trombocita. Kori{}ene su stan-dardne metode i komercijalni reagensi. Rezultati suupore|ivani izme|u grupa i pokazali su da su lipidniparametri bili u okviru referentnog opsega, dok su FVII,D-d, PAI-1 i fibrinogen bili zna~ajno pove}ani(p<0,001) u G2. Nije bilo zna~ajnih razlika izme|uostalih parametara hemostaze, iako su svi oni bili po-ve}ani u G2. Ovi nalazi ukazuju da, i pored regulisanihostalih faktora rizika, aktivirana fibrinoliza i koagulacijakao posledice sistemske inflamacije predstavljajudodatni rizik koji mo`e dovesti do nepovoljnog ishoda idaljih komplikacija.

NATRIURETSKI PEPTIDI - OCENA DIJAGNOSTI^KOG DOPRINOSA

ODRE\IVANJA MO@DANOG NATRIURETSKOG PEPTIDA

R. Kova~evi}, M. Miri}, O. Stojanovi}

Institut za kardiovaskularne bolesti »Dedinje«,Beograd, Srbija

Sr~ane pretkomore i komore sinteti{u i izlu~uju ne-koliko natriuretskih hormona, uglavnom kao odgovorna pove}anje intrakardijalnog pritiska. Sr~ani hormoni,

HAEMOSTATIC PARAMETERS IN PATIENTS TO BE SUBJECT

TO CORONARY ENDARTERECTOMY

V. Subota, S. Mandi}-Radi}, I. Petrovi}, \. Radak

Institute of Medical Biochemistry MMA, Institute of CVD »Dedinje«,

Health Care Institute for Employees of the Ministry of Internal Affairs, Belgrade, Serbia

While traditional approaches to predicting arterialocclusion rely on behavioural and lipid related risk fac-tors, the concept that impaired fibrinolysis and coagu-lation are risk factors for atherosclerosis is strongly sup-ported by many epidemiologic studies. Chronic inflam-mation plays an important role in the initiation and pro-gression of atherosclerosis. The aim of our study wasto compare the changes in haemostatic parameters inrelation to elevated levels of C-reactive protein (CRP),as a systemic inflammation marker. The studied popu-lation comprised 50 patients divided into two groups,G1, consisting of 25 patients (CRP 5 mg/L), and G2(CRP 5 mg/L). There were no patients with haemosta-tic disturbances and peripheral vascular diseases. Theparameters of lipid status: triglycerides, total choles-terol, HDL, LDL, Lp(a) and apolipoproteins A and B,were determined. The haemostatic parameters: fibrino-gen, protein C, coagulation factor VII, D-dimer, 2--antiplasmin, plasminogen, plasminogen activator inhi-bitor -1, Von Willebrand factor, fibrin monomer, plate-lets aggregation with ADP and epinephrine as ago-nists, and the number of platelets were measured.Standard methods and commercial reagents wereused. The results were compared between the groupsand showed that lipid parameters were within the reco-mmended range, but FVII, D-d, PAI-1 and fibrinogenwere significantly increased (p<0.001) in G2. Therewere no significant differences in the other parametersof haemostasis, but all of them were elevated in G2.With other risk factors in control, these findings indicatethat impaired fibrinolysis and coagulation as conse-quences of inflammation are additional risk factors foran unfavourable outcome and further complications.

NATRIURETIC PEPTIDES – EVALUATION OF THE DIAGNOSTIC CONTRIBUTION

OF BRAIN NATRIURETIC PEPTIDE

R. Kova~evi}, M. Miri}, O. Stojanovi}

Institute of Cardiovascular Diseases »Dedinje«,Belgrade, Serbia

Both atria and ventricles synthesize and secrete se-veral natriuretic peptide hormones ’ largely in res-ponse to increased intracardiac pressure. The pro-

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atrijumski natriuretski peptid (ANP) i mo`dani natri-uretski peptid (BNP) i njihovi prohormonski »metaboli-ti« doprinose kardiovaskularnoj homeostazi. Natriuret-ski peptidni sistem najvi{e se aktivira u ventrikularnojdisfunkciji. Merenje koncentracije mo`danog natriuret-skog peptida (BNP-a) u plazmi moè koristiti u postav-ljanju dijagnoze sr~ane insuficijencije. Cilj istraìvanjabilo je utvr|ivanje dijagnosti~kog doprinosa odre|iva-nja BNP-a u plazmi u pore|enju sa dijagnosti~kim do-prinosom rendgen snimka i ultrazvu~nog pregleda udijagnostikovanju sr~ane insuficijencije. KoncentracijeBNP-a u plazmi odre|ivane su imunofluorescentnimtestom (POCT Triage, BIOSITE Laboratories) kod 62bolesnika sa sr~anom insuficijencijom (dobi 55,82 ±9,09 godina, 74,19% mu{karaca, ishemijskom n=30 idilatativnom kardiomiopatijom n=32, NYHA funk-cionalnom klasom II n=33, i klasom III n=29, LVEF27,77 ± 4,35%). Ocena dijagnosti~kog doprinosa me-renja koncentracije BNP-a u komparaciji sa dijagno-sti~kim doprinosom rendgenskog snimka grudnogko{a i eho dijagnostikom utvr|ivana je diskrimina-tornom analizom vrednosti LVEF (parametri eho dijag-nostike), kardiotorakalnog indeksa (parametri rendgendijagnostike) i koncentracije BNP-a, sa teìnom bolestiprema NYHA klasifikaciji. Rezultati diskriminatorneanalize potvr|uju da klasifikovanje bolesnika premaNYHA klasifikaciji sa verovatno}om p=0,903 zavisi odvrednosti LVEF, odnosno da eho dijagnosti~ka meto-da ta~no odre|uje teìnu oboljenja prema NYHA klasi-fikaciji u 90,3% slu~ajeva. Rezultati diskriminatorneanalize pokazuju da klasifikovanje bolesnika premaNYHA klasifikaciji sa verovatno}om p=0,806 zavisi odvrednosti kardiotorakalnog indeksa, odnosno da rend-gen dijagnosti~ka metoda ta~no odre|uje teìnu obo-ljenja prema NYHA klasifikaciji u 80,6% slu~ajeva.Rezultati diskriminatorne analize potvr|uju da klasi-fikovanje bolesnika prema NYHA klasifikaciji saverovatno}om p=0,871 zavisi od vrednosti BNP-a,odnosno da merenje njegove koncentracije, kao dijag-nosti~ka metoda, ta~no odre|uje teìnu oboljenjaprema NYHA klasifikaciji u 87,1% slu~ajeva. Rezultatidiskriminatorne analize navedenih parametara ukazujuna to da je ve}i dijagnosti~ki doprinos eho dijagnostike(90,3% ispitanika klasifikovano korektno) od merenjakoncentracije BNP-a (87,1% ispitanika klasifikovanokorektno), dok je dijagnosti~ki doprinos rendgen sni-manja manji (80,6% ispitanika klasifikovano korektno).

ducts of the heart, atrial natriuretic peptide (ANP) andbrain natriuretic peptide (BNP), and possibly theirprohormone »metabolites«, contribute to cardiovascu-lar homeostasis. The natriuretic peptide system is acti-vated to its highest level in ventricular dysfunction.Plasma concentration of BNP could be of use in thediagnosis of heart failure. Our aim was to determinethe diagnostic contribution of BNP plasma measure-ment in comparison with the diagnostic contributionof X ray imaging and ultrasound examination in thediagnostics of heart failure. Plasma concentration ofBNP was measured by the POCT Triage immunofluo-rescent assay (BIOSITE Laboratories) in 62 heart fai-lure patients (aged 55.82 ± 9.09 years, 74.19% male,n=30 ischemic, n=32 dilated cardiomiopathy, n=33NYHA functional class II, n=29 class III, LVEF 27.77 ±4.35%). Assessment of the diagnostic contribution ofBNP plasma concentration measurement in compari-son with the diagnostic contribution of chest X rayimage and ECHO methods was determined using dis-criminatory analysis of LVEF (echo diagnostic para-meters), cardiothoracic index (X ray parameters) andBNP plasma concentration, with severity of diseaseexpressed according to the NYHA classification. Re-sults of discriminatory analysis confirmed that grada-tion of patients using the NYHA classification withprobability p=0.903 depends on the LVEF value, thatis, the echo diagnostic method correctly classifies theseverity of disease according to the NYHA classifica-tion in 90.3% of cases. Results of discriminatory analy-sis showed that classification of patients according tothe NYHA with the probability p=0.806 depends onthe value of the cardiothoracic index, that is, the X raydiagnostic method correctly classifies the severity ofdisease according to the NYHA classification in 80.6%of cases. Results of discriminatory analysis confirmedthat classification of patients according to the NYHAwith probability p=0.871 depends on the BNP value,that is, measurement of BNP plasma concentrationcorrectly classifies the severity of disease in 87.1% ofcases. Results of discriminatory analysis of the men-tioned parameters showed that the echo diagnosticcontribution (90.3% of patients classified correctly)was larger than the contribution of BNP measurement(87.1% of patients classified correctly), while the diag-nostic contribution of X ray images was lower (80.6%of patients classified correctly).


TARTARAT-REZISTENTNA KISELA FOSFATAZA I OSTEOKALCIN

KOD PACIJENATA SA OSTEOPOROZOM I OSTEOPENIJOM

J. Mehanovi} Nikoli}1, J. Lalo{-Milju{2

1Zavod za biohemiju, Medicinski fakultetUniverziteta u Banjoj Luci

2Klini~ki centar Banja Luka

Mjerenjem kataliti~ke aktivnosti kisele fosfatazerezistentne na tartarat (TRKP) u serumu procjenjujese osteoklasti~na aktivnost, po{to je ko{tane }elijesecerniraju tokom resorpcije. Osteokalcin je nekola-genski peptid koji u~estvuje u procesu mineralizacijekostiju. Kori{ten je u ranom prepoznavanju primarneosteoporoze. Cilj je bio da se utvrdi da li postoji razli-ka u kataliti~koj aktivnosti TRKP, kao i u serumskojkoncentraciji osteokalcina, u ispitanika sa osteoporo-zom i osteopenijom. Prva grupa (n=60) imala je T-scor mineralne gustine kosti (BMD) ≤’2,3, {to se,prema standardima WHO, defini{e kao osteoporoza.Druga grupa (n=60) imala je T-scor izme|u ’0,4 i’1,7, {to prema standardima WHO odgovara sma-njenoj ko{tanoj masi (osteopeniji). Kataliti~ka aktiv-nost TRKP mjerena je kineti~kom metodom firmeBIOMERIEUX (Njema~ka), na analizatoru Mira Cobasplus (Njema~ka), BMD je odre|en ultrazvu~nomtehnikom, a koncentracija osteokalcina tehnikomECL, firme ROCHE (Njema~ka), na analizatoru Elec-sys 2010 (Njema~ka). Rezultati ukazuju na zna~ajnopove}anje kataliti~ke aktivnosti TRKP u serumu ispi-tanika sa osteoporozom, u odnosu na grupu sa oste-openijom (p<0,001). Za osteokalcin je utvr|ena sta-tisti~ki zna~ajna razlika izme|u dvije grupe ispitanika(p<0,01). Smatramo da odre|ivanje kataliti~ke aktiv-nosti TRKP u serumu, kao i koncentracije osteokalci-na, uz druge biohemijske markere ko{tane pregrad-nje, mo`e da unaprijedi dijagnostiku osteoporoze.

TARTRATE-RESISTANT ACID PHOSPHATASE AND OSTEOCALCIN IN PATIENTS WITH OSTEOPOROSIS

AND OSTEOPENIA

J. Mehanovi} Nikoli}1, J. Lalo{-Milju{2

1Department of Biochemistry, School of Medicine,University of Banja Luka, Bosnia and Herzegovina

2Clinical Center, Banja Luka, Bosnia and Herzegovina

A way to estimate the activity of osteoclasts is tomeasure the catalytic activity of tartrate-resistant acidphosphatase (TRAP), as bone cells secrete it duringresorption. Osteocalcin (OC) is a non-collagen pep-tide involved in the bone mineralization process. It isused in the early diagnosis of primary osteoporosis.The aim of our work was to determine if there was adifference in both the catalytic activity of the TRAPand the serum concentration of OC in patientsaffected by osteoporosis and osteopenia. The bonemineral density T-score (BMD) of the first group ofpatients (n=60) was not higher than ’2.3, defined bythe WHO as osteoporosis. The T-score of the secondgroup (n=60) was found to be between ’0.4 and’1.7, defined by the WHO as osteopenia. The cata-lytic activity of TRAP was determined by the kineticmethod proposed by the BIOMERIEUX Company(Germany), using a Mira Cobas plus analyzer(Germany). The BMD was determined by an ultra-sound technique. The ROCHE ECL technique andthe Elecsys 2010 analyzer (Germany) were used tomeasure the OC concentration. The results show asignificant increase in the catalytic activity of TRAP inthe osteoporosis patients sera when compared withthe group with osteopenia (p<0.001). Statisticallysignificant difference was found between the groupsin the case of OC (p<0.01). We conclude that theTRAP catalytic activity in serum, as well as OC con-centration measurement, together with other bio-chemical markers of bone turnover, can be useful inthe diagnosis of osteoporosis.

444

NEOPHODNOST PRIDR@AVANJA STANDARDNIM USLOVIMA PRI

PREGLEDU URINA (SEDIMENTA)

S. \. Jovanovi}, N. Pri{i}, M. Jankovi}

Hemolab, Medicinska laboratorija,Sremska Kamenica, Novi Sad, Srbija

U radu je dokazana neophodnost pridràvanjastandardnim uslovima pri kompletnom pregleduurina, odn. sedimenta. Analizom 31 patolo{kog uzor-ka prvog jutarnjeg urina upore|ene su razlike u brojuizbrojanih elemenata (}elije okruglog epitela, epitelne}elije, leukociti, sveì eritrociti, izlu~eni eritrociti i hi-jalini cinidri) u koli~inama od 5 i 10 ml urina. Ostaliparametri kao {to su oksalati, amorfni urati i fosfati,bakterije i sluz su tako|e pra}eni, ali nisu broj~anoevidentirani. Dobijeni su slede}i rezultati (ukupan brojelemenata): okrugli epitel ’ 3 (5 ml) prema 14 (10ml); epitelne }elije ’ 78 (5 ml) prema 132 (10 ml);leukociti ’ 202 (5 ml) prema 385 (l0 ml); sveì eritro-citi ’ 38 (5 ml) prema 63 (10 ml); izlu~eni eritrociti ’14 (5 ml) prema 29 (10 ml); hijalini cilindri ’ 1 (5 ml)prema 6 (10 ml). Budu}i da su razlike izme|u 5 i 10ml kod 6 »kriti~nih« parametara uglavnom dvostru-ke, smatrali smo da nema potrebe za statisti~komobradom podataka. Jasno je da ovi rezultati imajudijagnosti~ki i terapeutski zna~aj za sve koji se baveovom problematikom.

THE NECESSITY OF ADHERING TO STANDARD CONDITIONS IN

URINE (SEDIMENT) INVESTIGATIONS

S. \. Jovanovi}, N. Pri{i}, M. Jankovi}

Hemolab, Medicinska laboratorija,Sremska Kamenica, Novi Sad, Serbia

The necessity to appreciate the standard condi-tions (10 ml) in complete urine (sediment) investiga-tions is approved in the paper. In 31 pathologic sam-ples of the first morning urine, the differences in thenumber of investigated parameters (round epithelialcells, plate epithelial cells, leukocytes, erythrocytes,segregated erythrocytes, hyalin cylinders) are com-pared in the samples of 5 and 10 ml of urine. Otherparameters like oxalates, amorph urates and phos-phates, bacteria and mucus, are monitored as well,but not presented in numbers. Following results havebeen achieved (total number of elements): roundepithel cells ’ 3 (5 ml) to 14 (10 ml); plate epithelialcells ’ 78 (5 ml) to 143 (10 ml); leukocytes ’ 202 (5ml) to 385 (10 ml); fresh erythrocytes ’ 38 (5 ml) zo63 (10 ml); segregated erytrocytes ’ 14 (5 ml) to 228(10 ml); hyalin cylinders ’ 1 (5 ml) to 6 (10 ml). Ta-king into account that differences between 5 and 10ml in 6 »critical« parameters are nearly double, statis-tic evaluation of data seemed to be unnecessary. Theresults are of major diagnostic and therapeutic impor-tance for everyone involved in the matter.

Sek c i j a 3S AV R E M E N I A S P E K T IA T E R O T R O M B O T S K E

B O L E S T I

S e s s i o n 3C U R R E N T A S P E C T S

O F T H EA T H E R O T H R O M B O T I C

D I S E A S E

LIPIDI I ATEROSKLEROZA

M. \eri}1, E. Stoki}2, B. Vu~kovi}1, S. Koji}-Damjanov1, V. âbarkapa1

1Instutut za laboratorijsku medicinu2Klinika za endokrinologiju, dijabetes i bolesti

metabolizma, Klini~ki centar - Novi Sad, Novi Sad, Srbija

Lipidi imaju zna~ajnu ulogu u aterogenezi, po~evod inicijalnih promena, te se danas najvi{e razmatranjihova prediktivna vrednost za koronarnu bolestsrca, kao i mogu}nosti novih terapijskih pristupa.Centralno mesto zauzimaju LDL ~estice, jer je samoza redukciju LDL-holesterola dokazano da redukujemorbiditet i mortalitet udruèn sa aterosklerozom.Osim regulacije ekspresije LDL-receptora, velikiklini~ki zna~aj imaju oksidisani LDL (ox-LDL) i maleguste LDL (sdLDL) koje su produkt intravaskularnogremodelovanja trigliceridima bogatih lipoproteina,dok egzaktna uloga anti-oxLDL antitela u progresijiateroskleroze i klini~ki zna~aj ostaju nejasni. Posled-njih godina, me|utim, potpunije upoznavanje bazi~-nih mehanizama uklju~enih kako u aterosklerozu,tako i u metaboli~ku sudbinu lipida i lipoproteina,isti~e i zna~ajnu ulogu ostalih lipoproteinskih ~estica,i to, ne samo u progresiji, ve} i u inicijaciji atero-skleroze i aterotromboze.

INTERAKCIJA IZME\U OKSIDATIVNOGSTRESA I INFLAMATORNIH BIOMARKERA

U ATEROSKLEROZI

V. B. \or|evi}1, T. Cvetkovi}1, M. Deljanin-Ili}3, V. ]osi}2, L. Zvezdanovi}2,

S. Kundali}2, S. Madi}2, I. Stojanovi}1

1Institut za biohemiju, Medicinski fakultet, Ni{, Srbija

2Centar za medicinsku biohemiju,Klini~ki centar ’ Ni{, Srbija

3Institut za kardiovaskularne bolesti »Ni{ka Banja«, Ni{ka Banja, Srbija

Brojni rezultati pokazuju da postoji uska vezaizme|u inflamacije, oksidativnog stresa i aterosklero-

LIPIDS AND ATHEROSCLEROSIS

M. \eri}1, E. Stoki}2, B. Vu~kovi}1, S. Koji}-Damjanov1, V. âbarkapa1

1Instutute of Laboratory Medicine2Clinic for Endocrinology,

Diabetes and Metabolic Diseases, Clinical Center - Novi Sad, Novi Sad, Serbia

Lipids play a pivotal role in the atherogenesis,starting from initial changes. Their predictive value incoronary heart disease and development of noveltherapeutic strategies is an increasingly addressedissue nowadays. LDL particles are fundamental, be-cause reduction of LDL-cholesterol was proven toreduce morbidity and mortality associated with ath-erosclerosis. Besides the regulation of LDL-receptorexpression, significant clinical importance is assignedto oxidized LDL (ox-LDL) and small dense LDL(sdLDL) that are generated through intravascularremodelling of triglyceride-rich lipoproteins, while theexact role of anti-oxLDL antibodies in atherosclerosispropagation and their clinical significance still remainunclear. In recent years, however, better understan-ding of the basic mechanisms involved in atheroscle-rosis development, as well as in the metabolic fate oflipids and lipoproteins, emphasizes the crucial role ofother lipoprotein particles not only in the propaga-tion, but also in the initiation of atherosclerosis andatherothrombosis.

THE INTERACTION BETWEEN OXIDATIVE STRESS AND BIOMARKERS

OF INFLAMMATION IN ATHEROSCLEROSIS

V. B. \or|evi}1, T. Cvetkovi}1, M. Deljanin-Ili}3, V. ]osi}2, L. Zvezdanovi}2,

S. Kundali}2, S. Madi}2, I. Stojanovi}1

1Institute of Biochemistry, Faculty of Medicine, Ni{, Serbia

2Centre for Medical Biochemistry, Clinical Centre ’ Ni{, Serbia

3Institute of Cardiovascular Diseases »Ni{ka Banja«, Ni{ka Banja, Serbia

A number of data demonstrated that there is aclose relation between inflammation, oxidative stress



448

ze. Inicijalni doga|aj u aterogenezi je disfunkcija iliaktivacija endotela. Ona moè biti izazvana mehani~-kim, hemijskim, infektivnim ili imunolo{kim faktori-ma, {to ukazuje da gotovo svi faktori rizika za atero-sklerozu mogu dovesti do endotelne disfunkcije. Toizaziva kaskadu inflamatornih reakcija u kojimau~estvuju monociti, makrofagi, T limfociti i }elijeglatkih mi{i}a. Ove }elije i endotel nadalje produkujuadhezione molekule, citokine, faktore rasta i meta-loproteinaze, prolongiraju}i aterogenezu. Aktivacijaenzima koji produkuju oksidanse, kao {to su NADPHoksidaza i azot-monoksid sintaza (NOS), dovodi dooksidativnog stresa i posledi~ne oksidativne modi-fikacije LDL, a oxLDL potom moè aktivisati endotel-ne }elije. Oksidativno modifikovani LDL preuzimajumakrofagi putem »scavenger« receptora. Rezultattoga je akumulacija holesterola u makrofagima i stva-ranje penastih }elija koje predstavljaju bazu atero-skleroze. îtav proces se cikli~no ponavlja, dovode}ido formiranja uznapredovalih ateroskleroti~nih prom-ena koje karakteri{e jezgro sa~injeno od lipida inekroti~nog tkiva pokriveno fibroznom kapom.

HEMOSTAZNI SISTEM U GENEZIATEROTROMBOZE

A. Lu~i}

Novi Sad, Srbija

Ateroskleroza i arterijska tromboza su dva direk-tno me|usobno zavisna patolo{ka procesa i stoganaziv aterotromboza na najbolji mogu}i na~inizraàva su{tinu ovog sloènog sistemskog oboljenja.Aterotromboza u progresivnom kontinuitetu dovodido dramati~nog naru{avanja integriteta arterijskihkrvnih sudova velikog i srednjeg promera i izazivadelimi~ni ili potpuni prekid protoka krvi u obolelimkrvnim sudovima. U patogenezi aterotromboze, he-mostazni sistem je sa svoje tri osnovne komponente:trombocitima, koagulacijskim i fibrinoliznim ~inio-cima, endotelskim }elijama i dubljim strukturamaunutra{nje povr{ine zida krvnog suda, neposredno iprvenstveno odgovoran za nastanak tromboze.Osnovni preduslov nastanka arterijske tromboze jeporeme}aj funkcije endotela ili naru{avanje njegovogintegriteta. U aterotrombozi, u svim stadijumima ra-zvoja aterosklerotskih promena na intimi krvnogsuda, taj osnovni preduslov je u celosti ispunjen. Naj-drasti~niji pokreta~ki ~inilac arterijske tromboze pred-stavlja ruptura vulnerabilne ili nestabilne ateromskeplo~e. Sadràj krupnog lipidnog jezgra ateroma, ras-cepljeni tanki fibrozni pokriva~, obilje van}elijskogmatriksa, brojna prisutnost zapaljenjskih }elija i visoksadràj tkivnog ~inioca (TF) izazivaju eksplozivnu akti-vaciju trombocita i koagulacijskog sistema. Signalniprenosni mehanizmi koji se pokre}u vezivanjem spe-

and atherosclerosis. The initial event in atherogene-sis is some form of endothelial dysfunction or activa-tion. It can be triggered by mechanical, chemical,infectious or immunological insults, indicating thatalmost all risk factors for atherosclerosis could pro-mote endothelial dysfunction. This triggers a cascadeof inflammatory reactions, in which monocytes, ma-crophages, T lymphocytes and smooth muscle cellsparticipate. These cells and the endothelium produceadhesion molecules, cytokines, growth factors andmetalloproteinases, thus prolonging atherogenesis.An activation of oxidative producing enzymes such asNADPH oxidases and nitric oxide synthase (NOS)leads to oxidative stress and subsequent oxidativemodification of LDL, and oxLDL can in turn activatethe endothelial cells. Oxidatively modified LDL isuptaken by macrophages via scavenger receptors.This results in the accumulation of cholesterol withinthe macrophages and the formation of foam cells, ahallmark of atherosclerosis. The process continueswith repeated cycles leading to the characteristicadvanced lesions with the core of lipids and necrotictissue which is covered by a fibrous cap.

HEMOSTATIC SYSTEM IN ATHEROTHROMBOSIS

A. Lu~i}

Novi Sad, Serbia

Atherosclerosis and arterial thrombosis are twodirectly interdependent pathologic processes and theterm atherothrombosis covers most fully the essenceof this complex systemic disease. Atherothrombosisin continual progression leads to a dramatic distur-bance of the large and medium-sized arteries integri-ty and provokes partial or complete blood flow inter-ruption in the diseased vessels. In the pathogenesisof atherothrombosis, the hemostatic system with itsthree basic components: platelets, coagulation andfibrinolytic factors, endothelial cells and the deeperstructure of the inner layer of blood vessels, is main-ly and directly responsible for the occurence anddevelopment of thrombosis. The main prerequisite ofarterial thrombosis is the endothelial dysfunction, aswell as a breach of the endothelial integrity. In athe-rothrombosis, during each phase of the developmentof atherosclerotic changes on the intima of the bloodvessel, this basic cause is fully realized. The mostpowerful trigger of arterial thrombosis is the ruptureof the vulnerable or unstable atherosclerotic plaque.Large lipid core of the disrupted plaque, splitting ofthe thin fibrous cap, high density of inflammatorycells, abundant extracellular matrix and high contentof the tissue factor (TF), promote the explosive acti-vation of platelet and clotting mechanisms. Signaltranduction mechanism, initiated upon the binding


cifi~nih agonista za vezne membranske receptore napovr{ini trombocita stimuli{u izlazak kalcijuma iz tu-bularnog sistema trombocita i izazivaju promenu ob-lika trombocita i pra`njenje sadràja njihovih granula.Adhezija i aktivacija trombocita i proces njihove me-|usobne agregacije su direktne posledice prepozna-vanja trombogenog podru~ja u krvnom sudu od stra-ne ovih }elija. Budu}i da reaktivnost trombocita zavisiod brojnih adhezivnih me|ureakcija, ve}ina glikopro-teina membrane trombocita su receptori adhezivnihproteina. Po svojoj strukturi oni uglavnom pripadajuintegrinima, selektinima, i glikoproteinima bogatimleucinom. Punu funkcionalnost ovih receptora omo-gu}ava njihova neposredna udruènost sa dinami~-nim lipidnim skupinama, sastavljenim iz sfingolipida iholesterola, koje slobodno plutaju unutar te~ne frak-cije }elijske opne trombocita. Raspad ateromske plo-~e izaziva i burnu aktivaciju koagulacijskog sistemapod uticajem TF-a koji u kofaktorskoj poziciji vezujeFVII/FVIIa i uve}ava kataliti~ku aktivnost FVIIa vi{e odmilion puta. TF se u krvi nalazi vezan za cirkuli{u}emikro~estice stvorene u monocitima, a u zna~ajnimkoli~inama se osloba|a i iz aktiviranih endotelskih}elija i }elija u okruènju krvnog suda. P-selektin, kojise nalazi u membrani trombocitnih alfa-granula kao iu skladi{nim granulama endotela, preme{ta se uprocesu aktivacije na povr{inu }elija a moè se na}i iu cirkulaciji. Ovaj adhezivni receptor pokre}e processtvaranja mikro~estica u monocitima koje sadrè TF,istovremeno ih zadràvaju}i na mestu stvaranja trom-bina i fibrina. O posebnoj sloènosti mehanizamaaktivacije hemostaznog sistema govori i vi{estruko irazli~ito delovanje trombina. Njegova osnovna funk-cija je stvaranje fibrina, ali vezivanjem za trombomo-dulin on pokre}e aktivaciju proteina C, a tako|e sti-muli{e i osloba|anje tkivnog aktivatora i inhibitoraaktivatora plazminogena iz endotelskih }elija. Na tajna~in trombin, osim {to je najuticajniji pokreta~ akti-vacije trombocita, ima i klju~nu ulogu u odràvanjusloène ravnoteè po~etnih protromboznih i posledi~-nih antitromboznih i tromboliti~kih doga|anja. Zna-~ajnu ulogu u aktivaciji hemostaznog sistema imaju ipokreta~i i medijatori zapaljenjskog procesa. Direk-tne posledice zapaljenja su stvaranje i osloba|anjeTF-a u endotelskim }elijama i monocitima, kao i akti-vacija trombocita i P-selektina, ~ime se tako|e stimu-li{e stvaranje TF. Mnogostruka povezanost hemo-staznog sistema sa etiopatogenezom aterotrombozenedvosmisleno ukazuje na neophodnost da se u pre-venciji i le~enju ove sloène bolesti, koja pouzdanoima i zapaljenjske karakteristike, u punom obimu ko-riste i sve raspoloìve terapijske mere koje mogu po-mo}i u spre~avanju, prekidu i kontroli svakog oblikaaktivacije hemostaznog sistema.

of a specific agonist to membrane-spanning recep-tors on the platelet surface, stimulates the dischargeof calcium from the platelet dense tubular systemand promotes the contraction of the platelet, with thesubsequent release of its granule contents. The initialrecognition of a damaged vessel wall involves adhe-sion and the activation and aggregation of plateletswith each other. Because the platelet function andreactivity depend on numerous adhesive interactions,most of the glycoproteins on the platelet membranesurface are receptors for adhesive proteins. Accor-ding to their structure, these glycoproteins mainlybelong to integrins, selectins and leucin-rich glyco-protein family. The full effect of these receptors isobtained by its close association with dynamicassemblies of sfingolipids and cholesterol floatingwithin the fluid fraction of the platelet membrane,which are designated by the descriptive term »rafts«.During plaque rupture, the clotting mechanism isexplosively activated by the strong influence of TFwhich, as a cofactor, binds coagulation FVII/FVIIaand enhances the catalytic activity of FVIIa by morethan one million times. In circulatory blood, TF ispresent on the microparticles derived from monocy-tes. It is also released in a large amount from the acti-vated endothelial and subendothelial cells. P-selectin,which is localized in the membranes of platelet alpha--granules and in the storage granules of endothelialcells, upon the activation of platelets and endothelialcells translocates within seconds to the cell surface,and can also be found in circulation. This adhesivereceptor has a significant role in the production andrecruitment of monocyte-derived microparticles con-taining TF at the site of thrombin and fibrin forma-tion. The best way to grasp the special complexity ofthe hemostatic system activation is to recognize themultilateral and different thrombin action. Its basicfunction is the catalytic transformation of fibrinogeninto fibrin, but at the thrombogenic site it binds tothrombomodulin, initiating the activation of proteinC, and stimulates the successive release of both tis-sue plasminogen activator and plasminogen-activa-tor inhibitor type 1 from endothelial cells. Thus, be-sides the fact that it is the main promoter of plateletactivation, thrombin plays a pivotal role in mainta-ning the complex balance of initial prothromboticand subsequent antithrombotic and thrombolyticpathways. Inflammatory stimuli and mediators alsohave a significant role in the hemostatic system acti-vation. Direct effects of inflammation are the forma-tion and releasing of TF-form endothelial cells andmonocytes, as well as platelet and P-selectin activa-tion. The multilateral connection of the hemostaticsystem with the etiopathogenesis of atherothrombo-sis undoubtedly points out the necessity that, in treat-ment of this complex disease which reliably has in-flammatory characteristics as well, all available thera-peutic procedures for the prevention, interruptionand control of the hemostatic system activationshould be used immediately and permanently.

450

INSULINSKA REZISTENCIJA I ATEROSKLEROZA

D. D. Mici}

Centar za metaboli~ka oboljenja u endokrinologiji,

Institut za endokrinologiju, dijabetes i bolesti metabolizma,

Klini~ki Centar Srbije, Beograd, Srbija

Uvo|enje abdominalne gojaznosti kao klju~nekomponente metaboli~kog sindroma ukazalo je dadisregulacija masnog tkiva mo`e imati glavnu uloguu patogenezi insulinske rezistencije i ateroskleroze.Suvi{ak masnih kiselina u cirkulaciji kreira na perife-riji insulinsku rezistenciju kroz omogu}avanje dodat-nih supstrata i modifikovanjem nishodnog insulin-skog signala. Bolesnici sa insulinskom rezistencijomimaju endotelijalnu disfunkciju sa parcijalnim ili kom-pletnim gubitkom balansa izme|u vazokonstrikcije ivazodilatacije, faktora koji promovi{u ili inhibirajurast, proaterogenih i antiaterogenih i prokoagulan-tnih i antikoagulantnih faktora. Insulin-rezistentneosobe mogu imati parcijalni blok u delovanju insuli-na kroz PI(3)K putanju u skeletnim mi{i}ima i endo-telijalnim }elijama sa o~uvanom MAPK putanjom, {tomo`e delimi~no da objasni njihov pove}ani kardio-vaskularni rizik.

INSULIN RESISTANCE AND ATHEROSCLEROSIS

D. D. Mici}

Center for Metabolic Disorders in Endocrinology,

Institute of Endocrinology, Diabetes and Diseases of Metabolism,

Clinical Center of Serbia, Belgrade, Serbia

Introduction of abdominal obesity as a crucialpart of the metabolic syndrome pointed out that adi-pose tissue dysregulation might play a main role inthe pathogenesis of insulin resistance and athero-sclerosis. Excess of fatty acids in circulation createsresistance in periphery insulin by the added substrateavailability and by modifying insulin downstream sig-nalling. Patients with insulin have endothelial dys-function with a partial or complete loss of balancebetween vasoconstrictors and vasodilatators, growthpromoting and inhibiting factors, proatherogenic andantiatherogenic, and procoagulant and anticoagulantfactors. Insulin resistant individuals may have a par-tial block to insulin action through PI(3)K pathway inskeletal muscle and endothelial cells with an intactMAPK pathway that may partially explain their incre-ased cardiovascular risk.

Sek c i j a 4BIOHEMIJSKI MARKERI

OBOLJENJA

Se s s i o n 4BIOCHEMICAL MARKERS

OF THE DISEASES


BIOMARKERI OKSIDANTNOG STRESA U BRONHIJALNOJ ASTMI

V. ]osi}1, I. Stankovi}2, M. Ran~i}2, L. Zvezdanovi}1, S. Kundali}1, V. \or|evi}1

1Centar za medicinsku biohemiju, Klini~ki centar »Ni{«, Ni{, Srbija

2Klinika za plu}ne bolesti, Klini~ki centar »Ni{«, Ni{, Srbija

Veruje se da oksidantni stres u~estvuje u inicijaci-ji i razvoju bronhijalne astme (BA). Uostalom, pato-fiziologija BA odlikuje se ogromnom produkcijomreaktivnih kiseoni~kih vrsta (ROS) i reaktivnih azotnihvrsta (RNS), uglavnom od strane inflamatornih }elijavazdu{nih puteva astmati~ara. ROS i RNS igrajuva`nu ulogu u remodeliranju vazdu{nih puteva, kao iu orkestriranju vrste inflamatornog odgovora. Oksi-danti uti~u na specifi~nu ravnoteù Th1/Th2 citokinai zajedno sa Th2 citokinima i Th2 indukovanim }eli-jama mogu uzrokovati mnoge specifi~nosti tipi~ne zaastmu. Oni indukuju bronhokonstrikciju, sekrecijumukusa, deluju na vaskulaturu vazdu{nih puteva ipove}avaju hiperreaktivnost prema pojedinim ago-nistima. Ovaj ~lanak ispituje neophodnost evaluacijeoksidantnog stresa u BA kori{}enjem pouzdanih bio-markera koji omogu}uju podesno pra}enje oksi-dantnog stresa. Poèljno je odre|ivanje prooksidana-ta i antioksidanata, i to sistemskih i lokalnih, u speci-fi~nim medijumima plu}a kao {to su bronhoalveo-larni lavat (BAL), sputum, izdahnuti vazduh i konden-zat izdahnutog vazduha. Ovi biomarkeri vazdu{nihputeva mogu biti reprezentativni indikatori de{avanjana povr{ini vazdu{nih puteva, na inicijalnom mestudelovanja oksidanata. Ispitivanje biomarkera va`no jei za utvr|ivanje mogu}ih ciljeva delovanja antioksi-dantnih suplemenata koji mogu biti u stanju da nor-malizuju oksidantnu/antioksidantnu ravnoteù.

BIOMARKERS OF OXIDANT STRESS IN BRONCHIAL ASTHMA

V. ]osi}1, I. Stankovi}2, M. Ran~i}2, L. Zvezdanovi}1, S. Kundali}1, V. \or|evi}1

1Centre of Biochemistry, Clinical Centre »Ni{«, Ni{, Serbia

2Clinic for Lung Disease, Clinical Centre »Ni{«, Ni{, Serbia

Oxidant stress is believed to contribute to boththe initiation and development of bronchial asthma(BA). As such, the pathophysiology of BA is charac-terised by the large generation of reactive oxygenspe-cies (ROS) and reactive nitrogen species (RNS),predominantly by inflammatory cells of asthmatic air-ways. These species play an important role in remo-delling airways and in orchestrating the type of infla-mmatory response. Oxidants influence the specificbalance of Th1/Th2 cytokines, and together withTh2-cytokines and Th2 induced cells can causemany of the features typical of asthma. They inducebronchoconstriction, mucus secretion, effects on air-way vasculature, and increase airway responsivenessto several agonists. This review discusses the neces-sity of oxidant stress evaluation in BA using reliablebiomarkers, which provide an appropriate tool forstudying oxidant stress. It is desirable to determineprooxidants and antioxidant systemic and localparameters in lung specific media such as bron-choalveolar lavage (BAL), sputum, exhaled air andbreath condensate. These airway biomarkers may berepresentative indicators which could show theprocesses occurring on the airway surface, the initialsite of oxidation. Thus, the examination of biomark-ers is essential for establishing the potential target forantioxidant supplementation that would be able tonormalise the oxidant/antioxidant imbalance.


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BIOMARKERI U KARCINOMU DOJKE

S. Filipovi}, A. Filipovi}, I. Pej~i}, S. Vrbi}, Z. Stanojevi}, I. Mi{i}

Medicinski fakultet, Ni{, SrbijaKlinika za onkologiju,

Klini~ki centar »Ni{«, Ni{, Srbija

Rak dojke je bolest abnormalnog rasta i razvojado tada normalnog tkiva dojke. Tumorska }elija na-staje kada }elija vi{e ne prepoznaje biolo{ke kontrol-ne mehanizme. Za bolje razumevanje biologije tumo-ra, }elijskog ciklusa, angiogeneze, apoptoze, neopla-sti~ne transformacije, fenomena progresije i metas-taziranja veoma su zna~ajni biomarkeri kao prognos-ti~ki i prediktivni faktori. Biomarkeri raka dojke imajuposebnu vrednost u proceni agresivnosti bolesti i ra-noj detekciji progresije.

ZNAÂJ CITOKINA U DIJAGNOSTICIAUTOIMUNSKIH OBOLJENJA

L. Zvezdanovi}1, V. \or|evi}1, V. ]osi}1, T. Cvetkovi}3, S. Kundali}1, A. Stankovi}2

1Centar za medicinsku biohemiju, Klini~ki centar ’ Ni{, Srbija

2Institut za prevenciju, le~enje i rehabilitacijusr~anih i reumatskih bolesnika »Zelengora«,

Ni{ka Banja, Srbija3Institut za biohemiju,

Medicinski fakultet, Ni{, Srbija

Autoimunske bolesti odlikuju se autoimunoreak-cijama usmerenim protiv {iroko rasprostranjenihsopstvenih determinanti. Mnogi citokini u~estvuju uregulisanju aktivnosti i zahvatanju organa kodrazli~itih autoimunskih oboljenja. Poznato je da nekatkiva odràvaju vrlo visoku »ulaznu barijeru« u pogle-du razvoja imunoposredovane inflamacije, {to vodiimunoj privilegovanosti putem generisanja specijali-zovane mikrosredine. Postoje razli~iti obrasci sintezecitokina u pojedinim autoimunskim oboljenjima kao{to su reumatoidni artritis, dijabetes tipa 1, sistemskilupus eritematodes i multipla skleroza, pri ~emutreba ista}i razliku izme|u citokina kao markerafenotipa i citokina kao medijatora inflamacije io{te}enja tkiva. U ve}ini autoimunskih oboljenja,ravnoteà izme|u proinflamatornih i antiinflama-tornih citokina odre|uje stepen i pro{irenost infla-

BIOMARKERS IN BREAST CANCER

S. Filipovi}, A. Filipovi}, I. Pej~i}, S. Vrbi}, Z. Stanojevi}, I. Mi{i}

Faculty of Medicine, University of Ni{, Ni{, Serbia

Clinical Centre »Ni{« - Clinic of Oncology, Ni{, Serbia

Breast cancer is a disease characterized by abnor-mal growth and development of normal breast tissue.When the growth and development of a normal cellno longer obey biological control mechanisms,tumour cells begin to appear. An understanding ofnormal biological processes relating to cell growth,such as the cell cycle, angiogenesis, and apoptosis,and of other abnormal processes, such as neo-plas-tic transformation, tumour progression, and metas-tasis, would aid not only in identifying tumour mar-kers, but also in determining the best uses of thesetumour markers for diagnosis and treatment ofpatients with cancer. Biomarkers are improving ourunderstanding of the biology and management ofbreast cancer.

THE SIGNIFICANCE OF CYTOKINES INDIAGNOSIS OF AUTOIMMUNE DISEASES

L. Zvezdanovi}1, V. \or|evi}1, V. ]osi}1, T. Cvetkovi}3, S. Kundali}1, A. Stankovi}2

1Centre of Medical Biochemistry, Clinical Centre – Ni{, Serbia

2Institute for Prevention, Treatment and Rehabilitation of Cardiac and Rheumatic Patients »Zelengora«,

Ni{ka Banja, Serbia3Institute of Biochemistry,

Faculty of Medicine, Ni{, Serbia

Autoimmune diseases are characterized by auto-immune reactions against one's own widespread de-terminants. Many cytokines are involved in activityregulation and organ involvement in various autoim-mune diseases. It is well known that some tissuesmaintain a very high »entry barrier« concerning thedevelopment of immune-mediated inflammation,which leads to the state of »immune privilege«through the generation of specialized microenviron-ment. There are different patterns of cytokine syn-thesis in particular autoimmune diseases such asrheumatoid arthritis, type I diabetes, systemic lupuserythematosus and multiple sclerosis, and worthstressing is the difference between cytokines as phe-notype markers and cytokines as inflammation andtissue damage mediators. In most autoimmune dis-eases the balance between proinflammatory and


macije i mo`e voditi evidentnim klini~kim efektima.Tako je kod bolesnika sa SLE dobijen zna~ajanporast TNF-a i IL-10 u svim, a posebno u neurolo{kojmanifestaciji bolesti. Razumevanje osnovnih meha-nizama kontrole diferencijacije T }elija predstavlja putka strategiji modulacije fenotipa citokina i spre~avan-ja tkivnog o{te}enja i autoimunskih bolesti i, nar-avno, promovi{e i za{titu od istih.

MARKERI TIREOIDNE AUTOIMUNOSTI

N. Gligorovi}

Klini~ki centar Crne Gore, Podgorica, Crna Gora

Autoimune tireoidne bolesti (AITB), naj~e{}iendokrinolo{ki poreme}aj kod ljudi, dovode do o{te-}enja }elija i promjene funkcije `lijezde humoralnim i}elijski posredovanim mehanizmima. Tri glavnaantigena uklju~ena u razvoj ovih bolesti su: tireope-roksidaza (TPO), tireoglobulin (TG) i receptor tireo--stimuliraju}eg hormona (TSH-R). Opisani su i drugiantigeni, ali za sada nije utvr|en njihov klini~ki zna~aj.Laboratorijski testovi koji bi se koristili za procjenu}elijski posredovanih mehanizama kod AITB jo{uvijek nisu dostupni. S druge strane, procjena humo-ralnog odgovora, tj. odre|ivanje tireoidnih antitijela,izvodi se u ve}ini klini~kih laboratorija. Vrijednostiovih parametara od zna~aja su za klasifikaciju, ireflektuju aktivnost i progresiju bolesti {titne `lijezde.U novije vrijeme, odre|ivanje TPO antitijela koristi seza procjenu rizika za razvoj AITB. Mjerenje tireoidnihantitijela jo{ uvijek prati niz problema vezanih zarazlike u osjetljivosti i specifi~nosti metoda, kao iodsustvo adekvatne standardizacije. Uprkos tome,njihovo odre|ivanje od velikog je zna~aja za niz kli-ni~kih situacija.

DIJAGNOSTIKA NASLEDNIH MALIGNITETA

K. Stankov

Zavod za biohemiju, Klini~ki centar ’ Novi Sad, Srbija

Na{e razumevanje etiologije nastanka predispozi-cije za nasledna oboljenja je poslednjih godina izu-zetno napredovalo. Taj napredak omogu}en je presvega naglim razvojem molekularne genetike i ispiti-vanja genoma, kao i njihovom primenom u humanojgenetici. Malignitet je specifi~an oblik kompleksnog

antiinflammatory cytokines determines the extentand spread of inflammation and can lead to conspic-uous clinical effects. In SLE patients, for instance, weobserved a significant elevation of TNF-a and IL-10in all, but especially in neurologic disease form.Understanding of the fundamental mechanisms of Tcell differentiation control is the road to the strategyof cytokine phenotype modulation and prevention oftissue damage and autoimmune diseases, promotingnaturally the protection from them.

MARKERS OF THYROID AUTOIMMUNITY

N. Gligorovi}

Clinical Center of Montenegro, Podgorica, Montenegro

Autoimmune thyroid diseases (AITD), the mostcommon endocrine disorders affecting humans,cause cellular damage and alter the thyroid glandfunction by humoral and cell-mediated mechanisms.Three principal thyroid autoantigens are involved inthe development of AITD: thyroperoxidase (TPO),thyroglobulin (TG) and thyroid stimulating hormonereceptor (TSH-R). Other autoantigens have also beendescribed, but as yet their diagnostic role in thyroidautoimmunity has not been established. Laboratorytests that determine the cell-mediated aspects of theautoimmune process are not currently available.However, tests of the humoral response, i.e. thyroidautoantibodies, can be assessed in most clinical lab-oratories. Thyroid autoantibodies are valuable forclassification and reflect disease activity and progres-sion. Lately, TPO antibodies assays have been usedfor assessing the risk of developing AITD. Thyroidautoantibodies measurement is hampered by met-hod specificity and sensitivity problems, as well assuboptimal standardization. Despite that, autoanti-body tests have inherent clinical utility in a number ofclinical situations.

DIAGNOSTICS OF HEREDITARY MALIGNANCIES

K. Stankov

Department of Biochemistry, Clinical Center »Novi Sad«, Novi Sad, Serbia

Significant advances have occurred in our under-standing of the cancer etiology in the last decade, asa consequence of the generalized use of molecularbiology techniques in human genetics. Cancer is aform of a complex genetic disease. Most forms ofcancer are characterised by the accumulation of

456

genetskog oboljenja. Ve}inu tipova malignih obolje-nja karakteri{e akumulacija razli~itih genetskih alte-racija koje uti~u na gene sa specifi~nim patogenimpotencijalom, koji su specifi~ni za svaki tip malignite-ta. Kod ve}ine malignih tumora te genetske alteraci-je odvijaju se na nivou somatskih }elija, me|utimneke od njih se prenose preko germinativnog epitelai imaju ulogu u naslednoj predispoziciji za nastanakmaligniteta. Budu}i izazov u genetici malignitetapredstavlja identifikacija gena sa visokom prevalenci-jom alela koji doprinose smanjenju ili pove}anjurizika za nastanak maligniteta.

PRIMJENA BIOMARKERA U ISPITIVANJUZRELOSTI PLU]A FETUSA

D. Popovi}-Pribilovi}, V. Miketi} , G. Matovi}

Centar za klini~ko-laboratorijsku dijagnostiku,Ginekolo{ka klinika, Klini~ki centar Crne Gore,

Podgorica, Crna Gora

U procesu maturacije, alveolarne epitelne }elijeprodukuju povr{inski aktivan materijal koji se sastojiuglavnom od fosfolipida, a u znatno manjoj mjerisadr`i proteine, neutralne lipide i ugljene hidrate.Pulmonalni surfaktant sinteti{u alveolarni pneumoci-ti tipa II i »pakuju« u vidu lamelarnih tijela. Prijeporo|aja, lamelarna tijela difunduju kroz bronhijalnostablo u amnionsku te~nost. Parametri koji ukazujuna zrelost plu}a mogu se posmatrati i kao odnoskoli~ine surfaktanata i drugih komponenti u amnion-skoj te~nosti (sfingomijelin i albumin). U ovoj studijiodre|ivani su odnos surfaktanta prema albuminu(S/A) i broj lamelarnih tijela (LB) u amnionskoj te~no-sti kao predskaziva~i (prediktori) zrelosti fetalnihplu}a. Istra`ivanje je obuhvatilo 90 trudnica ~ija jegestaciona starost bila od 32 do 40 nedjelje. Uzorciamnionske te~nosti dobijeni su amniocentezom i fil-trirani kroz specijalne filtere. Odnos S/A odre|ivan jetestom Fetal Lung Maturity II (FLM) na TDX apa-ratu (ABBOTT). Vrijednost (broj) LB odre|ivana je nahematolo{kom broja~u, na kanalu za trombocite, naaparatu Cell Dyn 3700 (ABBOTT). Srednja vrijednostS/A bila je 55,11 mg/g (min = 6,96 mg/g, max =112,00 mg/g). Odnos surfaktant’albumin manji od39,00 mg/g ukazuje na pojavu respiratornog distressindroma (RDS) sa vjerovatno}om od 94%. Srednjavrijednost LB bila je 69,4 × 109/L (min = 4,2 ×109/L, max = 199,0 × 109/L). Vrijednost lamelarnihtijela od 33 × 109/L ili manja ukazuje na pojavu RDSsa vjerovatno}om od 93%. Odnos S/A raste sa gesta-cionom staro{}u (r = 0,373, p<0,005), kao i brojlamelarnih tijela (r = 0,934, p<0,001). Ova dva testakoreliraju jedan sa drugim r = 0,341, p<0,005. Kod90 trudnica koje su se porodile u okviru 72 ~asaodnos surfaktant-albumin i vrijednost lamelarnih tije-

different genetic alterations affecting genes from aset of genes with pathogenic potential, which is spe-cific for each tumour entity. While in the majority ofmalignant tumours these changes are somaticallyacquired, some mutations are transmitted throughthe germline and account for an inherited tumourpredisposition. The next frontier in cancer genetics isto find genes with high prevalence alleles conferringa low increase or decrease of cancer risk.

APPLICATION OF BIOMARKERS INEVALUATION OF FETAL LUNG MATURITY

D. Popovi}-Pribilovi}, V. Miketi} , G. Matovi}

Center for Clinical-Laboratory Diagnostics,Department of Obstetrics and Gynecology,Clinical Center of Montenegro, Podgorica,

Montenegro

In the process of maturation, alveolar epithelialcells produce surface-active material (surfactant)which consists mainly of phospholipids and, althoughto a significantly lesser degree of neutral lipids, pro-teins and carbohydrate. Pulmonary surfactant is syn-thesized in alveolar type II granular pneumocytes and»packed« as lamellar bodies. Before delivery, thelamellar bodies diffuse through the bronchial tree intothe amniotic fluid. Parameters which indicate thematurity of lungs are also observed in the ratio againstthe quantity of surfactant with other components ofthe amniotic fluid (sphingomyelin and albumin). Thestudy concerns the determination of surfactant-to-al-bumin ratio and lamellar body count in the amnioticfluid as predictors of fetal lung maturity. The researchinvolved 90 pregnant women with gestational agefrom 32 to 41 weeks. The samples of the amnioticfluid were obtained by amniocentesis and filtratedthrough special filters. The surfactant-to-albumin ratiowas measured by the Fetal Lung Maturity II (FLM) test,on TDX instrument (ABBOTT). The lamellar bodycount was measured on a hematology cell counter bythe platelet channel, on the Cell Dyn 3700 instrument(ABBOTT). The average value of ratio S/A was 55.11mg/g (min = 6.96 mg/g, max = 112.00 mg/g). Thesurfactant-to-albumin ratio of less than 39.00 mg/gpredicts respiratory distress syndrome with the proba-bility of 79%. The average value of LB was 69.4 ×109/L (min=4.2 × 109/L, max = 199.0 × 109/L). La-mellar body concentration of 33 × 109/L or less pre-dicts respiratory distress syndrome with the probabilityof 86%. The surfactant-to-albumin ratio increased withgestation (r = 0.373, p<0.005), as did lamellar bodyconcentration (r = 0.934, p<0.001). The two testscorrelated with each other, r = 0.341, p<0.005. In 90


la korektno ukazuju na pojavu RDS u {est slu~ajeva.Postoji visok stepen pozitivne korelacije izme|u ge-stacione starosti i odnosa surfaktant albumin, kao ivrijednosti lamelarnih tijela.

BIOHEMIJSKI MARKERI ISHODA TRUDNO]E U FETALNOJ KRVI

A. Nikoli}-\or|evi}

Institut za laboratorijsku medicinu, Klini~ki centar – Novi Sad, Srbija

Tokom poslednjih deset godina postignut je zna-~ajan napredak u oblasti pobolj{anja maternalno-fe-talnog zdravlja. Biohemijska i hematolo{ka ispitivanjafetalne krvi obuhvataju veliki broj parametara i vr{e seiz umbilikalne krvi nakon dijagnosti~ke kordocenteze(radi genetskog ispitivanja) ili iz pre-transfuzionoguzorka fetalne krvi. U ovom radu je dat prikaz neko-liko dijagnosti~kih markera analiziranih u fetalnoj krvi:endotelina-1, leptina, beta-2-mikroglobulina i infla-matornog markera interleukin-6. Endotelin-1 je va-zokonstriktor na ~iju indukciju uti~u pove}anje ven-skog krvnog pritiska i stanje stresa. Studija je obu-hvatila uzorke pre i posle transfuzije fetalne krvi uslu~ajevima intrauterine transfuzije kod Rh-aloimuni-zovanih hemoliti~kih fetalnih anemija. Pove}anje fe-talnog intravaskularnog volumena nakon fetalnetransfuzije eritrocita sa visokim hematokritom podièvrednosti nivoa endotelina-1 u fetalnoj krvi nakon ini-cijalne ali ne i nakon ponovljene transfuzije. Beta-2-mikroglobulin je proteinski molekul prisutan na}elijskoj povr{ini, povezan sa glavnim histokompati-bilnim kompleksom. Nivo beta-2-mikroglobulina jetako|e pra}en u slu~ajevima intrauterine transfuzijekod Rh-aloimunizovanih fetalnih anemija pre i posletransfuzije. Koncentracije beta-2-mikroglobulina zna-~ajno su vi{e kod fetusa sa prethodnom transfuzijomu odnosu na neanemi~ne fetuse. Odre|ivanje beta-2-mikroglobulina moè se pokazati korisnim u identifi-kaciji fetusa sa potencijalno ozbiljnijim efektom Graft-versus-Host reakcije na }elijske transplantate. Pove-}an nivo beta-2-mikroglobulina nakon transfuzije su-geri{e imunomodulatorni efekat intrauterine transfu-zije (leukocitnih antigena donora) na fetalni imuniodgovor. Leptin je cirkuli{u}i hormon koji koordiniraunos i potro{nju energije u organizmu. U ovoj studiji,leptin je pra}en u fetalnoj i maternalnoj krvi nakongenetskog »skrininga« u normalnoj euploidnoj trud-no}i i trudno}i sa Daunovim sindromom. U normal-noj trudno}i, fetalne vrednosti leptina su zna~ajnoniè u odnosu na maj~inu krv, ali se nivo tokomgestacije pove}ava. Daunov sindrom povezan je sazna~ajno niìm vrednostima u fetalnoj krvi. Mogu}e je

patients delivered within 72 hours the surfactant-to--albumin ratio and the concentration of lamellar bodycorrectly predicted six cases of respiratory distress syn-drome. There was a high degree of positive correlationbetween gestational age and surfactant-to-albuminratio, as well as lamellar body concentration.

BIOCHEMICAL MARKERS OF ADVERSEPREGNANCY OUTCOMES IN FETAL BLOOD

A. Nikoli}-\or|evi}

Institute of Laboratory Diagnostics, ClinicalCenter »Novi Sad«, Novi Sad, Serbia

Within the past decade, significant progress hasbeen made with regard to improving maternal andnewborn health. Biochemical markers in fetal bloodare assessed after diagnostic cordocentesis (whichwas primarily collected for genetic screening), orimmediately prior to fetal transfusion. Concentrationsof the following diagnostic markers are measured inthis study: endothelin-1, leptin, beta-2-microglobulinand the inflammatory marker IL-6. Endothelin-1 is apotent vasoconstrictor, induced by rising venouspressure and rising shear stress. Subjects involved inthis study included women with anti-DRh alloimmu-nized pregnancies, and fetal blood sampled pre- andpost-transfusions. Rapid expansion of fetal intravas-cular volume by intravenous transfusion of packedred blood cells with a high hematocrit enhances fetalendothelin levels. Beta-2-microglobulin is a ubiqui-tous cell surface protein, associated with the majorhistocompatibility complex. It is a potential marker ofGraft-versus-Host Disease. The median concentra-tions of beta-2-microglobulin are significantly higherin fetuses with prior transfusions compared with non-anemic fetuses. Evaluation of fetal beta-2-micro-globulin might prove useful in identifying fetuses withpotentially severe Graft-versus-Host or Host-versus-Graft reaction to cell transplants. Leptin is a recentlydiscovered circulating hormone that coordinatesenergy intake and expenditure in adults. Leptin levelsin the umbilical cord blood positively correlate withneonatal birth weight, suggesting a role in adiposehomeostasis in utero. In this study, leptin levels weremeasured in fetal and paired maternal plasma in thesecond half of gestation, in pregnancies complicatedwith Down syndrome and euploid pregnancies. Ineuploid pregnancies, fetal leptin levels were signifi-cantly lower than in corresponding maternal values,but increased across gestation. Down syndrome wasassociated with significantly lower fetal leptin levels. Itis possible that lower fetal leptin levels could reflectthe persistent immaturity of the pattern of placentalpeptide hormone synthesis in fetal Down syndrome.Recent evidence strongly implicates the inflammato-

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da ni`e vrednosti leptina odra`avaju postoje}u ne-zrelost hormonske sinteze na nivou placente koja jeprisutna u fetalnom Daunovom sindromu. Novijestudije ukazuju da antiinflamatorni odgovor na intra-uterinu infekciju predstavlja najzna~ajniji razlog pre-vremenog poro|aja, te o{te}enja fetalnih organa.Inflamatorni citokin interleukin-6 pra}en je kao po-tencijalni marker prevremenog poro|aja kod prisutneinfekcije amnionske te~nosti kao najzna~ajnijegfaktora rizika. Vrednost IL6 >11 pg/mL ozna~ena jekao cut-off vrednost povezana sa pove}anim rizikomprisustva sistemskog inflamatornog odgovora.

ry response to intrauterine infection in the pathoge-nesis of neonatal brain and lung injury. The frequen-cy and clinical significance of systemic inflammatoryresponses were defined by elevated plasma inter-leukin-6 concentrations in fetuses with preterm labor.A fetal plasma interleukin-6 cut-off value of 11 pg/mgwas used to define the presence of a systemic infla-mmatory response.

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