ACTA MEDICA BULGARICA
Transcription
ACTA MEDICA BULGARICA
MEDICAL UNIVERSITY − SOFIA amb 1/2012 ACTA MEDICA BULGARICA vol. XXXIX This journal is indexed in Global Health Database, in Bulgarian Medical Literature Database and in Scopus Central Medical Library Editor in chief Prof. V. Mitev, MD, Ph. D. DSc Editorial board Prof. K. Tsachev, MD, Ph. D., DSc Prof. M. Marinov, MD, Ph. D., DSc Prof. D. Ziya, MD, Ph. D., DSc Prof. N. Lambov, Mag. Ph., Ph. D. Prof. W. Bossnev, MD, Ph. D., DSc L. Tacheva, MD CONTENTS Kr. Petrov, G. Kobakov, M. Manova, К. Mitov, A. Savova and G. Petrova. Influence of the choice of volatile anesthetics on liver enzymes after surgical liver resections .......................3 M. Manova, A. Stoimenova, L. Peikova, P. Peikov and G. Petrova. Patent protection policy of the therapeutic groups аngiotensin II receptor antagonists ..............................................9 D. Obreshkova, D. Tsvetkova and K. Ivanov. Most used combined multisupplements containing l – arginine ....................................................................................................................19 A. Savova, A. Stoimenova, M. Manova and G. Petrova. Pharmacotherapy costs of osteoporosis and related fractures in Bulgaria ...........................................................................31 M. Velizarova, D. Popova, E. Hadjiev, N. Dimitrova, I. Dimova, D. Toncheva and K. Tzatchev. Evaluation of molecular-cytogenetic Aberrations and overall survival in myeloid antigen positive adult acute lymphoblastic leukemia .....................................................................40 St. Sopotensky and Al. Cervenjakov. Adapted surgical thoracoscopic Heller’s myotomy in the treatment of achalasia ..........................................................................................................47 Т. Тurnovska, St. Kostianev, B. Мarinov and St. Mandadzhieva. Different low levels of air pollution and respiratory functions an 8-year natural experiment .........................................55 Kr. Todorova, S. Hayrabedyan, J. Dineva, I. Vangelov, D. Zasheva, V. Penchev, G. Nikolov, M. Mollova and M. Ivanova. Cumulus biomarker evaluation for human oocyte quality prediction ............................................................................................................................70 P. Nenoff, M. Peter, G. Tchernev, G. Mulyowa, E. Amerson, U. Paasch and J. Ananiev. lichen striatus in Uganda – case reports and update of clinical picture, differential diagnosis and pathogenesis: first description of patients in sub-Saharan-Africa ..........................77 Ju. Ananiev, X. Baraliakos and G. Tchernev. Reactivation of subacute cutaneous lupus erythematodes under the clinical picture of rowell syndrome ..............................................87 E. Boteva and D. Iovchev. The sizes of pulp chambers of molars with severe root curvatures – in vitro comparative study .........................................................................................92 E. Boteva and D. Yovchev. Efficiency of working length detection and irrigation during preparation of curved root canals........................................................................................97 N. Dimitrov, P. Tsekov and B. Matev. Arthroscopic surgery in cases with an aging locked posterior dislocation of the shoulder .................................................................................104 ACTA MEDICA BULGARICA 1/2012 Editor in Chief: Prof. V. Mitev, MD, Ph. D., DSc Scientific editor: Prof. W. Bossnev, MD, Ph. D., DSc Editor of the English text: A. Papazian, MD Art editor and computer design: D. Alexandrova Organizing secretary: M. Vankova Publisher’s sheets: 8.3 Printer’s sheets: 6.25 Format: 70 x 100/16 Issued by the Central Medical Library 120 INFLUENCE OF THE CHOICE OF VOLATILE ANESTHETICS ON LIVER ENZYMES AFTER SURGICAL LIVER RESECTIONS Kr. Petrov1, G. Kobakov1, M. Manova2, К. Mitov2, A. Savova2 and G. Petrova2 1 Specialized Oncology Hospital for Active Therapy “Dr. Marko Markov”, Varna, Bulgaria 2 Medical University Sofia, Faculty of Pharmacy, Bulgaria Summary. The purpose of this study was to evaluate the possible superiority in pharmacological protective effect of sevoflurane compared to isoflurane in patients with liver segmental or lobe resections, through examination of postoperative changes in the liver enzymes ALT and AST and the impact of the choice of anesthetic on surgical hospital charges. It is a prospective study based on the examination of surgical patients. A retrospective cost study analysis after approval of local ethics committee was also performed. Patients with surgical liver resections were divided in two groups according to the main volatile anesthetic used − isoflurane (n = 25) and sevoflurane (n = 17) group. All patients were tested for both liver enzymes AST and ALT before and after the surgery. The health care resources used during the anesthesia were collected. Mean time of operation, minimal alveolar concentration (MAC), average anesthetic quantity used, fresh gas flow, and cost for maintaining anesthesia were calculated. The cost for maintaining anesthesia was compared with the surgical hospital charges. No statistically significant difference has been observed between the two groups according to patients’ age, type of liver resection, as well as hospital stay (р > 0.05). Comparing the postoperative levels of AST and ALT, we found that their levels were higher in the group of patients on isoflurane. Levels of both ALT and AST were significantly lower, and decreased more rapidly in patients receiving sevoflurane than those receiving isoflurane from 1st to 5th postoperative day. The decrease was almost twice faster in the group on sevoflurane and close to normal physiological levels. All hospital costs appear to be equal in both groups except the cost of maintaining anesthesia that represents 1.67 per cent of hospital charges in the group on sevoflurane and 0.41 per cent in the group on isoflurane. In conclusion, for patients that are going to have liver resection, the preferred main anesthetic in the complex of balanced anesthesia should be sevoflurane, which decreases the AST and ALT levels significantly. For both Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 3 anesthetics the total cost for maintaining anesthesia is an insignificant part of the surgical hospital charges and the choice of anesthetic should be based on clinical results rather than on economical ones. Key word: anesthesia, sevoflurane, isoflurane, anesthesia cost, liver resections, ALT, AST S INTRODUCTION urgical liver resections are related to a high risk of hemorrhage, which has a negative influence on postoperative recovery, overall patient survival, and quality of life [1, 2]. A clamping maneuver (Pringle maneuver) [3] is used as a routine practice to prevent hemorrhage, but it is related to ischemic liver injury, which leads to postoperative increase in liver enzymes and could lead to death or complications [4]. Nowadays, in order to decrease liver stress, liver preconditioning through short preclamping is performed (5-10 minutes) followed by a recovery of the blood stream and consecutive clamping [5, 6]. There are a limited number of studies on the pharmacological protection of the liver during this process. Volatile anesthetics isoflurane and sevoflurane have been studied as protectors of the myocardium in cardiovascular surgery and ultrasound abnormalities after myocardial ischemia. [7, 8]. Beck-Schimmer et al. performed a randomized controlled trial in order to determine the protective effect of sevoflurane and propofol and to confirm this effect in patients with liver resection. [9] In cases of cirrhosis sevoflurane possess a higher protective effect [10], as well as in neurosurgical interventions [11]. The current study aims to evaluate the possible pharmacological better protective effect of sevoflurane compared to isoflurane in patients with liver segmental or lobe resections, through examination of the changes in the liver enzymes ALT and AST and its impact on surgical hospital charges. Primary endpoints of the study were to examine if a statistically significant difference in the levels of liver enzymes in both groups of patients – using sevoflurane or isoflurane anesthesia will occur, and what is the presumed economic effect of the anesthetic on the hospital charges within the concrete hospital context. MATERIALS AND METHODS It is a prospective study based on surgical patients and a retrospective cost study analysis conducted in one hospital in Varna after approval of local ethics committee. All patients with surgical liver resections were observed during the period January 2009 − June 2010. Patients are divided in two groups according to the volatile anesthetic used − isoflurane group (n = 25) and sevoflurane group (n = 17). All patients have been operated by the same surgical and anesthesiological team. 4 Influence of the choice of volatile... The choice of volatile anesthetic is unintentional. The available at the moment of operation medicinal product was taken. Both liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were tested in all patients before the operation and at first, second, third, and fifth postoperative days. The health care resources used during the anesthesia were collected, as well as mean time of operation, minimal alveolar concentration (MAC), average anesthetic quantity used, fresh gas flow, and cost for anesthesia maintenance were calculated. The cost for anesthesia maintenance was compared with the surgical hospital charges. The descriptive statistics, ANOVA analysis, nonparametric Wilcoxon, and Man Whitney tests were performed for the evaluation of statistically significant differences in the enzyme levels before the operation and after that in the days of follow-up. RESULTS Patients’ distribution according to gender, age, type of liver resection and average hospital stay is shown on Table 1. According to ASA physical status classification system all patients belonged to 2nd or 3rd category [12]. Pringle maneuver was performed to all patients. No statistically significant difference was found in the patients’ age, length of hospital stay or type of liver resection between the two groups (р > 0.05) (Table 1). Due to the lack of statistically significant differences in the basic patients’ characteristics we can consider both groups of patients as selected unintentionally. Table 1. Demographic characteristics of the operated patients a b Variable Sevoflurane group Isoflurane group N 17 25 Male / Female 6 / 11 11 / 14 Average age in years (SD) 62 (5.3) 62 (5.7) Hospital length of stay in days (SD) 6.5 (1.12) 6.5 (1.16) Type of resection − lobe − segment 2 Lecta / 1 Dectb 14 1 Lecta / 2 Dectb 22 Lect – left oriented Dect – right oriented General anesthesia with endotracheal intubation was used in both groups with either isoflurane or sevoflurane. Induction of anesthesia was conducted with sodium thiopental, the muscles relaxation was ensured with suxametonium, and as depolarizing agent pancuronium bromide was used. The premedication was performed with fentanyl and promethazine hydrochloride, and fentanyl was used Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 5 to control the pain during surgery. After the surgery procedure patients were left to spontaneous awakening. All operations were successful without lethal exits till the moment of hospital discharge. On Table 2 are presented the results of the AST and ALT tests before and after the surgery. No statistically significant difference has been observed among the initial level of AST and ALT. Levels of both ALT and AST were significantly lower, and decreased more rapidly in patients receiving sevoflurane than those receiving isoflurane from 1st to 5th postoperative days. The decrease is almost twice faster in the group on sevoflurane and close to normal physiological levels. The average duration of the operation was found to be equal in both groups with MAC for sevoflurane varying from 0.7 to 2.5 MAC for isoflurane and from 1.15 to 1.25 MAC respectively. The average supplied anesthetic volume was 0.5% higher in the sevoflurane group (Table 3). According to Table 3 the average volatile concentrations of isoflurane and sevoflurane are 1.5% and 2.0%, respectively and these are the concentrations when the 3rd stage anesthesia has been achieved. Since the anesthetic potency expressed as minimum alveolar concentration (MAC) of these agents are 1.15% and 1.71% respectively [13, 14], average concentrations of isoflurane and sevoflurane expressed as the ratio to MAC are 1.31 and 1.16, respectively, suggesting that the concentration of isoflurane is higher than that of sevoflurane. The possible explanation of this fact is that in this particular study the isoflurane group has required more anesthetic to achieve the 3rd stage anesthesia. All hospital costs appear to be equal in both groups of patients and thus the cost for maintaining anesthesia was calculated to be four times higher in the group on sevoflurane (Table 3). The cost of anesthesia maintenance as a part of the surgical hospital charges represents 1.67 per cent in the group on sevoflurane and 0.41 per cent in the group on isoflurane. Table 2. AST and ALT test levels (UL/L) Enzymes AST ALT * Anesthetic Day before the surgery and of follow up – Mean enzymes level (SD) 0 day – before the surgery 1 day after surgery 2 day 3 day 5 day after surgery after surgery after surgery isoflurane 23.72 (5.84) 411.60* (60.94) 324.40* (65.14) 233.96* (70.67) 126.92* (26.85) sevoflurane 22.24 (6.72) 328.24* (39.54) 233.35* (60.18) 141.94 (59.37) 87.94 (10.56) isoflurane 22.91 (5.75) 416.56* (40.07) 315.88* (48.45) 222.48* (43.69) 108.24* (12.02) sevoflurane 22.24 (6.72) 342.53* (31.02) 238.18* (40,04) 136.235* (23.86) 88.77* (10.33) p < 0.05 6 Influence of the choice of volatile... Table 3. The cost of anesthesia Mean time of operation in minutes (SD) Minimal alveolar concentration (%) Average anesthetic quantity (%) Fresh gas flow (l/min) Total cost of anaesthesia maintenance (BGN) % of surgical hospital charges Sevoflurane group 180 (SD 90-270) from 0.7 to 2.5 2.0 3 53.55 1.67 Isoflurane group 180 (SD 90-270) from 1.15 to 1.25 1.5 3 13.25 0.41 DISCUSSION Our results clearly show that in the sevoflurane group levels of ALT and AST are recovering faster to acceptable physiological values. The ALT and AST levels are considered as an indicator for liver damage and their faster recovery in the sevoflurane group suggest easier liver recovery in that group. Our results confirm those of BeckSchimmer Beatrice et al. [9] and Nishiyama et al. [10, 11] for the probable protective effect of sevoflurane in patients with liver resections. In contrast to Nishiyama [10], we are examining patients with different indications for surgical resection and not only cirrhotic ones. We can consider that the probable protective effect does not depend on the initial disease. Also, in contrast with Beck-Schimmer, we do compare the sevoflurane and isoflurane, аnd not the sevoflurane and propofol, thus confirming sevoflurane as an anesthetic with a potential protective effect on the liver during resection surgery. The fact that all patients have been operated by the same surgical and anesthesiological team is also important for eliminating the influence of the human factor on the performance of surgery and anesthesia. This confirms the pharmacological protection of sevoflurane [15]. Comparison of these undoubtfully positive clinical results with the relative share of the cost for anesthesia maintenance shows that even higher, sevoflurane cost is almost diminishing compared to total surgical hospital charges. Thus, if sevoflurane is chosen as a preferred anesthetic for patients with surgical liver resections, that will not have a considerable economic impact on total hospital’s charges. On the opposite, it will ensure faster achievement of the physiological levels of ALT and AST. Benefit economic results with sevoflurane were previously established for patients with laparoscopic cholecistectomy [16]. The usage of sevofluranee will decrease the unnecessary expenditures due to the higher level of AST and ALT, and will increase patients’ quality of life, supporting faster recovery of the liver function. CONCLUSION For patients that are going to have liver resection the preferred main anesthetic in the complex of balanced general anesthesia should be sevoflurane which decreases the AST and ALT levels statistically significantly. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 7 For both anesthetics the total cost of anesthesia maintaining is an insignificant part of the surgical hospital charges and the choice of anesthetic should be based on clinical results rather than on economical ones. REFERENCES 1. G o z z e t t i , G. et al. Liver resection without blood transfusion. – Br. J. Surg., 82, 1995, 1105-1110. 2. K o o b y , D. A. et al. Influence of transfusions on perioperative and long-term outcome in patients following hepatic resection for colorectal metastases. – Ann. Surg., 237, 2003, 860-869. 3. V a n d e r B i l t , J. D. et al. European survey on the application of vascular clamping in liver surgery. – Dig. Surg., 24, 2007, 423-435. 4. C l a v i e n , P. A. et al. Strategies for safer liver surgery and partial liver transplantation. – N. Engl. J. Med., 356, 2007, 1545-1559. 5. P e t r o w s k y , H. et al. A prospective, randomized, controlled trial comparing intermittent portal triad clamping versus ischemic preconditioning with continuous clamping for major liver resection. – Ann. Surg., 244, 2006, 921-928. 6. S e l z n e r , N. et al. Protective strategies against ischemic injury of the liver. – Gastroenterology, 125, 2003, 917-936. 7. M u l l e n h e i m , J. et al. Isofluranee preconditions myocardium against infarction via release of free radicals. – Anesthesiology, 96, 2002, 934-940. 8. Ta n a k a , K. et al. Mechanisms of cardioprotection by volatile anesthetics. – Anesthesiology, 100, 2004, 707-721. 9. B e c k - S c h i m m e r , B. et al. A Randomized controlled trial on pharmacological preconditioning in liver surgery using a volatile anesthetic. – Ann. Surg., 248, 2008, 909-918. 10. N i s h i y a m a , T., T. Fujimoto et K. Hanaoka. A Comparison of liver function after hepatectomy in cirrhotic patients between sevofluranee and isofluranee in anesthesia with nitrous oxide and epidural block. – Anesth. Analg., 98, 2004, 990-993. 11. N i s h i y a m a , T., T. Yokoyama et K. Hanaoka. Liver function after sevofluranee or isofluranee anaesthesia in neurosurgical patients. – Can. J. Anaesth., 8, 1998, 753-756. 12. H a y n e s , S. R. et P. G. Lawler. An assessment of the consistency of ASA physical status classification allocation. – Anaesthesia, 3, 1995, 195-199. doi:10.1111/j.1365-2044.1995.tb04554.x 13. S t e v e n s , W. D. et al. Minimum alveolar concentrations (MAC) of isoflurande with and without nitrous oxide in patients of various ages. – Anesthesiology, 42, 1975, № 2, 197-200. 14. K a t o h , T. et K. Ikeda. The minimum alveolar concentration (MAC) of sevoflurane in humans. – Anesthesiology, 66, 1987, № 3, 301-303. 15. S t e f a n o v , C. et al. Volatile anesthesia in thyroid gland surgery of patients with preliminary thyreostatic usage. – Anesthesiology and intensive case, 38, 2008, № 1, 27-31. 16. S t e v a n o v i c , P. et al. Low fresh gas flow balanced anesthesia versus target controlled intravenous infusion anesthesia in laparoscopic cholecystectomy: a cost-minimization analysis. – Clin. therap., 9, 2008, 1714-1725. ¨ 8 Address for correspondence: Guenka I. Petrova Faculty of Pharmacy Medical University of Sofia 2 Dunav str. Sofia 1000, Bulgaria + 35 92 9236 545 + 35 92 987 987 4 e-mail: [email protected] Influence of the choice of volatile... PATENT PROTECTION POLICY OF THE THERAPEUTIC GROUP АNGIOTENSIN II RECEPTOR ANTAGONISTS M. Manova1, A. Stoimenova1, L. Peikova2, P. Peikov2 and G. Petrova1 1 Faculty of Pharmacy, Medical University – Sofia Department of Social Pharmacy and Pharmacoeconomics 2 Department of Pharmaceutical Chemistry Summary. The goal of the current study is to analyze the patent protection status of medical products included in the therapeutic class аngiotensin II receptor antagonists or sartans. The analysis covered the period 1995-2008. A three – step internet patent database search methodology was applied in searching EPO, Orange book and Patent watch. Applications were systematised per IPC code, territory, manufacturer and date of patent issuing. The patent activity depends on the time of expiration of the main patent. The highest number of patents granted for IPC class A61K is due to the fact that this class is related with the therapeutic possibility of the formulations. Per territory protection activity is similar in regard to the prefered countries and follows the market dynamics. For some of the products additional factors might play an important role. Our study shows that a variety of factors are influencing the patent protection policy, such as the time of discovery, product application, disease priority, technologies etc. It appears that the therapeutic competition is more important than the generic one related to establishment of patent profile of particular INN. This is in contrast with the beliefs that the patent protection policy affects mainly generic companies. Key words: Angiotensin II receptor antagonists, patent protection, patent policy O INTRODUCTION riginal medicinal products ensure a new therapeutic option for poorly treated conditions or diseases, while the generic medicines support the sustainability of healthcare provision and contribute to maintaining a control over the pharmaceutical expenditures. The patent protection policy of pharmaceutical manufacturers is a critical milestone for both types of manufacturActa Medica Bulgarica, Vol. XXXIX, 2012, № 1 9 ers (innovative or generic) in terms of market penetration, product lifecycle and patients’ access to effective and affordable medicines [1, 2, 3]. After the endorsement of the TRIPS agreement, the influence of the pharmaceuticals patent protection policy on peoples’ access to medicines started to be widely discussed in the pharmaceutical literature [4, 5]. Researchers are studying the pharmaceuticals patent protection policy from different aspects. Some are trying to evaluate the real life protection of the basic patent. Others are studying the patent policy of the companies or differences among the countries and their influence on the generic entries [6, 7]. Lots of studies are focusing on the effect of the generic medicines entry on the pricing and reimbursement of medicines [8, 9], and on the utilization of medicines after the patent expiration [10, 11]. The public health concerns lead to the introduction of a measure to make access to patented medicines more flexible [12]. Researchers from the WHO published series of articles on the implementation of TRIPS agreement in the developing countries with the aim to promote series of measures for encouraging the compulsory licensing, exemptions from TRIPS in the national legislation, generic manufacturing and measures that support the generic medicines dispensing [13, 14. 15, 16]. The systematic analysis of the changes in the patent legislation is permanently ongoing and professionals are advocating for better public health through the measures facilitating the access to medicines. This is one of the key tasks for generic pharmaceutical industry and international organizations [17, 18, 19]. The other key task is the analysis of the patent drug policy of originating companies and their influence on generic pharmaceutical industry. Some studies consider that the generic pharmaceutical industry is characterized by a simultaneous entry, rather than sequential [20]. Thus for the generic pharmaceutical companies it is more attractive being the first entering the market. The mathematical models have been created to calculate the aggregated demand and supply features influencing the generic medicines market [21]. From the pharmaceutical perspective, the real life of a new medicine starts after the establishment of its therapeutic posology during the clinical trials and granting the international non-proprietary name (INN) through the World Health Organisation (WHO) procedures [22]. Before that, the product is not recognized by the pharmaceutical companies as a potential competitor due to usage of coded names instead of the INN. Due to this fact, the patent search and detection of any additional protection policy of the originator or therapeutic competitors is sometime complicated and not pharmaceutically reasonable. During the last three decades a tremendous progress has been made in the hypertension therapy through the introduction of new therapeutic classes [23, 24]. The discovery of a variety of new molecules from the groups of ACE inhibitors and 10 Patent protection policy... angiotensin II receptor antagonists or sartanes empowered the physicians in their attempts to attack the complex cardiovascular events in various ways. The fast development of new molecules has led to the development of whole new drug families and increased the competitiveness, thus posing challenges in front of the originator and generic manufacturers to create sophisticated patent protection on one side, and penetrate quickly the market and increase patients’ access to low – priced generics, on the other [25, 26, 27]. The goal of the current study is to analyze at a worldwide level the patent protection status of the medicines in the therapeutic class of angiotensin II receptor antagonists and respective patent strategies for the period 1995-2008. The main questions, discussed in this study are as follows: 1. What types of companies (originators and generics) are claiming the patents for the INNs from the observed groups? Is there any difference among the originators and generics companies patents claims? 2. What is the intensity of patent policy before and after the first patent expiration? 3. What is the specificity of the patent policy according to the IPC classification and territory? What is the time lag for the appearance of the first therapeutic competitors’ patent claims within the observed groups of medicines? MATERIALS AND METHODS The three three-step step internet patent database search methodology was applied for publications during the period 1995-2008. The first step was searching the “Orange book” for clarifying the date of issuing of the first patent [28] and “Drug patent watch” database for checking the date of valid patents expiration [29]. The second step was searching the worldwide patent database via the options provided in the European patent database [30] (EPO) by using as a key word the INNs of the observed medicines in the therapeutic group. The third search step was an expanded search via INPADOC patent family system of the European database for clarifying the additional publications of the patents per territory. The INPADOC system connects EPO with more than 70 countries and legal status data for more than 40 patent authorities. The information for the appearing patents was then systematized according to the following criteria: description title of the patent, inventor, applicant, IPC class, publication number of patent/publication date, and all INPADOC publications connected with the first description title. Out of all INNs we considered those with valid main patents that were not expired till the beginning of 1990, as well as those new products that are still under clinical trials investigation. Candesartan and olmesartan were not included in the analysis due to their latter appearance in the therapeutic group (Table 1). Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 11 Table 1. Patents claims per observed INNs Therapeutic Group Sartans INN Number of patents in the worldwide database Number of patents after the INPADOC search eprosartan losartan valsartan irbesartan telmisartan 25 44 50 31 44 50 100 152 48 94 The collected information was analyzed by classifying the patents according to the IPC classification code in following groups – formulation patents (C07D – chemical active substance), formulation or process patents chemical or pharmaceutical technology (C07C, C07D, C07K, C07F, C12K), application patents (A61K – preparations for medical dental or toilet purposes), therapeutic activity (A61Ptherapeutic activity of medicinal preparations or chemical compounds). Most of the patents are for more than one IPC code or subgroup due to the complexity of structures or process. For all patents the year of first publication was compared with the year of the first patent issued, territory covered, and date of patent expiration for particular INN. RESULTS Angiotensin receptor blockers (ARBs), also known as angiotensin II receptor antagonists or sartanes, modulate the renin-angiotensin-aldosterone system and thus decrease the blood pressure. The tetrazole group is a main part of the chemical structure of losartan, irbesartan, olmesartan, candesartan and valsartan. In addition, losartan, irbesartan, olmesartan, candesartan, and telmisartan include one or two imidazole groups [31, 32]. Sartanes patent policy Historically, within the group of sartanes, the first patent was granted to eprosartan, followed by losartan, valsartan, irbesartan, and telmisartan. The time lag for the next therapeutic alternatives and the time lag for next therapeutic competitor appearance is very short (7 days to 1 year and 9 months), as shown in Table 1. Twenty five patents were obtained for eprosartan during the studied period. Out of them, 17 were the originator company patents, covering a variety of innovations in the field of chemical composition and process for production (n = 4), area of application (n = 16) and therapeutic activity (n = 5). The first patent from generic manufacturer was obtained during 2006 (IPC-A61K, application), 16 years after the first patent granted. Three other generic companies own patents for the application, obtained during 2006-2008, and two other for novel formulation and process of production. 12 Patent protection policy... Losartan’s first patent was granted 1 year and 7 months after eprosartan's first patent. Out of 44 patents for losartan, 14% (n = 6) were from the originator Merck&Co, and 86% (n = 38) from the generic companies. There were 3 originator patents for formulation or process; 6 for application, and 1 for therapeutic activity respectively, including overlapping claims for more than one IPC category. The fist patent claim from generic company appears in 2001 (11 year after the first patent was granted) and it was for formulation, process, application, and therapeutic use. Among the patents from generic companies prevail those for application (A61K – 68%), followed by formulation or process (C – 50%) and therapeutic activity (A61P – 24%) claims. The total percentage is higher than 100%, because some patents are for several IPC categories. Valsartan is the molecule, protected with the highest number of patents despite of the fact that it is not the first one discovered, but probably is the most used one during the investigated period. The first patent for valsartan appears in 1992 and covers 3 areas of protection: formulation, application and therapeutic activity. Ciba Geigy (the company marketed the product first) owns 5 patents (2 for formulation and process, 5 for application and 1 for therapeutic activity). For some areas of protection more than one patent is granted. After the merge with Novartis, 25 patents were issued on the basis of the company claims (2 for process and formulation, 15 for application and 3 for therapeutic activity). Some of these patents are for valsartan combinations, since 2003. The first patent issued to generic companies appears in 2002 for application. Similar is the case with irbesartan, where the first patent belongs to Bristol Myers (for formulation and application) and after the agreement with Sanofi for joint marketing cooperation, the patent policy became the priority of both companies. Sanofi has been granted on total of 7 patents (4 for application and 2 for therapeutic activity). All other patents are granted to generic companies and 14 of them are formulation or production process patents, 12 for application and 6 for therapeutic activity. First generic competitor patent was granted in 2003 for application and therapeutic activity. Telmisartan first patent was issued in 1994 year to Boehringer Ingelheim, possessing 22 patents for this INN (6 for formulation or production process, 14 for application and 6 for therapeutic activity). The first patent for generic producer became available in 1999 for benzimidasole containing medicaments and process for preparation. Typically for the group, the patents are granted either for therapeutic activity or application, very often combined with diuretics, produced by other innovators, owners of the patents. Аll of the patents are protected with European patents, USA, Australian patents and countries out of the WTO. The intensity of patent protection policy for the analysed INNs within the group is presented in Figure 1. During the first five years, the patent activity was slow and Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 13 started to increase tremendously around the time of expiration of the first patent granted. The only exclusion is losartan due to the fact that it is already with expired main patents. 50 45 number of patent claims per INN 40 35 30 25 20 15 10 5 0 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 year losartan valsartan telmisartan irbesartan eprosartan Fig. 1. The intensity of patent protection policy for the observed sartans Within sartanes group, 20 to 42 patents were granted for application (A61K), 5 to 14 for therapeutic application (A61P) and next are for formulation or process (C07C and C07D) − Figure 2. 100% 5 8 11 7 14 80% 32 60% 20 42 19 36 40% 0 0 1 0 20% 0 17 25 19 8 16 0% eprosartan losartan valsartan C07D C07C A61K irbesartan telmisartan A61P Fig. 2. Number of granted patents per IPC classes for the observed sartans 14 Patent protection policy... DISCUSSION AND LIMITATIONS The discussion follows the answers of the study questions and additional comments are added for the differences. Innovative and generic companies have very high patent activity. The time of the peak of the patent activity is different for both types of companies. Innovative companies became very active when the time for main patent expiration draws near. This result confirms the conclusions of other authors [25-27]. Such a patent policy leads to changes in the marketing authorization procedures, when a reference product is necessary for bioavailability analysis. It was permitted, in case the first original substance is lacking, to be used the so called European reference product that is any essentially similar product with a valid marketing authorization in Europe [33]. In the beginning of the observed period and for the older products patents for application (A61K) were usually granted to the generic companies. During the latest years after 2000 we observed that the generic companies started to follow the originators policy and to increase the researches and protection of different types of derivatives like salts, intermediates, polymorphs form, etc. of the main formulation near to patent expiration period. This is due to the solubility of the derivatives that is important for technology process of the dosage forms, or probably due to the results of the clinical trials for derivatives bioavailability. Thus the competition was transferred to therapeutic competitor that is exactly the situation observed in our study. The highest number of patents granted for IPC class A61K (preparations for medical dental or toilet purposes) is probably due to the fact that this class is related to the possibility of the formulations to treat not only one symptom. Hypertension is a complex disease that is treated with a variety of medicines, as well as with combinations and all possible area of applications or combinations have been granted patents. For example, a patent for irbesartan is granted for all possible pharmaceutical compositions, observed for the studied medicines. Therapeutic activity (A61P) started to be protected since 1998 which could explain the small number of patents granted in this IPC class. Per territory protection activity is similar with regard to the preference countries and follows the market dynamics and emergence. All products have been claimed for protection in the USA, EPO, India, China, Australia, Canada, and Japan. The countries out of WTO (Russia, South Africa etc.) became patent priority in the last 10 years. The therapeutic competitors are more important than the generic ones within the sartans group. The first therapeutic alternatives appear earlier than the generic competitors. Thus the real life time of the main patent is therapeutically shortened and market monopoly is limited not on the basis of competition but due to the intensive work of the other innovators. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 15 For some of the products, additional factors might play an important role in formulating the company patent protection policy. The permanent decrease in the number of patents for losartan could be due to the market reasons. Further studies are necessary to explore the effects of the market indicators on the patent protection policy. The originality of this work is in the simultaneous comparison at a level of the two contemporary CV groups, and analysis of the influence of their patent protection at national policy level. Specificity in the behavior of the originator companies is that they are more oriented towards protection of therapeutic combinations among the active substance and possible therapeutically compatible products, as well as towards the protection of new therapeutic use. The generic manufacturers are oriented towards the protection of new pharmaceutical formulations of the main active substance, as well as towards protecting the changes in processes of synthesis or manufacturing. The similarities refer to the fact that both types of companies became more active in patent protection in a little while before the main patent expire. CONCLUSIONS Our study shows that a variety of factors are influencing the patent protection policy of pharmaceutical manufacturers, such as is the time of discovery, application of product, disease priority, technologies etc. We found that therapeutic competition is more important than the generic ones for creating the patent profile of particular INN, which is in contrast with the beliefs that the patent protection policy affects mainly generic companies. We also confirmed the results of similar studies that around the date of main patent expiration usually the activity of both originators and generic companies for granting new patents is increasing, probably due to the attempts to increase market monopoly of the product or to prevent other companies from market penetration. REFERENCES 1. M a g a z z i n i , L., F. Pammolli et M. Riccaboni. Dynamic Competition in Pharmaceuticals: Patent Expiry, Generic Penetration, and Industry Structure. – The Eur. J. Health Econom., 2, 2004, № 5, 175-182. 2. F i o n a , M. Scott Morton, Entry Decisions in the Generic Pharmaceutical Industry. – In: NBER (National Bureau of Economic Research) Working Paper series, № 6190, Cambridge, 8, 1997. 3. F o x , P. D. Prescription Drug Benefits: Cost Management Issues for Medicare. – Health care financing review, 25, 2003-2004, № 2, 7-21. 4. A g r e e m e n t on Trade-Related Aspects of Intellectual Property (1994)LT/UR/A-1C/IP/1art 1. 5. C o h e n , J. C. et al. TRIPS, the Doha Declaration and increasing access to medicines: policy options for Ghana. – Globalisation and health, 1, 2005, 17. 6. J a n n u z z i , A. Vasconcellos Alexandre, de Souza Cristina. Cad. Saúde Pública, 24, 2008, № 6, 1205-1218,. 16 Patent protection policy... 7. C o r r e a , C. M. A guide to pharmaceutical patents, The South Centre, 1, 2008, ISBN 92-9162-032-7. 8. S i b b a l d , B. Drug patent protection: How long is long enough? – CMAJ, 9, 2001, № 1, 1331. 9. E s s , S. M., S. Schneeweiss et T. D. Szucs . European health care policies for controlling drug expenditure. – Pharmacoeconomics, 21, 2003 № 2, 89-103. 10. M a n s l e y , E. C. et al. The utilization of medicines beyond patent expiration. – Forum for Health Economics and Policy, 11, 2008, № 2, 11-18. 11. M o s s i a l o s , E., M. Mrazek et T. Walley. Regulating pharmaceuticals in Europe: striving for efficiency, equity and quality. European Observatory on Health Systems and Policies, Oxford University Press, 2004. 12. H o p k i n s , V. Analysis of international patent protection and global public health. http://www. princeton.edu/~jpia/pdf2006/JPIA%2006_1%20chapter%20copy%205.pdf (Accessed 15 July 2009). 13. C o r r e a , C. M. Implementing TRIPS in developing countries. Third World Economics 189. 1998, 16-31. http://www.twnside.org.sg/title/ment-cn.htm (accessed March 25, 2005). 14. C o r r e a , C. M. Integrating Public Health Concerns Into Patent Legislation In Developing Countries, 2000. http://www.southcentre.org/ publications/publichealth/publichealth.pdf (accessed March 24, 2005). 15. C o r r e a , C. M. Implications of the Doha Declaration on the TRIPS Agreement and Public Health. World Health Organization: Essential Drugs and Medicines Policy Series 12, 2002a. http://www. who.int/medicines/areas/policy/WHO_EDM_PAR_2002.3.pdf (accessed March 25 2005). 16. C o r r e a , Carlos M. 2002b. Public and Intellectual Property Rights, Global Social Policy, 3, 2002, 261-278. 17. C o r r e a , C. M. Recent International Developments In the Area of Intellectual Property Rights. Universidad de Buenos Aires ICTSD-UNCTAD Dialogue, 2nd Bellagio Series on Development and Intellectual Property, 2003. http://w w w .iprsonline.org/unctadictsd/bellagio/docs/Correa_Bellagio2.pdf (accessed March 28, 2005). 18. W o r l d Trade Organisation. Developing country group’s paper. TRIPS: Council discussion on access to medicines. June 20. http//:www.wto.org/English/tratop_e/trips_e/paper_develop_w296_e. htm (accessed Fabruary 25, 2006) 19. D i r a c h , J. New safe medicines faster: A new concept for drug development. – EUFEPS. 27 Feb/ JDi; 1-5 20. F i o n a , M. Scott Morton, “Entry Decisions in the Generic Pharmaceutical Industry”. – In: NBER (National Bureau of Economic Research) Working Paper series, № 6190, Cambridge, 1997, 8. 21. G a s c o n , F. et al. On macroeconomic characteristics of pharmecutical generics and the potential for manufacturing and consumption under fuzzy conditions. – Artificial Intell in med., 41, 2007, 223-235. 22. W H O . Executive Board, 115 session. International Nonproprietary Names: revised procedure. Documents EB112/3 and EB112/2003/REC/1, summary record of the first meeting, section 4. http://apps.who.int/gb/ebwha/pdf_files/EB115/B115_11-en.pdf (Accessed 16 June 2009). 23. F y h r q u i s t , F. et O. Saijonmaa. Renin-angiotensin system revisited. – J. Intern. Med., 264, 2008, № 3, 224-36. 24. R a i z a d a , M. K. et A. J. Ferreira. ACE2: a new target for cardiovascular disease therapeutics. – J. Cardiovasc Pharmacol., 50, 2007, № 2, 112-119. 25. H o w a r d , L. Use of patents in drug lifecycle management. – J.Gener. Med., 4, 2007, 230-236. 26. H o w a r d , L. Fluvastatin and atorvastatin: A comparison of patent protection (Part 1). – J. Gener. Med., 4, 2007, 302-305. 27. H o w a r d , L. Fluvastatin and atorvastatin: A comparison of patent protection (Part 2). – J. Gener. Med., 5, 2007, 85-90. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 17 28. O r a n g e book: approved drug products with therapeutics equivalence evaluation. ttp://www.accessdata.fda.gov/ (Assess trough January 2006 – July 2009) 29. h t t p ://www.drugpatentwatch.com (Assess trough January 2006 – July 2009) 30. w w w .espacenet.com (Accessed trough January 2006 – July 2009) 31. R o s s i , S. et al. Australian Medicines Handbook 2006. Adelaide; 2006. 32. L e v y , B. I. How to explain the differences between renin angiotensin system modulators. – Am. J. Hypertens., 18, 2005, 134S–141S. 33. C o u n c i l Directive 2004/27/EC of the European Parliament and of the Council of 31 March 2004 amending Directive 2001/83/EC on the Community code relating to medicinal products for human use. Official Journal L, 136, 30/4/2004, 34-57. ¨ ª 18 Address for correspondence: Assoc. Prof. Assena Stoimenova, Ph. D., MPH Faculty of Pharmacy Department of Social Pharmacy and Pharmacoeconomics Medical University – Sofia 2 Dunav str., 1000 Sofia, Bulgaria 02/92 36 589 02/ 987 9874 0887 749 665 e-mail: [email protected] Patent protection policy... MOST USED COMBINED MULTISUPPLEMENTS CONTAINING L – ARGININE D. Obreshkova1, D. Tsvetkova1 and K. Ivanov2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University – Sofia, Bulgaria 2 Department of Pharmaceutical Sciences, Faculty of Pharmacy, Medical University – Plovdiv, Bulgaria 1 Summary. L – Arginine is an essential, proteinogenic amino acid and is involved in metabolic pathways, such as protein degradation, synthesis of creatine, citrulline, L – ornithine, L – glutamate, agmatine, proline, polyamines, urea, etc. The aims of the current study were: 1) to collect data for manufactured combined supplements, containing L – Arginine; 2) to analyse the collected data; 3) to estimate the number of multipreparations with L – Arginine, according to their form; 4) to summarize which are the most common combined supplements with L – Arginine. The performed observational study showed that in most cases L – Arginine was combined with: L – Citrulline, L – Glutamic acid, L – Lysine, L – Ornitine and Vitamin B6. Key words: L – Arginine, HPLC, accuracy, precision, linearity T INTRODUCTION he consumption of food supplements and their importance in people’s daily life are constantly increasing. [11, 12] According to the Natural Medicines Comprehensive Database the effectiveness ratings for L – Arginine are as follows [7]: I) possibly effective for: 1) chest pain associated with coronary artery disease and bladder inflammation [8, 13]; 2) lowering blood pressure in young cardiac transplant recipients by vasodilatation [1, 10] and in patients with gestational hypertension [3]; 3) elimination of extra fluids in congestive heart failure; 4) preventing of: a) heart and circulatory diseases; b) loss of effect of nitroglycerin in people with angina pectoris [13]; c) inflammation of the digestive tract in premature infants [8, 13]; 5) reducing: a) wound healing, recovery time and the Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 19 number of infections after surgery in combination with ribonucleic acid and eicosapentaenoic acid; b) cramping pain and weakness in the legs associated with blocked arteries (intermittent claudication); 6) improving: a) immune system in combination with hydroxymethylbutyrate (HMB) and glutamine; b) kidney function in kidney transplant patients taking cyclosporine; c) small – vessel coronary endothelial function in humans [6, 10]; 7) increasing: a) nitric oxide levels in patients with interstitial cystitis [2]; b) body weight; c) erectile function [5]; d) total antioxidant status [13]; ІІ) possibly ineffective for heart attack and pre – eclampsia [10]; III) promising evidence to rate effectiveness for: 1) improving: breast cancer (in combination with chemotherapy), immune system in people with head and neck cancer, lung inflammation [8], migraine headache in a combination with ibuprofen [9], senile dementia [13]; 2) preventing from: sepsis [8], common cold, diabetic foot ulcers, male infertility, sickle cell disease [13]. The aims of the current study were as follows: 1) collection of data for manufactured combined supplements, containing L – Arginine; 2) analysis of summarized data; 3) estimation of the part of different dosage multipreparations with L – Arginine; 4) summarizing the most common combined supplements with L – Arginine. MATERIALS AND METHODS We used the following approach in the evaluation of trade multisupplements with L – Arginine: 1) collection of data for manufactured combined supplements, containing L – Arginine; 2) analysis of the summarized data; 3) estimation of the number of multipreparations with L – Arginine, according to their form; 4) summariring the most common combined supplements with L – Arginine. RESULTS AND DISCUSSION I. Assessment of trade multisupplements with L – Arginine. An observation of data for all authorized for sale supplements in the world, containing L – Arginine, is described. The investigation has been made through the existing electronical database of medical sourses [4]. The study covers analysis of collected data, comprising the results for the part of distribition of different multicomponents with L – Arginine and estimation of percentage of the most common combinations. The collected data include 345 trade multisupplemets, containing L – Arginine. From results pointed out in Tables 1 – 9, it is obvious, that L – Arginine is manufactured in different dosages and formulations: powders (Table 1, Table 2, Table 3); effervescent sachets, solutions, infusions, gels, coated tablets and effervescent tablets (Table 4); tablets (Table 5, Table 6) and capsules (Table 7, Table 8). 20 Most used combined multisupplements... Table 1. Multisupplements containing L – Arginine – powders 1. Ageless Foundation Ultra Max Gold, 22 x 17.4 g 26. BPI Sports 1.M.R, 80 g 2. All American EFX K – Otic, 32 x 14.28 g 27. BPI Sports 1.M.R, 8 x 28 g 3. All Max Nutrition Arginine, 400 g 28. BSN NO – Xplode, 20 x 20.5 g 4. All Max Nutrition Arginine, 1000 g 29. BSN NO – Xplode, 50 x 20.5 g 5. All Max Nutrition Hema Novol, 240 g 30. Carlson L – Arginine, 1000 g 6. All Max Nutrition Iso Flex, 30 x 30 g 31. Cellucor C4 Extreme, 30 x 6.5 g 7. All Max Nutrition Muscle Prime, 50 x 20 g 32. Champion Adrenol 8, 40 x 20.5 g 8. Amino Vital, 480 g 33. Champion Gly Pro XTS Complete Stack, 20 x 20.7 g 9. Amino Vital Fast Charge, 30 x 5 g 34. Cheap Supplements Arginine, 250 g 10. Amino Vital Pro, 20 x 24 g 35. Controlled Labs Purple Wraath, 12.3 g 11. AST Anabolic Rush, 38 x 26.25 g 36. Controlled Labs Purple Wraath, 45 x 12.3 g 12. Axis Labs Smash Fully Loaded, 30 x 20 g 37. Controlled Labs Purple Wraath, 90 x 12.3 g 13. Betancourt Nutrition Bullnox Androrush, 35 x 18.1 g 38. CTD Labs L – Arginine, 454 g 14. Betancourt Nutrition Bullnox Bull Rush, 5 x 18.2 g 39. Cyto Sport Monster Pump, 30 x 40 g 15. Betancourt Nutrition Bull Rush Recelerator, 28 x 31 g 40. Dynamize ISO – 100, 28 g 16. Beverly Int. Up – Lift, 15 x 22 g 41. Dynamize ISO – 100, 32 x 28 g 17. Bio Quest Fusion Force, 40 x 24.5 g 42. Dymatize Xpand Xtreme Pump, 14 x 20 g 18. Bio Rhythm Arnge Krush, 19.5 g 43. Dymatize Xpand Xtreme Pump, 40 x 20 g 19. Bio Rhythm Arnge Krush, 780 g 44. EAS Pro Science Armor, 14 x 3 g 20. Bio Rhythm O2 Positive, 700 g 45. Elite Delivery Technologies Elite – KXS,68 x 5.5 g 21. Blue Star Nutraceuticals Extreme Rush, 850 g 46. Epic Performance 4 – Nitro Tropic, 40 x 20.5 g 22. Body Fortress Super Advanced Creatine High Performance, 30 x 48 g 47. 4 Ever Fit Detonator, 65 x 20 g 23. Body Fortress Super NOS Blast, 45 x 20 g 48. Full Combat Pre Combat, 50 x 20 g 24. Bodystrong L – Arginine, 1000 g 49. Garbage Garbage, 14 x 12.5 g 25. Bodystrong L – Arginine, 2000 g 50. Garbage Nuclear Garbage, 14 x 15 g Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 21 Table 2. Multisupplements containing L – Arginine – powders 1. Gaspari Nutrition Super Pump 250, 20 g 26. MHP Dark Rage, 20 x 44.7 g 2. Gaspari Nutrition Super Pump 250, 280 g 27. MHP Trac, 425 g 3. Gaspari Nutrition Super Pump 250, 400 g 28. MHP Trac Extreme – NO, 775 g 4. Gaspari Nutrition Super Pump 250, 800 g 29. Millennium Sport RPG IBCAA, 500 g 5. Genetic Breakthrough Nutrition Plazmosis, 30 x 18.8 g 30. Montiff Pure L – Arginine Base Powder, 150 g 6. German American Technologies Jet Fuse NOX, 40 x 20.5 g 31. MPR Pump, 30 x17.6 g 7. German American Technologies Jet Mass, 40 x 20.5 g 32. MRI Anabolic Switch, 20 x 45 g 8. GNC Pro Performance® Arginine, 400 g 33. MRI Black, 800 g 9. Higher Power BCAA AKG, 150 g 34. MRI Black, 68 x 20 g 10. Higher Power BCAA AKG, 120 x 2.5 g 35. MRI NO2 Ripcuts, 20 x 12.8 g 11. IDS Beta NOX, 680 g 36. Muscle Fortress Muscle Spike, 45 x 7.5 g 12. Infinite Labs Juggernaut, 40 x 20 g 37. Muscle FX Hydrobolic FX, 75 x 30 g 13. Isatori Morph Mega Drive, 30 x 20.84 g 38. Muscle Gauge Nutrition Huge Impact, 35 x 66 g 14. Iso Flex – Whey Protein Isolate, 75 x 30 g 39. Muscle Gauge Nutrition Massive Growth, 225 g 15. John Scott’s Nitro Cell Drive, 30 x 48 g 40. Muscle Pharm Assault, 800 g 16. Kal L – Arginine Unflavored, 120 g 41. Muscle Pharm Bullet Proof, 380 g 17. LA Muscle LA Whey, 38 x 60 g 42. Muscle Pharm Combat Powder 52 x 25g 18. L – Arginine Plus, 30 x 12 g 43. Muscle Pharm Recon, 40 x 30 g 19. LG Sciences Postal, 1170 g 44. Muscleology Nitro – Pro, 1360 g 20. Life Extension Arginine Ornithine, 150 g 45. Muscle Tech Anabolic Halo Hardcore Pro, 40 x 23 g 21. Live Long Nutrition AAKG, 100 g 46. Muscle Tech Cell – Tech Hardcore Pro, 20 x 100 g 22. Magnum Nutraceuticals Serum, 40 x 17 g 47. Muscle Tech Intra Vol, 32 x 30 g 23. Metagenics Arginine Plus Magnesium, 644 g 48. Muscle Tech NaNO Vapor Hardcore Pro Series, 50 x 18 g 24. Metabolic Diet Power Drink, 616 g 49. Muscle Tech Nitro Amino FX Pro Series, 385 g 25. Met – Rx Protein Revolution, 15 x 77 g 50. Muscle Tech Nitro – Tech Hardcore Pro, 27 x 33 g 22 Most used combined multisupplements... Table 3. Multisupplements containing L – Arginine – powders 1. Myogenix Hyper Shock Tactical Nitric Oxide Pre – Workout, 40 x 12 g 28. Sci Fit Muscle Smoothie, 18 x 75 g 2. Nimbus Nutrition Performance Protein, 900 g 29. Sci Fit Super Nova, 908 g 3. Now AAKG Power, 200 g 30. Seroyal/Pharmax – L – Glutamine L – Arginine, 250 g 4. Now Arginine Power, 454 g 31. SNI Nitric Blast, 600 g 5. Now Arginine Power Super Stack, 50 x 20 g 32. Sourse Naturals L– Arginine Free, 100 g 6. Now Pro – GH, 600 g 33. Sports One Equalizer, 720 g 7. NRG – X Labs Anabolic Raptor, 900 g 34. Sports One Solid Mass, 720 g 8. Nu Care Nitric Blast, 550 g 35. Sport Pharma Gain Max, 16 x 170 g 9. Nu Care Nitric Blast Extreme, 550 g 36. Star Chem Armageddon, 920 g 10. Nutra Bio L – Arginine Ajinomoto, 150 g 37. Star Chem Momentum, 40 x 11 g 11. Nutra Bio L – Arginine Ajinomoto, 500 g 38. Swanson Premium -- Arginine AKG Lemon Flavored, 350 g 12. Nutrabolics Anabolic Window, 12 x 92 g 39. Syntrax Innovations Nitrous, 300 g 13. Nutrabolics Hemorush, 50 x 20 g 40. Thermo Life Pump – Bol, 42 x 11 g 14. Nutrabolics NOZ I.V., 630 g 41. Top Secret Nutrition N.O., 30 x 11 g 15. Nutraceutics Vivax, 20 x 8.6 g 42. Twinlab Brewers Yeast, 31 x 16 g 16. Nutrex Hemo – Rage Black, 45 x 20 g 43. Twinlab Mass Fuel Xtreme, 18 x 150 g 17. Nutrex Hemo – Rage Black Ultra Concentratе, 90 x 3.2 g 44. Ultra Max Gold, 22 x 17.4 g 18. Olympian Labs A – AKG, 90 g 45. Universal Intra – Aid, 32 x 25 g 19. Panthera Pharma VME Vaso Muscle Expander, 28 x 32 g 46. Universal Shock Therapy, 50 x 21 g 20. Pride Nutrition Dominate, 800 g 47. Universal Shock Therapy, 400 g 21. Pride Nutrition Retaliate, 780 s 48. Universal Storm, 80 x 9.4 g 22. Prima Force AAKG, 250 g 49. USP Jack 3, 250 g 23. Pro Argi – 9 Plus, 150 g 50. USP Jack 3, 45 x 5.55 g 24. Professional Supplements Hyperbolic 10, 36 x 27.7 g 51. Vitol Russian Bear, 292 g 25. S.A.N. Myotein, 1202 g 52. Xero Limits EPOg – Blast, 20 x 14.7 g 26. S.A.N. V – 12 Magnum, 50 x 11 g 53. Xyience NOX – CG3, 13 x 1.7 g 27. Sci Fit Kreation, 690 g 54. Zipfizz Energy Drink Mix, 3 x 11.4 g 55. Zipfizz Energy Drink Mix, 20 x 11.4 g Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 23 Table 4. Multisupplements containing L – Arginine – effervescent sachets, solutions, infusions, gels, coated tablets and effervescent tablets Effervescent Sachets 1. Newton Everett Biotech Isotropin Rejuvenation, 60 eff. sachets 3. 2. Nutraceutics Vivax, Tropical Fruit Flavor, 20 eff. sachets Nutraceutics Medi Tropin, 60 eff. sachets Solutions 1. Aminogame 1500, 492 ml 10. Now Awe Slim Liquid Weight Management – Slim, 946 ml 2. ANSI Thermo Hydroxadrine Nitric Xplosion, 591 ml 11. Pharma Gen X Liquid Mojo, 240 ml 3. BSN Endorush Xtreme Strength, 118 ml 12. Premier Nutrition Nitro Shot, 54 ml 4. Cyto Sport Fast Twitch RTD, 240 ml 13. Sargenor 1000 mg/5 ml 5. Cyto Sport Fast Twitch RTD, 591 ml 14. Strength Systems USA Nitric Blast, 120 ml 6. IDS Beta NOX, 103 ml 15. Tonotyl, 10 ml/100 mg 7. iSatori Liquid Morph+, 92 ml 16. Trionix TRX – 7, 60 ml 8 Liquid Amino 2000, 946 ml. 17. Twinlab Amino Fuel Liquid, 450 ml 9. MMUSA Stratos, 150 ml 18. VPX Black Pearl RTD, 240 ml Infusions 1. SciFit Nitrox Infusion, 30 ml 1. AST Myog – D, 120 softgels 2. Axis Labs Hemodraulix, 180 liquid softgels Gels 3. Shocker Nutrition N.O. Extreme, 180 softgel Coated tablets 1. Met – Rx Xtreme Nitro Pump NOS, 180 tabl. Effervescent tablets 1. MDR Vital Factors, 10 eff. tabl. 2. MDR Vital Factors, 40 eff. tabl. 3. Millennium Sport Nitro Ceps, 120 eff. tabl. 24 Most used combined multisupplements... Table 5. Multisupplements containing L – Arginine – tablets 1. All Max Nutrition Hema Novol, 240 tabl. 21. Doubled – T Sports NO Beta, 30 tabl. 2. Amino 1000, 100 tabl. 22. Doubled – T Sports NO Beta, 180 tabl. 3. Amino 2000: L – Arginine 285 mg/L – Glutamic acid 1990 mg / L – Lysine 1040 mg, 365 tabl. 23. Doubled – T Sports NO Beta, 360 tabl. 4. Amino 2300, 325 tabl. 24. Elite Delivery Technologies Nitricd – X O2, 180 tabl. 5. Anabolic Agents Nanoxide Delivery, 240 tabl. 25. Enzymatic Therapy HDL Booster, 120 tabl. 6. ANSI Thermo Hydroxadrine Nitric Xplosion, 100 tabl. 26. Gaspari Nutrition Plasma Jet, 160 tabl. 7. ASN Maxabol II, 100 tabl. 27. German American Technologies Sonic Pump, 180 tabl. 8. Betancourt Nutrition Bullnox Androrush, 35 tabl. 28. Inner Armour Amino 2000, 350 tabl. 9. Betancourt Nutrition Bullnox Androrush, 175 tabl. 29. IronMagLabs Nitro4, 90 tabl. 10. Beverly Int. Mass, 500 tabl. 30. Isatori Morph GXR – 3, 90 tabl. 11. Beverly Int. Muscle Synergy, 240 tabl. 31. Isatori Morph GXR – 3, 180 tabl. 12. Biochem Max – Amino, 180 tabl. 32. ISS Research Satur 8 Nitric Oxide, 180 tabl. 13. Body Fortress Super NOS Pump, 90 tabl. 33. Jarrow Formula Arginine 1000 mg, 100 tabl. 14. Brain Pharma Happy Pills, 60 tabl. 34. Kal Amino Acid Complex 1000 mg, 100 tabl. 15. BSN Epozined – O2 NT, 180 tabl. 35. LA Muscle Vasculator, 90 tabl. 16. BSN Nitrix, 180 tabl. 36. L – Arginine/L – Citrulline Complex, 60 tabl. 17. Cel Edged – NO, 180 tabl. 37. L – Arginine/L – Citrulline Complex, 120 tabl. 18. Champion Adrenol 8, 90 tabl. 38. L – Arginine/L – Citrulline Complex, 240 tabl. 19. Cobal – M: L – Arginine 30 mg/ L – Glutamic acid 100 mg, 10 tabl. 39. Life Extension GH Pituitary Support Day Formula, 120 tabl. 20. Doubled – T Sports Nitro Mine, 360 tabl. 40. Mega – Pro Amino 2200 mg, 325 tabl. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 25 Table 6. Multisupplements with L – Arginine – tablets 1. Mega – Pro Super Amino, 90 tabl. 21. Panthera Pharma Primal Pump, 180 tabl. 2. Met – Rx Hardcore Amino 3000, 180 tabl. 22. Pure Essence Virility For Men, 60 tabl. 3. Metabolic Diet Antiox Version 4, 180 tabl. 23. STS Pro Muscle Nitric, 90 tabl. 4. Metabolic Diet GH Boost Version 4, 150 tabl. 24. Twinlab Amino Fuel, 60 tabl. 5. Metabolic Diet Lipo Flush, 120 tabl. 25. Twinlab Amino Fuel, 150 tabl. 6. Metabolic Diet Testo Boost, 120 tabl. 26. Twinlab Amino Fuel, 250 tabl. 7. Metabolic Diet Thermocell 35, 120 tabl. 27. Twinlab Nitric Fuel, 180 tabl. 8. Michael’s Testosterone Factors, 90 tabl. 28. Ultimate Nutrition Amino 2000, 325 tabl. 9. Now AAKG 3500 mg, 180 tabl. 29. Ultimate Nutrition Amino Gold 1000 mg, 250 tabl. 10. Now L – Arginine 500 mg, 120 tabl. 30. Ultimate Nutrition Amino Gold 1500 mg, 325 tabl. 11. Now L – Arginine 1000 mg, 120 tabl. 31. Ultimate Nutrition Super Amino 2000 mg, 150 tabl. 12. NPS Vir Max E2, 60 tabl. 32. Ultimate Nutrition Super Amino 2000 mg, 330 tabl. 13. Nutrabolics Hemotropin, 90 tabl. 33. Universal 100 % Amino, 200 tabl. 14. Nutrifacts Complete: L – Arginine 10 mg/ L – Glutamic acid 100 mg/L – Lysine 50mg/ Vitamin B6 3 mg, 50 tabl. 34. Universal 100 % Amino, 400 tabl. 15. Optimum Superior Amino 2222 mg, 160 tabl. 35. Universal Amino 2700 mg, 120 tabl. 16. Prolab Amino 2000 mg, 325 tabl. 36. Universal Amino Tech, 375 tabl. 17. Source Naturals L – Arginine/L – Citrulline Complex, 120 tabl. 37. Universal GH Max, 180 tabl. 18. Spectrient: L – Arginine 9 mg/L – Glutamic acid 31 mg/L – Lysine 5 mg/Vitamin B6 5 mg, 100 tabl. 38. Vita Life 100 % Amino 2000 mg, 250 tabl. 19. Sport Pharma Amino Max, 325 tabl. 39. Vitol Russian Bear, 140 tabl. 20. Super Amino 2000: L – Arginine 37 mg/ L – Lysine 212 mg/L – Glutamic acid 334 mg, 365 tabl. 40. Xero Limits Eponox, 180 tabl. 26 Most used combined multisupplements... Table 7. Multisupplements with L – Arginine – capsules 1. Ageless Foundation Ultra MAX Gold, 90 caps. 21. Fusion Bodybuilding Sleeping – Giant, 240 caps. 2. Applied Nutriceuticals RPM, 110 caps. 22. Healthy’N Fit Advanced GH Enhancers, 90 caps. 3. Applied Nutriceuticals RPM, 240 caps. 23. Healthy’N Fit Advanced GH Enhancers, 180 caps. 4. Beast Sports Nutrition Predator Rapid Release, 240 caps. 24. Labrada Re Charge, 200 caps. 5. Beast Sports Nutrition Super Test, 180 caps. 25. L – Arginine 500 mg/B6, 50 caps. 6. Beverly Int. GH Factor, 180 caps. 26. L – Arginine 500 mg/B6, 100 caps. 7. Big Nutrition Ada Lift, 90 caps. 27. L – Arginine 500 mg/L – Citrulline 250 mg, 120 caps. 8. Bio Rhythm Big Blue, 200 caps. 28. L – Arginine 500 mg/L – Ornithine 250 mg, 100 caps. 9. Controlled Labs White Blood, 90 caps. 29. L – Arginine 1000 mg/L – Ornithine 1000 mg, 300 caps. 10. Country Life – Arginine/L – Ornithine, 180 caps. 30. L – Arginine 500 mg/L – Ornithine 500 mg/ B6, 60 caps. 11. Daily Wellness Company Argin Max For Men, 180 caps. 31. L – Arginine 500 mg/L – Ornithine 500 mg/ B6, 90 caps. 12. Daily Wellness Company Argin Max For Women, 180 caps. 32. L – Arginine 500 mg/L – Ornithine 500 mg/ B6, 180 caps. 13. Dymatize Energized Xpand, 240 caps. 33. LG Sciences I – GH – 1, 100 caps. 14. EST Plasmatic, 96 Lipof – Matic gels, 96 caps. 34. Magnum Nutraceuticals Ac – Bomb, 180 caps. 15. 4 Ever Fit AKG2, 240 caps. 35. MHP Anadrox, 112 caps. 16. 4 Ever Fit L – Arginine 500 mg, 180 caps. 36. MHP Anadrox, 224 caps. 17. FTH Nutraceuticals Healthy Heart PM, 120 caps. 37. Mus – L – Blast 2000 Supplement Facts: L – Arginine 325 mg/L – Glutamic acid 1855 mg, 100 caps. 18. Fusion Bodybuilding Agent – M, 120 caps. 38. Muscle Fortress Vasoc – Pump Hot Start, 180 caps. 19. Fusion Bodybuilding Shut – Eye, 120 caps. 39. Muscle Juice Supplement Facts: L – Arginine 371.25 mg/L – Glutamic acid 2743.25 mg, 100 caps. 20. Fusion Bodybuilding Sleeping – Giant, 120 caps. 40. Muscle Tech Alpha Amino Prototype 216, 120 caps. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 27 Table 8. Multisupplements with L – Arginine – capsules 1. Muscle Tech Myo Shock HSP, 140 caps. 21. Sci Fit Kreation, 120 caps. 2. Muscle Tech NaNO Vapor Hardcore Pro Series, 150 caps. 22. Sci Fit Kreation, 240 caps. 3. Muscle Tech NaNO X9, 180 caps. 23. Sci Fit L – Arginine 500, 100 caps. 4. Muscle Tech NaNO X9 Hardcore, 30 caps. 24. Six Star Muscle Professional Strength Nitric Oxide Overdrive, 100 caps. 5. Natrol L – Arginine, 1000 mg, 50 caps. 25. Snac Aerobitine, 120 caps. 6. Now Arginine/Citrulline, 120 caps. 26. SNS Arginine E2 Matrix - 180 Caps 7. Now Arginine/Ornithine, 250 caps. 27. Sports One Equitest, 120 caps. 8. Now Arginine Power Super Stack, 120 caps. 28. Sports One Ethylsterol, 120 caps. 9. Now Tri – Amino, 120 caps. 29. Sports One HGHD – XS, 120 caps. 10. Nutrabolics Skin-Bursting Stack, 60 capss 30. Super Amino 2222: L – Arginine 90 mg/ L – Glutamic acid 370 mg, 100 caps. 11. Olimp AAKG Extreme - Arginine − 120 Caps 31. Super Amino 4800: L – Arginine 181.5 mg/L – Lysine 271.5 mg/ L – Glutamic acid 640.5 mg, 100 caps. 12. Olympian Labs Arginine/Ornithine, 100 caps. 32. T-Max Dual Anabolic System 90 caps 13. Palo Alto Labs Paravol, 60 caps. 33. TSN Labs NO3X Extreme A – AKG, 550 mg, 120 caps. 14. Pro Blend 55 Supplement Fact, L – Arginine 818.5 mg/L – Glutamic acid 4709.4 mg, 100 caps. 34. TSN Labs NO3X Extreme A – AKG, 800 mg, 200 caps. 15. Pro Card Nutrition GH Accelerator, 100 caps. 35. Twinlab L – Arginine 500 mg, 100 caps. 16. Pro Card Nutrition Kre Oxi Pump, 120 caps. 36. Twinlab L – Arginine/L – Ornithine, 100 caps. 17. Pro Fight Amino Sports 4500 mg, 180 caps. 37. Ultimate Nutrition Amino Gold 1000 mg, 250 caps. 18. Pro Rx Labs X Fuel, 180 caps. 38. Ultimate Nutrition Arginine Power, 100 caps. 19. S.A.N. CM2 Nitrate, 240 caps. 39. Ultimate Nutrition Arginine/Ornithine/ Lysine, 100 caps. 20. Sci Fit Endurd – O2 + Kreation, 120 caps. 40. Ultimate Nutrition Arginine/Pyroglutamate/ Lysine, 750mg, 100 caps. 41. Vigor Labs Chainsaw, 30 caps. The distribution of dosage multisupplements, containing L – Arginine as percentage from all analyzed 345 preparations is summarized in Table 9: powders: 44.93%, capsules: 23.48%, tablets: 23.19%. Data for tablets and capsules are analyzed and the percentage of most commonly saled combinations with L – Arginine: L – Citrulline, L – Glutamic aicd, L – Lysine, L – Ornitine and Vitamin B6 are obtained. 28 Most used combined multisupplements... Table 9. Distribution of number of multisupplements, containing L – Arginine, according to their form N: Form of multisupplements 1. Powders Number % from all 345 multisupplements 155 44.93 2. Effervescent sachets 3 0.87 3. Tablets 80 23.19 4. Coated tablets 1 0.29 5. Effervescent tablets 3 0.87 6. Capsules 81 23.48 7. Solutions 18 5.22 8. Infusions 1 0.29 9. Gels 3 0.87 10. Total 345 100.0 CONCLUSION An observational study based on information in medical sourses for multisupplements with L – Arginine shows different trade form preparations: powders, effervescent sachets, tablets, coated tablets, effervescent tablets, capsules, solutions, infusions, gels. From the assessment of the results it is obvious, that in the most common cases L – Arginine is combined with: L – Citrulline, L – Glutamic acid, L – Lysine, L – Ornitine and Vitamin B6. REFERENCES 1. B o d e – B o g e r , S. M. et al. L – Arginine – induced vasodilation in healthy humans: pharmacokinetic-pharmacodynamic relationship. – Br. J. Clin. Pharmacol., 46, 1998, 489-497. 2. E h r e n , I., J. O. Lundberg , J. Adolfsson et N. P. Wiklund. Effects of L – Arginine – treatment on symptoms and bladder nitric oxide levels in patients with interstitial cystitis. – Urology, 52, 1998, 1026-1029. 3. F a c c h i n e t t i , F. et al. Volpe. L – arginine supplementation in patients with gestational hypertension: a pilot study. – Hypertens. Pregnancy., 26, 2007, № 1, 121-130. 4. h t t p ://naturaldatabase.therapeuticresearch.com/nd/Search.aspx?cs=&s=ND&fs=ND&pt=103&i d=54659. 5. L e b r e t , T., J. M. Hervéa, P. Gornyb, M. Worcelc et H. Botto. Efficacy and safety of a novel combination of L – Arginine Glutamate and Yohimbine Hydrochloride: a new oral therapy for erectile dysfunction. – Eur. Urology, 41, 2002, 608-613. 6. L e r m a n , A. et al. Long – term L – Arginine improves small – vessel coronary endothelial function in humans. – Circulation, 97, 1998, 2123-2128. 7. M c K e v o y , G. K., ed. A. H. F. S. Drug Information. Bethesda, MD: American Society of Health – System Pharmacists, 1998. Top of Form 8. M u r a d , F. Discovery of some of the biological effects of nitric oxide and it’s role in cellular signaling. Nobel Lekture. Biosciences Reports, 19, 1999, 133-154 and Les Prix Nobel, 1998, (the Nobel Prizes,1998), 1999, 273-307. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 29 9. S a n d r i n i , G. et al. Effectiveness of ibuprofen – arginine in the treatment of acute migraine attacks. – Int. J. Clin. Pharmacol. Res., 18, 1998, 145-150. 10. S c o t t , L. D. et al. Effect of oral L – arginine on oxidant stress, endothelial dysfunction and systemic arterial pressure in young cardiac transplant recipients. – The Am. J. Cardiol., 94, 2004, № 6, 828-831. 11. S t o i m e n o v a , A. Food supplements in Central and Eastern European countries. – Acta Medica Bulgarica, 37, 2010, № 1, 71-77. 12. S t o i m e n o v a , A. et al. Educational project on food supplements for community pharmacists. – Acta Medica Bulgarica, 37, 2010, № 2, 51-57. 13. Ta p i e r o , H., G. Mathé, P. Couvreur et K. D. Tew. Dossier: Free amino acids in human health and pathologies. – I. Arginine. Biomed Pharmacother., 56, 2002, 439-445. 30 Address for correspondence: Dobrina Tsvetkova Department of Pharmaceutical Chemistry Faculty of Pharmacy Medical University – Sofia Bulgaria e-mail: [email protected] Most used combined multisupplements... PHARMACOTHERAPY COSTS OF OSTEOPOROSIS AND RELATED FRACTURES IN BULGARIA A. Savova, A. Stoimenova, M. Manova and G. Petrova Department of Social Pharmacy and Pharmacoeconomics, Faculty of Pharmacy, Medical University Summary. The objective of the study is to investigate and calculate the direct medical costs of osteoporosis, the indirect ones, as well as the family costs in Bulgaria. A top down retrospective cost study has been developed. Epidemiology data for osteoporosis has been derived from two published reports. The information on the prices of medicines, patient co-payment, level of reimbursement, hospitalization cost and average hospital stay is based on the official national sources. Official information provided by the National Health Insurance Fund is that in 2009 only 2,143,046 BGN for 5,950 treated patients were paid. The annual cost of femoral fractures should be 8 million BGN, and 800,000 BGN for rehabilitation. The rest of the fractures are mild and account for 26,950,000 BGN. The average disability loses account for approximately 23,105,472 BGN. The direct medical cost exhibits the proportion of 2 million for medicines to 36 million BGN for treatment of fractures. The major conclusions from our analysis are that the patients with osteoporosis in Bulgaria are not adequately treated in terms of reimbursement coverage and patients carry the greater part of financial and social burden. Key words: cost study, pharmacotherapy, osteoporosis, health policy, medicines policy O INTRODUCTION steoporosis is a chronic condition characterised by bone fragility resulting in bone fracture. Bone fracture is associated with pain and decreased quality of life. Osteoporosis affects 1 in every 8 persons over of age. One in every 3 women and one in every 5 men over the age of 50 years develop osteoporosis and will suffer fracture in their lifetime. Fragility fracture is the clinically apparent and relevant outcome in osteoporosis. In Europe every 30 seconds one person experiences fracture, but there is no clear evidence of the proActa Medica Bulgarica, Vol. XXXIX, 2012, № 1 31 portion of osteoporotic ones [5, 16]. The risk of a subsequent fracture in the same year increases fivefold, when women have already had vertebral fracture due to osteoporosis. Fractures increase the health care cost of patients with osteoporosis. When the risk of fractures increases over 40 per cent, the cost doubles [15]. It is estimated that there are 180,000 osteoporosis-related symptomatic fractures annually in England and Wales. Of these, 70,000 are hip fractures, 25,000 are clinical vertebral fractures and 41,000 are wrist fractures [17]. The economic burden of osteoporosis is tremendous. Burge et al. estimated the cost of osteoporotic fractures as $ 19 billion in the USA in 2005 year [4]. Out of them 56 per cent are hospitalization cost due to fractures, and 37 per cent are covered by the health insurance institutions. The health economic analysis shows that in 2010 year the costs of osteoporosis in Europe will double to 40 million Euro in comparison to 2000 year. It is expected that fracture expenditure be 80 milliard Euro till 2050 year when the proportion of fractures of women and men will be 2 to 1 [6, 19]. According to the type of care health care costs are distributed to 42 per cent for medical cares, 26 per cent - for medicines, 24.7 per cent – for visiting the GP, 5.8 per cent - for additional insurance. The indirect costs of fracture disability are almost equal to the direct health care costs, and in some countries they are twice as much. The direct unit costs associated with non-vertebral osteoporotic fractures in five European countries (France, Italy, Spain, UK, Belgium) range for hip fractures between 8,346 Euro and 9,907 Euro; for others – between 890 and 3,262 Euro [2]. The average cost for hospitalization of women is 5,548 euro and for the men 6,834 Euro. The interest in cost of illness studies in Bulgaria has been increasing recently, but there is no cost study of osteoporosis therapy [13]. The overall world economic burden of osteoporosis and the lack of studies on health care costs in relation to this chronic disease in Bulgaria stimulate our interest in the topic. The objective of the study is to investigate and calculate the direct medical costs of osteoporosis, the indirect ones as well as the family costs in Bulgaria. The point of view is that of the health insurance fund, society, and the patients with a time horizon of one year. MATERIALS AND METHODS A top down retrospective cost study has been developed. First, an interview with the chairman of the Association of osteoporotic patients in Bulgaria focusing on cost drivers was taken. The interview comprises of open ended questions on the reimbursed medicines, co-payment contribution, coverage of hospitalization and rehabilitation after fractures, and medical devices. The answers were used to create a cost-structure model. Epidemiology data for osteoporosis in Bulgaria has been derived from two published reports [1, 3]. The information on the prices of medicines, patient co-pay- 32 Pharmacotherapy costs of osteoporosis... ment, level of reimbursement, hospitalization cost, average hospital stay is based on the official national sources or normative and administrative acts as presented in Table 1. The population and average income data has also been obtained from the official statistical sources – Table 1. [7-12, 14, 18]. Table 1. Sources of information Variable Source of information Epidemiology [10] , [11] Reimbursed medicines, prices and co payment Positive drug list [12] Expenditures for reimbursed medicines Database of the health insurance fund [13] Hospital charges for fractures and rehabilitation procedures National framework contract [14] Population National Statistical Institute [15] Hospital stay National Health Information Institute [16] Income National Social Insurance Fund [17] The direct medical costs are calculated as a sum of the yearly cost of pharmacotherapy, cost of fractures, and rehabilitation costs. The yearly cost of pharmacotherapy is provided by the National Health Insurance Fund (NHIF). Cost of fractures is calculated by multiplying the epidemiology information from the published studies by the NHIF charges for fracture. The rehabilitation cost is calculated by multiplying the cost for 10 days rehabilitation by the number of patients. There are 2 negotiated charges in case of fracture in the National framework contract and these are 2,035 BGN for femoral fracture, and 385 BGN (Bulgarian Levs, National currency) for any other fracture. In addition, 10 days of rehabilitation procedures are paid 20 BGN per day [14]. The indirect cost of productivity losses and premature death was calculated by multiplying the average number of days lost due to the event (hospital stay, or mortality statistics) by the lowest monthly or yearly income per capita based on information on the population structure and payment [17]. Out of the cost contributed by the patients, only the cost for co-payment for medicines was calculated. The exchange rate for 2009 is 1,956 BGN (Bulgarian levs, national currency) equal to 1 Euro. RESULTS Epidemiology information about osteoporosis in Bulgaria Two main national reports on osteoporosis and related fractures in the country were found. According to the expert opinion of the Bulgarian endocrinologist Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 33 society, published in 2004, 92,000 women had at least one osteoporotic vertebral fracture, over 4,000 of them had femoral fractures every year and 800 would die due to complications within one year [10]. The second study was presented by the Expert Group of “National Program for Osteoporosis Limitting”. This is a prospective study of 426,000 women with osteoporosis over 50 years of age during the 2005-2010 period. The number of femoral fractures reported in the study is 30,436 (1, 9% of 1,601,919 being the total number of women over 50 years of age, according to demographic statistics), of vertebrale – 36,844 (2, 3%), of the wrist – 145,774 (9, 1%). The absolute risk of fracture within ten years calculated in the study reaches >3 per cent for the people over 65 years of age, and more than 20 percent - over 70 [11]. The hospital stay after femoral neck fracture is 30-35 days [16]. The huge difference in the number of fractures in the two studies is probably due to the fact that the first one is an expert opinion focusing on the age group over 60 years of age, while the second one is a prospective study focusing on the age group over 50 years of age. Both studies could be considered as a basis for establishment of the lower and upper boundaries of osteoporosis cost. Cost structure model The interview with the Chairman of the Patients’ Osteoporosis Association led to the creation of the following cost structure model – Fig. 1. Ambulatory pharmacotherapy – 25% reimbursed by the National Health Insurance Fund (NHIF) Direct Medical Costs Hospitalization due to fractures – Paid by the NHIF after invoice issued by hospitals Rehabilitation - 10 days paid by NHIF Disability losses – carried by society Indirect costs Premature death – carried by society Ambulatory pharmacotherapy – 75% co-payment Home care after fracture - if necessary Costs to patients and their families Tests - which are not covered by NHIF. Medical devices – implants, crutch and etc. Rehabilitation – additional days if necessary Fig. 1. Costs structure model for osteoporosis therapy in Bulgaria 34 Pharmacotherapy costs of osteoporosis... Three main groups of costs were derived from the interview and included in the model. Direct medical costs are assigned to medicines, fracture therapy and rehabilitation. The National Health Insurance Fund reimburses 25 per cent of the medicines for osteoporosis therapy, which are included in the national positive drug list. It also pays the previously negotiated hospital charges for different types of fractures, as well as 10 days of rehabilitation procedures following every fracture. The patients and their families pay 75 per cent of the cost of medication therapy, home care if necessary, clinical tests for bone density evaluation, medical devices, and additional rehabilitation procedures if necessary. The indirect costs to society are determined by the days off work due to disability (30-35 day) and premature death. Cost calculations Cost of pharmacotherapy Тhe medicinal products that are included in the positive drug list and reimbursed for osteoporosis therapy are presented in Table 2 [10]. There are 7 INN (International Nonproprietary Name) of medicines reimbursed. Out of them 2 have 3 competitors each (risendronate and alfacalcitriol), and one is provided in two dosage forms (ibandronate). Table 2. Annual treatment costs for one patient with reimbursement alternatives in 2009 year INN DDD Monthly cost per DDD (BGN) Annual cost per DDD per day (BGN) Reimbursement Level Patient copayment Alendronic acid tabl Ibandronic acid tabl 10 mg 23.88 279.36 69.84 209.52 5 mg 64.62 775.44 193.86 581.58 Ibandronic acid inj 5 mg 221,18 884,72 221,18 663.52 Risedronate sodium tabl 5 mg 29.98 359.76 89.94 269.82 30.01 360.12 90.03 270.09 69.34 832.08 208.02 624.06 Alendronate /Colecalciferol tabl 10 tabl 47.7 572.40 143.10 429.30 Zoledronic acid inj 5 mg 806.84 806.84 201.71 605.13 Strontium ranelat tabl 2g 89.63 1075.56 268.89 806.67 Alfacalcidol tabl 1 mcg 14.24 170.88 42.72 128.16 9.74 116.88 29.22 87.66 21.28 255.36 63.84 191.52 INN – International Nonproprietary Name DDD – Defined Daily Dose BGN – Bulgarian Leva (national currency) Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 35 If every patient receives one defined daily dose (DDD) per day the annual cost of pharmacotherapy per patient ranges between 116.88 BGN and 1075.56 BGN at pharmacy prices. The reimbursement level is within the range of 29.22 BGN to 268.89 BGN. If all 426,000 women with osteoporosis will take the cheapest medicine (Alendronic acid) then the theoretical total costs for pharmacotherapy should be 72,539,280 BGN. According to the existing reimbursement level of 25 per cent the costs for the National Health Insurance Fund will be 18,134,820 BGN. In 2009 only 5,950 patients who received pharmacotherapy are included in the database of the National Health Insurance Fund on the average (the number of patients varies between 3,952 and 7,064 people during different months). These are just 10 per cent of the patients with osteoporosis diagnosed by the Bulgarian Expert Group. Official information provided by the National Health Insurance Fund is that in 2009 only 2,143,046 BGN for 5,950 treated patients were paid. The rest of the diagnosed patients are either not suitable for reimbursement, or use medicines which are not included in the positive drug list. Cost of fractures Because no separate information on the reasons for fractures is available, we calculated the cost of osteoporosis fractures based on the health insurance charges and epidemiology data for their prevalence. It is accepted that 50 per cent of the women and 25 per cent of the men over the age of 60 years will have at least one fracture due to osteoporotic bone damages in their lifetime. In Bulgaria the total number of population over age 60 is 1,835,749, of which 771,858 men and 1,063,890 women. Out of them 1,110,839 will have at least one fracture in their lifetime (917,875 in the women and 192,964 in the men). When remaining lifespan is 15 years on the average, the annual number of fractures is 74,056 and over 4,000 are on the femoral neck. Thus the annual cost of femoral fractures should be 8,140,000 BGN (4,000 х 2,035 BGN). The cost of rehabilitation of those patients is paid by the National Health Insurance Fund and should be 800,000 BGN (4,000 х 10 x 20 BGN). If we accept that the rest of the fractures are mild and are paid applying the smallest charge of 385 BGN, their cost should be 26,950,000 BGN (70,000 х 385 BGN). Indirect costs The average disability losses of 74,056 patients for a month in plaster will be 23,105,472 BGN (312 BGN is the lowest monthly income). The real costs are higher because those for the recovering period are higher and incomes differ. If the patient is paid at least 10 days for rehabilitation at 20 BGN per procedure, a further 14,811,200 BGN costs will be generated. The loss of premature death will be at least 30,000,000 BGN. (800 persons х 10 years remaining life х 3,750 BGN lowest annual income). 36 Pharmacotherapy costs of osteoporosis... On the basis of these approximate calculations it is possible to present the structure of total annual costs for osteoporosis in Bulgaria – Table 3. Тable 3. Structure of total osteoporosis costs in Bulgaria in 2009 Type of costs Main point Costs Total costs Direct medical costs − medicines − fractures − rehabilitation 2 143 046 35 090 000 800 000 38 033 046 Indirect costs − productivity losses − losses of premature death 23 105 472 30 000 000 53 105 472 Costs to patients and their families − − − − − 6 429 138 n.a. n.a. n.a. n.a. medicines rehabilitation home cares medical devices additional tests 6 429 138 DISCUSSION Although the calculations are illustrative and provide information only about the minimum costs that the society, the National Health Insurance Fund, and the patients with osteoporosis have to cover, they provide useful information about the costs structure. The costs structure follows the world tendencies with main driver’s hospitalization due to fractures for medical cost. It is also clear that the patients with osteoporosis in Bulgaria are not properly treated in terms of unified coverage, the treatment is sub-financed, and patients carry the heavier burden. There is a lack of prevention programs at a national level and not all patients are treated. On the other hand the National Health Insurance Fund has paid for all fractures and thus it is not clear what the savings in case of a prevention program could be. The present ratio between resources paid for medicines and for fractures from the National Health Insurance Fund is 2 million to 36 million BGN. It is obvious that the medicines are not the greater part of the cost. A better preventive program focusing on increasing the amount of medicines reimbursement could lead to the decrease of fractures expenditure. We have not calculated the possible decrease in the expenditures due to better pharmacotherapy. Studies indicate that proper pharmacotherapy could decrease the relative risk of fractures to 62-35 per cent [18]. There are serious doubts that the current low level of reimbursement is one of the major obstacles for better patients’ compliance with the pharmacotherapy [19]. In the future it is expected that the costs of osteoporosis will increase and this tendency is also applicable to Bulgaria. Thus it is necessary to point out that the reimbursement policy should be changed and patients should receive higher level of reimbursement. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 37 It should also be noted that we have not calculated all costs carried by the patients. Except the co - payment for medicines they cover expenditures for home care, additional rehabilitation procedures, tests, and medical devices. Thus, resources which are paid by the patients could become equal to those paid by National Health Insurance Fund. In this case the logical question is why those persons are obligatorily health insured and how they benefit from that. CONCLUSION The major conclusions from our analysis are that the patients with osteoporosis in Bulgaria are not adequately treated in terms of reimbursement coverage, the treatment is not financed well enough and patients carry the greater part of osteoporosis financial and social burden. The direct medical cost shows a ratio of 2 million for medicines to 36 million BGN for treatment of fractures. Therefore appropriate medication therapy is essential to reduce the cost of fractures treatment. REFERENCES 1. B o r i s s o v a , A. M. et al. Report of the working group at the National program for osteoporosis limitation in Bulgaria (2005-2010 year) (In Bulgarian). 2. B o u e e , S. et al. Estimation of direct unit costs associated with non-vertebral osteoporotic fractures in five European countries. – Rheumatol. Int., 26, 2006, 1063-1072. 3. B u l g a r i a n Endocrinology society, recommendations in good practice for osteoporosis therapy, Sofia 2004 (In Bulgarian). 4. B u r g e s et al. Incidence and economic burden of osteoporosis –related fractures in the United States, 2005-2025. – J. Bone Miner Res., 22, 2007, 465-475. 5. E u r o p e a n Parliament Osteoporosis Interest Group and EU Osteoporosis Consultation Panel. Osteoporosis in Europe: Indicators of progress, 2004. 6. F l e u r e n c e , R. L., C. P. Iglesias et D. J. Torgerson. Economic evaluations of interventions for the prevention and treatment of osteoporosis: a structured review of the literature. – Osteoporos. Int., 17, 2006, 29-40. 7. h t t p ://portal.nap.bg/ospage?id=188 (Assessed March 2010). 8. h t t p ://www.mh.government.bg/Articles.aspx?lang=bgBG&pageid=384&categoryid=1355 (Assessed March 2010). 9. h t t p ://www.nchi.government.bg/statistika6.html (Assessed March 2010). 10. h t t p ://www.nhif.bg/web/guest/45 (Assessed March 2010). 11. h t t p ://www.nhif.bg/web/guest/58 (Assessed March 2010). 12. h t t p ://www.nsi.bg/otrasal.php?otr=19 (Assessed March 2010). 13. I v a n o v a , A. D. et G. I. Petrova. Hypertension and Common Complications — Analysis of the Ambulatory Treatment Cost. – Cent. Eur. J. Public. Health, 17, 2009, № 4, 223-230. 14. M e a d o w s , E. S. et al Cost-effectiveness of preventative therapies for postmenopausal women with osteopenia, BMC Women’s Health, 2007, 7, 6. 15. M e l t o n , L. J. et al. Fractures attributable to osteoporosis: report from the National Osteoporosis Foundation. – J. Bone Miner. Res., 12, 1997, 16-23. 38 Pharmacotherapy costs of osteoporosis... 16. M e l t o n , L. J. et al. How many women have osteoporosis? – J. Bone Miner. Res., 7, 1992, 1005-1010. 17. N a t i o n a l Institute for Health and Clinical Excellence (NICE). TA161 Osteoporosis—secondary prevention including strontium ranelate: guidance. 29 October 2008a. Available at: http://guidance. nice.org.uk/TA161/Guidance/pdf/English Accessed 5 October 2009. 18. O b e r e n d e r , P. et J. Zerth. The search for good compliance: economic aspects of a conveyed combination pharmaco-therapy, exemplified by an osteoporosis therapy. – Eur. J. Health Econ., 9, 2008, 127-136. 19. S t e i n , K. V. et al.. Ökonomische Konzepte zur Erfassung der Krankheitskosten von Osteoporose: Österreich im internationalen Vergleich. – Wien Med. Wochenschr, 159, 2009, № 9-10, 253-261. Address for correspondence: A. Savova Faculty of Pharmacy, Department of Social Pharmacy and Pharmacoeconomics Medical University 2 Dunav Str. 1000 Sofia, Bulgaria e-mail: [email protected] Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 39 EVALUATION OF MOLECULAR-CYTOGENETIC ABERRATIONS AND OVERALL SURVIVAL IN MYELOID ANTIGEN POSITIVE ADULT ACUTE LYMPHOBLASTIC LEUKEMIA M. Velizarova1, D. Popova2, E. Hadjiev3, N. Dimitrova4, I. Dimova5, D. Toncheva5 and K. Tzatchev1 1 Department of Clinical Laboratory and Clinical Immunology, Alexandrovska University Hospital 2 Department of Clinical Laboratory and Immunology, Military Mediacal Academy 3 Clinic of Hematology, Alexandrovska University Hospital 4 National Oncological Hospital, Bulgarian National Cancer Registry 5 Departments of Medical Genetics, Medical University – Sofia Summary. Leukemic cells from a significant number of adults with acute lymphoblastic leukemia (ALL) show aberrant co-expression of myeloid-associated markers. We studied the incidence and relations of myeloid-antigen (MyAg) expression to molecular-cytogenetic features of ALL and to outcome. Leukemic blasts from 33 newly diagnosed adults with untreated ALL were examined for myeloid surface antigen expression. The simultaneous expression of lymphoidassociated antigens and myeloid-associated antigen (CD33, CD13, CD15) on leukemic cells was detected by a standard two-color direct immunofluorescent assay. As a result, MyAg(+) ALL was established in 45% of the B-cell lineage. Immunologic subtyping of B-ALLs revealed an association between common B phenotype and coexpression of myeloid antigens – 53% of MyAg(+)ALL (P < 0.05). Various cytogenetic abnormalities, associated with MyAg(+) ALLs, were detected, including t(9;22), 11q23 abnormalities, del 4p, and del 12p. No differences in complete remission rate (p = 0.51) and overall survival (p = 0.75) were observed between MyAg(+) and MyAg(-) patients. In conclusion, a high incidence of poor prognostic chromosomal aberrations was recorded in MyAg (+) cases, but the aberrant myeloid antigen expression was not shown to have an impact on the outcome of B-ALL. Key words: cytogenetic aberrations, myeloid antigens, adult ALL, survival 40 Evaluation of molecular-cytogenetic... I INTRODUCTION mmunologic characteristics of adult acute lymphoblastic leukemia (ALL) show considerable differences in terms of presentation, molecular-cytogenetic patterns and clinical outcomes. After recent developments in molecular genetics and immunophenotyping of ALL, various subtypes were defined with different prognostic significance [1, 2, 3, 6, 7]. Aberrant co-expression of myeloid-associated markers on lymphoblasts is a well-known phenomenon and in ALL it has been reported in 10-47% of cases [1, 2, 4]. The prognostic value of aberrant myeloid antigens MyAg (+) expression is still controversial. Although the few early adult ALL studies had shown an inferior outcome for myeloid antigen positive patients [4], the recent data demonstrated no prognostic correlation using high-dose chemotherapies [2, 3, 13,14, 18]. The aim of this study was to correlate MyAg expression with clinical, hematologic and biological parameters, and to analyze their impact on the treatment response and prognosis in a small series of adult ALL. PATIENTS AND METHODS Patients The present study included 33 patients with de novo adult acute lymphoblastic leukemia (ALL) for whom a complete set of clinical, immunophenotypic and molecular-cytogenetic information was available. The patients were treated according to the GET-LALA-94 (Groupe d‘Etude et de Traitement de la Leucémie Aiguë Lymphoblastique de l’Adulte) [17] ALL protocol for adult. Diagnosis of ALL was based on the French-American-British (FAB) classification system’s morphological and cytochemical criteria, and on lymphoid immunophenotype. A complete remission (CR) was defined as 5% or less blast cells in normocellular or hypercellular bone marrow, a normal peripheral and differential blood count and exclusion of extramedullary disease. Remission time was defined as time from diagnosis to remission. A resistant disease (RD) was accepted if CR was not achieved after three courses of induction therapy. Overall survival was measured from the time of treatment study to the time of death. Immunophenotyping For immunophenotyping, leukemic cells obtained from fresh bone marrow or peripheral blood samples, collected in EDTA-containing tubes, were analyzed. Surface, cytoplasmic, and nuclear antigens were detected via a standard 2-color direct immunofluorescence assay using a broad panel of commercially available lymphoid and myeloid-associated monoclonal anti- Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 41 bodies (MoAbs). According to the European Group for Immunophenotyping of Leukemia (EGIL) [1], the stage of B-lineage acute leukemias was defined as follows: pro-B-ALL (BI): CD19+ / CD22+ / cyCD79a+ / CD10- / cyIg- / sIg-; common B-ALL (BII): CD10+ (CALLA+) / cyIg- / sIg-; pre-B-ALL (BIII): CD10+/- / cyIg+ / sIg-; mature B-ALL (BIV): sIg+. T-lineage ALL was characterized based on CD1a, CD2, CD3, CD4, CD5, CD7 and CD8 cell marker expression. Lineage assignment was based on the positivity of at least two lineage-specific antigens. Myeloid markers (CD13, CD14, CD15, and CD33) were also tested. Markers were considered positive when present on more than 20% of the blast cells. Cytogenetics and fluorescent in situ hybridization (FISH) Metaphases from short-term (24 hours) and long-term (48 hours) bone marrow cultures were prepared according to standard methods. G-banded chromosomes were classified following the International System for Human Cytogenetic Nomenclature [10]. A minimum of twenty G-banded metaphases were required to consider the case evaluable. FISH analysis was performed on cytogenetic preparations obtained from bone marrow cells. Direct labeling locus-specific probes (Vysis, Ltd.) were used for MLL gene rearrangements, bcr/abl gene fusion, and C-MYC rearrangements. The size of genetically abnormal clones was determined after analyzing at least 100 successfully hybridized cells. Statistical analysis Statistical analysis was performed taking into account gender, age, white blood cell count, hemoglobin level, platelet count, presence or absence of CD13, CD33 and CD15 antigens, cytogenetic and FISH data. Three- and 5-year survival was estimated using the life table’s method. Kaplan-Meier [12] curves were constructed for CR time and survival; a Log rank test was used to compare these curves in both cytogenetic groups. Comparison of quantitative variables between patient groups was performed using one-way analysis of variance. Comparison of qualitative data was performed using the chi-square test and t-test. All statistical analyses were 2-sided. P values < 0.05 were considered statistically significant. RESULTS Clinical features The clinical and biological characteristics of the 33 patients at presentation are shown in Table 1. 42 Evaluation of molecular-cytogenetic... Table 1. Presenting biological and laboratory features of adult ALL patients, according to myeloid antigen expression Parameters All cases MyAg(+) Total number (n /%) 33/100 15/45 18/55 0.67 Gender-M/F 19/14 13/2 6/12 0.002 Age-years median (range) 40.8 (18-74) 39..5 (18.69) 42 (19-74) 0.78 WBC x109/l median (range) 16.5 (1.7-300) 42.2 (1,7-300) 21.6 (1.8-90) 0.009 Hb g/l median (range) 91.1 (49-161) 93.7 (49-161) 88.4 (58-149) 1 PLT x10 /l median (range) 81 (7-214) 86 (7-196) 76 (5-214) 0.43 Bone marrow blasts median (range) 82 (30-100) 86 (34-100) 78 (30-100) 0.46 Immunophenotype B-lineage/T-lineage 28/5 15/0 13/5 0.03 Extramedulary involvement (n / %) 8/24.2 6/40 2/11 0.002 CR rate (n/%) 22/66.7 12/80 10/55.6 0.51 Resistant Disease (n / %) 14/42.2 7/46.7 7/39 0.38 Time to remission (median, months) 2.3 2.3 1.9 0.75 3-years OS (%) 45.2 26.7 18.5 0.57 5-years OS (%) 38.5 20.0 18.5 0.83 Overall survival (median, months) 12.33 11.1 12.4 0.75 9 MyAg (-) P-value Immunophenotyping A total of 5 cases (15.2%) were diagnosed as pro-B ALL, 17 (52%) were common ALL, 2 (6.0 %) were pre-B ALL, 4 (12.0%) were mature-B ALL and 5 (15.2%) were T-ALL. Immunologic subtyping of B-ALLs revealed an association between common B phenotype and coexpression of myeloid antigens − 53% of MyAg(+) ALL (P < 0.05). In 15 cases (45%), myeloid antigens (CD13, CD15 and CD33) were expressed. B-ALL expressing MyAg were found in all stages of cell maturation. Cytogenetic and molecular analysis A wide variety of cytogenetic abnormalities were observed in the patients with myeloid-antigen-positive ALL (Tabl. 2). Non-random and random cytogenetic aberrations were found in 11 of 15 (73.3%) of MyAg(+) ALL and in 6 of 18 (33.3%) of MyAg(-) ALL. Cytogenetic abnormalities that have been associated with MyAg(+) ALLs were common, including hyperdiploidy, t(9;22)/bcr-abl, 11q23/MLL abnormalities, t(8;14)/C-MYC, del 4p, and del 12p. All cases with complex karyotype (more than 5 aberrations) expressed aberrant myeloid antigens. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 43 Table 2. Cytogenetic and molecular aberrations in adult ALL patients All cases N (%) MyAg(+)ALL N (%) MyAg (-) ALL N (%) P-value Abnormalities of cell ploidy Normal Diploid 16 (48.5) 4 (26.7) 12 (66.7) 0.02 Pseudodiploid (46, abnormal) 13 (49.4) 7 (46.7) 6 (33.3) 0.2 Hyperdiploid > 46 4 (12.1) 4 (26.7) 0 0.02 Non-random 12 (36.3) 8 (53.3) 4 (22.2) 0.002 Random 5 (15..2) 3 (20.0) 2 (11.1) 0.14 Normal diploidy t(9;22)/bcr-abl 16 (48.5) 5 (15.2) 4 (26.7) 4 (26.7) 12 (66.7) 1(5.5) 0.02 0.001 t(11q23)/MLL 2 (6.1) 1(6.7) 1(5.5) t(8q24)/C-MYC 4 (12.1) 3 (20.0) 1 (5.5) t (1;19)/E2A-PBX1 1(3.0) 1 (6.7) 0 complex karyotype 2 (6.1) 2 (11.1) 0 Structural abnormalities Differences in CR rates and overall survival CR was estimated in 22 of 33 ALL cases (66.7%) without significant difference between MyAg (+) and MyAg (-) patients (Tabl. 1). The estimated 3- and 5-years survival for patients with ALL was 18.5 % for those without myeloid-antigen expression and 26.7% and 20% respectively for those with myeloid-antigen expression (p = 0.57 for 3-years survival, p = 0.83 for 5-years survival). Overall survival showed minimal differences without clinical and statistical significance (fig. 1) − 11.1 months for MyAg(+) vs. 12.4 months for MyAg (-) cases, p = 0.75 (Tabl. 1, fig. 1). S urvival F unc tions 1,0 Cum Survival 0,8 0,6 0,4 MyAg (+) ALL 0,2 MyAg (-) ALL 0,0 0,00 20,00 40,00 60,00 80,00 Months Fig. 1. Kaplan-Meier analysis of overall survival (months) in adult ALL patients with and without MyAg expression: MyAg(+) – 11,1months (95% CI: 8.39-13,86) and MyAg(-) – 12.4 months (95% CI: 3.59 – 21.27). 44 Evaluation of molecular-cytogenetic... DISCUSSION This study compared the biology and laboratory characteristics, molecularcytogenetic data, the achieved complete remission rate and overall survival of 33 adults with newly diagnosed acute lymphoblastic leukemia according to myeloid antigen expression on leukemic cells. The overall incidence of MyAg expression in our study (45%) is in line with the data reported in the literature [1, 2, 13]. The presence of MyAg was correlated with a number of clinical and biological data. The MyAg(+) cases were significantly more frequent in males than in females (87% vs. 33%, p = 0.002). MyAg (+) ALL is characterized with higher values of white blood cells (42.2 x 109/l vs. 21.6 x 109/l, p = 0.009) and higher incidence of extramedullary involvement (40% vs. 11%, p = 0.002), compared to MyAg (-) B-ALL. For immunophenotypic data, there was a significant difference in the incidence of aberrant MyAg expression between B-lineage ALL and T-ALL subgroups (p = 0.03). In this study we found a high incidence of structural chromosomal aberrations (73.3% vs. 33.3% in MyAg(-) cases, p = 0.02) and a lot of them were with poor prognostic significance- t(9;22)/bcr-abl, t(8q14)/C-MYC, t(1;19)/E2A-PBX1 and complex karyotype. Hyperdiploidy was found in 4 of 15 MyAg (+) ALL, all with structural changes. Leukemic blasts in all t(9;22)/bcr-abl (+) cases expressed Blineage specific markers and aberrant myeloid markers in 4 of 5 cases. According to some authors [13, 15, 16] the frequency of myeloid antigen expression in bcr-abl (+) cases is higher but further studies will be required to determine the association of specific karyotype abnormalities with aberrant myeloid expressions. A lot of published data demonstrated no prognostic correlation of aberrant myeloid expression using high-dose chemotherapies [2, 3, 11, 13, 14, 18]. In our study the presence of aberrant MyAg did not affect the achievement of CR and overall survival; no differences were found between MyAg(+) and MyAg(–) cases in OS at 3 and 5 years. The median time to achieve remission shows no significant difference for both groups − 2.3 months for MyAg(+) and 1.9 months for MyAg(–) patients (p = 0.75). No statistical differences in RD were recorded, but the percentage of resistant patients was higher in MyAg(+) ALL compared to MyAg(–) (46.7% vs. 39%, respectively). Even though the myeloid-lineage antigen expression is associated with a high incidence of non-random genetic abnormalities, it lacks a prognostic value in adult ALL. This has clinical implications in terms of therapeutic decisions [8] (e.g. antiCD33 treatment), but the expression of MyAg per se is not related to short or longterm prognostic significance. REFERENCES 1. B e n e , M. C. et al. Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL). – Leukemia, 9, 1995, № 10, 1783-1786. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 45 2. B h u s h a n , B. et al. Aberrant phenotypes in childhood and adult acute leukemia and its association with adverse prognostic factors and clinical outcome. – Clin. Exp. Med., 10, 2010, № 1, 33-40. 3. C z u c z m a n , M. S. et al. Value of immunophenotype in intensively treated adult acute lymphoblastic leukemia: cancer and leukemia Group B study 8364. – Blood, 1, 1999, № 93, 3931-3939. 4. D r e x l e r , H. G., E. Thiel et W. D. Ludwig. Review of the incidence and clinical relevance of myeloid antigen- positive acute lymphoblastic leukemia. – Leukemia, 5, 1991, № 8, 637-645. 5. F i e r e , D. Myeloid surface antigen expression in adult acute lymphoblastic leukemia. – Leukemia, 4, 1990, № 9, 664-666. 6. H o e l z e r , D. et N. Gokbuget. Recent approaches in acute lymphoblastic leukemia in adults. – Crit. Rev. Oncol./Hematol., 36, 2000, № 1, 49-58. 7. H o e l z e r , D et al. Acute lymphatic leukemia in the adult. Diagnosis, risk groups and therapy. – Internist (Berl), 43, 2002, № 10, 1212-1216. 8. H o e l z e r , D. Novel antibody-based therapies for acute lymphoblastic leukemia. – Hematology Am. Soc. Hematol. Educ. Program, 2011, 2011, 243-249. 9. H u r , M. et al. Immunophenotypic and cytogenetic changes in acute leukaemia at relapse. – Clin. Lab. Haematol., 23, 2001, № 3, 173-179. 10. I S C N : An international system for human cytogenetic nomenclature. Shaffer LG, Tommerup N, eds. S Karger, Basel 2005. 11. Y e n e r e l , M. et al. Myeloid antigen expression provides favorable outcome in patients with adult acute lymphoblastic leukemia: a single-center study. – Ann. Hematol., 81, 2002, № 9, 498-503. 12. K a p l a n , E. et P. Meier. Nonparametric estimation from incomplete observations. – J. Am. Stat. Assoc., 53, 1958, № 282, 457-481. 13. P u i , C. H. et al. Reappraisal of the clinical and biological significance of myeloid-associated antigen expression in childhood acute lymphoblastic leukemia. – J. Clin. Oncol., 16, 1998, № 12, 3768-3773. 14. P u t t i , M. C. et al. Expression of myeloid markers lacks prognostic impact in children treated for acute lymphoblastic leukemia: Italian experience in AIEOP-ALL 88-91 studies. – Blood, 92, 1998, № 3, 795-801. 15. Ta b e r n e r o , M. D. et al. Adult precursor B-ALL with BCR/ABL gene rearrangements displays a unique immunophenotype based on the pattern of CD10, CD34, CD13, and CD38 expresssion. – Leukemia, 15, 2001, № 3, 406-14. 16. T h o m a s , D. A. Philadelphia Chromosome-Positive Acute Lymphocytic Leukemia: A New Era of Challenges. – Hematology, 2007, 2007, № 1 ,435-443. 17. T h o m a s , X. et al. Outcome of treatment in adults with acute lymphoblastic leukemia: analysis of the LALA-94 trial. – J. Clin. Oncol., 22, 2004, № 20, 4075-4086. 18. V i t a l e , A. et al. Absence of prognostic impact of CD13 and/or CD33 antigen expression in adult acute lymphoblastic leukemia.Results of the GIMEMA ALL 0496 trial. – Haematologica, 92, 2007, № 03, 342-348. 46 Address for correspondence: Dr Milena Velizarova, PhD Department of Clinical Laboratory and Clinical Immunology University Alexander’s Hospital 1 G. Sofiiski str. 1431 Sofia, Bulgaria +359 2 92 30 916 e-mail: [email protected] Evaluation of molecular-cytogenetic... ADAPTED SURGICAL THORACOSCOPIC HELLER’S MYOTOMY IN THE TREATMENT OF ACHALASIA St. Sopotensky and Al. Cervenjakov University hospital “N. I. Pirogov”, 1st Surgery Clinic Summary. The aim of this study is to describe our technique of video-assisted thoracoscopy without fundoplication for surgical treatment of achalasia. The method is developed by surgeons from University hospital “N.I. Pirogov” and applied in 30 patients with achalasia during the years 2002-2010. It is a video-assisted thoracoscopic method applicable for benign processes of the cardia, involving the use of fibrin glue. The technique comprises two main stages: video-assisted thoracotomy (VATS) – cardiomyotomy and application of fibrin glue “TISSUCOL”. The first characteristic of the thoracoscopic approach is that it is not very common for non – invasive techniques. An advantage of its application is that it is associated with improved recovery period and lack of any symptoms of recurrence. We did not have surgical mortality. The frequency of the postoperative heartburn was significantly lower than that in the case of an open procedure and could be managed by medicines relatively easily, which allows the avoidance of the antireflux surgical procedure. This is an advantage in such cases in which preliminary therapeutic approaches had been applied. The third specificity is the application of the fibrin glue as a sealant. To our knowledge this is the first technique in which the glue is applied during the thoracoscopic myotomy. The adapted surgical thoracoscopic Heller’s myotomy in the treatment of achalasia, elaborated by our team, is a safe and efficacious method for surgical treatment of patients with achalasia. Key words: Achalasia, adapted Hellers’ myotomy, VATS, thoracoscopic surgery A INTRODUCTION chalasia was first recognized 300 years ago and was called cardiospasm at the beginning because of the functional obstruction of the esophageal sphincter. Thomas Willis described the first successfully treated case in 1674 by rigid dilatation with a sponged-tipped whalebone. This approach remained successful for nearly 25 years [1]. Lendrum proposed the contemporary concept about this disease in 1937, as a syndrome involving the failure Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 47 of relaxation of the lower esophageal sphincter (LES) upon swallowing and the loss of esophageal peristalsis [2]. Achalasia is a rare disease with an incidence of 0.6-1 per 100,000 people that affects patients mostly at 20-40 years of age [3]. Surgical treatment of achalasia was introduced by Ernest Heller in 1913, consisting of anterior and posterior myotomy across the LES performed via thoracotomy [4]. Recently, the laparoscopic technique has become the contemporary surgical approach to this disease, achieving excellent results [5, 6]. Video-assisted thoracoscopy was first reported from Pelligrini et al. in 1992 [7]. It was recognized as a safe and efficacious method used by many surgeons [8, 9, 10]. Minimally – invasive surgical methods, both laparscopic and thoracoscopic, are permanently developing with the aim to improve the techniques and to decrease the risk for patients. The aim of this study is to describe our technique of video-assisted thoracoscopy without fundoplication for surgical treatment of achalasia. METHOD The method is developed by surgeons from the University hospital “N. I. Pirogov” and applied in 30 patients with achalasia during the years 2002-2010. It is a video-assisted thoracoscopic method applicable for benign processes of the cardia, involving the use of fibrin glue. The technique comprises of two main stages – video-assisted thoracotomy (VATS) – cardiomyotomy and application of fibrin glue “TISSUCOL”. RESULTS The team consists of a surgeon, assistant, anesthesiologist, anesthesiological nurse and a surgical nurse. In the beginning the patients are led in general endotracheal anesthesia with the use of a double lumen tube. The method aims at disuniting the muscles of the LES. The patient is being placed in the right lateral position with the left hand up (Fig. 1). The position of the thoracoports is shown in Fig. 1. Fig. 1. Ports placement during the surgical procedure 1 port − 10 mm posterior ancillary line in 4th-5th intercostal space for optics. 2nd port − 10 mm anterior ancillary line in 6th intercostal space for working instruments. 3rd port − 5 mm anterior ancillary line in 7th - 8th intercostal space for working instruments 4th port − 5 mm following the anterior ancillary line for retractore. st 48 Adapted surgical thoracoscopic Heller's.... Firstly, the camera port is placed in the collapsed lung and next, under the visual control of the camera, the other ports are placed. The lig. pulmonale is released with the support of a dissector and a grasper (Fig. 2). Fig. 2. Release of the lig. pulmonale After the cranial ecartation of the pulmonary parenchyma, the pleura, that is covering the distal part of the esophagus is dissected and the access to the esophagus is opened. The esophagus is mobilized with the support of a small gauze pellet. Through distal approach the lower legs of the diaphragm is accessed and a proximal space of 8 – 10 cm is released (Fig. 3). Fig. 3. Opening of the parietal pleura and mobilization of the esophagus Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 49 Fig. 4. Myotomy With the grasper the longitudinal muscles of the esophagus are caught and raised above the place of stenosis. At the place of the dilatated part of the esophagus the fibers are disunited thus reaching the circular fibers, which are also intercepted and disunited. The submucosa is reached and through the disunited muscle fibers the mucous membrane is prolapsed. Cardiomyotomy is accomplished through the left lateral surface of the esophagus (Fig. 4). The myotomy continues in a distal direction through the narrowed place, the mucous membrane is detached with the support of the gauze pellet in a blunt way, and upper musculature is disunited with the dissector and grasper. After reaching the stomach musculature the myotomy continues proximally 2-3 cm above the narrow section. Fig. 5. Access to the stomach by ecartiration of the diaphragm 50 Adapted surgical thoracoscopic Heller's.... Fig. 6. Fibrogastroendoscopic inspection of the myotomy Intraoperatively, a fibrogastroendoscopic inspection of the effectiveness of the myotomy passing through the stomach is carried out. A hydro-aero test is carried out via inprompt in the esophagus for eventual lesions of the mucous (Fig. 6). Completed myotomy is shown in Fig. 7. Fig. 7. Completed myotomy In the esophagus a nasogastric tube is placed for 24 to 48 hours. Cardiomyotomy is covered by 2 mm fibrin glue “Tissucol” (Fig. 9). At the 3rd port tube drainage Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 51 of 18-20 sheriers is set for 48 hours. The next thoracoports are sealed hermetically layer by layer. The application of the fibrin glue aims to protect the mucosa from prolapsing, stimulate haemostasis, and cover eventual microscopic lesions. Fig. 8. Application of the fibrin glue “TISSUCOL” After completing the surgery a radiography is carried out to control the result (Fig. 9). Fig. 9. Control radiography after the surgery 52 Adapted surgical thoracoscopic Heller's.... Thoracal drainage is active for 24 hours. Water intake is allowed at second day and soft food at 4th day of surgery. The procedure was performed on 30 patients with achalasia without complication during the surgery, and before the hospital discharge. All patients were prescribed proton pump inhibitors. The hospital length of stay was 5 days. DISCUSSION Our video-assisted minimally invasive thoracoscopic surgical method for benign diseases of the esophagus with fibrin glue application possess the following characteristics and advantages. As surgical approach it is an adapted Hellers’ myotomy in a non-invasive manner. The first characteristic is the thoracoscopic approach that is not very common for non-invasive techniques [6, 11, 12, 13]. It confirms the conclusions of other authors that the minimaly invasive techniques via thorax or abdomen are safer than the open procedures because they allow to control the surgery process and lead to a decrease in hospital stay, as well as faster recovery to normal life. A second advantage that was observed was the light recovery and lack of any recurrence symptoms. We did not have surgical mortality. The frequency of the postoperative heartburn is significantly lower than that in the case of open procedure and could be managed by medicines relatively easily, which allows the avoidance of an the antireflux surgical procedure. Some authors consider that the thoracoscopic method increases patients satisfaction and improves long term results [11]. The minimally invasive techniques are preferable due to the effective release of the esophagus, shorter hospital stay, and lower number of patients with post operative reflux. Our results are similar to these conclusions. The second specificity of out techniques is the patients orientation (right lateral), which allows working at the left side of the esophagus. This is an advantage in cases where preliminary therapeutic approaches have been applied. The third specificity is in the application of the fibrin glue. To our knowledge this is the first technique in which the glue in applied during the thoracoscopic myotomy. The application of fibrin glue allows protecting the mucous membrane without any other additional surgical manipulations. In the original instructions for use, the Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 53 glue must be applied via trickle, while we apply it under pressure. This approach allows a uniform distribution of the glue on the mucous surface. CONCLUSION The adapted surgical thoracoscopic Heller’s myotomy in the treatment of achalasia, elaborated by our team, is a safe and efficacious method for the surgical treatment of patients with achalasia. REFERENCES 1. C a d e , R. J. et C. J. Martin. Thoracoscopic cardiomyotomy for achalasia. – ANZ J. Surg., 66, 1996, 107-109. 2. C h a m p i o n , J. K. Nissa Delisle, Tracey Hunt Comparison of thoracoscopic and laproscopic esophagomyotomy with fundoplication for primary motility disorders. Eur. J. Cardio-thoracic Sur. 16 (Suppl. 1), 1999, S34-S36. 3. C o d i s p o t i , M. et al. Clinical results of thoracoscopic Heller’s myotomy in the treatment of achalasia. – Eur. J. Cardio-thoracic Sur., 24, 2003, 620-624. 4. H e l l e r , E. Extramukoze kardioplastic beim chronishen spasmus mit dilatation des oesophagus. – Mitt. Grenzeb. Med. Chir., 27, 1913, 141-149. 5. L e e , J. M., C. H. Wang, P. M. Huang et al. Enduring effects of thoracoscopic Heller Myotomy for treating achalasia. – World J. Surg., 28, 2004, 55-58. 6. L e n d r u m , F. C. Anatomic features of the cardiac orifice of the stomach with special reference to cardiospasm. – Arch. Intern. Med., 59, 1937, 474-511. 7. M a y b e r r y , J. F. et J. Rhodes. Achalasia in the city of Cardiff from 1926 to 1977. – Digestion, 20, 1980, 248-252. 8. P a t t i , M. G. et al. Comparison of medical and minimally invasive surgical therapy for primary esophageal motility disorder. – Arch. Surg., 130, 1995, 609-616. 9. P e l l e g r i n i , C. et al. Thoracoscopic esophagomyotomy. Initial experience wiшh a new approach for treatment of achalasia. – Ann. Surg., 216, 1992, 291-296. 10. R a i s e r , F. et al. Heller myotomy via minimal access surgery: an evaluation of anti-reflux procedures. – Arch. Surg., 131, 1996, 593-598. 11. S c o t t , H. J. et R. D. Rosin. Thoracoscopic laser Heller's myotomy. J. Royal Soc. Med., 87, 1994. 12. Ta t u m , R. P. et C. A. Pellegrini. How do I do it: Laparoscopic Heller Myotomy with Toupet Fundoplication for Achalasia. – J. Gastrointest. Surg., 13, 2009, 1120-1124. 13. W i l l i s , T. Pharmaceutice Rationalis Sive Diatribe de Medicamentorum Operationibus in Human Corpore. London, England: Hagae Comitis, 1674. 54 Address for correspondence: St. Sopotensky First Surgery Clinic University hospital "N. I. Pirogov" 21 Totleben blvd. 1606 Sofia e-mail: [email protected] Adapted surgical thoracoscopic Heller's.... DIFFERENT LOW LEVELS OF AIR POLLUTION AND RESPIRATORY FUNCTIONS: AN 8-YEAR NATURAL EXPERIMENT Т. Тurnovska1, St. Kostianev2, B. Мarinov2 and St. Mandadzhieva2 2 1 Department of Hygiene, Ecology & Epidemiology, Department of Pathological Physiology, Medical University – Plovdiv, Bulgaria Summary. The objective of this study was to perform a full functional examination of the external breathing of two cohorts healthy children, living in the same areas, but under the influence of different low levels of air pollution. The following methods were used: First cohort (I) 27 boys at age 10.47 ± 0.49 y and 38 girls at age 10.29 ± 0.46 y were examined in years 1996, 1997, 1998, 1999; Second cohort (II) – 34 boys at age 10.47 ± 0.51 y and 27 girls at age 10.37 ± 0.49 y had lived in conditions of lower level of pollution; they had visited the same schools as the children of the First cohort and were examined in years 2001, 2002, 2003 and 2004. All 8 series of the study were conducted in the Pulmonary Function Testing Laboratory of the Medical University of Plovdiv. The results obtained in the present investigation suggest that the average levels of respiratory indices have significantly lower average values in the first cohort compared to the second one: VC%pred (Vital capacity as a % of predicted value): 94.99 (I) vs. 99.88% (II), P = 0.000; FVC%pred (Forced vital capacity as a % of predicted value): 92.09 (I) vs. 100.94% (II), P = 0.000; FEV1%pred (Forced expiratory volume per 1 s as a % of predicted value): 100.40 (I) vs. 109.09 (II), P = 0.000, etc. The following conclusions were made: 1. The restrictive effect of air pollution on respiratory functions has been established, even when it is below the admissible levels. 2. The pubertal stimulus in development contributes to the compensation of the lower lung function parameters from early childhood, but some unfavorable tendencies continue to persist. Key words: children, low levels of air pollution, pulmonary functions Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 55 T INTRODUCTION he epidemiological investigations carried out during the last years show unfavorable health effects caused by the influence of atmospheric pollutants even when they are in concentrations about or below the operative national standards and the levels recommended by the World Health Organization [1, 2, 3, 6, 12, 22, 30]. The spectrum of biological reaction in these cases includes different elements depending on the level of exposure, environmental conditions, physiological peculiarities of the concerned contingent etc., which requires the application of different evaluation indices. One of the most sensitive among them is the analysis of respiratory functions, [17, 19] especially in children and adolescents. Investigating the influence of low-degree atmospheric pollution in a prospective cohort study of 1001 children from two regions of Krakow, Jedrychowski et al. [28] found a slower increase of respiratory functions in children from the more polluted region. Gauderman et al. [24] carried out a prospective study of 1759 tenyear old children from 12 municipalities in South California, including annual examination of the respiratory indices for a period of eight years. After they eliminated the influence of some obscuring and modifying factors, the authors found a deficit in the increase of the Forced expiratory volume in 1 sec (FEV1) depending on the exposure to nitrogen dioxide, acid aerosols, dust with diameter of particles 2.5 μm and simple carbon. Something more, the retardation in the increase of respiratory functions was recorded until the age of 18. This gives a reason for the authors to conclude that the contemporary, relatively low levels of atmospheric pollution have a chronic unfavorable effect on lung development in children, resulting in a statistically significant deficit in FEV1, persisting up to the age of 18. During the years of the transition of Bulgaria to market economy (after year 1989), many industrial manufactures, sources of massive atmospheric pollution, were closed down, others significantly decreased the volume of their output. As a result of the recession in economy and the drop in industrial manufacture, accompanied by diminishing electric power consumption, the ecological situation in the most industrialized and polluted regions of the country ameliorated [11]. Taking advantage of the unique opportunity to conduct an epidemiological experiment under natural conditions we aimed at performing a full functional study of the external breathing of two cohorts healthy children, living in the same areas, but under the influence of different low levels of air pollution. MATERIAL AND METHODS Study design Town, air pollution and subjects As the appropriate settlement was chosen the town of Dimitrovgrad (54,000 people before crisis and 41,000 in 2007 y). It was one of the most industrialized 56 Different low levels of air pollution... and heavily polluted areas before the transition to market economy. After the beginning of the crisis in 1990, the production of phosphorus fertilizers, phosphorus and sulfuric acids, sodium silicofluoride, dimethylsulfate, phenylamine, heating energy, asbestos-cement products, etc. was fully discontinued and the volume of output of the other productions was greatly reduced. This led to an abrupt decrease in the air-pollutant levels, as we described it in our previous publication [26]. The air pollution in Dimitrovgrad was controlled at three permanent stations for manual tests as a part of the National Air Pollution Monitoring System during the investigated period, but First station stopped working after 1998. The following pollutants have been analyzed according to the Bulgarian standards: TSPM (total suspended particulate matter), SO2 (sulphur dioxide), NO2 (nitrogen dioxide), H2S (hydrogen sulphide), Pb (lead aerosols) and HF (hydrogen fluoride – up to 1993 (because of stopping the production of phosphorus fertilizers after 1990 and phosphorus acid production – after 1992, that was the main source of HF). Probes were taken according to the Uniform National System for Air Pollution Control in Bulgaria – during the first 10 days of every month, 4 times a day (on 08.00 h, 10.00 h, 14.00 h and 16.00 h) 30 minutes duration for gaseous pollutants and 8 hours non-stop for dust and lead aerosols (since 08.00 h to 16.00 h). The mean year values of the pollutants in the city as a whole are calculated as arithmetic means of three control stations’ data. In the year 1994, an additional automated control station post was included in the air pollution monitoring system, which station post was put into full operation in the year 1995, but because of different reasons during the period of crisis – with a number of omissions in the following years. For instance, with a requirement of a minimum of 90% registered data, the percentage of recorded data for some years varies between 20 and 45%; in the year 2000, only 211 samples were provided for particulate matter under 10 μm (PM10), of a realizable number of 35040. All this hinders, or makes the data processing and the calculation of correct average-annual concentrations for the separate indicators impossible and unreliable. For that reason, as well as to be able to fully juxtapose the data with similar records from the times prior to the period of crisis, the results used in the article are those from the station posts with manual sample-taking. For participation in the study 100 children (50 boys and 50 girls) for each of the two cohorts were invited. First cohort (I) at age 10,40 ± 0,55 y (X ± SD) y in the first cycle were examined in years 1996, 1997, 1998, 1999; Second cohort (II) at age 10,38 ± 0,50 in the first cycle had lived in conditions of lower level of pollution; they had visited the same schools as the children of the First cohort and were examined in years 2001, 2002, 2003, 2004. Due to different individual reasons the number of investigated children varied in the different cycles between 8 and 17%. Also, the children who had had respiratory disease during the last 12 months were excluded from the analysis. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 57 Equipment and methods All 8 series of the study were conducted at the Pulmonary Function Testing laboratory of Medical University of Plovdiv in the first ten days of the month of May in 1996-1999 and 2000-2004 respectively, by the same team. Complete anthropometric measurements were performed before the actual test procedures. Pulmonary function testing was carried out with a diagnostic system Masterscreen Diffusion™ (Jaeger E, Wuerzburg, Germany) in a seated position with a nose clip in the following order: 1. Slow spirometry; 2. Measurement of diffusion capacity; 3. Forced expiration; 4. Maximal inspiratory and expiratory pressures. Spirometric reference values for European children, Quanjer et al, previously validated for the Bulgarian population by Kostianev and Iluchev, were used [8, 10]. Diffusing capacity (transfer factor – TL,co) was calculated as a mean value of two Single-Breath measurements. TL,co values are given unadjusted for hemoglobin. Each child’s parents were asked to fill in one and the same questionnaire, requesting information on some indices characterizing domestic conditions and child’s health status that might be confounders: Domestic heating, Passive smoking, Mothers’ education (in years), Diseases in the past (Asthma included). Prior to the test procedures, a written informed consent was obtained from a parent or guardian and the associated risks and benefits were clarified. The procedures used in this study had been approved by the Institutional Ethics Committee at the Medical University of Plovdiv. The following statistical methods were used for processing the data: Descriptive analysis, Fisher’s exact test and χ2 (for a check on hypotheses about the existence of a connection among the class variables), non-parametric test of Shapiro-Wilk (for conducting a check on the type of distribution), non-parametric test of Kruskal-Wallis (for checking the hypotheses of the difference among several independent measurements), non-parametric test of Man-Whitney (for a check on hypotheses about the difference between two independent measurements), Stepwise multiple regression analysis. RESULTS The levels of systematically controlled air pollutants are presented in Table 1. The data shows that the average concentrations of all air pollutants, with the exception of that of H2S, are higher in the first four-year period compared to those in the second one. However, this is not a permanent situation and it is obviously due to the peak level of pollution in the year 2001. In order to substantiate it we calculated the arithmetic average value of the annual concentrations for the three years (2002, 2003 and 2004). It is 8 times less (0.0005 mg/m3) than the average one for the full 4-year period (0.004 mg/m3). Therefore, the average H2S pollution had a value many times below the limit value during 3/4 of the studied four-year period. The origin of the established peak pollution has not been specified by now. 58 Different low levels of air pollution... It may be possibly due to a broadside emission of pollutants, due to non-rhythm of production in the crisis period or to a transmission of an unknown source, etc. Table 1. Average levels of air pollutants Pollutants TSPM Pb SO2 NO2 H 2S NH3 Years N Mean SD SE 1996-1999 428 0.163 0.032 0.0016 2001-2004 512 0.126 0.007 0.0003 1996-1999 428 0.112 0.02 0.001 2001-2004 512 0.073 0.018 0.0008 1996-1999 428 47.928 6.604 0.3192 2001-2004 512 31.623 5.473 0.2419 1996-1999 428 16.28 2.289 0.1106 2001-2004 512 13.485 1.844 0.0815 1996-1999 428 0.002 0.001 0.0001 2001-2004 512 0.004 0.007 0.0003 1996-1999 428 0.053 0.012 0.0006 2001-2004 512 0.051 0.032 0.0014 P 0.000 0.000 0.000 0.000 0.000 0.159 Abbreviations: TSPM – Total suspended particulate matter; SO2 – Sulphur dioxide; NO2 – Nitrogen dioxide; H2S – Hydrogen sulphide; Pb – Lead aerosols Figures 1A and 1B present the height of the two contingents by sex as main anthropological characteristic significant for the level of respiratory functions. The analysis of the results shows there are no statistically significant differences in the average height between the two cohorts and during the 4 cycles of the study. Their average values correspond to Bulgarian standards (Regulation No 2/04.02.2003 of Health Ministry in Bulgaria). cm cm 165 160 155 150 145 140 135 130 165 160 155 150 145 140 135 130 2 cohort 1st cohort 1 2 3 4 Fig. 1A Height among boys during the 4 cycles of investigation Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 2nd cohort 1st cohort 1 2 3 4 Fig.1B Height among girls during the 4 cycles of investigation 59 The results of the mean levels of respiratory indices are presented in Table 2 and the Alveolar ventilation – in Fig. 2. Table 2. Mean levels of pulmonary function values Indices Cohorts N Mean SD 1 92 2.5 0.38 2 100 2.58 0.39 Sig. Mean VC (L) 1st cycle 2nd cycle 3rd cycle 4th cycle 2nd cycle 3rd cycle 4th cycle 1 95 2.85 0.41 2 93 2.85 0.47 1 101 3.2 0.47 2 99 3.11 0.55 1 87 3.55 0.5 2 93 3.52 0.62 1 92 2.34 0.36 2 100 2.52 0.39 1 2 66 92 2.69 2.79 0.38 0.47 0.178 0.94 0.248 0.715 2nd cycle 3rd cycle 4th cycle 1 99 3.09 0.45 2 1 98 87 3.01 3.4 0.54 0.5 2 93 3.45 0.63 1 92 2.16 0.32 2 100 2.3 0.34 0.002 0.122 0.231 0.546 2nd cycle 3rd cycle 4th cycle 60 9.3 99.88 9.61 100.01 11.17 102.26 9.83 101.83 11.66 103.3 10.26 105.35 11.14 104.39 11.99 0 0.142 0.345 0.574 92.09 9.93 100.94 11.51 95.67 100.62 9.28 10.25 97.37 11.87 96.7 99.26 11.07 11.47 99.93 12.96 0 0.002 0.681 0.715 FEV1%pred 1 66 2.49 0.36 2 92 2.54 0.42 1 100 2.83 0.44 2 98 2.74 0.48 1 87 3.11 0.48 2 93 3.15 0.56 1 92 4.38 0.93 2 100 4.73 0.85 1 66 5.21 0.93 2 92 6.33 0.45 0.004 0.381 0.181 0.621 PEF (L) 1st cycle 94.99 FVC%pred FEV1 (L) 1st cycle Sig. VC%pred FVC (L) 1st cycle SD 100.4 11.56 109.09 11.05 103.12 16.24 108.49 10.71 105.71 14.07 104.85 12.15 108.21 14.72 108.74 13.7 0 0.013 0.646 0.8 PEF%pred 1 99 5.27 1.24 2 98 5.57 1.02 1 2 87 93 6.48 6.5 1.23 1.08 0.007 0.34 0.203 0.926 89.15 17.8 98.03 14.97 97.23 16.6 101.53 12.63 106.31 20.31 106.38 13.43 103.48 103.97 20.02 14.39 0 0.067 0.072 0.851 Different low levels of air pollution... Indices Cohorts N Mean SD Sig. MEF50 (L) 1st cycle 2nd cycle 3rd cycle 4th cycle 2nd cycle 3rd cycle 4th cycle 1 92 2.86 0.7 2 100 3.06 0.72 1 66 3.28 0.75 2 92 3.37 0.72 1 100 3.69 0.93 2 98 3.53 0.76 1 87 3.87 1 2 93 4.04 0.87 0.051 0.426 0.205 0.239 2nd cycle 3rd cycle 4th cycle 1 90 1.25 0.47 2 100 1.47 0.48 1 66 1.44 0.56 2 92 1.56 0.53 1 100 1.78 0.65 2 98 1.7 0.57 1 86 1.83 0.65 2 93 2.03 0.66 0.001 0.173 0.404 0.036 2nd cycle 3rd cycle 4th cycle 92.33 21.05 100.78 20.37 96.03 21.94 102.3 18.16 100.54 24.02 98.41 18.6 99.78 25.08 104.41 20.42 0.005 0.052 0.485 0.175 77.95 27.77 93.72 27.67 83.21 31.12 92.3 27.49 93.9 33.07 91.67 28.24 91.71 30.46 101.4 29.78 0 0.055 0.611 0.034 TLCO%pred 1 90 5.66 0.8 2 99 5.52 1.07 1 66 6.54 0.92 2 92 6.43 1.34 1 100 7.52 1.19 2 98 7.63 1.37 1 2 87 93 9.25 9.14 1.7 1.36 0.332 0.525 0.575 0.516 TLC (L) 1st cycle Sig. MEF25%pred TL.co (mmol.min-1.kPa-1) 1st cycle SD MEF50%pred MEF25 (L) 1st cycle Mean 87.77 10.25 86.87 12.54 93.76 10.35 93.55 13.53 98.82 11.93 98.59 13.28 114.88 113.2 15.85 12.86 0.594 0.916 0.654 0.653 TLC%pred 1 92 3.04 0.36 2 99 3.44 0.47 1 66 3.7 0.51 2 92 3.77 0.58 1 100 4.14 0.56 2 98 4.15 0.64 1 87 4.54 0.64 2 93 4.51 0.69 Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 0 0.42 0.901 0.757 89.64 8.19 103.04 10.23 98.99 9.38 102.82 8.39 99.21 10.44 102.06 9.64 101.66 11.34 100.81 9.45 0 0.008 0.047 0.583 61 Indices Cohorts N Mean SD Sig. Mean RV (L) 1st cycle 2nd cycle 3rd cycle 4th cycle 2nd cycle 3rd cycle 4th cycle 1 82 0.61 0.15 2 99 0.88 0.24 1 66 0.85 0.23 2 92 0.93 0.28 1 100 1.01 0.25 2 98 1.05 0.27 1 87 1.09 0.27 2 93 1.09 0.34 1 91 1.63 0.45 2 97 1.97 0.55 1 65 1.95 0.46 2 92 2.14 0.64 1 100 2.16 0.47 2 98 2.27 0.57 1 87 2.4 0.5 2 93 2.47 0.67 0 0.085 0.403 0.934 2nd cycle 3rd cycle 4th cycle 82.32 21.19 120.69 33.49 103.04 25.53 115.73 31.77 111.29 25.56 118.33 28.54 111.96 25.59 112.76 30.59 0 0.006 0.069 0.849 FRC%pred 0 0.036 0.13 0.429 VCin (L) 1st cycle Sig. RV%pred FRC (L) 1st cycle SD 101.93 30.6 126.24 38.76 106.33 27.18 122.47 36.11 107.37 22.91 116.18 27.73 111.18 23.62 113.47 26.81 0 0.002 0.016 0.544 VCin%pred 1 92 2.44 0.37 2 99 2.54 0.4 1 66 2.85 0.42 2 92 2.85 0.49 1 100 3.12 0.52 2 98 3.11 0.54 1 87 3.45 0.55 2 93 3.42 0.58 0.065 0.986 0.896 0.697 93.26 9.71 99.63 11.23 99.18 10.83 100.58 9.7 96.89 12.78 98.73 11.61 99.82 13.01 98.36 12.44 0 0.395 0.29 0.445 Abbreviations: VS − Vital capacity, VC%pred − Vital capacity as a % of pedicted value; FVC − Forced vital capacity, FVC%pred − Forced vital capacity as a % of predicted value; FEV1 − Forced expiratory volume in 1 sec, FEV1%pred − Forced expiratory volume in 1 s as a % of predicted value; PEF − Peak expiratory flow, PEF%pred − Peak expiratory flow as a % of predicted value; MEF50 − Maximal expiratory flow at 50% of forced vital capacity, MEF50%pred − Maximal expiratory flow at 50% of forced vital capacity as a % of predicted value; MEF25 − Maximal expiratory flow at 25% of forced vital capacity, MEF25%pred − Maximal expiratory flow at 25% of forced vital capacity as a % of predicted value; TL,co − Diffusing capacity (transfer factor), TL,co%pred − Diffusing capacity as a % of predicted value; TLC − Total lung capacity, TLC%pred − Total lung capacity as a % of predicted value; RV − Residual volume, RV%pred − Residual volume as a % of predicted value; FRC − Functional residual capacity, FRC%pred − Functional residual capacityas as a % of predicted value; VCin − Vital capacity in inspiration, VCin%pred − Vital capacity in inspiration as a % of predicted value 62 Different low levels of air pollution... 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 2 cohort 1st cohort 1 P = 0,000 (1); NS (2); NS (3); NS (4) 2 3 4 Fig. 2. Alveolar ventilation The presented data show that the average levels of all indices correspond to the referential values. It is rather impressive that most indices have significantly lower average values in the first cohort compared to the second one. The best manifested differences are found out in the first 1-2 cycles of the research. In the 3rd and 4th cycle the values remain greater in the second cohort but without statistical reliability. An exception is only the Tiffneau index (FEV1/VC %), which is greater in the second cohort during all the four cycles of the study, as in the third one the difference is not significant – Fig. 3. % 90 89 88 87 86 2 cohort 85 1st cohort 84 1 2 3 4 P = 0,000 (1); 0,002 (2); NS (3); 0,015 (4) Fig. 3. Index of Tiffneau (FEV1/VC%) It is possible that other factors also have a negative influence on respiratory functions – passive smoking [4, 15], type of heating at home [7, 21, 23], presence of pets at home [20, 25], mothers’ and fathers’ education, which is one of the criteria Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 63 64 Constant B Passive Type of Mother’s Father’s Pets TSPM smoking heating education education -0.153 0.157 x x x x 0 0 Pb 4.354 VC x 0 Sig. VC%pred 115.101 x x x x x x x 0 Sig. 4.241 -0.136 0.143 FVC x x x x x 0 0.001 0 Sig. FVC%pred 109.152 x x x x x x x 0 Sig. 3.869 -0.08 0.146 FEV1 x x x x x 0 0.035 0 Sig. FEV1%pred 117.534 x x x x x x x 0 Sig. 94.267 -1.27 -0.485 Tiffneau x x x x x 0 0.004 0 Sig. 4.7 -0.148 0.196 MEF50 x x x x x 0 0.022 0 Sig. -1.642 MEF50%pred 115.662 x x x x x x 0 0.001 Sig. 8.989 -0.41 -0.256 PEF x x x x x 0 0.05 0.006 Sig. 2.287 -1.994 PEF%pred 118.444 x x x x x 0 0 0 Sig. 11.478 -0.376 0.822 -0.111 -0.105 Tlco x x x 0 0.001 0 0 0.019 Sig. -2.153 -5.558 -1.418 TL.co%pred 146.457 x x x x 0 0.029 0 0.002 Sig. 6.428 -0.113 0.201 -0.059 VA x x x x 0 0.021 0 0 Sig. 6.58 -0.122 0.21 -0.06 TLC x x x x 0 0.014 0 0 Sig. TLC%pred 111.181 x x x x x x x 0 Sig. 1.875 0.062 -0.032 RV x x x x x 0 0 0 Sig. -3.422 RV%pred 184.969 -4.666 -4.872 x x x x 0 0.048 0.026 0 Sig. 3.4 0.069 FRC x x x x x x 0 0 Sig. 120.291 4.1 FRC%pred x x x x x x 0 0 Sig. 3 3 Changes in lung functions are: 10 mg/m за TSPM, SO2, NO2, H2S и NH3, но 10 мg/m for Pb Indices NO2 x x x x -0.407 0 -0.416 0 -2.356 0 -0.125 x 0 -4.118 -15.605 0.021 0.023 -0.236 x 0 x -0.556 0 x x x x x x x x x x x x x H2S R2 0.047 0.018 -0.23 0.038 0 x -0.167 0.178 0 x 0.036 x x x x 9.941 0.106 0.04 0.157 29.702 0.541 28.018 0.263 0.565 0.41 0.211 0.555 0.407 -0.083 0.161 0 0.041 1.227 16.56 3.683 0.274 12.934 0.441 0.07 0.033 0.113 x 0.802 5.514 0 0 0 0 0 0 0 0 Sig. 0 0 0 0 0 0 0 0 0 0 0 0.017 21.229 0.001 0.171 0.044 0.12 x -0.04 0.216 0.432 11.178 0.465 0.034 13.257 0.358 x 0.048 0.379 0.049 x 0.47 0.066 10.618 0.365 Adjusted SEE R2 -0.141 0.363 0 -0.153 0.369 0 -0.969 0.068 0 -0.158 0.384 0 NH3 -24.535 x x 0.074 0.001 -1.59 -0.242 x 0.447 0.023 0 -30.361 -18.965 x 0.28 0 0.007 -0.16 x x 0.412 0 -0.165 x x 0.415 0 -14.827 x x 0.109 0.001 x -0.759 0.005 x -0.43 0 -0.384 -0.385 0 0.014 -2.061 x 0 -0.408 -0.495 0 0.002 -2.726 x 0 -0.365 -0.444 0 0.002 -2.738 x 0 SO2 Table 3. Influence of main additional factors on pulmonary function used for the assessment of socioeconomic status [29]. Dong et al. (2008) in a study of 10784 children summarize that the different factors of home environment have essential unfavorable influence on respiratory health of children [5]. In our study, the children from the two cohorts are statistically balanced according to sex, age, height, body mass and they live in almost equal geographic and weather conditions. Therefore we accept that the necessary pre-requisites have been fulfilled for correct investigation of the effect of air pollutants, passive smoking, type of heating at home, presence of pets, mothers’ and fathers’ education, which were included in the regression model. The results are presented in the Table 3. Different low levels of air pollution... They show that all respiratory indices are reliably dependant on the levels of atmospheric pollutants and the above extra factors, but to a different extent for certain indices. For example, the negative influence of nitrogen dioxide has a clear restrictive effect on almost all respiratory functions. It can be supplemented by the distinct influence of ammonia, nitrogen dioxide and lead aerosols without minimizing the influence of dust and hydrogen sulfide. Regression coefficients for the different parameters vary from -0.111, Sig.0.000 (TL,co – Diffusing capacity) to -5.558, Sig.0.000 (TL,co%pred – Diffusing capacity as a % of predicted value) for TSPM; from -0.125, Sig.0.000 (RV – Residual volume) to -4.118, Sig.0.021 (RV%pred – Residual volume as a % of predicted value) for SO2; from -0.385, Sig.0.014 (VC – Vital capacity) to -30.361, Sig.0.000 (TL,co%pred) for NO2, etc. The data from the analysis of obscuring factors show that passive smoking has leading significance among them for the level of respiratory functions. Second factor in significance ranks the parents’ educational level, especially the fathers’ education – the regression coefficients range from 0.062, Sig.0.000 (RV) to 4.100, Sig.0.000 (FRC%pred – Functional residual capacity as a % of predicted value); Pets – from -0.410, Sig.0.050 (PEF – Peak expiratory flow) to -2.153, Sig.0.029 and Type of heating – from -0.148, Sig.0.022 (MEF50 – Maximal expiratory flow at 50% of forced vital capacity) to -4.666, Sig.0.026 (RV%pred). The influence of the polluting type of fuel and growing up of pets is manifested on comparatively few indices in our study, but obviously it cannot be neglected. In some literary sources it is pointed out that boys are the more sensitive sex to environmental factors, including atmospheric pollution as well. Jedrychowski et al [28] for instance found that the average levels of respiratory functions are reliably lower in boys living in the polluted region compared to controls, while for the girls the result is not reliable. On the contrary, Oftedal et al. [14] found that the early and continuous exposure to atmospheric pollution with dust and nitrogen dioxide is related to reduction of PEF and FVC (Forced vital capacity) for girls particularly. We did not find statistically significant differences for any of the respiratory indices according to sex: boys First cohort vs. boys Second cohort and girls First cohort vs. girls Second cohort. Hibbert et al. [18] emphasize that the rate of increase in respiratory functions for boys and girls is different in the different age periods, but in our case the groups we examined were statistically balanced in this respect. DISCUSSION In this study we find that low-degree atmospheric pollution has a clearly expressed restrictive effect on respiratory functions of 10-year old students. This effect gradually decreases in the consequent 3 years but its influence on the Tiffneau index persists during the whole period (10-14 years old). These data support the conclusions of Gauderman еt al., Wang et al., etc. for the unActa Medica Bulgarica, Vol. XXXIX, 2012, № 1 65 favorable effect of low-degree atmospheric pollution on the level of respiratory functions [9, 24]. The great advantage of our study is that the two contingents which we investigated are balanced not only by sex and age but also they live in absolutely identical geographic and weather conditions, they visit one and the same schools, the qualities of drinking water do not differ, etc. Thus, in our opinion, the influence of different levels of low-degree atmospheric pollution is more clearly manifested. As a disadvantage we record the lack of special study on the nutrition of the investigated children but we have no reasons to believe there are essential differences between the investigated groups with regard to nutrition, unhealthy habits, living conditions, etc., since they are homogenous in ethnics and race. The relative proportions of the children from both cohorts, living in conditions of passive smoking, for example, do not vary reliably (45.89% – First cohort vs. 54.11%, NS). Special attention was given to heating. The use of solid, liquid or gaseous fuels in the children of the second cohort has a reliably higher relative proportion than that in the first one, 37.96% vs. 24.72, P = 0.0479. It is important to note that despite the use of fuels polluting the environment, the children from the second cohort display higher functional levels of external breathing. In our opinion it means that the polluting type of heating at home, though in general it plays the role of aggravating factor, has a secondary significance in the particular case. The atmospheric pollution in the town environment, even when low-degree, has a primary significance for the restrictive effect of respiratory functions in children. The results from our study do not give us reasons to specify a particular pollutant as a cause for the more unfavorable functional indices of the children from the first group. It is obvious that the degree of effect of particular pollutants on respiratory indices is different which supports the conclusion of Schindler et al. [9] that “dust effect cannot be separated from that of nitrogen dioxide and the rest pollutants”. No matter the degree of participation of pollutants, it is important that all indices, excluding the Tiffneau index, balance up to the 4th cycle of the study. Probably, the intensive growing up of children in pre-puberty and puberty age contributes to a great extent to compensation for the lower levels of respiratory functions in the first group of children. It is probably due to the continuous lung development – more than 80 percent of the alveoli arise postnatally [13, 16]. The results obtained by us convincingly support the conclusions of Avol et al. [27] based on a 10-year study according to which the changes of atmospheric pollution in adolescents has an essential significance for the increase of their respiratory functions. For example, when children move to municipalities with different degree of atmospheric pollution, it reflects on their respiratory functions even in the end of the respective year. The children who had moved to municipalities of lower atmospheric pollution showed increase of their functional indices, and those who 66 Different low levels of air pollution... had moved to municipalities of higher levels of atmospheric pollution showed lower respiratory indices. The authors concluded that the stay in environment with different degree of atmospheric pollution, even when not continuous, leads to changes in indices of external respiration. We believe that our results signify that the air pollution, even in the determined low concentrations, has a restrictive effect on the respiratory functions of children. In our opinion it has a great practical significance for the proper organization of the prophylactic measures in growing ups from towns and villages with polluted atmosphere. CONCLUSIONS As a result of our investigation the following conclusions were drawn: 1. The average levels of respiratory indicators correspond to the referential values in both groups, in the four trials of the study. 2. The restrictive effect of air pollution on respiratory functions has been established, even when it is below the admissible levels. 3. The pubertal stimulus in development contributes to the compensation of the lower lung function parameters from early childhood, but some unfavorable tendencies continue to persist. Acknowledgements We thank the mayors of the town of Dimitrovgrad – Mr. Naydenov, Mr. Gospodinov and Mr. Hadzhiivanov, the director of the Regional Environmental Agency – Mr. Iliev, the heads of division “Ecology” in municipality of Dimitrovgrad – Mr. Dimitrov and Mr. Mitkov, the head of division “Education” – Mrs. Ivanova and the directors, teachers and health care providers in schools “P. Slaveikov”, “A. Konstantinov” and “H. Botev”. REFERENCES 1. A n n u a l reports of the Council of Ministers of the Republic of Bulgaria on environmental situation in 1994 and 1999 y – Green books 1995 and 2001 y (In Bulgarian). 2. A r e n a , V. C. et al. A retrospective investigation of PM10 in ambient air and cardiopulmonary hospital admissions in Allegheny County, Pennsylvania: 1995-2000. – J. Occup. Environ. Med., 48, 2006, 38-47. 3. A v o l , E. L. et al. Respiratory Effects of Relocating to Areas of Differing Air Pollution Levels. – Am. J. Respir. Crit. Care. Med., 164, 2001, № 11, 2067-2072. 4. B r u n e k r e e f , B., D. W. Dockery et M. Krzyzanowski. Epidemiologic studies on short-term effects of low levels of major ambient air pollution components. – Environ Health Perspect. 103, 1995, (Suppl. 2), 3-13. 5. C h e n , C. et al. Longitudinal study on cat allergen exposure and the development of allergy in young children. – J. Allergy. Clin. Immunol., 119, 2007, № 5, 1148-1155. 6. C o h e n , A. J. et al. Urban air pollution. In: Ezzati M, Lopez AD, Rodgers A, Murray CJL, editors. Comparative quantification of health risks. – World Health Organization, Geneva, 2004, 2, 13531433 [http://www.who.int/publications/cra]. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 67 7. C o h e n , A. J.. et al. The global burden of disease due to outdoor air pollution. – J. Toxicol. Environ. Health A, 68, 2005, 1301-1307. 8. C o r b o , G. M. et al. Effect of gas cooking on lung function in adolescents: modifying role of sex and immunoglobulin E. – Thorax, 56, 2001, № 7, 536-540. 9. D ’ A m a t o , G., G. Liccardi et M. D’Amato. Environment and development of respiratory allergy. II. Indoors. – Monaldi Arch. Chest Dis., 49, 1994, № 5, 412-420. 10. D i e t e r t , R. et al. Workshop to identify critical windows of exposure for children’s health: immune and respiratory systems work group summary. – Environ Health Perspect. 108, 2000, № 3, 483-490. 11. D o n g , G. H. et al. Effects of housing characteristics and home environmental factors on respiratory symptoms of 10,784 elementary school children from northeast China. – Respiration, 76, 2008, № 1, 82-91. 12. G a u d e r m a n , W. J. et al. The effect of air pollution on lung development from 10 to 18 years of age. – N. Engl. J. Med., 351, 2004, № 11, 1057-1067. 13. H i b b e r t , M. et al. Gender differences in lung growth. – Pediatric Pulmonology, 19, 1995, № 2, 129-134. 14. J e d r y c h o w s k i , W., E. Flak et E. Mroz. The adverse effect of low levels of ambient air pollutants on lung function growth in preadolescent children. – Environ Health Perspect, 107, 1999, № 8, 669-674. 15. K a s a m a t s u , J., M. Shima, S. Yamazaki et al. Effects of winter air pollution on pulmonary function of school children in Shenyang, China. – Int. J. Hyg. Environ. Health, 209, 2006, № 5, 435-444. 16. K a t s o u y a n n i , K. Health effects of air pollution in southern Europe: are there interacting factors? – Environ Health Perspect, 103, 1995, (Suppl. 2), 23-27. 17. K o s t i a n e v , S. S. et D. H. Iluchev. Is the problem concerning the children’s reference spirometric values in Europe solved? – Pediatria, 1996, XXXL:37-8 (in Bulgaria). 18. M a t t h e w , J. et R. Crapo Socioeconomic Status and Lung Function. – Chest., 132, 2007, 1608-1614. 19. M c C o n n e l l , R. et al. Dog ownership enhances symptomatic responses to air pollution in children with asthma. – Environ Health Perspect, 114, 2006, № 12, 1910-1915. 20. M o s h a m m e r , H. et al. Low levels of air pollution induces changes of lung function in a panel of schoolchildren. – Eur. Respir. J., 27, 2006, 1138-1143. 21. M o s h a m m e r , H., H. P. Hutter et M. Neuberger. Gas cooking and reduced lung function in school children. – Atmospheric Environment. 40, 2006, № 18, 3349-54. 22. O f t e d a l , B. et al. Residental air pollution and lung function in schoolchildren. – Epidemiology. 19, 2008, № 1, 129-137. 23. P i n k e r t o n , K. E. et J. P. Joad. Influence of air pollution on respiratory health during perinatal development. – Clin. Exp. Pharmacol. Physiol., 33, 2006, № 3, 269-272. 24. P o p e III, C., D. Bates et M. Raizenne. Health Effects of Particulate Air Pollution: Time for Reassessment? – Environ. Health Perspect., 103, 1995, № 5, 472-480. 25. Q u a n j e r , P. H. et al. Spirometric reference values for white European children and adolescents: Polgar revisited. – Pediatr Pulmonol., 19, 1995, 135-142. 26. R i n n e, S. T., E. J. Rodas, M. L. Rinne, et al. Use of biomass fuel is associated with infant mortality and child health in trend analysis. − Am. J. Trop. Med. Hyg., 76, 2007, № 3, 585-591. 27. S c h i n d l e r , C. et al. Short-term variation in air pollution and in average lung function among never-smokers. The Swiss Study on Air Pollution and Lung Diseases in Adults (SAPALDIA). – Am. J. Respir. Crit. Care Med., 163, 2001, № 2, 356-561. 68 Different low levels of air pollution... 28. S c h m i t z b e r g e r , R. et al. Effects of air pollution on the respiratory tract of children. – Pediatr. Pulmonol. 15, 1993, № 2, 68-74. 29. T u r n o v s k a , T. et B. Marinov. The influence of air pollution during intrauterine development and early childhood on respiratory functions at later age. – Int. J. Hyg. Envir. Heal., 212, 2009, № 5, 519-532. 30. W a n g , X. et al. Pulmonary function growth velocity in children 6 to 18 years of age. – Am. Rev. Respir. Dis., 148, 1993, 1502-1508. ª Address for correspondence: Assoc. Prof. Tanya Turnovska, MD, PhD, DMSc Department of Hygiene, Ecology & Epidemiology Medical University − Plovdiv 24 Lozengrad Str. 4000 Plovdiv, Bulgaria 359 32 602-543, 359 32 651-222 +359 886 838-989 e-mail: [email protected] Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 69 CUMULUS BIOMARKER EVALUATION FOR HUMAN OOCYTE QUALITY PREDICTION Kr. Todorova1, S. Hayrabedyan1, J. Dineva1, I. Vangelov1, D. Zasheva1, V. Penchev2, G. Nikolov2, M. Mollova1 and M. Ivanova1 Institute of Biology and Immunlology of Reproduction, Bulgarian Academy of Sciences 2 Medical Center “REPROBIOMED” 1 Summary. Selecting oocytes with normal developmental competence is of great importance in assisted reproductive technology. We suspect that the expression of specific genes in cumulus cells would affect the developmental competence of the oocyte and these genes may be used as prognostic indicators for successful in vitro procedure. We investigated cumulus cells samples (n = 39) from women who had a reproductive problem, and were participating in ICSI procedures, as well as cumulus cells samples (n = 9) from women who were from male factor sterility couples. The total number of women been examined was 16. In our study we used quantitative real-time PCR to estimate the differential gene expression levels of sex determining region Y-box (sox9), myb, vascular endothelial growth factor (VEGFA), N-cadherin (CDH2) gene transcripts, normalized to phosphoglycerate kinase (PGK1). We observed increased levels in gene expression of SOX9 transcripts in women with primary sterility compared to women from male factor sterility couples. 2D PAGE showed differential spots, subject to software analysis that revealed putative targets based on spots Mr and pI. Our study suggests that these genes could be putative candidate markers for non-invasive assessment oocyte competence. Key words: Cumulus oocyte complex, SOX9, MYB, VEGFA, NCDH, 2D PAGE O INTRODUCTION varian follicle development and maturation are complex processes, including endocrine and paracrine regulations between oocytes and surrounding cumulus (CCs) and granulosa cells (GCs). Oocytes also promote cumulus and granulosa cell proliferation, differentiation and apoptosis, 70 Cumulus biomarker evaluation for human... suggesting that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte [1]. In this way, the study of the CCs transcriptomic profile offers the opportunity, by a non-invasive method, to predict oocyte competence because bidirectional traffic between CCs and the oocyte is very important for the acquisition of this competence [7]. The aim of our study was to evaluate genes, which expression levels in case of changes affect the oocyte fertility competence. We investigated sex determining region Y-box (sox9), MYB, vascular endothelial growth factor (VEGFA), N-cadherin (CDH2) and phosphoglycerate kinase 1 (PGK1). SOX9 is implicated in the early events leading to sex determination. Mutation in the SOX9 gene is associated with genital defects [11]. C-myb oncoprotein was found to be correlated with fertilization, being immunohisotchemically localized in mice cumulus cell nuclei [14]. VEGFa stimulates preantral follicle growth in rat ovary [3]. VEGF-related growth factors and receptors are regulators of angiogenesis and disease progression in ovarian cancer [4, 6]. N-cadherin regulates ovarian epithelial cell survival and it is involved in primordial follicle formation in perinatal hamster ovary [10, 13]. PGK1 is a transferase enzyme that has been considered as housekeeping gene for the cumulus cells. PGK is found in all living organisms and its sequence has been highly conserved throughout evolution [9]. MATERIALS AND METHODS We investigated cumulus cells samples (n = 39) from women who were with a reproductive problem, and were participating in ICSI procedures or cumulus cells samples (n = 9) from women who were from male factor sterility couples, the latter noted as the healthy women group. The total number of women was 16. The average age of women was 30 years. In our study we investigated 3 samples from every women. After separation of the CCs from each oocyte, the specimens were individually collected and immediately frozen in liquid nitrogen. In the experiments the cells were used in standard concentration of 1x104 cells per well. In our study we used quantitative real-time PCR to estimate the differential gene expression levels of SOX9, MYB, VEGFA, CDH2 gene transcripts, normalized to PGK1, in the cumulus cells surrounding acquired oocytes from ICSI-targeted primary and secondary sterility IVF patients and healthy women group. RT-PCR: Fastlane Cell RT-PCR Kit (Qiagen, USA) and RT-PCR Cycler (Agilent Technologies MX3005P, Stratagene, USA) were used in this study. Real-time reverse transcription PCR was used for differential transcript level expression of all studied genes in CCs of patients (with primary and secondary sterility) and comparative group of healthy women with male factor infertility. The mRNA transcript expression levels of all studied genes were normalized towards transcript levels of the cumulus cells specific housekeeping gene PGK1. RNA extraction and reverse Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 71 transcription to cDNA were done using Fastlane Cell RT-PCR Kit (Qiagen, USA), with 500 ng template DNA per reaction, and subsequent qPCR reaction using the master-mix of QuantiTect SYBR Green RT-PCR kit (Qiagen, USA). The following primer sequences, produced by Biomers (Germany) were used: Sox9 Fw: 5’ – gag gaa gtc ggt gaa gaa cg – 3’, Re: 5’ – atc gaa ggt ctc gat gtt gg – 3’; Myb Fw: 5’- aag tct gga aag cgt cac ttg – 3’, Re: 5’- aca tct gtt cga ttc ggg aga ta – 3’; Vegfa Fw: 5’ – cct tgc tgc tct acc tcc ac – 3’, Re: 5’ – cca tga act tca cca ctt cg – 3’; N-cadherin Fw: 5’ – tgg gaa tcc gag gaa tgg – 3’, Re:5’ – tgc aga tcg gac cgg ata ct – 3’; Pgk1 Fw: 5’- att agc cga gcc agc caa aat ag – 3’, Re: 5’- tca tca aaa acc cac cag cct – 3’. The normalized expression levels of the genes, expressed as fold change compared to healthy women group expression, were subject to ANOVA analysis. 2D PAGE: The isoelectrofocusing was performed using strips with pH gradient 3-10. The pharmalytes (pH range 3-10, Pharmacia Fine Chemicals) were added to the rehydratation buffer. The protein samples were loaded on the strips and the isoelectrofocusing was carried out on Multiphore apparatus (Pharmacia Biotech) in two steps: 30 min at 500V, 14mA, 15W and 90 min at 2000V. The strips were equilibrated in equilibration buffer in two steps and the proteins were distributed by their molecular weights using SDS-PAGE method (Laemley) on 12% PAGE. The gels were silver stained, using silver staining kit (Amersham). The results are presented after the processing of the gel images using software for 2D PAGE analysis. RESULTS Cumulus cells specimens (n = 39) obtained from women treated for primary or secondary sterility and from healthy women group (n = 9) were subject to RT-qPCR. SOX9, MYB, VEGFA and NCDH genes mRNA transcripts were amplified and their levels were relatively expressed as fold change compared to healthy women group expression levels, thus the need for quantitative calibration was avoided. The three groups described were designated as sterility 0, 1 and 2 (0 – healthy women group, 1 – primary sterility, 2 – secondary sterility). We found that SOX9 was upregulated in the primary sterility investigated group. These data are represented as box-andwhisker graph as well (Fig.1a). We found that MYB, VEGFA and NCDH were downregulated in the primary and secondary sterility group (Fig. 1b, c, d). All studied genes were normalized towards PGK besides comparative fold-change expression (ΔΔCt) estimation in sterile groups and healthy women group. Silver stained gels from healthy women (gel 1), secondary sterility patients (gel 2), and primary sterility patients (gel 3), were subject to image software analysis. Corresponding spots from each gel were auto numbered and spots intensity scatterplot showed several significant over expressed putative protein targets (Fig. 2). 2D PAGE showed differential spots, subject to software analysis that revealed putative targets based on spots Mr and pI. The resulting differences are represented in Table 1. 72 Cumulus biomarker evaluation for human... SOX9: F(2;69) = 67,9693; p = 0.0000; KW-H(2;72) = 44,0344; p = 0.0000 MYB: F(2;69) = 30,8159; p = 0.0000; KW-H(2;72) = 35,1903; p = 0,00000 0,6 1,5 0,4 1,0 0,2 0,5 0,0 -0,2 0,0 -0,4 -0,5 -0,6 -0,8 -1,0 MYB SOX9 -1,0 -1,5 -1,2 -2,0 -1,4 -1,6 -2,5 -1,8 Median -3,0 25%-75% -2,0 Non-Outlier Range -3,5 Outliers 0 Extremes -2,2 1 0 2 classp = 0.0000; VEGFA: F(2;69) =sterility 374,9073; KW-H(2;72) = 63,027; p = 0.0000 1 Median 25%-75% Non-Outlier Range Outliers Extremes 2 class CDH2: F(2;69) =sterility 64,5698; p = 0.0000; KW-H(2;72) = 52,405; p = 0.0000 0,4 0,5 0,2 0,0 0,0 -0,2 -0,5 -0,4 -1,0 -0,6 -0,8 -1,5 VEGFA CDH2 -1,0 -1,2 -2,0 -1,4 -2,5 -1,6 -1,8 -3,0 Median -2,0 25%-75% -2,2 Non-Outlier Range 0 Outliers -3,5 1 0 2 1 sterility class Median 25%-75% Non-Outlier Range Outliers 2 sterility class Fig. 1. The three groups described were designated as “sterility” (0 – healthy women group, 1 – primary sterility, 2 – secondary sterility). 1a: SOX9 was upregulated in the primary sterility investigated group. 1b: MYB, 1c: VEGFA and 1d: NCDH were downregulated in the primary and secondary sterility investigated groups pI : 3, 5 4, 5 5, 2 5, 6, 6, 85 55 85 7, 8, 8, 8, 35 15 4565 9, 3 pI: 3, 5 4, 5 5, 2 5, 85 6, 6, 55 85 7, 8, 35 15 8, 8, 45 65 9, 3 pI: 3, 5 4, 5 5, 2 5, 85 6, 6, 55 85 7, 8, 35 15 8, 8, 45 65 9, 3 250 kDa 250 kDa 250 kDa 150 150 150 75 75 50 50 37 37 15 15 10 10 75 50 37 15 10 Fig. 2. Silver stained gels from healthy donors (gel 1), secondary sterility patients (gel 2), and primary sterility patients (gel 3), were subject to image software analysis. Corresponding spots from each gel were auto numbered and spots intensity scatterplot showed several significant over expressed putative protein targets (for list of putative candidates see Table 1) Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 73 DISCUSSION The quality of the oocyte is largely dependent on its follicular environment. The primary criterion for oocyte selection in the human fertility clinic is morphological characteristics of the oocytes based on its nature. Many studies reported that granulosa cell gene expression is associated with oocyte health and development [1]. The maintenance of the ovarian phenotype is an active process throughout life. It was considered that the gender depends solely on the arrangement of chromosomes – embryo’s default route to gender determination would result in a female unless a transcription factor gene known as SRY was located on the Y chromosome. This long-held assumption that the development of female traits is the default pathway of nature is no longer valid. The adult ovary has plasticity at cellular level and ‘‘terminally’’ differentiated gonadal cell types can switch to a new fate. FOXL2 mutations in the human germline in females lead to autosomal dominant syndrome (BPES) associated with premature ovarian failure [2]. Foxl2 was implicated to participation in a true adult lineage reprogramming in vivo: upon conditional loss of Foxl2 in the adult ovary the two major female-specific somatic cell lineages switch their cell fate: granulosa cells, are reprogrammed into testis-specific Sertoli-like cells and steroidogenic theca cells start to produce testosterone at male-like levels in blood [12]. We found that male main Sry target sox9 was upregulated in the primary sterility investigated group. The presence of SOX9 74 Cumulus biomarker evaluation for human... in primary sterility patients suggests for an impaired function in the cumulus cells of these women. We hypothesized that SOX9 might be used as a novel predictor of oocyte quality, at least in some cases. Other genes, subjects of our investigation showed downregulated levels in the primary and secondary sterility investigated groups. C-myb oncoprotein was found to be correlated with fertilization, being immunohistochemically localized in mice cumulus cell nuclei. C-myb antisenseoligodeoxynucleotides are able to inhibit the rate of fertilization in vitro in a dosedependent way, suggesting that c-myb expression is required for this process [14]. VEGFa stimulates preantral follicle growth in rat ovary [3]. It was demonstrated that VEGF promotes the early development of bovine embryo in the presence of cumulus cells [8] and also suppresses ovarian granulosa cell apoptosis in vitro [5]. Our data showed that VEGFa has the main role in human cumulus cells. N-cadherin regulates ovarian epithelial cell survival trough cell contact mediated mechanism [10]. N-cadherin is involved in primordial follicle formation and FSH regulation in perinatal hamster ovary [13]. CONCLUSION Our study suggests that the investigated genes could be putative candidate markers for non-invasive assessment oocyte competence. Further studies are necessary to confirm our suggestion. Acknowledgements: This study was conducted using research equipment purchased by funding under 7th EU FP ReProForce REFERENCE 1. A s s o u , S. et al. A non-invasive test for assessing embryo potential by gene expression profiles of human cumulus cells: a proof of concept study. – Mol. Hum. Reprod., 14, 2008, № 12, 711719. 2. C r i s p o n i , L. et al. The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. – Nat. Genet., 27, 2001, № 2, 159-166. 3. D a n f o r t h , D.R. et al. Vascular endothelial growth factor stimulates preantral follicle growth in the rat ovary. – Biol. Reprod., 68, 2003, № 5, 1736-1741. 4. D u y n d a m , M.C.A. et al. Vascular Endothelial Growth Factor-165 Overexpression Stimulates Angiogenesis and Induces Cyst Formation and Macrophage Infiltration in Human Ovarian Cancer Xenografts. – Am. J. Pathol., 160, 2002, № 2, 537-548. 5. K o s a k a , N. et al. Vascular endothelial growth factor (VEGF) suppresses ovarian granulosa cell apoptosis in vitro. – Biochem. Biophys. Res. Commun., 363, 2007, № 3, 733-737. 6. K u m a r a n , G.C. et al. Antiangiogenic drugs in ovarian cancer. – Br. J. Cancer., 100, 2009, № 1, 1-7. 7. L i , W. et al. Localization and activity of lysyl oxidase within nuclei of fibrogenic cells. – PNAS, 94, 1997, № 24, 12817-12822. 8. L u o , H. et al. Vascular endothelial growth factor (VEGF) promotes the early development of bovine embryo in the presence of cumulus cells. – J. Vet. Med. Sci., 64, 2002, № 11, 967-971. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 75 9. M a r k u s , M.A. et al. Refining the overall structure and subdomain orientation of ribosomal protein S4 delta41 with dipolar couplings measured by NMR in uniaxial liquid crystalline phases. – J. Mol. Biol., 292, 1999, № 2, 375-387. 10. P e l u s o , J.J. N-cadherin-mediated cell contact regulates ovarian surface epithelial cell survival. – Biol. Signals Recept., 9, 2000, № 2, 115-121. 11. H a r d i n g , R. and Bocking, A.D. Fetal Growth and Development – Academic and Professional Books – Cambridge University Press. Cambridge University Press. 2001, 209-212 p. 12. U h l e n h a u t , N.H. et al. Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. – Cell., 139, 2009, № 6, 1130-1142. 13. W a n g , C. and Roy, S.K. Expression of E-cadherin and N-cadherin in perinatal hamster ovary: possible involvement in primordial follicle formation and regulation by follicle-stimulating hormone. – Endocrinology, 151, 2010, № 5, 2319-2330. 14. W u , L.-feng et al. Study of effect and mechanism of c-myb on the fertilization in mouse. – Ch. J. Appl. Physiol., 21, 2005, № 1, 81-5. 76 Address for correspondence: Krassimira Todorova Institute of Biology and Immunology of Reproduction Bulgarian Academy of Sciences 73 Tsarigradsko chaussee Blvd. 1113 Sofia, Bulgaria +359 894371404 e-mail: [email protected] Cumulus biomarker evaluation for human... LICHEN STRIATUS IN UGANDA – CASE REPORTS AND UPDATE OF CLINICAL PICTURE, DIFFERENTIAL DIAGNOSIS AND PATHOGENESIS: FIRST DESCRIPTION OF PATIENTS IN SUB-SAHARAN-AFRICA P. Nenoff1, M. Peter2, G. Tchernev3, G. Mulyowa2, E. Amerson4, U. Paasch5 and J. Ananiev6 1 Laboratory for Medical Microbiology, Mölbis, Germany Skin Clinic, Department of Dermatology, Mbarara University of Science and Technology, Mbarara, Uganda, Africa 3 Policlinic of Dermatology and Venerology, University Hospital Lozenetz, Academic Educational Hospital of the Saint Kliment Ohridski University, Medical Faculty, Sofia, Bulgaria 4 Department of Dermatology and Venerology, University of California, San Francisco, USA 5 Department. of Dermatology, Venerology and Allergology, University of Leipzig, Leipzig, Germany 6 Department of General and Clinical Pathology, Medical Faculty, Trakia University, Stara Zagora, Bulgaria 2 Summary. Lichen striatus is a rare, benign, self-limited, mostly asymptomatic, inflammatory condition of unknown etiology, typically occurring in childhood. It is rare in adults. The unilateral papular lesions have a linear morphology following the lines of Blaschko. The most commonly involved sites of the lichenoid lesions are the limbs. Differential diagnosis should consider other blaschkoid and papular dermatoses, such as linear psoriasis, linear atopic dermatitis, linear lichen planus, inflammatory linear verrucous epidermal nevus (ILVEN), and, in particular, the so-called “Blaschkitis”. A 30-year old HIV-negative patient from Uganda suffered from lichen striatus of his left arm, chest, and palm. Diagnosis was made based on the characteristic clinical picture and medical history of the disorder. A 13-year old Ugandan boy showed strand-like papular hyperpigmented and hypopigmented lesions of his right arm. This is the first description of lichen striatus in Sub-Saharan-Africa, in particular in Uganda. It is suggested that lichen striatus could be found much more frequent in tropical and subtropical countries however not published. Key words: Lichen striatus; linear papular dermatosis; Blaschkitis; Uganda; Sub-Saharan Africa, tropical dermatosis Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 77 L INTRODUCTION ichen striatus (LS, Synonym: Dermatitis linearis) is an acquired dermatosis with a characteristic morphology following Blaschko´s lines [1-3]. In the English literature the acronym BLAISE is sometimes used (Blaschko Linear Acquired Inflammatory Skin Eruption). LS represents a self limited linear dermatosis affecting predominantly children between 5 and 15 years. LS requires symptomatic treatment only [4-6]. LS is known to affect adults rarely. Kennedy and Rogers [7] found a preponderance of cases of LS in the preschool-age group (average 3 years old) This contradicts what is said above (that it predominantly affects people from 5-15) – could say “affecting predominantly children between 5 and 15 years, although one report found a preponderance of cases occurred in a preschool-aged group (average 3 years old). Onset of the disease in the spring and summer months support the hypothesis of an environmental agent, possibly an infection, in the etiology of the condition. The female to male ratio was found to be 1.6 : 1 [7]. Until now, there are reports on lichen striatus from several European countries, e. g. Italy, Germany, UK, Serbia, from the United States, Australia, from Turkey, Israel, Korea, and Japan. Long ago, in 1963, El-Nasr and El-Hefnawi [8] reported on lichen striatus in Egypt, North Africa. However, until now there are no reports on lichen striatus in tropical and subtropical countries of Sub-Saharan Africa. CLINICAL PICTURE The initial papules are typically discrete and pale red. Later, deep red brown and sometimes hyperpigmented papules with a diameter of approximately 1-3 mm develop in a linear fashion, following Blaschko’s lines, on previously healthy skin. Individual papules are either smooth or show a fine scaling. Hyperpigmentation may occur, particularly in pigmented skin. Over several weeks, the papules may gradually become confluent, arranged in 1 to 3 cm broad stripes. The morphology is typically unilateral, on the upper or lower limbs, sometimes spreading to the upper trunk. Adjacent Blaschko´s lines can become involved. The lesions are commonly subjectively asymptomatic, although in some patients mild pruritus may occur. Nail involvement rarely occurs, manifesting as longitudinal ridging or onychodystrophy. Exceptionally rarely, LS may be limited to the nails [9-11]. Recently, unilateral LS with bilateral onychodystrophy has been described [12]. The course of LS is characterized by progression of the cutaneous lesions over a few weeks, followed by stabilization, with the lesions lasting several months. Regression takes up to one year. Resulting postinflammatory hypopigmentation may be seen after the dermatosis resolves [13, 14]. 78 Lichen striatus in Uganda... CASE REPORTS Patient 1 In February, 2010, a 30-year old male presented to the Skin Clinic, Department of Dermatology, Mbarara University of Science and Technology in Mbarara, Uganda, East Africa, complaining of 3 months of slightly pruritic skin lesions exclusively involving the left arm and the left side of the trunk. In the medical history, the patient had no atopic diseases, and no asthma. Both HIV and syphilis serology were negative. On the left upper arm until the antecubital fossa red brown and hyperpigmented papules of a different size were seen. The lesions appeared grouped and confluent, forming a relatively broad stripe over the entire left crook of the arm to the axilla. Some of the papules were hyperpigmented with a velvety surface and dry hyperkeratosis, while others were silvery to whitish with mild scaling. A pityriasis-like scaling was seen (Fig. 1 a-d). Dense hyperpigmented and black lesions with a peripheral inflammatory redness were discernible. The mucous membranes of mouth and tongue were free of symptoms, in particular no signs of lichen planus or Wickham´s striae were present. At the lower arm and antecubital fossa fewer papules were found, these were smaller, flat, slightly hyperpigmented, and also arranged as stripe. The same was found at the chest. On the left side stripes of papules were seen, too. In addition, discrete and flat hyperpigmented papular lesions were apparent at the left palm. Laboratory investigation (complete blood count, urinalysis, stool for ova and parasites) revealed no sign of infection or inflammation. Treatment using topical corticosterid ointment (hydrocortisone 1% cream) twice daily was started. 4 weeks later a slight improvement was to be seen. Further treatment was recommended. And the patient has been seen after two months once again. It was planned to change the local therapy of bethametasone containing creme for 4 weeks and to see the patient again. a) b) Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 79 c) d) Fig. 1. a) A 30-year old male from Mbarara, Uganda, suffering from lichen striatus. On the left upper arm (crook) papules in different size were seen. The papules were red brown stained, and, predominantly, dark pigmented. The lesions appeared grouped and showed confluence, forming a relatively broad stripe over the entire left crook of the arm to the axilla. b) The brown papules showed a dark hyperpigmented velvety surface, particular a marked and dry hyperkeratosis was seen. Some of the papules were silvery to whitish with a slightly scaling surface. A pityriasis-like scaling ruff could be demonstrated by dermatoscopy. c) At the lower arm crook fewer papules were found, these were smaller, flat, slightly hyperpigmented, however, arranged as stripe, too. d) The same pigmented papulous lesions were apparent was found at the chest. Patient 2 A 13-year old HIV-negative boy from Mbarara, Uganda, attended the Mbarara Skin Clinic. For 2 months, he had suffered from non-pruritic skin lesions of his right arm. Examination revealed lichenoid red-brown to dark brown slightly hyperkeratotic papules formed in a linear array of 1 to 3 cm width (Fig. 2 a and 2b). a) b) Fig. 2 a) A 13-year old HIV negative boy from Mbarara, Uganda, Noticeable were stripe formed lichenoid red brown to dark brown slightly hyperkeratotic papules on his right arm forming a linear array of 1 to 3 cm width. b) At the lower arm the stripe has cleaved in two strand-like formations. There was no sign of Wickham´s striae or umbilical depression of papules. In particular, both hyperpigmented and hypopigmented parts of the papulous stripes were found. 80 Lichen striatus in Uganda... At the lower arm the stripe cleaved in two strand-like formations with both hyperand hypopigmentation. There was no sign of Wickham´s striae or umbilical depression of papules. Dermatoscopically, molluscum contagiosum was excluded. Based on the characteristic histopathological picture, clinical morphology and the medical history the diagnosis of LS was made. Betamethasone containing cream twice daily led to negligible improvement. It was planned to change the local treatment to clobetasol, propionate crème 0,05% after two months with the purpose for more significant improvement. DIFFERENTIAL DIAGNOSIS Based on the typical histopathological picture, medical history and clinical morphology, some other linear dermatoses could be easily distinguished from LS [14, 15], including linear psoriasis vulgaris, linear atopic eczema, linear porokeratosis Mibelli, lichen nitidus, and rarely linear Darier’s disease [16]. In addition, very rarely, linear Lichen ruber planus occurs [17]. Blaschkitis, a significant differential diagnosis, is affecting predominantely adult patients. Inflammatory epidermal nevus (ILVEN…inflammatory linear verrucous epidermal nevus) can be distinguished from LS in particular by marked elevation and hyperkeratosis. Furthermore, ILVEN occurs as congenital malformation presenting at birth or in early childhood. ILVEN is characterized by pruritus, and, notably, unlike LS, no spontaneous resolution is observed. The histology of ILVEN is quite different from that of LS. ILVEN exhibits alternating para- and orthokeratosis together with acanthosis and a discrete superficial lymphocytic infiltrate [11]. HISTOLOGY Remarkable, LS exhibits an epidermal hyperkeratosis. Moreover, focal parakeratosis, a mild spongiosis with lymphocytic exocytosis, even some dyskeratotic cells in the stratum granulosum are visible [12, 18]. In the dermis, either focal interface dermatitis or a frank lichenoid lymphocytic infiltrate may be present, affecting both the dermo-epidermal junction and the appendages. In approximately 50% of cases colloid bodies are found. A superficial and deep perivascular and peri-appendageal infiltrate is characteristic, differentiating LS from other lichenoid dermatoses. ETIOLOGY AND PATHOGENESIS OF LICHEN STRIATUS Triggering factors and predisposition Although the etiology of LS is unknown, the observation that LS occurs in siblings and children suffering from atopic eczema, and furthermore the increased occurrence in spring and summer, underlines the importance of genetic, infectious Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 81 and environmental factors as potential triggers for this relatively rare disease [7, 19, 20]. Taniguchi et al. [21] confirmed the correlation between both atopy and pruritus and LS in a study with 89 patients suffering from LS in Brazil. Because of the increased occurrence of LS in atopic children, it is plausible that an external stimulus (such as an allergen or an infectious agent) results in a loss of immunologic tolerance in a mutated fetal clone of keratinocytes. A T-cell mediated immune response results, followed by an inflammatory reaction restricted to the progeny of the mutated clone of cells [22]. In Australia, Shepherd et al. [23] described a 16-year old boy who developed a LS of his arm and thumbnail (onychodystrophy) following a trauma by a pineapple leaf. Yaosaka et al. [24] reported LS occurring simultaneously in a mother and son. Brennand et al. [25] observed LS in the third trimester of a pregnant woman. LICHEN STRIATUS AND THE BLASCHKO´S LINES There is a strong correlation of LS to the so-called Blaschko´s lines. Along these lines, which were first described and drawn by Blaschko in approximately 1900, several different naevoid skin conditions and acquired (inflammatory) cutaneous disorders develop on the human skin and mucous membranes [26]. Blaschko´s lines do not follow any known nervous, vascular, or lymphatic structures of the skin. In addition to keratinocytes, skin appendages, melanocytes, blood vessels, and subcutaneous fat, may be involved in the morphological manifestations of the dermatoses which follow Blaschko´s lines. Many of the naevoid lesions are lifelong (e. g. linear nevus sebaceous, unilateral nevoid teleangiectasia); however, many of the acquired skin disorders have a relatively short duration of weeks to months or a few years (e. g. LS, linear psoriasis). The linear manifestation along the Blaschko´s lines is possibly a kind of human “mosaicism”. This term stands for the concept that certain specific cells or groups of cells react differently from other cells due to chromosomal abnormalities. Based on the pattern affecting Blaschko´s lines, it is possible that, due to a polyzygotic somatic mutation, a cutaneous mosaic has developed which is responsible for the development of the lichenoid skin alterations [22]. Due to an acute trigger (immunological reaction or infection), the expression of novel membrane antigens may be possible after methylation and demethylation of silent gene segments. LICHEN STRIATUS AND BLASCHKITIS There is disagreement in the literature regarding how these two terms should be used, and whether they actually represent the same disorder or two distinct entities. As Hofer noted [27] Grosshans and Margot in 1990 introduced the term “adult blaschkitis”, which refers to LS occurring in adult patients [28, 29]. They concluded that LS doesn´t occur as rarely in adults. Therefore, it should be denominated in the same way as in children, simply as LS (and not „adult blaschkitis“). Today, the term 82 Lichen striatus in Uganda... “blaschkitis” is used to refer to an acquired linear dermatosis with spread along the Blaschko´s lines affecting predominantly adults. Recently, Denk and Flux described a 2 1/2 year-old girl with a linear papular dermatosis following the Blaschko´s lines on her trunk. Based on the characteristic morphology and the course the diagnosis of blaschkitis was made [30]. The authors suggested that their case supports the hypothesis that blaschkitis is an entity of its own. It has been proposed that multiple grouped papules along the lines of Blaschko on the trunk, rarely at the limbs, are distinctive of blaschkitis. In contrast the lesions of LS might be hypopigmented – as in patient 2 presented here – and they exhibit epidermal change. The surface of the 2-4 mm scaling papules is frequently velvety, slightly hyperkeratotic, and sometimes scaling. LS overwhelmingly affects children and adolescents. LS exhibits a characteristic morphology with one or more narrow stripes ranging from singly-arranged papules with confluence to a dense linear formation, predominantly on the limbs. However, blaschkitis, as mentioned before, is a cutaneous disorder affecting adults which develops predominantly on the trunk, and rarely on the limbs. The recurrence rate of blaschkitis is relatively high. Both entities, blaschkitis and LS, tend to regress spontaneously and eventually resolve. PATHOGENESIS OF LICHEN PLANUS/LICHENOID SYNDROMES There is no scientific explanation regarding the etiology and pathogenesis of lichen planus and other more rare occurring variants of lichenoid dermatoses, such as LS, keratosis lichenoides chronica (KLC) or Graham-Little-Piccardi-LassueurSyndrome (GLPLS). Although some pathogenic differences exist regarding the triggering or induction of LS when compared to KLC and GLPLS, “antigen mimicry” may play a central role for the initial triggering of these inflammatory dermatoses. In theory, LS may develop along the lines of Blaschko due to genetic mosaicism, with the characteristic clinical picture developing only in the affected cells after an external trigger such as a vaccination or infection [31, 32]. PATHOGENESIS OF LICHEN STRIATUS The characteristic morphology occurring along the lines of Blaschko – which should be considered as locus minoris resistentiae – underlines the topographic and pathogenetic concept of this dermatosis. Interestingly, variants of lichen planus, e. g. LS, seem to be triggered by external factors (vaccination and infections) in a similar way as in lichenoid syndromes (GLPLS/KLC) [31-35]. Although sometimes the lesions generalize, the morphology remains monomorphic. Therefore, in case of LS the “epitope spreading” seems not play a role, distinguishing it from KLC and GLPLS, which show a more heterogenic or polymorphic clinical picture. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 83 LS occurring after vaccination against hepatitis has been described, which may be an example of a cross reaction in the form of “antigen mimicry” [33, 35-37]. Generalized and atypical forms of LS are described which may indicate a possible external triggering in young patients [36, 37]. Therefore, it is conceivable that immunizations during the childhood could play a significant role for the clinical manifestations of lichenoid dermatoses, generally, and in particular for LS. In LS the distinct genetic background seems to determine the typical monomorphic clinical picture, at least in part. The medical hypothesis of “antigen mimicry” seems to be supported by the prevalence of lichenoid exanthema, e. g. Gianotti-Crosti-Syndrome in children after EBV (Epstein-Barr virus) infection, coxsackie A 16 virus infection, parainfluenza virus and ECHO virus and also infections by poliomyelitis virus, Bordetella pertussis or Clostridium tetani (tetanus). TREATMENT As a general rule, treatment of LS is not necessary because the skin disorder is mostly asymptomatic, and furthermore, it has a tendency for spontaneous regression. In case of mild pruritus, topical corticosteroids may be used temporarily. It should be emphasized that even ultrapotent topical steroids or intralesional injected corticosteroids do not achieve a significant regression or cure of the lesions of LS. However, the symptoms of pruritus and dryness of the skin might be influenced by the ointment itself (the oil or water-soluble base) [14, 38]. Successful application of the topical calcineurin inhibitors tacrolimus and pimecrolimus, in persistent and strong pruritic lesions of LS has been reported [3943]. Kim et al. used tacrolimus in LS and onychodystrophy successfully [38]. CONCLUSIONS LS seems to be a special form of lichen planus. At present, it is not clear whether there are external factors which are responsible for the onset of the phenotypic manifestations of lichen planus and LS, in particular in the tropics. Studies on representative groups of patients are missing. In addition, there are no sufficient special observations in affected children, in particular in the phase after a vaccination or immunization. This is the first description of patients suffering from lichen striatus in SubSaharan-Africa, in particular in Uganda. Despite this fact it is suggested that occurrence of lichen striatus could be much more frequent in tropical and subtropical countries however not published. REFERENCES 1. H a p p l e , R. Mosaizismus und epidermale Nävi. In: Braun-Falco O, Plewig G, Wolff HH, Burgdorf WHC, Landthaler M (Hrsg.). Dermatologie und Venerologie. Springer Verlag Heidelberg, 5. Auflage 2005, 1201-1214. 84 Lichen striatus in Uganda... 2. O d o m , R. B., W. D. James et T. G. Berger. Andrew´s diseases of the skin. Clinical dermatology. Chapter 12: Lichen planus and related conditions. 2000, 9th ed., W. B. Saunders Company, Philadelphia, London, New York, St. Louis, Sydney Toronto, 266-283. 3. S i l v e r , S. G. et V. C. Y. Ito. Benign epithelial tumors. Chapter 84, Section 11. – In: Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Katz SI. Fitzpatrick´s dermatology in general medicine. McGraw-Hill Medical Publishing Division New York Chicago, etc., 1, 6th edition. 767-785. 4. To d a , K., H. Okamoto et T. Horio. Lichen striatus. – Int. J. Dermatol., 25, 1986, 584-585. 5. W e s t o n , W. L., A. T. Lane et J. G. Morelli. Papulosquamous disorders. – In: Color textbook of pediatric dermatology. 2002, 3rd ed., Mosby/Elsevier Science St. Louis, 119-143. 6. P a t r i z i , A. et al. Lichen striatus: clinical and laboratory features of 115 children. – Pediatr. Dermatol., 21, 2004, 197-204. 7. K e n n e d y , D. et M. Rogers. Lichen striatus. – Pediatr. Dermatol., 13, 1996, 95-99. 8. E l - N a s r and El-Hefnawi. Lichen striatus. – J. Egypt. Med. Assoc., 46, 1963, 1271-1282. 9. K a u f m a n n , J. Lichen striatus with nail involvement. – Cutis, 14, 1974, 232-234. 10. K o r p , D. L. et B. A. Cohen. Onychodystrophy in lichen striatus. – Pediatr. Dermatol., 10, 1993, 359-361. 11. To s t i , A. et al. Nail lichen striatus: clinical features and long-term follow-up of five patients. – J. Am. Acad. Dermatol., 36, 1997, 908-913. 12. A l - N i a i m i , F. A. et N. H. Cox. Unilateral lichen striatus with bilateral onychodystrophy. – Eur. J. Dermatol., 19, 2009, 511. 13. G i a n o t t i , R. et al. Lichen striatus – a chameleon: an histopathological and immunohistological study of forty-one cases. – J. Cutan. Pathol., 22, 1995, 18-22. 14. S o r i , T. et al. Hypopigmentary disorders in children in South India. – Indian J. Dermatol., 56, 2001, 546-549. 15. T i l l y , J. J., B. A. Drolet et N. B. Esterly. Lichenoid eruptions in children. – J. Am. Acad. Dermatol., 51, 2004, 606-624. 16. H a p p l e , R. Mibelli revisited: a case of type 2 segmental porokeratosis from 1893. – J. Am. Acad. Dermatol., 62, 2010, 136-138. 17. H e r d , R. M., K. M. McLaren et R. D. Aldridge. Linear lichen planus and lichen striatus - opposite ends of a spectrum. – Clin. Exp. Dermatol., 18, 1993, 335-337. 18. Z h a n g , Y. et N. S. McNutt. Lichen striatus. Histological, immunohistochemical, and ultrastructural study of 37 cases. – J. Cutan. Pathol., 28, 2001, 65-71. 19. D i L e r n i a , V., G. Ricci, A. Bonci et A. Patrizi. Comment on: Lichen striatus and atopy. – Int. J. Dermatol., 25, 1986, 584-585. Int J Dermatol 1991; 30: 453-454 20. P a t r i z i , A. et al. Simultaneous occurrence of lichen striatus in siblings. – Pediatr. Dermatol., 14, 1997, 293-295. 21. Ta n i g u c h i Abagge, K. et al. Lichen striatus: description of 89 cases in children. – Pediatr. Dermatol., 21, 2004, 440-443. 22. S c h n o p p , C. Differentialdiagnose lichenoider Hautveränderungen. – Pädiatrie Hautnah, 3, 2005, 138-141. 23. S h e p h e r d , V., K. Lun et G. Strutton. Lichen striatus in an adult following trauma. – Australas J. Dermatol., 46, 2005, 25-28. 24. Y a o s a k a , M. et al. Lichen striatus affecting a mother and her son. – J. Am. Acad. Dermatol., 53, 2005, 352-353. 25. B r e n n a n d , S., S. Khan et A. H. Chong. Lichen striatus in a pregnant woman. – Australas J. Dermatol., 46, 2005, 184-186. 26. J a c k s o n , R.. The lines of Blaschko: a review and reconsideration: Observations of the cause of certain unusual linear conditions of the skin. – Br. J. Dermatol., 95, 1976, 349-360. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 85 27. H o f e r , T. Lichen striatus in adults or ‘adult blaschkitis’?. There is no need for a new naming. – Br. J. Dermatol., 148, 2003, 587-590. 28. G r o s s h a n s , E. et L. Margot. Blaschkite de l´adulte. – Ann. Dermatol. Venreol., 117, 1990, 9-15. 29. Ta i n e b , A., A. El-Youbi et E. Grosshans. Lichen striatus: a Blaschko linear acquired inflammatory skin eruption. – J. Am. Acad. Dermatol., 25, 1991, 637-642. 30. D e n k , K. et K. Flux. Blaschkitis in children - a new entity? – J. Dtsch. Dermatol. Ges., 9, 2011, 48-49. 31. Tc h e r n e v , G. et P. Nenoff. Lichen ruber planus and lichenoid like pathologies of the skin: commuting between investigation of apoptotic pathways, immunologic parameters and simple dermatologic prevention? – Acta Medica Bulgarica, 36, 2009, 70-81. 32. Tc h e r n e v , G. et P. Nenoff. Antigen mimicry followed by epitope spreading: a pathogenetic trigger for the clinical morphology of lichen planus and its transition to Graham-Little-PiccardiLassueur syndrome and keratosis lichenoides chronica – medical hypotheses or reality? – Anais Brasileiros de Dermatol., 84, 2010, 682-688. 33. D r a g o s , V., L. Mervic et B. Zgavec. Lichen striatus in a child after immunization. A case report. – Acta Dermatovenerol. Alp. Panonica Adriat., 15, 2006, 178-180. 34. M i t t a l , R. et al. Multiple lichen striatus – an unusual presentation. – Indian J. Dermatol. Venereol. Leprol., 67, 2001, 204. 35. H a f n e r , C., M. Landthaler et T. Vogt. Lichen striatus (blaschkitis) following varicella infection. – J. Eur. Acad. Dermatol. Venereol., 20, 2006, 1345-1347. 36. K a r a k a s , M. et al. Lichen striatus following HBV vaccination. – J. Dermatol., 32, 2005, 506-508. 37. H w a n g , S. M., S. K. Ahn, S. H. Lee et E. H. Choi. Lichen striatus following BCG vaccination. – Clin. Exp. Dermatol., 21, 1996, 393-394. 38. K i m , G. W. et al. Lichen striatus with nail abnormality successfully treated with tacrolimus ointment. – J. Dermatol., 36, 2009, 616-617. 39. F u j i m o t o , N., S. Tajima et A. Ishibashi. Facial lichen striatus: successful treatment with tacrolimus ointment. – Br. J. Dermatol., 148, 2003, 587-590. 40. S o r g e n t i n i , C., M. A. Allevato, M. Dahbar et H. Cabrera. Comment on. – Br. J. Dermatol., 148, 2003, 587-590 Lichen striatus in an adult: successful treatment with tacrolimus. Br J Dermatol 2004; 150: 776-777 41. C a m p a n a t i , A. et al. Lichen striatus in adults and pimecrolimus: open, off-label clinical study. – Int. J. Dermatol., 47, 2008, 732-736. 42. Te j e r a - V a q u e r i z o , A. et al. Adult blaschkitis (lichen striatus) successfully treated with topical tacrolimus. – Actas Dermosifiliogr., 100, 2009, 631-632. 43. V u k i c e v i c , J. et al. Unilateral multiple lichen striatus treated with tacrolimus ointment: a case report. – Acta Dermatovenerol. Alp. Panonica Adriat., 18, 2009, 35-38. 86 Address for correspondence: Associated Professor Georgi Tchernev Specialist in Dermatology and Venerology University Hospital Lozenetz, Academic Educational Hospital Saint Kliment Ohridski University Medical Faculty Policlinic of Dermatology and Venerology 1 Koziak street 1407 Sofia, Bulgaria 00359 885 588 424 e-mail: [email protected] Lichen striatus in Uganda... REACTIVATION OF SUBACUTE CUTANEOUS LUPUS ERYTHEMATODES UNDER THE CLINICAL PICTURE OF ROWELL SYNDROME Ju. Ananiev1, X. Baraliakos2 and G. Tchernev3 Department of General аnd Clinical Pathology, Medical Faculty, Stara Zagora, Bulgaria 2 Rheumazentrum Ruhrgebiet Herne, Ruhr-University Bochum, Germany 3 Policlinic of Dermatology and Venerology, Saint Kliment Ohridski University, University Hospital Lozenetz-Academic Teachning Hospital of the Saint Kliment Ohridski University, Medical Faculty, Sofia, Bulgaria 1 Summary. A patient is presented with a history of subacute cutaneous lupus erythematodes (SCLE), appearing immediately after an intensive exposure to UVA rays. The relapse of the diseases was established in the form of the so-called “Rowell syndrome”. Literature data relating to the pathogenesis and the existence of this rare clinical variant of SCLE is contradictory to date. The paper emphasizes critical key points relating to the diagnostics of the Rowell syndrome and its precise restriction by Erythema Exudativum Multiforme (EEM) as well as the possibility for a parallel manifestation of both conditions. Key words: Rowell syndrome, subacute cutaneous Lupus erythematodes, target lesions, EEM, SSA R owell syndrome (RS) is a rare entity, in which patients with lupus erythematosus (LE) develop lesions of erythema multiforme (EM). Scholtz first described the simultaneous appearance of LE with EM in 1922 [1]. In 1963, Rowell et al. reported four discoid LE (CDLE) female patients with EM-like lesions who were associated with a speckled anti-nuclear antibodies (ANA) pattern, positive rheumatoid factor (RF), and antibodies to a saline extract of human tissues [2]. Because EM patients do not typically show accompanying immunologic characteristics or anti-SjT antibodies, this entity was classified with the distinct disease term Rowell syndrome [3, 4]. Zeitouni et al. proposed the following diagnostic criteria for RS in 2000: major criteria include SLE, DLE, or SCLE; EM-like lesions (with and/or without mucosal involvement); and an ANA with a speckled pattern. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 87 Minor criteria include chilblains, anti-Ro or anti-La antibody, and a positive RF [3]. All three major criteria and at least one minor criterion are required for the diagnosis of RS [3, 4]. A review of 18 case reports of RS between 1963 and 2000 showed that the speckled ANA pattern was the most consistent feature of RS and is described in about 88% of the cases, whereas RF is the least preserved feature and is present in only 41% [5]. Although chilblains had been described in all four of Rowell’s original cases, this feature was found in only five of the 15 cases reported between 1982 and 2008 [5]. We report about a female patient, aged 72 years, who noticed a reddening, blushing, appearing mainly in the areas submitted to sunbathing for the first time about 12 years ago. One year later, again after sunbathing, she experienced a new episode of reddening which resulted to referral to the hospital. An elevated RF and ANA, together with a slight increase of the antibodies against a double-chain DNA, slightly decreased С3 and С4 fractions of the complement were recorded. The histological examination of the skin proved the viability of SCLE, while the systemic involvement was not established. After a short-term treatment with glucocorticoids in restricted doses of 30 mg daily for 20 days and a three months treatment with 250 mg of chloroquine daily, a complete remission could be reached, which lasted for 10 years. Five years after the start of the disease the patient had a deep vein thrombosis in one of her legs. Examination status. During clinical examination, erythematous suculent confluating plaques in the sphere of the hairy part of the head were found. There were slightly raised erythemato-edematous plaques with an area of 3-4 cm in the face, which showed a tendency to confluation. Furthermore, we found polycyclic bizonal configurated, slightly raised skin lesions on the back, which, in some places, showed a tendency to confluation, the so-called “target lesions” (Figs. 1, 2). In the area of the flexion surfaces of the upper limbs, small erythematous plaques with a diameter of 5-6 cm were observed. Well-expressed deformations in the metacarpophalangeal joints and the knee joints were also found. Fig. 1, 2. Typical double contour cockade in the area of the back, characteristic with еrythema exudativum multiforme in a patient with subаcute cutaneous lupus erythematodes. Case report is from the oral presentation of Dr Georgi Tchernev at the Berliner Dermatological days in the University Clinic for Dermatology and Venerology Campus Benjamin Franklin 2003/2004 under the leadership from Prof Constantin Orfanos. Fotos also from the University Archiv of UKBF, Departments of Dermatology, Venerology and Allergology 88 Reactivation of subacute cutaneous lupus... Paraclinics: Altered clinical-chemical and immunological indicators: erythrocyte sedimentation rate-68/82, leucocites -2,99.109/l (leucopenia), RF-147, anti SSA-positivity АNА-titre 1:260, anti-cardiolipin antibodies 10,8 Mpl/Е/ml, /norm < 6 Mpl/Е/ml. Within normal values were: red blood line differential blood picture, indicators of the protein and lipid metabolism, blood sugar, creatinine and creatinine clearance, tumor markers СЕА, СА-19-9, antibodies against the double chain DNA, SSВ / antibodies against La/ SCL70 и Jo1. Apparative examinations: normal ECG and conventional radiogrpah of the lung.. The ultrasound of the abdomen showed cholelitiasis but no other pathological findings. Light gradation: a persistent reaction to UVA rays after 72 hours as well as suspicion of a persistent reaction to UVB 72 hours after the radiation. The histological investigation of a 6 mm biopsy of the erythema plaque localized on the back confirmed the clinical picture of a subacute cutaenous lupus erythematodes. Course of the disease: After the confirmation of the diagnosis of SLE in combination with skin lesions similar to EEM and the adoption of the diagnosis Rowell syndrome, daily treatment with chloroquine 250 mg and prednisolone 30 mg was started as well as the local application of triamcinolon 0.05% cream. Due to the persistence of the skin plaques, the prednisolone dose was increased to 60 mg daily for a period of one week. This resulted to a rapid decrease of the skin infiltrates as well as the reddening during the next 3-4 days after which the dose was gradually decreased. During treatment, high blood sugar levels were found, reaching values of 10 mmol/l, which was accepted as latent diabetes and was treated with diet and Glimepirid 1 mg. Due to the fast positive effect on the skin, the prednisolone dose was reduced to 30 mg/daily. As a further adverse event, a mouth candidosis was observed, which was locally treated with Nystatin suspension. Ambulatory consultation was planned with a vessel surgeon in view of the need to start a therapy with anticoagulants relative to the increased anticardiolipin antibodies. DISCUSSION The association between LE and erythema exudativum multiforme has been described for the first time in 1922 by the German dermatologist Scholz [1]. In those days it wasn’t been yet fully clear whether the described condition relates to different skin diseases or to rather a separate disease in itself [1]. In 1963, during the examination of 120 patients with SCLE, Rowell found that 4 of them had additional skin lesions which conform to the typical EEM as well as positive immunological indicators, namely elevated ANAs, positive RF and SSA / anti Ro [2]. This constellation is marked as the Rowell syndrome since then. As of that period literature has quoted between 20 and 30 cases of this rare syndrome. The incidental appearance of EEM in patients with the skin or systemic form of LE may lead to difficulties related to the differential diagnosis due to which the basic factors causing EEM should be taken into consideration. These are a) chemical drugs (acetosalicic acid, barbiturates, ibuprofen, methotrexate, minoxidil, furosemide, penicillin, phenylbutazon, arsenic, quinine), b) viral and bacterial infections Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 89 (HSV,EBV,VZV, measles, hepatitis B and C, enteroviruses, streptococcus, trichomonas infections, Yersinina, Treponema pallidum, Corrynebacterium diphteriae) or c) fungal infections (Trichophitia profunda, histoplasmosis, coccydiomycosis). This report indicates that in case of a hospitalization of a patients with subacute cutaneous lupus erythematodes, all these findings need to be excluded. On the other hand, given the clinical manifestation of the typical skin lesions characteristic of ЕЕМ and the simultaneous existence of systemic and subcutaneous lupus erythematodes, a possible relapse of LE relapse should also be excluded as a differential diagnosis. When the combination of immunologically confirmed LE and the clinical availability of the target lesions, characteristic of the EEM/elevated ANA and positive RF and SSA, then the disease would be defined as the Rowell syndrome [1]. It is remarkable that in some rare cases, an EEM manifestation is possible in patients with SCLE, which demands additional extended screening programs. What is beneficial in these cases is that the two diseases are well treated by the systemic immunosupressive drugs although the duration of the immunosupression and the types of immunosupressives drugs, which have to be applied, is different in both of the diseases. This is what demands the exact identification of the disease so that a specific, schemebased therapy may be prescribed to guarantee against possible relapses. Additional activity markers, coming to confirm the LE diagnosis or the SCLE, are the availability of positive АNA, antibodies against the double-chain DNA (evidencing the damages of the parenchymatous organs and a bad prognosis), decrease of С3 and С4 fractions of the complement and the available clinical symptoms. In spite of the proposed criteria, the differentiation of the two diseases quite often remains difficult and cannot be fully clarified. The major issue here is whether the case is a true EEM in a patient with a systemic and subacute cutaneous lupus erythematodes where EEM is provoked by a drug, streptococcus infection, after an infection with herpes labialis or genitalis, which at the point of clinical manifestation of the so called target lesions cannot be established as a clinically manifest infection. Obviously, these cases are rare. The second possibility is that the lesions of the EEM type have to be interpreted as an atypical manifestation of the auto-immune disease. In view of the lack of specific features that distinguish RS from LE, Kuhn et al. suggested that Rowell syndrome is probably not a distinct entity and is now widely considered to be a variant of SCLE [6]. Other authors have been very critical toward the criteria, which substantiate the existence of this rare syndrome [7, 8]. In the case of our patient, definite evidence was given to prove the clinical existence of SCLE with a trend to transfer to the systemic form the course of which includes extensive remissions and typical cutaneous and immunological alterations. During the reactivation of the disease new cutaneous lesions were clinically observed, which in combination with the established immunological changes, were typical for Rowell syndrome. In conclusion, we must say that the described case is a female patient with the rare diagnosis of a Rowell syndrome, which improved rapidly after the pre90 Reactivation of subacute cutaneous lupus... scribed treatment with Resochin and the systemic and local treatment with corticosteroids. There are many cases where the clinical manifestation of the cutaneous form of lupus erythematosus could be linked to another immunological, infectious-allergic or purely infectious disease [9]. In these cases of exceptional importance in the process of placing the diagnosis is the precise interpretation of the anamnesis, the actual clinical picture, histopatological findings and the immunological parameters. Financial support There was no financial support or other benefits from commercial sources for the work reported on in the manuscript, or any other financial interests that any of the authors may have, which could create a potential conflict of interest or the appearance of a conflict of interest with regard to the work REFERENCES 1. S c h o l t z , M. Lupus erythematosus acutus disseminatus haemorrhagicus. – Arch. Dermatol. Syphilol., 6, 1922, 466. 2. R o w e l l , N. R., J. S. Beck et J. R. Anderson. Lupus erythematosus and erythema multiforme-like lesions. – Arch. Dermatol., 88, 1963, 176-180. 3. Z e i t o u n i , N. C. et al. Redefining Rowell’s syndrome. – Br. J. Dermatol., 142, 2000, 343. 4. S h a d i d , N. H. et al. Lupus erythematosus associated with erythema multiforme: Rowell’s syndrome. – Int. J. Dermatol., 46, 2007, 30. 5. K h a n d p u r , S. et al. Rowell’s syndrome revisited: report of two cases from India. – Int. J. Dermatol., 44, 2005, 545. 6. K u h n , A. et al. Clinical manifestations of cutaneous lupus erythematosus. – J. Dtsch. Dermatol. Ges., 6, 2008, 48. 7. M o d i , G. M. et al. Lupus erythematosus masquerading as erythema multiforme: does Rowell syndrome really exist? – Dermatol. Online J., 15, 2009, № 2, 5. 8. S h t e y n g a r t s , A. R, M. R. Warner et C. Camisa. Lupus erythematosus associated with erythema multiforme: does Rowell’s syndrome exist? – J. Am. Acad. Dermatol., 40, 1999, (5 Pt 1), 773-777. 9. O b e r m o s e r , G., R. D. Sontheimer et B. Zelger. Overview of common, rare and atypical manifestations of cutaneous lupus erythematosus and histopathological correlates. – Lupus., 19, 2010, № 9, 1050-1070. Address for correspondence: Associated Prof Dr Georgi Tchernev MD, PhD Sofia University Saint Kliment Ohridski, Policlinic for Dermatology and Venerology University Hospital Lozenetz, Academic Teaching Hospital of the Saint Kliment Ohridski University, Medical Faculty 1 Koziak street 1407 Sofia, Bulgaria 00359 885 588424 e-mail: [email protected] Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 91 THE SIZES OF PULP CHAMBERS OF MOLARS WITH SEVERE ROOT CURVATURES – IN VITRO COMPARATIVE STUDY E. Boteva1 and D. Iovchev2 Department of Conservative Dentistry, 2Department of Oral Rentgenology and Oral Diagnostics, Faculty of Dental Medicine, Sofia, Bulgaria 1 Summary. The macromorphology of pulp chambers has been studied in the last few decades, but there is a lack of knowledge on the sizes of the pulp chambers of molars. The aim of the present study was to measure the size of the pulp chambers of upper and lower molars with different root curvatures and to compare them with the same dimensions in molars without root curvatures. Seventy-seven upper and lower molars, matured, fully mineralized and sound were selected in the following groups: two groups, including upper and lower teeth, respectively and three groups, divided as follows: with straight roots up to 25-30º, with severe curvatures up to 45º and with abnormalities 45º-90º from the axial axis. Three dimensions of the crowns were measured for each tooth in mm: one mesio-distal and two bucco-lingual dimensions, one of then being measured from the top of buccal cusp to the top of the mesio-lingual (palatal) cusp. All teeth were submitted to X-rays and were photographed after opening the pulp chambers. Measurements of the pulp chambers were made after opening with horizontal cuts, made 1 mm apically from the equator with a diamond blend. The two buccolingual dimensions were refered to as L1 and L2, the mean value – as L and the mesio-distal – as MD, sizes were measured in the widest part of the pulp chamber with an endodontic file and endoblock in mm. A careful approach to these sizes of the pulp chambers of the molars with severe curvatures can safe hard dental tissues during endocavity and pulp chamber preparation .These findings are important for the prevention of crown and root fractures, and teeth loses and for lowering the use of posts and pins and the use of crowns and bridges in young age groups. Key words: endodontics, curved root canals, pulp anathomy 92 The sizes of pulp chambers.... I n the last few decades the macromorphology of pulp chambers has been studied [1]. There is a serious lack of knowledge on the sizes of the pulp chambers of molars in different dimensions and non – existing information on the sizes of pulp chambers of teeth with roots with severe curvatures and root abnormalities [2, 3, 9, 13, 15]. This is an important matter for the proper sizes of endodontic cavities and for the prevention of iatrogenic errors [4, 11, 12]. All sizes of the endodontic cavities are usually defined as distance between cusps or mm from the buccal and lingual walls or from the respective walls in relation to the type of the tooth and the sex and age of the patient, not in any relation to the size and anatomy of the particular teeth roots. Very little information is available and completely out of date are data referring some sizes of the crowns and roots of molars in one book of Wetzel form year 1947 [15]. In the dental literature it has been established that there is relation between the size and the shape of the crown and the size and the shape of the pulp chamber in young age and the age changes related to the reduction of the pulp chamber parameters [1]. Unfortunately this fact is not always considered during endodontic cavity preparations, which often leads to iatrogenic errors [5, 6]. A practical review of the sizes of clinical crowns in the last 40 years shows a significant reduction of the mesio-distal and bucco-lingual dimensions of the molars. An important matter is the preparation of the pulp chamber on teeth with massive enamel and dentine loses. From the literature review and the online cross-database search of the data available from the last 20 years, only 8 articles can be related to the macromorphology of the molars. Five of these studies were in vitro studies with a large variation of the number of cases from 5 up to 700 root canals. Only in two of these studies, the sizes of the pulp chambers were measured. Only in one study differences were observed between “young” and “old” teeth [1]. AIM OF THE STUDY The aim of the present study was to measure the range and mean dimensions of the pulp chambers of upper and lower molars with different root curvatures and to compare them with same dimensions of molars without root curvatures. MATERIALS AND METHODS Teeth: Seventy-seven upper and lower molars, left and right teeth. All teeth were matured, fully mineralized and sound. Groups: Two groups, including upper and lower teeth, respectively, and three groups, including teeth with straight roots up to 25-30º, with severe curvatures up to 45º and with abnormalities 45º- 90º from the axial axis were examined. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 93 Measurements of the clinical crown: Three dimensions were measured for each tooth in mm: mesio-distal, from the approximal marginal ridge, bucco-lingual, from the top of the buccal cusp to the top of the mesio-lingual (palatal) cusp. The height of the crown was also measured from the buccal side (H), from the enamel border to the middle part of the line between the cusps. X-Ray: All teeth were submitted to X-ray examination and were photographed after opening the pulp chambers. Measurements of the pulp chambers: Pulp chambers were measured under the following technology: 1. The pulp chambers were opened with horizontal cuts made, 1 mm apically from the equator with a diamond blend. 2. After polishing the ridges, the final size of the chamber was 2 mm bellow the equator. 3. Both buccolingual dimensions were measured as L1 and L2, and the mean value – as L, and the mesio-distal dimension – as MD, sizes were measured in the widest part of the pulp chamber. 4. This measurements are performed with endodontic file and endoblock in mm. Exclusion criteria: non-vital teeth, massive tooth loses, teeth with root caries, incisors and premolars, teeth with hypoplasia and non-caries enamel defects and not matured teeth. RESULTS Table 1. Sizes of the pulp chambers of molars Size Teeth L1 L2 MD Up to 25-30º n=20 Upper Lower 5.58 4.37 4.91 3.62 3.16 3.50 30-45º n=20 Upper Lower 5.01 4.80 4.14 4.15 2.57 3.30 45-90º n=37 Upper Lower 5.14 4.15 4.05 3.55 3.05 3.25 As it is presented in Table 1, all dimensions of the upper molars and most of the lower molars from the group of teeth with root canal curvatures, are smaller than the sizes of the pulp chambers of teeth with straight roots. Table 2. Sizes of the clinical crowns of molars Type of tooth Upper Teeth n=161 Lower teeth n=125 94 Dimension ВL МD H ВL МD H Mean 6.5 8.1 4.5 5.3 9.9 5.2 Range (mm) 5.8 – 7.8 7.9 – 9.4 4.0 – 6.2 4.0 – 8.0 8.0 – 13.0 3.5 – 7.0 The sizes of pulp chambers.... DISCUSSION The importance of these findings is related to the fact that the bucco-lingual size of the crowns and the pulp chambers are very similar. On the other hand, reducing the preparation of hard dental tissues in this area is very important and directly related to the lower amount of active axial root surface bellow this area in teeth with severe root canal curvatures. The lack of available data on the size of the pulp chambers of molars in the literature is a fact. This is the explanation why after endodontic treatment the most common mistake is remaining pulp tissue in retentive lodges in the pulp chamber which becomes a source of infection and periapical lesions, a complication that represents 19.8% of all indications for endodontic retreatments in the Faculty of Dental Medicine in Sofia, shown in our previous study. Not accurate exposure of pulp chambers, failures in working length estimation and poor preparation of root canals are some other mistakes commonly mode [14, 16]. Overdone preparation of cavity walls and crown fractures, mostly as a result of overdone preparation of medial and distal walls followed by the use of posts and pins, represents 18.2% of all reasons for endodontic treatments. In many literature sources it is proved that nearly in 50% of all endodontic treatments there are failures, especially when it is considered that with age all pulp chambers lower their sizes and orifices migrate up on cavity walls [6,12]. CONCLUSIONS 1. There is a need of up-to-date knowledge not only on the pulp anatomy but also on the pulp chamber sizes and crown sizes of molars with curved roots. 2. A careful approach to these sizes can lead to preservation of hard dental tissues during endocavity and pulp chamber preparation . 3. The use of smaller pulp chambers in molars with curvatures can be important in the prevention of crown and root fractures, as well as teeth loses and in lowering exrtactions, use of posts and pins and the use of crowns and bridges in young age groups. REFERENCES 1. B j o r n d a l , L. et al. External and internal macromorphology in 3D- reconstructed maxillary molars using computerized X-ray microtomography. – Int. Endod. J., 32, 1999, 3-9. 2. B o t u s h a n o v , P. Cariology and operative dentistry. Autospektar, Plovdiv, 2000. 3. B o t u s h a n o v , P. Endodontics – theory and practice. Autospektar, Plovdiv, 1998, 401-418. 4. E l e f t h e r i a d i s , G. I. et T. P. Lambrianidis. Technical quality of root canal treatment and detection of iatrogenic errors in an undergraduate dental clinic. – Int. Endod. J., 38, 2005, 725-734. 5. G l i c k m a n , G. et al. The crisis in endodontic education: current perspectives and strategies for change. – JOE, 31, 2005, № 4, 225-261. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 95 6. H a y e s , S. J. et al. An audit of root canal treatment performed by undergraduate students. – Int. Endod. J., 34, 2001, 501-505. 7. H u l s m a n , M. et F. Stryga. Comparison of root canal preparation using different automated devices and hand instrumentation. – JOE, 19, 1993, № 3, 141-145. 8. I o d w a y , B. et M. Hulsmann. A comparative study of root canal preparation with NiTi – TEE and K3 rotary NiTi instruments. – Int. Endod. J., 39, 2006, № 1, 71-80. 9. I n d j o v , B. Fundaments of cavity preparation, Indjident, Sofia, 2006, 109-113. 10. M o n c a d a , G. et al. Increasing the longevity of restorations by minimal intervention: A two year clinical trial. – Operative Dentistry, 33, 2008, № 3, 258-264. 11. Q u a l t r o u g h , A. J., J. M. Whitworth et P. M. Dummer. Preclinical endodontology: an international comparison. – Int. Endod. J., 32, 1999, 406-414. 12. S o n n t a g , D. et al. Pre-clinical endodontics: a survey amongst German dental schools. – Int. Endod. J., 41, 2008, 863-868. 13. S t r a n s k i , D. Child dental Health. Мedicine and Physical activity, Sofia, 1959, 85. 14. S c h a f e r , E. et al.. Roentgenographic investigation of frequency and degree of canal curvatures in human permanent teeth. – JOE, 3, 2002, 211-216. 15. W e t z e l , G. Anathomy, hystology and embrinologyof teeth. Teeth and dentition., Alma mater, Sofia, 1947, 13-51. 16. W i l c o x , L. R. et M. L. Swift. Endodontic retreatment in small and large curved canals. – JOE, 17, 1991, № 7, 313-315. 96 Address for correspondence: Assoc. Prof. Dr. Ekaterina Boteva Deptartment of Conservative Dentistry Faculty of Dental Medicine 1 Georgy Sofiisky str. Sofia, Bulgaria +359888328327 e-mail: e_boteva@ abv.bg The sizes of pulp chambers.... EFFICIENCY OF WORKING LENGTH DETECTION AND IRRIGATION DURING PREPARATION OF CURVED ROOT CANALS E. Boteva1 and D. Yovchev2 2 1 Department of Conservative Dentistry Department of Imaging and Oral Diagnostics, Faculty of Dental Medicine, Sofia, Bulgaria Summary. Curved root canals are a challenge for instrumentation, preparation, irrigation and obturation. The aim of the present study was to find the working length and irrigation efficiency in root canals with curvatures 30º-45º and in root canals with anatomical abnormalities 45º-90º. Sixty-eight human, matured, extracted molars with 201 root canals were included in the study. Molars were placed in three groups in relation to the angle between the root and the axis. The first group of molars – straight – 25º-30º, was the control group, n = 14 teeth – 45 root canals; second group – 25-30º to 45º, n = 22 teeth with 66 root canals; third group – 45º to 90º (n = 31 teeth with 96 root canals). Measurements: mesio-distal buccal size of the chamber in its largest part and both bucco-lingual sizes – the mesio dimension was refered to as L1 and the distal one – as L2. Root canal preparation: extraction of the root pulp with K endofiles number 6, 8, 10, with Step Down and Balance force techniques. Canals length was measured rentgenologically using intraoral radiography, preparation continued after X-ray analysis of the level of penetration of irrigants with contrast solution of diluted Urograffin 66%. Regime of active irrigation: same for all groups with 2 % H2O2 and 1% NaOCl and paper points drying. To follow up the results a fourth radiography was used and a second one with Urograffin. The applied criteria for working length and for penetration of the irrigant were as follows: 3 – whole working length, 2-1 mm shorter than the working length, 1-2 mm shorter than the working length and 0 – more than 2 mm shorter than the working length. X-ray – working length detection of molars with root canal curvatures was more accurate, compared with straight roots, due to curved canals in straight roots and inadequate instrumentation. The active irrigation was more efficient in curved root canals, because in straight canals most of the irrigant was lost back in the mouth or Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 97 periapically. In straight root canals only moisturizing (Miller pins) the canal could be more effective and less dangerous. Key words: endodontics, working length, curved root canals C INTRODUCTION urved root canals are a well known challenge for instrumentation, preparation, irrigation and obturation. Root curvatures with abnormalities over 45º are not investigated from this point of view at all. Iatrogenic errors are often associated with these teeth. In the last 15 years, between 1995 and 2010 only 13 articles are related to this problem, excluding those with extreme methodology as the use of 6, 25% NaOCl . All published articles are researches from in vitro studies and surprisingly the curvatures are from 20º up to 40º. Teeth with curvatures between 25º and 45º, as we found in our previous studies, are from 16 to 19% of all teeth, and with 45º are more than 1.3%. The number of experimental cases in most of the studies varies between 30 and 135 teeth, where the average numbers are 59-62 [1-14]. In most studies the preparation technique is reported to be Step Down [1-9, 11, 12]. Different instrumentations with hand files and machine rotary files are used [6, 10, 11, 13, 14]. Apical preparation size varies from № 10-25 to № 40-45, in relation to the degree of curvature. In these papers differences are not only seen in the irrigation regimes, but also in the type of medication. NaOCl is used as 2.5% – 4.5% – 5% [3, 7, 10] аnd in a combination with – EDTA [5, 6]. In all regimes H2О2 is used for irrigation. The follow-up methods are X-ray [3], SEM [8], bioluminiscense [7, 9], intraoperative microscopy [2], stereomicroscopy [13], and light microscopy [6]. In most of the cases the results were predictable and in most cases a logical result was obtained from the design of the experiment. Summarizing the results, it could be concluded that the type of preparation is a major factor for the degree of remove of the smear layer and the penetration of irrigants and medicines. The highest efficiency is 70% in one group in one article. In most articles not full penetration of irrigants has been found among curvatures between 30-40º. The aim of the present study was to find the working length and irrigation efficiency in root canals with curvatures 30º-45º and in root canals with anatomical abnormalities 45º-90º. MATERIALS AND METHODS Teeth: Sixty-eight human matured extracted molars with 201 root canals were included in the study. All teeth were from the same geographical region. Groups: Molars were separated in three groups in relation to the angle between the root and the axis. First group – straight – 25º-30º as a control group, n = 98 Efficiency of working length detection... 14 teeth with 45 root canals, second group 25-30º to 45º (n = 22 teeth with 66 root canals) and third group – 45º to 90º (n = 31 teeth with 96 root canals). Measurements: Mesio-distal buccal size of the chamber in its largest part and both bucco-lingual sizes – mesio as L1 and distal as L2, and the average of the two – as L, were measured. Root canal preparation: Starts with opening of the orifices with manual Orifice Openers and extraction of the root pulp with K endofiles number 6, 8,1 0, with Step Down and Balance force techniques. Root canal length was measured rentgenologically with K files and the preparation continuous after X-ray analysis of the level of penetration of irrigants with contrast solution of diluted Urograffin 66%. Regime of active irrigation: Same for all groups with 2 % H2O2 and 1% NaOCl and paper points drying. The aim of root canal preparation was, even in roots with 90º curvatures, the apical stop to be number 20-25, and for the rest of the teeth – 30-40. To follow up the results a fourth radiography was used, as well as a second one with Urograffin. Instrument fractures and canal blockage were registered, too. X-ray regimes: Dental X-ray unit– Phot – XII (Takara Belmont corp, Japan) and intraoral digital sensor Eva (Dent-X Co.) were used. All radiographies were exposed under the same conditions – exposure settings – 60 kv, 7mA, time 0.04 s, etc. Irrigation measurements scale: The applied criteria for working length (WL) and for penetration of the irrigant were as follows: 3 – whole working length, 2-1 mm shorter than the working length, 1-2 mm shorter than the working length and 0 – more than 2 mm shorter than the working length. All measurements were performed from one examiner, three times with at least three days intervals in between. RESULTS Out of total of 207 root canals, four were not found – 2%. Seven instruments were fractured, 4 in group 2 and 3 in group 3, and none in the control group. In seven canals dentine debries formed intracanal blockages, from them 3 in group 1 – the control group, 1 in group 2 and 3 in group 3. Table 1. WL and number of samples in the tested groups of upper and lower molars GROUPS 3 – WL 2WL – 1 mm 1WL – 2 mm 0WL – > 2 mm Controls n = 45 26 7 − − 2 1 5 1 30º to 45º n = 66 42 7 4 7 1 − 3 2 45º to 90º n = 96 42 36 − − 1 − 6 − Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 99 Tаble 2. Penetration of the irrigant GROUPS WL 3-0 mm WL 2-1 mm WL 1-2 mm WL 0<3 mm Controls n = 45 17 3 − 21 30º to 45º n = 66 43 1 9 13 45º to 90º n = 96 65 9 8 14 As is shown in Table 1 most mistakes are made in the control group, nearly in one third of the cases. This fact can be explained in three different ways. One explanation is the more frequent ramifications, second – the difficulties related to the most accurate choice of the size of the instrument being used, when the teeth are extracted and the third one is the excessive cutting, made more often in straight root canals. In all in vitro studies there was a lack of data on age and sex of the teeth and anthropometrical data. In the experimental groups, this percentage was 10% and 9%, or three times less. The same trends persisted when irrigants were tested. In the control group, in 50% of the cases, the irrigant was not present in the canal system (Table 2). Still high but lower are the cases of root canals with curvatures, 33% in group 2 and 20% in group 3. Figures 1-3 show X-rays of canals with irrigant and 7-12 X-rays of WL of different groups. a) Correct and incorrect working length b) Correct and incorrect (not sufficient) penetration Fig. 1. (a and b) Group 1 Straight root canals 100 Efficiency of working length detection... a) Correct and incorrect working length b) Correct and incorrect penetration of the irrigant Fig. 2 (a and b) Group 2 – Curved root canals a) Correct and incorrect WL b) Correct and incorrect penetration of the irrigant Fig. 3.(a and b) Group 3 – Root canals with severe curvatures and abnormalities Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 101 DISSCUSSION Under the conditions of this study, designed on the basis of a realistic clinical approach to difficult teeth, it is hard the role of irrigants in root canal preparation to be favoured. In straight canals, especially in young patients, the extrusion of irrigants periapically is more often. In the clinics, this leads to toxic periodontitis and later to chronical periapical lesions. In curved canals the penetration of irrigants is more difficult, which has its positive and negative trends. Practically, the more severe the curvature is, the more the instrumentation is related to moisturizing the internal root canal surface and to the use of new flexible files with proper sizes. These findings are matching a few other studies [3, 7, 10]. Non-effective irrigation is the irrigation even in curved canals only with 24-28º [7] and 30-33º [3]. Division of teeth in groups of lower and upper teeth in this study was found to be unefficient and without significant differences, even trends and was not found in the literature. CONCLUSIONS X-ray WL detection of molars with root canal curvatures is more accurate, compared with straight roots, due to curved canals in straight roots and more often inadequate instrumentation in them. The active irrigation is more efficient in curved root canals, because in straight canals most of the irrigant is losted back in the mouth or periapically. In straight root canals only moisturizing (Miller pins) the canal can be more effective and less dangerous. REFERENCES 1. B a u g h , D. et J. Wallace. The role of apical instrumentation in root canal treatment: a review of the literature. – JOE, 31, 2005, 333-340. 2. B i n g – F a n et al. Negotiation of C shaped canal system in mandibular second molars. – JOE, 35, 2009, 1003-1008. 3. B r o n n e c , F., S. Bouillaguet et P. Machtou. Ex vivo assessment of irrigant penetration and renewal during the final irrigation regimen. – Int. Endod. J., 43, 2010, 663-672. 4. D i n g - M i n g , H et al. Study of the progressive changes in canal shape after using different instruments by hand in simulated S shaped canals. – JOE, 33, 2007, 986-989. 5. L i u , S. B. et al. Cleaning effectiveness and shaping ability of rotary ProTaper compared with rotary GT and manual K-Flexofile. – Amm. J. of Dent., 19, 2006, 353. 6. L u m l e y , P. J. Cleaning efficiency of two apical preparation regimes following shaping with hand files of greater taper. – Int. Endod. J., 33, 2000, 262-265. 7. N g u y , D. et C. Sedgley. The influence of canal curvature on the mechanical efficiency of root canal irrigation in vitro using real-time imaging of bioluminescent bacteria. – JOE, 32, 1077-1080. 8. R o d i g , T., M. Hulsmann et G. Kahlmeier. Comparison of root canal preparation with two rotary NiTi instruments ProFile 04 and GT Rotary. – Int. Endod. J., 40, 2007, № 7, 553-562. 102 Efficiency of working length detection... 9. S c h a f e r , E., M. Erler et T. Dammaschke. Comparative study on the shaping ability and cleaning efficiency of rotary Mtwo instruments. – Int. Endod. J., 39, 2006, 203-212. 10. S c h a f e r , E. et M. Vlosis. Comparative investigation of two rotary NiTi instruments: ProTaper versus RaCe.Part 2. Cleaning effectiveness and shaping ability in severely curved root canals of extracted teeth. – Int. Endod. J., 37, 2004, 239-248. 11. S c h n a i d e r , S. W. A comparison of canal preparations in straight and curved canals. OralSurg, – Oral Med. and Oral Pathogy., 32, 1971, 271-275. 12. S e d g l e y , C. M. et al. Real time imaging and quantification of bioluminescent bacteria in root canals in vitro. – JOE, 30, 2004, 893-898. 13. W u , M. K. et P. R. Wesselink. Efficacy of three technics in cleaning the apical portion of curved root canals. – Oral Surg., Oral Med., Oral Pathol., Oral Radiol. and Endod., 79, 1995, 492-496. 14. Y o s h i m i n e , Y., M. Ono et A. Akamine. The shaping effects of three NiTi Rotary instruments in simulated S shaped canals. – JOE, 31, 2005, 333-340 . Address for correspondence: Assoc. Prof. Dr. Ekaterina Boteva Department of Conservative Dentistry Faculty of Dental Medicine 1 Georgy Sofiisky str. 1431 Sofia, Bulgaria ++359888328327 e-mail: e_boteva@ abv.bg Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 103 ARTHROSCOPIC SURGERY IN CASES WITH AN AGING LOCKED POSTERIOR DISLOCATION OF THE SHOULDER N. Dimitrov, P. Tsekov and B. Matev Clinic of Surgery of the upper limb USHATO – “Prof. B. Boychev” Summary. The shoulder joint is most vulnerable to dislocation. Anterior dislocation is common, while the posterior one is very rare – about 4% of all dislocations of the shoulder. Because of this, the diagnosis is frequently missed at the initial examination – McLaughlin called the situation a “diagnostic trap”. The surgical treatment of posterior dislocations initially aimed at reduction of the joint. However, this is not sufficient for aging dislocations. Stability is to be provided. In recent years, the promotion of arthroscopic treatment is becoming increasingly important, especially given for its indisputable advantages. In the Department of Surgery of the Upper Limb in the University Specialized Hospital for Active Treatment in Orthopedics USHATO – “Prof. B. Boychev” for the period 2005-2011, were operated 12 patients (8 men and 4 women – a total of 12 shoulders). All had persistent posterior dislocation of the shoulder as a result of injury, after a lapse of between 3 and 8 months – an average of 4.5 months. In all cases an osteohondral defect from 25 to 45% (average 30%) located on the front surface of the head of the humerus was set. Surgery: Seven patients were first operated through arthroscopic reduction and then stability was restored by transposition of m.subscapularis in the osteohondral defect. Five patients were operated without prior arthroscopic reduction – only through open surgery. Follow-up period was from 6 months to 6 years (3 years averagely). The reported results indicate that abduction, flexion and internal rotation increased much more rapidly and to a larger volume in patients in which first arthroscopic reduction was made, compared with those where it was held via conventional surgery, due to its larger volume and greater operational trauma. These results were also confirmed using two post-operative function of the shoulder joint scoring systems: Murley Constant Score and UCLA. Key words: posterior locked luxation of the shoulder, arthroscopic-assisted reduction, stability 104 Arthroscopic surgery in cases with an aging... T INTRODUCTION he shoulder joint is most vulnerable to dislocation. Anterior dislocation is common, while the posterior one is very rare – about 4% of all shoulder dislocations. In 50-80% of cases the diagnosis is omitted at the initial examination due to lack of experience and distinct clinical features – McLaughlin called the situation a “diagnostic trap”. Therefore, the time between injury and initiation of treatment is quite long. As a result, almost always the patient has already spent more than 3-4 weeks of painful awaiting for the solution of his problem and all, even minimal chances for conservative treatment, have been released long ago. The first part of the article includes a brief historical, as well as an up-to-date review of the various types of surgery. Some basic pathoanatomical, clinical and radiographic findings, characteristic for posterior dislocation, have also been presented. Our study is presented in the second part of this paper. HISTORY In 1839, Sir Ashley Cooper for the first time describes the posterior dislocation of the shoulder joint. In 1855, such injuries were found in a patient after an epileptic seizure. Further on, in 1937, Thomas describes such a patient after an electrical shock. PATHOANATOMY Posterior dislocation is often presented with “locked shoulder” with the humeral head perched on the posterior glenoid. More than 50% of cases have an associated impacting osteohondral defect on the anteromedial underside of the head of the humerus – the so called ‘reverse Hill – Sachs lesion’ or ‘McLaughlin – leasion’. This determines the persistent posterior instability. Depending on the size of this defect, different surgical treatment options are indicated. Pathoanatomical features of posterior dislocation of the shoulder are shown in Figures 1a and 1b. Fig. 1. (a, b) Pathoanatomical features of posterior dislocation of the shoulder Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 105 ETIOLOGY AND CLINICAL SIGNS Some of the most common causes of posterior dislocation are high energy trauma, epileptic seizures and electric trauma. Clinical Signs 1. Inconclusive clinical signs are: − loss of contour of the deltoid; − posterior position; − soft tissue anterior “gap”; − the relative prominence of the proc. coracoideus; − elevation – no more than 90°; − very limited external rotation; − flexible limited internal rotation. 2. Diagnosis is difficult due to the simulated clinical signs of “frozen shoulder”. 3. The standard views of the shoulder, which are used after injury, are required to make the diagnosis of a posterior dislocation. These include an anteroposterior (AP) view and an axillary view. The true AP view is difficult to interpret. Therefore, radiography has limited utility, but would facilitate the initial diagnosis. Surgery (history) 1. In 1952 McLaughlin and Hill – Sachs developed a surgical method in which a transposition of m. subscapularis and the lesser tuberosity into the humeral defect is made. It is indicated for patients with lesion size 20-40% of the volume of the head of the humerus. 2. Later on, Neer modified the method, making an osteotomy of the lesser tuberosity and then fixing it in the defect, along with the adjacent insertion of m. subscapularis. 3. Hawkins et al. showed success in a series of 4 patients with a defect between 20% and 40%. 4. Walch reported success in 6 patients using this method on identical humeral defects – less than 50%. 5. Finkelstein demonstrated very good results in 7 patients with a defect between 20% and 45%. All of these studies included patients whose surgery was performed 4 weeks after the trauma. All are adamant that intervention can be undertaken up to 6 months after injury, but success would be significantly lower. Checcia concluded, that one could count on success up to 2 years between injury and intervention, with very good and excellent results achieved. Despite the good results, complications may arise due to the extensive nature of the operation in the aging of dislocations and the surgical trauma of the open adhesiolisis needed in order to achieve the reduction. 106 Arthroscopic surgery in cases with an aging... Checchia shows osteochondritis reported at 3 years follow-up. One should bear in mind the difficulties in any future joint endoprosthesis, because of the limitation of internal rotation after these interventions. SURGERY The aim of surgery is the reduction of the joint. However, this is not enough in the case of aging dislocation. Inner stability is to be provided. Depending on the size of the osteohondral defect, limitation of trauma, and the experience and capabilities of the surgical team, various surgical interventions are known, which could be divided into anatomical and nonanatomical: 1. Open reduction and posterior plication of the capsula following posterior stabilization in patients with defects up to 20%; 2. Transposition of m. subscapularis into the defect with sutures through bone channels (with flaw 20-40%); 3. Neer’s modification – osteotomy of the lesser tuberosity and fixation into the defect (Fig. 2a and 2b); 2a 2b Fig. 2 (a, b) Neer’s modification – (a) osteotomy of the lesser tuberosity and (b) fixation into the defect 4. Plication of m. subscapularis and fixing it into the defect by means of anchors without its desinsertion (Charalambous, Ravenscroft) (Fig. 3a and 3b); Fig. 3 (a, b) Plication of m. subscapularis and fixing it into the defect by means of anchors without its desinsertion (Charalambous, Ravenscroft) Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 107 5. Filling the defect by osteohondral graft (Fig. 4); Fig. 4. Filling the defect by osteohondral graft 6. Filling or raising osteohondral impaction for smaller defects (Engel et al.) (Fig. 5a and 5b). The authors indicate that there is a risk of instability of the raised cartilage, which is vulnerable and can easily collapse; Fig. 5 (a, b) Filling or raising osteohondral impaction for smaller defects (Engel et al.) 7. Recovery of the shape of the head with a bone graft fortified with reconstruction of the posterior capsule (introduced for the first time by Dobousset); 8. Filling the defect with spongios allograft (Gerber – a series of 9 shoulders with a deficit of over 50% – as an alternative to endoprothesis); 9. Transposition of proc. coracoideus defect in more than 50%; 10. In significant defects (50-60%) – endoprosthesis. ARTHROSCOPIC SURGERY In recent years, the promotion of arthroscopic treatment, has a growing importance in shoulder surgery, in particular, in restoring stability to the shoulder. Arthroscopy is of great importance in the treatment of aging posterior locked dislocations. Its great advantages are: 108 Arthroscopic surgery in cases with an aging... − more accurate view of the joint without extensive open access. − much more extensive for a good while sparing adhesiolysis: 1. anterior – front release of fibrosis which provides space for reduction; 2. posterior – a strong reduction of rigidity, which can provide a sufficient volume of abduction and external rotation necessary for reduction without tension – which limits further increase of the defect during the intervention. − a sufficient posterior stabilization by means of anchors, a placation of posterior capsule, when the defects are less than 15-20%. − arthroscopic- assisted reduction in aging posterior locked dislocations limits the trauma of the intervention and thus has a great advantage as a first stage of the operation. − subsequent stabilization at more than 20% defects is achieved through minimal surgical approach. MATERIALS AND METHODS In the Department of Surgery of the Upper Limb in the University Specialized Hospital for Active Treatment in Orthopedics USHATO – “Prof. B. Boychev” for the period 2005 – 2011, were operated 12 patients (8 men and 4 women – a total of 12 shoulders). All had persistent posterior dislocation of the shoulder as a result of injury, after a lapse of between 3 and 8 months – an average of 4.5 months. In all cases an osteohondral defect from 25 to 45% (average 30%) located on the front surface of the head of the humerus was set. In all cases the humeral head “mounted her” posterior glenoid was visualized by CT or MRI examinations. Clinical findings were as follows: − abduction: − 70° – very painful in 5 patients; − elevation: − 50° – 4 patients; − 80° – in 6 patients; − 90° – in 1 patient; − 100° – in 1 patient; − external rotation: − up to pelvis – springy in 8 patients; − impossible in 4 patients. Radiographic findings: − abnormal contour overlaps the joint profit in AP view (Fig 6a); − impossible axillary view (Fig 6b). Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 109 6a 6b Fig. 6 (a, b) An anteroposterior (AP) view (a) and an impossible axillary view (b) The CT scan shows a middle range deficit of the defect of the humeral head in 10 patients (Figures 7a, 7b, 7c and 7d). 7a 7c 7b 7d Fig. 7 (a, b, c, d) CT scan of the humeral head defect 110 Arthroscopic surgery in cases with an aging... MRI shows the same range deficit in 2 patients (Fig. 8 a, b, c, d). 8a 8b 8c 8d Fig. 8 (a, b, c, d) MRI of the same humeral head defect in 2 patients Operatively: − 7 patients were operated by: − I stage 1. arthroscopic adhaesiolysis by anterior, posterior and inferior capsulotomy; 2. arthroscopic assisted reduction; 3. arthroscopic posterior stabilisation in 3 patients. − II stage – to restore stability by transposition of m. subscapularis in the osteohondral defect; − 5 patients were operated without prior arthroscopic reduction – only through the restoration of stability through transposition of m. subscapularis in the osteohondral defect. Surgical techniques: I. Arthroscopic surgical techniques: 1. Arthroscopic diagnosis (Fig. 9 a,b,c,d): − lateral position of the patient; − anterior and posterior adhaesiolysis; − demonstrate the size of the defect of humeral head. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 111 9a 9b 9c 9d Fig. 9 (a, b, c, d) Arthroscopic diagnosis 2. Arthroscopic assisted reduction (Fig. 10 a and b). 10a 10b Fig. 10 (a, b) Arthroscopic assisted reduction 3. Arthroscopic posterior stabilisation by means of anchors, extracapsular sutures, or plication of the posterior capsule (Fig. 11 a, b, c, d). 112 Arthroscopic surgery in cases with an aging... 11a 11b 11c 11d Fig. 11 (a, b, c, d) Arthroscopic posterior stabilisation by means of anchors, extracapsular sutures, or plication of the posterior capsule Thus was prepared the second stage – minimally invasive surgery. Postoperative results: Up to 4-th week – brace in abduction: − abduction 15°; − neutral in terms of rotation; − flexion of 5°-10° 5-th week – passive movements: − circumduction; − abduction – up to 60°; − flexion up to 80°; − external rotation up to 0°; − no internal rotation. After 5 th week – start of active movements: − increase the amount of abduction and flexion; − external rotation to 20°; − without internal rotation. After 2 months – stretching – exercises. Minimum period of kinesitherapy – 3 months. Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 113 RESULTS The follow-up period was between 6 months and 6 years – an average of 3 years. Table 1. Shows the increase in the volume of motions with time: Table 1. Shoulder range of motion – functional outcome at 4th week, 12th week and 1 year Study Min. Max. x S Passive abduction 4w 12 w 1y 65 135 160 80 180 180 71 157 170 12.4 8.7 9.1 Active abduction 4w 12 w 1y 45 120 145 95 160 180 84 142 160 13.7 9.5 8.4 Passive flexion 4w 12 w 1y 65 120 160 100 160 180 89 135 175 12.1 10.2 8.4 Active flexion 4 w. 12 w 1y 65 120 155 110 165 180 97 145 170 10.8 7.5 6.3 Internal rotation 4w 12 w 1y 5 30 70 5 35 90 5 31 80 9.1 5.2 3.4 External rotation 4w 12 w 1y 0 35 50 25 60 80 15 45 60 6.1 7.2 5.9 In patients with prior arthroscopic reduction, abduction (Fig. 12a), flexion (Fig. 12b) and internal rotation (Fig. 12c) increased as follows: Fig. 12 a. abduction 114 12b. flexion 12c. internal rotatio Arthroscopic surgery in cases with an aging... In patients without prior arthroscopic reduction, abduction, flexion and internal rotation grew more slowly and for a longer period (Fig. 13 a,b,c,d): Fig. 13. (a, b, c, d) In patients without prior arthroscopic reduction, abduction, flexion and internal rotation grew more slowly and for a longer period X-ray – postoperatively – 5th month (Fig. 14 a, b): 14a 14b Fig. 14 (a, b) X-ray findings five months after the sugrery Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 115 X-ray – postoperatively – 6th year (Fig. 15 a,b): 15a 15b Fig. 15 (a, b) X-ray 6th year CT – postoperatively (Fig. 16 a,b): 16a 16b Fig. 16 (a, b) CT scan − postoperative results Clinically – postoperatively – 5 months (Pic. 1 a, b, c): Pic. 1 (a, b, c) 116 Arthroscopic surgery in cases with an aging... − Clinical – postoperatively – 6 years (Pic. 2 a, b. c): Pic. 2 (a, b, c) − Murley Constant Score Mean Constant Murley Score – 19p – pre-operatively. Mean Constant Murley Score – 82p – postoperatively – after 2 months in 7 patients after prior arthroscopic reduction Mean Constant Murley Score – 61p – postoperatively – after 2 months in 5 patients without a prior arthroscopic reduction − UCLA Average UCLA – Score – 8p – preoperatively Average UCLA – Score – 25p – postoperatively – after 2 months in 7 patients after prior arthroscopic reduction Average UCLA – Score – 18p – postoperatively – after 2 months, in 5 patients without a prior arthroscopic reduction Similarity between our results and the reports of some authors was found: − Peter Bock and Rainer Kluger – 5 years follow up in 6 patients – CS – 88.2p (average) – 2 – with excellent, 4 – with good results. − Haukins – 2 years. follow-up in 4 patients – CS – 80p average). − Walch – 3 years. follow-up in 6 patients ( up to 50% defect) – CS – 75p (average) − Finkelstein – 3 years. follow-up in 7 patients – CS – 78p (average) − Christopher and Craig – 4 years. follow-up in 9 patients – UCLA (21-29p) − Gavrilidis and Magosch – 4-years-follow-up in 11 patients – CS – 72p (average) − Complications: − expressed arthrosis in 1 patient, who, however, has very good movements; − very limited external rotation up to 20° in 1 patient DISCUSSION Arthroscopic surgical treatment of aging posterior locked dislocation of the shoulder: Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 117 − stores the maximum and the anatomy of the joint which is so altered: 1. by arthroscopic adhaesiolysis; 2. by means of most gentle reduction without tension, arthroscopically secured. − provides accurate arthroscopic assessment of the size of the defect of the head of the humerus and helps select the most accurate plan for the second stage of the intervention; − provides another opportunity for stabilization through by means of anchors or plication of the posterior capsule, which may be sufficient for small defects – to 15-20%; − minimizes the surgical access to the second stage of intervention, because of lack of need for large, open adhaesiolysis as a purpose of the reduction; − shortening the time for the second stage – open surgery – reducing the possibility of complications; − permits abduction, flexion and internal rotation to be increased much more rapidly and to much larger volume in patients in which first arthroscopic reduction was made, compared with those where it was held only via conventional surgery. CONCLUSION Arthroscopic surgical treatment of aging posterior locked shoulder dislocations can enter into consideration after an accurate indication of its performance in the presence of an operating team, familiar with the method. The reported results indicate that abduction, flexion and internal rotation increased much more rapidly and to much larger volume in patients in which first arthroscopic reduction was made, compared with those, where it was held via conventional surgery, because of its larger volume and greater operational trauma. REFERENCES 1. B l a s i e r , R. B. et J. K. Burkus.Management of posterior fracture-dislocations of the shoulder. – Clin. Orthop. Relat. Res., 232, 1988, 197-204 . 2. C h e n g , S. L. et al. Treatment of locked posterior fracture-dislocations of the shoulder by total shoulder arthroplasty. – J. Should. Elb. Surg., 6, 1997 , № 1, 11-17 . 3. G e r b e r , C. et S. M. Lambert. Allograft reconstruction of segmental defects of the humeral head for the treatment of chronic locked posterior dislocation of the shoulder. – J. Bone. Jt. Surg., 78, 1996, № 3, 376-382 . 4. B r o w n , R. J. Bilateral dislocation of the shoulders. – Injury, 15, 1984, № 4, 267-273. 5. K e l l y , J. P. Fractures complicating electro-convulsive therapy and chronic epilepsy. – J. Bone Joint Surg. Br., 36-B, 1954, № 1, 70-79 . 6. M e s t d a g h , H. et al. Traumatic posterior dislocation of the shoulder in adults. Apropos of 25 cases. – Ann. Chir., 48, 1994, № 4, 355-363. 7. H a w k i n s , R. J. et al. Locked posterior dislocation of the shoulder. – J. Bone Joint Surg., 69, 1987, № 1, 9-18 . 118 Arthroscopic surgery in cases with an aging... 8. C o o p e r , A. On the dislocations of the os humeri upon the dorsum scapulae, and upon fractures near the shoulder joint. – Guy’s Hosp. Rep., 4, 1839, 265-284 . 9. E n g e l h a r d t , M. B. Posterior dislocation of the shoulder: report of six cases. – South. Med. J., 71, 1978, № 4, 425-427. 10. D o r g a n , J. A. Posterior dislocation of the shoulder. – Am. J. Surg., 89, 1955, № 4, 890-900. 11. R o w e , C. R. et B. Zarins. Chronic unreduced dislocations of the shoulder. – J. Bone Joint Surg., 64, 1982, № 4, 494-505. 12. H i l l , N. A. et L. H. Mc. Locked posterior dislocation simulating a ‘frozen shoulder’. – J. Trauma, 3, 1963, 225-234 . 13. M c L a u g h l i n , H. L. Dislocation of the shoulder with tuberosity fracture. – Surg. Clin. North. Am., 43, 196, 31615-1620. 14. I r l e n b u s c h , L. et al. Possibilities for the operative treatment of traumatic posterior shoulder dislocation. – Unfallchirurg, 111, 2008, № 6, 464-468. 15. O ’ C o n n o r , S. J. et A. S. Jacknow. Posterior dislocation of the shoulder. – AMA Arch. Surg., 72, 1956, № 3, 479-491. 16. W i l s o n , J. C. et K. F. Mc. Traumatic posterior dislocation of the humerus. – J. Bone Joint Surg. Br., 31A, 1949, № 1, 160-172. 17. H u g h e s , M. et C. S. Neer II. Glenohumeral joint replacement and postoperative rehabilitation. – Phys. Ther., 55, 1975, № 8, 850-858. 18. M c L a u g h l i n , H. Posterior dislocation of the shoulder. – J. Bone Jt. Surg., 24-A-3, 1952, 584-590. 19. G a v r i i l i d i s , I. et al. Chronic locked posterior shoulder dislocation with severe head involvement. – Int. Orthop., 34, 2010, № 1, 79-84. 20. S e y b o l d , D. et al. A new method of closed reduction of locked posterior shoulder dislocation. – In: Paper presented at the Congress of the German Association of Shoulder and Elbow Surgery (DVSE), 2007. 21. I t o i , E. et al. Position of immobilization after dislocation of the shoulder. A cadaveric study. – J. Bone Jt. Surg., 81, 1999, № 3, 385-383. 22. K i r c h h o f f , C. et al. Traumatic posterior shoulder dislocation: diagnosis and therapy. – Unfallchirurg, 110, 2007, № 12, 1059-1064. 23. M a l g a i g n e , J. F. Traité des Fractures et Luxationes. – JB. Bailliere, Paris, 1855. 24. D i k l i c , I. D. et al. Treatment of locked chronic posterior dislocation of the shoulder by reconstruction of the defect in the humeral head with an allograft. – J. Bone Joint Surg. Br., 92, 2010, № 1, 71-76. 25. S p e r l i n g , J. W. et al. Shoulder arthroplasty for locked posterior dislocation of the shoulder. – J. Should. Elb. Surg., 13, 2004, № 5, 522-527. 26. S p e n c e r , E. E. Jr et J. J. Brems. A simple technique for management of locked posterior shoulder dislocations: report of two cases. – J. Should. Elb. Surg., 14, 2005, № 6, 650-652. ª Address for correspondence: B. Matev, DM Clinic of Surgery fo the Upper Limb USHATO − "Prof. B. Boychev" 56 Nikola Petkov blvd. 1614 Sofia, Bulgaria 02/81-81-622 0888 46 11 75 e-mail: [email protected] Acta Medica Bulgarica, Vol. XXXIX, 2012, № 1 119