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2 Published and copyright by: Medical Assotiation of Zenica-Doboj Canton; Address: Zenica, 72000, Bulevar kralja Tvrtka I 4, Bosnia and Herzegovina; tel./fax: ; ljkozedo@bih.net.ba, web site: http// For ordering information please contact: Tatjana Žilo, ljkozedo@bih.net.ba; Access to this journal is available free online trough: The Journal is indexed by MEDLINE, EMBASE (Exerpta Medica), Scopus, EBSCO; ISSN DTP by: Graphic and web design studio B Panel Zenica, Zmaja od Bosne bb, info@bpanel.ba, tel , ; Printed by: Printed by: GRAFORAD d.o.o. Zenica, Zmaja od Bosne bb, graforad@bih.net.ba, tel fax: Printing supported by the Federal Ministry of Education and Science (Federalno ministarstvo obrazovanja i nauke, BiH)

3 Medicinski Glasnik Official Publication of the Medical Association of Zenica-Doboj Canton Bosnia and Herzegovina Editor-in-chief Selma Uzunović Zenica, Bosnia and Herzegovina MANAGING EDITOR Tarik Kapidžić Zenica, Bosnia and Herzegovina Editors Adem Balić, Tuzla, Bosnia and Herzegovina Dubravka Bartolek, Zagreb, Croatia Branka Bedenić, Zagreb, Croatia Asja Čelebić, Zagreb, Croatia Josip Čulig, Zagreb, Croatia Filip Čulo, Mostar, Bosnia and Herzegovina Jordan Dimanovski, Zagreb, Croatia Branko Dmitrović, Osijek, Croatia Ines Drenjančević, Osijek, Croatia Harun Drljević, Zenica, Bosnia and Herzegovina Davorin Đanić, Slavonski Brod, Croatia Lejla Ibrahimagić-Šeper, Zenica, Bosnia and Herzegovina Tatjana Ille, Belgrade, Serbia Vjekoslav Jerolimov, Zagreb, Croatia Mirko Šamija, Zagreb, Croatia Sven Kurbel, Osijek, Croatia Snježana Pejičić, Banja Luka, Bosnia and Herzegovina Belma Pojskić, Zenica, Bosnia and Herzegovina Besim Prnjavorac, Tešanj, Bosnia and Herzegovina Asja Prohić, Sarajevo, Bosnia and Herzegovina Velimir Profozić, Zagreb, Croatia Radivoje Radić, Osijek, Croatia Amira Redžić, Sarajevo, Bosnia and Herzegovina Suad Sivić, Zenica, Bosnia and Herzegovina Sonja Smole-Možina, Ljubljana, Slovenia Vladimir Šimunović, Mostar, Bosnia and Herzegovina Adrijana Vince, Zagreb, Croatia Jasmina Vraneš, Zagreb, Croatia Živojin Žagar, Zagreb, Croatia Secretary: Tatjana Žilo; Proofreaders: Aras Borić (Bosnian, Croatian, Serbian), Glorija Alić (English)

4 MEDICINSKI GLASNIK Official Publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina Volume 12, Number 1, February 2016 Free full-text online at: and (DOAJ, Directory of Open Access Journals) REVIEW 1 Cystic fibrosis: model of pathogenesis based on the apical membrane potential Beatrica Kurbel, Saša Rapan, Sven Kurbel 7 The assessment of acid-base analysis: comparison of the traditional and the modern approaches Jasna Todorović, Jelena Nešovic-Ostojić, Aleksandar Milovanović, Predrag Brkić, Mihailo Ille, Dušan Čemerikić 19 Clonus: definition, mechanism, treatment Ismail Boyraz, Hilmi Uysal, Bunyamin Koc, Hakan Sarman Original article 27 Human dental pulp mesenchymal stem cells isolation and osteoblast differentiation Moustafa Alkhalil, Amer Smajilagić, Amira Redžić 33 Inflammatory cytokine gene polymorphism profiles in Turkish patients with ulcerative colitis İlhami Gök, Fahri Uçar, Orhan Ozgur 40 False-positive 18-fluorodeoxyglucose positron emission tomography computed tomography (FDG PET/CT) scans mimicking malignancies Zehra Yasar, Murat Acat, Hilal Onaran, Akif Ozgül, Erhan H. Dincer, Erdogan Cetinkaya, Nurdan A. Korkmaz 47 Human West Nile virus infection in Bosnia and Herzegovina Sead Ahmetagić, Jovan Petković, Mirsada Hukić, Arnela Smriko-Nuhanović, Dilista Piljić 52 Incidence and etiological agents of genital dermatophytosis in males Asja Prohić, Mersiha Krupalija-Fazlić, Tamara Jovović Sadiković 57 Surgical therapy for pilonidal sinus in adolescents: a retrospective study Tamer Sekmenli, Ilhan Ciftci 61 Impacts of education level and employment status on health-related quality of life in multiple sclerosis patients Selma Šabanagić-Hajrić, Azra Alajbegović 68 Importance of Herrings classification in predicting the outcome of aseptic necrosis of the femoral head Žarko Dašić, Miroslav Kezunović, Goran Pešić, Vesna Bokan, Mira Jovanovski 73 A retrospective review of 139 major and minor salivary gland tumors Marija Trenkić Božinović, Dragan Krasić, Vuka Katić, Miljan Krstić 79 Effects of hormone replacement therapy on depressive and anxiety symptoms after oophorectomy Danijela D. Ðoković, Jelena J. Jović, Jelena D. Ðoković, Marinela Ž. Knežević, Slavica Djukić- Dejanović, Dragana I. Ristić-Ignjatović 86 Mechanical prosthetic valve disease is related with an increase in depression and anxiety disorder Yasemin Turker, Kurtulus Ongel, Mehmet Ozaydin, Yasin Turker, Funda Yildirim Bas, Mehmet Akkaya

5 93 Emotional profile and risk behaviours among tattooed and non-tattooed students Matea Zrno, Maja Frencl, Dunja Degmečić, Ivan Požgain 99 The effect of anger management levels and communication skills of Emergency Department staff on being exposed to violence GozdeYildiz Das, Ilknur Aydin Avci Letter to Editor Retraction 105 Occurrence and morphological characteristics of cataracts in patients treated with general steroid therapy at the Cantonal Hospital Zenica Jasmin Zvorničanin, Edita Zvorničanin 107 Increased counts and degranulation of duodenal mast cells and eosinophils in functional dyspepsiaa clinical study Shijun Song, Yan Song, Haishan Zhang, Gaiqin Li, Xiaopei Li, Xiaohong Wang, and Zhen Liu Medicinski Glasnik is indexed by MEDLINE, EMBASE (Exerpta Medica), EBSCO, Scopus, and Directory of Research Journals Indexing (DRJI)

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7 REVIEW Cystic fibrosis: model of pathogenesis based on the apical membrane potential Beatrica Kurbel 1, Saša Rapan 2, Sven Kurbel 3 1 Department of Anesthesiology, Zagreb University Hospital, Zagreb, 2 Department of Orthopedic Surgery and 3 Department of Internal Medicine, University Hospital, Osijek; Croatia ABSTRACT Corresponding author: Saša Rapan School of Medicine, University Josip Juraj Strosmayer, J. Huttlera 4, Osijek, Croatia Phone: ; Fax: ; sasa.rapan@gmail.com A simple model of cystic fibrosis (CF) is proposed, based on the apical membrane (ApM) potential. The ApM of epithelial cells is highly permeable to sodium and activation of CFTRs makes it permeable to chloride. Calculated ApM potentials of cells with activated cystic fibrosis transmembrane conductance regulators (CF- TRs) are between the sodium and chloride Nernst values and thus allow rapid absorption of both ions in exocrine glands. In CF patients the potential is near the sodium Nernst value and thus more salt is left in the ducts. Simulation predicts that the sodium driving force increases more than 3.5 times if the ApM permeability for Cl- increases from 5-94% of the sodium permeability. In pancreatic ductal cells basolateral sodium bicarbonate cotransporters (pnbc1) allows influx of bicarbonates with sodium. Bicarbonates are exchanged for intraductal chloride by anion exchanger 1 (AE1) in the ApM. Activated CFTRs let some chloride to leak back to ducts, followed by water that dilutes ductal proteins. Replenished intraductal chloride allows more bicarbonate secretion. In CF patients, pancreatic water and bicarbonate secretion is limited by the intraductal chloride pool. Key words: membrane potential, cystic fibrosis, pnbc1, AE1 Original submission: 21 August 2014; Revised submission: 23 November 2014; Accepted: 21 December Med Glas (Zenica) 2015; 12(1):1-6 1

8 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION This paper on electrophysiological aspects of cystic fibrosis is based on the previous paper about importance of chloride (Cl - ) membrane traffic and Donnan effect of cytoplasmic proteins (1). Cystic fibrosis (CF) is an inherited disease caused by dysfunctional chloride channels, cystic fibrosis transmembrane conductance regulator (CFTR) in various epithelial cells (2). Since this disease is often diagnosed by measuring increased negativity of the transepithelial potential, calculating the basolateral and apical membrane potentials by using the Goldman s equation calculators seemed suitable for the model of pathogenesis (3,4). BASIC ASSUMPTIONS BEHIND THE PROPOSED MODEL The model of cystic fibrosis pathogenesis presented here uses several assumptions. The CFTR is among other regulators of the ductal pancreatic cell function: anion exchanger 1 (AE1), responsible for the exchange of chloride for bicarbonate (HCO 3- ), sodium-hydrogen antiporter 1 (NHE-1), encoded by the SLC9A1 gene, pnbc1 that imports two bicarbonate ions and one sodium (Na + ) in the cell (5). Dysfunctional CFTRs in patients with cystic fibrosis affect several organs and tissues. Genes for all these structures of ion traffic are variably expressed in all epithelial cells affected by cystic fibrosis (6,7), but high expressions of CFTR and pnbc1 genes seem unique for pancreas. Membrane ion traffic is governed by electric fields, concentration gradients and ion specific membrane permeability. All cell membranes act as diffusion bottlenecks and if ions accumulate near the membrane, their electric charges alter diffusion of other ions (5). If a membrane allows only one ion to diffuse along its concentration gradient the diffusion will continue until the membrane reaches the Nernst value of that ion. Further ion traffic depends on Brownian kinetic that washes away ions near the membrane. Cell membranes are permeable to more than one ion and the actual membrane potential is calculated by the Goldman equation (3,4). Cells permeable to potassium (K + ) and chloride ions normally have membrane potential somewhere between the respective Nernst values. An example: the resting potential in neurons is near the potassium Nernst potential and traffic of K + is opposed by a strong electric force. Traffic of ions across the membrane is intensive during the action potential. The apical membrane (ApM) of epithelial cells faces the ductal lumen. Due to presence of ENaCs, ApM is highly permeable to sodium and activation of CFTRs makes it permeable to chloride. Except in some kidney cells, ApMs of various epithelial cells are probably less permeable to potassium than their basolateral membrane (BlM). This means that the actual ApM potential depends mainly on Na + and Cl - entering the cell along their concentration gradients. Since these two ions have very different Nernst values, the expected intermediary membrane potential would allow both ions to enter with ease. In normal individuals and in CF patients transepithelial potential is negative. Since the outer epithelial surface consists of closely arranged apical membranes, this potential has to be related to the actual potential of apical membranes, although the measured transepithelial potential is probably smaller than the actual ApM potential. THE PROPOSED ELECTROPHYSIOLOGICAL MODEL OF THE APICAL MEMBRANE POTENTIAL Table 1 shows model predictions of the apical membrane potentials in cells with and without functional CFTRs. The Goldman s equation calculators (3,4) are used to simulate transepithelial voltage in normal ducts with increased Na permeability through ENaCs, and high Cl - permeability through activated CFTRs. For the 1 st, basolateral column values of the generic cell setting in the calculator (3) were used. Sodium and potassium permeability values were interchanged in the remaining two columns that show the apical membrane potentials. Without functional CFTRs (3 rd column with low chloride permeability) the apical membrane potential is so near the sodium Nernst value that diminished sodium traffic depends on Brownian mediated diffusion of cations away from the apical membrane. With functional CFTRs (2 nd column with chloride permeability that is 94% of sodium permeability), the apical membrane potential is in the middle between the 2

9 Kurbel et al. Membrane potential model of cystic fibrosis Table 1. Simplistic model of ion traffic of epithelial cells of normal individuals and patients with cystic fibrosis, calculated by Goldman s equation (3,4) Parameters of Goldman s equation Sodium Potassium Chloride Nernst potentials (mv) Electrochemical driving force (mv) Basolateral membrane Apical membrane permeability in epithelial cells Epithelium with camp activated CFTRs Epithelium without camp activated CFTRs, as in CF patients PNa [Na+]out [Na+]in PK [K+]out [K+]in PCl [Cl-]out [Cl-]in ENa EK ECl VDF,Na VDF,K VDF,Cl VDF,Na ratio of Na+ traffic: activated CFTRs / non-activated CFTRs 3.56 Goldman s equation results (mv) Predicted potential between intraductal content & IF (assumed to be mv) IF-(apical-basolateral) Comments both K+ and Cl- easily cross since the basolateral potential is intermediary to their Nernst values Na+ and Cl- both easily cross since the apical membrane potential is intermediary to their Nernst values low influx of Na+ due to membrane potential close to the Na+ Nernst value and almost no Cl- or K+ influx due to low permeability Cl - and Na + Nernst values, so the electric field is allowing the maximal traffic of these two ions. The model predicts that permeability values of sodium and chloride need to be almost equal to allow the maximal influx of these ions through the apical membrane. Predicted potential difference between intraductal content and interstitial fluid (IF) is also calculated. It is assumed that this potential is the source of the measured negative transepithelial potential and, as expected, potential is more negative in epithelial cells with dysfunctional CFTRs. Apical membranes of epithelial cells are highly permeable to sodium (ENACS), and probably some potassium. Permeability to Cl - through CF- TRs depends on activation by camp (5). Chloride that enters through functional CFTRs maintains an intermediary apical membrane potential that allows rapid absorption of both ions (Table 1). In this way, activation of CFTRs modulates salt absorption in various exocrine glands. Epithelial cells in exocrine glands of patients with cystic fibrosis are in a specific situation. Their apical membranes cannot modulate the Cl - permeability via CFTRs and only the small potassium permeability keeps the membrane potentials from hitting the sodium Nernst value that would further compromise Na + diffusion. When the ApM is compared with the IF potential of 0 mv, a more negative difference can be predicted in these patients (Table 1), leading to more negative intraductal potentials. Altered permeability to chlorine makes the apical membrane potential so high that this reduces sodium transport and more salt is left in the ducts. Based on simulation in Table 1, the sodium driving force increases more than 3.5 times if the apical membrane permeability for Cl - increases from 5 to 94% of the sodium permeability. The remaining question is why respiratory tract mucosa is the most damaged epithelial tissue in cystic fibrosis patients when similar transepithelial potentials are found in sweat glands and in salivary glands without much damage. A plausible explanation is that it is a consequence of the failed salt reabsorption from the evaporating fluid that covers respiratory mucosa. Each day a normal adult exhales some 400 ml of pure water, and near one half of it comes from the respiratory airways. The only way to prevent accumulation of hypertonic residues after evaporation is quickly to reabsorb salt before evaporation. This desalinization is done via combined action of ENACs and CFTR in normal individuals. In CF patients this process is compromised and some minerals from 150 to 200 ml of evaporated IF similar fluid 3

10 Medicinski Glasnik, Volume 12, Number 1, February 2015 remain on mucosal surfaces after water evaporation. The hypertonic ductal content forces some water to remain in ducts due to osmosis and this salty microenvironment is prone to infection and biofilm formation by respiratory bacteria (8). The presented model interpretation of CFTR function in pancreatic ductal cells is complex. Basolateral membranes in these calls contain pnbc1 that carries two bicarbonate ions with sodium, and each action allows three osmotically and electrically active particles to enter the ductal cell. Excess sodium is taken away by Na + / K + pumps but the faith of bicarbonates is variable. Bicarbonates can interact with intracellular H + ions and form carbonic acid that can interact with carboanhydrase and leave the slightly more alkaline cell as CO 2. Alternatively, bicarbonates can leave the cell in exchange for intraductal Cl - via AE1 structures in the apical membrane. Due to this trade, the overall sum of chloride and bicarbonates concentrations in the ductal content remain almost the same until secretion of bicarbonates is stimulated through opening of CFTRs in the apical membrane. The AE1 action is probably bidirectional, as is described in erythrocytes during the chloride shift (5). This means that bicarbonates leave the cell if intraductal Cl - is higher than the cytoplasmic level. Nevertheless, when the pool of intraductal Cl - is exhausted, the AE1 stops and both bicarbonate and chloride levels in pancreatic ducts and in ductal cells become similar, meaning that without replenishing of the ductal Cl - pool, pancreatic juice is limited in the bicarbonate content and thus in the enzymatic activity. Normally, due to various stimuli, intracellular camp opens CFTRs on the apical membrane and some influxed Cl - leak back to ducts, thus allowing more bicarbonates to be secreted by AE1. The consequence is that the sum of Cl - and bicarbonates increases during the bicarbonate secretion. This is in concordance with the report that pancreatic secretion in two dogs shows more than a 100-fold variation (9), while concentrations of sodium and of potassium were independent of secretory rates. At low secretory rates, bicarbonate levels dropped to values equal to, or lower than plasma concentration, while the concentration of chloride varied inversely with that of bicarbonate. It is here proposed that by returning the influxed Cl - ions, the CFTRs add some water to the ductal content, since Cl - and bicarbonates are both osmotically active and this added water dilutes proteins secreted by acinar pancreatic cells, allowing the diluted and highly alkaline juice to leave pancreas. In patients with CF, the model predicts that the basal secretion produces juice with limited concentration of bicarbonates until the ductal chloride pool is so exhausted that Cl - concentration is near the cytoplasmic level. This interpretation suggests that accumulated intracellular excess of bicarbonates cannot leave to the duct due to AE1 function failure, and this can lead to cellular alkalosis, via carboanhydrase action. It is well known that cellular alkalosis increases Donnan effect of cytoplasmic proteins due to increased number of protein-bound charges in alkaline ph. The model predicts that this might block further influx of bicarbonate ions via pnbc1, despite the sodium gradient and increased affinity for cations by the Donnan. This means that the ion traffic across the ductal cell apical membrane would be halted until the ductal chloride pool is replenished by the acinar cells. This interpretation is in concordance with reports that the defect in agonist-stimulated ductal bicarbonate secretion in patients with CF is predominantly due to decreased NBC-driven bicarbonate entry at the basolateral membrane, in the absence of functional CFTR (10). The consequence is that without CFTRs pancreas lacks camp modulated secretion of bicarbonates, chloride and water needed to dilute proteins in the pancreatic juice below the dangerous concentration of enzyme activation within the gland. POSSIBLE EXTRAPOLATIONS OF THE PRESENT- ED MODEL Simplistic electrophysiological model of CF changes in ion traffic in various cells presented here clearly suggest that ion traffic is seriously altered in CF patients who are homozygous for the mutation. The remaining question is what can be expected in heterozygous individuals without evident CF. Their phenotype needs to carry some survival advantage that allowed the CF mutation to spread in European nations. Estimated mutation incidences range from 1:200 in northern Sweden, 1:143 in Lithuanians, 1:38 in Denmark to 4

11 Kurbel et al. Membrane potential model of cystic fibrosis high values in Italy, France, Switzerland, British Isles, Germany and Greece (11,12). On the other hand, Saamis and Finnish have the lowest rates in Europe (13), while the highest incidences have been found in some disparate locations such as Ireland, Romania, Slovakia and Bulgaria (14). It is certain that the overall information is blurred by continuous human migrations during the last 50 Ky. This period is crucial due to the estimate that the most common CF linked mutation is possibly no more than 52 Ky old (15). The model presented here is possibly related to the CF incidence interpretation proposed by M. Lubinsky (16) that involves complex interactions between climate, pathogens and human physiology. The climate factors that are considered are temperature, latitude and altitude, the probable pathogen is tuberculosis while the physiological mechanisms involve vitamin D availability and arterial hypertension. The basic idea is that altered Cl- transport suppresses tuberculosis and alleviates the risk of hypertension caused by salt ingestion. On the other hand, low vitamin D availability, due to scarce sun exposure, increases the chances for tuberculosis and hypertension. This vitamin D and CFTR mutation link is based on the distribution of CF mutations with a high incidence among people from northern latitudes with low vitamin D levels in food and low insolation, except among Inuits, who have rich vitamin D sea food diet. On the other hand, cold climate, high altitude and vitamin D deficiency can all increase risk of arterial hypertension that becomes a very strong selective pressure during pregnancy. Thus, the heterozygote individuals for the CF mutation might have been more resistant to hypertension in this setting due to increased salt losses. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interest: none to declare REFERENCES 1. Kurbel S. Donnan effect on chloride ion distribution as a determinant of body fluid composition that allows action potentials to spread via fast sodium channels. Theor Biol Med Model 2011; 8: Rosenstein BJ, Zeitlin PL. Cystic fibrosis. Lancet 1998; 351: Wright SH. Generation of resting membrane potential. Adv Physiol Educ 2004; 28: The Nernst/Goldman Equation Stimulation (01 August 2012) 5. Barrett KE, Barman SM, Boitano S, Brooks HL. Ganong s Review of Medical Physiology, (Lange Basic Science). (23th Ed.). New York, USA Mc- Graw-Hill Medical, Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA, Block D, Zhang J, Soden R, Hayakawa M, Kreiman G, Cooke MP, Walker JR, Hogenesch JB. A gene atlas of the mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci USA 2004; 101: Wu C, Orozco C, Boyer J, Leglise M, Goodale J, Batalov S, Hodge CL, Haase J, Janes J, Huss JW 3rd, Su AI. BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources. Genome Biol 2009; 10:R Goldman MJ, Anderson GM, Stolzenberg ED, Kari UP, Zasloff M, Wilson JM. Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 1997; 88: Bro-Rasmussen F, Killmann SA, Thaysen JH. The composition of pancreatic juice as compared to sweat, parotid saliva and tears. Acta Physiol Scand 1956; 37: Shumaker H, Amlal H, Frizzell R, Ulrich CD 2nd, Soleimani M. CFTR drives Na + -nhco - cotransport 3 in pancreatic duct cells: a basis for defective HCO - secretion in CF. Am J Physiol 1999; 276:C Wennberg C, Kucinskas V. Low frequency of the delta F508 mutation in Finno-Ugrian and Baltic populations. Hum Hered. 1994; 44: Bobadilla JL, Macek M Jr, Fine JP, Farrell PM. Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening. Hum Mutat 2002; 19: Wennberg C, Kucinskas V. Low frequency of the delta F508 mutation in Finno-Ugrian and Baltic populations. Hum Hered. 1994; 44: Farrell PM. The prevalence of cystic fibrosis in the European Union. J Cyst Fibros 2008; 7: Wiuf C. Do delta F508 heterozygotes have a selective advantage? Genet Res 2001; 78: Lubinsky M. Hypothesis: Cystic fibrosis carrier geography reflects interactions of tuberculosis and hypertension with vitamin D deficiency, altitude and temperature. Vitamin D deficiency effects and CF carrier advantage. J Cyst Fibros 2012;11:

12 Medicinski Glasnik, Volume 12, Number 1, February 2015 Model mehanizma oštećenja u cističnoj fibrozi temeljen na apikalnom membranskom potencijalu duktalnih stanica Beatrica Kurbel 1, Saša Rapan 2, Sven Kurbel 3 1 Klinika za anesteziologiju, Klinički bolnički centar Zagreb, Zagreb, 2 Odjel za ortopediju i 3 Klinika za unutarnje bolesti, Klinički bolnički centar Osijek, Osijek; Hrvatska SAŽETAK U radu je prikazan jednostavni model oštećenja u bolesnika s cističnom fibrozom (CF), temeljen na membranskom potencijalu apikalne membrane (APM) duktalnih stanica koja je normalno vrlo propusna za natrij, a u slučaju aktivacije CFTR kanala postaje propusna za klorid. U modelu izračunati APM potencijali stanica s normalnom aktivnošću CFTR kanala pokazuju vrijednosti između Nernstovog potencijala natrija i Nernstovog potencijala klora, što znači da oba iona prolaze s lakoćom i apsorbiraju se iz vodova egzokrinih žlijezda. U bolesnika s CF-om, izračunati apikalni membranski potencijal je blizu Nernstove vrijednosti za natrij, što znači da električno polje priječi apsorpciju natrija a time i više soli ostaje u vodovima žlijezda. Simulacija predviđa da pokretačka snaga apsorpcije natrija raste više od 3,5 puta, ako se APM propusnost za klor povećava u rasponu od 5% do 94% propusnosti za natrij. U stanica vodova gušterače, bazolateralne membrane sadrže natrij/bikarbonata kotransporter (pnbc1) koji omogućuje prolaz bikarbonata zajedno s natrijem. Bikarbonati se razmjenjuju za intraduktalni klorid preko anionskog izmjenjivača 1 (AE1) na apikalnoj membrani. Aktivni CFTR kanali dozvoljavaju povrat klorida iz stanice nazad u vodove pankreasa. Klorid prati voda koja razrjeđuje sadržaj u vodovima. Recirkuliranje klorida u vodove omogućuje veću sekreciju bikarbonata. U bolesnika s cističnom fibrozom, sekrecija bikarbonata i vode je ograničena intraduktalnom količinom klorida koja se ne obnavlja kroz CFTR. Ključne riječi: membranski potencijal, cistična fibroza, pnbc1, AE1 6

13 REVIEW The assessment of acid-base analysis: comparison of the traditional and the modern approaches Jasna Todorović 1, Jelena Nešovic-Ostojić 1, Aleksandar Milovanović 2, Predrag Brkić 3, Mihailo Ille 4, Dušan Čemerikić 1 1 Department of Pathological Physiology, 2 Insitute of Occupational Health, 3 Department of Medical Physiology, 4 Department of Surgery; School of Medicine, University of Belgrade, Belgrade, Serbia ABSTRACT Corresponding author: Jelena Nešović Ostojić Department of Pathological Physiology, School of Medicine Dr. Subotića 1/II, Belgrade, Serbia Phone: ; Fax: ; E mail: jelnes@eunet.rs Three distinct approaches are currently used in assessing acid-base disorders: the traditional - physiological or bicarbonate-centered approach, the base-excess approach, and the modern physicochemical approach proposed by Peter Stewart, which uses the strong ion difference (particularly the sodium chloride difference) and the concentration of nonvolatile weak acids (particularly albumin) and partial pressure of carbon dioxide (pco 2 ) as independent variables in the assessment of acid-base status. The traditional approach developed from the pioneering work of Henderson and Hasselbalch and the base-excess are still most widely used in clinical practice, even though there are a number of problems identified with this approach. The approach works well clinically and is recommended for use whenever serum total protein, albumin and phosphate concentrations are normal. Although Stewart s approach has been largely ignored by physiologists, it is increasingly used by anesthesiologists and intensive care specialists, and is recommended for use whenever serum s total protein, albumin or phosphate concentrations are markedly abnormal, as in critically ill patients. Although different in their concepts, the traditional and modern approaches can be seen as complementary, giving in principle, the same information about the acid-base status. Key words: acid-base balance, anion gap, strong ion difference, bicarbonate, base excess, nonvolatile weak acids, strong ion gap Original submission: 03 September 2014; Revised submission: 21 November 2014; Accepted: 18 December Med Glas (Zenica) 2015; 12(1):7-18 7

14 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION Historically, two different conceptual approaches have been evolved among clinicians and physiologists for interpreting acid-base phenomena. The traditional or bicarbonate-centered approach relies qualitatively on the Henderson- Hasselbalch equation, whereas the modern or strong ion approach utilizes either original Peter Stewart equations or its simplified version derived by Peter Constable (1,2). The strong ion and bicarbonate-centered approaches are qualitatively identical even in the presence of non-bicarbonate buffers. The traditional approach for interpreting acid-base disorders developed from the pioneering work of Henderson and Hasselbalch is still the most widely used in clinical practice. One of the advantages of this approach is that it is relatively easy to understand and to apply in clinical situations (3). The strong ion model offers a novel insight into the pathophysiology of mixed acid-base disorders and is mechanistic (4). The newest approach has steadily gained acceptance especially among critical-care physicians and anesthesiologists (5,6). In this review we will discuss the advantages and disadvantages of Stewart s method of acid-base balance compared with traditional bicarbonate-based approach, what will help better understanding in the complexity of acid-base balance. THE TRADITIONAL APPROACH In the early 1900s sufficient laboratory and observational evidence had been accumulated to define the influence of carbon dioxide on ph and to suggest a role for serum bicarbonate in characterizing acid-base disorders. Henderson recognized that carbon dioxide and bicarbonate were key elements of carbonate mass action, as shown by his famous equilibrium equation (7). Hasselbalch reformulated this equation by introducing the negative logarithmic ph notation and by applying Henry s law to generate the pco 2 term. A buffer is substance that has ability to bind or release hydrogen ions (H + ) in solution, thus keeping the ph of the solution relatively constant despite the addition of considerable quantities of acid or bases (8). Bicarbonate is the most important buffer in biological system at constant pco 2, and Henderson-Hasselbalch equation provides a simple relationship among the respiratory parameter pco 2, the non-respiratory parameter bicarbonate, and overall acidity parameter, ph (9). But a change in bicarbonate concentration does not reflect the total amount of non-carbonic acid or base, because there are non-bicarbonate buffers, especially albumin and hemoglobin (10). Even more important is the fact that bicarbonate concentration is not independent of variation in pco 2. As pco 2 increases carbonic acid is buffered by non-bicarbonate buffers and the bicarbonate concentration increases. An elevated bicarbonate concentration may therefore erroneously be interpreted as a metabolic alkalosis when respiratory acidosis is the cause (11). So, this equation lists pco 2 and bicarbonate as independent predictors of ph while, in fact, these variables are interdependent. Consequently, this equation merely serves as a description of a patient s acid-base state but does not provide insight into the mechanism of the patient s acid base disorder (12,13). The traditional approach to clinical acid-base interpretation (bicarbonate-centered approach) is based on Lowry-Bronsted theory, wherein acids are defined as substances capable of donating protons, and the centrality of the bicarbonate buffer system in whole body acid-base homeostasis, given that it is composed of a volatile and nonvolatile buffer pair. Two bicarbonatecentered approaches evolved: the comparative HCO 3 / pco 2 approach (14) and the base excess (BE) approach (15). The comparative HCO 3 / pco 2 approach Qualitatively, this approach starts with the assumption that the components of the HCO 3 - CO 2 equilibrium reaction are in equilibrium with non-bicarbonate buffers (albumin, phosphate, hemoglobin) (3). Two bicarbonate-centered approaches evolved: the comparative HCO 3 / pco 2 approach (14) and the base excess (BE) approach (15). The HCO 3 / pco 2 approach has been criticized (2,11) for being qualitative in nature and incapable of quantifying acid or base loads that result in metabolic acid-base disorders. In particular, because body compartments consist of multiple buffers, it is argued that HCO 3 buffer is only one of several buffers that were protonated by an H + load (16). Therefore, the HCO 3 would underestimate the actual total body acid bur- 8

15 Todorović et al. Assessment of acid-base analysis den as, for example, in a patient with keto-acidosis (17). Additional criticism is the fact that a component of the change in the HCO 3 is due to a shift in the HCO 3 -CO 2 equilibrium reaction as a result of the compensatory ventilatory response (altered pco 2 ) that occurs in patients with metabolic acid-base disturbances (18). Moreover, the compensatory ventilatory induced alteration in the pco 2 causes a change in renal NH 4 and HCO 3 and titrate-able acid excretion. This results in a further change of the [HCO 3 ] (which is independent of the acid-base load and independent of the change due to the shift in the HCO 3 -CO 2 equilibrium reaction) (19, 20). Finally, a disadvantage is that the HCO 3 / pco 2 ratio expected in acute respiratory acid-base disorders depends on the number of proton binding sites on non-bicarbonate buffers (albumin, hemoglobin, phosphate) (2). Thus, the bicarbonate concentration may be used as a screening parameter of a non-respiratory acid-base disturbance when respiratory disturbances are taken into account (2). One possibility to solve this problem is to measure the bicarbonate concentration at a standard condition (when pco 2 is 40 mm Hg) - value that we call standard bicarbonate, or to use the sum of bicarbonate and non-bicarbonate buffer anions value that we call buffer base (changes in pco 2 would not affect the buffer base concentration as the rise in bicarbonate concentration associated with a rise in pco 2 is matched with a fall in concentration of other buffer anions) (11). Disagreement about the best parameter to describe acid-base balance in the body has dominated this area of physiology for more than three decades (19). The Henderson-Hasselbalch equation does not satisfactorily explain why the apparent value of pk 1 in plasma depends on ph, protein concentration and Na + concentration as well as the fact that only a non-linear relationship exists between log pco 2 and ph in vivo (markedly in acidic plasma) (2, 21). So, this equation (especially HCO 3 / pco 2 ratio) is criticized because of its qualitative nature and impossibility to quantify the acid or base excess that exists in acid base balance disorders (20). So, this approach can only be accurately applied to human plasma at approximately normal ph, protein concentration, and Na + concentration (22). Base excess (BE) approach Dissatisfactions with the Henderson-Hasselbalch approach prompted Singer and Hastings to propose in 1948 that plasma ph may be determined by two independent factors, pco 2 and net strong ion charge as the difference between all of the cations (termed total base) and anions (termed total fixed acid). They introduced the concept of buffer base (BB), (23) as the sum of all plasma buffer anions, i.e. bicarbonate plus the non-volatile weak acid buffers (albumin, phosphate) (3,24). These parameters (standard bicarbonate and buffer base) suggested as measures of metabolic acid-base disturbance are now obsolete. It is shown that a change in buffer base corresponds to a change in the metabolic component of acid-base balance, and yields the base excess (BE) methodology (25-28). Base excess is traditionally calculated from Van Slyke equation as developed by Siggard-Anderson (29). In the late 1950s, Siggaard-Andersen and colleague, at a fixed temperature and partial pressure of carbon dioxide, measured the plasma bicarbonate concentration and compared the difference between this value and a reference (30). When corrected by a constant, this difference yields the base excess (BE) as a more sensitive measure of metabolic imbalance. Clinically, this base excess represents the amount of acid per unit volume that must be added to achieve a normal ph (27). Blood base excess was introduced to replace plasma [HCO 3 ] with a measure of the metabolic component that is independent from the respiratory component, and incorporates the effect of hemoglobin as a buffer (30). Base excess represents the amount of acid or alkali that must be added to 1l of oxygenated blood exposed in vitro to a pco 2 of 40 mm Hg to achieve the average normal ph of 7.40 (30). Acid is required when blood ph is higher than 7.40 (positive BE or base excess), whereas alkali is needed when blood ph is lower than 7.40 (negative BE or base deficit). Under normal conditions, the average blood BE is zero (11). Criticism of this method followed soon (18, 31, 32). For example, the laboratory BE value represents the net effect of all metabolic acid-base abnormalities. Therefore, the effect of coexisting metabolic acidosis and alkalosis may lead to falsely suggest that no acid-base abnormality exists. Furthermore, this BE does not propose 9

16 Medicinski Glasnik, Volume 12, Number 1, February 2015 an etiology for the acid-base disorder once an abnormality is discovered (33). During in vitro blood titration, any CO 2 induced increase in plasma concentration of bicarbonate is attended by an equivalent decrease in the anionic charge of non-bicarbonate buffers (mainly hemoglobin). This comes from the binding of H + released from carbonic acid, and as a result blood base excess remains constant. Base excess becomes controversial because in vivo base excess is altered by purely respiratory changes. This occurs because blood freely exchanges ions with interstitial fluid, which contains little or no protein buffer. Therefore, as PaCO 2 changes in vivo, whole blood base excess changes measurably as bicarbonate and other ions equilibrate between blood and the interstitial space. Thus, primary PaCO 2 change in living organism causes base excess to move in the opposite direction, despite demonstrable in vitro CO 2 invariance. When the PaCO 2 is varied in vivo as a result of hypoventilation or hyperventilation, blood base excess does not remain constant because a concentration gradient for bicarbonate develops between blood and interstitial compartment. Accordingly, bicarbonate is removed from the plasma into the interstitial fluid in hypercapnia resulting in a negative base excess, whereas bicarbonate is added to plasma from the interstitial fluid in hypocapnia causing positive base excess (1,5,34). When the pco 2 is varied in vivo by CO 2 inhalation or hyperventilation, not only blood but all extracellular fluid is equilibrated with the new pco 2. When pco 2 increases ph tends to decrease more in the poorly buffered interstitial fluid than in the well buffered blood. H + therefore tends to diffuse from the interstitial fluid into the blood where they are buffered in the erythrocytes. This addition of H + to the blood is registered as a fall in whole blood base excess, while the plasma base excess rises slightly. The actual ionic movements involve a diffusion of bicarbonate ions from the erythrocytes to the plasma and interstitial fluid in exchange for chloride ions. However, the base excess of the total extracellular fluid remains constant, during acute pco 2 changes in vivo. It is not possible to obtain a sample of average extracellular fluid (including erythrocytes). However, a blood sample diluted threefold (1 + 2) with its own plasma may serve as a model of extracellular fluid. This pitfall was addressed by introducing the extracellular base excess or standard base excess by Siggaard-Andersen (11), as a measure of the metabolic component that is modeled by diluting the blood sample threefold with its own plasma or estimated by using the blood base excess at a hemoglobin concentration of 50 g/l. Thus, standard base excess or extracellular base excess is modeled from the existing PaCO 2 and ph, at a hemoglobin concentration of approximately 50 g/l, to replicate the mean extracellular hemoglobin concentration and does appear to have acceptable CO 2 invariance in vivo, although it is less than perfect. The base excess equation was modified to standardize the effect of hemoglobin on CO 2 titration in order to improve the accuracy of the base excess in vivo. Base excess of such model of the extracellular fluid may be calculated using the Van Slyke equation and now it represents the most relevant measure of a metabolic acid-base disturbance. Standard base excess is therefore roughly the corrective dose of sodium bicarbonate in mmol per liter of extracellular fluid. Currently, many blood gas analyzers calculate standard base excess from measured ph, pco 2 and hemoglobin. Modern ph-blood gas analyzers calculate the extracellular base excess and present the result with the same ease as they present the actual bicarbonate concentration. Determination requires an arterial blood sample and a modern ph blood gas analyzer. Total CO 2 (bicarbonate) measured in venous plasma using an electrolyte analyzer or multi-purpose chemical analyzer may be used as screening parameter in patients without respiratory disorders. Base excess is numerically identical with the delta buffer base of Singer and Hastings: the change in buffer base from the value at ph 7.4 and pco 2 40 mm Hg. However, standard base excess still yields results that are slightly unstable as pco 2 changes. Furthermore, the equation assumes normal concentration of non-bicarbonate buffers (albumin and phosphate), and when albumin or phosphate is decreased, a common scenario in the critically ill patients, standard base excess will result in even more instability (4,11,23,35). Even that standard base excess became (and remains) an optional computation that could be printed by most commercial blood gas analyzers, Bill Schvartz and Arnold Relman in Boston 1967 continued to advocate for the use of the actual bicarbonate concentration in assessing a metabolic component of acid- 10

17 Todorović et al. Assessment of acid-base analysis base status. Discussions in letters to the editors of several journals were called the great trans- Atlantic acid-base debate by John Bunker, and Boston and Copenhagen school were unreconciled (4,11,23,35). So, in a traditional approach the metabolic component of acid-base physiology is based on the analysis of plasma concentrations of bicarbonate and standard base excess. Both are usually used in clinical practice and their calculations are included in all blood gas analyzers. Despite these complexities, the comparative HCO 3 / pco 2 and BE approaches evolved historically as two alternative bicarbonate-centered approaches for diagnosing clinical acid-base disturbances (2,36). The anion gap method An additional diagnostic contribution in assessing metabolic component of acid-base analysis, the anion gap method, was eventually introduced. The anion gap is defined as the difference between unmeasured plasma anions and the unmeasured plasma cations. A normal anion gap is 12±4 mmol/l. Since normally the total unmeasured anions exceed the total unmeasured cations, there is an anion gap. Under normal conditions, the bulk of the serum anion gap (approximately 80%) is due to the sum of the anionic charges on circulatory proteins (albumin is the most abundant of circulating proteins). The charge on albumin at ph 7.4 contributes ~66% of the total net charge calculated by the anion gap, with the remainder composed of phosphate, urate, lactate, ketone bodies, and sulfate (37). Usually proteins behave as anions, contributing about 13 mmol/l to the unmeasured anion pool (ph-independent protein charge is 3.7 mmol/l, ph-dependent protein charge is 10.3 mmol/ L, ph-dependent phosphate charge is 1.0 mmol/l, the net protein charge of human plasma = = 13 mmol/l) (37). Therefore, changes in the concentration of serum albumin would be expected to alter the serum anion gap. For each 10 g/l decrement in the serum concentration of albumin, the serum anion gap was decreased by 2.5 mmol/l, and needed to be corrected to compensate for abnormal albumin concentrations (thus, for example, significant ketoacidosis, could be missed in a diabetic patient with hypoalbuminemia): corrected anion gap = observed anion gap 0.25 x ([normal albumin g/l] - [abnormal albumin g/l]), (38). This corrected anion gap can unmask an organic acidosis that was previously undetected in the setting of hypoalbuminemia (33). So, changes in the anionic effect of albumin will alter both the anion gap and the base excess. The presence of organic acid (XAH), which dissociates to form the anionic species XA while consuming bicarbonate results in increased unmeasured anions (UA) and a subsequently increased anion gap. Therefore, an increase in the calculated anion gap compared to the institutional reference, suggests the presence of an organic metabolic acidosis. Thus, when anion gap acidosis exists, the increase in the anion gap should qualitatively mirror the fall in bicarbonate. Disruption of this expected relationship is indicative of certain mixed acid-base disorders (39). The small size of the anion gap is most likely to result from a reduced concentration of the normal unmeasured anion albumin in critically ill patients. Globulins do not have a significant charge contribution compared to albumin since their pk a is much greater than plasma ph (K a is the effective equilibrium dissociation constant for plasma weak acids). As myeloma proteins have isoelectric points >7.4 they become positively charged in the serum and behave as cations. In this way they may lead to a reduced anion gap by creating an excess of positively charges ions. That has to be counterbalanced by an increase in anions, mainly chloride. This explains why in about 30% of myeloma or gammapathies the anion gap could be <3 mmol/l (40). THE MODERN APPROACH In late 70s and early 1980s Peter Stewart proposed that the generalized Arrhenius definition of acid (substance that, when dissolved in water, produces increased concentration of H + ) with Naunyn s ideas (acid-base status was partly determined by electrolytes, particularly sodium - base forming and chloride - acid forming), is more useful to outline physiology than the Bronsted-Lowry definition (acid-donate proton, base acceptor of proton), (41). In 1978 Stewart questioned the traditionally accepted approach used to analyze acid-base chemistry. He modeled a solution which contained a complex mixture of ions of constant charge over the physiologi- 11

18 Medicinski Glasnik, Volume 12, Number 1, February 2015 cal ph range (Na +, K +, Ca 2+ ), nonvolatile proton donor/acceptors which transfer H + within the physiological ph range (albumin, phosphate, hemoglobin, metabolizable organic compounds), and the volatile bicarbonate-co 2 buffer system 2- composed of CO 2, HCO 3, H 2 CO 3, and CO 3. According to requirements of electro neutrality, the law of conservation of mass and certain equilibrium constants, Stewart solved a fourth-order polynomial equation for calculating the H + concentration. His analysis did not depart from the traditional approach to acid-base chemistry as a result of the equations that are derived. Pivotal to the Stewart formulation was the categorization of certain species as being dependent or independent variables in relationship to their purported role in determining and modifying the H + concentration of solution. At first, H +, OH -, HCO 3, 2- and CO 3 were categorized as dependent variables (the mass balance of these species in a solution or specific body fluid compartment could not per se affect the H + concentration). Finally, he contended that H + concentration was a function of three variables: strong ion difference (SID) (the difference in the net charge of fixed cations and anions fully dissociated in solution), partially dissociated weak acids (albumin, phosphate) (A TOT ), and the partial pressure of carbon dioxide (pco 2 ) of the solution (2). According to the principle of electro neutrality, SID is balanced of the weak acids (albumin, phosphate) and CO 2. Therefore, SID can be defined either in terms of strong ions or in terms of the weak acids and CO 2 offsetting it. Of note, the SID defined in terms of weak acids and CO 2, termed as the effective SID and is identical to the buffer base term coined by Singer and Hastings over half a century ago. So, changes in standard base excess also represent changes in SID (42). Stewart hypothesized that water dissociates into H + and OH - to a greater or a lesser extent when (SID), (A TOT ) or pco 2 change. Aqueous solutions contain a virtually inexhaustible source of H +. Although pure water dissociates only slightly into H + or OH -, electrolytes and CO 2 produce powerful electrochemical forces that influence water dissociation. SID has a powerful electrochemical effect on water dissociation, and hence on H + concentration. As SID becomes more positive, H +, a weak cation, decreases - (and ph increases) in order to maintain electrical neutrality. Strong ions cannot be created or destroyed to satisfy electro neutrality but H + ions are generated or consumed by changes in water dissociation (30). This method emphasizes the role of water dissociation as a proton source and the association of water to consume protons as the driving force behind changes in blood ph. The relative balance of plasma positive and negative charges, including those on serum proteins, most notably albumin, drives water dissociation and association by the law of mass action (43). Stewart approach puts water dissociation at the center of the acid-base states of body fluids. ph of a body fluid is a function of water dissociation modified by pco 2, other weak acids and certain electrolytes (5,44). To date there have been no empirical observations that confirm water dissociation as the mechanism whereby (SID), (A TOT ) or pco 2 affect ph. Thus, the disadvantage of Siggaard-Andersen approach is that it implies adding or introducing H + to the solution, which is impossible. The more general Stewart s approach explains the acid base variations over more valuable physical basis. This physicochemical approach can be helpful in understanding mechanisms barely understandable using traditional approach (45). The Stewart approach is a very general physicochemical method that uses charge and mass balance to deduce an expression for proton concentration. Similarly, the base excess method is another very general physicochemical approach, but one that uses proton balance to calculate changes in proton concentration by using the Van Slyke equation (46). Thus, Stewart challenged the traditional bicarbonate-based method of diagnosis and treating acid-base disorders and proposed an approach based primarily on charge differences between strong cations and anions. He suggested that three independent factors, the pco 2, the strong ion difference, and the total non-volatile weak acid concentration may be used to analyze the causes of acid-base disorders. These three factors control other variables including hydrogen ion concentration and bicarbonate concentration which are dependent variables. Increases in pco 2 and total weak acid concentration increase acidity. Decreases in strong ion difference increase acidity. This approach may better explain the me- 12

19 Todorović et al. Assessment of acid-base analysis chanism of acid-base physiology and disorders than the Henderson-Hasselbalch approach (44). On the basis of Stewart s definition H + and bicarbonate are dependent variables whose concentrations are determined by three independent variables, namely SID, pco 2 and A TOT. SID therefore can be calculated as the difference between fully dissociated cations and anions which do not participate in proton transfer reactions (aprote ions - neither donate or accept H + ) and by the principle of electro neutrality this difference is equal to the sum of bicarbonate and the non-bicarbonate anions, which represent total charges contributed of all non-bicarbonate buffers, primarily, albumin and phosphate, and in whole blood, hemoglobin. SID is, therefore, the same as buffer base concept introduced by Singer and Hastings more than 5 decades ago. Apparent SID and effective SID When abnormal anion is present, a gap will appear between SID calculated by the difference between strong ions (the so-called apparent SID ) and calculated by the addition of bicarbonate and non-bicarbonate buffers (so called effective SID). This difference, named strong anion gap (SIG), is a marker for the presence of an abnormal anion (23). The SIG indicates the presence of unmeasured strong anions if its value is positive, the normal value of the SIG is zero. The SIG is similar in concept to the anion gap, once the latter has been corrected for anionic contribution of albumin and phosphate (3). A positive SIG value represents unmeasured anions (such as ketoacids, urate, sulfate, citrate, pyruvate, acetate and gluconate) that are present in the blood. Apparent SID is referred to as apparent (SID a- ) with the understanding that some unmeasured pp ions might be also present. It represents the difference between the charge of measured strong cations (sodium, potassium, calcium, magnesium) and strong anions (chloride, lactate, sulfate, ketoacids, nonesterified fatty acids and many others) that are completely dissociated in biological solutions. Currently, (SID) is routinely measured from [Na + ] + [K + ] + [Ca 2+ ] + [Mg 2+ ] - [Cl - ] - [lactate - ]. It is always positive in plasma and in healthy humans - its value is 40 to 42 mmol/l, while it is often quite different in critically ill patients. The effective SID (SID eff ) represents the effect of the corrected pco 2 and the weak acids, albumin and inorganic phosphate, on the balance of electrical charge in plasma, where HCO 3 is in mmol/l, albumin in g/l and phosphate in mmol/l. All the independent variables are present in millimolar concentrations and their interaction with water dictates the amount of free H +, the concentration of which is in order of nanomoles. The difference between the calculated apparent SID and effective SID constitutes the strong ion gap, SIG. In healthy humans, the SIG should be theoretically equal to zero (electrical charge neutrality). If this is not the case, there must be unmeasured charges to explain this ion gap. A positive SIG value represents unmeasured anions (such as ketoacids, urate, sulfate, citrate, pyruvate, acetate and gluconate) that are present in the blood and account for the measured ph, the measured levels of strong and weak ions, and the need to maintain electro neutrality (46). Unlike the classic parameters used to calculate the anion gap, in calculating SIG, the effect of albumin, phosphate and lactate is subtracted out. Therefore, SIG compared with the anion gap is caused by shorter list of unmeasured ions including ketone bodies, sulfate and uraemic anions (2). Calculating the effective SID takes into account the role of weak acids (carbon dioxide, albumin and phosphate) in the balance of electrical charges of plasma water (47). Once weak acids are qualitatively taken into account, the difference between apparent and effective SID should be zero, unless there are unmeasured charges (anions). Such charges are then described by the strong ion gap, SIG (SIG = apparent SID effective SID). Bicarbonate is considered separately because this buffer system is an open system in arterial plasma. Rapid changes in pco 2 and hence arterial bicarbonate concentration can be rapidly induced through alteration in respiratory activity. In contrast, the non-bicarbonate buffer system is a closed system containing relatively fixed quantity of buffers. Nonvolatile buffer ion (A - ) represents a diverse and heterogeneous group of plasma buffers consisting primarily of dissociable imidazole and α-amino groups on plasma proteins with a smaller contribution from phosphate-containing weak acids and citrate. On the basis of information stated above, plasma contains three types of charged entities: SID +, 13

20 Medicinski Glasnik, Volume 12, Number 1, February 2015 HCO 3 and A -. The requirement for electro neutrality dictates that at all times [SID + ] equals the sum of [HCO 3 ] and nonvolatile [A - ], such that [SID + ] - [HCO 3 ] - [A - ] = 0. (The dissociation reaction for a weak acid-conjugate base-pair, HA and A -, is HA =H + +A - ) (48). Partially dissociated weak acids (albumin, phosphate), (A TOT ) Plasma proteins provide the major contribution to A TOT, and therefore, plasma protein concentration independently affects acid-base balance. One major departure from the traditional approach is classification of acid-base disorders as a result of alteration in A TOT. A TOT, representing all non-bicarbonate buffers pairs (HA + A - ), is made up of charges contributed primarily by serum proteins (mainly albumin) with phosphate and other buffers playing a minor role. The sum of [HA] and [A - ] (called A TOT by Stewart) therefore remains constant through conservation of mass. The normal value of the total negative charges on plasma non-bicarbonate buffers is [A TOT ] = [A - ] + albumin [A- ] + globulin [A- ] = 16.6 mmol/l (49). phosphate On the basis of this classification, an increase in serum protein would result in metabolic acidosis while a decrease would cause metabolic alkalosis. Siggaard-Andersen and Fogh-Andersen have suggested that changes in protein concentration should not be considered as acid-base disorders, and have considered the Stewart approach problematic in that regard (11). Dissatisfaction with the Henderson-Hasselbalch approach prompted Singer and Hastings to propose in 1948, that plasma ph was determined by two independent factors, pco 2 and net strong ion charge, equivalent to the strong ion difference (SID + ). Stewart later proposed that third variable, the total plasma concentration of nonvolatile weak acids (A TOT ), also, exerted an independent effect on plasma ph. Thus, by combining equations for conservation of charge, conservation of mass, and dissociation equilibrium reactions, Stewart developed a polynomial equation, relating H + concentration [H + ] to three independent variables (pco 2, [SID + ] and [A TOT ]) and five constants (48). These three independent variables may change the hydrogen concentration in water (i.e. the acid-base equilibrium). The strong ion difference is regulated by the kidney, weak acid concentration primarily by liver, and pco 2 by lung (48). An independent variable is defined as one that influences the system but is not influenced by the system. The term system refers to any single aqueous compartment (i.e. plasma). The Stewart s approach states that ph is primarily determined by several independent variables (which change primarily and independently of one another: by pco 2, strong ion difference (SID) and nonvolatile weak acids). This physicochemical approach might identify altered individual component of complex acid-base abnormalities and provide insights to their underlying mechanisms (35). The relation between strong ion difference (SID) and standard base excess (SBE) The SID a must be counterbalanced by an equal and opposing charge termed the effective strong ion difference (SID e ) (normal approximately -40 to -42 mmol/l). The SID e negative charge principally stems from the dissociated moieties of plasma proteins (-78% albumin) and phosphate (-20%). The sum of these weak acids is known as A TOT since they exist in a dissociated form (A - ), as well as an associated form (AH). When the SID a and SID e are equal the plasma ph is exactly 7.4 at a pco 2 of 40 mm Hg. Bicarbonate is dependent variable and does not determine the ph (47). According to the principle of electro neutrality, SID a is balanced of the weak acids and CO 2, such that SID can be defined either in terms of strong ions (SID a ) or in terms of the weak acids and CO 2 (SID e ) offsetting it. Of note, the SID defined in terms of weak acids and CO 2, which has been subsequently termed the effective SID is identical to the buffer base term coined of Singer and Hastings over half a century ago. So, changes in the standard base excess also represent changes in SID. The difference in electrical charge between strong cations and strong anions is called strong ion difference. In normal plasma this amount is about 42 mmol/l. Indeed, to reach the electro neutrality, 42 mmol/l of negative charged ions, are required. These are basically the bicarbonate (HCO 3 ) and negative charged form of weak acids (A - ), primarily albumin plus an extremely small amount of hydroxyl (OH - ). The sum of [HCO 3 ] + [A - ] which equals the strong ion difference was called buffer base by Singer and Hastings, and 14

21 Todorović et al. Assessment of acid-base analysis later by Siggaard-Andersen. Indeed, a big difference between the traditional approach (SBE) and the Stewart s approach is that the first considers what happens inside buffer base domain. As an example, in the traditional model (SBE) the normal buffer base (42 mmol/l, as the normal [SID]) may decrease for 10 mmol/l if the (A - ) and (HCO 3 ) are consumed by adding 10 mmol/l of H +. In this case the actual buffer base is 32 mmol/l and the difference between the actual buffer base and the ideal buffer base is equal to -10 mmol/l, and is called base excess. In the Stewart model the same problem is considered from another point of view. If a strong ion is added to the system the ratio between strong cations and strong anions will change. As an example, by adding 10 mmol/l of lactate the strong ion difference decreases from 42 mmol/l to 32 mmol/l. The space available for A - and HCO 3 and OH - decreases, indeed part of A - will become AH, part of HCO 3 will become H 2 CO 3 and part of OH - will become H 2 O. As the product of H + and OH - is constant, a decrease of OH - will correspond to an increase of H +. i.e. acidosis (45). So, the disadvantage of standard base excess approach is that it implies adding or introducing H + to the solution, which is impossible. The more general Stewart s approach explains the acid-base variations on a more valuable physical basis. So, the physicochemical Stewart s approach can be helpful in understanding mechanisms barely understandable using traditional approach. The mystery of dilutional acidosis during repletion of extracellular fluid deficit can be better explained by the modern approach. The mechanism is obviously not bicarbonate dilution as explained by the traditional approach (otherwise why would the proton donors not be diluted at the same time?). Therefore, from Stewart s perspective an alternative explanation for dilutional acidosis needed to be developed. His mechanistic explanation is based on strong ions and the maintenance of electro neutrality. It was believed that positive or negative charges (i.e. changes of the concentrations of strong ions) influence the dissociation of water (50). In the context of dilutional acidosis, this means dilution of plasma (which has a positive SID of 39 mmol/l) by water or another solution with SID of zero decreases the SID, i.e. diminishes the surplus of positive charges. However, the decrease in SID demands compensation by a positive charge. This is suggested by increased water dissociation with generation of a positively charged H +. This newly generated H + then causes acidification of the solution, i.e. dilutional acidosis (50). Interestingly, hypertonicity makes solutions more acidifying, as more water is drained from the intracellular space, which ultimately contributes to the final equilibrium (51). Changes in SID, SIG and A TOT in acid base disorders Respiratory disorders, in the modern approach as in the traditional approach, are due to change in pco 2, whereas metabolic disorders are due to alterations in either SID or A TOT. SID is decreased in metabolic acidosis and increased in metabolic alkalosis. By calculating SIG, one can further classify metabolic acidosis. In hyperchloremic metabolic acidosis both effective and apparent SID decreases equally, as the increase in chloride is counterbalanced by an equal decrease in the bicarbonate concentration. SIG therefore remains at or near zero. In anion gap metabolic acidosis, apparent SID does not change (as chloride concentration is unchanged), but effective SID decreases (as a result of a decrease in bicarbonate concentration) and SIG therefore becomes positive, reflecting high levels of unmeasured anions such as lactate and ketoanions (52). One major departure from the traditional approach is classification of acidbase disorders as a result of alteration in A TOT. On the basis of this classification, an increase in serum protein would result in metabolic acidosis and a decrease, metabolic alkalosis (23). CONCLUSION Three distinct approaches are currently used in assessing acid-base disorders, the traditional or physiological approach pioneered by Van Slyke, the base-excess method, developed by Astrup, and the physicochemical approach, proposed by Stewart. The last and newest approach has steadily gained acceptance especially among criticalcare physicians and anesthesiologists (5). The traditional approach to interpreting acid-base disorders developed from the pioneering work of Henderson and Hasselbalch and is still most widely used in clinical practice. An advantage of this approach is that it is relatively easy to understand and to apply in common clinical situations (3). 15

22 Medicinski Glasnik, Volume 12, Number 1, February 2015 We conclude that the physiological or traditional approach remains the simplest, most rigorous, and most serviceable approach to assessing acid-base disorders. Clinically, the traditional approach is intuitive in nature and is supported by a large body of robust empirical observations. The true relevant acid-base quantities are the arterial ph, the arterial pco 2, and the extracellular base excess. Determination requires an arterial blood sample and a modern ph blood gas analyzer. Total CO 2 (bicarbonate) measured in venous plasma using an electrolyte analyzer or multipurpose chemical analyzer may be used as screening parameter in patients without respiratory disorders (101). The strong ion model offers a novel insight into the pathophysiology of a mixed acid-base disorders and is mechanistic (4). The traditional approach should be abandoned only if proponents of Stewart s approach could provide clear empirical observations supporting its superiority as a clinical tool in diagnosis and treating patients with acid-base disorders. The dependence of pk (carbonic acid) on ph 1 and protein concentration is a major anomaly for the Henderson-Hasselbalch equation because the dissociation constant for equilibrium reactions should not be influenced by changes in reactants (hydrogen ion activity = ph) or by anything else (including protein) except temperature and ionic strength, the latter being determined primarily by the sodium concentration. The change in [SID + ] from normal is equivalent to the base excess value assuming a normal, nonvolatile buffer ion concentration (normal albumin, globulin and phosphate concentrations) (1). If serum total protein, albumin and phosphate concentrations are approximately normal, then acid-base status should be evaluated using blood ph, pco 2 and extracellular base excess concentration, which is the traditional Henderson-Hasselbalch approach. The presence of unidentified anions should be investigated by calculating anion gap. If albumin concentration is abnormal, the anion gap can be corrected based on the albumin concentration, as this corrected anion gap can unmask an organic acidosis that was previously undetected in the setting of hypoalbuminemia. However, if serum total protein, albumin and phosphate concentrations are markedly abnormal, as in critically ill patients, then acid-base status should be evaluated using blood ph, pco 2, measured [SID + ] and A TOT. The presence of unidentified strong ions should be investigated by calculating the SIG. Traditional approach works well clinically and is recommended for use whenever serum total protein, albumin and phosphate concentrations are normal. Although Stewart s approach has been largely ignored by physiologists, it is increasingly used by anesthesiologists and intensive care specialists, and is recommended for use whenever serum total protein, albumin or phosphate concentrations are markedly abnormal, as in critically ill patients. Although different in their concept, the traditional and modern approaches can be seen complementary giving, in principle, the same information about the acid-base status. FUNDING This work was supported by grants from the Ministry of Education, Science and Technological Development of the Republic of Serbia. TRANSPARENCY DECLARATION Competing interests: None to declare. REFERENCES 1. Constable PD. Clinical assessment of acid-base status: comparison of the Henderson-Hasselbalch and strong ion approaches. Vet Clin Pathol 2000; 29: Kurtz I, Kraut J, Ornekian V, Nguyen MK. Acidbase analysis: a critique of the Stewart and bicarbonate-centered approaches. Am J Physiol 2008; 294:F Sirker AA, Rhodes A, Grounds RM, Bennett ED. Acid-base physiology: the traditional and the modern approaches. Anaesthesia 2002; 57: Kellum JA. Disorders of acid-base balance. Crit Care Med 2007; 35: Morgan TJ. The Stewart approach-one clinician s perspective. Clin Biochem Rev 2009; 30: Kishen R, Honore PM, Jacobs R, Joannes-Boyau O, De Waale E, De Regt J, Van Gorp V, Boer W, Spapen HD. Facing acid-base disorders in the third millennium the Stewart approach revisited. Int J Nephrol Renovasc Dis 2014; 7: Henderson LJ. Concerning the relationship between the strength of acids and their capacity to perserve neutrality. Am J Physiol 1908; 21:

23 Todorović et al. Assessment of acid-base analysis 8. Henderson LJ. The theory of elecroneutrality regulation in the animal organism. Am J Physiol 1907; 18: Van Slyke DD. Studies of acidosis I. The bicarbonate concentration of the blood plasma; its significance and its determination as a measure of acidosis. J Biol Chem 1917; 30: Hughes R, Brain MJ. A simplified bedside approach to acid-base: fluid physiology utilizing classical and physicochemical approaches. Anesthesia & Intensive Care Medicine 2013; 14: Siggaard-Andersen O, Fogh-Andersen N. Base excess of buffer base (strong ion diference) as mesure of a non-respiratory acid-base disturbance. Acta Anaesthesiol Scand 1995; 39(Suppl. 106): Hickish T, Farmery AD. Acid-base physiology: new concepts. Anesthesia & Intensive Care Medicine 2012; 13: Wilcox C, Cemerikic D, Giebisch G. Differential effects of acute mineralo- and glucocorticosteroid administration on renal acid elimination. Kidney Int 1982; 21: Schwartz WB, Relman AS. A critique of the parameters used in the evaluation of acid-base disorders whole-blood buffer base and standard bicarbonate compared with blood ph and plasma bicarbonate concentration. New Enlg Med 1963; 268: Siggaard-Andersen O. The acid-base status of the blood (4th Ed.). Baltimore, MD: William and Wilkins, Hubble SMA. Acid-base and blood gas analysis. Anesthesia & Intensive Care Medicine 2007; 8: Severinghaus JW. Case for standard-base excess as the measure of nonrespiratory acid-base imbalance. J Clin Monit 1997; 7: Siggaard-Andersen O, Wimberley PD, Fogh-Andersen N, Gothgen IH. Measured and derived quantities with modern ph and blood gas equivalent circulation algorithm with S4 equations. Scand J Clin Lab Invest 1988; 48: Kurtz I. Acid Base Case Studies Victoria, Canada: Trafford. 20. Madias MA, Schwartz WB, Cohen JJ. The maladaptive renal response to secondary hypocapnia during chronic HCl acidosis in the dog. J Clin Invest 1997; 60: Cemerikic D, Wilcox C, Giebisch G. Intracellular potential and K + activity in rat kidney proximal tubular cells in acidosis and K + depletion. J Membr Biol 1982; 69: Berend K. Acid-base pathophysiology after 130 years: confusing, irrational and controversial. J Nephrol 2013; 26: Rastegar A. Clinical utility of Stewart s method in diagnosis ans management of acid-base disorders. Clin J Am Soc Nephrol 2009; 4: Wooten EW. Analytic calculation of physiological acid-base parameters in plasma. J Appl Physiol 1999; 86: Astrup P, Jorgensen K, Siggaard-Andersen O. Acidbase metabolism: New approach. Lancet 1960; 1: Siggaard-Andersen O. The ph-log PCO 2 blood acidbase nomogram revised. Scand J Clin Lab Invest 1962; 14: Grogno AW, Bzles PH, Hawke W. An in vivo representation of acid-base balance. Lancet 1976; 1: Severinghaus JW. Acid-base balance nomogram a Boston-Copenhagen detente. Anesthesiology 1976; 45: Siggaard-Anderson O. The Van Slyke equation. Scand J Clin Lab Invest 1977; 146: Kellum JA. Determinants of blood ph in health and disease. Crit Care 2000; 4: Gonzales NC, Clancy RL. Intracellular ph regulation during prolonged hypoxia in rats. Respir Physiol 1986; 65: Roos A, Thomas LJ. The in-vitro and in-vivo carbon dioxide dissociation curves of true plasma. Anesthesiology 1977; 28: Fidkowski C, Helstrom J. Diagnosis metabolic acidosis in the critically ill: bridging the anion gap, Stewart and base excess methods. Can J Anest 2009; 56: Greenbaum J, Nirmalan M. Acid-base balance: Stewart s physicochemical approach. Curr Anaesthesia Crit Care 2005; 16: Dubin A, Menises MM, Masevicius FD, Moseinco MC, Kutscherauer DO, Ventrice E, Laffaire E, Estenscoro E. Comparison of three different methods of evaluation of metabolic acid-base disorders. Crit Care Med 2007; 35: Geoffrey C, Aiken A. History of medical understanding and misunderstanding of acid-base balance. J Clinical Diagnostic Research 2013; 7: Staempfli H, Constable PD. Experimental determination of net protein charge and A tot and K a of nonvolatile buffers in human plasma. J Appl Physiol 2003; 95: Kraut JA, Madias NE. Serum anion gap: its uses and limitations in clinical medicine. J Am Soc Nephrol 2007; 2: Emmett M. Anion gap interpretation: the old and the new. Nat Clin Pract Nephrol 2006; 2: Story DA. Bench-to-bedside review: a brief histor of clinical acid-base. Crit Care 2004; 8: Correy HE. Stewart and beyond: new models of acid-base balance. Kidney Int 2003; 64: Kellum JA. Clinical review: Reunification of acidbase physiology. Crit Care 2005; 9: Kaplan LJ, Kellum JA. Initial ph, base deficit, lactate, anion gap, strong ion. Crit Care 2004; 32: Smuszkiewicz P, Szrama J. Theoretical principles of fluid management according to the physicochemical Stewart approach. Anaesthesiol Intensive Ther 2013; 45: Gattinoni L. Carlesso E, Maiocchi G, Polli F, Cardingher P. Dilutional acidosis: where do the protons come from? Intensive Care Med 2009; 35: Nguyen BV, Vincent JL, Hamm JB, Abalin JH, Carre JL, Nowak E, Ahmed MO, Arvieux CC, Gueret G. The reproducibility of Stewart parameters for acid-base diagnosis using two central laboratory analyzers. Anesth Analg 2009; 109: Naka T, Bellomo R. Bench-to-bedside review: treating acis-base abnormalities in the intensive care unit-the role of renal replacement therapy. Crit Care 2004; 8: Constable PD. A simplified strong ion model for acid-base equilibria: application to horse plasma. J Appl Physiol 1997; 83:

24 Medicinski Glasnik, Volume 12, Number 1, February Matousek S, Handy J, Rees SE. Acid-base chemistry of plasma consolidation of the traditional and modern approaches from a mathematical and clinical perspectives. J Clin Monitor Comp 2011; 25: Doberer D, Funk GC, Kirchner K, Schneeweiss B. A critique of Stewart s approach: the chemical mechanism of dilutional acidosis. Intensive Care Med 2009; 35: Story DA, Morimatsu H, Bellomo R. Strong ions, weak acids and base excess: a simplified Fencl- Stewart approach to clinical acid-base disorders. Brit J Anaesthesia 2004; 92: Androgue HJ, Gennari FJ, Galla JH, Madias NE. Assessing acid-base disorders. Kidney Int 2009; 76: Acido-bazne analize: poređenje tradicionalnog i modernog pristupa Jasna Todorović 1, Jelena Nešović-Ostojić 1, Aleksandar Milovanović 2, Predrag Brkić 3, Mihailo Ille 4, Dušan Čemerikić 1 1 Institut za patološku fiziologiju, 2 Institut za medicinu rada, 3 Institut za fiziologiju, 4 Hirurgija; Medicinski fakultet Univerziteta u Beogradu, Beograd, Srbija SAŽETAK Danas se koriste tri posebna pristupa u proceni acido-baznih poremećaja: tradicionalni ili fiziološki ili bikarbonatni pristup, metoda baznog ekcesa i moderni fizikalno-hemijski pristup predložen od Stewarta koji koristi razliku glavnih jona (naročito natrija i hlorida), koncentraciju neisparljivih slabih kiselina (osobito albumina) i parcijalni pritisak ugljen dioksida (pco 2 ) kao nezavisne varijable u analizi acido-baznog statusa. Tradicionalni pristup kojeg su predložili Henderson i Hasselbalch i metoda baznog ekcesa, uprkos brojnim nedostacima, još uvek su najšire korišćeni u kliničkoj praksi. Ovaj pristup se naročito preporučuje u kliničkoj praksi kada su koncentracije ukupnih proteina, albumina i fosfata u serumu normalne. Iako je Stewartova metoda bila uglavnom ignorisana od strane fiziologa, nju sve više primenjuju anesteziolozi i lekari intenzivne nege prilikom lečenja teško bolesnih pacijenata kod kojih su koncentracije ukupnih proteina, albumina i fosfata u serumu poremećene. Iako različite u svom konceptu, tradicionalna i moderna metoda mogu se posmatrati kao komplementarni pristupi koji upotpunjuju informisanost o acido-baznom statusu. Ključne reči: acido-bazna ravnoteža, anjonski zjap, razlika glavnih jona, bikarbonati, bazni eksces, neisparljive slabe kiseline, zjap glavnih jona 18

25 REVIEW Clonus: definition, mechanism, treatment Ismail Boyraz 1, Hilmi Uysal 2, Bunyamin Koc 1, Hakan Sarman 3 1 Physical Medicine and Rehabilitation, Abant Izzet Baysal University, Bolu, 2 Neurology, Akdeniz University Hospital, Antalya, 3 Orthopeadics Department, Abant Izzet Baysal University, Bolu; Turkey ABSTRACT Corresponding author: Ismail Boyraz Physical Medicine and Rehabilitation Hospital, Abant Izzet Baysal University Aibu Ftr Hospital Karacasu, Bolu 14100Turkey Phone: ; Fax: ; boyraz@yahoo.com Clonus is involuntary and rhythmic muscle contractions caused by a permanent lesion in descending motor neurons. Clonus may be found at the ankle, patella, triceps surae, wrist, jaw, biceps brachii. In general, clonus may occur in any muscle with a frequency of 5-8 Hz and the average period of oscillations of the ankle clonus is approximately ms. Plantar flexion (PF) comprises 45% of the period, dorsifleksion (DF) comprises 55% of the period. The first beat is always longer, with the time shortening in continuing beats and becoming stable in the 4th or 5th period. The exact mechanism of clonus remains unclear. Two different hypotheses have been asserted regarding the development of clonus. The most widely accepted explanation is that hyperactive stretch reflexes in clonus are caused by self-excitation. Another alternative explanation for clonus is central generator activity that arises as a consequence of appropriate peripheral events and produces rhythmic stimulation of the lower motor neurons. The durations of clonus burst were found longer than the durations of Soleus medium-latency reflex (MLR). There is a similarity in their nature, although the speed and cause of the stretch of triceps surae differ in the MLR and the clonus, and there is a sufficient period of time for group II afferents and for other spinal mechanisms to be involved in the clonus, together with Ia afferents. Clonus can be treated by using baclofen, applying cold, botox or phenol injections. Key words: botulinum toxin, spasticity, upper motor disorder, gait disorder Original submission: 04 September 2014; Revised submission: 22 December 2014; Accepted: 05 January Med Glas (Zenica) 2015; 12(1):

26 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION Clonus is involuntary and rhythmic muscle contractions caused by a permanent lesion in descending motor neurons and it is usually considered to be a result of oscillations in the group Ia spinal stretch reflex (Figure 1). Clonus is accompanied by spasticity and other findings of reflex excitability (1). Spasticity is defined as an increased resistance to stretching caused by disorders involving the upper motor neurons, and clonus is characterized by exaggerated brain stem and spinal reflexes resulting in increased muscle tone and involuntary spasms. Although closely linked, clonus is not seen in all patients with spasticity (2).Clonus does not occur if the muscle is excessively hypertonic (2). Any mechanism or pharmacological drug suppressing increased reflexes and muscle tone is also prone to block the clonus (2). Severe clonus can interrupt sleep and prevent the transfer capability of the patient and result in fatigue that can decrease work performance of an individual (3). It can also interfere with the posture and gait of the patient (4). Clonus can also occur in normal individuals. The plantar flexion power is low in normal individuals (5). Clonus may be found at the ankle, patella, triceps surae, wrist, jaw, biceps brachii (6-8). Jaw jerk is due to supranuclear lesion of the trigeminal nerve and it may occur in Amyotrophic Lateral Sclerosis (6).Wrist clonus in patients with hemiplegia was notably described in lectures published in 1883 by the French neurologist Jean-Martin Charcot, who called the phenomenon provoked trepidation, the patients, on raising the paralyzed arm, often experience trembling similar to that which Figure 1. Ankle clonus Soleus rectified EMG and position of the ankle are displayed superimpose. Soleus muscle activity can be seen after ankle dorsiflexion occurs in the lower limb under like circumstances (7). But the wrist-phenomenon, provoked or spontaneous, is much more uncommon. In general, clonus may occur in any muscle with a frequency of 5-8 Hz and the average period of oscillations of the ankle clonus is approximately ms (9). Plantar flexion (PF) comprises 45% of the period, and dorsiflexion (DF) comprises 55% of the period (9). It has been shown that the duration of the dorsiflexion was 88.63±10.83 ms, and the duration of the PF was 71.75±6.73 ms (9). The DF and PF comprised 55.17±3.9% and 44.83±3.9% of one clonus beat, respectively (9). The first beat is always longer, with the time shortening in continuing beats and becoming stable in the 4th or 5th period. Measured the refractory period only in the triceps surae muscle is ms. This period may differ for other muscle groups with different central stretch reflex organizations, thereby resulting in different maximum clonus frequencies (9). In order to reach an understanding of clonus, it is essential to consider not only reflex path length but also muscle contraction and relaxation times, muscle load, muscle spindle activity and central excitability, all of which play a role in clonus (7,9). Dimitrijevic et al. have shown that clonus occurred in the presence of a lesion involving a large portion of the lateral corticospinal tract(2). This observation was based on the histopathological evaluation of specimens from patients with a lesion in the central nervous system (CNS). They reported that the frequency of clonus was constant in each muscle and the frequency of clonus did not show a tendency toward a change over time (2). Rapid onset exteroceptive stimulations in sufficient intensity can induce clonic discharge in the muscle and not only via type Ia afferent fibers (9). Painful stimuli and cold are the leading cutaneous stimuli giving rise to and sustaining clonus. The cutaneous stimulation of the unaffected side can also produce clonus. The stimulations causing polysynaptic flexor or extensor reflexes are susceptible to produce clonus via nonspecific descending facilitations produced by the Jendrassik maneuver. The stimuli activating these pathways can stop clonus (5). Clonus may even occur in the absence of any movement in the extremity. The amplitude of clonus induced and sustained by stretch can decrease and become attenuated over time. Cutaneous sti- 20

27 Boyraz et al. Clinical aspect of clonus mulation triggered by scratching skin over the muscle will provide sufficient input to the spinal cord to maintain the amplitude of clonus (2). Bernhard and Therman showed that proprioceptive inputs generated with the movement of the limbs trigger rhythmic discharges from the motor units in decerebrate cats (10). Gottlieb and Agarwal showed that pharmacological agents increasing the discharge from stretched muscle fibers could produce clonus in healthy individuals. They reported that clonus in normal individuals shares common features with those in spastic patients and possesses a limited band of frequency, and it is independent from the loading on the extremity (11).Struppler observed these findings using iv succinylcholine injection, and Marsden, Meadows, and Hodgson used IV adrenalin injections (12,13). CLONUS MECHANISM The exact mechanism of clonus remains unclear. Two different hypotheses have been asserted regarding the development of clonus. The most widely accepted explanation since the pioneering studies by Denny-Brown ( ) is that hyperactive stretch reflexes in clonus are caused by self-excitation (14). Szumski et al. observed that a few beats of clonus occurred after tendon tap in the wrist flexors and clonus was sustained by the Jendrassik maneuver. They concluded that the spindles involved in clonus were abnormally sensitive and dynamic fusimotor neurons were important motor neurons involved in eliciting clonus (2). Szumski and Hagbarth showed the discharge of Ia afferent fibers before clonic bursts on electromyographi (EMG) and these discharges were not activated during muscle contraction. They concluded that muscle spindles were stretched during muscle relaxation and repeated oscillatory movement elicited EMG activity (5). Janell et al. reported that clonus would not be elicited if reflex responses were not generated against a stretch (3). Rack et al. observed that the frequency of soleus EMG activity could be regulated by loading and loaded oscillatory movements in spastic patients, and they concluded that self-sustaining oscillation of stretch reflex pathway resulted in clonus. In spastic subjects, motoneuron firing threshold may decrease to a level in which the spindle afferent output elicited during muscle lengthening is now sufficient to reach threshold for motoneuron firing (16). This shift in threshold can be thought of as an effective increase in the feedback gain since the same amount of afferent input in the spastic case will result in higher motoneuron activation than in a normal threshold level (17,18). According to control theory, instability may arise in a system with a high feedback gain and significant delays, conditions both present in the ankle muscles of spastic subjects (13). Hidler et al have clearly shown that both movement frequency and EMG burst frequency can be altered, and so we can only speculate that the loads used in the mentioned studies were not sufficient to perturb the system onto a different limit cycle orbit (19). Clonus was of shorter duration when more muscles were activated. In contrast, clonus was persistent when EMG activity was largely confined to the synergistic triceps surae muscles (20). Iansek found a linear relationship between the frequency of clonus and the distance between spinal cord and the muscle. Mathematically, reflex oscillation latency was found to be predominant in determining frequency, and if there was a central spinal pacemaker, it would predict the frequency of clonus regardless of the length of the reflex pathway (21). The findings that are parallel to pure peripheral self-re-excitation mechanisms are preferably coupled with high reflex arc gain (shift in threshold of motoneuron activation). Possible factors involved in the regulation of clonus frequency are length of reflex arc; the frequency of clonus can increase with the decrease in activation latency of la afferent fibers; factors such as the mass and viscosity of the muscles can affect the frequency of clonus by changing the activity latency of spindle relaxation (21). The idea that central mechanisms may be involved was not adopted in observations where clonus was attributed to peripheral mechanisms. The frequency of clonus changed by changing the mechanical load on the joint. The rhythmic oscillations occurring in stretched muscles in some animal preparations are assumed to be analogous to clonus, and these oscillations were inhibited by the blockade of peripheral afferent fibers (22). Unsuccessful utilization of the signals from muscle spindles and Golgi tendon organs complicates imaging and regulation of muscle length 21

28 Medicinski Glasnik, Volume 12, Number 1, February 2015 and power and autogenic reflex pathways play a major role in motor control in humans (4,23,24). The stretch reflex is a primary autogenic reflex and the negative feedback arc is the first line of active resistance when the body interacts with the environment. In normal conditions, the gains in reflex pathways were shown to be minimal. The functional behavior of the reflexes changes significantly with increasing excitability of motor neurons. It is believed that clonus with rhythmic or oscillatory contractions could occur in distal limbs where there is a change in the excitability of CNS associated with concurrent neurological disorders and when there is an increased tendency toward instability (2,4,23). Hidler et al. hypothesized the coexistence of both conditions for the occurrence of clonus: reflex pathway delay (involving distal extremity muscles, displaying slow twitch properties), and increasing motor neuron excitability (decrease in motor neuron excitability threshold). These two phenomena disrupt the stability of motor neurons. The high incidence of orderly motor unit recruitment in human skeletal muscles that, due to spinal trauma, are under no voluntary control from higher centers suggests that spinal systems also dominate the stereotyped excitation of human motoneurons during clonus. Thus, any changes in spinal neuron excitability, synaptic inputs, or muscle properties due to injury were appropriate to preserve an orderly pattern of motor unit recruitment, as found during voluntary contractions of muscles innervated from the level of injury (12,13). Orderly recruitment of motor units during clonus is ordered by size of unit excitability. Afferent activity from the previous contraction and the level of spinal excitation were adequate to recruit most of the units during every contraction but were insufficient to increase their firing rates. None of these peripheral or spinal factors were sufficient to markedly disrupt the recruitment order of pairs of motor units during clonus (4). The reason for this lengthened delay in spasticity may be the sensitivity of muscle spindles or changes in the passive features of the muscle. Increase of viscoelasticity of passive tissues enlarges the clonus receptive area (shaded); that is, it increases the amount of combinations of motor unit pool threshold and gain that will result in clonus (24).Cook et al. showed that ankle dorsi-flexor remained reactively silent during the emergence of clonus, and the blockade of the peroneus communis nerve did not affect the amplitude and duration of oscillation (25). The character of the input-output relationship in motor neurons can be defined by the Gaussian cumulative distribution function. Accordingly, the synaptic current scale is linearly correlated with the spindle firing rate. The functional pattern of motor neurons is determined by both motor unit recruitment and modulation rate. The single major reason for the delay in the generation of the monosynaptic reflex arc is neural conduction time in the reflex pathway. The delays in the negative feedback pathway possess a destabilizing effect on the behavior of the system. The frequency of oscillation decreases with increasing conduction delay (1). Another reason for the delay in the reflex pathway is the contractile features of the muscle. These delays are caused by Ca dynamics, myofilament cross bridges, elasticity of the muscle fibers, and tendon compliance. In pathological conditions, slow-twitch muscle fibers can be replaced by fast-twitch muscle fibers. The input-output behavior in the muscle is similar to that in low pass filtering. Low pass filtering in the muscle or the delays in the reflex pathway due to conduction delays will affect reflex stability (24). It is believed that clonus and spasticity share a common pathway; therefore, their co-occurrence on most, if not all, occasions is not surprising. The neuroaxial lesions such as stroke or spinal cord injury result in a net inhibition in segmental neurons. The balance of synaptic input to the motor neurons would change in favor of net excitation. It was reported that the muscle was continuously active due to on-off signal during rotational movement, and high tonic activity can be responsible for this condition. The oscillatory behavior observed in clonus is similar to closed arc oscillations seen in negative feedback control encompassing high feedback gains accompanied by significant delays. Hagbarth et al. recorded medial gastrocnemius Ia afferent muscle spindle discharges during clonus caused by the stretch before muscle stretch and not during muscle activation. While spindle activity is expected during muscle stretch, the 22

29 Boyraz et al. Clinical aspect of clonus observation of muscle spindle activation in medial gastrocnemius is not surprising during clonus elicited by fast stretch of PF; however, it was suggested that this would not be proven if spindle activation directly elicited or maintained clonus. No positive correlation was found between the number and frequency of power and spindle discharges following clonic EMG bursts. They reported that hyperexcitability of the stretch reflex is not centrally related for certain (26). If repeated muscle stretch and the resulting muscle spindle activation elicit clonus, tibialis anterior muscle spindle activity and subsequent EMG activity should have been formed in a pattern following the activity of medial gastrocnemius. Hagbarth et al. did not record this from the tibialis anterior (26). Janell et al. suggested that the synchronous discharge of muscle spindle afferents of antagonistic muscles would be unlikely during DF-PF of the ankle joint, although muscle spindle activation was not measured directly (3). When synchronous activation of plantar flexors and tibialis anterior during clonus was demonstrated, the inconsistency with the origin of the stretch reflex was not taken into consideration. Cook et al reported tibialis anterior EMG activity synchronous with PF that could not be eliminated by tibialis anterior nerve blockade, and they concluded that the observed tibialis anterior EMG activity could have been caused by crossconvergence due to PF (27). In addition, successive plantar-dorsiflexion EMG was not observed during clonus. They concluded that antagonistic activity was not necessary to elicit clonus and it was attributed to the repeated reflex stretch of plantar flexors. According to the results of the stimulation data, the investigators ruled out tibialis anterior and supported repeated stretch reflex as the cause of clonus (l). Cook et al. provided alternative explanations, suggesting that the activity observed in tibialis anterior was not caused by plantar flexors, but may have been caused by incomplete nerve blockade (19). Hidler and Rymer observed tibialis anterior EMG activity synchronous with soleus and medial gastrocnemius activity during clonus, and they attributed tibialis anterior EMG activity to shortening reaction. The shortening reaction is defined as the EMG response in the shortened muscle commonly observed in patients with Parkinson s disease. The shortening reaction in the ankle has been rarely observed in patients with first motor syndrome (12%) and the rate was uncommonly compared to disabled subjects (23). Attempts have been made to change the frequency of clonic oscillatory burst patterns in order to test the stretch reflex and central oscillatory theories. If clonus correlates with the stretch, externally applied motion frequency affects the frequency of clonus. Rack et al. observed rhythmic EMG activity with various frequencies in response to ankle loading (16). Hidler and Rymer reported that the increase in the applied moment loading produced a greater stretch on the plantar flexors, and this resulted in early EMG response with higher frequency (1). It was reported that clonus could be re-established (reset) with the stimulation of the soleus H-reflex in the time frame between two successive clonic beats (28). Peripheral events are estimated to regulate afferent output, and such observations are commonly reported. On the other hand, there is no sufficient evidence to suggest that clonic EMG was only caused by the recurrent stretch reflex. The observation of oscillatory EMG activity in the absence of synchronous repetitive peripheral inputs supports the role of oscillatory neurons in the spinal cord that can be activated by many afferent events (19). Another alternative explanation for clonus is central generator activity that arises as a consequence of appropriate peripheral events and produces rhythmic stimulation of the lower motor neurons (9). Walsh reported that clonic EMG frequencies of plantar flexors remained unchanged (14). In their study, Dimitrijevic et al. evaluated clonus EMG records, ankle angle, and pressure applied to the soles, and they investigated whether the silent period between two beats of clonus was caused by loading on the spindles or by the central refractory period (2). The attempts failed to change the frequency of clonus. The refractory period was approximately 100 msec and the excitatory period was approximately 60 msec, and accordingly cyclic changes in centrally regulated excitability constitute the basis for clonus and determine its frequency. They indicated that periodicity could be modified only for a short period by Ia inputs while transforming from the refractory period to excitatory period (2). According 23

30 Medicinski Glasnik, Volume 12, Number 1, February 2015 to Dimitrijevic, the central generator is a transistor providing a functional organization, and it is made up of segmental reflex activity influenced by peripheral, propriospinal, suprasegmental mechanisms, proprioceptive volleys from the limb, and the movement of the muscle and parts of the limb. The features of the central generator include cyclic, regular activation at a fixed phase (2). Brune and Schenck examined H-reflex volleys between two clonic bursts and reported a refractory period between EMG bursts. They attributed the cessation of motor neuron activity at the beginning of the silent period to the refractory state of the motor neurons with the inhibition of Renshaw cells after firing and lack of stimulation from spindle afferents at the rest of the period (29). Strupler, Burg, and Erbel suggested that recurrent inhibition produced by Renshaw cells and autogenic inhibition by Golgi afferents played a role in the refractory phase of the motor neurons and not only spindle unloading (30). Nathan measured the refractory period only in the triceps surae muscle ( ms). He proposed that this period may differ for other muscle groups with different central stretch reflex organizations, thereby resulting in different maximum clonus frequencies (31). Wachholder and Altenburger showed that the latency of the first clonic beat was same as the stretch reflex. This time relationship did not persist in sustained clonus. Therefore, they expressed that clonus was triggered by the stretch and rhythmic discharge was maintained by the central factors (32). The characteristic feature of clonus is synchronous motor discharge. It was reported that synchronous discharge occurred despite the input from asynchronous spindles to the clonus, muscle geometry, and the contribution of peripheral muscle factors such as the relaxation rate of the muscle (31). This indicates that the reflex is rigidly controlled over time and in the spatial extent in the motor unit pool. It was asserted that the discrepancy between peripheral factors and synchronized motor unit response indicates that central mechanisms play a major role (3,5). It was reported that peripheral input is essential for the re-activation of cyclic bursts and the overall activity is controlled by spinal mechanisms. The intermittent discharge of clonus is suggested to be caused by the periods of refractoriness, which is due to the inhibition of motor neurons and/or interneurons. The prolonged period of refractoriness is caused by Renshaw cells. The results of Janell et al.and Walsh support the interaction between many peripheral events and central mechanisms to elicit clonus (3,33). Despite the lack of an input that would produce a stretch in the muscles, bilateral clonic EMG activity was prominent in the proximal and distal limbs in the standing position without bearing weight. Clonus has been observed in the hamstring muscles following the development of clonus in the vastus medialis, vastus lateralis, and rectus femoris muscles while loading in the standing position and clinically after clonus in the ankle. The coactivation of the muscles between the limbs may have played a role after spinal cord injury, but the co-activation of antagonistic muscles in the same limbs also point to the convergence of the interneurons. A synchronous and bilateral muscle stretch in agonist and antagonist muscles seems unlikely (3). TREATMENT OF CLONUS Clonus can be treated by using baclofen, applying cold, botox or phenol injections (7, 9, 34-37). Several studies in the literature have reported that centrally active antispastic drugs do not have significant effects on clonus; however, some studies have shown that baclofen has more dramatic effects than other drugs. Tizanidine selectively blocks group II pathways, which have a role in spasticity but has no effect on clonus (38-41). In a study by Bassett and Lake on patients with upper motor neuron lesions, spasticity and clonus both decreased with the application of wet towels wrapped in crushed ice and with submergence in cold water (42). Measurable functional improvement has been reported in association with decreased spasticity after cold application. Knutsson who studied the kinematics of spastic gait before and after cold application, reported that a decrease in spasticity of antagonistic spastic plantar flexors paralleled an increase in the late oscillation phase during dorsiflexion (43). Hedenberg on the other hand, tested upper extremity functions of patients with hemiplegia before and after submergence in cold water and after cold application and noted significant improvements in functional capacities (44). Dimitrijevic et al. reported no 24

31 Boyraz et al. Clinical aspect of clonus changes in clonus frequencies with cold application (2). Miglietta showed that the longer the period of cold application, the longer it took for clonus to recur. The average periods of recurrence of clonus observed after 10, 20, and 30 minutes of cold application were 28 (range, 15 to 45 minutes), 48 (range, 10 minutes to 2 hours), and 85 minutes (20 minutes to 6 hours), respectively (40,45). Cold application induced prolonged inhibitory effects on clonus. In response to cryotherapy, Boyraz et al. showed persistence of H and T reflexes with prolonged latencies, as well as decreases in the stimulation threshold and H/M ratio, but with a marked inhibitory effect on clonus. There is a persistence of ankle clonus inhibition even after a cooled muscle has returned to body temperature. This phenomenon could be explained by an increase in the threshold of the nerve fiber and/or a relatively prolonged refractory period. The prolonged effect of the cold supports the presence of spinal neuroplasticity and adaptation in individuals with neurologic impairments (35). Thevenon showed that clonus affected the first metatarsal, since it was selectively triggered by extension of the first metatarsophalangeal joint. To treat clonus, they applied injecting botulinum toxin into the peroneus muscles but failed. To stop clonus through selective neurotomy of the gastrocnemius and soleus, Thevenon performed neurotomy of the branches of the superficial fibular nerve that innervated the peroneus brevis and peroneus longus. After the surgery, clonus of the first metatarsal was no longer observed (35). Botulinum toxin has a role in treating ankle clonus in neurological patients, where it interferes in gait and may improve walking speed and level of dependence on others (33). The treatment of clonus and spasticity may be obtained by using centrally and peripherally effective mechanisms simultaneously. Clonus was considered to be a common presentation of the intrinsic oscillation of the spinal neural network after a reduction in sensorial input related to loading and chronic loss of supraspinal input. The spinal networks can be activated by numerous stimulations including interventions during voluntary movements, nociceptive synapses, and cutaneous synapses. Due to the presence of limited motor pools to elicit voluntary movements after severe spinal cord injury, the attempts mostly result in generalized motor patterns. In most cases with spinal cord injury, chronic unloading occurs not only as a result of the absence of supraspinal input, but also due to a lack of stepping and standing. Synchronous oscillatory motor output could be a re-organization of the neural network as a response to chronically changing afferent and supraspinal inputs, and therefore the same stimulus before injury did not cause the activation of the entire network. It must be investigated as to whether repetitive afferent information regarding stepping would re-modify the clonic motor firing pattern. Better results in the treatment of clonus and spasticity may be obtained by using centrally and peripherally effective mechanisms simultaneously. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interest: none to declare. REFERENCES 1. Hidler JM, Rymer WZ. A simulation study of reflex instability in spasticity: origins of clonus. IEEE Trans Rehabil Eng 1999; 7: Dimitrijevic MR, Nathan PW, Sherwood AM. Clonus: the role of central mechanisms. J Neurol Neurosurg Psychiatry 1980; 43: Beres-Jones JA, Johnson TD, Harkema SJ. Clonus after human spinal cord injury cannot be attributed solely to recurrent muscle-tendon stretch. Exp Brain Res 2003; 149: Wallace DM, Ross BH, Thomas CK. Motor unit behavior during clonus. J Appl Physiol (1985) 2005; 99: Szumski AJ, Burg D, Struppler A, Velho F. Activity of muscle spindles during muscle twitch and clonus in normal and spastic human subjects. Electroencephalogr Clin Neurophysiol 1974; 37: Iodice R, Manganelli F, Dubbioso R, Ruggiero L, Santoro L. Teaching video neuroimages: clonus of the lower jaw: an old sign that comes back. Neurology 2014; 82:e Biotti D, Vighetto A. Images in clinical medicine. Upper limb clonus. N Engl J Med 2013; 369:e Walsh EG, Wright GW. Patellar clonus: an autonomous central generator. J Neurol Neurosurg Psychiatry 1987; 50:

32 Medicinski Glasnik, Volume 12, Number 1, February Uysal H, Boyraz I, Yagcioglu S, Oktay F, Kafali P, Tonuk E. Ankle clonus and its relationship with the medium-latency reflex response of the soleus by peroneal nerve stimulation. J Electromyogr Kinesiol 2011; 21: Bernhard CG, Therman PO. Rhythmical activity of motor units in myotatic reflexes. Acta Physiol Scand 1947; 14: Gottlieb GL, Agarwal GC. Physiological clonus in man. Exp Neurol 1977; 54: Struppler A, Schulte F, Scheininger R, Kukku M. Eine elektromyographische Untersuchung bei Spastik und Rigor. Journal of Neurology 1961; 183: Marsden CD, Meadows JC, Hodgson HJ. Observations on the reflex response to muscle vibration in man and its voluntary control. Brain 1969; 92: Walsh EG. Clonus: beats provoked by the application of a rhythmic force. J Neurol Neurosurg Psychiatry 1976; 39: Beres-Jones JA, Johnson TD, Harkema SJ. Clonus after human spinal cord injury cannot be attributed solely to recurrent muscle-tendon stretch. 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33 ORIGINAL ARTICLE Human dental pulp mesenchymal stem cells isolation and osteoblast differentiation Moustafa Alkhalil 1, Amer Smajilagić 1, Amira Redžić 2 1 Department for Oral and Cranio-Maxillo-Facial Surgery, Hamad Medical Corporation, Doha, Qatar, Weill Cornell Medical College, Doha, Qatar 2 Institute for Biology and Human Genetic, School of Medicine University of Sarajevo, Sarajevo, Bosnia and Herzegovina ABSTRACT Aim This study was focused on the isolation and characterization of mesenchymal stem cells (MSCs) from human dental pulp (DPSC). Corresponding author: Amer Smajilagić Department for Oral and Cranio-Maxillofacial Surgery, Hamad Medical Corporation P. B. 3050, Doha, Qatar Phone: ; Fax: ; smajilagica@gmail.com Original submission: 10 September 2014; Revised submission: 22 September 2014; Accepted: 29 October Methods The study was performed in the Department for Oral and Cranio-Maxillo- Facial Surgey Hamad Medical Corporation, Doha, Qatar and Weill Cornell Medical Colleague Doha, Qatar, in period Dental pulp was extracted from premolars and third molars of 19 healthy patients. The pulp was digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase for 1 hour at 37C. After filtration, cells were cultured in Dulbecco s Modified Eagle Medium (DMEM Low Glucoses) with 20% Fetal Bovine Serum (FBS), 2mM L-glutamine and antibiotics (100 U/ ml penicillin, 100 ug/ml streptomycin) at 37 C under 5% CO2. Cultures were treated with osteoinductive medium for differentiation MSC in to the osteoblast cell line. Staining with Alizarin red were used for the detection of the osteoblast production and calcification new formed tissue. Results On the total of three out of 19 patients it was possible to isolate DPMSCs after 2 to 3 weeks: in one patient it was not possible to expand MSCs because of infection, and in other two patients positive Alizarin red staining reaction showed osteogenic differentiation capability and strong mineralization in vitro. Conclusion The main advantage of using DPSC is absence of morbidity. MSCs could be isolated noninvasively from teeth, routinely extracted in the clinic and discarded as medical waste. Standardization of clinical and laboratory protocols for DPMSCs isolation and team work coordination could lead to significantly improved result. Key words: regenerative medicine, bone, tissue engineering, osteoblast Med Glas (Zenica) 2015; 12(1):

34 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ. Those involved in regenerative medicine place more emphasis on the use of Stem Cells to produce tissues (1). Stem cells have two main properties: pluripotency, e.g. formation of all ectoderm, endoderm and mesoderm cells layers, and self renewal (2,3). Depending on the origin there are embryonic stem cells and somatic stem cells which include mesenchymal stem cells, hematopoietic stem cells and neural stem cells. Mesenchymal stem cells (MSCs) are able to differentiate into the different cell types such as osteoblast, chondroblast myoblast, adipoblast and even beta- pancreatic cells (4). These cells could be isolated from the variety of tissues and expanded in vitro. Such produced MSC may have the use as valuable tools for regenerative medicine (5,6). Mesenchymal stem cells of human origin were isolated and have been used for the preclinical studies and clinical trials (7,8). The most common source is bone marrow (9-11), fat tissue (12,13) and umbilical cord blood (14-16). However, isolation of the MSCs from those sources is performed by aspiration and is often an invasive and painful procedure for donors, associated with morbidity. Umbilical cord cells are only available immediately after the delivery so they have limited availability. Because of those disadvantages, identification of other sources for MSCs isolations is of great importance (17). This study is focused on the isolation and characterization of MSCs from human dental pulp (DPSC) (18). An important issue is presented indicating that there is a significant diversion in the MSCs isolation protocols from different tissues (19), which makes it difficult to compare results between laboratories and verify success of clinical trials. The aim of this study was isolation and cultivation of MSCs from the human dental pulp as a future model for regenerative medicine application with the intention of establishing unique and effective clinical and laboratory protocol, characterization and differentiation in the osteoblast cells phenotype. MATERIALS AND METHODS For isolation of human dental pulp mesenchymal stem cells (DPMSCs), the extracted teeth of the total of 19 patients treated at the Department for Oral and Cranio-Maxillofacial Surgery, Hamad Medical Corporation, Doha, Qatar were used. In collaboration with the Laboratory for Stem Cells Research Weill Cornel Medical College, Doha, Qatar, the study was performed in the period All patients were healthy based on documented evidence found in their medical files. All patients or their legal representatives signed an informed consent form, following the guidelines approved by the Ethic Committee of the Medical Research Center, Hamad Medical Corporation, Doha, Qatar. All removed teeth used in the study were intact and medically indicated for the extraction, either for orthodontic reasons or causing health problems. Teeth were removed using local anesthesia with 2% xylocain/adrenaline, under standard conditions, and after the removal they were cleaned with 70% alcohol swab topically. Extracted teeth were placed into the sterile plastic tubes, and tubes were put into ice, stored between 0-4 C, for 6-18 hours overnight. This time delay was necessary whilst waiting for transportation to the stem cell laboratory and isolation procedures. All the teeth had fully developed roots and using Luers forceps, in the laboratory the roots were separated from the crown. After the separation an extirpation needle was used to remove dental pulp. After extirpation pulp tissues were digested in a solution contest from 4 ml of DMEM/low Glc, 100U/mL penicillin, 100 ug/ml streptomycin, and 3 mg/ml enzymes collagenase type I and 4 mg/ml dispasae for 60 minutes et 37 C. Then, cells were digested in the media and centrifuged for 10 min at 1200 rpm. The pellet was washed twice in PBS, resuspended in to the 6 ml cell culture medium and placed in T25 flasks. The flasks were incubated in 5% CO 2 at 37 C. The medium was changed twice weekly. The cells cultivate medium were contest of: DMEM/Low Glc with 20% fetal bovine serum (FBS) 2 mm L-glutamine, 100U/mL penicillin and 100 ug/ ml streptomycin. When the cells in 24 well plate reached 50-60% confluency, the medium in each flask was replaced using fresh osteogenic differentiation media contest: ascorbic 28

35 Alkhalil et al. h-mscs dental pulp isolation acid 50 ug/ml, b-glycerol phosphate 10 mm, Dexamethason 10-7 in DMEM/low Glu and 10% FBS, 2mM L-glutamine, 100U/mL penicillin and 100ug/mL streptomycin. Osteogenic medium was changed twice weekly, and after 2 weeks the cells were differentiated and stained with 2% Alizarin, for the detection of osteoblast production and calcification. The Alizarin method for the staining was performed according the procedure: washing the cells 2x with PBS, 10 min with 70% ice cold ehanol, washing with distilled H 2 O, application 10 min Alizarin red at RT, 5 times washing with distilled H 2 O, add PBS and after 15 minute checking under a microscope to see the calcification. RESULTS Dental pulps were extirpated from the extracted teeth, respectively for each individual patient from the total of 19 patients included in study (6 males, 13 females), for the purpose of isolation and cultivation of MSCs. In 16 of them permanent premolars teeth were extracted, according to orthodontist indication in therapeutic purpose, and in the other three patients permanent molars were extracted due to health reasons. The average age of the patients was 13 years (range 9-22 years). From the total of 19 patients, it was possible to detect and isolate MSCs in three of them. The MSCs were detected by visualization colonies on electronic microscope and their specific spherical like cluster shape (Figure 1). The time between extirpation pulp tissue and DPMSCs isolation was variable. In one patient time the duration between isolation and detection of MSCs was 7 days, on another it took 13 days, and on the third 24 days. Two patients were females and one patient was male (average age of 13 years). After visualization, colonies started to grow rapidly in the cell media. After isolation in one patient, an infection appeared and destroyed all the cells. The other two patients (one male, one female) dental pulps were extirpated from the extracted premolars, and showed DPMSCs isolation after 2 weeks, colonies were expanded and when they reached confluences 50-60% they were exposed to the osteogenic differentiation media for next two weeks (Figure 2). A) B) Figure 2. A) Expanded stem cells in culture, single pulp extracted premolar tooth, day 24 after isolation on male patient; B) Expanded stem cells in culture, single pulp extracted premolar tooth, day 30 after isolation on female patient. On both isolated cultures, cells are adherent, elongated in spindle shape with thin expansion (phase contrast microscopy) (Mehteb M, 2010) Figure 1. Colonies of the aggregated stem cells on the 13 th day of culture in a 13-year old male patient, isolated from single dental pulp of the removed premolar tooth. Cells were adherent and formed aggregates. In total 7 colonies of stem cells were detected (Mehteb M, 2010) Two weeks after the exposure to osteoinductive factors, Alizarin red staining of DPMSCs culture demonstrated positive reaction. Long term growth cultures of DPMSCs (between 4-6 weeks) 29

36 Medicinski Glasnik, Volume 12, Number 1, February 2015 in vitro, demonstrated the capacity to form Alizarin red-positive mineralization nodules with high levels of calcium (Figure 3). Figure 3. Adherent layers of cultured DPMSCs are shown (with Alizarin Red staining as a measure of calcium accumulation after induction with Ascorbic acid 50ug/ml, b-glycerol phosphate 10mM, Dexamethason 10-7 in DMEM/low Glu and 10% FBS, 2mM L-glutamine), capacity to form Alizarin Red-positive condensed nodules with high levels of calcium (Mehteb M, 2010) DISCUSSION Human tissues have different potential for regenerative properties. It appears that stem cells and their biology may play an integral role in that regenerative process. Multipotent mesenchymal stem cells (MSCs) were first discovered by Friedenstein 1976 (9), who isolated them from bone marrow. Based on this protocol, MSCs from other tissues were searched for isolation and cultivation. Several loci within human body were isolated as a source for the MSCs isolation: bone marrow, cartilage tissue, fat tissue, and umbilical cord blood (11, 13-15, 20). However, accessibility can be limiting. Mesenchumal stem cells from dental tissue were isolated by Gronthoss 2000 (18) from the pulp tissue. Those cells showed capability to differentiate into the osteoblasts, odontoblasts, adipocytes, and neural cells (21). The results of this study have shown ability of ex vivo expanded human DPMSCs taken from permanent teeth to differentiate into osteoblasts and produce in vitro a mineralized bone like tissue, detected by using a specific calcium deposit staining Alizarin red, similar to some of the previous research (18,19,21). The aim of this study was to isolate (from each individual) DPMSCs as autologous cells model, potential clinical tools which can be used as alternative to conventional medicine, free of any immune response and no ethical dilemma that we have in embriogenic stem cells research. For example, as supplement for bone grafting in conventional surgery, or for use in any clinical situations where the regenerative process is damaged (22). In systematic diseases, such as osteoporosis, therapies are mostly oriented towards suppression osteoclast function but no one or very few are oriented towards osteoblast proliferation (23). Comparing with isolation from other sources of MSCs, DPMSCs are easily accessible tissue resource (24). Most important advantage of this MSCs source is absence of morbidity and no need for additional surgical procedures. Harvesting process of donor tissue is done when a patient comes to have a tooth removed for health or therapeutic reasons, and instead of throwing tooth in the garbage, it should be placed in the process of MSCs isolation and cultivation. Such isolated DPMSCs could be also cryo-preserved and stored for future treatment of that person if required (25). Comparing with DPMSCs, an umbilical cord blood and amniotic fluid are limited MSCs sources, as they are available only after delivery (25) Dental pulp as MSCs source offers multiple opportunities for their isolation throughout life. Some research indicates that the DPMSCs are detectable in humans up to the age of 30 years, which is in accordance with results of this study where the average age of successful isolations was 13 years (26-28). Some investigations show that stem cells can be obtained from the dental pulps of subjects between 30 and 45 years of age using specific antibodies for stromal stem cells (29). Our results also indicate that h-dpm- SCs possess high proliferation ability and great potential for isolation of large numbers of cells, suggesting that they are a useful source for stem cell based therapy. In conclusion, individual autologous stem cells from dental pulp were isolated in only three out of 19 patients, and in two of them osteoblast cells line and calcification tissue were produced. Main disadvantages of this study include the low rate of the DPMSCs isolation and a long time period between pulp tissue isolation and DPMSCs visualization and cultivation. Our findings suggest the main reason for such results was the long time between tooth extraction and 30

37 Alkhalil et al. h-mscs dental pulp isolation pulp tissue extirpation for DPMSCs isolation. Probably, the overnight delay of more than 10 hours could lead to the MSCs and other cells dying. Even that result of 15% successful isolation in such conditions indicates that dental tissue is a great potential reservoir of stem cells, with a high rate of survival. Improvement in the standardization of clinical and laboratory protocols, and better coordination and team work could significantly improve this process. FUNDING This research was supported by the Medical Research Office, Hamad Medical Corporation, Doha, Qatar (Project Project No: 8121/08 Mesenchymal Stem Cells Isolations and Cultivation as Experimental Bone Tissue Engineering System ) and Qatar, Doha, Weill Cornel Medical School, Laboratory for Stem Cells Research and Senior Researcher Mehteb Meleki. TRANSPARENCY DECLARATIONS Competing interest: none to declare. REFERENCES 1. Arthur A, Zannetino A, Gronthos S. The Therapeutic applications of multipotential m esenchymal/stromal stem cells in skeletal tissue repair. J Cell Physiol 2009; 218: Kassem M. Mesenchymal stem cells: biological characteristics and potential clinical applications. Cloning Stem Cells 2004; 6: Abdallah BM, Kassem M. Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther 2008; 15: Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 2004; 8: Graziano A, d Aquino R, Laino G. Papaccio G. Dental pulp stem cells. A promising tool for bone regeneration. Stem Cell Rev 2008; 4: Iohara K, Zheng L, Wake H, Ito M, Nabekura J, Wakita H, Iohara K, Zheng L, Wake H, Ito M, Nabekura J, Wakita H, Nakamura H, Into T, Matsushita K, Nakashima M. A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp. Stem Cells 2008; 26: Yamada Y, Ueda M, Hibi H, Nagasaka T. Translational research for injectable tissue- engineered bone regeneration using mesenchymal stem cells and platelet-rich plasma-from basic research to clinical case study. Cell Transplant 2004; 13: Yamada Y, Nakamura S, Ito K, Kohgo T, Hibi H, Nagasaka T. Injectable tissue-engineered bone using autogenous bone marrow-derived stromal cells for maxillary sinus augmentation: clinical application report from a 2-6-year follow-up. Tissue Eng Part A 2008; 14: Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 1976; 4: Caplan AI. Mesenchymal stem cells. J Orthop Res 1991; 9: Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997; 276: Mizuno H, Zuk PA, Zhu M, Lorenz HP, Benhaim P, Hedrick MH. Myogenic differentiation by human processed lipoaspirate cells. Plast Reconstr Surg 2002; 109: Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002; 13: Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells 2003; 21: Mareschi K, Biasin E, Piacibello W, Aglietta M, Madon E, Fagioli F. Isolation of human mesenchymal stem cells: bone marrow versus umbilical cord blood. Haematologica 2001; 86: In t Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings G Claas FH, Fibbe WE. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004; 22: Huang GT, Gronthos S, Shi S. Mesenchymal Stem Cells Derived from Dental Tissues vs. Those from Other Sources: Their Biology and Role in Regenerative Medicine. J Dent Res 2009; 88: Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 2000; 97: Gronthos S, Zannettino ACW. A method to isolate and purify human bone marrow stromal stem cells. In: Prockop DJ, Bunnell BA, Phinney DG, editors. Mesenchymal stem cells: methods and protocols. Totowa, NY: Humana Press, 2008; Young HE, Steele TA, Bray RA. Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec 2001; 264: R d Aquino R, Graziano A, Sampaolesi M, Lainol G, Pirozzi G, De Rosa, R d Aquino R, Graziano A, Sampaolesi M, Lainol G, Pirozzi G, De Rosa, Papaccio G. Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ 2007; 14: Graziano A, d Aquino R, Laino G, Papaccio G. Dental pulp stem cells: a promising tool for bone regeneration. Stem Cell Rev 2008; 4:65. 31

38 Medicinski Glasnik, Volume 12, Number 1, February Undale AH, Westendorf JJ, Yaszemski MJ, Khosla S. Mesenchymal stem cells for bone repair and metabolic bone diseases. Mayo Clin Proc 2009; 84: Nakamura S, Yamada Y, Katagiri W, Sugito T, Ito K, Ueda M. Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp. J Endod 2009; 35: Papaccio G, Graziano A, d Aquino R, Graziano MF, Pirozzi G, Menditti D, De Rosa A, Carinci F, Laino G Long-term cryopreservation of dental pulp stem cells (SBP- DPSCs) and their differentiated osteoblasts: a cell source for tissue repair. J Cell Physiol 2006; 208: Suchaneka J, Soukupb T, Visekb B, Ivancakova R, Kucerovac L, Mokryb J. Dental Pulp Stem Cells and their characterization. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2009; 53: Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A. Stem cell properties of human dental pulp stem cells. J Dent Res 2002; 81: Batouli S, Miura M, Brahim J, Tsutsui TW, Fisher LW, Gronthos Set, Batouli S, Miura M, Brahim J, Tsutsui TW, Fisher LW, Gronthos SRobey PG, Shi S. Comparison of stem-cell-mediated osteogenesis and dentinogenesis. J Dent Res 2003; 82: Laino G, D Aquino R, Graziano A, Lanza V, Carinci F, Naro F, Pirozzi G, Papaccio G. A new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue. J Bone Miner Res 2005; 20:

39 ORIGINAL ARTICLE Inflammatory cytokine gene polymorphism profiles in Turkish patients with ulcerative colitis İlhami Gök 1, Fahri Uçar 2, Orhan Ozgur 3 1 Department of Bioengineering, Faculty of Engineering & Architecture, Kafkas University Kars, 2 Departments of Medical Biology & Genetics, School of Medicine, Akdeniz University, Antalya, 3 Divisions of Gastroenterology, School of Medicine, Karadeniz Technical University, Trabzon; Turkey ABSTRACT Aim To investigate IL-1α, IL-1β, IL-1R, IL-4RA, TGF-β, TNF-α and IFN-γ, genes polymorphism in Turkish patients with ucerative colitis (UC). Methods An analysis was carried out at Trabzon Karadeniz Technical University Medicine Faculty Gastroenterology polyclinics between March 2005 and May 2011 on 51 patient with UC (cases) and 100 healthy individuals (controls). PCR-SSP and cytokine gene panel (Helderberg kits) based techniques for analysis of gene polymorphisms were used. Corresponding author: İlhami Gök Department of Bioengineering, Faculty of Engineering & Architecture, Kafkas University Kars, Turkey Phone: ; Fax: ; dnzgoki@gmail.com Original submission: 28 October 2014; Revised submission: 18 December 2014; Accepted: 05 January Results Changes in allelic frequencies of each of the investigated eight cytokine genes polymorphisms in patient with ulcerative colitis were found. Among the allelic genes analyzed here, the highest statistically significant change was observed in the position TNF-α -308 G/A (339.7%). The following increases were observed in IL-IR mspa T/C variation (179.4%), IFN-γ 5644A/T variation (77.4%), and in IL-1β -511T/C SNPs (35.9% ). In other analyzed genes, allelic changes were found to be decreasing for TGF- β codon10c / T (-71.9%), IL4RA G / A (-47.3 %), and for IL- 1α -889T / C (-37.7%). The lowest negative change (-25.9%) was observed in the allele frequency in IL- 1β 3962T / C (p<0.000). In addition, there were changes in genotypic frequencies investigated seven gene polymophic site and only one of cytokine gene IL-1β 3962TT/TC/CC was not changed. Conclusion Genes polymorphism is not itself the only determining factor for clinical diagnoses. However, it can be used in the clinical diagnosis of UC in order to determine the low level or high level variations in cytokine gene polymorphisms. Key words: colitis, ulcerative, cytokines, genetic polymorphisms, PCR-SSP, Turkey Med Glas (Zenica) 2015; 12(1):

40 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION Today, ulcerative colitis (UC), which as a common disease, brings serious problems particularly such as risk of developing toxic megacolon and colon cancer in developed countries, as well as in regions with high level of well-being (1). Although it can be seen at any age, it is rather known as a disease of young adults, and does not discriminate on gender (2). The prevalence of UC in normal population is % and the incidence / The incidence in the age group is 7-11/100,000, and the prevalence more than 15% in those over the age of sixty (1-3). Many environmental factors are involved in the pathogenesis of UC, but disease-related multifactorial genes have not yet been fully identified (4). In order to clarify the exact cause of UC inflammation, the genotypic level of cytokine genes should be studied indifferent population and we should increase UC samples. Field research on autoimmune diseases has been performed but data regarding the results are conflicting (4-6). The genes that encode proinflammatory cytokines could be activated in an important phase of relationships of autoimmune factors with the genes which might be susceptible to UC as in other genetic factors, contribution of mediator molecules to the functioning of the genes (5). It manages the changing level of transcription and translation in cytokine gene, and detecting the autoimmune response by cells may promote inflammation or can act as a trigger for inflammation (6). In this context, interleukin-1 receptor antagonist (IL-1RA) and interleukin-1 (IL-1) gene family that functions at opposite effects as regulator proinflammatory have come to the fore in the relationship between polymorphic genes and alleles especially in patients with UC (7-8). It can regulate the relationship with UC disease, the formation of IL-IRA in different parts of the gene encoding the allelic polymorphisms by these findings. Identification of a polymorphic region variable can modify the frequency of allele one and allele two with the number of repetitions (9). Also, the two alleles of the gene IL-IRA can affect (GM-CSF)-stimulated monocytes as colony-stimulating factor for in vitro production of granulocytes (10). A close connection of IL-1 gene family with another gene is known as IL-IRA encoding the endogenous gene antagonist (11-12). Proteins encoded by the genes interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β ) are known to play a role in UC formation. The causes of substantially individual differences of in vivo production of cytokines is that IL-1 protein domains are managed as hereditary (13). Another comment T cells differentiation in the formation of inflammation is synergistic participation of IL-1α production on polymorphisms (14). So far, the studies conducted in patients with UC have been considerable and there are conflicting results about the relationship between cytokine polymorphisms and the disease (15). Also, promoter regions of cytokine gene as inflammation trigger can be held responsible for the formation of inflammation (16). A common emphasis in linkage disequilibrium of autoimmune diseases as UC, alleles of the genes encoding IL-1α include existing heterogeneous polymorphic region (17). Conflicting results were found between disease groups with gene IL-1RA and healthy controls (18). Therefore, IL-1α, gene polymorphism, either alone or in combination with IL-lRA can be tested by including in the pathogenesis and by method cross-matching alleles and genotypes in patients with UC by IL-IRA genotype frequency analyses (19). Environmental agents that cause activation of inflammation in patients with UC, especially nuclear antigens, auto-reactive B cells and T cells can accelerate the formation of autoantibodies and may cause different immune response in different tissues (20). Basically, as the functioning of cytokine genes is variable, antagonist relationship was observed among IL-1α, tumor necrosis factor alpha (TNF-α), transforming growth factor (TGF-β) and interferon gamma (IFN-γ) (21). However, cytokine production varies during the stage of the disease (22). In UC animal models, TH1 cytokines vary as predominate at the onset of disease and then unstable in clinical stage in favor of the TH2 (19-21). The presence of different histone peptide stimulates T lymphocytes in patients with UC during clinical stage and can affect the stages of the disease. Then, at a certain point in time, Th1 cytokines are secreted, and in later periods, Th2 cytokine secretion comes to the fore (22). Most studies have shown higher TNF-α in UC patients and that secretion is high during illness (23). However, different research groups have observed that TNF is at normal and low levels in patients (8). In additi- 34

41 Gök et al. Cytokine genes in ulcerative colitis on, levels of IFN-γ have been identified as variable in patients, IFN- γ gamma levels vary depending on affected organ (6). Cytokine TGF-β at plasma levels in immune regulation systems were reduced in patients with UC (3,15). In recent years, the evidence gathered is that cytokine production is under genetic control. Polymorphisms which have the promoter and coding region in most cytokine genes are associated with the level of cytokine production (24). In this sense, accepted cytokines are cytokine genes as TGF-β, TNF-α, IFN polymorphisms and the IL-1, IL-4RA genes (8,18). Conflicting results and allele differences between populations and genetic variability are emphasized in observations in patients with UC (10). Among them, TNF-α 308 A/G polymorphism is associated with autoimmune diseases at a high rate (21,22). TNF-α production is considerably higher in Colombia populations with UC susceptibility (24). The aim of this study is to investigate various polymorphisms genes IL-1α, IL-1β, IL-1, IL-4RA, TGF-β, TNF-α and IFN-γ in Turkish population patients with UC by genomic techniques. PATIENTS AND METHOD Patients and genomic DNA extraction This research was conducted among the Black Sea Region population which geographically corresponds to the northern Turkey. Fifty-one individual cases in the study were selected from the patients applying to the Farabi Hospital Gastroenterology (School of Medicine), Karadeniz Technical University polyclinics as to whom the inflammatory bowel diagnosis was set, and 100 healthy individuals as control group (one patient controlled with two healthy individuals). The inflammatory bowel disease diagnosis was determined using the physical examination, radiological, endoscopic, and pathological test criteria according to signs and symptoms of the patients. The classifications of cases (UC) were made according to the settlement of the lesions in the digestive organs and behavior type of the illness in the inflammatory bowel patients. From each of 51 UC patients and 100 healthy individuals included to the study, 8 ml blood was taken to the EDTA tube. From the peripheral blood samples, the DNA isolation was conducted with the method of Salting-Out (25).The optical density of the concentration of DNA obtained in this stage was read in the spectrophotometer (Shimadzu, Japan) in 260 nm wave length. The isolated DNA samples were kept at -80 o C. The genomic analyses were completed in Farabi Hospital Hematology Department tissue typing Laboratory School of Medicine, Karadeniz Technical University. PCR-SSP genotyping PCR-SSP cytokine gene panel (Cytokine Kits, Heiderlberg, Germany for the determination of the cytokine gene polymorphisms was used in the determination of 16 polymorphic varieties of 7 different cytokines: interleukin-1alpha (IL-1α), interleukin-1beta (IL- 1β), interleukin-1reseptor (IL-1R), interleukin-4 anti-receptor (IL- 4RA), tumor growth factor beta (TGF-β), and tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). The Cytokine Kits gene is composed of 3 different subcomponents with the primers. For the PCR-SSP amplification of each individual, the master-mix and DNA mixture having total volume of ml Genomic DNA ( ng/ml) and Taq Pol (5U/m L) was prepared. The prepared master mixes were slightly mixed in the vortex and were distributed to 16 wells in the plate with the final volume being 10 m L for each patient. For the following ones given for the cytokine genes, the beginning denaturation was amplified at 94 o C for 2 min, denaturation was amplified at 94 o C for 10 sec, annealing-extension was amplified at 65 o C for 1 min 10 cycles, the final denaturation was amplified at 94 o C sec, annealing was amplified at 61 o C for 50 sec, the extension was Table1. Heidelberg Cytokine genotyping CTS-PCR-SSP Tray Kit Corresponding Specific Size of Cytokine genes am- PCR am- Alleles specificity and positions Alleles/ plicon plification genotype (bp) (bp Interleukin-1a T at pos -889 T bp Interleukin -1a C at pos -889 C bp Interleukin -1b T at pos T bp Interleukin -1b C at pos C bp Interleukin -1b C at pos -511 C bp Interleukin -1b T at pos -511 T bp Interleukin -1R T at pos mspa T bp Interleukin -1R C at pos mspa C bp Interleukin -4RA G at pos G bp Interleukin -4RA A at pos A bp Transforming T At pos kodon 10 growth factor -b TC bp Transforming growth factor -b C at pos kodon 10 C bp Tumor necrosis factor -a T at pos Gg bp Tumor necrosis factor -a A at pos -308 Ag bp Interferon - γ A at pos UTR5644 A bp Interferon -γ T at pos UTR5644 T bp pos, Bases G, C, T, A at position corresponding genotype or alleles 35

42 Medicinski Glasnik, Volume 12, Number 1, February 2015 amplified at 72 o C for 30 sec, 30 cycles and hold was amplified at 4 o C in indefinite PCR cycle programme (26). For the implementation of the amplified PCR products in the electrophoresis, 2% agarose gel was prepared. The samples were executed at 75V, 50 A for 35 minutes by being loaded to 16 well gel for each patient. The results were examined in the transilluminator at UV and the wells having double bands from the samples available in each well were accepted as positive and photographed. The changes in the cytokine gene polymorphisms of the patients were determined as a result of the comparison with the control groups within information provided in the chart in the Cytokine Kits system analysis as given in Table 1. In total, we studied 16 allelic locations of 8 different polymorphic regions of 7 cytokine genes in UC Statistical Analysis Allelic frequencies of all cytokine genes were estimated by allele counts and expressed as a percentage, Chi-square test (χ 2 ) with Yates correction, Odd s ratio, and relative risk. Frequencies were also tested for Hardy-Weinberg equilibrium (HWE) by calculating allele frequencies using the Pearson s Chi-square test (χ 2 ) with 1 freedom. Findings were considered significant with two-tailed Fisher s exact tests (p<0.001). RESULTS From the 51 patients, 27 (53%) were females and 24 (47%) were males, with the age ranging from 18 to 69 (mean 49.3 years). Of 100 healthy controls, 47 (47%) were females and 53 (53%) were males, with the age ranging from 18 to 65 (mean Table 2. Demographic characteristics and clinical features of patients with ulcerative colitis (UC) and healthy controls Clinical findings Ulcerative colitis (n=51) Controls (n=100) Sex (males/ females) (No of patients) 24/27 53/47 Age (years) (43.9) (18-65) Course of the diseases (years) mean (range) 2.3 (1-8) negative Disease location (distal part/ total colitis) (No of patients) 36/15 negative Disease severity (mild/severe) (No of patients) 41/10 negative 39.1 years). Evolution of the disease was 1-8 years. Distal colitis was present in 36 (70.5%) patients, and 15 (29.5%) patients were without distal colitis. According to clinical course of disease, in 41(42%) patients mild form was diagnosed, 10 (30%) patients were with severe continual activity. None of the patients received treatment with cyclosporine or infliximab (Table 2). In total, 8 sites of 16 different polymorphic regions of seven cytokine genes in UC patients were analyzed. Of the 8 sites examined, a total of all 8 (100%) sites in the UC patients showed the changes in allele frequencies compared to the control group with statistically significant differences. Among the allelic genes analyzed here, the highest statistically significant change was observed in the position TNF-α -308 G/A (339.7%). The following increases were observed in IL-IR mspa T/C variation (179.4%), IFN-γ 5644A/T variation (77.4%), and in IL-1β -511T/C SNPs (35.9% ). In the other analyzed genes, allelic changes were found to be decreasing for TGF- β codon10c / T (-71.9%), IL4RA G / A (-47.3 %), and for IL- 1α -889T / C ( -37.7%). The lowest negative change (-25.9%) was observed in the allele frequency in IL- 1β 3962T / C (p<0.000) (Table 3). Table 3. The allele frequencies of cytokine polymorphisms in ulcerative colitis (UC) patients and controls Cytokine Position Alleles No (%) of patients with ulcerative colitis (n=51) No (%) of healthy controls (n=100) p Odd ratio 95% CI IL-1-α -889 C 55 (53.92) 32 (32) T 47 (46.08) 68 (68) IL-1-β 3962 C 44 (43.14) 30 (30.00) T 58 (56.86) 70 (70.00) IL-1-β -511 C 54 (52.94) 67(67.00) T 48 (47.06) 33 (33.00) IL-1R mpsa C 37 (36.27) 79 (79.00) T 65 ( (21.00) IL-4RA 1902 A 45 (44.12) 20 (20.00) G 57 (55.88) 80 (80.00) TGF-β Codon 10-2 C 54 (52.94) 12 (12.00) T 48 (47.06) 88 (88.00) TNF-α -308 A 35 (34.31) 87 (87.00) G 67 (65.69) 13 (13.00) IFN-γ UTR5644 A 55 (53.92) 77 (77.00) T 47 (46.08) 23 (23.00)

43 Gök et al. Cytokine genes in ulcerative colitis Table 4.The genotypes frequencies of cytokine polymorphism in ulcerative colitis (UC) patients and controls Genotype distribution Cytokine Position Genotypes No (%) of patients with ulcerative colitis (n=51) No (%) of controls (n=100) p IL-1-α -889 IL-1-β -511 IL-1-β 3962 IL-1R mpsa IL-4RA 1902 TGF-β Codon 10-2 TNF-α -308 IFN-γ UTR5644 TT 17 (0.333) 46 (0.462) TC 13 (0.255) 44 (0.435) CC 21 (0.412) 10 (0.102) TT 16 (0.314) 11 (0.108) TC 16 (0.314) 44 (0.442) CC 19 (0.373) 45 (0.448) TT 18 (0.353) 49 (0.490) TC 22 (0.431) 42 (0.420) CC 11 (0.216) 9 (0.090) CC 6 (0.118) 62 (0.624) TT 20 (0.392) 5 (0.045) TC 25 ( (0.331) GG 16 (0.314) 64 (0.640) GA 25 (0.49) 32 (0.320) AA 10 (0.196) 4 (0.040) CC 23 (0.451) 2 (0.052) TC 8 (0.157) 21 (0.211) TT 20 (0.392) 77 (0.774) GG 23 (0.451) 1 (0.069) GA 21 (0.412) 23 (0.226) AA 7 (0.137) 76 (0.756) AA 10 (0.196) 59 (0.592) AT 35 (0.686) 36 (0.355) TT 6 (0.118) 5 (0.052) For the genotypic polymorphisms of eight gene regions examined, there were statistically significant changes in seven genes: interleukin-1alpha (IL-1α), interleukin-1reseptor (IL-1R), interleukin-4 anti-receptor (IL- 4RA), tumor growth factor beta (TGF-β),and tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). There was one gene only with no changes in gene region, IL-1β 3962TT/TC/CC. In other regions, a statistically significant change compared to the control group was observed (p<0.001) (Table 4). DISCUSSION In the last 10 years, in order to determine the UC epidemiological and genetic aspects, several surveys were carried out in Turkey with the participation of 1107 inflammatory bowel diseases (IBD) patients in different cities (27). In these studies, the incidence of UC in Turkey was determined as 4.4/100,000. It is emphasized that UC incidence in Turkey is lower than that in North and West Europe (Norway and England, 30/100,000) (6,11), and it is at a level closer to that in the Middle East countries, e. g., the prevalence of 4.1/100,000 in population of Lebanon (22). In the present study, cytokine genes were studied by genomic analyses in which the variation of polymorphic sites was at the allelic level (28). Accordingly, for the genetic polymorphisms investigated for all cytokine gene sites most susceptible to the disease, highest rate was observed in TNF-α -308 G in UC patients compared to controls, and minor changes at allelic level in 3962T C, IL-1β, and IL-1β -511T C. In our research, it was determined IL-1β gene alleles between 54% and 69% in UC patients, and same gene alleles between 30% and 70% in controls. In the research of Latino et al. (2013) in the Italy population, TGF 1 gene polymorphism T allele frequencies have been shown as between 0.26 and 0.46 of all UC patients. The T allele frequency was between 0.30 and 0.35 in controls (21). In the analysis of Weersma et al. from the Netherlands in 2007, in the analysis of the IL-1 gene, it has been found that polymorphism rates in healthy individuals was 6%-57% and % in UC patients ( 28). In our results, IL-1 gene codon polymorphisms T allele frequencies were found in 23%-58 % in the control group and 15% -77% in UC patients. In this sense, our rate of TGF-1 gene is observed higher than that of the European rate. The clinical presentation of a very different courses of UC, which is a chronic disease, can bring along uncertainty in the diagnosis of the disease. Also, genes and regulatory environmental agents showing multifactorial inheritance can affect the course of UC (13,29). Environmental impact-induced cytokine genes may be unstable in protein synthesis in reducing or increasing direction (15). Stimulated monocytes can be shown as reason for criteria for the production of protein in the ILlα gene. Therefore, the cytokine gene is included 37

44 Medicinski Glasnik, Volume 12, Number 1, February 2015 within the specific allele determining factor (30). In IL-1α allele frequencies there were similarities between Turkish and the Netherlands populations for healthy controls, but we could not compare them with the frequency in same gene of Caucasian populations (26-28). A European research group has shown no statistically significant difference between patients and healthy controls in UC genotypic frequencies. They were reported as inflammation and inflammatory periods in patients with UC, and it is suggested that the detection of the polymorphism in the Netherlands population is widely studied whereas other genetic technology like gene sequencing is not. (4) It will be required to conduct additional studies using other genetic technologies like gene sequencing. According to Ferguson et.al (6) the TNF-α polymorphisms were defined by a G to A substitution. A significant decrease in the frequency of the TNF-α A allele was observed in UC patients compared with further controls in our study. Opposite to Ferguson et al, we observed that the TNF-α gene G to A alelles were increased (17) Yamamoto-Furusho et al. (2011) investigated proinflammatory cytokines genes (IL-1 β and IL-R), and they found a decreasing frequency of genotypes in UC patients as compared with healthy controls (2,4), which is similar with the results of the presented study. In the regulation of intestinal inflammation, it is known that IL-1α can be effective, it can be predicted that changes in cytokine polymorphisms can lead to an imbalance in certain parts of the intestinal mucosal (23). Thus, the effect of cytokines, which are heteromorphic may be due REFERENCES 1. Ahirwar DK, Kesarwani P, Singh R, Ghoshal UC, Mittal RD. 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46 ORIGINAL ARTICLE False-positive 18-fluorodeoxyglucose positron emission tomography computed tomography (FDG PET/CT) scans mimicking malignancies Zehra Yasar 1, Murat Acat 2, Hilal Onaran 3, Akif Ozgül 3, Erhan H. Dincer 4, Erdogan Cetinkaya 3, Nurdan A. Korkmaz 5 1 Department of Chest Diseases, School of Medicine, Abant Izzet Baysal University, Bolu, Turkey, 2 Department of Chest Diseases, School of Medicine, Karabuk University, Karabuk, Turkey, 3 Pulmonary Division, Yedikule Chest Diseases and Surgery Teaching and Research Hospital, Istanbul, Turkey, 4 Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, University of Minnesota, MN, USA, 5 Department of Nuclear Medicine, School of Medicine, Abant Izzet Baysal University, Bolu, Turkey ABSTRACT Aim 18-Fluorodeoxyglucose (FDG) positron emission tomography computed tomography (PET/CT) is an imaging modality that is often used to help differentiate benign from malignant pulmonary lesions and it has been shown to be more efficacious than conventional chest computed tomography (CT). However, some benign lesions may also show increased metabolic activity which can lead to false-positive PET findings. We aim to illustrate false positive findings of PET scan that simulate lung cancer in a variety of diseases. Corresponding author: Zehra Asuk Yasar Department of Chest Diseases, Abant Izzet Baysal University School of Medicine Gölköy Yerleşkesi,Gölköy-Bolu, Turkey Phone: ; Fax: ; Original submission: 08 September 2014; Revised submission: 25 December 2014; Accepted: 08 January Methods Patients referred to Yedikule Chest Diseases and Surgery Teaching and Research Hospital with increased FDG uptake for which histological results were available over a 2-year period ( ) were reviewed. Seven patients with false-positive PET/CT findings were reported in this study. Results The majority of lesions showing increased metabolic activity were due to malignant diseases. However, increased 18 F-FDG uptake was also seen in benign lesions such as active pulmonary inflammation or infection, granulomatous processes and fibrotic lesions. Conclusion. The integration of clinical history, morphologic findings of lesions on the CT component, and metabolic activities of PET/CT scan can help reduce false interpretations. Interventional procedures may be needed for tissue confirmation for differential diagnosis. Key words: false positive, FDG, PET scan, mimicking malignancies Med Glas (Zenica) 2015; 12(1):

47 Yasar et al. Falsely FDG-PET positive pulmonary lesions INTRODUCTION Fluorine-18 fluoro-2-deoxyglucose (18F-FDG) positron emission tomography combined with computed tomography (PET/CT) is a useful test to evaluate malignancies (1). Mechanism of 18F- FDG uptake in malignant tissues depends on the metabolic activity of the lesion, e.g. the extent of uptake is proportional to the number of malignant cells and their proliferative activity (1,2). Malignant lesions have been shown to have elevated expression of the glucose transporter (GLUT- 1) and tend to have increased metabolic activity evidenced by increased FDG uptake (2). Notwithstanding the controversial views, SUVs of 2.5 or greater have been used as a cutoff value indicative of malignancy (1-3). However, inflammatory diseases may also show increased uptake of 18F-FDG and cause false-positive PET scan results, necessitating further investigations to rule out malignant conditions (3,4). The rationale underlying this belief is that activated macrophages and neutrophils in inflammatory tissue use glucose as an energy source for chemotaxis and phagocytosis, whereas fibroblasts use glucose for proliferation (3). The aim of this study was to describe benign lung lesions with increased 18F FDG uptake that simulate malignancies at series of seven cases, and their imaging characteristics that may help in differentiating them from malignant metastases. PATIENTS AND METHODS This study retrospectively analyzed seven patients in order to describe false-positive 18F-FDG uptake increase according to PET/CT scans and to review the diagnosis and management of the patients with increased uptake on PET/CT scan. This study included 7 patients with suspected lung cancer (patients with pulmonary lesions of ambiguous nature) who were referred to Yedikule Chest Diseases and Surgery Teaching and Research Hospital clinic over 2 years ( ). The presented data were collected from patients records: patient s age and sex, clinical features on presentation, results of investigations including X-ray, CT, PET/CT, treatment and outcome. There were three women and four men, in the age between 27 and 78 years. The metabolic activity of the lesions determined by the chest CT were evaluated for F-18 FDG PET/CTF-18 FDG uptake. The patients underwent 18F-FDG PET/CT examination using a multidetector CT integrated high-resolution PET/CT scanner (Biograph, Siemens Medical Solutions, USA Inc.). They fasted for 6 h before receiving an intravenous injection of MBq of 18F-FDG. The PET/CT acquisitions started 60 min after tracer injection. The PET scan (6 7 fields of view, 3 min/field) and low-dose non-enhanced CT scan were acquired from the skull to the mid-thigh (5). As a limit value for lesions suspected of neoplastic nature, SUVmax higher than 2.5 was accepted. In case of hypermetabolic lesions, histological or cytological examinations of the material obtained from biopsies (CT-guided fine needle aspiration cytology, FNAC), endobronchial ultrasound-guided transbronchial aspiration (EBUS-TBNA),video-assisted thoracic surgery (VATS) biopsy and lobectomy, were diagnosed. RESULTS AND DISCUSSION Case 1: Pulmonary tuberculosis and tuberculous lymphadenopathy A 57-year-old female presented with dyspnea, cough and weight lost on exertion. She has a 20-pack-year smoking history and continues to smoke. Family history is positive for her father with lung cancer who was heavy smoker. There is history of loss of appetite. Physical examination is normal. Laboratory data revealed normal blood count, erythrocyte sedimentation rate (ESR) rate 33/1h. The sputum smear was negative for acid fast bacillus (AFB) testing. A chest X-ray done for evaluation of dyspnea and cough on exertion revealed mediastinal enlargement, and there was heterogeneous density at the right upper lobe (RUL). The CT chest revealed a patchy consolidation in the right upper lobe and also hilar, mediastinal lymph node enlargement. The 18F-FDG PET/CT scan showed increased uptake in parenchymal right upper lobar lesion (peak standardized uptake value [SUV] = 3.9) and in mediastinal, hilar lymph node (peak SUV = 6.5) (Figure1). Given the patient s high risk based on tobacco use and family history of lung cancer, fiberoptic bronchoscopy was performed and no endobronchial lesion was found. EBUS- TBNA biopsy of mediastinal and hilar lymph nodes was performed. Pathology of both lymph 41

48 Medicinski Glasnik, Volume 12, Number 1, February 2015 nodes revealed chronic inflammation with granulomatous formation, no evidence of malignancy. Mycobacterium tuberculosis was demonstrated in bronchoalveolar lavage and was sus ceptible to all the tested anti-tuberculous agents. Tuberculoma is one of the most well-known diseases that show intense FDG uptake. Active granulomatous processes such as tuberculosis have been reported to accumulate FDG (1-4, 6). In tuberculosis, granulomatous lesions are mainly composed of lymphocytes and macrophages, which use 18F-FDG as an energy source (7). Activated inflammatory cells have markedly increased glycolysis. The hexose monophosphate shunt is stimulated by phagocytosis, with increases of times that of baseline values, which is the cause of high FDG uptake (6). Tuberculous lymphadenopathy can be understood in the same manner as tuberculoma in the lung parenchyma. Figure 1. Tuberculous lymphadenitis in a 57-year-old woman. In the mediastinal window setting, axial transverse CT scan shows lymph node enlargement (left). Increased 18F-FDG uptake is observed in lesion on PET image (peak standardized uptake value = 6,5) (right) (Yedikule EAH, 2013) Case 2: Sarkoidosis A 27-year-old nonsmoking male presented with a 1-month history of cough, weight loss and increasing shortness of breath. He had no history of fever, night sweats, chest pain, palpitation, arthralgia or skin rash. There was no history of allergy or systemic disease. On admission, he was afebrile with a respiratory rate of 22. The physical examination was unremarkable. Chest radiograph on admission showed mediastinal enlargement and bilateral millimetric nodules in the upper and middle lung zone. The laboratory tests including the complete blood count, biochemical and tumor markers were within normal limits. The CT chest revealed hilar, mediastinal lymph node enlargement and numerous millimetric-dimensional parenchymal nodules predominantly viewed in the upper and middle zone. Heterogeneous and numerous milimetric hypodense areas with unclear borders seen in liver parenchyma and spleen. Quanti FERON Gold blo- od test for tuberculosis was negative. The sputum smear was negative for AFB. Malignancies were suspected clinically and PET-CT scan was done for diagnosis. On PET-CT, mediastinal lymph nodes, paranchimal nodules and liver and spleen showed fluorodeoxyglucose (FDG) uptake with a maximum standardized uptake value (SUVmax) of 2.2, 4.8 and 8.8 both liver and spleen, respectively (Figure 2). Since malignancy could not be excluded by PET-CT scan, EBUS-TBNA biopsy of mediastinal and hilar lymph nodes was performed. Pathology of both lymph nodes revealed chronic inflammation with granulomatous formation, no evidence of malignancy. After that, steroid therapy was proposed and the patient s complaints regressed. Sarcoidosis is a chronic multisystem disorder. It can be characterized in affected organs by an accumulation of T lymphocytes and mononuclear phagocytes, noncaseating epithelioid granulomas, and derangements of the normal tissue architecture (8). The etiology is unknown, but it is thought to be caused by exaggerated cellular immune responses (8). Pathologically, the first manifestation of the disease is an accumulation of mononuclear inflammatory cells, mainly CD4+ T helper 1 lymphocytes and mononuclear phagocytes, in the affected organ. This inflammatory process is followed by the formation of granulomas, aggregates of macrophages and their progeny, epithelioid cells, and multinucleated giant cells (8). Actively granuloma-forming parenchymal lesions or nodes show increased 18F- FDG uptake because activated lymphocytes and macrophages contribute to increased glucose use in the corresponding lesions (9). Figure 2. Sarcoidosis in 27- year-old man: in the lung window setting, axial transverse CT scan shows small millimetric nodules (left). Increased 18F-FDG uptake is observed in lesion on PET image (peak standardized uptake value = 4.8) (right) (Yedikule EAH, 2013) Case 3: Pulmonary actinomycosis A 56-year-old woman was admitted with the complaints of intermittent cough with blood-tinged sputum for 2 years, fatigue and weight loss. She smoked cigarettes 1 pack a day for over 20 years. 42

49 Yasar et al. Falsely FDG-PET positive pulmonary lesions Respiratory examinations found no cervical lymphadenopathy, musculoskeletal disorder or other abnormalities. The remainder of her physical exam was unremarkable. Laboratory data revealed normal blood count, ESR, C-reactive protein (CRP), and renal and liver functions. Sputum examinations (three consecutive samples) were negative for acid-fast bacilli, malignant cells, or fungal elements. The contrast-enhanced computerized tomography (CT) showed a speculated mass, 14.17mm in diameter, in the right lower lobe, which was highly suspected as a malignancy. The FDG PET/ BT revealed a hypermetabolic lesion over the right lower lobe of the lung, with a maximum SUV of 5.2 which favors a malignancy (Figure 3). Bronchoscopy revealed normal airways and mucosa and bronchoalveolar lavage was negative for malignant cells or fungal elements. CT-guided FNAC was done from the right lung mass lesion. The smears were prepared and stained with haematoxylin and eosin, periodic acid Schiff (PAS), and Giemsa stains. The smears revealed radiating filamentous colonies of Actinomyces in a background of neutrophilic exudates; PAS stain also showed Actinomyces colonies, resulting in the confirmation of the diagnosis of pulmonary actinomycosis. The patient was treated with intravenous penicillin for a month and then given oral penicillin for six months. The patient responded well to the treatment. Pulmonary actinomycosis is a rare bacterial lung disease and causes lung cavities, lung nodules, and pleural effusion (10). Radiographic and clinical features of pulmonary actinomycosis varied and could mimic a wide spectrum of benign and malignant diseases (11). Mabeza et all. reported that up to 25% of cases with thoracic actinomycosis were initially misdiagnosed as malignancy (12). In general, high uptake on FDG PET suggests that the nodule contains active and proliferative lesions such as lung cancer, infiltration tumor, tuberculoma, and pulmonary mycosis (13). Figure 3. Pulmonary actinomycosis in a 56- year-old woman: axial transverse CT scan shows a spiculated mass, 14.17mm in diameter, in the right lower lobe (left). Increased 18F-FDG uptake is observed in lesion on PET image (peak standardized uptake value = 5.2) (right) (Yedikule EAH, 2013) Case 4: Reumatoid nodules A 65-year-old woman with a history of rheumatoid arthritis (RA) presented with dyspnea on exertion. The RA was diagnosed 16 years earlier and has been treated with salisilazosülfapiridin and prednisolon since the time of diagnosis. She has a 15-pack-year smoking history. Family history is positive for lung cancer. There is history of loss of appetite and weight over a period of 6 months. Physical examination including joint examination is normal except for decreased breath sounds. Laboratory data revealed normal blood count, ESR,CRP, and renal and liver functions. The CT chest revealed a mm noncalcifiednodule in the right lower lobe and bilateral millimetric nodules. The 18F-FDG PET/CT scan showed moderately increased uptake with a maximum SUV of 4.3 in the right lower lobe (RLL) nodule (Figure 4). Subsequent VATS biopsy of the right lower and middle lobe nodules was performed. Pathology of both nodules revealed chronic inflammation with necrotizing granulomatous formation consistent with rheumatoid nodules and no evidence of malignancy. Fungal and acid-fast bacilli stains and cultures were negative. Figure 4. Reumatoid nodule in 65- year-old woman: axial transverse CT scan shows mm non-calcified nodule in the right lower lobe (left).increased 18F-FDG uptake is observed in lesion on PET image (peak standardized uptake value = 4.3) (right) (Yedikule EAH, 2013) Reports of the use of PET and PET/CT in extraarticular RA are limited to subcutaneous nodules, lymph nodes, and the lung (14,15). Gupta et al. described a patient with RA found to have mild increased uptake in pulmonary nodules on PET scan (16). Histological examination of these nodules revealed the presence of rheumatoid nodules. On the other hand, Rodr ıguez et al. described two patients with RA in whom pulmonary nodules showed increased SUV on 18F-FDG PET scan (17). Biopsy of the nodules demonstrated bronchogenic carcinoma developing wi- 43

50 Medicinski Glasnik, Volume 12, Number 1, February 2015 thin preexisting rheumatoid nodules. Clinicians need to keep in mind that rheumatoid nodules can have increased activity on PET scan in the management of lung nodules in rheumatoid arthritis. Case 4: Anthracosilicosis A 78-year-old asymptomatic man was admitted to our hospital for evaluation of an abnormal shadow on chest roentgenogram. He has 45-packyear smoking history and did not have history of industrial exposure. On admission, physiological and laboratory examinations, including tumor markers, were within normal limits. Chest X-ray showed an abnormal mass in right middle field of the lung and mediastinal enlargement. Chest CT revealed an approximate 1.8cm sized mass in the right middle lob, and enlarged mediastinal lymph nodes. The FDG-PET showed high uptake with a maximum SUVmax 9.1 in hilar and mediastinal nodes and 1.5 in the RML mass lesion which favors a malignancy. Since malignancy could not be excluded by PET-CT scan, EBUS-TBNA biopsy of mediastinal and hilar lymph nodes was performed. Pathological findings showed that contained polarizable material suggestive of silica with focally contained fine anthracotic pigments, and negative for malignancy. The silicosis with anthracotic pigments in mediastinal lymph nodes might be caused by inhalation of irritant dusts and attendant distortion of local lymphatic vessels (18). One clinicopathologic form of this reaction is fibrosis, while the other form consists of aggregates of particle-laden macrophages with minimal or no accompanying fibrosis, a reaction that is typically seen with inert dusts such as iron, tin, and barium (18). The FDG-PET studies have revealed increased uptake in pneumoconiosis and progressive massive fibrosis. Some of this uptake is perhaps related to the presence of inflammatory cells such as macrophages, as well as fibroblasts (19). Case 5: Vanishing tumor A 75 -year-old man with operated sigmoid colon tumor presented with cough of several days duration but reported no chest pain, dyspnea, fever, or hemoptysis. He also had hypertension. There was a 60 pack-year smoking history, but no alcohol or illicit drug use. A chest radiograph obtained during evaluation demonstrated homogenous densities in the right hemithorax. Laboratory studies were remarkable for a normal leukocyte count, a hemoglobin level of 10 g/dl. The CT scan (mediastinal windows) reveals homogenous, spherical density within the right lower lobe. The opacity is surrounded by a pleural rim and lie along the expected location of the oblique fissure. Malignancies were suspected and PET- CT scan was done for diagnosis. The FDG PET/ BT revealed a hypermetabolic lesion over the right lower lobe of the lung of the patient, with a maximum SUV of 8-10 which favors a malignancy. Since malignancy could not be excluded by PET-CT scan, CT-guided biopsy was planned for diagnosis. While carrying out this procedure, we recognized that the lession had regressed. An echocardiogram obtained and demonstrated left ventricular hypertrophy and mild systolic dysfunction; there was no pericardial effusion. The patient responded well to diuretic treatment. Vanishing tumor refers to the transient localized collection of pleural fluid in the interlobar fissures, usually in association with congestive heart failure from various causes (20). Vanishing tumor is a phenomenon predominantly occurring in the right hemithorax (21). The pathogenesis of vanishing tumors involves the adhesion and obliteration of the pleural space due to pleuritis, thus preventing the free accumulation of fluid (21). In this setting, whenever hydrostatic and/or oncotic forces produce fluid at the pleural surface beyond the resorptive ability of the pleural lymphatics, a localized pleural effusion that is recognized as a vanishing tumor may result. This hypothesis is supported by the finding of adhesive pleuritis at autopsy in cases of known vanishing tumors (22,23). Case 6: Aspergillosis A 68-year-old man operated for ampullary carcinoma 2 years before presented with cough of several days duration. He also had hypertension. There was a 40 pack-year smoking history, but no alcohol use. Chest CT revealed an irregularly shaped lung nodule approximately 11.17mm in diameter in the right upper lobe. The FDG uptake at PET showed that the SUVmax was 5.4. Radiologists did not diagnose the nodule as an aspergilloma. The pathological examination of the FNAC was negative for malignant cells, and the cultures were negative by bronchoscopic 44

51 Yasar et al. Falsely FDG-PET positive pulmonary lesions examinations. Because lung cancer was strongly suspected, video-assisted thoracic surgery was performed. An upper lobe wedge resection was performed, including the tumor in the right upper lobe. In the specimen, the tumor was necrotic and a pathological examination during operation had shown no evidence of malignancy. The final pathological examination showed the presence of an aspergilloma. The postoperative evolution was therefore favorable. Recently, FDG-PET accumulation in cases of pulmonary aspergillosis mimicking lung cancer was reported (24), and in the three cases reported in that study, the FDG uptake during PET scans showed an SUVmax ranging from 4.0 to 8.3 suggesting a tendency for high FDG accumulation in 10 cases. Case 7: Pulmonary nocardiosis A 35-year-old male presented with cough and expectoration with episodes of haemoptysis for 2 years. He took an antitubercular treatment for six months. With antitubercular treatment fever had subsided but the amount of sputum and haemoptysis had continued. Two months ago he referred to a general physician with low grade fever associated with productive cough and received some medication without any improvement. His condition became worse. Chest x-ray showed infiltrations in right upper lobe with cavity formation and CT revealed the presence of areas of consolidation with air bronchograms and cavitary lesions containing air and infiltration beginning from the apical segment lying to anterior segment of right lower lobe. The FDG PET/BT revealed a hypermetabolic lesion over the right upper lobe of the lung of the patient, with a maximum SUV of which favors a malignancy (Figure 5). So due to findings he Figure 5. Pulmonary nocardiosis in 35- year-old man: axial transverse (mediastinal windows) CT scans reveals cavitary lesions in anterior segment of right lower lobe (left). Increased 18F-FDG uptake is observed on PET image (peak standardized uptake value = ) (right) (Yedikule EAH, 2013) received ceftazidim and ciprofloxacin. But he had no improvement in respiratory symptoms. Several sputum samples were collected and tested for the presence of acid-fast bacilli, but all smears were negative. The patient then underwent bronchoscopy and aspirated material was negative for tuberculosis, fungi (including Pneumocystis jirovecii), and malignancy. The FNAC was done from the right lung lesion. Aspirated material was negative for tuberculosis and malignancy. Because of progressive worsening of clinical status, right upper lobectomy was performed. On gram staining, the organism appeared as gram-positive, beaded, coccoid, thin branching filaments. Modified Ziehl-Neelsen staining showed many branching acid-fast bacilli, consistent with the morphology of Nocardia species. The patient was started on trimethoprim-sulfamethoxazole and improved remarkably both clinically and radiographically. Pulmonary nocardiosis is an infection caused by gram-positive aerobic bacilli belonging to genus Nocardia. Pulmonary nocardiosis occurs through inhalation and most often affects patients presenting with immunosuppression due to AIDS, neoplasia, as well as kidney or bone marrow transplantation (25). The diverse radiological manifestations of pulmonary nocardiosis reflect its ability to cause both suppurative and granulomatous infection (8). Actively granuloma-forming parenchymal lesions or nodes show increased 18F-FDG uptake because activated lymphocytes and macrophages contribute to increased glucose use in the corresponding lesions (9). In conclusion, metabolic imaging with FDG- PET is beginning to play an important role in the management of malignancies. However, benign and malignant lesions may have overlaps. Benign focal lung lesions can simulate lung cancer with increased 18F-FDG uptake. The integration of clinical history, morphologic findings of lung parenchymal lesions on the CT component, and metabolic activities on the PET component of integrated PET/CT can help reduce false interpretations. Interventional procedures may be needed for lesions showing increased 18F FDG uptake on PET for tissue confirmation irrespective of their morphology on CT. 45

52 Medicinski Glasnik, Volume 12, Number 1, February 2015 We must keep in mind that not all increased 18F- FDG uptakes should be considered malignant. These cases exemplify the need of clinicians to exercise clinical and critical thinking skills to consider the broad diagnostic possibilities of lung lesions presenting as a malignancy. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare REFERENCES 1. Kostakoglu L, Agress H, Jr., Goldsmith SJ. Clinical role of FDG PET in evaluation of cancer patients. Radiographics 2003; 23: Mochizuki T, Tsukamoto E, Kuge Y, Kanegae K, Zhao S, Hikosaka K, Hosokawa M, Kohanawa M, Tamaki N. FDG uptake and glucose transporter subtype expressions in experimental tumor and inflammation models. J Nucl Med 2001; 42: Bakheet SM, Saleem M, Powe J, Al-Amro A, Larsson SG, Mahassin Z. F-18 fluorodeoxyglucose chest uptake in lung inflammation and infection. Clin Nucl Med 2000; 25: Turkington TG, Coleman RE. Clinical oncologic positron emission tomography: an introduction. Semin Roentgenol 2002; 37: Ohira H, Tsujino I, Ishimaru S, Oyama N, Takei T, Tsukamoto E,Miura M, Sakaue S, Tamaki N, Nishimura M. Myocardial imaging with 18F-fluoro-2-deoxyglucose positron emission tomography and magnetic resonance imaging in sarcoidosis. Eur J Nucl Med Mol Imaging 2008; 35: Goo JM, Im JG, Do KH, Yeo JS, Seo JB, Kim HY, Chung JK. Pulmonary tuberculoma evaluated by means of FDG PET: findings in 10 cases. Radiology 2000; 216: Amrein PC, Larson SM, Wagner HN, Jr. An automated system for measurement of leukocyte metabolism. J Nucl Med 1974; 15: Crystal RG. Disorders of the immune system, connective tissue, and joints, sarcoidosis. In: Braunwald E FA, Kasper DL,Hauser SL, Longo DL, Jameson JL (Eds). Harrison s principle of internal medicine (15th ed.). NewYork: McGraw-Hill 2001: Brudin LH, Valind SO, Rhodes CG, Pantin CF, Sweatman M, Jones T, Hughes JM. Fluorine-18 deoxyglucose uptake in sarcoidosis measured with positron emission tomography. Eur J Nucl Med 1994; 21: Klapholz A, Talavera W, Rorat E, Salsitz E, Widrow C. Pulmonary actinomycosis in a patient with HIV infection. Mt Sinai J Med 1989; 56: Kim TS, Han J, Koh WJ, Choi JC, Chung MJ, Lee JH, Shim SS, Chong S.Thoracic actinomycosis: CT features with histopathologic correlation. AJR Am J Roentgenol 2006; 186: Mabeza GF, Macfarlane J. Pulmonary actinomycosis. Eur Respir J 2003;21: Chang JM, Lee HJ, Goo JM, Lee HY, Lee JJ, Chung JK, Im JG. False positive and false negative FDG- PET scans in various thoracic diseases. Korean J Radiol 2006; 7: Bagga S. Rheumatoid lung disease as seen on PET/ CT scan. Clin Nucl Med 2007; 32: dos Anjos DA, do Vale GF, Campos Cde M, do Prado LF, Sobrinho AB, da Cunha AL, Santos AC. Extraarticular inflammatory sites detected by F-18 FDG PET/CT in a patient with rheumatoid arthritis. Clin Nucl Med 2010; 35: Gupta P, Ponzo F, Kramer EL. Fluorodeoxyglucose (FDG) uptake in pulmonary rheumatoid nodules. Clin Rheumatol 2005; 24: Rodriguez P, Romero T, Rodriguez de Castro F, Hussein M, Freixinet J. Bronchogenic carcinoma associated with rheumatoid arthritis: role of FDG-PET scans. Rheumatology (Oxford) 2006; 45: Speizer FE. Environmental lung diseases. In: Braunwald E FA, Kasper DL, Hauser SL, Longo DL, Jameson JL(Eds). Principle of internal medicine (15th ed.). NewYork: McGraw-Hill2001: Alavi A, Gupta N, Alberini JL, Hickeson M, Adam LE, Bhargava P, Zhuang H. Positron emission tomography imaging in nonmalignant thoracic disorders. Semin Nucl Med 2002; 32: Rabinowitz JG, Kongtawng T. Loculated interlobar air-fluid collection in congestive heart failure. Chest 1978; 74: Stark P, Leung A. Effects of lobar atelectasis on the distribution of pleural effusion and pneumothorax. J Thorac Imaging 1996;11: Pereira W, Jr., Kovnat DM, Snider GL. Subcostal loculated pleural effusion in congestive heart failure: an unusual case of vanishing tumor. Am J Med Sci 1975;269: Van Gelderen WF. Vanishing pleural fluid collections in cardiac failure simulating lung tumours. Australas Radiol 1994; 38: Baxter CG, Bishop P, Low SE, Baiden-Amissah K, Denning DW. Pulmonary aspergillosis: an alternative diagnosis to lung cancer after positive [18F]FDG positron emission tomography. Thorax 2011; 66: Martinez Tomas R, Menendez Villanueva R, Reyes Calzada S, Santos Durantez M, Valles Tarazona JM, Modesto Alapont M, Gobernado Serrano M. Pulmonary nocardiosis: risk factors and outcomes. Respirology 2007; 12:

53 ORIGINAL ARTICLE Human West Nile virus infection in Bosnia and Herzegovina Sead Ahmetagić 1, Jovan Petković 1, Mirsada Hukić 2, Arnela Smriko-Nuhanović 1, Dilista Piljić 1 1 Clinic for Infectious Diseases, University Clinical Centre Tuzla, Tuzla, 2 International Burch University, Sarajevo; Bosnia and Herzegovina ABSTRACT Aim To describe the first two cases of West Nile virus (WNV) neuroinvasive infections in Bosnia and Herzegovina. Methods At the Clinic for Infectious Diseases of the University Clinical Centre Tuzla, Bosnia and Herzegovina (B&H), specific screening for WNV infection was performed on patients with neuroinvasive diseases from 1 August to 31 October Serum samples were tested for the presence of WNV IgM and IgG antibodies using enzyme-linked immunosorbent assay (ELISA); positive serum samples were further analyzed by detection of WNV nucleic acid of two distinct lineages (lineage 1 and lineage 2) in sera by RT-PCR. Corresponding author: Arnela Smriko-Nuhanović Clinic for Infectious Diseases, University Clinical Centre Tuzla Trnovac bb, Tuzla, Bosnia and Herzegovina Phone: ; Fax: ; asmriko@hotmail.com Original submission: 14 August 2014; Revised submission: 24 October 2014; Accepted: 18 December Results Three (out of nine) patients met clinical criteria, and two of them had high serum titre of WNV specific IgM antibodies (3.5 and 5.2). Serum RT-PCR testing was negative. Conformation by neutralization testing was not performed. Both cases represented with encephalitis. None of these cases had recent travel history in WNW endemic areas, or history of blood transfusion and organ transplantation, so they represented autochthonous cases. Conclusion Although there were no previous reports of flavivirus infections in B&H, described cases had high titre of WNV specific antibodies in serum, and negative flavivirus-vaccination history, they were defined as probable cases because recommended testing for case confirmation was not performed. The West Nile virus should be considered a possible causative pathogen in this area, probably in patients with mild influenza-like disease of unknown origin and those with neuroinvasive disease during late summer and early autumn. Key words: neuroinvasive infections, encephalitis, flaviviruses Med Glas (Zenica) 2015; 12(1):

54 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION The genus Flavivirus comprises 53 viruses, and many of them are human pathogens of concern. In Europe, many flaviviruses are endemic (West Nile, Usutu, tick-borne encephalitis viruses) or occasionally imported (dengue, yellow fever viruses) (1). West Nile virus (WNV) is transmitted in an avian cycle by ornithophilic mosquitoes, chiefly of the genus Culex. Mammals can also be infected, but are considered dead end hosts because viraemia is generally too low to infect mosquitoes (2). West Nile virus was first identified in the West Nile district of Uganda in 1937 in a woman who presented with a mild febrile illness (3). During the next decades, the virus spread through Africa and Asia. It was first described in Europe in the 1960s when seropositive animals or virus isolates were reported in France, Portugal and Cyprus (4). West Nile virus has historically been considered less pathogenic in humans than dengue virus or yellow fever virus; however, more virulent genotypes have emerged since Isolates from Israel (1998), Hungary (2003), or North America (1999) belonging to the Israeli-American cluster of WNV lineage 1a are highly pathogenic in birds and mammals, and lineage 2 viruses have caused an increasing number of WNV outbreaks in Europe since 2008 (2). In Europe, WNV has mainly been reported in central and south-eastern Europe, regions in which WNV infections and virulence have recently increased, and the implicated viruses have spread to new areas, including Bulgaria and Greece in 2010, Albania and Macedonia in 2011, and Croatia, Serbia, and Kosovo in 2012 (1,2,5). The first laboratory-confirmed cases of the West Nile virus neuroinvasive infection in Croatia were diagnosed in September 2012 in three eastern Croatian counties, although specific antibodies to West Nile virus in humans, horses, and European brown bears have been previously detected (6-9). Also, in Serbia first outbreak of West Nile virus infection was reported in humans in August to October 2012, and evidence of detected virus activity in horses, wild birds and mosquitos (10-12). There are no reports of WNV or other flaviviruses activities in humans or animals in Bosnia and Herzegovina. PATIENTS AND METODS At the Clinic for Infectious Diseases University Clinical Centre Tuzla, Bosnia and Herzegovina (B&H), specific screening for WNV infection was performed on patients with neuroinvasive diseases with negative CSF bacterial cultures and negative serological tests for other common causes of bacterial, viral or protozoan meningitis and/or encephalitis (rubella, measles, mumps, varicella-zoster viruses, adenovirus type 3, Epstein-Barr, herpes simplex virus-1, herpes simplex virus-2, cytomegalovirus, coxackie B, coxsackie A7, echovirus type 7 viruses, Treponemaa pallidum, Borrelia burgdorfer sensu stricto, Borrelia garinii, Borrelia afzelii, Haemophilus influenza, Listeria monocytogenes 1/2a and 4b, Toxoplasma gondii) from 1 August to 31 October There was no data about specific WNV screening performed before in medical centres in B&H. The study had been approved by the Research Ethics Committee of the University Clinical Centre Tuzla. Case definition The case definition and case classification were established according to European Union case definitions for West Nile fever (13). Persons with fever ( 37.5 C) and meningitis and/or encephalitis were included. Meningitis was defined as presence of fever, clinical signs of meningeal inflammation (including headache, nuchal rigidity, Kernig s sign or Brudzinski signs, photophobia or phonophobia), and the presence of cerebrospinal (CSF) pleocytosis (>5 leucocytes/mm 3 ), elevated protein levels (>0.45 g/l; normal range g/l) and normal ( mmol/l, 50-60% serum glucose levels) or mildly decreased ( mmol/l) CSF glucose level. Encephalitis was defined as the presence of fever, encephalopathy (decreased or altered level of consciousness, lethargy or personality change) and/or focal neurological signs (weakness, cranial nerve palsy), seizures or movement disorders (tremor, parkinsonisam, ataxia), and the presence of CSF pleocytosis (>5 leucocytes/mm 3 ), elevated protein levels (>0.45 g/l), and normal ( mmol/l, 50-60% serum glucose levels) or mildly decreased ( mmol/l) CSF glucose level. Laboratory criteria for a probable case were the presence of WNV-specific antibody response in serum (IgM or IgM and IgG). Laboratory criteria for a case conformation were isolation of WNV from blood or CSF, detection of WNV nucleic acid in blood or CSF, WNV specific antibody 48

55 Ahmetagić et al. Human WNV infection in B&H response (IgM) in CSF, WNV IgM high titre and detection of WNV IgG, and conformation by neutralisation. Serology and molecular tests Serum samples were tested at the Institute of Clinical Microbiology, University Clinical Center of Sarajevo, for the presence of WNV IgM and IgG antibodies using the West Nile Virus IgM Capture DxSelect TM ELISA and West Nile Virus IgG DxSelect TM ELISA kits (Focus Diagnostics, Cypress, California, USA). Serum samples, which tested positive in the ELISA, were further analyzed by detection of WNV nucleic acid of two distinct lineages (lineage 1 and lineage 2) in sera by RT-PCR. Results were confirmed at the Institute of Microbiology and Immunology, Medical Faculty University of Ljubljana, Slovenia. Diagnostic tests for WNV isolation from blood, specific CSF testing (WNV isolation, specific antibody response, or detection of nucleic acid), and conformation by neutralization were not performed. Patient data Patient s demographic characteristics (age, sex, region of residence), travel history, blood transfusions, transplants, and vaccination status, comorbidity, presenting symptoms and signs, clinical findings, hematology and blood biochemistry analysis, CSF cell count and biochemical analysis were recorded, along with serology and molecular diagnostic tests results, as well as the outcome at hospital discharge. RESULTS During the period 01 August to 31 October 2013 only three patients (out of nine) met clinical criteria, and two of them were identified as probable cases according to the case definition (Table 1); conformation by neutralization testing was not performed. Table 1. Results of serology and molecular testing of two patients with neuroinvasive West Nile virus infection Case Sample type Day of sampling after illness onset IgM ELISA (titre) IgG ELISA (titre) RT-PCR 1 Serum 20 Positive (3.5) Positive (2.2) Negative 2 Serum 12 Positive (5.2) Negative (0.27) Negative First case had onset of symptoms at the beginning of August, and second case at the beginning of September None of these cases had recent travel history in areas where WNV was endemic, nor the history of blood transfusion and organ transplantation, and had negative flavivirus-vaccination history (demographic characteristics and comorbidities are shown in Table 2). They were both represented with encephalitis (self-reported symptoms and clinical signs are shown in Table 3). Table 2. Demographic characteristics and comorbidities of two patients with neuroinvasive West Nile virus infection Case Age (years) Sex City area Immunosuppression Hypertension including diabetes Other chronic illness 1 84 Male Tuzla No Yes Vertigo Chronic bronchitis 2 68 Female Kladanj No Yes Hypertensive heart disease Table 3. Self-reported symptoms and clinical signs of two patients with neuroinvasive West Nile virus infection Signs and symptoms Case 1 Case 2 Fever 37.5 C + + Neurological manifestation + + Fatigue + + Consciousness impairment + + Rash + - +, present; -, absent; Neurological manifestation, including neurological deficit and consciousness impairment were recorded on day 10 after symptom onset in both cases. Neurological manifestations in case 1 included horizontal nystagmus, positive Romberg s sign, positive Kernig s sign, neck and extremities muscle stiffness with lively deep tendon reflexes, and urine retention. In this case qualitative (confusion, disorientation) and quantitative (somnolence) consciousness impairment were present, and mood changes in form of anxiety. Neurological manifestations in case 2 included ataxia, horizontal nystagmus, positive Romberg s sign, nuchal rigidity, and diminished deep tendon reflexes, with qualitative (confusion, disorientation) and quantitative (somnolence) consciousness impairment, and incoherent speech. Cerebrospinal fluid analysis results of these two cases are shown in Table 4. None of these patients developed respiratory failure requiring mechanical ventilation. Both patients recovered by the time they were discharged, case 2 had complete recovery, and case 1 still had 49

56 Medicinski Glasnik, Volume 12, Number 1, February 2015 mild horizontal nystagmus, positive Romber s sign and urine retention. Table 4. Cerebrospinal fluid analyses results of 2 patients with neuroinvasive West Nile virus infection Parameter Normal value/range Case 1 Case 2 WBC (per mm3) < WBC differential No predominance 60% L 62% N Protein (g/l) < Glucose (mmol/l) mmol/l WBC, white blood cells; L, lymphocytes; N, neutrophiles; DISCUSSION Taking in consideration that large WNV outbreaks happened in Eastern Croatia (6) and Serbia (10) in 2012, we analyzed appearance of the human neuroinvasive disease in B&H, performing a screening for patients hospitalized throughout summer and autumn 2013 for WNV infection with the aim to investigate presence of the virus in this location. Although only patients with neuroinvasive disease were tested, of whom two were positive, we assumed that there were more patients with WNV infection who had influenzalike febrile illness who were not tested, or who did not seek medical help due to the symptoms being too mild or were treated in primary care. This is a single-center study at regional level, and a large study at state level should be planned so that the incidence rates can be calculated. Both presented patients had clinical signs of encephalitis that could not be distinguished from the patients with encephalitis with a different etiology (14), but large sample case-control studies might reveal specific characteristic of encephalitis due to WNV infection (14). Serological diagnosis of flavivirus infections is complicated by the antigenic similarities among the Flavivirus genus because most flavivirus antibodies are directed against the highly immunogenic envelope protein, which contains both flavivirus cross-reactive and virus-specific epitope (1). Serological assay results should thus be interpreted with care and confirmed by comparative neutralization test using a panel of viruses known to REFERENCES 1. Beck C, Jimenez-Clavero MA, Leblond A, Durand B, Nowotny N, Leparc-Goffart I, Zientara S, Jourdain E, Lecollinet S. Flavivirus in Europe: Complex circulation patterns and their consequences for the diagnosis and control of West Nile Disease. Int J Environ Res Public Health 2013;10: circulate in Europe (1,15). Although there have been no previous reports of other flavivirus infections in B&H, the cases presented in this report had high titre of WNV specific antibodies in serum, and had negative flavivirus-vaccination history, and accordingly, they were defined as probable because we were not able to perform recommended testing for case conformation. Knowledge of flavivirus diversity at the local level is essential before rigorous serological surveys can be undertaken (1). Veterinary and entomologic investigations related to WNV and other flaviviruses have not been performed in B&H, so we can only assume from the published regional data (6-12) the circulating pattern of the virus in this area. Systematic research is needed to understand epidemiology and ecology of WNV in B&H. It is very important to develop a project to detect WNV in mosquitoes in urban regions and near rivers in various parts of the state, as well as surveillance of human cases. Considering the vectors and recent floods, a number of public health measures can be undertaken (intensifying activities to reduce the number of mosquitos in environments using insecticide, destroying mosquito habitat, systematic extermination of larvae and adult form of larvae and adult form of mosquitoes, education of the population on how to avoid or decrease the risk of being bitten by potentially infected mosquitoes through posters, leaflets, television, and newspapers) (16). These actions should help avoid outbreaks of human WNV infection and raise awareness of healthcare providers about emergence of the virus in B&H. In patients with mild influenza-like disease of unknown origin and those with neuroinvasive disease during late summer and early autumn, WNV should be considered a possible causative pathogen. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare. 2. Reiter P. West Nile virus in Europe: understanding the present to gauge the future. Eurosurvaill aspx?articleid= (3 Jun 2014) 3. Sithburn KC, Hughes TP, Burke AW Paul JH. A neutropic virus isolated from the blood of a native of Uganda. Am J Tro Med 1940; 20:

57 Ahmetagić et al. Human WNV infection in B&H 4. Murgue B, Murri S, Triki H, Deubel V, Zeller H. West Nile in Mediterranean basin: Ann N Y Acad Sci 2001; 951: Nowotny N, Bakonyi T, Weissenbock H, Seidel B, Kolodziejek J, Sekulin K, Lussy H. West Nile virus infection in Europe general features. Medica Sciences 2013; 39: Merdić E, Perić L, Pandak N, Kurolt IC, Turić N, Vignjević G, Stolfa I, Milas J, Bogojević MS, Markotić A. West Nile virus outbreak in humans in Croatia, Coll Antropol 2013; 37: Madić J, Huber D, Lugović B. Serologic survey for selected viral and rickettsial agents of brown bears (Ursus arctos) in Croatia. J Wildl Dis 1993; 29: Madić J, Savini G, Di Gennaro A, Monaco F, Jukić B, Kovac S, Rudan N, Listes E. Serological evidence for West Nile virus infection in horses in Croatia. Vet Rec 2007; 160: Barbić L, Listeš E, Katić S, Stevanović V, Madić J, Starešina V, Labrović A, Di Gennaro A, Savini G. Spreading of West Nile virus infection in Croatia. Vet Microbiol 2012; 159: Popović N, Milošević B, Urošević A, Poluga J, Lavadinović L, Nedelijković J, Jevtović D, Dulović O. Outbreak of West Nile virus infection among humans in Serbia, August to October Euro Surveill (11 July 2014) 11. Petrović T, Blazquez AB, Lupulović D, Lazić G, Escribano-Romero E, Fabijan D, Kapetanov M, Lazić S, Saiz J. Monitoring West Nile virus (WNV) infection in wild birds in Serbia during 2012: first isolation and characterisation of WNV strains from Serbia. Euro Surveill 2013; 31:18. pii: Lupulović D, Martín-Acebes MA, Lazić S, Alonso- Padilla J, Blázquez AB, Escribano-Romero E, Petrović T, Saiz JC. First serological evidence of West Nile virus activity in horses in Serbia. Vector Borne Zoonotic Dis 2011; 11: European Center for Disease Prevention and Control (ECDC). EU case definition. Stockholm: ECDC. aspx (10 October 2013) 14. Sejvar JJ. Clinical manifestations and outcomes of West Nile virus infection. Viruses 2014; 6: Sambri V, Capobianchi MR, Cavrini F, Charrel R, Donoso-Mantke O, Escadafal C, Franco L, Gaibani P, Gould EA, Niedrig M, Papa A, Pierro A, Rossini G, Sanchini A, Tenorio A, Varani S, Vázquez A, Vocale C, Zeller H. Diagnosis of West Nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies. Viruses 2013; 5: Radivojević S, Maris S, Ljubić B, Obrenović J. First detection of West Nile fever in human population in the territory of Belgrade. Proceedings Third International Epizootiology Days and XV Serbian Epizootiology Days, Niš/Serbia, May Faculty of veterinary medicine Belgrade, University of Belgrade, Department for infectious disease of animals and bee diseases, Belgrade. FVM; p vet.bg.ac.rs/uploads/file/seminari_simpozijumi/epizootioloski.dani.2013.zbornik.pdf (20 April 2014) Humana infekcija virusom Zapadnog Nila u Bosni i Hercegovini Sead Ahmetagić 1, Jovan Petković 1, Mirsada Hukić 2, Arnela Smriko-Nuhanović 1, Dilista Piljić 1 1 Univerzitetski klinički centar Tuzla, Tuzla, 2 International Burch University, Sarajevo; Bosna i Hercegovina SAŽETAK Cilj Prikazati prva dva slučaja neuroinvazivne infekcije uzrokovane virusom Zapadnog Nila (WNV) u Bosni i Hercegovini (BiH). Metode U Klinici za infektivne bolesti Univerzitetskog kliničkog centra Tuzla (BiH), u periodu od 1. avgusta do 31. oktobra godine, kod pacijenata kojima je postavljena dijagnoza neuroinvazivne infekcije provedena su specifična testiranja na WNV infekciju. Uzorci seruma testirani su na prisustvo specifičnih WNV IgM i IgG antitijela pomoću enzimsko-imunološkog testa (ELISA), a pozitivni uzorci seruma su dalje testirani ispitivanjem prisustva WNV nukleinske kiseline za dvije različite linije (linija 1 i linija 2) pomoću RT-PCR-a. Rezultati Samo tri pacijenta (od ukupno devet) zadovoljavala su postavljene kliničke kriterije, od kojih su dva imala visoke titrove WNV specifičnih IgM antitijela (3,5 i 5,2) u serumu. RT-PCR testovi iz istog seruma bili su negativni. Potvrdni test neutralizacije nije urađen. Oba slučaja imala su kliničku sliku encefalitisa. Pacijenti nisu boravili u područjima gdje je virus Zapadnog Nila endemičan, niti su primali transfuzije krvi ili transplantaciju organa, tako da predstavljaju autohtone slučajeve. Zaključak Iako nije bilo ranije prijavljenih flavivirusnih infekcija u BiH, u oba slučaja su u serumu nađena specifična WNV antitijela u visokom titru i u oba slučaja nije bilo prethodne vakcinacije protiv flavivirusa; oba slučaja su ostala nepotvrđena jer nije provedeno preporučeno testiranje za potvrdu slučaja. Virus Zapadnog Nila je prisutan u ovoj regiji te ga uvijek treba razmotriti kao potencijalnog patogena u pacijenata sa simptomima blage influence nepoznatog porijekla ili neuroinvazivne bolesti koji se javljaju u kasno ljeto i ranu jesen. Ključne riječi: neuroinvazivne infekcije, encefalitis, flavivirusi 51

58 ORIGINAL ARTICLE Incidence and etiological agents of genital dermatophytosis in males Asja Prohić, Mersiha Krupalija-Fazlić, Tamara Jovović Sadiković Department of Dermatovenerology, University Clinical Center Sarajevo, Sarajevo, Bosnia and Herzegovina ABSTRACT Aim To determine the incidence and etiological agents of dermatophytosis of male genitalia in Sarajevo area, Bosnia and Herzegovina, during a 5-year period ( ). Methods A total of 313 male patients with confirmed dermatophyte infection elsewhere in the body was analyzed. All samples (skin scrapings and hairs) were treated with lactophenol to detect a possible presence of fungal elements and then cultured on Sabouraud glucose agar. Dermatophytes species were identified based on macroscopic and microscopic morphology. Corresponding author: Asja Prohić Department of Dermatovenerology, University Clinical Center Sarajevo Bolnička 25, Sarajevo, Bosnia and Herzegovina Phone: ; Fax: ; asjaprohic@hotmail.com Results Dermatopyte infection of penis and/or scrotum was confirmed by positive cultures in 17 (5.4%) patients, of which four had lesions on the penis alone, five had lesions on scrotum and eight patients had lesions on both penis and scrotum. Majority of patients, 12 (70.0%) belonged to the age group Fifteen patients (88.2%) had associated foci of dermatophyte infection, but the inguinal area was most frequently affected, in 10 (66.6%) patients. Microsporum canis was the most frequent dermatophyte found on culture, in 10 (58.8%) patients. Conclusion Dermatophytosis of male genitalia is a rare entity, occurring more often in young males and the main causative species is Microsporum canis. Key words: tinea, male genitalia, incidence, etiology Original submission: 14 October 2014; Accepted: 24 October Med Glas (Zenica) 2015; 12(1):

59 Prohić et al. Dermatophytosis of the male genitalia INTRODUCTION Dermatophyte (tinea) infections of penis and scrotum are relatively rare compared with those involving the inguinal area (1). The infection usually extends from the inguinal area to the scrotum and uncommonly to the penis, but rarely occurs on the glans or prepuce (2). Different factors such as occlusive clothing, systemic diseases and a reservoir of dermatophytes at any other body site are considered to contribute to the occurrence of infections, which mainly affect young males (1,3,4). The incidence of tinea in males is traditionally considered to be very low (4-6). On the other hand, some studies report it as quite common (7-9). Moreover, there are different results regarding the causative dermatophyte species in this particular body site in males (4,7,9) These controversial opinions regarding the rarity of this entity incited us to undertake an investigation to find out incidence and etiological agents of male genitalia in individuals with dermatophytosis of the skin in population of Sarajevo region, Bosnia and Herzegovina. PATIENTS AND METHODS During a five-year period ( ), clinical presence of dermatophytosis of the penis and/ or scrotum has been studied in 313 male patients who reported the Department of Dermatovenerology, University Clinical Center Sarajevo, Bosnia and Herzegovina, with dermatophytosis elsewhere in the body. The samples were taken from a prominent border of the lesions and examined in the Mycological Laboratory of the Institute of Microbiology, Parasitology and Immunology, Sarajevo University Clinical Center. No patient gave any history of diabetes, tuberculosis or any other immunosuppressive condition in the recent past. Mycological examination consisted of direct microscopy and culture identification of causative agents. All scrapings were examined in 10% potassium hydroxide (KOH) solutions directly under the microscope and cultivated on Sabouraud glucose agar with added chloramphenicol and cycloheximide. The cultures were incubated at 27 o C for up to 6 weeks and observed weekly for evidence of growth. The identification of dermatophytes was based on their macroscopic and microscopic characteristics of the colonies (10). RESULTS Among 313 male patients who attended the Department of Dermatovenerology, University Clinical Center Sarajevo, Bosnia and Herzegovina, with dermatophytosis elsewhere in the body, KOH preparations of the skin scraping from the lesions showed mycelia of dermatophyte in 17 (5.4%) patients. The causative dermatophyte species was identified by positive cultures in 17 (5.4%) patients. Microsporum (M.) canis was the most frequent dermatophyte isolated, in 10 (58.8%), followed by Epidermophyton (E.) floccosum, in five (29.4) and Trichophyton (T.) mentagrophytes var. interdigitalis in two (11.8%) patients. Majority of the patients with positive culture findings, 12 (70.0%) belonged to the age group years, followed by years age group, three (17.7%) patients. No patients belonged to age group below 10 or above 51 years of age (Table 1). Table 1. Age distribution of patients with genital dermatopytosis Age group (years) No (%) of patients (5.8) (70) (17.7) (5.8) 51 and above 0 Of the 17 patients with positive culture findings, penile dermatophytosis was found in four (23.5%) patients, scrotal lesions in five (29.4%) patients, both penile and scrotal lesions in eight (47.0%) patients. None of the patients had lesions on the glans or prepuce. Wives of the six married patients (35.3%) were examined and found to be free from genital infection. Solitary involvement of the penis or scrotum without the involvement of the crural region or any other regions was found in two (11.8%) patients. Fifteen (88.2%) patients showed tinea infection at other anatomical sites of whom ten (66.7%) patients presented with tinea cruris, four (26.7) with tinea unguium and one (6.7) patient with tinea pedum (Table 2). Table 2. Sites of involvement in patients with genital dermatopytosis Site of the lesion No (%) of patients Penis 4 (23.5) Scrotum 5 (29.4) Penis and scrotum 8 (47.0) Other sites of dermatophytosis 15 (88.2) Solitary genital involvement 2 (11.8) 53

60 Medicinski Glasnik, Volume 12, Number 1, February 2015 DISCUSSION Dermatophytosis of male genitalia has been regarded as a rare entity since its first description in 1860 by Ferdinand Ritter von Hebra ( ), a founder of the Vienna School of Dermatology (11). So far the same opinion has been supported by many other investigators (4-6). A study conducted in Italy has shown low frequency because the authors reported nine cases of male tinea genitalis over a period of fifteen years (4). In another study from India, of 2200 patients with confirmed dermatophytosis, approximately 1% had penile involvement (8). Some authors attributed rarity of the genital involvement to capric acid and some fungistatic serum factor and sebum (12). Contrary to this, some studies from different researchers showed that dermatophytosis of male genitalia is quite common, particularly from tropical countries, where the reported incidence was 21% (7). Similarly, Gupta and Banerjeer reported six cases of dermatophytes of male genitalia in a short period of three months (9). Our observation shows an incidence of 5.4% of all examined patients to be a relatively rare entity. The low incidence in our study, but also in aforementioned studies, may be due to difference in climate and socioeconomic condition. Climate plays an important role and the higher incidence of the infection in the tropics has been noted during the rainy season of a year when humidity was more than 95% (7). The low incidence could be also attributed to the fact that the disease could go unnoticed because the clinical manifestations are slight and healing is often spontaneous (9). In this study most of the patients with penile and/ or scrotal dermatophytosis (70.37%) belonged to the age group 21-40, which is in concordance with the majority of the studies, which found that young males were most susceptible to the dermatophyte infection of genital region (7,13). Surprisingly no patients belonged to age group below 10 or above 50 years of age which was also abserved by Pandey et al. (14) and Vora et Mukhopadhyay (7). Further investigations are required particularity in this age group. The presence of tinea infection at other anatomical sites may serve as a reservoir of infection. Especially tinea cruris, tinea pedis and toenail onychomycosis are common sources of infection of male genitalia (7,8,14,15). In our study, the lesions situated on penis and scrotum were preceded by dermatophytosis in inguinal area in 58.8% patients, which indicates that the infection probably started in the crural region and spread later to the genitalia. Glans penis involvement is considered even rarer (16), whereas dermatophytosis of the prepuce has not been reported in the literature. In our case series, none of the patients had lesions on glans and/or prepuce, which supports earlier reports. Solitary involvement of the penis or scrotum without the involvement of any other regions was found in 11.8 % males, which indicates the possibility that penis and scrotum may be infected with dermatophytosis de novo without involvement of any other area (2,6,17). Our study has confirmed zoophilic dermatophyte M. canis as being the most common species isolated from genital area. However, most studies reported T. rubrum as a common pathogen (4,7,9,15,17,18), although E. floccosum and T. mentagrophytes var. interdigitale have also been isolated from the genital region (4,7,17). High isolation rate of M. canis from the genital region (58.8%) correlates with the high frequency of this species in our population in general (19). In the last fifteen years, an increasing incidence of zoophilic dermatophytes, especially M. canis has been observed in many regions in Europe (20). Generally, the prevalence of M. canis in our population is one of the highest in Europe (90.4) (20) and is comparable only with rates reported from Italy (90.5) (21), Grace (84.5%) (22) and Spain (63.5%) (23). The high prevalence of this dermatophyte species can be attributed to the increase in the number of domestic animals particularly cats living outside homes and consequently an increase in the phenomenon of animals stray and semistray (24). Presumably stray cats are the major reservoir and carriers of M. canis. It is widely accepted that dermatophytes are keratinophilic in nature and they invade their host by enzymatic digestion of keratin. This can explain the rarity of tinea infections on male genitalia, since anatomically the glans penis and inner surface of the prepuce are covered with non-keratinized epithelium. Overweight, occlusion, high moisture, inadequate hygiene, diabetes, previous antibiotic treatment and immunosuppression are common predisposing factors. 54

61 Prohić et al. Dermatophytosis of the male genitalia In tinea of the genital region individual treatment approach is needed, considering both local and systemic susceptibility factors. Topical antifungal treatment is usually sufficient in acute infections. In many cases, systemic treatment with either azoles or terbinafin may be needed in cases of widespread dermatophytic infection (25). To conclude, our results suggest that dermatophytosis of male genitalia with an incidence of 5.4% is rare mycotic infection of this particular REFERENCES 1. Nenoff P, Krüger C, Schaller J, Ginter-Hanselmayer G, Schulte-Beerbühl R, Tietz HJ. Mycology - an update Part 2: Dermatomycoses: Clinical picture and diagnostics. J Dtsch Dermatol Ges 2014; 12: Dekio S, Jidoi J. Tinea of the glans penis. Dermatologica 1989; 178: Aridogan IA, Izol V, Ilkit M. Superficial fungal infections of the male genitalia: a review. Crit Rev Microbiol 2011; 37: Romano C, Ghilardi A, Papini M. Nine male cases of tinea genitalis. Mycoses 2005; 48: Arnold HL, Jr, Odom RB, James WD. Andrews Diseases of the Skin-Clinical Dermatology (8th ed.). Philadelphia: WB Saunders Co., Palleschi GM, Guadagni R, Difonzo E, Panconesi E. Tinea of the penis: a rare occurrence. Internat J Dermatol 1986; 25: Vora NS, Mukhopadhyay AK. Incidence of dermatophytosis of penis and scrotum. Indian J Dermatol Venerol Leprol 1994; 60: Kumar B, Talwar P, Kaur S. Penile tinea. Mycopathologia 1981; 75: Gupta R, Banerjee U. Tinea of the penis. Ind J Dermatol Venereol Leprol 1992; 58: Moriello KA. Diagnostic techniques for dermatophytosis. Clin Tech Small Anim Pract 2001; 16: La Touche CJ. Scrotal dermatophytosis : an insufficiently documented aspect of tinea cruris. Brit J Dermatol 1967; 79: Pavitran K. Dermatophytosis of the scrotum, penis and lip. Ind J Dermatol Venereol Leprol 1987; 53: Phadke SN, Gupta DK, Agarwal S. Dermatophytosis in Jabalpur. Ind J Pathol Bacteriol 1973; 16:42. body site. The infection affects more often young males, occurring more often on the penis and scrotum at the same time. Zoophyte species M. canis is the most frequently isolated dermatophyte. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare 14. Pandey SS, Chandra S, Guha PK, Kaur P, Singh G. Dermatophyte infection of the penis. Association with particular undergarments. Internat J Dermatol 1981; 20: Pielop J, Rosen T. Penile dermatophytosis. J Am A Dermatol 2001; 44: D Antuono A, Bardazzi F, Andalou F. Unusual manifestation of dermatophytosis. Int J Dermatol 2001; 40: Das JK, Sengupta S, Gongopadhyay A. Dermatophyte infection of the male genitalia. Indian J Dermatol 2009; 54: Mukhopadhyay AK. Trichophyton rubrum infection of the prepuce. Indian J Dermatol Venereol Leprol 2005; 71: Prohić A. Prevalence of zoophilic dermatophytes in Sarajevo area. Med Arh 2003; 57: Skerlev M, Miklić P. The changing face of Microsporum spp. infections. Clin Dermatol 2010; 28: Romano C. Tinea capitis in Siena, Italy. An 18-year survey. Mycoses 1999; 42: Frangoulis E, Athanasopoulou B, Katsambas A. Etiology of tinea capitis in Athens, Greece - a 6-year ( ) retrospective study. Mycoses 2004; 47: del Boz J, Crespo V, Rivas-Ruiz F, de Troya M. A 30-year survey of paediatric tinea capitis in southern Spain. J Eur Acad Dermatol Venereol 2011; 25: Proverbio D, Perego R, Spada E, Bagnagatti de Giorgi G, Della Pepa A, Ferro E. Survey of dermatophytes in stray cats with and without skin lesions in Northern Italy. Vet Med Int 2014; 2014: Pires CA, Cruz NF, Lobato AM, Sousa PO, Carneiro FR, Mendes AM. Clinical, epidemiological, and therapeutic profile of dermatophytosis. An Bras Dermatol 2014; 89:

62 Medicinski Glasnik, Volume 12, Number 1, February 2015 Incidenca i etiološki agensi genitalne dermatofitije kod muškaraca Asja Prohić, Mersiha Krupalija-Fazlić, Tamara Jovović Sadiković Klinika za kožne i spolne bolesti, Klinički centar Univerziteta u Sarajevu, Sarajevo, Bosna i Hercegovina SAŽETAK Cilj Utvrditi incidencu i etiološke agense genitalne dermatofitoze kod muškaraca na području Sarajeva, u petogodišnjem periodu (od do godine). Metode Analizirano je ukupno 313 muškaraca s potvrđenom dermatofitnom infekcijom druge lokalizacije. Svi uzorci (ljuske s kože i dlake) preparirani su u laktofenolu radi otkrivanja eventualno prisutnih gljivičnih elemenata i zatim kultivirani na Sabouraud glukoza agaru. Rezultati Dermatofitna infekcija penisa i/ili skrotuma potvrđena je pozitivnom kulturom kod 17 (5,4%) pacijenata, od kojih je četiri imalo promjene samo na penisu, pet na skrotumu, dok je njih osam imalo promjene na penisu i skrotumu. Većina pacijenata, 12 (70%), pripadala je dobnoj skupini od 21 do 40 godina. Petnaest (88,2%) pacijenata imalo je dermatofitnu infekciju druge lokalizacije, od kojih je najčešće bilo zahvaćeno ingvinalno područje, 10 (66,6%). Microsporum canis bio je najčešće izolirani specijes u kulturi, 10 (58,8%). Zaključak Dermatofitoza muških genitalija je rijetka pojava, češće prisutna kod mlađih muškaraca, a glavni uzročnik je Microsporum canis. Ključne riječi: tinea, genitalije muškarca, incidenca, etiologija 56

63 ORIGINAL ARTICLE Surgical therapy for pilonidal sinus in adolescents: a retrospective study Tamer Sekmenli, Ilhan Ciftci Department of Pediatric Surgery, School of Medicine, Selcuk University, Konya, Turkey ABSTRACT Aim To investigate optimal surgical management of pilonidal sinus (PS) in pre-adolescent and adolescent periods in which it is less common. Methods A retrospective study based on 25 adolescent patients with PS disease that were treated at the Department of Pediatric Surgery, Selcuk University, Konya, Turkey, between 2010 and 2013 was conducted. Corresponding author: Ilhan Ciftci Selcuk Universitesi Tip Fakultesi Cocuk Cerrahisi A. D. Konya, Turkey Phone: ; Fax: ; driciftci@yahoo.com Results Among 25 patients with PS disease 17 (68%) were males and eight (32%) females, with a mean age of ± 1.2 years. According to the body mass index (BMI), 17 (68%) of the cases were in the normal range, 4 four (16%) of each were overweight, and obese, respectively. Twenty cases (80%) were managed with total excision and primary closure, while five (20%) cases underwent Limberg flap repair. Four patients were managed with the Limberg procedure due to very large sinuses, while a single patient underwent the procedure due to a recurrence following primary repair. Conclusion Factors that predispose patients to developing PS include an above average BMI, significant body hair, and prolonged time in a seated position. Elevated BMI as a risk factor for PS is a growing concern given the rise in obesity. Although there are various treatment modalities, total excision and primary closure has demonstrated successful outcomes. Key words: hair, surgery, adolescent, follow-up Original submission: 30 September 2014; Revised submission: 04 November 2014; Accepted: 17 December 2014 Med Glas (Zenica) 2015; 12(1):

64 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION Pilonidal sinus (PS) was first described in 1883 by Mayo, and further characterized by Hodges in 1880 (1,2). Pilonidal disease is an acquired skin condition that develops in the sacrococcygeal area or other hair-bearing areas, and is characterized by an epithelialized tract (the sinus) generally containing hair (3,4). Although PS can occur at any age, it is known to be less common during the pre-adolescent and adolescent periods (5). Pilonidal disease can have a dramatic impact on activities of daily living and quality of life (5). In the past, it was thought to be a congenital skin disease, but it is now considered to be an acquired condition that results from burrowing of loose hair shafts into vulnerable skin (5). Although pilonidal disease is not uncommon in adolescents, there are very few publications concerning PS in this age group. There is an ongoing debate regarding the optimal surgical management of PS. Various approaches to treatment have been suggested, ranging from conservative, nonsurgical treatments, to extensive resections (1,6,7). There is no clear evidence to identify the best method, and the majority of the literature pertains to children (6,7). The fact that there are multiple surgical procedures in use suggests that the optimal technique remains to be determined (6,7). The aim of this study was to describe our experience with PS in adolescents with specific attention to complications and management strategies. PATIENTS AND METHODS Adolescents patients who were treated for PS at the Pediatric Surgery Department, Selcuk University Medicine Scholl Hospital between January 2010 and June 2013 were retrospectively analyzed. Patients were categorized according to sex, age, body mass index (BMI), amount of body hair, and daily duration of sitting. Patients were managed with either excision and primary closure or Limberg flap repair. Patients who presented with infected PS were treated 7 to 10 days with antibiotics after drainage, if necessary. The degree of hirsutism was quantified using the Ferriman-Gallwey scale and prolonged sitting was defined as four or more hours per day. RESULTS Adolescents with pilonidal disease who presented between January 2010 and June 2013, were evaluated. Seventeen (68%) patients were males and eight (32%) were females, with a mean age of ± 1.2. According to the BMI, 17 (68%) patients were in the normal range (average BMI 24.8), four (16%) were considered overweight (average BMI 28.2), and four (16%) were classified as obese (average BMI 31). Regarding body hair, 12 (48%) patients had a moderate amount, while 13 (52%) had a high degree of body hair. Eighteen (72%) patients reported spending four or more hours a day in a seated position. Twenty (80%) patients were managed with total excision and primary closure, while five (20%) underwent Limberg flap repair. The Limberg procedure was chosen for five patients: four had particularly large sinuses and one had a recurrence after initially being managed with primary repair. No recurrences have been identified in follow-up. DISCUSSION Pilonidal disease is a common, acquired disease that mainly affects active young adults and adolescents (2). This study is limited to sacrococcygeal pilonidal pathology although the disease process can occur elsewhere, particularly around the finger webbing in hair dressers and shearers and breasts of wool handlers in shearing sheds (2). Hair (wool) insertion is the essential cause of the disease (5). The peak incidence of pilonidal disease is in years of age (2). Studies have shown that 38% of PS patients have a family history of pilonidal disease. Caucasians get PS more frequently than other races (8). In our study 50% of patients had normal body weight and 37% were overweight. Patients with a high body mass index have an increased risk of recurrence after surgery (9). Weight excess may be a risk factor for PS (10,11). It is notable that one patient in our study who had a recurrence after primary closure was overweight. Patients with repeated local trauma and occupations that require prolonged sitting have a higher prevalence of pilonidal disease (10). Of our patients, 68% had normal BMI, the daily sitting rate of more than 58

65 Sekmenli et al. Pilonidal sinus in adolescents four hours was 72%, and more than half of the patients in our series had a high degree of body hair. As the whole patient series in this study consisted of students sitting for extended periods while studying, time seated is considered to be a risk factor. Prolonged local trauma for PS has been shown to be a risk factor (12). Three factors are thought to combine in the pathogenesis of PS: loose hair, some force causing penetration of hair into the skin, and vulnerability of the skin to insertion of the hair at the natal cleft (13). One hair penetrating the dermis may create an environment in which other hairs can penetrate more easily. The resulting foreign body reaction develops into PS (13). Lifestyle changes may address the first two factors. Hair removal may help, and weight loss results in a natal cleft that is not as deep and has less friction (13). One study demonstrated successful management of pilonidal disease on an outpatient basis, because hospital admissions were decreased by 78% when patients began instituting a conservative strategy that emphasized hygiene and meticulous shaving (14). There are different treatment approaches, and although medicine treatment is an option, it is not preferred as it takes a long time and its efficacy is hard to establish (14). In medicine therapy for PS the use of phenol has been proposed in the literature (15,16). A disadvantage of using phenol is prolonged treatment. Surgical intervention is initially aimed at removing pits and sinuses, and debriding any infected tissue by excision (17). One large series of 78,924 American soldiers during the Second World War reported hospitalization time of 55 days, mainly due to complications resulting from a wide excision (18). This is because wide excision typically results in large defects that require healing by secondary intention. This resulted in an open wound that was, theoretically, susceptible to further invasion by loose hairs (13). We have used the technique of en bloc excision of the sinus or sinuses with the surrounding healthy tissue down to the presacral fascia and primary closure of the wound. It is an uncomplicated technique that can be easily learned as it does not require the creation of flaps. There are several advantages to primary closure. The procedure is easy to perform and does not require significant experience. As such, it can be performed quickly. In addition, there is little postoperative pain, early wound healing, and an early return to normal activities. Two of the major complications that can occur after primary closure are wound site infection and wound dehiscence. Neither of these complications, however, occurred in our patient series. The average healing period of PS was days following excision or marsupialization and leaving the site completely open. During this period, dressing changes should be performed regularly with the defect expected to heal with granulation tissue. In this case, the result has a poor cosmetic appearance. When flap methods are compared with primary closure, considerable cosmetic scar tissue develops in the gluteal area. This was demonstrated in the study in which the Limberg flap method was used and displeasure regarding the cosmetic outcome was expressed and considered to be a significant disadvantage (19). In conclusion, until now, the etiology of pilonidal disease has not been clarified although it has been discussed in many studies (1,2,10,11). In this study, excessive hair growth, obesity, and prolonged sitting have increased the incidence of PS. Adolescents with these risk factors ought to be examined routinely. In surgical treatment, the benefits of primary closure include ease of performance, minimal postoperative pain, early wound healing, and early return to work, with acceptable recurrence rates. Thus, excision and primary closure may be the preferred approach in adolescents with PS. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare 59

66 Medicinski Glasnik, Volume 12, Number 1, February 2015 REFERENCES 1. Humphries AE, Duncan JE. Evaluation and management of pilonidal disease. Surg Clin N Am 2010; 90: Lee PJ, Raniga S, Biyani DK, Watson AJ, Faragher IG, Frizelle FA. Sacrococcygeal pilonidal disease. Colorectal Dis 2008; 10: Mentes O, Bagci M, Bilgin T, Ozgul O, Ozdemir M. Limberg flap procedure for pilonidal sinus disease: results of 353 patients. Langenbecks Arch Surg 2008; 393: Ersoy E, Devay AO, Aktimur R, Doganay B, Ozdoğan M, Gündoğdu RH. Comparison of the short-term results after Limberg and Karydakis procedures for pilonidal disease: randomized prospective analysis of 100 patients. Colorectal Dis 2009; 11: Hull TL, Wu J. Pilonidal disease. Surg Clin N Am 2002; 82: Daphan C, Tekelioglu MH, Sayilgan C. Limberg flap repair for pilonidal sinus disease. Dis Colon Rectum 2004; 47: Akin M, Gokbayir H, Kilic K, Topgul K, Ozdemir E, Ferahkose Z. Rhomboid excision and Limberg flap for managing pilonidal sinus: long-term results in 411 patients. Colorectal Dis 2008; 10: de Parades V, Bouchard D, Janier M, Berger A. Pilonidal sinus disease. J Visc Surg. 2013; 150: Varnalidis I, Ioannidis O, Paraskevas G, Papapostolou D, Malakozis SG, Gatzos S, Tsigkriki L, Ntoumpara M, Papadopoulou A, Makrantonakis A, Makrantonakis N. Pilonidal sinus: a comparative study of treatment methods. J Med Life 2014; 7: Sakr M, El-Hammadi H, MoussaM, Arafa S, Rasheed M. The effect of obesity on the result of Karydakis technique for the management of chronic pilonoidal sinus. Int J Colorectal Dis 2003; 18: Arda IS, Güney LH, Sevmiş S, Hiçsönmez A. High body mass index as a possible risk factor for pilonidal sinus disease in adolescents. World J Surg 2005; 29: Søndenaa K, Andersen E, Nesvik I, Søreide JA.Patient characteristics and symptoms in chronic pilonidal sinus disease. Int J Colorectal Dis 1995; 10: Karydakis GE. Easy and successful treatment of pilonidal sinus after explanation of its causative process. Aust N Z J Surg 1992; 62: Armstrong JH, Barcia PJ. Pilonidal sinus disease. The conservative approach. Arch Surg 1994; 129: Girgin M, Kanat BH. The results of a one-time crystallized phenol application for pilonidal Sinus disease. Indian J Surg 2014; 76: Gulpinar K, Pampal A, Ozis SE, Kuzu MA. Nonoperative therapy for pilonidal sinus in adolescence: crystallised phenol application, report of a case. BMJ Case Rep 2013; 3: Petersen S, Koch R, Stelzner S, Wendlandt TP, Ludwig K. Primary closure techniques in chronic pilonidal sinus: asurvey of the results of different surgical approaches. Dis Colon Rectum 2002; 45: Abramson D. Outpatient management of pilonidal sinuses: Excision and semiprimary closure technique. Milit Med 1978; 143: Eryılmaz, R, Sahin M, Alimoglu O, Daşıran F. Surgical treatment of sacrococcygeal pilonidal sinus with the Limberg transposition flap. Surgery 2003; 134:

67 ORIGINAL ARTICLE Impacts of education level and employment status on healthrelated quality of life in multiple sclerosis patients Selma Šabanagić-Hajrić, Azra Alajbegović Department of Neurology, Clinical Center of the University of Sarajevo ABSTRACT Aim To evaluate the impacts of education level and employment status on health-related quality of life (HRQoL) in multiple sclerosis patients. Corresponding author: Selma Šabanagić-Hajrić Department of Neurology, Clinical Center, University of Sarajevo Bolnička 25, Sarajevo, Bosnia and Herzegovina Phone: ; Fax: ; E mail: selmahajric@gmail.com Original submission: 08 September 2014; Revised submission: 07 October 2014; Accepted: 22 October Methods This study included 100 multiple sclerosis patients treated at the Department of Neurology, Clinical Center of the University of Sarajevo. Inclusion criteria were the Expanded Disability Status Scale (EDSS) score between 1.0 and 6.5, age between 18 and 65 years, stable disease on enrollment. Quality of life (QoL) was evaluated by the Multiple Sclerosis Quality of Life-54 questionnaire (MSQoL-54). Mann-Whitney and Kruskal-Wallis test were used for comparisons. Linear regression analyses were performed to evaluate prediction value of educational level and employment status in predicting MSQOL-54 physical and mental composite scores. Results Full employment status had positive impact on physical health (54.85 vs ; p<0.001) and mental health (59.55 vs ; p<0.001) composite scores. Employment status retained its independent predictability for both physical (r 2 =0.105) and mental (r 2 =0.076) composite scores in linear regression analysis. Patients with college degree had slightly higher median value of physical (49.36 vs ) and mental health composite score (66.74 vs ) comparing to others, without statistically significant difference. Conclusion Employment proved to be an important factor in predicting quality of life in multiple sclerosis patients. Higher education level may determine better QOL but without significant predictive value. Sustained employment and development of vocational rehabilitation programs for MS patients living in the country with high unemployment level is an important factor in improving both physical and mental health outcomes in MS patients. Key words: QOL, physical health, mental health Med Glas (Zenica) 2015; 12(1):

68 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION Multiple sclerosis (MS) is a chronic and unpredictable neurological disease varying from a mild course with minimal disabilities to a rapidly progressing or fluctuating course resulting in severe disabilities (1). The associations between employment status and the incidence and prevalence of chronic diseases and mortality have been previously discussed (2-4). Evidence regarding whether two different pathways, employment health status or health status employment, actually do exist in the chronically ill patients has been described in an extensive review by Clougherty (2). Based on the current literature, employment status is analyzed in relation to disease-specific, therapeutic, psychosocial and socioeconomic factors with special emphasis on the vocational status of MS patients (5). On cognitive testing, unemployed individuals performed significantly worse on measures assessing information processing speed, verbal learning and memory and executive functioning (6). While the physical disability is well recognized in MS patients, it does not necessarily reflect all aspects which patients should consider as important once in their life. Although fatigue, depression, and physical disability are usually experienced aspects of MS patients, it is also documented that cognitive, emotional, and psychological functions contribute to their quality of life (QoL) (7). Today, the Neurology Quality-of-Life Measurement Initiative is a standardized approach based on extant items used for measuring QoL across common neurologic conditions, including multiple sclerosis, for both adults and children (8,9). Using QoL measures may provide clinicians with the information regarding the general health status of MS patients who might otherwise go unrecognized with high recommendations for using them in clinical practice (10). Several questionnaires have been developed to evaluate aspects of health related quality of life (HRQoL) in MS patients. There is a distinction between studies using generic HRQoL inventories, and the studies with disease specific instruments, such as the Multiple Sclerosis Quality of Life-54 questionnaire (MSQoL-54), which comprises the SF-36 and 18 additional MS specific items (11). A large number of studies have demonstrated that patients with MS have a poorer HRQoL than persons from the general population, as MS occurs in people during the peak years of employment (12,13). The disease significantly impacts the ability to remain in the workforce with data suggesting high rates of unemployment and early retirement in MS patients (14,15). Considering that employment is a central aspect of one s standard of living or for forming relationships, of individuals personality and self-esteem, loss of paid work is associated with financial, social and mental health implications for patients and their families, leading to a significant decline of patients quality of life (QoL) (16-18). Data indicates that majority of US MS patients (90%) were employed before the diagnosis, and about 60% continued to work at the time of the diagnosis (13). Only 20-40% of patients with MS, however, remain employed following the diagnosis (19,20). It is shown that onset of early retirement was about three years after receiving a diagnosis of multiple sclerosis at an average age of 38 years (21) and rates of early retirement due to MS are about 40% (22,23). Previous studies showed that unemployed patients reported low scores in some HRQoL domains, such as physical role limitation, physical function, and emotional limitation (24). In addition, education seems to have some impact on HRQoL. High school or college graduates had higher physical health composite scores than patients with a low educational level (25). The aim of this study was to evaluate the effects of education level and employment status on HRQoL in MS patients at different stage of the disease treated at the Clinical Center of the University of Sarajevo, Bosnia and Herzegovina. A purpose of the study was to evaluate importance of sustained employment and development of vocational rehabilitation programs for MS patients living in the country with high unemployment level. This is the first study of this type conducted in Bosnia and Herzegovina. The results are expected to provide important information to medical professionals and people from an immediate environment how to best participate in treating and taking care for persons with MS. PATIENTS AND METHODS Study design This independent, observational, cross-sectional study included 100 consecutive patients with 62

69 Šabanagić-Hajrić et al. Education and employment on QoL in MS multiple sclerosis treated at the Department of Neurology, Clinical Center University of Sarajevo, Bosnia and Herzegovina, during the period January 1st to July 15th Inclusion criteria for the study were: clinically defined diagnosis of MS according to previously published recommendations (26), Expanded Disability Status Scale (EDSS) score between 1.0 and 6.5 (27), 18 years of age or older and patients who were able to give written informed consents. Exclusion criteria were cognitive deterioration (Mini Mental Status Test Score <26) (28), and the presence of any acute somatic or neurological disease. Having evaluated the protocol the Ethics Committee of the Clinical Center of the University of Sarajevo gave an ethical consent to have the study the performed. Each patient gave an informed written consent for use of the results obtained for publication before the enrollment. Instruments Patients reported their QoL using a self-administered questionnaire. The disease-specific Multiple Sclerosis Quality of Life-54 (MSQoL-54) questionnaire developed by Vickrey et al. was used (11); it was translated and adapted to the Bosnian language (29). This questionnaire consists of 18 MS-specific dimensions and ratings for overall QoL (MS-18 module), in addition to the generic QoL features of the Short-Form 36-Item Health Survey Questionnaire (SF-36) (30) to obtain the MSQoL in reference to the following domains: physical health composite score (PHCS), mental health composite score (MHCS), physical function (PF), role limitation-physical (RP), emotional wellbeing (EWB), mental health (MH), role limitation-emotional (RE), bodily pain (BP), energy (EN), health perception (HP), social function (SF), change in health (CH), health distress (HD), cognitive function (CF), sexual function (SxF), satisfaction with sexual function (SSxF), and overall quality of life (OQoL). The MSQoL-54 item results are transformed linearly to scores, and final scores are calculated by averaging items within the scales. Statistical analyses Statistical analyses were performed for patients satisfying all the inclusion criteria. Demographic parameters and other baseline characteristics were summarized. The Linkert method was adopted to assemble MSQoL-54 scale scores and the raw scores were transformed into scales. Mann-Witney and Kruskal Wallis tests were used for comparisons between sociodemographic and clinical characteristics and QOL scores. Linear regression analyses were performed to evaluate prediction value of educational level and employment status in predicting MSQOL-54 physical and mental composite scores, with a significance level of p<0.05. RESULTS The mean age of patients at enrolment was / years; 69 (69%) patients were females and 31 (31%) were males. The majority of patients, 64 (64%) were married. The mean disease duration at enrolment was 9.39+/ years. The majority of patients, 72 (72%) had relapsing-remitting MS at the time of enrolment, 25 (25%) had secondary progressive type while only three (3%) patients had primary progressive type of the disease. Table 1. Multiple Sclerosis Quality of Life questionnaire (MSQOL)-54 scores according to employment status Median value of physical health composite score (25th-75th percentile) MSQQOL-54 domain Employed Unemployed p (n=38) (n=62) Physical function ( ) ( ) Role limitationsphysical ( ) ( ) <0.001 Role limitations emotional ( ) ( ) <0.001 Pain ( ) ( ) Emotional wellbeing ( ) ( ) Energy ( ) ( ) Health perceptions ( ) ( ) <0.001 Social function ( ) ( ) <0.001 Cognitive function ( ) ( ) <0.001 Health distress ( ) ( ) <0.001 Overall quality of life ( ) ( ) Sexual function ( ) ( ) Satisfaction with sexual function ( ) ( ) Change in health ( ) ( ) Physical health composite score ( ) ( ) <0.001 Mental health composite score ( ) ( ) <

70 Medicinski Glasnik, Volume 12, Number 1, February 2015 The mean EDSS score of all patients at enrolment was 3.57+/ In terms of education, 67 (67%) of patients had a high school degree, 23 (23%) had a college degree while only 10 (10%) had primary education. Regarding occupation, 38 (38%) patients were employed, six (6%) were students, 25 (25%) patients were unemployed, and 31 (31%) retired. Employed patients scored significantly higher than unemployed patients in all MSQOL-54 domains (Table 1). Employed patients had statistically significant higher median value of physical health composite score (66.70 vs ; p<0.001) and mental health composite score (72.60 vs ; p<0.001) comparing to unemployed patients. Patients with college degree scored better than those with secondary and primary education in most domains, e.g. role limitations emotional, body pain, emotional well-being, health perceptions, social function, cognitive function, health distress, overall quality of life, satisfaction with sexual function, change in health-ch, physical health composite score-phcs, mental health composite Table 2. Multiple Sclerosis Quality of Life questionnaire (MSQOL)-54 scores according to education level MSQOL-54 domain Physical function Role limitationsphysical Role limitations emotional Pain Emotional well-being Energy Health perceptions Social function Cognitive function Health distress Overall quality of life Sexual function Satisfaction with sexual function Change in health Physical health composite score Mental health composite score Median (25th-75th percentile) High school degree (n=23) Primary and secondary school (n=77) p ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) score-mhcs, but without statistically significant difference (p>0.05). Patients with secondary and primary education scored better than the group with college degree only in sexual function domain, also without statistically significant difference (p>0.05) (Table 2). Patients with college degree had slightly higher median value of physical health composite score (49.36 vs ) and mental health composite score (66.74 vs ) comparing to unemployed patients, without statistically significant difference (p>0.05). Stepwise linear regression analysis examining the prediction value of demographic and clinical characteristics of all patients in predicting MSQOL-54 physical and mental health composite scores is shown in Table 3. Employment status proved its independent predictability of physical health composite score, PHCS (r 2 =0.105) and mental health composite score, MHCS (r 2 =0.076). Table 3. Stepwise linear regression analysis: predictors of quality of life on the MSQOL-54 MSQOL-54 Significant predictors Eta squared R2 Presence of pain EDSS MSQOL-physical Sphincteric disorders Employment status Disease type Patient age Presence of pain MSQOL-mental Disease type Employment status EDSS Eta squared, correlation ratio; EDSS, enabled disability status scale; R2, coefficient of determination; DISCUSSION The age and employment status of patients in this study is consistent with the fact that MS often strikes young adults and working people at the top of their careers and that increasing disability makes finding a job becomes more difficult (31). According to the literature, 67% of those diagnosed with MS for less than 5 years change job status, and 45% switch to a different field; approximately 21% of patients with MS for less than 5 years are unemployed, versus 92% of those with MS for at least 30 years (32). Patients with multiple sclerosis with good selfrated health are more likely to be employed, even after adjusting for age, gender, education, functional disability, disease duration, depression and 64

71 Šabanagić-Hajrić et al. Education and employment on QoL in MS anxiety (33). Studies with similar percentage of employed patients to this study showed statistical significant influence of employment on both physical and mental health composite scores (1,11). Employed patients in this study scored significantly higher than unemployed patients in all MSQOL-54 domains. In other studies with higher level of employment, employed patients did not scored significantly better in health perception and emotional well-being (18), and did not score significantly better in mental health domains at all (34). Those findings implicate higher influence of unemployment on mental health in this study that could be partially explained by poor mental health status among unemployed patients. Results from German study showed that in patients with minor motoric impairment, depressive symptoms seem to have a major impact on employment status (5). The lack of flexible working hours, inability to have flexible resting times at work, lack of understanding from colleagues and employers, as well as the personal attitude were main non-disease-specific reasons for early retirement (5). Greater physical and cognitive disability, progression of disease, longer disease duration, and older age are considered to be the factors associated with unemployment (35). Other studies on employment in MS have shown that those patients who remain employed have better selfreported quality of life (1,36). Being employed was also positively associated with physical HRQOL (37) with findings that for MS patients, regardless of disability level, increased physical activity is related to better HRQOL in terms of energy, social functioning, mental and physical health (38). The results of this study clearly suggest the importance of sustained employment after the MS diagnosis. The condition of being employed with a social role and salary seems to improve the QoL of patients diagnosed with MS. Employment rate among patients with MS is variable, and can partly depend on economic status of different countries, as demonstrated by previous studies (24, 39-41). Majority of patients in this study had a high school and college degree, which is in accordance with data from the literature indicating that more than 50% of MS patients have high school and college degree, while less than 5% have primary education only (42). In this study there was no statistically significant difference in HRQOL between patients with different educational level, although the patients with college degree scored better than those with secondary and primary education in most domains except in sexual function. This could be partially explained by stronger awareness of the disease and better coping ability with the challenges of a chronic disease among the patients with higher education level but still not significant and with no independent predictability in the group of patients with low employment rate. In an Italian study, educational level was shown to be a significant and independent predictor for the physical health composite score, pain, mental health composite score, physical health, role limitation - physical, cognitive function, social function, role limitation emotional domains of the MSQoL-54 (18). Less education was proved to be negatively associated with mental HRQOL in other studies that were done in different countries (33,43). Taking these findings into account, a neurologist should encourage a patient to sustain from his/her usual activity, either employment or studying. The clinician should stress the assertion that patients who continue working or studying have a better QoL (44). Mean scores for pain, role limitation emotional, and social function domains of the MSQoL-54 were generally not clinically different among 185 patients with MS, as well as in the US population as a whole (44). Results from different studies have shown effects that coping skills of MS patients have on social and emotional adjustment and emphasized the need to provide better information to the MS patients about the nature and evolution of multiple sclerosis. In conclusion, sustained employment and development of vocational rehabilitation programs for MS patients living in the country with high unemployment level is an important factor in improving both physical and mental health outcomes in MS patients. FUNDING No specific funding was received for this study TRANSPARENCY DECLARATIONS Competing interests: none to declare 65

72 Medicinski Glasnik, Volume 12, Number 1, February 2015 REFERENCES 1. Krokavcova M, Nagyovac I, Rosenberger J, Gavelova M, Middel B, Gdovinova Z, Groothoff JW, van Dijk JP. Employment status and perceived health status in younger and older people with multiple sclerosis. Int J Rehabil Res 2012; 35(Suppl 1): Clougherty JE, Souza K, Cullen MR. Work and its role in shaping the social gradient in health. Ann NY Acad Sci 2010; 1186: Minis MA, Kalkman JS, Akkermans RP, Engels JA, Huijbregts PA, Bleijenberg G, Oostendorp RA, van Engelen BG. Employment status of patients with neuromuscular diseases in relation to personal factors, fatigue, and health status: a secondary analysis. J Rehabil Med 2010; 42: Mackenbach JP, Stirbu I, Roskam AJR, Roskam AJR, Schaap MM, Menvielle G, Mall Leinsalu M, Kunst AE. For the European Union Working Group on Socioeconomic Inequalities in Health. Socioeconomic inequalities in health in 22 European countries. N Engl J Med 2008; 358: Kern S, Kuhn M, Ziemssen T. 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73 Šabanagić-Hajrić et al. Education and employment on QoL in MS 32. Dennett SL, Castelli-Haley J, Oleen-Burkey MK. The impact of multiple sclerosis on patient employment: a review of the medical literature. JHP 2007; Krokavcova M, Nagyova I, Van Dijk JP, Rosenberger J, Gavelova M, Middel B, Szilasiova J, Gdovinova Z, Groothoff JW. Self-rated health and employment status in patients with multiple sclerosis. Disabil Rehabil 2010; 32: Solari A, Filippini G, Mendozzi L. Ghezzi A, Cifani S, Barnieri E, Baldini S, Salmaggi A, Mantia LL, Farinotti M, Caputo D, Mosconi P. Validation of Italian multiple sclerosis quality of life 54 questionnaire. J Neurol Neurosurg Psychiatry 1999; 67: Grima DT, Torrance GW, Francis G, Rice G, Rosner AJ, Lafortune L. Cost and health related quality of life consequences of multiple sclerosis. Mult Scler 2000; 6: Strober LB, Christodoulou C, Benedict RH, Westervelt HJ, Melville P, Scherl WF, Weinstock-Guttman B, Rozvi S, Goodman AD, Krupp LB. Unemployment in mul tiple sclerosis; the contribution of personality and disease. Mult Scler 2012; 18: Buhse M, Banker WM, Clement LM. Factors associated with Health-related quality of life among older people with multiple sclerosis. Int J MS Care 2014; 16: Marck CH, Hadgkiss EJ, Weiland TJ, van der Meer DM, Pereira NG, Jelinek GA. Physical activity and associated levels of disability and quality of life in people with multiple sclerosis: a large international survey. BMC Neurol 2014; 14(Suppl 1): Gruenewald DA, Higginson IJ, Vivat B, Edmonds P, Burman RE. Quality of life measures for the palliative care of people severely affected by multiple sclerosis: a systematic review. Mult Scler 2004; 10: Hakim EA, Bakheit AM, Bryant TN, Roberts MW, McIntosh-Michaelis SA, Spackman AJ Martin JP, McLellan DL. The social impact of multiple sclerosis: a study of 305 patients and their relatives. Disabil Rehabil 2000; 15: Amato MP, Ponziani G. Quantification of impairment in MS: discussion of the scales in use. Mult Scler 1999; 5: Chwastiak L, Ehde DM., Gibbons LE, Mark Sullivan M,Bowen JD, KraftGH. Depressive symptoms and severity of illness in multiple sclerosis: epidemiologic study of a large community sample. AM J Psychiatry 2002; 159: Idiman E, Uzunel F, Ozakbas S, Yozbatiran N, Oguz M, Callioglu B, Gokce N, Bahar Z. Cross-cultural adaptation and validation of multiple sclerosis quality of life questionnaire (MSQOL-54) in a Turkish multiple sclerosis sample. J Neurol Sci 2006; 240(Suppl 1-2): Pittock SJ, Mayr WT, McClelland RL, Jorgensen NW, Weigand SD, Noseworthy JH, Rodriguez M. Quality of life is favorable for most patients with multiple sclerosis: a population-based cohort study. Arch Neurol 2004; 61: Utjecaj stupnja obrazovanja i statusa zaposlenosti na kvalitet života oboljelih od multiple skleroze Selma Šabanagić-Hajrić, Azra Alajbegović Neurološka klinika, Klinički centar Univerziteta u Sarajevu SAŽETAK Cilj Evaluirati utjecaj stupnja obrazovanja i statusa zaposlenosti na kvalitet života oboljelih od multiple skleroze. Metode U ispitivanje je uključeno 100 pacijenata s dijagnosticiranom multiplom sklerozom koji su liječeni na Neurološkoj klinici Kliničkog centra Univerziteta u Sarajevu. Kriteriji za uključenje bili su skor Proširene skale stupnja onesposobljenosti (EDSS) između 1,0 i 6,5, dob između 18 i 65 godina, te stabilni tok bolesti. Kvalitet života je evaluiran uz pomoć upitnika kvaliteta života oboljelih od multiple skleroze (MSQoL-54). Mann-Whitneyev i Kruskal-Wallisov test su korišteni za usporedbe. Linearna regresiona analiza korištena je za procjenu signifikantnih prediktora fizičkog i mentalnog zbirnog skora kvaliteta života (MSQOL-54). Rezultati Zaposleni pacijenti imali su više zbirne skorove fizičkog (54,85 vs ; p<0,001) i mentalnog zdravlja (59,55 vs. 45,90; p<0,001). Zaposlenost se potvrdila kao neovisni prediktivni faktor fizičkog (r 2 =0,105) i mentalnog (r 2 =0,076) zbirnog skora kvaliteta života. Pacijenti koji imaju visoko obrazovanje imali su nešto veće srednje vrijednosti fizičkog (49,36 vs. 45,30) i mentalnog zbirnog skora (66,74 vs. 55,62) kvaliteta života u usporedbi s ostalim, ali bez statističke značajnosti. Zaključak Zaposlenost se pokazala kao značajan faktor pozitivnog utjecaja na kvalitet života oboljelih od multiple skleroze. Viši stupanj obrazovanja može utjecati na bolji kvalitet života, ali bez značajne prediktivne vrijednosti. Zadržavanje statusa zaposlenosti i razvoj profesionalnih rehabilitacionih programa za pacijente oboljele od multiple skleroze koji žive u zemlji s visokim stupnjem nezaposlenosti jeste važan faktor u poboljšanju njihovog kako fizičkog, tako i mentalnog zdravlja. Ključne riječi: kvalitet života, fizičko zdravlje, mentalno zdravlje 67

74 ORIGINAL ARTICLE Importance of Herrings classification in predicting the outcome of aseptic necrosis of the femoral head Žarko Dašić 1, Miroslav Kezunović 1, Goran Pešić 1, Vesna Bokan 2, Mira Jovanovski 3 1 Clinic for Orthopedics and Traumatology, 2 Center for Physical Medicine and Rehabilitation; Clinical Center Podgorica, 3 Montenegro Ministry of Health; Podgorica, Montenegro ABSTRACT Aim To highlight the importance of values of the Herring s classification in the treatment planning of Legg-Calve-Perthes disease (LCPD). Method The charts of 14 patients in a period of 4 years ( ) were retrospectively reviewed. Inclusion criteria was unilateral LCPD and contralateral healthy hip. The patients were divided into three Herring groups according to radiographic images (A, B and C). For all patients the acetabulum/head index (AHI) was determined. Corresponding author: Žarko Dašić Clinic for Orthopedics and Traumatology, Clinical Center Podgorica Ljubljanska bb, Podgorica, Montenegro Phone: ; Fax: ; zarko.dasic@kccg.me Results The youngest patient was 4.9 years and the oldest 9.11 years; male patients were dominant (male:female 11:3). The right hip side was more affected comparing to the left one (8:6). The distribution of patients in Herring groups was three in the Group A, six in the Group B and five patients in the Group C. The AHI index was lowest in the group C. Patients in the group C were treated surgically. Conclusion Herrings classification predicts patients with extensive changes and suggests what kind of treatment should be applied. Key words: Perthes disease, Herring s classification, treatment Original submission: 16 June 2014; Revised submission: 05 November 2014; Accepted: 20 November Med Glas (Zenica) 2015; 12(1):

75 Dašić et al. Herrings classification INTRODUCTION Since Legg-Calve-Perthes-disease (LCPD) is a chronic disease, it has a characteristic cyclic flow. Perthes disease is not only an aseptic necrosis of the bone, but also subchondral fracture, which leads to deformities of the head and neck of the femur (1,2). The disease occurs in three pathological stages: the initial stage, the stage of fragmentation and the reparation stage. Definite changes in the proximal femur in this disease depend on several factors that influence the course of the disease (3). Out of these, the most important are patient age, degree of involvement of the epiphysis, sex, containment of the head within the acetabulum, premature closure of the growth zones, changes in the metaphysis, increased body weight and prolonged restriction of movement in the hip (1,3,4). Deformity that occurred as final outcome of the LCPD caused altered morphology of the hip and related conditions for the emergence of early degenerative hip changes (osteoarthritis). A significant number of methods (2,3,5,6) for evaluating the results of LCPD treatment are described: Heyman and Heradon (calculated epiphysis index, head and neck index, acetabulum index and acetabulum-head index), Mose (estimated AP and lateral radiographs, which are made in Lowenstein s position), Catterell s classification in 4 stages and one of the last modern classifications by Herring which consists in determining the side (lateral) collapse of the epiphysis in the process of fragmentation of the containment of the head within the acetabulum in the final stage of the disease. According to Stolberg, there are three types of congruency between the femoral head and the acetabulum: spherical congruency, non-spherical congruence and non-spherical incongruence (7-10). Spherical congruence does not cause degenerative changes; non-spherical congruence leads to degenerative changes at a later age, and non-spherical incongruence to early degenerative changes in the hip joint, before the end of the fifth decade (11). Montenegro is an area with disorders of the hip joint during childhood, highly represented. Good diagnosis and adequate treatment are conditions for full functional recovery. The importance of Herring classification has not been studied in this area and this study can serve as a further contribution to clinical practice. The aim of this study was to highlight the importance of Herring s classification values in the treatment planning and outcome of Legg-Calve- Perthes disease. PATIENTS AND METHODS A retrospective analysis of X-ray images in 14 patients treated at the Clinic for Orthopedics and Traumatology of the Clinical Center Podgorica, Montenegro, in the period from 2004 to 2008 was conducted. All patients had unilateral occurrence of the LCPD and all patients were at the stage of complete restitution of the disease. X-rays of the pelvis at the final stage of the fragmentation of the disease with definite changes in the proximal femur were analyzed. Inclusion criteria were healthy contra lateral hip as healthy material for comparison. Image analysis of the fragmentation phase included a division and definition of the three pillars of the femoral head; the determination of the collapse degree of the lateral pillar (determined by measuring the distance h (the distance between the epiphysis cracks and the highest point of the epiphysis in the lateral column). This distance was determined in both: the diseased and the healthy opposite hip, and it was expressed in mm. According to Herring s classification (12,13) patients were divided into 3 groups. In the Group A (Herring A) lateral pillar height of the affected hip was identical with the height of the lateral pillar of the healthy hip, in the Group B (Herring B) lateral pillar of the affected hip reached a height of more than 50% of the healthy hip, and in the G group C (Herring C) the height of the lateral pillar of the affected hip was less than 50% of the height of the lateral pillar of the healthy hip. Evaluation of the head containment within the acetabulum was performed by determining the acetabulum/head index (AHI) and the Heyman- Herndon index: the ratio between horizontal diameters of the head covered by acetabulum and horizontal diameter of the head. The study was approved by the Ethics Committee of the Clinic for Orthopedics and Traumatology, Clinical Center Podgorica. RESULTS From the total number of 14 patients, three (21.42%) were females and 11 (78.58%) were 69

76 Medicinski Glasnik, Volume 12, Number 1, February 2015 males. The youngest patient was 4.9 and the oldest one was 9.11 years of age (Table 1). According to the localization of the LCPD the right hip dominated in eight (57.14 %) patients. The highest number of patients with LCPD were in the age group 6-8 years, eight patients (57.14%), while the smallest representation of LCPD was in the age group of 5 years, one patient (7.14%) (Table 1). Table 1. Patients with Legg-Calve-Perthes disease (LCPD) according to sex, localization and age distribution No (%) of patients Gender Side Total Age distribution Left Right Males (28.57) (50.01) (78.58) (7.14) (42.85) (28.57) Females Total (14.28) 6 (42.85) (7.14) 8 (57.15) (21.42) 14 (100) 1 (7.14) (14.28) 8 (57.15) (7.14) 5 (35.71) The largest representation of LCDP was in males on the right hip at the age of 6-8 years. According to the Herring classification in the group A there were three (21.43%) patients, in group B six (42.86%) and in group C five (35.71%) patients; gender distribution was approximately uniform, A: B: C for males was 3: 4: 4 (Table 2). Table 2. Distribution of patients according to Herring groups No (%) of patients Herrings classification Group Males Females Total A 3 (21.43) 0 3 (21.43) B 4 (28.57) 2 (14.29) 6 (42.86) C 4 (28.57) 1 (7.14) 5 (35.71) Total 11 (78.57) 3 (21.43) 14 Evaluation of the head containment within the acetabulum showed the AHI value in group A of 0.8, in the group B 0.7, and in the group C 0.6 (Figure 1). Figure 1. Acetabulum/Head Index and Heyman-Herndon index according to Herring groups DISCUSSION For the disease evaluation and treatment planning examination (14), the presence of (containment) of the femoral head within the acetabulum is an important factor. Calculation and determination of AHI (acetabulum-head-herndon Hayman index) derives from the need to timely detect and assess risk patients, in order to plan their treatment and to start it on time (15,16). Treatment started timely avoids the risk of pronounced changes in the proximal femur, and thus significantly reduces non-spherical incongruence (17). Measurement of the epiphyseal collapse of the diseased hip in the studied patients showed significant differences compared with measurements of the same parameter of the healthy hip in all three groups classified according to Herring. Park et al. (18) suggested highest reliability of the Herring classification stating that patients who are older than 8 years and belong to group A, may be considered for surgical treatment. In our study, group A (three males) with AHI of 0.8 were treated conservatively. The results of this study showed that the AHI in patients decreased from group A to group C, and that the containment of the femur head was smallest when the degree of the epiphysis collapse was higher. In group B there were six patients with AHI index 0.7, and in group C five patients had AHI index 0.6. This difference between groups suggests that the Herrings classification in predicting the outcome of treatment of LCPD has a basis, since the group (group A) patients with AHI index 0.8 had the smallest changes in the lateral column of the fragmentation stage and that in group (group C) patients with AHI index 0.6, changes were the biggest. Patients from group C were treated as high- risk patients and an active surgical treatment was engaged for them to achieve good results. In the prospective multicenter Herring et al. study (17) the lateral pillar classification and age of onset of the disease strongly correlated with the outcome of the treatment of LCPD. Patients in groups B and C had a better outcome with operative treatment and the age of eight years. In our study, according to the age of disease onset, more than half of the patients were in the group 6-8 years, which correlates with the results of Herring (17). Recent studies 70

77 Dašić et al. Herrings classification show that the Herring classification is significant in predicting the outcome of treatment in children under six years of age (19). Examination of the degree of collapse of the outer epiphysis pillar and the containment of the head in the acetabulum was biggest in group C, where the collapse of the epiphysis was most pronounced, in group B it was moderate, while in the group A it was least pronounced, and accordingly, the patients in group C were candidates for active surgical treatment. Patients in the Herring groups A and B were treated conservatively. A limitation of this study is the small number of patients; larger number of patients would enable comparison of Herring groups of different ages. There is no single method of treatment of Perthes disease. The results of this study indicate that the application of Herring s classification provides an appropriate division of patients with Perthes disease. Herrings classification provides a good estimate of the degree of change in the epiphysis and adequate distribution of patients into three groups. Also, it evaluates patients with extensive changes and poor prognosis, thus indicating the need of adequate treatment. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare. REFERENCES 1. Stulberg S D, Cooperman DR, Wallensten R. The natural history of Legg Calve - Perthes Disease. J Bone Joint Surg Am 1981; 63: Catterall A, Pringle J, Byers PD, Fulford GE, Kemp HBS, Dolman CL, Bell HM, McKibbin B, Ralis Z, Jensen OM, Lauritzen J, Ponseti IV, Ogden J. A review of the morphology of Perthes Disease. J Bone Joint 1982; 64: Salter RB. The present status of surgical treatment for Legg-Calve-Perthes disease. J Bone Joint Surg Am 1984; 66: Ippolito E, Tudisco C, Farseti P. Long-term prognosis of Legg Calve Perthes Disease developing during adolescence. J Pediatr Orthop 1985; 5: Cristensen F, Soballe K, Ejsted R, Luxhøj T. The Catterall classification of Perthes`s disease: an assessment of reliability. J Bone Joint Surg Am 1986; 68: Ippolito E, Tudisco C, Farseti P. The long-term prognosis of unilateral Perthes Disease. J Bone Joint Surg Am. 1987; 69: Kollitz KM1, Gee AO. Classifications in brief: the Herring lateral pillar classification for Legg- Calvé-Perthes disease. Clin Orthop Relat Res 2013; 471: Mukherjee A, Fabry G. Evaluation of the prognostic indices in Legg Calve Perthes Disease: statistical analysis of 116 hips. J Pediatr Orthop 1990; 10: Wenger DR, Ward WT, Herring JA. Current concepts review: Legg Calve Perthes Disease. J Bone Joint Surg Am 1991; 73: Urlus M, Stoffelen D, Fabry G. Hinge abduction in avascular necrosis of the hip: diagnosis and treatment. J Pediatr Orthop 1992; 1: Crutcher JP, Staheli LT. Combined osteotomy as a salvage procedure for severe. J Pediatr Orthop 1992; 12: Herring JA, Neustadt JB, Williams JJ, Early JS, Browne RH. The lateral pillar classification of Legg Calve Perthes Disease. J Pediatr Orthop. 1992; 12: Herring JA, Williams JJ, Neustadt JB, Early JS. Evolution of femoral head deformity during the healing phase of Legg Calve Perthes Disease. J Pediatr Orthop 1993; 13: Ritterbush JF, Shantharam SS, Gelinas C. Comparison of Lateral Pillar Classification and Catterall Classification Legg Calve Perthes Disease. J Pediatr Orthop 1993; 13: Schepers A, Robertson AF. Legg-Calvé-Perthes disease. The results of a prospective clinical trial comparing the outcomes of surgery and symptomatic treatment for patients presenting at age 5 years or younger. SA Orthop J 2011; 10: Sales de Gauzy J, Kerdiles N, Baunin C, Kany J, Darodes P, Cahuzac JP. Imaging evaluation of subluxation in Legg-Calvé-Perthes disease: magnetic resonance imaging compared with the plain radiograph. J Pediatr Orthop 1997; 6: Herring JA, Kim HT, Browne R. Legg-Calve-Perthes disease. Part II: Prospective multicenter study of the effect of treatment on outcome. J Bone Joint Surg Am 2004; 86: Park MS, Chung CY, Lee KM, Kim TW, Sung KH. Reliability and stability of three common classifications for Legg-Calvée-Perthes disease. Clin Orthop Relat Res 2012; 470: Gent A, Antapur P, Mehta RL, Sudheer VM, Clarke NM. Predicting the outcome of Legg-Calve-Perthes disease in children under 6 years old. J Child Orthop 2007; 1:

78 Medicinski Glasnik, Volume 12, Number 1, February 2015 Značaj Herringove klasifikacije u predviđanju ishoda aseptičke nekroze glave butne kosti Žarko Dašić 1, Miroslav Kezunović 1, Goran Pešić 1 Vesna, Bokan2, Mira Jovanovski 3 1 Klinika za ortopediju i traumatologiju, 2 Centar za fizikalnu medicinu i rehabilitaciju; Klinički centar Podgorica 3, Ministarstvo zdravlja; Podgorica, Crna Gora SAŽETAK Cilj Ukazati na značaj vrijednosti Herringove klasifikacije u planiranju liječenja Legg-Calvé-Perthesove bolesti (LCPB). Metode Retrospektivnom studijom je obuhvaćena grupa od 14 ispitanika, u periodu od četiri godine ( ). Uključujući kriteriji bili su unilateralna LCPB i kontralateralni zdravi kuk. Analizom radiografskih snimaka ispitanici su podijeljeni u tri Herringove grupe (A, B i C). Kod svih ispitanika je određen acetabulum/head index (AHI). Rezultati Najmlađi ispitanik imao je 4,9 godina, a najstariji 9,11 godina; u polnoj strukturi muški pol je dominirao (muškarci:žene, 11:3). Desna strana je bila češće zahvaćena u odnosu na lijevu (8:6). Zastupljenost pacijenata u Herringovim grupama bila je tri pacijenta u grupi A, šest u grupi B i pet pacijenata u grupi C. AHI indeks bio je najmanji u grupi C. Pacijenti iz grupe C su operativno liječeni. Zaključak Herringova klasifikacija omogućava prepoznavanje pacijenata s opsežnim promjenama i sugeriše vrstu liječenja. Ključne riječi: Perthesova bolest, Herringova klasifikacija, liječenje 72

79 ORIGINAL ARTICLE A retrospective review of 139 major and minor salivary gland tumors Marija Trenkić Božinović 1, Dragan Krasić 2,3, Vuka Katić 2, Miljan Krstić 2,4 1 Ophthalmology Clinic, University Medical Center, 2 University of Niš, School of Medicine, 3 Clinic of Maxillofacial Surgery, 4 Institute of Pathology, University Medical Center; Niš, Serbia ABSTRACT Aim To describe demographic and histomorphological characteristics of 139 patients with epithelial salivary gland tumors in the Southeastern Serbia population. Methods A total number of 139 patients with epithelial tumors arising in major and minor salivary glands in the period was evaluated. After standard tissue proceeding, the routine haematoxylin-eosin (HE) and histochemical alcian blue-periodic acid-schiff (AB - PAS) methods were used for histomorphological examination. Corresponding author: Marija Trenkić Božinović Department of Ophthalmology, University Medical Center Dr. Zorana Djindjica 48, Niš, Serbia Phone ; Fax: ; marija.trenkic@gmail.com Results Among 139 patients, 102 (73.38%) had benign, and 37 (26.62%) malignant tumors. The majority of tumors were localized in the parotid gland, in 117 (84.17%) patients. Among benign tumors there were 50 (49.02%) pleomorphic adenoma, 48 (47.06%) Warthin s tumor, two (1.96%) myoepithelioma, and two (1.96%) oncocytoma. In the group of malignant tumors the most common was mucoepidermoid carcinoma, in 12 (32.43%) patients, carcinoma ex pleomorphic adenoma in six (16.22%), adenoid cystic carcinoma in five (13.51%), and oncocytic carcinoma in three (8.11%) patients. Conclusion Benign tumors were more common than malignant ones, with predominance of pleomorphic adenoma. Malignant tumors are less common than benign in the large salivary glands, and more common in the minor salivary glands. Histochemical AB-PAS method helps in the diagnosis of mucinous salivary gland carcinoma. Key words: benign, carcinoma, epidemiology, histopathology. Original submission: 31 July 2014; Revised submission: 30 October 2014; Accepted: 16 December Med Glas (Zenica) 2015; 12(1):

80 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION Salivary gland tumors can show a striking range of morphological diversity among different tumor types and sometimes within an individual tumorous mass. In addition, hybrid tumors, dedifferentiation and propensity for some benign tumors to progress to malignancy can confound histopathological interpretation. These features, together with the relative rarity of a number of tumors (1), can sometimes make diagnosis difficult. There is some geographic variation and among different ethnic groups according to the place of residence (2-5). Therefore, the global annual incidence, when all salivary glands tumors were considered, varied from cases per population (6). The frequency of malignant salivary neoplasm range from cases per population (7). Between 64% and 80% of all primary tumors occur in the parotid gland, 7% 11% in the submandibular glands, and 9% 23% occur in the minor glands (6,8). Females are more frequently affected (2). The mean ages of patients with benign and malignant tumors is 46 and 47 years, respectively (3,9). The aim of this study was to investigate both demographic and histomorphological characteristics of epithelial salivary gland tumors of 139 patients over a period of three years, and to compare findings with results of other studies. The purpose of this paper is to contribute to more accurate diagnosis of a lesion of salivary glands. PATIENTS AND METHODS The study included 139 patients with epithelial salivary gland tumors arising in the major and minor salivary glands, selected from the medical files of the University Hospital, Department of Maxillofacial Surgery, and the Institute of Pathology of the University Medical Center Niš, Serbia, from the beginning of 2010 to the end of The following parameters were analyzed: patient age and gender, distribution of tumors in relation to malignancy (benign or malignant), as well as the localization in salivary glands (minor glands, parotid gland, submandibular glands, sublingual glands). After standard tissue proceeding, routine haematoxylin-eosin (HE) and histochemical alcian blue-periodic acid-schiff (AB - PAS) methods were used for histomorphological examination. The results were statistically analyzed using descriptive and quantitative analysis, the arithmetic mean (X) and standard deviation (SD). The difference in the average values was calculated using the t- test for two independent samples. The association between the two marks was measured using the χ 2 test. The Pearson s rank correlation test was used to determine a relation between the associated parameters. The threshold for statistical significance was taken at p RESULTS During the span of 3 years, 139 cases of salivary glands tumors were diagnosed. Among these, 73 (52.52%) patients were females (56 benign and 17 malignant), and 66 (47.48%) were males (46 benign and 20 malignant); 102 (73.38%) were benign and 37 (26.62%) malignant tumors (Tables 1-3). The mean age of the patients was 51.2 ± years for benign tumors, and ± years for malignant tumors. In the group of benign tumors, the youngest patient was a 16-year-old female with pleomorphic adenoma in the right parotid gland. The oldest patient was 80-year-old male with pleomorphic adenoma of the left parotid gland. In the series of malignant tumors, the youngest patient was also female, 43-year old, with mucoepidermoid carcinoma of the right submandibular gland. The oldest patient was a 81-yearold male, who was diagnosed with adenoid cystic carcinoma of the right submandibular gland. Localization of tumors in the major (parotid, submandibular and sublingual glands) and minor salivary glands is presented in Table 1. Examining the correlation between types of tumors (benign or malignant) and their localization in the salivary glands (minor glands, parotid gland, submandibular glands, sublingual glands) a statistically significant difference was found (p< ) (Table 1). Table 1. Distribution of tumors in the salivary glands Type of tumor Minor glands No (%) of patients Parotid gland 3 97 Benign (2.94%) (95.10%) Malignant (29.73%) (54.05%) (16.22%) Total (10.07%) (84.17 %) (5.76 %) Submandibular Sublingual glands glands Total p (1.96%) (73.38%) < (26.62%)

81 Trenkić Božinović et al. Major and minor salivary gland tumors Table 2. Histological types and other characteristics of benign salivary gland tumors No (%) of patients Type of tumor Gender Localization Type of salivary gland Male Female Right Left Parotid Submandibular Sublingual Minor Total Pleomorphic adenoma 14 (28.0) 36 (72.0) 25 (50.0) 25 (50.0) 47 (94.0) 1 (2.0) 0 2 (4.0) 50 (49.02) Warthin s tumor 30 (62.5) 18 (37.5) 29 (60.42) 19 (39.58) 47 (97.92) 1 (2.08) (47.06) Myoepithelioma 1 (50.0) 1 (50.0) 1 (50.0) 1 (50.0) 2 (100.0) (1.96) Oncocytoma 1 (50.0) 1 (50.0) 2 (100.0) 0 1 (50.0 ) (50.0) 2 (1.96) Total 46 (45.1) 56 (54.9 ) 57 (55.88) 45 (44.12) 97 (95.1) 2 (1.96) 0 3 (2.94) 102 The most frequent tumors originated from the parotid gland (97 benign and 20 malignant), followed the submandibular gland (2 benign and 6 malignant) and minor salivary glands (benign 3 and malignant 11). The majority of tumors, both benign and malignant, was localized in the parotid gland, 117 (84.17%). The most frequent tumors in minor salivary glands were malignant, 11 (78.57%). Benign lesions were localized in parotid gland, in 97 (95.10%) patients (69.78% of all tumors), and only 20 (54.05%) malignant lesions (14.39% of all tumors) were localized in the parotid gland. No tumor was implicated from sublingual mayor salivary gland. There was no statistically significant association between tumor type and gender (p>0.05). Patients with malignant tumors were significantly older than patients with benign tumors (p<0.05). In Table 3. Histological types and other characteristics of malignant salivary gland tumors Type of tumor (Ca) No (%) of patients Gender Type of salivary gland Total Sub- Sublingual Male Fimale Parotid mandi- bular Minor Mucoepidermoid (58.33) (41.67) (58.33) (33.33) (8.34) (32.43) Ca ex PA 0 0 (33.33) (66.67) (66.67) (33.33) (16.22) Adenoid cystic (60.0) (40.0) (100.0) (13.51) Oncocytic 0 0 (66.67) (33.33) (66.67) (33.33) (8.11) Myoepithelial Cystadeno Squamous cell Basal cell adeno Mucinous adeno Salivary duct Small cell type Total Ca, carcinoma 1 (50.0) 2 (100.0) 1 (50.0) 1 (100.0) 1 (100.0) 20 (54.05) 1 (50.0) (50.0) 2 (100.0) 2 (100.0) 2 (100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (45.95) (54.05) (16.22) 0 2 (5.41) 2 (5.41) 2 (5.41) 2 (5.41) 1 (2.70) 1 (2.70) 1 (2.70) (29.73) both, benign and malignant tumors, there was no significant difference considering the localization (right/left) (p>0.05 ). Among 102 (73.38%) benign tumors, there were 50 (49.02%) pleomorphic adenoma, 48 (47.06%) Warthin s tumor, and myoepithelioma and oncocytoma, two (1.96%) of each (Table 2). The ma- Figure 1. A) Pleomorphic adenoma: epithelial cells arranged in strands in mucoid background (HE, x 200); B) Warthin s tumor: double-layered columns of cells with intervening lymphoid tissue (HE, x 200); C) Myoepithelioma: clear cell variant with mucoid surrounding stroma (AB- PAS, x 200); D) Oxyphilic adenoma: large polyhedral cells with finely granular cytoplasm; colagenous stroma is minimal PAS positive (AB- PAS, x 300) (Katić, V, 2014) A) B) C) D) 75

82 Medicinski Glasnik, Volume 12, Number 1, February 2015 jority of the benign tumors were located in the parotid glands, in 97 (95.10%) cases. The most common histological types (overall prevalence was 62.16%, e.g., 23 cases) were mucoepidermoid carcinoma which were reported in 12 (32.43%) cases, carcinoma ex pleomorphic adenoma in six (16.22%) cases, adenoid cystic carcinoma in five (13.51%) cases, followed by oncocytic carcinoma, in three (8.11%), myoepithelial carcinoma in two (5.41%), cystadenocarcinoma in two (5.41%), squamous cell carcinoma in two (5.41%), basal cell adenocarcinoma in two (5.41%) cases. Small cell carcinoma, salivary duct carcinoma, and mucinous adenocarcinoma were uncommon, in one (2.70%) case each, respectively. Most of the tumors in the minor salivary glands were malignant, 11 (78.57%) (Table 1 and 3). The most common histologic appearance of benign and malignant tumors is presented in Figure 1 and 2. Figure 2. A) Mucoepidermoid carcinoma: both solid and multicystic pattern, with syalomucin in cystic component (AB-PAS, x 200); B) Carcinoma ex pleomorphic adenoma: poorly differentiated adenocarcinoma (HE, x 200); C) Adenoid cystic carcinoma: multiple cystic spaces filled with acid mucin (AB- PAS, x 250) (Katić, V, 2014) A) B) C) DISCUSSION This paper describes the epidemiological and histomorphological features of 139 epithelial tumors of the salivary glands, with reference to their surgical treatment. Our results are similar to other reports in relation to age, sex and localization of the tumors (2,9,10). In this series, the most common benign tumor was pleomorphic adenoma, localized mostly in the parotid glands, mostly affecting women. Pleomorphic adenoma is a benign tumor, but recurrence appears very often (10). Recurrences can be explained by the growth of the tumor around the facial nerve, which complicates its surgical extirpation (11,12), or they arise as complications of multicentric growth of pleomorphic adenoma (1). The recurrence increases the risk to malignant alteration of pleomorphic adenoma (11,13). Warthin s tumor was more common in our series, localized in the parotid gland without malignant alteration. In some reports, this is one of rare variants of epithelial salivary gland tumors (14). The discrepancy in the frequency could be explained by geographical, racial factors, as well as aggravated differential diagnosis with metastatic adenocarcinoma tumors in the lymph nodes, that is induced by its mixed lymphoid - glandular structure (3,4,15-17). Cancers of the salivary glands are less common than benign forms in this study. A higher incidence of malignant tumors, compared to the results of other authors (14,18), could be explained by the profile of patients treated in our institution of tertiary level, as pointed out by the others (19). The microscopic pattern of malignant salivary gland tumors is sometimes very similar to benign tumors, therefore, the differentiation is difficult (6,8,20). The specific criteria of malignancy include anaplasia, infiltration of the capsule to surrounding tissue or the absence of a capsule, multiple foci of necrosis and hemorrhage, lymphangio invasion, as well as perineural invasion inside the tumor. The most important characteristic of malignant tumor is the involvement of the regional lymph nodes, that we found in our malignant salivary gland tumors (7). According to the results of this study, the most common was mucoepidermoid carcinoma, reported in 12 cases. Contrary to the literature that 76

83 Trenkić Božinović et al. Major and minor salivary gland tumors mucoepidermoid carcinoma is frequently located in the minor salivary glands (20), we have found that the mucoepidermoid carcinoma is more frequently presented in the parotid gland and submandibular glands. Carcinoma ex pleomorphic adenoma was second in frequency, which is directly related to the long-standing pleomorphic adenoma and its recurrences. Inexplicable manifestation of metastasizing benign mixed tumor with local or distant metastasis ( metastasizing pleomorphic adenoma ) has been described in the new WHO histological classification of tumors of the salivary glands (1). Differential diagnosis of the metastatic adenocarcinoma of surrounding organs from primary salivary gland adenocarcinoma is very difficult (21,22). Due to these characteristics, it is emphasized that pathognomonic finding of primary adenocarcinoma of the salivary gland is the presence of polymorphic adenoma or healthy salivary gland tissue in its vicinity (23). Adenoid cystic carcinoma reported in this study was localized in the small salivary glands, oncocytic carcinoma in the parotid glands, and in the minor salivary glands. In terms of localization and frequency, this is in accordance with data from the literature (24,25). Other cancers, myoepithelial carcinoma, cystadenocarcinoma, squamous cell carcinoma and basal cell carcinoma were far less frequently found in this study. Myoepithelial carcinoma and cystadenocarconoma were discovered in the minor salivary glands, while squamous and basal cell adenocarcinoma were found in the parotid gland (5,25). In conclusion, this study is the first epidemiological study on salivary gland tumors performed on the population from Southeastern Serbia, based on the 2005 WHO tumor classification. The findings of this study contribute significantly to the awareness of clinical and pathological features of salivary gland tumors in our region and can improve our understanding of significant differences in the global distribution of salivary gland tumors which have been reported. Although, the reason for these differences remains unclear, further investigations specifically searching for the possible causes, are greatly encouraged. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATIONS Conflict of interest: none to declare. REFERENCES 1. Everson JW, Auclair P, Gnepp DR, El-Naggar AK. Tumors of salivary glands. In: Barnes L, Eveson JW, Reichart P, Sidransky D, editor. Pathology and genetics of head and neck tumours. Lyon: IARC Press, 2005: de Oliveira FA, Duarte EC, Taveira CT, Máximo AA, de Aquino EC, Alencar Rde C, Vencio EF. Salivary gland tumor: a review of 599 cases in a Brazilian population. Head Neck Pathol 2009; 3: Shashinder S, Tang, IP, Velayutham P, Prepageran N, Gopala KG, Kuljit S, Anura MM, Chong SY. A review of parotid tumours and their management: a ten-year-experience. Med J Malaysia 2009; 64: Vuhahula EAM. Salivary gland tumors in Uganda: clinical pathological study. Afr Health Sci 2004; 4: Gbotolorun OM, Arotiba GT, Effiom OA, Omitola OG. Minor salivary gland tumours in a Nigerian hospital: a retrospective review of 146 cases. Odontostomatol Trop 2008; 3: Ellis GL, Auclair PL, Gnepp DR. Surgical Pathology of Salivary Glands. Philadelphia: WB Saunders, McHugh JB, Visscher DW, Barnes EL. Update on selected salivary gland neoplasms. Arch Pathol Lab Med 2009; 133: Eneroth CM. Salivary gland tumors in parotid gland, submandibular gland, and the palate region. Cancer 1971; 27: Lukšić I, Virag M, Manojlović S, Macan D. Salivary gland tumours: 25 years of experience from a single institution in Croatia. Craniomaxillofac Surg 2012; 40: Guzzo M, Locati LD, Prott FJ, Gatta G, McGurk M, Licitra L. Major and minor salivary gland tumors. Crit Rev Oncol Hematol 2010; 74: Katabi N, Gomez D, Klimstra DS, Carlson DL, Lee N, Ghossein R. Prognostic factors of recurrence in salivary carcinoma ex pleomorphic adenoma, with emphasis on the carcinoma histologic subtype: a clinicopathologic study of 43 cases. Hum Pathol 2010; 41: Shing Howe To V, YuWai Chan J, Tsang RKY, Wei WI. Review of salivary gland neoplasms. ISRN Otolaryngol 2012; 2012: Argyris PP, Pambuccian SE, Cayci Z, Singh C, Tosios KI, Koutlas IG. Lacrimal gland adenoid cystic carcinoma with high-grade transformation to myoepithelial carcinoma: report of a case and review of literature. Head Neck Pathol 2013; 7:

84 Medicinski Glasnik, Volume 12, Number 1, February Mejía-Velázquez CP, Durán-Padilla MA, Gómez- Apo E, Quezada-Rivera D, Gaitán-Cepeda LA. Tumors of the salivary gland in Mexicans. A retrospective study of 360 cases. Med Oral Patol Oral Cir Bucal 2012; 17: Shishegar M, Ashraf MJ, Azarpira N, Khademi B, Hashemi B, Ashrafi A. Salivary gland tumors in maxillofacial region: a retrospective study of 130 cases in a southern Iranian population. Patholog Res Int 2011; 2011: Lawal AO, Adisa AO, Kolude B, Adeyemi BF, Olajide MA. A review of 413 salivary gland tumours in the head and neck region. J Clin Exp Dent 2013; 5: Bello IO, Salo T, Dayan D, Tervahauta E, Almangoush A, Schnaiderman-Shapiro A, Barshack I, Leivo I, Vered M. Epithelial salivary gland tumors in two distant geographical locations, Finland (Helsinki and Oulu) and Israel (Tel Aviv): a 10-year retrospective comparative study of 2,218 cases. Head Neck Pathol 2012; 6: Shukla NK, Hazarika S, Deo S, Kar M, Kumar S, Samaiya A, Sharan R, Rath GK. Salivary gland tumours: profile and management at a tertiary cancer centre. J Indian Med Assoc 2011; 109: Seethala RR. An update on grading of salivary gland carcinomas. Proceedings of the 2009 North American Society of head and neck pathology companion meeting (Boston, MA). Head Neck Pathol 2009; 3: Seethala RR. Histologic grading and prognostic biomarkers in salivary gland carcinomas. Adv Anat Pathol 2011; 18: Uro-Coste E update in salivary gland tumoral pathology. Ann Pathol 2009; 29: Cheuk W, Chan JK. Advances in salivary gland pathology. Histopathology 2007; 51: Seethala RR, Hunt JL, Baloch ZW, Livolsi VA, Leon Barnes E. Adenoid cystic carcinoma with high-grade transformation: a report of 11 cases and a review of the literature. Am J Surg Pathol 2007; 31: Pons-Vicente O, Almendros-Marqués N, Berini- Aytés L, Gay-Escoda C. Minor salivary gland tumors: a clinicopathological study of 18 cases. Med Oral Patol Oral Cir Bucal 2008; 13: Becerril-Ramírez PB, Bravo-Escobar GA, Prado- Calleros HM, Castillo-Ventura BB, Pombo-Nava A. Histology of submandibular gland tumours, 10 years experience. Acta Otorrinolaringol Esp 2011; 62: Retrospektivni pregled 139 tumora malih i velikih pljuvačnih žlezda Marija Trenkić Božinović 1, Dragan Krasić 2,3, Vuka Katić 2, Miljan Krstić 2,4 1 Klinika za očne bolesti, Klinički centar Niš, 2 Medicinski fakultet, Univerzitet u Nišu, 3 Klinika za maksilofacijalnu hirurgiju, Niš, 4 Institut za patologiju, Klinički centar Niš; Niš, Srbija SAŽETAK Cilj Opisati demografske i histomorfološke karakteristike 139 slučajeva epitelijalnih tumora pljuvačnih žlezda u populaciji jugoistočne Srbije. Metode Analizirano je 139 pacijenata s epitelnim tumorima malih i velikih pljuvačnih žlezda operisanih na Klinici za maksilofacijalnu hirurgiju u Nišu, u periodu od do godine. Posle standardne obrade tkiva za histomorfološko ispitivanje korišćena je rutinska hematoksilin-eozin (HE) metoda i histohemijska AB-PAS metoda. Rezultati Studija je obuhvatila 139 slučajeva, odnosno 102 (73,38%) benigna tumora i 37 (26,62%) malignih lezija. Većina tumorâ bila je lokalizovana u parotidnim žlezdama, u 117 (84,17%) slučajeva. Među benignim tumorima bilo je 50 (49,02%) slučajeva pleomorfnog adenoma, 48 (47,06%) Warthinovog tumora i po dva slučaja (1,96%) mioepitelioma i oncocitoma. U grupi malignih tumora najčešći je bio mukoepidermoidni karcinom, u 12 (32,43%) slučajeva, carcinoma ex pleomorphic adenoma u šest (16,22%), adenoidni cistični karcinom u pet (13,51%) i oncocitic carcinoma u tri (8,11%) slučaja. Zaključak Benigni tumori pljuvačnih žlezda su češći nego maligni, uz dominaciju pleomorfnog adenoma. Maligni tumori su se ređe od benignih javljali u velikim pljuvačnim žlezdama, a češće su bili lokalizovani u malim pljuvačnim žlezdama. Histohemijska AB-PAS metoda pomaže u dijagnozi mucinoznih karcinoma pljuvačnih žlezda. Ključne reči: benigni, maligni, epidemiologija, histopatologija. 78

85 ORIGINAL ARTICLE Effects of hormone replacement therapy on depressive and anxiety symptoms after oophorectomy Danijela D. Ðoković 1,2, Jelena J. Jović 3, Jelena D. Ðoković 1, Marinela Ž. Knežević 1, Slavica Djukić- Dejanović 1,2, Dragana I. Ristić-Ignjatović 1,2 1 School of Medicine, University of Kragujevac, 2 Psychiatric Clinic, Clinical Center Kragujevac; Kragujevac, 3 Department of Preventive Medicine, University of Prishtina-Kosovska Mitrovica, Kosovska Mitrovica; Serbia ABSTRACT Aim To assess the effect of hormone replacement therapy on postoperative depression and anxiety symptoms. Corresponding author: Jelena J. Jović Department of Preventive Medicine, University of Prishtina-Kosovska Mitrovica, Kosovska Mitrovica; Serbia Anri Dinana BB, Kosovska Mitrovica 38220, Serbia Phone: ; Fax: jovic.jelena@gmail Original submission: 15 April 2014; Revised submission: 17 June 2014; Accepted: 24 October Methods In observational prospective study 80 women divided into two groups were evaluated: women who received estrogen and androgen replacement therapy after hysterectomy with bilateral oophorectomy before onset of menopause (35-45 years old) and a control group that consisted of perimenipausal women (45-55 years old). Hormone replacement therapy began one week after surgery. The severity of depression and anxiety was evaluated through the use of Hamilton Depression Rating Scale and Hamilton Anxiety Rating Scale. Subjects from the study group were interviewed right after the surgical treatment, one, two and three months later. Subjects from the control group were interviewed only once. Results The women who underwent surgery had a statistically significantly higher score in Hamilton Depression Scale (p<0.001) and Hamilton Anxiety Scale (p=0.002) compared to the control perimenopausal women. There was a significant reduction of depressive and anxiety symptoms during hormone replacement therapy. Statistically significant difference in depressive score was found immediately after one month of hormone replacement therapy (first week/one month later: p=0.0057). Statistically significant difference in anxiety score appeared three months after the introduction of hormone therapy (first week/one month later: p=0.309; first week/two months later: p=0.046; first week/three months later: p<0.001). Level of serum luteinizing hormone was in correlation with depressive and anxiety score. Conclusion Estrogen-androgen replacement therapy may reduce the risk of psychiatric disorders developing in women with bilateral oophorectomy (indication for hysterectomy with oophorectomy was leiomyomata uteri). Keywords: hysterectomy, mood disorders, women health, HAMD, HAMA Med Glas (Zenica) 2015; 12(1):

86 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION In women with surgically removed uterus and ovaries before natural menopause, there is a sudden and drastic decrease in levels of ovarian hormones in the circulation which causes surgical menopause. Women who experienced this menopause have increased morbidity. They have a higher risk of developing cardiovascular (1), neurological (2,3) and psychiatric diseases (4,5) and osteoporosis (6) compared to referent population. Study of Rocca 2009 found that bilateral oophorectomy performed before the onset of menopause is associated with an increased long-term risk of depressive and anxiety symptoms, especially at younger age (4). The pattern of psychiatric disorder development is multifaceted. The possible role of hormone deprivation in occurrence of psychiatric symptoms in this population is not clear. The association between oophorectomy and increased risk of depressive and anxiety symptoms development may be explained by deficit in ovarian hormones and by the disruption of hypothalamic hypophysal ovarian axis (7). However, this association may be due to some undergoing psychological factors. Whether the abrupt onset of hormone imbalances will affect and how it will affect the mental and physical health of women depends on many factors (indications for surgery, mental and physical health of women before the operation, her sexual function, specific surgical procedure, age, marital status, socio-economic status, parenting) (8). The effect of the hormone does not cause behavior changes, but the likelihood of response to stimuli (9). It is known that hysterectomy only leads to the development of depressive and anxiety symptoms (10-12). However, results of some studies are inconsistent with these findings because there no association was found between surgical treatment and psychological symthoms (13,14) or any data that would imply that an intervention may improve psychological health (15,16). In most researches, it is not precise what the secretory function of ovary was. Also, age specific data are limited although some researches show that women who had undergone hysterectomy in young age had more severe psychological reaction (13). Altough there are many preclinical studies regarding the role of testosterone in depression and anxiety disorders, there are very few clinical studies on this subject (17). On the other hand, it is a known fact that women are twice as prone to anxiety disorders and depression than men. It is possible that sexual hormones are some of the key factors in the discrepancy between men and women, which leads to the possible role of testosterone and its possible protective benefits against anxiety and depression (18). The aim of our study is to determine whether hysterectomy with bilateral oophorectomy and consequent ovarian hormone disruption leads to an increase of anxiety and depression, and if it is possible eliminate or reduce those symptoms by hormone substitution therapy. The purpose of our research is to better understand these issues and improve mental health of women. PATIENTS AND METHODS Patients This observational prospective study was conducted during a two-year period at the Clinic of Gynecology and Obstetrics of the Clinical Centre Kragujevac, Clinic of Psichiatry of the Clinical Centre Kragujevac and Dispensary for Women of the Primary Health Care Centre Kragujevac. It evaluated 80 women who were divided into two groups: women who received estrogen and androgen replacement therapy after hysterectomy with bilateral oophorectomy before onset of menopause, and the control group that consisted of women in perimenopause. The first group included 40 women who were years old. The control group included 40 women aged Most of the women finished high school. Majority were from urban environments while 16.78% were from rural environments; 52 women were married, eight were single and 20 were divorced. By comparing the socio-demographic characteristics, (except for the age) no statistically significant difference between the two groups in any of the categories was found (p>0.001). The indication for hysterectomy with oophorectomy was leiomyomata uteri. The inclusion criteria was age between for the clinical and for the control group, no hormone therapy for one year prior, body mass index (BMI) 80

87 Đoković et al. Hormone therapy after oophorectomy <33kg/m2. The women with mental retardation, with known or suspected history of breast carcinoma, any malignant disease in the last 5 years, severe liver or renal disease, thromboembolic history or treatment with liver enzyme inducing medications or those that could have affected bone metabolism were excluded. Women did not use any antidepressants and anxiolytics. A written informed consent was obtained in all cases. The study was approved by the Ethics Committee of Faculty of Medical Sciences Kragujevac. Methods The severity of depressive and anxious symptoms was evaluated with the hetero-administered Hamilton Depression Rating Scale (HAM-D 21) (19) and Hamilton Anxiety Rating Scale (HAM-A) (20). In all patients diagnosis was confirmed by two experienced psychiatrists based on DSM-IV critera for depression/anxiety. Hormone replacement therapy began one week after the surgery. The therapy included estradiol valerianate-dehydroepiandrosterone enanthate at a 1:50 ratio (Gynodian Depo). Subjects from the study group were interviewed during the first week after surgery and then again during follow-up visits when they received hormonal therapy, four times in total. Subjects from the control group were interviewed only once. Blood was taken for analysis from patients on the same day the psychiatric interview was conducted. Statistical methods Data were expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used for testing variables normality. Because of non-normal distribution of data the statistical significance between the study groups was assessed by using the non-parametric Kruskal-Wallis Tests (three or more categories) and Mann-Whitney Tests (two categories). Wilcoxon Test was used to estimate if there were statistically significant differences among repeated measurements in the group with hormone substitution therapy. Linear correlation was used to examine relation between blood hormone level and HAMD and HAMA score. Statistical significance of p <0.001 was used. RESULTS Eighty women divided into two groups were evaluated: women who received estrogen and androgen replacement therapy after hysterectomy with bilateral oophorectomy due to leimyomas and the referent group of perimenopausal women. The first group included 40 women who were years old (mean age of 42±6.9). Control group included 40 women who were years old (mean age of 53±7.1). The women who had undergone surgery had a statistically significant difference in HAMD (p<0.001) and HAMA (p=0.002) score compared to women with physiological perimenopause (Table 1). It is apparent that women who had surgically undergone hysterectomy with bilateral oopherectomy had significantly more depressive symptoms than perimenopausal women. Anxiety symptoms were also more drastic in that group of women. Table 1. Hamilton Depression Rating Scale (HAMD) and Hamilton Anxiety Rating Scale (HAMA) scores in women who underwent hysterectomy with bilateral oophorectomy and perimenopausal women HAMD/HAMA Hysterectomy with oophorectomy* (n= 40) (mean± SD) Perimenopausa (n= 40) (mean± SD) HAMD score ± ± 3.71 <0.001 HAMA score ± ± *first week after surgery; SD, standard deviation Scores of depression (HAMD scale) in the first week (1), one month (2), two months (3) and three months (4) after the surgical procedure are shown in Table 2. Hormone replacement therapy began in the first week after the procedure, e.g., when the first interview with the subjects was conducted. Statistically significant difference in depressive score immediately after one month of hormone replacement therapy (the first week/one month later: p=0.0057) was found; the depressive score significantly reduced each month. Table 2. Hamilton Depression Rating Scale (HAMD) score in women in the study group at different time intervals after the surgical procedure HAMD scores during different time intervals* Xsr ± SD Xsr ± SD p ± 4.74 (n= 40) ± 4.74 (n= 40) ± 4.74 (n= 40) ± 4.67 (n= 40) ± 4.67 (n= 40) ± 4.17 (n= 40) ± 4.67 (n= 40) ± 4.17 (n= 40) ± 3.56 (n= 40) ± 4.17 (n= 40) ± 3.56 (n= 40) ± 3.56 (n= 40) *first week (1), one month (2), two months (3), three months (4); SD, standard deviation p

88 Medicinski Glasnik, Volume 12, Number 1, February 2015 Figure 1. Correlation between serum level of LH and HAMD score HAMD, Hamilton Depression Rating Scale; LH, luteinizing hormone Scores of anxiety (HAMA) in the first week (1), one month (2), two months (3) and three months (4) after the surgical procedure were shown in Table 3. The anxiety score was reduced one month after the hormone replacement therapy. Statistically significant difference in anxiety score appeared three months after the start of hormone therapy (after the subjects received the 3 rd dose of the therapy) (first week /one month later: p=0.309; first week /two months later: p=0.046; first week /three months later: p<0.001). Table 3. Hamilton Anxiety Rating Scale (HAMA) score in women in the study group at different time intervals after the surgical procedure HAMA scores during different time intervals* Xsr ± SD Xsr ± SD p ± (n= 40) ± (n= 40) ± (n= 40) ± (n= 40) ± (n= 40) 36.90± (n= 40) ± (n= 40) ± (n= 40) ±8.30 (n= 40) ± (n= 40) ±8.30 (n= 40) ±8.30 (n= 40) *first week (1), one month (2), two months (3), three months (4); SD, standard deviation The Pearsons linear correlation test showed existence of strong negative correlation between the level of serum luteinizing hormone and HAMD and HAMA score (Figures 1, 2). DISCUSSION Menopause is a universal and irreversible part of the overall aging process as it involves a woman s reproductive system. It is the final menstrual period which is diagnosed after 12 months of amenorrhea and is characterized by a myriad of symptoms that may include changes from regular, predictable menses, vasomotor and urogenital symptoms and sleep and mood dysfunction (21,22). Hormonal changes and clinical symptoms occur over a period leading up to menopause. This period is termed the climacteric or perimenopause or menopausal transition and it characteristically begins years before menopause, typically occurring between the ages of 45 and 55, with median age at inception of perimenopause 47.5 years (21-23). Until recently, perimenopause was not recognized as a period with a higher risk for new or repeated depression. It Figure 2. Correlation between serum level of LH and HAMA score HAMA, Hamilton Anxiety Rating Scale; LH, luteinizing hormone 82

89 Đoković et al. Hormone therapy after oophorectomy is still unclear whether and to what extent changes in estrogens, progesterone and androgens that occur in menopause period influence psychological status in women. The first important result of this study was the finding of a difference in HAMD and HAMA score between women who underwent surgery and the control group of perimenopausal women. Previous studies show that women who underwent hysterectomy with bilateral oophorectomy had more pronounced psychiatric symptoms (4,5,7). In the studies of women undergoing natural menopause an increase in depressive symptoms was demonstrated, generally revealed during perimenopause with a decrease in risk during postmenopausal years (5). Very interesting finding of this study is the indication of moderate depression according to average HAM-D value found in the control group, which was in line with some of the other researches (24). Previous research suggests that the estrogen deficiency caused by bilateral oophorectomy may be the initial step in a chain of causality that determines an increased long-term risk of depression or anxiety (4). Although the precise mechanisms are still unknown, depression during perimenopause is likely to occur due to the influence of estrogen to actions of serotonin and norepinephrine. A decline in estrogen concentrations may decrease levels of these hormones and thus contribute to depression (25,26). The hormonal changes induced by premenopausal bilateral oophorectomy are different from those occurring during natural menopause (27-29). In natural menopause, as well as after menopause, the ovary leads testosterone production. In widespread tissues and organs including the brain, testosterone is aromatized into estrone and estradiol, the most potent estrogen. Bilateral oophorectomy before menopause results not only in an abrupt drop in levels of circulating estrogen but also an abrupt drop in levels of circulating testosterone and in a disruption of the hypothalamicpituitary-ovarian axis with an increased release of the gonadotropins luteinizing hormone and follicle stimulating hormone (27-29) According to the correlation between hormonal status and score of depression and anxiety was found in this study, it is a question if deteriorated mental health in hysterectomized and oophorectomized women could be attributed only to hormonal abruption. These results can be explained by losing uterus and ovary as symbols of femininity and maternity in woman s life, which leads to psychological problems (28,29). The loss of the uterus and scarring after surgery may result in impairment of body image, which includes the perception of a loss of femininity and vitality (33,34). Impairment of body image has been found in oophorectomized women (35). Conserving their uterus may be important for gender identity, sexuality, marital relations, and selfesteem for many women in our population (36). A statistically significant decrease in depression and anxiety across post surgical time period has been found in this study. The scores for depression decreased significantly earlier (after first measure) and moved in a positive direction during the time compared to the anxiety score which did not change considerably for longer time (it decreased significantly after three months). Cohen 2011 showed that anxiety and depression after hysterectomy were highest in the immediate post-operative period and decreased significantly over the period of eight weeks (37). The results of this study have shown a correlation between the level of serum luteinizing hormone and score of depression and anxiety; the levels of LH had a negative correlation with the levels of depression and anxiety in the investigated groups of women. Similar results were obtained in a study (38) showing that psychiatric disorders should be considered in polycystic ovarian syndrome women in which significantly higher LH level has been found in the investigated group as compared to the control group. The main finding of this study is that postoperative estrogen therapy in combination with androgen therapy may decrease anxiety and depression in women who underwent hysterectomy with oophorectomy. There are some studies which examined the effect of estrogen replacement therapy on anxiety and depressive symptoms in such women, but results are inconsistent. The study of Rocca et al. (2008) found that the treatment with estrogen in women who are 50 years old and underwent bilateral oophorectomy at younger age did not modify the risk (4). Nathorst-Böös et al. (1993) reported less anxiety and depression and more well-being in oophorectomized women who received estrogen replacement therapy (39). There are two important mechanisms by which estrogen influences depression and depressivelike behavior: interactions with neurotrophic 83

90 Medicinski Glasnik, Volume 12, Number 1, February 2015 factors and influence on the serotonergic system (40). On the other hand, there are almost no studies that examine the effect of combined estrogenandrogen on anxiety and depressive symptoms. One of the shortcomings of this study is that the levels of hormones were determined solely on the basis of morning blood samples. Another shortcoming is that we did not have a group of women who underwent an operation, and who did not have a hormonal therapy. Furthermore, the control and clinical group were not compatible in terms of age. Therefore, there is a possibility that difference in the levels of depression and anxiety between these groups, in that case, would be much higher. All this should be taken into consideration in the future research. REFERENCES 1. Rivera CM, Grossardt BR, Rhodes DJ, Brown RD Jr, Roger VL, Melton LJ 3rd, Rocca WA. Increased cardiovascular mortality following early bilateral oophorectomy. Menopause 2009; 16: Rocca WA, Bower JH, Maraganore DM, Ahlskog JE, Grossardt br, de Andrade M, Melton LJ 3 rd. Increased risk of cognitive impairment or dementia in women who underwent oophorectomy before menopause. Neurology 2007; 69: Rocca WA, Bower JH, Maraganore DM, Ahlskog JE, Grossardt br, de Andrade M, Melton LJ 3 rd. Increased risk of parkinsonism in women who underwent oophorectomy before menopause. Neurology 2008; 70: Rocca WA, Grossardt BR, Geda YE, Gostout BS, Bower JH, Maraganore DM, de Andrade M, Melton LJ 3rd. Long-term risk of depressive and anxiety symptoms following early bilateral oophorectomy. Menopause 2008; 15: Mantani A, Yamashita H, Fujikawa T, Yamawaki S. Higher incidence of hysterectomy and oophorectomy in women suffering from clinical depression: retrospective chart review. Psychiatry Clin Neurosci 2010; 64: Gallagher JC. Effect of early menopause on bone mineral density and fractures. Menopause 2007; 14: Shuster LT, Rhodes DJ, Gostout BS, Grossardt BR, Rocca WA. Premature menopause or early menopause: long-term health consequences. Maturitas 2010; 65: Shifren JL, Avis NE. Surgical menopause: effects on psychological well-being and sexuality. Menopause 2007; 14: Pavlovic DM. Neuropsychology with the basics of behavioral neurology. Belgrade: Kaligraf, Carranza-Lira S, Murillo-Uribe A, Martínez Trejo N, Santos-González J. Changes in symptomatology, hormones, lipids, and bone density after hysterectomy. Int J Fertil Womens Med 1997; 42: Chung-Park M. Anxiety attacks following surgical menopause: a case report. Holist Nurs Pract 2005; 19: Even though our results show that appropriate hormone therapy can have a considerable effect on anxiety and depression in women with hysterectomy and bilateral oophorectomy, it is necessary to continue research in this field. It is also necessary to reduce the limitations of our study in future research. FUNDING Hereby authors would like to express gratitude to the Grant N and of Ministry of Science and Technological Development of The Republic Serbia, out of which this study was partially financed. TRANSPARENCY DECLARATION Competing interest; none to declare. 12. Helmy YA, Hassanin IM, Elraheem TA, Bedaiwy AA, Peterson RS, Bedaiwy MA. Psychiatric morbidity following hysterectomy in Egypt. Int J Gynaecol Obstet 2008; 102: Cooper R, Mishra G, Hardy R, Kuh D. Hysterectomy and subsequent psychological health: findings from a British birth cohort study. J Affect Disord 2009; 115: Cohen SM, Linenberger HK, Wehry LE, Welz HK. Recoveryy after hysterectomy: A year-long look. Webmed Central Obstetrics and Gynaecology (16 June 2014) 15. Carlson KJ, Miller BA, Fowler FJ Jr. The Maine Women s Health Study: I. Outcomes of hysterectomy. Obstet Gynecol 1994; 83: Lambden MP, Bellamy G, Ogburn-Russell L, Preece CK, Moore S, Pepin T, Croop J, Culbert G. Women s sense of well-being before and after hysterectomy. J Obstet Gynecol Neonatal Nurs 1997; 26: Mueller SC, Grissom EM, Dohanich GP. Assessing gonadal hormone contributions to affective psychopathologies across humans and animal models. Psychoneuroendocrinology 2014; 46C: McHenry J, Carrier N, Hull E, Kabbaj M. Sex differences in anxiety and depression: role of testosterone. Front Neuroendocrinol 2014; 35: Hamilton M. A rating scale for depression. J Neurol Neurosurg Psychiatry 1960; 23: Hamilton M. The assessment of anxiety states by rating. Br J Med Psychol 1959; 32: Butler L, Santoro N. The reproductive endocrinology of the menopausal transition. Steroids 2011; 76: Santoro N, Randolph JF Jr. Reproductive hormones and the menopause transition. Obstet Gynecol Clin North Am 2011; 38: McKinlay SM, Brambilla DJ, Posner JG. The normal menopause transition. Maturitas 1992; 14: Karaoulanis SE, Rizouli KA, Rizoulis AA, Angelopoulos NV. Lack of association of acute phase response proteins with hormone levels and antidepressant medication in perimenopausal depression. BMC Psychiatry 2014; 14:

91 Đoković et al. Hormone therapy after oophorectomy 25. Steiner M, Dunn E, Born L. Hormones and mood: from menarche to menopause and beyond. J Affect Disord 2003; 74: Halbreich U. Role of estrogen in postmenopausal depression. Neurology 1997; 48:S Morrison JH, Brinton RD, Schmidt PJ, Gore AC. Estrogen, menopause, and the aging brain: how basic neuroscience can inform hormone therapy in women. J Neurosci 2006; 26: Laughlin GA, Barrett-Connor E, Kritz-Silverstein D, von Muhlen DG. Hysterectomy, oophorectomy, and endogenous sex hormone levels in older women: the Rancho Bernardo Study. J Clin Endocrinol Metab 2000; 85: Rocca WA, Shuster LT, Grossardt BR, Maraganore DM, Gostout BS, Geda YE, Melton LJ 3rd. Longterm effects of bilateral oophorectomy on brain aging: unanswered questions from the Mayo Clinic cohort study of oophorectomy and aging. Womens Health (Lond Engl) 2009; 5: Simpson ER. Aromatization of androgens in women: current concepts and findings. Fertil Steril 2002; 77:S Lalinec-Michaud M, Engelsmann F, Marino J. Depression after hysterectomy: a comparative study. Psychosomatics 1988; 29: Drellich MG, Bieber I. The psychological importance of the uterus. J Nerv ment DIS 1958; 126: Bachmann GA. Psychosexual aspects of hysterectomy. Women s Health Issues 1990; 1: Sloan D. The emotional and psychosexual aspects of hysterectomy. Am J Obstet Gynecol 1978; 131: Bellerose SB, Binik YM. Body image and sexuality in oophorectomized women. Arch Sex Behav 1993; 22: Lalinec-Michaud M, Engelsmann F. Cultural factors and reaction to hysterectomy. Soc Psychiatry Psychiatr Epidemiol 1989; 24: Cohen SM, Hollingsworth AO, Rubin M, Bonnie MG, Thomas JS, Welz HK, Wehry LE. Psychosocial adaptation during recovery from hysterectomy. Webmed Central Obstetrics and gynaecology (16 June 2014). 38. Davari-Tanha F, Hosseini Rashidi B, Ghajarzadeh M, Noorbala AA. Bipolar disorder in women with polycystic ovarian syndrome (PCO). Acta Med Iran 2014; 52: Nathorst-Böös J, von Schoultz B, Carlström K. Elective ovarian removal and estrogen replacement therapy--effects on sexual life, psychological well-being and androgen status. J Psychosom Obstet Gynaecol 1993; 14: Borrow AP, Cameron NM. Estrogenic mediation of serotonergic and neurotrophic systems: Implications for female mood disorders. Prog Neuropsychopharmacol Biol Psychiatry 2014; 54: Efekti hormonske supstitucione terapije na simptome depresivnosti i anksioznosti kod žena posle uklanjanja jajnika Danijela D. Ðoković 1,2, Jelena J. Jović 3, Jelena D. Ðoković 1, Marinela Ž. Knežević 1, Slavica Djukić- Dejanović 1,2, Dragana I. Ristić-Ignjatović 1,2 1 Medicinski fakultet, Univerzitet u Kragujevcu; 2 Klinika za psihijatriju, Klinički centar Kragujevac, Kragujevac; 3 Katedra za preventivnu medicinu, Medicinski fakultet, Univerzitet u Prištini, Kosovska Mitrovica; Srbija SAŽETAK Cilj Proceniti uticaj efekata hormonske supstitucione terapije na postoperativne simptome depresije i anksioznosti. Metode U opservacionoj prospektivnoj studiji pratili smo 80 žena, podeljenih u dve grupe: žene pre početka menopauze (od 35 do 45 godina starosti) koje su posle operativnog uklanjanja materice i jajnika dobijale supstitucionu terapiju estrogena i androgena, i perimenopauzalne žene (starosti od 45 do 55 godina) koje su činile kontrolnu grupu. Hormonska supstituciona terapija započeta je nedelju dana nakon hirurške intervencije. Depresivnost i anksioznost je procenjivana Hamiltonovom skalom depresivnosti i Hamiltonovom skalom anksioznosti. Ispitanice iz studijske grupe intervjuisane su neposredno posle operacije, te mesec, dva i tri meseca kasnije. Ispitanice iz kontrolne grupe intervjuisane su samo jedanput. Rezultati Operisane žene, u poređenju s perimenopauzalnim ženama, imale su statistički značajno više skorove na Hamiltonovoj skali depresivnosti (p<0.001) i Hamiltonovoj skali anksioznosti (p=0.002). Tokom primene hormonske supstitucione terapije došlo je do značajne redukcije simptoma depresivnosti i anksioznosti. Statistički značajna razlika u depresivnosti ustanovljena je mesec dana nakon početka hormonske terapije (prva nedelja/mesec dana kasnije: p=0.0057). Statistički značajna razlika u pogledu anksioznosti utvrđena je tri meseca nakon početka hormonske terapije (nedelja/mesec dana kasnije: p=0.309; nedelja/dva meseca kasnije: p=0.046; nedelja/tri meseca kasnije: p<0.001). Nivo luteinizirajućeg hormona bio je u korelaciji sa skorovima depresivnosti i anksioznosti. Zaključak Estrogen-androgen supstituciona terapija može smanjiti rizik od razvoja simptoma depresivnosti kod žena kojima su uklonjeni jajnici (indikacija za histerektomiju s adneksektomijom bio je benigni miom materice). Ključne reči: histerektomija, poremećaji raspoloženja, žensko zdravlje, HAMD, HAMA 85

92 ORIGINAL ARTICLE Mechanical prosthetic valve disease is related with an increase in depression and anxiety disorder Yasemin Turker 1, Kurtulus Ongel 2, Mehmet Ozaydin 3, Yasin Turker 4, Funda Yildirim Bas 5, Mehmet Akkaya 6 1 Family Medicine Center, Duzce, 2 University of Katip Celebi, Faculty of Medicine, Department of Family Medicine, İzmir, 3 University of Suleyman Demirel, Faculty of Medicine, Department of Cardiology, Isparta, 4 Duzce University, Faculty of Medicine, Department of Cardiology, Duzce, 5 University of Suleyman Demirel, Faculty of Medicine, Department of Family Medicine, Isparta, 6 Bezm-i Alem University, Faculty of Medicine, Department of Cardiology, Istanbul; Turkey ABSTRACT Aim Patients with organic disease can present with psychiatric symptoms. We hypothesized that since patients with prosthetic heart valve require frequent hospital followup and are at higher risk for complications, the incidence of depression and anxiety is higher in these patients. Methods This cross-sectional study prospectively studied 98 consecutive patients with mechanical prosthetic heart valve. All patients fulfilled prosthetic heart valve evaluation form, Beck Depression Inventory (BDI) and Hamilton Anxiety Scale (HAS). Complete blood count, basic metabolic panel and echocardiogram results were collected for all the patients. Corresponding author: Yasemin Turker Family Health Center Duzce Provincial Directorate of Health, No. 3, Düzce, Turkey Phone: ; Fax: ; dryaseminturker@gmail.com Original submission: 02 July 2014; Revised submission: 18 August 2014; Accepted: 28 August Results Using the BDI, there were 26 patients (27%) with no depression, 20 (20%) with mild depression, 38 (39%) with moderate, 4 (4%) with severe and 10 (10%) patients with very severe depression. Avarege score was 18.3±11.4 on BDI and 19.1±11.1 on HAS. The depression level was positively associated with prothrombin time (p<0.001) and international normalized ratio (INR) level (p<0.001). Hamilton Anxiety Scale was significantly correlated with comorbidities (r: 0.344; p=0.002), blood transfusion (r: 0.370; p<0.001), obesity (r: 0.319; p=0.007) and Beck Depression Scale was correlated with comorbidities (r: 0.328; p=0.002), in patients with prosthetic heart valve disease. Conlusion Patients with prosthetic heart valve have higher prevalence of depression and higher scores of anxiety and depression. Early recognition and appropriate treatment of depression and anxiety may decrease the morbidity in prosthetic heart valve disease. Besides, use of new oral anticoagulant agents that do not need INR check, could decrease anxiety and depression in the future. Keywords: anxiety, depression, heart valve diseases, comorbidities, prothrombin time, international normalized ratio. Med Glas (Zenica) 2015; 12(1):

93 Turker et al. Valve disease and depression and anxiety INTRODUCTION Patients with organic disease can present with psychiatric symptoms. Depression is the most common psychiatric disorder accompanying organic disorders and is a significant clinical syndrome affecting both mortality and morbidity in those patients (1). Among the patients with organic disorders, the prevalence of major depression was found to be 5-10% in inpatients and 9-16% in outpatients (2). Patron et al. found that 48% patients were depressed any time during their life after cardiac surgery (e.g. heart valve surgery, coronary artery bypass graft) (3). Mechanical prosthetic heart valves are commonly used nowadays for the treatment of heart valve diseases with proven utility for increasing life expectancy and quality of life. Patients with prosthetic heart valves constitute 28% of the population with heart valve disease and they need to take anticoagulation therapy lifelong and international normalized ratio (INR) level check at regular intervals. Besides, these patients may develop structural heart disease over time (4). We hypothesized that since patients with prosthetic heart valve require frequent hospital followup and are at higher risk for complications, the incidence of depression and anxiety is higher in these patients. PATIENTS AND METHODS Study design The study was designed as cross-sectional and the patients with mechanical prosthetic heart valve, aged 18 or older, who were admitted to the Cardiology Outpatient Clinic, University of Suleyman Demirel, Faculty of Medicine, between March 2008 and August 2009, were consecutively enrolled. With a tolerance of 5% error, assuming alpha 0.05 and beta 0.20, it was found that 100 patients would be sufficient to detect a significant difference. Patients with mental retardation were excluded and all patients gave informed written consents. All patients completed prosthetic heart valve evaluation form, Beck Depression Inventory (BDI) and Hamilton Anxiety Scale (HAS). In prosthetic heart valve evaluation form the data including patient s age, gender, type of prosthetic valve, procedure date, need for repeated procedure, smoking status, comorbidities (hypertension, obesity, diabetes, chronic renal failure), history of bleeding, transfusion, cerebrovascular accident, myocardial infarction, congestive heart failure, pulmonary disease or gastrointestinal disease were collected. Patients recent complete blood count, basic metabolic panel and echocardiogram results were collected too. Beck Depression Inventory The Beck Depression Scale (BDS) is one of the most common scales used for assessment of mood disorders and depression, created by Aeron Beck in 1961 (5). It consists of 21 questions, 15 for emotional and 6 for somatic symptoms and filled by the patient. In BDS, scoring ranges from 0-3 on each question. The total score is calculated by summation of all the patient s responses, so the maximum possible sum is a score of 63. Higher scores on the scale indicate a greater level of depressive symptoms, BDS 0-11 indicates no depression, mild depression, moderate depression, severe depression and above 40 indicates very severe depression (5). Hamilton Anxiety Scale The Hamilton Anxity Scale measures the severity of anxiety level and symptom distribution. It is a 14-item test including mood and somatic symptoms administered by an interviewer. Each item is rated on a five-point Likert-type scale ranging from 0-4, so the sum of the score range is between The patients must complete the test within 72 hours from the admission (6). The study protocol was approved by the Ethics Committee of Suleyman Demirel University and all subject signed written consent forms. Statistical analysis Measured values are reported as mean ± standard deviation (minimum-maximum values). Oneway ANOVA, Kruskal Wallis, chi-square test and student test were used to compare group differences where appropriate. Spearman correlation test was used to identify correlation between continuous variables. Differences were considered significant at a p value of < RESULTS In the study 98 patients (54% males, n=53) with prosthetic heart valve presenting to Cardiology Outpatient Clinic were enrolled. The past dura- 87

94 Medicinski Glasnik, Volume 12, Number 1, February 2015 tion of the surgery for heart valve was 7.89 ± 5.7 years (1-25 year) before they were included into the study (Table 1). According to the BDS, there were 26 (27%) patients with no depression, 20 (20%) with mild depression, 38 (39%) with moderate, four (4%) with severe and 10 (10%) patients with very severe depression. Avarege score was 18.3 ± 11.4 on BDS and 19.1 ± 11.1 on HAS. Table 1. Characteristics of study participants Characteristic No (%) of patients/value at current visit Number 98 Age, years 53.6±10.9 Gender (male) 54 (54.5) AVR 10 (10%) MVR 75 (77) AVR and MVR 13 (13) Beck Depression Inventrory 18.3±11.4 No depression 26 (27) Mild depression 20 (20) Moderate depression 38 (39) Severe depression 4 (4) Very severe depression 10 (10) Hamilton Anxiety Scale 19.1±11.1 Cerebrovascular accident 3 (3) CHF 2 (2) COAH 2 (2) Gastrointestinal bleeding 4 (4) Epistaxis 19 (19.4) Gingival bleeding 15 (15.3) Blood transfusion 8 (8) Hypertension 29 (30) Diabetes mellitus 8 (8%) Obesity 9 (9) Arrhythmia 26 (26.5) Atrial fibrillation 19 (19.4) Atrial flutter 1 (1) Premature atrial contraction 5 (5) Premature ventricular contraction 1 (1) AVR, aort valve replacement; MVR, mitral valve replacement; COAH, chronic obtructive pulmanary disease; CHF, congestive heart failure Of those 98 patients, eight (8%) were 33-40, 31 (31%) 41-50, 36 (37%) 51-60, 13 (13%) and 10 (10%) were years of age. A comparison of patients by age groups did not show difference for either BDS (p=0.520) or HAS (p=0.469). Medical history of the patients was significant for cerebrovascular accident in three (3%) patients, congestive heart failure and chronic obstructive pulmonary disease in two (2%) patients each. Four patients had gastrointestinal bleeding, 19 had epistaxis and 15 patients had gingival bleeding after the surgery. Eight patients had received blood transfusion due to anemia or bleeding. Among the cardiac risk factors, hypertension was present in 29 (30%) patients, diabetes mellitus in eight (8%) and obesity in nine (9%) patients. Arrhythmia was detected in 26 patients, 19 had atrial fibrillation, one had atrial flutter, five had premature atrial contraction and one had premature ventricular contraction. Table 2 showed the comparison between depression level on BDS and patients complete blood count (CBC), coagulation parameters, fasting glucose, renal function test and echocardiographic findings. The depression level was positively correlated with prothrombin time and INR level. Prothrombin time was longer than expected in patients with mild and moderate depression (36±12.4 vs. 39±11.7, respectively), whereas it was shorter than expected in patients with very severe depression (21.8±8.2) (Table 2). Patients with severe depression were also found to have lower than expected INR levels (Table 2). However, BDS was not significantly correlated with prothrombin time (p=0.446) and INR (p=0.919) (in correlation analysis). Table 2. Comparison between depression level and laboratory and echocardiographic data Depression level Very No Mild Moderate Severe Paremeters severe (n = 26) (n = 20) (n = 38) (n = 4) (n = 10) p Hb 12.6 ± 13.1 ± 12.5 ± 13.3 ± 13 ± (g/dl) Glucose 108 ± 90 ± 93 ± 96 ± 108 ± (mg/dl) PT (sec) INR BUN (mg/dl) Cr (mg/dl) EF (%) LA (mm) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± < ± < ± ± ± ± EF, ejection fraction; Hb, hemoglobin; INR, international normalized ratio; LA, left atrium diameter; PT, prothrombin time; Cr, creatinine The depression levels were also compared with patients demographic and clinical features (Table 3). Patients with comorbidities accompanying prosthetic heart valve had more advanced degree of depression (p=0.005). Similary, chronic gastrointestinal disease was correlated with very severe stage of depression (p=0.001). Other parameters did not show significant difference. Patients with chronic diseases accompanying prosthetic heart valve were more depressive according to BDS (22.5 ± 13.3 vs ± 7.9; 88

95 Turker et al. Valve disease and depression and anxiety Table 3. Comparison between depression level and demographic and clinical features No (%) of patients with depression level Paremeters No Mild (n = 26) (n = 20) Moderate (n = 38) Severe (n = 4) Very severe (n = 10) Gender (male) 15 (58) 13 (65) 18 (47) 3 (75) 4 (40) Smoker 1 (4) 3 (15) (20) Comorbidities 8 (30.7) 8 (40) 17 (44.7) 2 (50) 10 (100) Heart Failure 2 (7.6) 1 (5) 3 (37.5) Arrhythmia 6 (23.07) 7 (35) 7 (18.4) 2 (50) 4 (40) DM 2 (7.6) 1 (5) 3 (7.8) 0 2 (20) Obesity 2 (7.6) 0 5 (13.1) 0 2 (20) HT 4 (15.3) 5 (25) 12 (31.5) 2 (50) 6 (60) CVA (7.8) Bleeding 7 (29.9) 10 (50) 19 (50) 0 2 (20) GIS disease (20) Repeat surgery 2 (7.6) 4 (20) 4 (10.5) GIS, gastro intestinal system; DM, diabetes mellitus; HT, hypertansion; CVA, cerebrovascular disease p = 0.001) and more anxious according to HAS (23.2 ± 11.7 vs ± 9.4; p = 0.001) than patients without comorbidities. The presence of hypertension was associated with higher scores for both depression (24.9 ± 10.2 vs ± 10.6; p = 0.01) and anxiety (24.9 ± 10.2 vs ± 10.6; p = 0.01). There was no significant difference between patients with and without diabetes for depression (p=0.272) and anxiety (p = 0.842). Gender difference, history of repeat procedure, cerebrovascular accident, congestive heart failure and smoking status were also not in correlation to depression and anxiety. History of bleeding by itself was not associated with depression and anxiety. However, while depression score was not different between patients with and without blood transfusion (p=0.235) Table 4. Comparison between type of heart valve surgery and clinical and laboratory data No (%) of patients/value Paremeters AVR (n=10) MVR (n=75) AVR and MVR (n=13) HAS 21.8 ± ± ± BDS 21.8 ± ± ± BDS level No depression Mild depression 4 (40) 14 2 Moderate depression 4 (40) 27 7 Severe depression Very severe depression 2 (20) 8 0 Gender (male) 9 (90) 37 (49.3) 8 (61.5) Age, years 61.0 ± ± ± Comorbidities 4 (40) 35 (46.7) 6 (46.2) PT (sec) 30.7 ± ± ± INR 2.6 ± ± ± AVR, aortic valve replacement; BDS, Beck Depression Scale; HAS, Hamilton Anxiety Scale; INR, international normalized ratio; MVR, mitral valve replacement; PT, prothrombin time p P due to anemia or bleeding, patients with transfusion history were more anxious (32 ± 4.2 vs ± 10.8; p < 0.001). Similarly patients with obesity were more anxious than non-obese ones (27.77 ± 7.83 vs ± 11.09; p=0.014). Patients with arrhyhthmia had similar depression scores compared to the patients without arrhythmia. Although the difference did not reach statistical significance, patients with arrhythmia tended to be more anxious than those without it (22.7 ± 12.7 vs ± 10.2; p=0.054). The presence of gastrointestinal disease in the patients was correlated with both high depression (45 ± 10.0 vs ± 10.8; p=0.001) and anxiety (39 ± 10.0 vs ± 10.8; p=0.01) scores. The Hamilton Anxiety Scale was significantly correlated with comorbidities (r: 0.344; p=0.002), blood transfusion (r: 0.370; p<0.001), obesity (r: 0.319; p=0.007) and the Beck Depression Scale (r: 0.660; p<0.001) in patients with prosthetic heart valve disease. The Beck Depression Scale was correlated with comorbidities (r: 0.328; p = 0.002) in patients with prosthetic heart valve disease. The Hamilton Anxiety Scale was not significantly correlated with PT (p=0.423) and INR (p =0.789). The type of heart valve surgery were aortic valve replacement (AVR) for 10 (10%) patients, mitral valve replacement (MVR) for 75 (77%) patients and both AVR and MVR for 13 (13%) patients. Table 4 showed the comparison between type of heart valve surgery and clinic and laboratory data. There were no significant differences in HAS, depression level and BDS between ARV, MVR and AVR and MVR. DISCUSSION In the present study, the level of depression and anxiety in patients with prosthetic heart valve and its relationship with the demographic and clinical features was investigated. A significant association between depression level and prothrombin and INR levels was found. The level of depression was more severe in patients having comorbidities and gastrointestinal disease. Besides, hypertensive patients were more anxious and depressive compared to non-hypertensive ones. Patients who had blood transfusion and obesity were more anxious. Chronic diseases are long-term diseases that may cause psychiatric problems in their course (7-9). 89

96 Medicinski Glasnik, Volume 12, Number 1, February 2015 Depression and anxiety disorder are the most common psychiatric diseases occurring in patients with somatic diseases and they usually coexist (10). Worsening in the quality of life, functional disability and direct biological effects of organic diseases are the main reasons for development of depression (11). Several studies have reported increased prevalence of depression and anxiety in patients with chronic disease as compared to patients without chronic disease (12-16). It was determined that patients undergoing heart operation experienced such physical and psychological problems as decrease in appetite, sleep disturbances, fatigue and activity intolerance, anxiety and depression within six months of being discharged (17). In our study, 27% of the patients with prosthetic heart valve did not have depression measured by the Beck Depression Inventory. In a study done by Bahar et al., age was reported to be correlated with higher anxiety and depression scores (15). On the contrary, Munir et al. found that majority of patients with anxiety and depression were aged between years (18). In the present study, we did not find significant difference in neither the Beck Depression Inventory nor Hamilton Anxiety Scale by patient s age. Female gender has been shown to be a risk factor for the development of depression. Some studies have demonstrated that women have higher average depression scores than males (19-20). Gilmour et al. found that females with depression had a higher risk for the development of heart disease while males did not (21). The relationship between psychiatric disorders and irritable bowel syndrome has been subject of interest for a long time (22). It has been shown that patients with irritable bowel syndrome have a higher incidence of depression and anxiety disorder (23). In addition, patients with depression have also been reported to have higher incidence of gastorinteestinal disease (24). Coexistance of depression and irritable bowel syndrome has been attributed to increased sympathetic activity. Our results are in accordance with previous studies, showing that patients with gastrointestinal diseases were more depressive and anxious. These patients were also more likely to be in very severe depression group in the Beck Depression Inventory groups. This study demonstrated a significant relationship between the depression levels and the prothrombin time and INR levels. Longer prothrombin time than expected in patients with mild and moderate depression was found, whereas it was shorter than expected in patients with severe depression. Additionally, INR levels were lower than expected in the very severe depression group. These findings may indicate that patients with more depression and anxiety have lower sense of responsibility to protect themselves from possible complications that may occur due to prosthetic heart valve disease. It is reasonable to think that these patients do not have regular prothrombin time and INR check and hence cannot reach target values. In the present study, the prevalence of depression and anxiety was higher in patients with comorbidities accompanying prosthetic heart valve disease. Besides, the level of depression was more severe in these patients compared to patients without comorbidities. Patients with chronic diseases are known to be more depressive and anxious (13,15,16). Since the study participants had chronic heart disease, comorbidities with this challenging heart problem make them more depressive and anxious. A meta-analysis reported a significantly higher risk for the development of hypertension in patients with high psychological stress levels. Moreover, it was claimed that this risk is comparable to wellknown risk facors for the development of hypertension such as obesity and lack of physical activity (25). Supporting the literature, patients with hypertension in this study were more depressive than without hypertension using the Beck Deprerssion Inventory. The Hamilton Scale anxiety scores were also higher in patients with hypertension. Patients requiring blood transfusion following bleeding or due to anemia experienced intense anxiety in this study. Since patients with prosthetic heart valve need frequent follow-ups over a long period of time, the need for admission to the hospital for blood transfusion probably makes them more aggressive and anxious. Several studies have demonstrated high prevelance of depression and anxiety in patients with obesity (26,27) pointing that in obese individuals, impaired body image and body dissatisfaction were observed, which might contribute to the development of anxiety and depression in this population. However, no correlation was reported between obesity and a level of anxity and depression. In our study, there was no difference in respect to depression between obese and non- 90

97 Turker et al. Valve disease and depression and anxiety REFERENCES 1. Katon W, Ciechanowski P. Impact of major depression on chronic medical illness. J Psychosom Res 2002; 53: Katon W, Schulberg H. Epidemiology of depression in primary care. Gen Hosp Psychiatry 1992; 14: Patron E, Messerotti Benvenuti S, Zanatta P, Polesel E, Palomba D. Preexisting depressive symptoms are associated with long-term cognitive decline in patients after cardiac surgery. Gen Hosp Psychiatry 2013; 35: Otto CM, Prosthetic valves. In: Otto CM (Ed.). Valvular Heart Disease. Philadelphia: WB Saunders, 1999: Burns DD. Feeling Good. The New Mood Therapy. New York: Morrow, Yazıcı MK, Demir B, Tanrıverdi N, Karaağaoğlu E, Yolaç P. Hamilton Anxiety Rating Scale: Interrater Reliabilty and Validity Study. Turk Psikiyatri Derg 1998; 9: Strain JJ, Lyons JS, Hammer JS, Fahs M, Lebovits A, Paddison PL, Snyder S, Strauss E, Burton R, Nuber G, et al. Cost Offset from a Psychiatric Consultationliaion Intervention with Elderly Hip Fracture Patients. Am J Psychiatry 1991; 148: obese patients whereas obese patients were found to feel more anxious. Prevalence of anxiety and depression is very common in patients with ischemic heart disease (28). Persistent symptoms of anxiety and depression increased substantially the risk of death in patients with ischemic heart disease (29). Patients with organic disease with major depressive disorder have longer in-hospital stays, fail to comply with treatment of the disease and medication they are prescribed, and have increased mortality and morbidy rates. Similar correlation is also found in patients with anxiety. Van Hout et al. reported that presence of anxiety disorder increases mortality in men, however, mortality in women is not increased (30). Garfield et al. reported that anxiety disorders, major depressive disorder, and co-occurring anxiety and major depressive disorder are associated with incident heart failure in large cohort (31). The depression and anxiety scores were not significantly different among the prosthetic valve type in this study. Relatively lower depression and anxiety detection in patients with both AVR and MVR may be associated with their younger age. Ther are several limitations of the present study. The number of patients across the different depression categories is unbalanced (only four patients in severe depression), therefore, a more balanced sample size across the depression categories would potentially allow for more robust comparison between these groups. The other limitation of study was that there was no control group of healthy individuals. Finally, patients did not have pre-surgery measurements of depression and anxiety level to compare the difference between pre and post surgery changes. In conclusion, it is crucial to identify depression and anxiety disorders of the patients with mechanical prosthetic valve disease, which can play an important role in treatment planning, and helps physicians predict the indication for treatment modalities and good long-term outcomes. Early recognition and appropriate treatment of depression and anxiety may decrease the morbidity in prosthetic heart valve disease. Besides, use of new oral anticoagulant agents that do not need INR check, could decrease anxiety and depression in the future. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATIONS Competing interest; none to declare. 8. Sim K, Rajasoorya C, Lam KN, Chew LS, Chan YH. High prevalence of psychiatric morbidity in a medical intensive care unit. Singapore Medical Journal 2001; 42: Gagnon LM, Patten SB. Major depression and its association with long-term medical conditions. Canadian Journal of Psychiatry 2002; 47: Hirschfeld RM. The Comorbidity of Major Depression and Anxiety Disorders: Recognition and Management in Primary Care. Prim Care Companion J Clin Psychiatry 2001; 3: Kılıcoglu A. Risk factors and etiology of depression among elderly: a review. Anatolian Journal of Psychiatry 2006; 7: Ozmen E, Taşkin EO, Ozmen D, Demet MM. Which psychiatric label is more stigmatizating? ruhsal hastalik or akil hastaligi. Turk Psikiyatri Derg 2004; 15: Altan L, Bingöl U, Sagirkaya Z, Sarandöl A, Yurtkuran M. Anxiety and depression in rheumatoid arthritis patients. Turk J Rheumatol 2004; 19: Hacıhasanoglu R, Yıldırım A. Depression among the elderly in Erzincan nursing home and influential factors. Turkish Journal of Geriatrics 2009; 12:

98 Medicinski Glasnik, Volume 12, Number 1, February Bahar A, Tutkun H, Sertbas G. The determination of the level of anxiety and depression of old people who live in the nursing home. Anadolu Psikiyatri Derg 2005; 6: Romao AP, Gorayeb R, Romao GS, Poli-Neto OB, dos Reis FJ, Rosa-e-Silva JC, Nogueira AA. High Levels of Anxiety and Depression have a Negative Effect on Quality of Life Women with Chronic Pelvic Pain. Int J Clin Pract 2009; 63: Jaarsma T, Kastermans M, Dassen T, Philipsen H. Problems of cardiac patients in early recovery. J Adv Nurs 1995; 21: Munir F, Khan HTA, Yarker J, Haslam C, Long H, Bains M, Kalawsky K. Self-Management of Health Behaviors Among Older and Younger Workers with Chronic Illness. Patient Educ Couns 2009; 77: Güz H, Yaman MA, Dilbaz N. Frequency of depression and psychiatric symptoms in elderly population with physical illness. Türkiye de Psikiyatri 2007; 9: Yakar T, Baran A, Güngör S, Altinsoy B, Yalcinsoy M, Can G, Akkaya E. The factors affecting Beck depression scale in asthmatic patients. Tuberk Toraks 2007; 55: Gilmour H. Depression and risk of heart disease. Health Rep 2008; 19: Creed F, Guthrie E. Psychological factors in the irritable bowel syndrome. Gut 1987; 28: Lydiard RB. Irritable bowel syndrome, anxiety, and depression: what are the links? J Clin Psychiatry 2001; 62(Suppl 8): Garakani A, Win T, Virk S, Gupta S, Kaplan D, Masand PS. Comorbidity of irritable bowel syndrome in psychiatric patients: a review. Am J Ther 2003; 10: Ruthledge T, Hogan BE. A quantitative review of prospective evidence linking psychological factors with hypertension development. Psychosom Med 2002; 64: Black DW, Goldstein RB, Mason EE. Prevalence of mental disorder in 88 morbidly obese bariatric clinic patients. Am J Psychiatry 1992; 149: Britz B, Siegfried W, Ziegler A, Lamertz C, Herpertz-Dahlmann BM, Remschmidt H, Wittchen HU, Hebebrand J. Rates of psychiatric disorders in a clinical study group of adolescents with extreme obesity and in obese adolescents ascertained via a population based study. Int J Obes Relat Metab Disord 2000; 24: Pająk A, Jankowski P, Kotseva K, Heidrich J, de Smedt D, De Bacquer D; EUROASPIRE Study Group. Depression, anxiety, and risk factor control in patients after hospitalization for coronary heart disease: the EUROASPIRE III Study. Eur J Prev Cardiol 2013; 20: Doering LV, Moser DK, Riegel B, McKinley S, Davidson P, Baker H, Meischke H, Dracup K. Persistent comorbid symptoms of depression and anxiety predict mortality in heart disease. Int J Cardiol 2010; 145: van Hout HP, Beekman AT, de Beurs E, Comijs H, van Marwijk H, de Haan M, van Tilburg W, Deeg DJ. Anxiety and the risk of death in older men and women. Br J Psychiatry 2004; 185: Garfield LD, Scherrer JF, Hauptman PJ, Freedland KE, Chrusciel T, Balasubramanian S, Carney RM, Newcomer JW, Owen R, Bucholz KK, Lustman PJ. Association of anxiety disorders and depression with incident heart failure. Psychosom Med 2014; 76:

99 ORIGINAL ARTICLE Emotional profile and risk behaviours among tattooed and non-tattooed students Matea Zrno 1, Maja Frencl 2, Dunja Degmečić 1,2, Ivan Požgain 1,2 1 School of Medicine, University of Osijek Josip Juraj Strossmayer, 2 Department of Psychiatry, University Hospital Centre; Osijek, Croatia ABSTRACT Aim To determine differences in emotional profile and frequencies of certain risk behaviours between tattooed and non-tattooed students. Methods One hundred students fulfilled personality assessment (trust, timid, depressive, distrust, aggressive, gregarious, controlled, uncontrolled) and questionnaire of socio-demographic data that also included some questions about possession of tattoo (time, place, motive) and about certain risk behaviours (court punishment, consummation of alcohol, psychoactive substances and cigarettes). Corresponding author: Maja Frencl Department of Psychiatry, University Hospital Centre Osijek, Huttlerova 4, Osijek, Croatia Phone: ; Fax: ; frenclmaja@gmail.com Original submission: 08 September 2014; Revised submission: 29 October 2014; Accepted: 04 December Results The total number of 35 (out of 100) students had a tattoo, and 67 wished to have a tattoo. There was no statistically significant difference in emotional profile between tattooed and nontattooed individuals, yet the differences were detected when the group of subjects who wanted a tattoo and those who did not want a tattoo were compared. Higher result on the aggression scale of and lower on control scale was gained by those with the wish for tattooing. Students with bigger tattoos (23) showed higher score on depression scale. Students in the tattooed group more frequently abused drugs and committed traffic offences compared to the students in non-tattooed group. Conclusion Results of this research as well as previous research show that the presence of a tattoo could be a rough indicator for possible emotional problems and risk behaviour, which could have significant implications in preventing these behaviours. Future studies are required on a larger and more representative sample as well as to clarify why young people decide to be tattooed. Key words: tattooing, personality, drug abuse, traffic offences Med Glas (Zenica) 2015; 12(1):

100 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION Main assumption in the theory of personality is that characteristics of personality are partly genetically predisposed and are manifested as individual differences in cognitive, emotional and behavioural functioning in different time and situations (1). Personality is manifested through behaviour, therefore researching connections between specific behaviour, emotions and personality characteristics can help in predicting the appearance of certain behaviours (1). This can be especially useful in the research of risk behaviours because of the prevention and treatment plans. Through history in most cultures tattooing had a negative meaning. Common attitude is that tattooed persons are different and that they have a certain pathology in personality (2-11). There are also some stereotypes connected to tattooing. People can perceive tattooed males as more dominant which means more attractive, and tattooed females as less healthy compared to nontattooed (2,3), more sexually promiscuous and heavier drinkers than non-tattooed females (3). Researches showed that a differences in personality between tattooed and non-tattooed individuals have existed. Studies found tattooed persons more extroverted, with a greater need to be unique and more prone to adventurous behaviours as compared to non-tattooed individuals (4). They also showed a more positive attitude towards tattoos in comparison with non-tattooed individuals. According to that research it is assumed that tattooing has become a sign of self-expression used by individuals to differ more from others (4). There are many studies conducted worldwide which show connection between tattooing and risk and violent behaviours. A few new studies (5-9) show that tattooing is connected with high-risk behaviours. Tattooed young people drink more alcohol drinks (5,6,7) and more frequently (6), use psychoactive substances more frequently (6,7), have higher incidence of school throw-outs (6), practice more insecure sexual intercourses (6,7), while suicidal behaviors and suicidal ideation were not related to tattoo status among university students (7). These differences occurred not only in young population, but in the population through the lifespan also (8). Tattooed students were also significantly more often depressed (6). Higher impulsivity, adventurism and risk behaviours in tattooed soldiers with PTSD were found (9), and more tattooed patients were diagnosed with antisocial personality disorder (10) compared to the non-tattooed. Non-psychiatric population study showed that tattooed individuals more often had unconventional sexual relationships, they were more extroverted, more prone to adventurist/risk behaviour and that they had less consciousness than non-tattooed individuals (11). Bender et al.(12) showed that impulsiveness has an indirect relationship to suicidal behavior, and that this relationship is mediated by painful and provocative events that include tattooing as well. Nevertheless, there are also researches that show that nowadays tattooing is only the expression of fashion and is not connected with higher rate of risk behaviour and personality differences (13,14). The motivation for different body modifications like tattooing and piercing was found in expressing self-consciousness, identity, and a wish to demonstrate autonomy and thereby to control one s own body (15). Although tattooing is planetary popular today and it does not stigmatize individuals as much as it used to, there is still an open question if there is connection between tattooing and different kind of disturbed or high-risk behaviour, and if tattooing can be a certain predictor of such behaviour. The aim of the study was to determine the differences in emotional profile and frequencies of certain risk behaviour between individuals with a tattoo and those without it. As having tattoo can be a rough indicator of emotional and behavioural problems, we hypothesized that students with a tattoo were more prone to emotional problems and risk behaviours. EXAMINEES AND METHODS The study was conducted among students of Josip Juraj Strossmayer University of Osijek. One hundred students were included (37 males, 63 females) between 19 and 30 years of age. Mean age was years (SD=2.53). Students participated in the research on voluntary basis, they were invited by an advertisement on information board of a few faculties. From the total number of 100 students, two (2%) were first year students, 38 (38%) second year, 18 (18%) third year, 10 (10%) fourth year, 94

101 Zrno et al. Tattoing: emotional profile and risk behaviour 15 (15%) fifth year, eight (8%) sixth year students, and nine (9%) students were at the point of finishing their study. Socio-demographic questionnaire was designed for this study and it included questions about age, sex, faculty and year of the study, eventual court punishment, consummation of alcohol, psychoactive substances and cigarettes, existence of a tattoo, wish for a tattoo and questions about the tattoo (place, motive, time of tattooing). In the second part of the study participants fulfilled Emotions Profile Index (PIE) assessment, which was supervised by a licensed psychologist. Participants completed questionnaires anonymously and voluntary, in groups or individually, in presence of the skilled person, and were allowed to ask questions and get additional explanations relating to questionnaire completeness. Written consent was obtained from the subjects for collecting the data and publication of the study. This study was approved by the Ethical Committee of School of Medicine, J.J. Strossmayer University of Osijek. Assessment PIE is designed for measurement of emotionality of participants according to the Plutchik multidimensional model of emotions (16). The theory assumes existence of 8 basic dimensions of emotions: Trust, Timid, Depressive, Distrust, Aggressive, Gregarious, Controlled, and Uncontrolled. Assessment PIE is composed of 62 questions which are pairs of expressions for characteristics of personality (total of 12), and the task for the participants was to choose which one of the two words describe him/her better (16). Results were presented as frequencies and percentages (for descriptive variables), means and standard deviations (for numerical variables). Furthermore, independent samples t-test was performed for testing differences in emotional dimension between tattooed and non-tattooed students, and Chi-square was performed for testing differences in frequencies of certain risk behaviours. In cases where expected frequency was lower than 5, Fisher exact test was used. Results were considered statistically significant on the level of risk equal to or less than 5% (p<0.05). RESULTS In the observed group (n=100), 35 (35%) had tattoo: six (17.1%) of them had 2 tattoos, two (5.7%) had 3 tattoos, two (5.7%) had 5 tattoos, and one (2.9%) had 6 tattoos. Considering the size of the tattoo, 12 (34.3%) had small tattoos, 23 (65.7%) had medium or large tattoos. More than half of the students, 18 (51.4%), responded that love for tattoos was their motivation to have it, personal connection with the motive of the tattoo was declared by five (14.3%), a moment decision was stated by three (8.6%), two (5.7%) had it done for memories, two (5.7%) because their close persons told them to do it, one (2.9%) out of curiosity, one (2.9%) because of wish to have the tattoo seen by everyone, one (2.9%) student said he/she did it for love. The remaining two students had different reasons like: a song, nothing, feeling that everyone has something, life road etc. Most of the subjects had their tattoos made at the age between 18 and 21, 16 (45.7%). The majority of the students, 31 (88.6%), were not sorry for having a tattoo, while four (11.4%) affirmed that they were sorry. There was no statistically significant difference in the distribution of the dimensions of emotions between the groups of tattooed and non-tattooed subjects. From the total number of subjects, 67 (67%) wished to have a tattoo. There was statistically significant difference in the distribution of the dimensions of emotions between those with andwithout a wish for tattooing. Statistically significant difference resulted in the distribution of dimension Controlled between those two groups (p=0.05). Students who had a wish to tattoo had lower result on scale Controlled (M=15.9; SD=4.04) compared to those who had no wish to tattoo (M=17.67; SD=4.11). Statistically significant difference resulted in the distribution of the dimension Aggressive (t=2.29; p<0.05). Students with the wish to tattoo had a higher result on the scale Aggressive (M=11.09; SD=5.77) compared to those without the wish for tattooing (M=8.36; SD=5.13). A statistically significant difference (p=0.02) in the personality dimension Depressive between students with small tattoo (n=12) and those with medium or big tattoo (n=23) was found. Students who had medium or big tattoos had higher score on the scale Depressive (M=7.74; SD=3.01) than those with small tattoos (M=5.00, SD=3.25) (Table 1). 95

102 Medicinski Glasnik, Volume 12, Number 1, February 2015 Table 1. Mean and standard deviation of participants answers on dimensions of aggression, control and depression considering wish for tattoo and size of tattoo Dimension of emotions Groups M (SD) Aggression With wish (5.77) Without wish 8.36 (5.13) Control With wish (4.04) Without wish (4.11) Depression Big tattoo 7.74 (3.01) Small tattoo 5.00 (3.25) M=Mean, SD=Standard deviation More tattooed students reported to use drugs (p<0.05) and have traffic offenses (p<0.05) than the non-tattooed ones. In the results distribution consummation of alcohol, cigarettes, other offenses (domestic violence, disturbing public peace, crime) there was no statistically significant difference in the frequency distribution between tattooed and non-tattooed students (Table 2). Table 2. Frequencies of specific risk behaviours in two groups of subjects No (%) of students Risk behaviour Answer With tattoo Without tattoo Punishment for traffic offence Drug abuse DISCUSSION Yes 5 (14.7) 0 No 29 (85.3) 66 (100) Yes 19 (55.88) 14 (21.21) No 15 (44.12) 52 (78.79) From the total number of students who participated in the study, 35% had a tattoo, and 67% had a wish for tattoo in the future. In the western civilization, the number of tattooed persons is growing (13, 17) possibly because of the promotion of the tattoos by celebrities in music, film industries, resulting in the transfer of tattooing from the edge of society to the main stream. No statistically significant difference occurred in dimensions of emotions between the two groups, but differences were found between the students with/without a wish to tattoo. It seems that students who wish to tattoo are more aggressive and have less control than students without a wish to tattoo. It is proposed that students with a wish to tattoo can be considered as the group of tattooed (they are still too young and financially dependent, but will have a tattoo done in the future); both groups have the same attitudes towards tattoos. Moreover, a significant difference in emotional profile between the groups with and without a wish for tattoo were found, which is in concordance with the results of the previous researches (4,9). However, it is important to notice, contrary to the findings of Tate and Shelton (14), that some differences in emotional dimensions found in our study emerged as important. Although in Tate and Shelton study differences in personality between tattooed and non-tattooed have emerged, mean scores for those variables fell within the range of standardized norms. In the presented study mean scores of tattooed students were either above (for Aggression) or below the average (for Control). That indicates some emotional problems in tattooed students that are not negligible. The size of the tattoo was also connected with the differences in the student s emotional profile in this study, students with a big tattoo were more depressed than those with a small tattoo. Similar results were detected in other studies too, where tattooed persons (males) were more depressed than non-tattooed (7). Furthermore, results of this research have shown that more tattooed compared with non-tattooed are involved in risk behaviour, such as consummation of drugs and traffic offences, but it was not confirmed that tattooed students drink alcohol more often than non-tattooed, like in some other studies (5-8). This could be explained by the fact that a 4-point scale for measuring frequency of drinking alcohol used in this questionnaire is not appropriate for this population. Both groups of students mostly answered that they occasionally drank alcohol. So the findings in this study just partly confirmed previous findings about tattoos and risk behaviour (5-8). Regarding the choice of the tattoo place we can conclude that our subjects wanted to have a possibility to hide the tattoo, showing that there is still stigma about tattoos. Hands were the most frequently used place for tattooing (31%) followed by the back (24%). Those are the places where tattoo is visible but also can be hidden. In one study, 68% of tattooed subjects admit that they hide their tattoos in special occasions, such as exams, certain festivities, and the main reason for that behaviour is because they consider them inappropriate (17). In this study 89% of the tattooed students were not sorry because they had a tattoo. New research among American population showed that 14% of tattooed persons want to have it removed 96

103 Zrno et al. Tattoing: emotional profile and risk behaviour because they were too young when they made a tattoo (21%), they did not like to be marked for the rest of the life (19%) and did not like it any more (18%) (18). The results in this study are not the same, probably because young population was investigated, so their first tattoo was not made long time ago, at the age between 18 and 21 (45.7%). Our results are similar to those of the study of Aslam and Owen (19), but in their study one third of subjects regreted their tattoos. Considering that fact and a short period from the tattooing in which they could feel regret, our results are expectable. The act of tattooing is rather painful, maybe persons who have tattoos are considered braver, more special, more capable compared to the nontattooed, and therefore this courage leads them to risk behaviours. According to the interpersonal theory of suicide, impulsive people often do painful and provocative things and have more capability for self-harm and over time they get accustomed to pain, which gives them more capability to suicidal behavior if they ever desire it (20). Maybe stable personality or just current immaturity of those who decide to have a tattoo is the reason, because later in their life they regret for this act. Most young people have tattoos done in the adolescent age, which is marked with emotional instability and less capacity for the anticipation of consequences later in life. To summarize, based on the results from our and other studies, although tattoos have become planetary popular and do not stigmatize individuals as in the past, they still carry and lead to some risk behaviour and possible markers of those behaviours, respectively. The presence of a tattoo in a person could be a rough indicator for possible risk behaviour, like drug abuse and traffic offences, and also for emotional problems like aggression and lack of behavioural control. This could have significant implications in therapeutic interventions and prevention. A similar study had not been performed in Croatia before and this one gives some interesting insights into tattooing, emotional profile and risk behaviours in this region. Future studies should be done on a larger and more representative sample, which will include young people with or without finished high school who are not going to the college. Also, it would be interesting to include in the study the population of broader age scale. Reasons for the tattooing should be determined with more details, with the aim to discover whether this behaviour has deeper meaning or is it just a part of fashionable behaviour. FUNDING No specific funding was received for this study. TRANSPARENCY DECLARATION Competing interests: None to declare REFERENCES 1. Hampson SE, Colman AM. Individual differences and personality. New York and London: Longman Publishing, Wohlrab S, Fink B, Kappler PM, Brewer G. Perception of human body modification. Pers Indiv Differ 2009; 46: Swami V, Furnham A. Unattractive, promiscuous and heavy drinkers: perception of women with tattoos. Body Image 2007; 4: Swami V, Pietsching J, Bertl B, Nader IW, Stieger S, Voracek M. Personality differences between tattooed and non-tattooed individuals. Psychol Rep 2012; 111: Healthday News for healthier living. Tattoos, Piercings Tied to Heavier Drinking in French Study. asp?aid= (25 November2012). 6. Yen CF, Hsiao RC, Yen JY, Yeh YC, Wang PW, Lin HC. Tattooing among high school students in southern Taiwan: the prevalence, correlates and associations with risk-taking behaviors and depression. Kaohsiung J Med Sci 2012; 28: King KA, Vidourek RA. Getting inked: Tattoo and risky behavioural involvement among university students. Soc Sci J 2013; 50: Heywood W, Patrick K, Smith AM, Simpson JM, Pitts MK, Richters J. Who gets tattoos? Demographic and behavioural correlates of ever being tattooed in a representative sample of men and women. Ann Epidemiol 2012; 22: Požgain I, Barkić J, Filaković P, Koić O. Tattoo and personality traits in Croatian veterans. Yonsei Med J 2004; 45: Cardasis W, Huth-Bocks A, Kenneth RS. Tattoos and antisocial personality disorder. J Pers Ment Health 2008; 2: Swami V. Written on the body? Individual differences between British adults who do and do not obtain a first tattoo. Scand J Psychol 2012; 53: Bender TW, Gordon KH, Bresin K, Joiner Jr TE. Impulsivity and suicidality: The mediating role of painful and provocative experiences. J Affec Disorders 2011; 129:

104 Medicinski Glasnik, Volume 12, Number 1, February Preti A, Pinna C, Nocco S, Mulliri E, Pilia S, Petretto DR, Masala,C. Body of evidence: Tattoos, body piercing and eating disorders symptoms among adolescents. J Psychosom Res 2006; 61: Tate JC, Shelton BL. Personality correlates of tattooing and body piercing in a college sample: The kids are alright. Pers Indiv Differ 2008; 45: Stirn A, Oddo S, Peregrinova L, Philipp S, Hinz A. Motivation for body piercing and tattoos: the role of sexual abuse and the frequency of body modification. Psychiat Res 2011; 190: Plutchik R, Kellerman H. Emotions profile index manual (PIE). Jastrebarsko: Naklada Slap, DeMello M. Not just for bikers anymore: Popular representation of American Tattooing. J Pop Cult 1995; 29: American Academy of Dermatology. Tattoos and body piercings. (25 November 2012). 19. Aslam A, Owen CM. Fashion change but tattoos are forever: time to regret Brit J Dermatol 2013; 169: Van Orden KA, Witte TK, Cukrowicz KC, Braithwaite SR, Selby EA, Joiner Jr. TE. The Interpersonal theory of suicide. Psychol Rev 2010; 117: Profil emocija i rizična ponašanja kod tetoviranih i netetoviranih studenata Matea Zrno 1, Maja Frencl 2, Dunja Degmečić 1,2, Ivan Požgain 1,2 1 Medicinski fakultet, Sveučilište J. J. Strossmayera u Osijeku, 2 Klinika za psihijatriju, Klinički bolnički centar Osijek, Osijek, Hrvatska SAŽETAK Cilj Utvrditi postoji li razlika u emocionalnom profilu i u kojim dimenzijama između tetoviranih i netetoviranih studenata, te ispitati učestalost javljanja određenih rizičnih ponašanja kod ove dvije skupine ispitanika. Metode U istraživanju su sudjelovali studenti koji su ispunili upitnik o ličnosti (povjerljivost, bojažljivost, depresivnost, nepovjerljivost, agresivnost, društvenost, kontroliranost, nekontroliranost) i učestvovali u anketi posebno osmišljenoj za ovo istraživanje, a koja je uključivala sociodemografska pitanja, pitanja o tetovažama (posjedovanje, želju za posjedovanjem, dob tetoviranja, lokaciju tetovaže, motiv), te učestalosti rizičnih ponašanja (sudsko kažnjavanje, konzumiranje alkohola, droge i cigareta). Rezultati Od ukupnog broja studenata, 35 (od ukupno 100) ih je imalo tetovažu, a 67 ih je imalo želju za tetoviranjem. Nisu pronađene statistički značajne razlike u emocionalnom profilu između tetoviranih i netetoviranih, ali su utvrđene razlike u agresivnosti i kontroliranosti između studenata sa željom za tetoviranjem i onih koji nemaju takve želje. Studenti s većim tetovažama (23) pokazali su veću depresivnost od onih s malim i srednjim tetovažama. U odnosu na rizična ponašanja, kod tetoviranih ispitanika utvrđena je češća zloporaba droga i kažnjavanje za prometne prekršaje. Zaključak: Postojanje tetovaže kod osobe mogao bi biti grubi indikator za mogućnost postojanja emocionalnih problema i rizičnog ponašanja što može imati implikacije u prevenciji takvih ponašanja. Potrebna su daljnja istraživanja na većem i reprezentativnijem uzorku. Ključne riječi: tetoviranje, osobine ličnosti, zloporaba droga, prometni prekršaji 98

105 ORIGINAL ARTICLE The effect of anger management levels and communication skills of Emergency Department staff on being exposed to violence GozdeYildiz Das 1, Ilknur Aydin Avci 2 1 School of Health, Amasya University, Amasya, 2 Nursing Department, Samsun School of Health, Ondokuz Mayis University, Samsun; Turkey ABSTRACT Aim To determine the effect of anger management levels and communication skills of emergency department staff on their frequency of being exposed to violence. Methods This cross-sectional study was conducted in the Training and Research Hospital, Istanbul, Turkey between 11 April and 15 October 2013 by using a questionnaire including descriptive features, anger management scale, and communication skills scale applied to 283 health personnel working in children and adult emergency department clinics. Corresponding author: Ilknur Aydin Avci Nursing Department, Samsun School of Health, Ondokuz Mayis University Atakum Kurupelit, Samsun, Turkey Phone: ; Fax: ; ilknura@omu.edu.tr Results Statistically significant differences were found between the health workers ages and their anger control levels, marital status and anger-in and anger control levels, working position and anger-in levels, and between anger-in, anger-out and anger control levels based on their level of education. Statistically significant differences were also found between age and communication levels based on the personnel s working position. Statistically significant difference between the anger-in subscale of health personnel based on their state of being exposed to violence was found (78.4% of the health workers had been exposed to violence). Conclusion In the in-service programs of institutions, there should be trainings conducted about anger management and effective communication techniques so that the health personnel can be aware of their own feelings and express anger in a suitable way Key words: anger management, communication, patients Original submission: 18 August 2014; Revised submission: 09 October 2014; Accepted: 22 October Med Glas (Zenica) 2015; 12(1):

106 Medicinski Glasnik, Volume 12, Number 1, February 2015 INTRODUCTION Anger, which has an important place among feelings, is generally related to familial, work-related, health related and legal problems. Anger is a negative mood state which varies in terms of intensity and continuity and associated with a sense of emotional elevation and being exposed to a wrong behavior (1). Anger is also related to an emotional reaction to restrictions, strains and intense stressors within the working environment (2). It is obvious that human health is under the physical and psychological effect of many-sided stresses which are related to working life. One of the most important sources of stress caused by working life is the relationships among people (3). It has been reported that a stressful working environment, not asking for ideas within the organizational process, absence of common goals and presence of rivalry among organizations cause nurses to become more inclined to conflict and to experience intense feelings of anger (2). Within the hospital environment, nurses frequently come across verbal anger expressions from patients and generally perceive these as a threat and thus, they experience anger either directly, by getting angry with the patient and reflecting this anger in their behaviors, or indirectly by getting away from the patient (4). Deterioration in nurses health will cause loss of workforce, economic loss for the organization and the country, an increase in accidents and risk for the healthcare workers or patients for whom they are offering service. Thus, it is very important to determine the risks which are related to the working conditions of nurses and to eliminate these risks or to try to minimize them. It is important to develop nurses skills to deal with the feelings of anger and to channel the energy felt directly anger and to use this energy to increase the quality of care (5,6). Hospital environment causes healthcare workers to violate boundaries and privacy. During the critical period caused by illness, the patient needs to belong to and connect to a person. Thus, it is very important that the patient can reach and trust his/ her doctor, nurse and other workers (7). Most of the time, patients and their relatives are in fear and anxiety since they do not know what to do. Because they think that their condition is more urgent than others and because the necessity of triage is not adopted by the society, they have an expectation of immediate treatment. This situation often causes conflict between the emergency department staff and relatives of the patient (8). Communication skills have an important place in nursing services. While providing service to patients and healthy people, nurses should expect to understand these people and to be understood by them. Healthy communication skills will help nurses to develop their interpersonal relationships and this will in turn cause an increase in the satisfaction of the people that they are providing service for. Thus, health professionals should have effective communication skills in order to perform this important mission (9). Deterioration of the health workers physical and psychological health will cause loss of workforce, a decrease in care, treatment and efficiency, economic loss for the organization and the country and risk for the people or patients for whom they are offering service. Thus, the risks which are caused by the working conditions of health workers should be determined and eliminated. While the studies done in this field have so far been oriented towards the reason of increasing violence regarding emergency staff, this study aims to determine the effect of anger management levels and communication skills of emergency department staff on their frequency of exposure to violence. EXAMINEES AND METHODS This cross sectional study was conducted in the Training and Research Hospital Provincial Directorate of Health, Istanbul, Turkey, in the period between11 April and 15 October The research group of the study consisted of 238 health personnel working at the Pediatric and Adult Emergency Clinics of the Training and Research Hospital. All the personnel working in these units were voluntarily included in the study: 107 nurses, 113 doctors and 63 other health personnel (laboratory technicians, radiology technicians, emergency technicians). Data collection The data was collected through a question form of descriptive characteristics, trait anger and anger management inventory and communication skills questionnaire. 100

107 Das et al. Anger management and communication skills The question form which was prepared by the researchers contained 19 questions: the individual and occupational descriptive characteristics of the health personnel, their exposure to violence in the emergency service and their anger, communication and behavior styles. Trait Anger and Anger Expression Inventory Trait Anger and Anger Expression Inventory is a self evaluation inventory that measures anger and anger expression. It was developed by Spielberger in 1983 (10) and its validity and reliability for Turkey were made by Özer in 1994 (11). The inventory has two main subscales, S1-Anger and Anger-Style, respectively. The inventory has a total of 34 items. The items do not measure the absence of anger, but they measure the presence of anger. Scoring of the scale is as follows: Almost never (1), Sometimes (2), Often (3) and Almost always (4). In the S1 scale, total score was obtained by adding up the scale interval scores of each item. The first 10 questions of the inventory included the items measured trait anger, 24 remaining items were about anger expression; 8 of these items are related to anger-out expression (be reflected out of anger), while 8 items are related to anger-in (be reflected in himself/herself of anger) expression and 8 items are related to anger-control. Alpha reliability coefficient of the inventory of 0.77 was used. Communication Skills Inventory Communication skills inventory is a 5-point Likert scale inventory developed by Ersanli and Balci in 1998 was used, in order to evaluate the communication skills level of individuals (12). Cronbach Alpha coefficient which was measured to determine the internal consistency of the inventory was 72. The scale was scored as always 5, never 1. The scale has a total of 45 items and the highest possible score was 225, while the lowest possible score was 45. Alpha reliability coefficient of the scale of 0.82 was used. The health personnel individually completed the question form, trait anger and anger management inventory and communication skills inventory and they filled in the forms by themselves. Data analysis Descriptive analysis was used for the comparison of quantitative data, Student t Test was used for the comparison of normally distributed parameters, One-way Anova test was used for intergroup comparisons, and Tukey HSD test was used for the determination of groups which caused difference. Pearson Correlation analysis was used for the evaluation of inter-parameter relationships. Ethical principles of the study The study was approved by the Ondokuz Mayis University Ethics Committee and the permission was taken also from Istanbul Provincial Directorate of Health and the Training and Research Hospital. Only health personnel who accepted to participate voluntarily were included in the study. RESULTS Descriptive characteristics of the participants The participants age varied between years, with a mean age of the participants being 29.23±5.94 years; 44.5% (n=126) of the participants were males, and 55.5% (n=157) were females. Participants (n=107) were nurses, 39.9 % (n=113) were doctors, 9.9% (n=28) were emergency medicine technicians and 12.3 % (n=35) were technicians (radiology or laboratory). With regard to marital status, 56.2% (n=159) of the health personnel were single, and 43.8 % (n=124) were married. High school graduates had 16.3% (n=46) of the personnel, 19.8% (n=56) had two-year degree, 45.2% (n=128) were doctors, while 18.7% (n=53) were postgraduates. While 25.1% (n=71) of the participants had children, 74.9% (n=212) did not have children. The time spent in the occupation ranged between 0.10 and 26 years, with the mean of 6.26±5.21 years, and the time spent in the emergency department was between 0.10 and 26 years, with the mean of 4.43±3.99 years; 38.9% (n=110) of the health personnel were willing to work at the emergency department while 61.1 % were unwilling. Violence related features Large majority of the participants, 78.4% (n=222) had been exposed to violence within the time they worked in the emergency department:

108 Medicinski Glasnik, Volume 12, Number 1, February 2015 % (n=144) had been exposed to verbal violence, and 9.5 % (n=21) had been exposed to physical violence, 22.5 % (n=50) had been exposed to both verbal and physical violence, 2.3% (n=5) had been exposed to all verbal, physical and sexual violence and 0.9 % (n=2) had been exposed to both verbal and sexual violence. Nine percent of the participants (n=20) had been exposed to violence by patients, 48.2% (n=107) by patients relatives, 34.2% (n=76) by both patients and patients relatives, and 8.3% (n=19) had been exposed to violence by the hospital administration (mobbing). Eleven percent (n=31) of the health personnel stated that they got angry frequently, and 42.4% (n=120) stated that they had a training for anger and anger management (Table 1). Table 1. Reasons for the participants exposure to violence (n=222) Reason Number (%) of participants Being late in treating a patient because of having too many patients 92 (41.44) Patients forcing the personnel to see a doctor although their situation is not urgent 49 (22.07) Not letting in relatives to the intervention room 44 (19.81) Referring a patient to another hospital 25 (11.26) Patient relatives reactions to the intervention 12 (5.40) Anger related features Statistically significant differences were found between the anger-in levels of the health personnel regarding their levels of education (p<0.01). The participants who had graduate degree had a higher level of anger-in comparing to others. Statistically significant differences were also found between the anger-out level of the health personnel regarding their level of education (p<0.05). The participants who had undergraduate degree had a high level of anger-out. Statistically significant differences were found between the anger control level of the health personnel regarding their level of education (p<0.01). The participants who had graduate degree had a higher level of anger control comparing to others. Statistically significant differences were not found between the trait anger level of the health personnel regarding their level of education (p>0.05) (Table 2). Communication related features Statistically significant difference was found between the health personnel s mean age and Table 2. Evaluation of the trait anger and anger style inventory scores of the health personnel by their levels of education Inventory score (Mean±SD) Trait anger / Two-year Undergraduate High school Graduate p anger style degree (n=107) (n=35) (n=113) (n=28) Trait anger 17.67± ± ± ± level Anger-in 14.63± ± ± ± Anger-out 13.56± ± ± ± Anger 19.48± ± ± ± control their mental communication skills and mental communication levels (p=0.009), their behavioral communication skills and behavioral communication levels (p=0.045), and between their general communication skills and general communication levels (p=0.02) (it found increasing by the age). Difference was not found between the health personnel s age and their emotional communication skills (p=0.081) (Table 3). Table 3. Relationship between the age of the health personnel and scores from communication skills scale Scores of communication Communication skill subscale Age Mean SD R p Mental communication Behavioral communication Emotional communication General communication SD, standard deviation; R, Pearson Correlation Coefficient The relationship between the participants anger levels and their communication skills No statistically significant relationship was found between the general communication scores and anger-out of the health personnel, while statistically significant relationship was found between health personnel trait anger, anger-in scores, and anger control scores (p=0.000, p=0.000 and p=0.000, respectively). It was found that as their general communication skills increased, health personnel trait anger, anger-in and anger-out levels decreased while their anger control levels increased (Table 4). Table 4. Relationship between the health personnel s anger control scale scores and their general communication skills scores Trait anger / anger style Scores of anger control subscales Mean SD General communication R Trait anger level p R Anger-in p R Anger-out p R Anger control p SD, standard deviation; R, Pearson Correlation Coefficient 102

109 Das et al. Anger management and communication skills DISCUSSION In this study, 64.9 % of the participants were found to have been exposed to verbal violence while 9.5 % to physical violence, 22.5 % to both verbal and physical violence and the rest of examinees had been exposed to all verbal, physical and sexual violence. Similar results were shown by Crilly et all. stating that 61% nurses were sworn at, 10% pushed, 3% of each hit or kicked (18). There are many reports about recorded violence among health workers at emergency departments (13-17). Behnam et al. found out that 75% of the health workers have been exposed to verbal violence and 21% to physical violence (13). Kowalenko et al. reported 48.1% of women and 51.9 % of men among emergency physicians had been exposed to workplace violence, most commonly to verbal violence (74.9%) (14). Wu et al. found out that 11% of the health workers had been exposed to physical violence, 26% to verbal violence and 1% to sexual violence (15), Canbaz et al. (2008) found 59.6 % of the health care workers had been exposed to verbal violence while 19.6% had been exposed to physical violence (16). There is a lot of stress and workload in the emergency service. At the same time, emergency service is also a very stressful place with lots of anxiety for patients and their relatives. Thus, emergency department attending physicians who have high anger management and communication problems may have tendency to violence in this environment. It has been found that 30-89% of health workers exposed to violence by patients, 9-82% to patient s relatives (14,19,20). The findings of this study are in parallel with the findings of In Kitaneh and Hamdan study showing that 20.8% and 59.6% health professionals were exposed to physical and non-physical violence, respectively (21). All this data show that even though the type and kind of violence to health workers vary, violence that can be caused by patients or patient relatives seems to have become a part of working life. There are many studies describing the reasons of health workers exposed to violence such as misunderstandings, medical reasons, patients and their relatives did not like the treatment, too waiting for too long, patient deaths, insufficient number of personnel, and late treatment of patients (10,15,19,20,22). The findings of the study are similar to those studies. In a study by Rosenstein and O Daniel which was conducted with 1500 nurses showed that as the anger level of the nurses increased, they had difficulties in concentration and communication, the problems among the team, about information exchange e.g., relationships in the work places had been negatively affected (23). Communication is a very important factor in anger management. The results of this study showed that healthcare workers with bad communication have higher anger levels. As the education level of the participants increased, the mean of anger management and the mean of anger-in among health personnel increased. In Balkaya s study with 756 healthcare workers which aimed to develop multi-dimensional anger inventory, it was found that high school graduates were more inclined to experience more anger problems in their interpersonal relationships when compared with primary education or university graduates (24). The findings of the study are in concordance with the findings of this study. According to the results of this study, behavioral control increases with age. This study found a statistically significant positive relationship between the participants age and their general communication skills and it was determined that as healthcare workers aged, their general communication skills level increased. This situation can be explained by the fact that as health workers get older, they will have a better knowledge from their experience and information and that they can more easily turn these into theoretical application. In this study, as the health workers get older, mental and behavioral communication skills increase while emotional communication skills decrease. Kaya et al. did not find a relationship between year old nurses and communication skills although their communication skills scores were the highest (25). In conclusion, in the in-service programs of institutions, training should be conducted about anger management and effective communication techniques so that the health personnel can be aware of their own feelings and express anger in a suitable way, and the reasons of 103

110 Medicinski Glasnik, Volume 12, Number 1, February 2015 violence and aggressiveness toward the health personnel should be researched and solutions should be developed. Similar studies should be made more extensively within the country. FUNDING This study was supported by Ondokuz Mayis University Scientific Research Fund TRANSPARENCY DECLARATION Competing interest: none to declare REFERENCES 1. Çivitçi N. Multidimensional school anger scale of the Turkish to adaptation: validity and reliability studies. PUJE 2007; 22: Thomas SP. Transforming Nurses Stress and Anger: Steps Towards Healing. New York: Springer, Weinstein N, Brown KW, Ryan MR. A multi-method examination of the effects of mindfulness on stress attribution, coping, and emotional well-being. J Res Pers 2009; 43: Doğan S, Güler H, Kelleci. Nurses approaches in theface of angry behaviors of patients. C.U. Journal of Nursing 2001; 5: Günüsen NP. Ustün B. Burn out in the nurses and doctors working in secondary healthcare services in Turkey: a literature review. DEUHY ED 2010; 3: Eslamin J. Fard SH. Tavakol K. Yazdani M. The effect of anger management by nursing staff on violence rate against them in the emergency unit. Iran J Nurs Midwifery Res 2010; 15(Suppl 1): Borum R. Improving the clinical practice of violence risk assessment: technology, guidelines, and training. Am Psychol 1996; 51: Büyükbayram A, Okçay H. The socio-cultural factors that affect violence in health care personnel. J Psychiatr Nurs 2013; 4: Kumcağız H, Yılmaz M, Balcı SÇ, Avcı IA. Nurses communication skills: example of Samsun Province. Dicle Tıp Dergisi 2011; 38: Spielberger CD, Jacobs S, Russell S, Crane RS. Assesment of anger: the state-trait anger scale. Advances in Personality Ass 1983; 2: Özer AK. Sürekli öfke ve öfke ifade tarzı ölçekleri çalışması. Turkish J Psych 1994; 31: Ersanli K, Balci S. Iletişim Becerileri Envanterinin Geliştirilmesi-Geçerlik ve Güvenirlik Çalışması. Türk Psikolojik Dan ve Reh Der 1998; 10: Behnam M, Tillotson R, Davis SM, Hobbs GR. Violence in the emergency department: a natıonal survey of emergency medicine residents and attending physicians. J Emerg Med 2009; 40: Kowalenko T, Walters B, Khare R, Compton S. Workplace violence: a survey emergency physicians in the State of Michigan. Ann Emerg Med 2005; 46: Wu S, Zhu W, Li H, Lin S, Chai W, Wang X. Workplace Violence and influencing factors among medical professionals in China. Am J Ind Med 2012; 55: Canbaz S, Dündar C, Dabak Ş, Sünter A, Pekşen Y, Çetinoğlu E. Samsun Hospital Emergency Department and emergency employees against violence: an epidemiological study. Ulus Travma Acil Cerrahi Dergisi 2008; 14: Gülalp B, Karcıoğlu Ö, Köseoğlu Z, Sarı A. Dangers faced by health personnel: experiences from Turkey s southern city office. Ulus Travma Acil Cerrahi Dergisi 2009; 15: Crilly J, Chaboyer W, Creedy D. Violence towards emergency department nurses by patients. Accid Emerg Nurs 2004; 12: Lin Y, Liu H. The impact of workplace violence on nurses in South Taiwan. Int J Nurs Stud 2005; 42: Rahmani A, Hassankhani H, Mills J, Dadashzadeh A. Exposure of Iranian emergency medical technicians to workplace violence: a cross-sectional analysis. Emerg Med Australas 2012; 24: Kitaneh M, Hamdan M. Workplace violence against physicians and nurses in Palestinian public hospitals: a cross-sectional study. BMC Health Serv Res 2012; 12: Alameddine M, Kazzi A, El-Jardali F, Dimassi H, Louf S. Occupational violence at Lebanese emergency departments: Prevalence, characteristics and associated Factors. J Occup Health 2011; 53: Rosenstein AH, O Daniel M. Disruptive Behavior and Clinical Outcome Perceptions of Nurses and Physicians. Am J Nurs 2005; 105: Balkaya F, Hisli Şahin N. Multidimensional Anger Scale. Türk Psikiyatri Dergisi 2003; 214: Kaya F, Özcan A, Yılmaz M. Comparing communication and empathic ability levels of nurses with patients perception of nursing care. JPHMP 2013; 1:

111 LETTER TO EDITOR Occurrence and morphological characteristics of cataracts in patients treated with general steroid therapy at the Cantonal Hospital Zenica Jasmin Zvorničanin 1, Edita Zvorničanin 2 1 Eye Clinic, University Clinical Centre Tuzla, 2 Institute of Public Health Tuzla Canton; Tuzla; Bosnia and Herzegovina Dear Editor, We read with interest the article by Čerim et al. regarding the occurrence and morphological characteristics of cataracts in patients treated with general steroid therapy (1). Similar to the results of previous studies, the authors found that the use of corticosteroids is associated with a higher incidence of cataract development and posterior subcapsular (PSC) cataract as most prevalent morphological type (2). Older age and heredity are the most important risk factors associated with different types of cataracts and females are at increased risk of cortical cataract (2,3). Myopia ( 1.0 D) and elevated intraocular pressure are also associated with an increased risk of nuclear and PSC cataracts (3). The major causal external risk factors influencing cataract formation include: smoking, excessive UV-B exposure, diabetes mellitus (DM) and steroidal treatment (2,3). There is also a significant relationship between the risk of cataracts and delivered corticosteroid dose (4). Lower monthly household income, lower education, hypercholesterolemia, hypertension and DM are independent risk factors for the development of any cataract type, while older age and DM are independent risk factors for the development of pure PSC (5). Elevated body mass index (BMI) and rapid weight gain may also increase the risk for age related cataract, especially PSC cataract (3,6). Other risk factors for PSC development also include hypertension, the use of amiodarone, thiazide diuretics, aspirin and vitamin E (2). For these reasons, we would kindly ask the authors to perform the correlations for age, gender, BMI, length and regimen of steroid use, cumulative steroid dose, the use of other systemic drugs, DM duration, spherical equivalent and intraocular pressure changes, with cataract occurrence and morphology between the groups. Without this information it would be difficult to hypothesize the direct steroid induced cataractogenesis, especially in the group on the steroid therapy >4 years, where all patients had iatrogenic diabetes. In these patients, it is the indirect impact of steroids on body metabolism that might initiate the cataractogenesis. These findings will significantly contribute to the paper s scientific value and contribution. Overall, we agree with Čerim et al. that general steroid therapy remains the important risk factor for cataract development and all patients should have regular ophthalmological control examinations. Other systemic risk factors such as BMI, DM, smoking history, duration of basic systemic disease and corticosteroid dose should be carefully monitored too. Corresponding author: Jasmin Zvorničanin Eye Clinic, University Clinical Center Tuzla Trnovac bb., Tuzla, Bosnia and Herzegovina Phone: ; fax: ; zvornicanin_jasmin@hotmail.com Med Glas (Zenica) 2015; 12(1):

112 Medicinski Glasnik, Volume 12, Number 1, February 2015 References: 1. Čerim A, Dizdarević A, Pojskić B. Occurrence and morphological characteristics of cataracts in patients treated with general steroid therapy at Cantonal Hospital Zenica.Med Glas (Zenica) 2014; 11: Robman L, Taylor H. External factors in the development of cataract. Eye (Lond) 2005; 19: Chang JR, Koo E, Agrón E, Hallak J, Clemons T, Azar D, Sperduto RD, Ferris FL 3rd, Chew EY; Age-Related Eye Disease Study Group. Risk factors associated with incident cataracts and cataract surgery in the Age-related Eye Disease Study (AREDS): AREDS report number 32. Ophthalmology 2011; 118: Weatherall M, Clay J, James K, Perrin K, Shirtcliffe P, Beasley R. Dose-response relationship of inhaled corticosteroids and cataracts: a systematic review and meta-analysis. Respirology 2009; 14: Rim TH, Kim MH, Kim WC, Kim TI, Kim EK. Cataract subtype risk factors identified from the Korea National Health and Nutrition Examination survey BMC Ophthalmol 2014; 14:4. 6. Ye J, Lou LX, He JJ, Xu YF. Body mass index and risk of age-related cataract: a meta-analysis of prospective cohort studies. PLoS One 2014; 9:e

113 RETRACTION Increased counts and degranulation of duodenal mast cells and eosinophils in functional dyspepsia- a clinical study Shijun Song, Yan Song, Haishan Zhang, Gaiqin Li, Xiaopei Li, Xiaohong Wang, and Zhen Liu The above article published in Medicinski Glasnik online on 26 June 2014 by the Medical Association of Zenica-Doboj Canton ( and in Volume 11, Issue 2, pages , has been retracted by agreement between the authors, the journal Editor-in-Chief, Professor Selma Uzunović, and the Medical Association of Zenica-Doboj Canton. The reasons for this retraction are as follows: The work reported in the paper was about the role of duodenal eosinophils and mast cells in the pathogenesis of functional dyspepsia. Most of the experiments were carried out by a former member of the authors team named Yuan Haipeng, who has left the team for more than two years. A high proportion of data in the paper had been reported in the doctoral dissertation of Yuan Haipeng in 2012, and the paper was published without the knowledge or permission of Yuan. Besides the data previously reported in the doctoral dissertation of Yuan Haipeng, the authors calculated the other data in the paper before the submission. However, it has come to the authors attention that they had made quite a few mistakes due to a loss of the original data, which was not described in details in the dissertation. REFERENCE Shijun Song, Yan Song, Haishan Zhang, Gaiqin Li, Xiaopei Li, Xiaohong Wang, Zhen Liu. Increased counts and degranulation of duodenal mast cells and eosinophils in functional dyspepsia- a clinical study. Med Glas (Zenica) 2014; 11(2): Med Glas (Zenica) 2015; 12(1):

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