Acta medica-1-11

MEDICAL UNIVERSITY − SOFIA

This journal is indexed in Global Health Database,in Bulgarian Medical Literature Database andin Scopus

ACTA MEDICABULGARICAambvol. XXXVIII

1/20

11

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

Acta Medica Bulgarica, Vol. XXXVIII, 2011, № 1 3

OSTEOCLASTOMA OF METACARPAL BONESH. Georgiev, B. Matev, N. Dimitrov and P. Georgiev

University Specialized Hospital for Active Treatment of Orthopaedics “Prof. B. Boychev” – Sofi a

Summary. Osteoclastoma is a locally aggressive tumor, very rarely located in bones and joints (locomotory system). In 1987, Matev presented his 20-year study of 502 tumors of the hand, where no osteoclastoma cases were observed. In our study which tracks a period of 20 years, we found and treated 3 cases of osteo-clastoma of the metacarpal bone and for the fi rst time in Bulgaria we are presenting them. We follow consecutively the clinical picture, the applied treatment and the results. The conclusions show that it is an extremely rare aggressive tumor, com-plicated to treat, so that early diagnosis and therapy is the key to a comprehensive treatment, for organ and hand-function preservation.

Key words: osteoclastoma, giant cell tumor, hand

Pain in the hand and the forearm is a frequent symptom of different dis-eases: over-tension conditions, sport traumas, neurological diseases etc. A chronic dull pain in young people localized in the hand area,

although rare, is also the fi rst symptom for primary bone cancer disease. Our study presents extremely rare cases of osteoclastoma of metacarpal bones, for the fi rst time described in Bulgaria.

Osteoclastoma is a local aggressive tumor, with strong inclination to recidi-vate, to give “benign” lung metastases or to transform into a real malignant blasto-ma. This is the reason for this tumor to be a specifi c clinical-X ray and pathological anatomy unit, fi led in a separate group of the bone cancer classifi cation [12, 13].

This giant cell tumor received this name and was described in 1818 by Sir Astley Cooper. In 1922, Steward introduced the British term osteoclastoma. Jaffe defi ned this primary bone tumor as quasi-malignant, because approximately 20% of the lesions are malignant, while 80% are benign. In 1940, the same author divided osteoclastoma in three stages depending on the histological picture [14]. It is assumed that the single-nucleus cells, the basic ones in the tumor, originate from the undifferentiated mesen-chymal cells or the supporting connective tissue of the marrow [7, 11, 12, 13, 16, 18].

Osteoclastoma of metacarpal bones4

CLINICAL MATERIAL

Three patients with osteoclastoma of the metacarpal bones have been diag-nosed and treated at “Prof. B. Boychev” University Ortopaedics Hospital – Sofi a, for over a twenty-year period. The patients were 34 years and 6 months old at the average, women prevailing by 2:1.

The clinical picture of the three cases was identical: complaints of a dull and constant pain localized in the arm. Presence of a swelling with hard consistence, painful at palpation. In some of the cases, the complaints were accompanied by a minimal reduction of the movements in the adjacent joint.

X-ray picture: Roentgenography of two surfaces showed metaepiphyseal os-teolytic focus without periostal reaction. In one of the cases, the lesion was con-fi ned to the bone, and the corticalis was strongly thinned and infl ated. In the other two cases, due to its aggressive character, the tumor had destroyed the cortex and spread over the adjacent soft tissues and joint. In one of the cases, the tumor had a characteristic X-ray cell-like look – “soap bubbles”. The lytic character was expressed in the other two cases.

Pathological anatomy picture: The intraoperative fi nding was a tissue of dark-red to brownish color with zones of yellow-grayish color resulting from occurred necroses. The tumor had soft consistency, was fragile and friable. The histologi-cal pictures showed presence of single-nucleus cells of round, oval and spindle shapes, as well as giant multi-nucleus cells situated in loose intercellular stroma.

The treatment strategy in every case was surgically individualized. In one of the cases, recidivating occurred.

Patient: K.T.K. – male, aged 41, with osteoclastoma of the fourth metacarpal bone on the right, and affected proximal 2/3 of the bone. Histology – Ist degree after Jaffe: Abundant giant multi-nucleus cells of osteoclastic type and sinlge-nucleus cells – oval and spindle-shaped, with nidus accumulation of xanthoma cells. Sev-eral consecutive stages of surgical treatment were applied: block resection of IV th metacarpal bone with auto-osteoplastics and carpo-metacarpal arthrodesis. The next stage included falangeal endoprosthesis with Matis bipolar joint. Fig 1.

A B C

A 3D – CT scan – primary diagnostic view; B Radiography after autoosteoplastics from fi bula; C Finally radiography – endoprosthesis of carpometacarpal joint

Fig. 1. Patient: K.T.K. – male, 41 age


Patient: D.O.S. – female, aged 25 with giant cellular tumor in the Vth meta-carpal bone. After biopsy, the histological diagnosis was osteoclastoma, locally aggressive of II-III degree: Soft tissues – skin with focused infl ammatory changes in the papillary layer of the derma and a blastoma – well restricted by the hypo-derma, and hypercellular due to the prevailing single-nucleus cells with big round vesicular nuclei and distinct small nuclei in part of them and presence of mitoses. Giant multi-nucleus cells of osteoclastic type. Focal bone formation and infi ltra-tion in the skeleton muscles as well as in the adjacent spongioid bone. Due to the aggressive character of the tumor, disarticulation of the whole fi fth ray of the right hand was performed. Two years later – no data for recidivating and “benign” lung metastases. Fig 2.

A B C

A Diagnostic primary radiography; B One year later radiography; C Postoperative radiography – Disarticulation of the V th ray

Fig. 2. Patient: D.O.S. – female, 25 age

Patient: Sht.A.D. – female, aged 38 with metacarpal osteoclastoma – Ist and IInd degree after block resection of the bone. Reoperated after six months fol-lowing recidivation. A larger block resection of the bone with aloosteoplastics was performed. Two years later – no data for recidivation.

DISCUSSION

The giant cell tumor is a primary neoplasma representing 5-8% of all primary malignant bone tumors and 15% of all primary benign bone tumors [19]. According to Andreev, the osteoclastoma rates at 6, 6% of all primary new formations and tumor-like diseases of the skeleton [1]. The most frequent localizations of affection are bones around the knee joint, the distal end of the radius, the proximal humerus, the proximal part of the femoral bone and so on in this order, with reducing fre-quency. Localization of the tumor in hand bones is quite rare, about 2-3% [1, 8, 16,


20]. Still rarer is osteoclastoma isolated only in metacarpal bones, less than half of all hand osteoclastomas [8]. In an extensive study by Matev and co., treating 502 patients with tumors of the hand, there is no case of osteoclastoma [3]. As a consequence, we treat our three cases not as exotic casuistic, but as an important differential diagnosis direction.

The age diapason of osteoclastoma is 20-40 years – the most active age. Frequency in males and females is almost identical, benign osteoclastomas prevail in females corresponding to 3:2, while malignant prevail in males at 3:1 [19]. All our three cases are typical of the known statistics.

The clinical expression of the tumor localized in metacarpal bones, especially at the beginning, is not typical – complaints of a dull, constant pain in the area of the hand. Complaints are similar as in light chronic hand trauma, pressure of n.medianus or n.ulnaris syndrome or mm. lumbricales et interosei insertionites. Only later swelling of hard consistence, painful at palpation and restriction of move-ments in the adjoining joint appear. Sometimes the tumor is discovered only after a pathological fracture occurs.

X-ray is basic for the diagnosis. The tumor is visualized as an eccentrically situated metaepiphyseal osteolytic focus. Periostal reaction is lacking. The lesion can be reduced to the bone, the corticalis could be strongly thinned and infl ated or due to its aggressive character, the tumor can destroy the cortex and spread over the adjoining soft tissues and joint [4, 21]. In about 60% of the cases, the lesions have a purely lytic picture, while in 40% the picture is “soap bubbles” type as an expression of the reactive bone trabeculi formed in the tumor [6, 9]. On this basis the M. Campanacci et al. classifi cation is built, which divides osteoclastomas into latent, active and aggressive forms, as well as the adopted in Bulgaria Andreev classifi cation, describing the tumors as quite, cell-like and osteolytic types [4, 5].

The histological diagnosis confi rms the X-ray and the intraoperative fi ndings and is basic for the adoption of the therapeutic plan.

Prognosis of the benign lesions is good, but only 10% of patients with the malignant form of osteoclastoma are still alive after the 5th year. That is why timely and adequate surgical treatment is of vital importance. Several methods for osteo-clastoma treatment are proposed: curetage, curetage and plombage with cortical bone or biomaterial, resection, resection with bone graft, amputation and disarticu-lation [2, 8].

The curetage is easily performable and organ-preserving intervention, but carries a big risk for recidivation – in some studies from 47 to 80% recidivated [9, 15]. Curetage as an independent procedure is recommended only for cases with very small lesions and unaffected cortex of the respective bone. We have not ap-plied this technique on our patients.

En block resection of the affected part of bone is the safest way to avoid recidives, leading to cure. M. Campanacci et al. report only 13% recidivated after resection [5, 6]. The necessity to further surgery interventions is taken into con-


sideration here, for replacing one of the joint poles which is also a joint surface, with an autotransplant and performing arthrodesis or replacing the defection with endoprosthesis. In two of our cases, we chose the surgical method of resection of affected bone, the fi rst – with autograft and arthrodesis to the wrist joint, followed by metacarpal-falangeal endoprosthesis and the second recidivated, imposing a wider resection and allotransplant.

Amputation or desarticulation of the entire ray of the hand is applied when the malignant form of osteoclastoma tumor has been proven and has affected a big part of the bone and the adjacent soft tissues [10]. Desarticulation of the entire Vth ray was performed in one of the cases of our clinical material with histological results for local aggressive osteoclastoma of II-III degree.

CONCLUSION

Osteoclastoma is an extremely rare tumor, which nevertheless provokes quite diffi cult issues to solve. For the fi rst time in Bulgaria we present three cases of osteoclastoma of the metacarpal bone. Prognosis, treatment and its results are directly dependent on early diagnosis and adequate therapy. We treat the pre-sented cases of osteoclastoma with metacarpal localization as a contribution to the differential diagnosis fi eld of hand diseases. Our cases demonstrate the necessity for early diagnosis thus giving the chance for organ-preserving surgery to keep the functions of the hand.

REFERENCESA n d r e e v , Iv. et R. Raychev. [Tumors of the bones.]. Sofi a, Med. i fi zk., 1993, 97-113. (in Bulgarian)1. A n d r e e v , Iv. et R. Raychev. [Operative treatment of the giant cell tumor of the bones.] – Ortop. 2. Traum., 10, 1973, № 3-4, 208. (in Bulgarian)M a t e v , Iv. [Tumors of the hand. – In: Diseases of the hand. Bossnev, V., Matev, I. (eds.)] Sofi a, 3. Med. i fi zk., 1989, 131-137. (in Bulgarian)H a d j i d e k o v , G., Iv. Andreel et L. Velikov. [Rentgenological particularities of osteolytic variant of 4. osteoclastoma of the long tubular bones.]. – Onkologiya (Sofi a), 7, 1970, № 3, 117. (in Bulgarian)C a m p a n a c c i , M., A. Guini еt R. Olmi. Giant cell tumors of bone. A Study of 209 cases with 5. long follow-up in 1130. – Ital. J. Orthop. Traumatol., 1, 1975, 249.C a m p a n a c c i , M. et al. Giant cell tumor of bone. – J. Bone Joint Surg., 6. 59A, 1987, 106-114.F u j i w a r a , Y. Tissue culture study of the giant-cell tumor of bone. – Arch. Jap. Chir., 7. 36, 1967, 803.G l i c e n s t e i n , J., J. Ohana et C. Leclercq. Tumors of the Hand. Berlin, Heidelberg, Springer-8. Verlag, 1988, 189-191.G o l d e n b e r g e r , R. R., C. J. Campbell et M. Bonfi glio. Giant cell tumor of bone. An analysis of 9. 218 Cases. – J. Bone Joint Surg., 52A, 1970, 619-663.G u p t a , S., A. Kumar et I. Gupta. Giant cell tumor of the fi rst metacarpal bone. – Hand, 10. 12, 1980, 288-292.H u t t e r , R. V. P. et al. Benign and malignant giant cell tumors of bone. A Clinical-pathological 11. analysis of the natural history of the disease. – Cancer, 15, 1962, 65.


J a c o b s , Th. et al. Giant cell tumor in Paget’s disease of bone. – Cancer, 12. 20, 1979, 742.J a f f e , H. L. Tumors and tumorous conditions of bones and joints. Philadelphia, Lea & Febiger, 13. 1958.J a f f e , H. L., L. Lichtenstein et B. Portis. Giant cell tumor of bone. Its pathologic appearance, 14. grading, supposed variants and treatment. – Arch. Pathol., 30, 1940, 993.R o c k , M.G. et al. Secondary malignant giant cell tumor of bone: Clinical pathological assess-15. ment of 19 patients. – J. Bone Joint Surg., 68A, 1986 1073-1079.S a i k i a , K. C. et al. A. Rare site giant cell tumors: report of two cases on phalanges of the fi nger 16. and review of literature. – J. Orthop. Traumatol., 10, 2009, № 4, 193-197.S c h a i o w i c z , F. Giant cell tumors of bone (Osteoclastoma). A pathological and histochemical 17. study. – J. Bone Joint Surg., 43-A, 1961, 1.S p j u t , H. J. et al. Tumors of Bone and Cartilage. 18. – In: Tumor Pathology. Washington, D. C., Armed Forces Inst. Pathology, 1971.Ya s u d a , T. et al. Multicentric diffuse-type giant cell tumor of the hand. – Mod. Rheumatol., 19. 18, 2008, № 1, 67-71.Y i p , K. M., P. C. Leung et S. M. Kumta. Giant cell tumor of bone. – Clin. Orthop. Relat. Res., 20. 323, 1996, 60-64.Yo c h u m , T. R. et L. J. Rowe. Essentials of skeletal radiology. Ed.2. Vol. 2. Baltimore, Williams 21. & Wilkins, 1996, 1054-1059.

Addresses for correspondence: Assoc.prof. Dr. Hristo Georgiev, PhD Clinic of Children’s Orthopaedics USHATO “Prof. B. Boychev” – Sofi a Sofi a, 56 N. Petkov Blvd.

00359887621513 е-mail: [email protected]


HEMOSTATIC MONITORING OF THE PERIOPERATIVE FIBRINOLYTIC ACTIVITY DURING LIVER

TRANSPLANTATIONA. Stancheva1, L. Spassov1 and K. Tzatchev2

1University Hospital “Lozenets”, Sofi a, Bulgaria2Medical University, Sofi a, Bulgaria

Summary. Rotation thrombelasthometric analysis (ROTEM®) is a global he-mostatic method, initiating the coagulation process both by contact surface activa-tion and by additional activating reagents. The graphical and numerical ROTEM® results refl ect the whole clot formation process and the next-coming fi brinolysis, as well as a great number of hemostatic and therapeutic factors, changing the kinetics of the hemostatic process.The performance of an extended perioperative fi brin-olytic and therapeutic control during liver transplantation becomes possible with the development of the rotation thrombelastometry method ROTEM®. Perioperative hemostatic monitoring was performed to 30 patients undergoing orthotopic liver transplantation (13 male – 42% and 17 female – 58%), age (mean ± SD): 21±17 years. A STA Compact instrument (Diagnostica Stago-Roche, France, chronometric method) was used to determine prothrombin time (PT/INR), activated partial throm-boplastin time (APTT) and fi brinogen (FIB). Rotation Thrombelastometry analyses were performed on ROTEM® analyzer (Pentapharm GmbH, Munich, Germany) in citrated blood, based on reagent activation of the hemostatic processes with throm-boplastin tissue factor (TF) for EXTEM test and aprotinin for APTEM test. The cor-relation between MCF and A15 parameters was assessed to explore the possibility for application of A15 during the monitoring. The correlation between MCF and A15 parameters was excellent for EXTEM, INTEM and FIBTEM tests (r=0,98 p<0,001). The highest percentages of patients from the general group with increased fi brin-olytic activity (33,33%) and with hyperfi brinolytic activity (8,7%) were determined during the anhepatic period R4. 16 % from the group with heavy coagulopathy displayed hyperfi brinolytic activity in the anhepatic period R4, while no patient from the moderate coagulopathy group developed hyperfi brinolysis during all periop-erative periods R1-R6. During the anhepatic period (R4), the percentage of adults with increased fi brinolytic activity was signifi cantly higher than the percentage of children (adults 41,66% and children 25,00%). Conclusions are that EXTEM and

Hemostatic monitoring of the perioperative...10

APTEM parameters could be used for determing the increased fi brinolytic and hy-perfi brinolytic activity during the preanhepatic, anhepatic and reperfusion periods (R3, R4, R5) of liver transplantation. Patients with considered preoperative heavy coagulopathy are more risky to develop hyperfi brinolysis during the preanhepatic and anhepatic periods ( R3-R4). The established excellent correlations between MCF and A15 parameters suggest the application of A15 as satisfactory for the monitoring of fi brinolytic activity.

Key words: rotation thrombelastometry ROTEM®, fi brinolysis, liver transplantation

INTRODUCTION

The perioperative hemostatic monitoring during liver transplantation in-cludes performing clinical-laboratory diagnostics of the arising coagul-opathies [4, 3], suggesting adequate medicamentous and substitutive

therapy [6] and following up the effect of the applied therapy [14].The possibilities of standard coagulation and fi brinolytic parameters for diag-

nostics of the perioperative states of hyperfi brinolysis or increased coagulability, are estimated as not suffi ciently quick or informative, with no prognostic value for hemostatic bleeding and necessity for hemotransfusion, when concerning the dy-namic coagulation changes during liver transplantation.

The performance of an extended perioperative fi brinolytic and therapeutic con-trol during liver transplantation becomes possible with the development of the ro-tation thrombelastometry method ROTEM® (3). The ROTEM® EXTEG and APTEM activated tests are used for the diagnostics of fi brinolitytic activity. Increased values of CT and CFT parameters of EXTEM , with normal values of APTEM indicate in-creased or hyperfi brinolytic activity and are treated with antifi brinolytics [1, 8, 9, 17].

The aim of the study was to assess the fi brinolytic and hyperfi brinolytic activity during the perioperative periods (R1-R6) of orthotopic liver transplantation by rota-tion thrombelastometric method ROTEM®.

MATERIALS AND METHODS

MaterialsPerioperative hemostatic monitoring was performed to 30 patients undergoing

orthotopic liver transplantation (13 male – 42% and 17 female – 58%), age (mean ± SD): 21±17 years.

The patients were classifi ed in the following groups:General group (n=30)1. According to age – up to 10 years (n=15); up to 48 years (n=15)2. According to the type of the preoperative coagulopathy – heavy coagul-3.

opathy (n=16); moderate coagulopathy (n=14)


(With heavy coagulopathy were considered patients with PT(INR) > 1,6; APTT > 1,5 ratio, PLT< 100 х 109/L, FIB < 1,0 g/l), one hour preoperatively [18].

Blood samples were collected according to a study design protocol:One hour preoperatively :•

the perioperative period (R1) Four times intraoperatively:•

10 min. after the intervention – the dissection period (R2)10 min. before clamping – the preanhepatic period (R3)10 min. before declamping – the anhepatic period (R4)30 min. after grafts’ reperfusion – the reperfusion period (R5)

Two hours postoperatively: • the postoperative period (R6)The correlation between MCF and A15 parameters was explored for EXTEM,

INTEM and FIBTEM tests in six groups: a reference group (n=20), the general group for liver transplantation (n=30), two groups according to age (n=15) and two groups – with heavy or moderate preoperative coagulopathy (n=15).

A STA Compact instrument (Diagnostica Stago-Roche, France, chronometric method) was used to determine prothrombin time (PT/INR) (STA Neoplastin®plus, Di-agnostica Stago, France) and activated partial thromboplastine time (APTT) (STA aPTT Diagnostica Stago, France, chronometric method). Levels of fi brinogen (FIB) were as-sayed according to Clauss technique (STA Fibrinogen, Diagnostica Stago, France).

Platelets (PLT) and red blood cells (RBC) were determined in EDTA plasma on Cell Dyn 3700 (Abbott Diagnostics, Germany, MAPSS technology).

Rotation Thrombelastometry analyses were performed on ROTEM® analyzer (Pentapharm GmbH, Munich, Germany) in citrated blood.

The fi brinolytic activity was assessed by ROTEM® method – a global hemo-static method for fast diagnostics of hemostatic disorders with acute bleeding, leading to hypercoagulation, hypocoagulation or hyperfi brinolysis [3]. Like classi-cal thrombelastography, ROTEM® analysis is expressed graphically (by a reaction curve – TEM-ograme) and numerically (by thrombelstometric parameters).

The new ROTEM® methodology is based on reagent activation of the global haemostatilc processes in a whole blood assay, using supplements like thromboplas-tin tissue factor (TF) for test EXTEM, contact activator (ellagic acid) for test INTEM, anti-platelet reagents (anti IIb/IIIa, cytochalasin D) for test FIBTEM, aprotinin for test APTEM, heparinase for test HEPTEM and ecarine activator for test ECATEM.

STATISTICAL ANALYSIS

Statistical analysis was performed using SPSS 17.0.1. software. To determine the perioperative fi brinolytic activity, the International Guidelines of the Expert Group on Rotation Thrombelastometry 2005-2009, Munich, Germany were followed.


Thrombelastometric indications for increased fi brinolytic activity were deter-mined in cases of:

APTEM_CT<EXTEM_CT ≥ ∆ 10%,

APTEM_MCF >EXTEM_MCF ≥ ∆ 10% ≥ ∆ 10%

(p<0,05)

Thrombelastometric indications for hyperfi brinolytic activity were determined in cases of:

АРТЕМ_СТ / ЕХТЕМ_CT < 0.75

АРТЕМ_MCF/ ЕХТЕМ_MCF > 1,25 or

АРТЕМ_CT < ЕХТЕМ_CT ≥ Δ 25%

АРТЕМ_MCF > ЕХТЕМ_MCF ≥ Δ 20%

>20%( MCF_ EXTEM < 35 mm; p < 0,05)

RESULTS

The correlations established between MCF and A15 parameters were excel-lent and very good for EXTEM, INTEM and FIBTEM tests in the reference group and in all groups of liver transplanted patients. (Tables 1, 2, 3).


Table 1. Correlation (r) between INTEM_MCF and INTEM_ A15 in different groups

Parameter Group

INTEM_ A15

Referencegroup

General patients’

group

Moderate coagulopathy

group

Heavy coagulopathy

groupChildren Adults

INTEM_MCF

Referencegroup

0,937***

General patients’ group

0,980***


group0,913***

Heavy coagulopathy

group0,986***

Children 0,947***

Adults 0,991***

*** – p < 0.001

Table 2. Correlation (r) between EXTEM_MCF and EXTEM_A15 in different groups

Parameter Group

EXTEM_ A15

Referencegroup

General patients’

group


group

Heavy coagulopathy


ЕХTEM_MCF

Referencegroup

0,878***

General patients’ group

0,981***


group0,955***

Heavy coagulopathy

group0,993***

Children 0,985***

Adults 0,988***

***– p < 0.001


Table 3. Correlation (r) between FIBTEM_MCF and FIBTEM_A15 in different groups

Parameter Group

FIBTEM_ A15

Referencegroup

General patients’

group


group

Heavy coagulopathy


FIBTEM_MCF

Referencegroup 0,830***

General patients’ group 0,892***


group0,819***

Heavy coagulopathy

group0,985***

Children 0,844***

Adults 0,901******– p < 0.001

The highest percentage of patients with increased fi brinolytic activity from the general group (33,33%) was determined in the anhepatic period (R4), with calcu-lated ROTEM parameters APTEM_CT < EXTEM_CT > Δ 10% and APTEM_A10 >EXTEM_A10 > Δ 10%.

During the anhepatic period (R4), in the group with heavy coagulopathy a sig-nifi cantly higher percentage of patients with increased fi brinolytic activity (46,15%) was determined, than in the group with moderate coagulopathy (18,8%). This per-centage decreased in the course of the operation for both groups – (30,7% and 28% (R5); 15,38% and 11,8% (R6) - and the difference was insignifi cant (fi g. 1).

Fig. 1. Patients from the corresponding groups with activated fi brinolysis during the perioperative peri-ods R1-R6 (APTEM_CT < EXTEM_CT > Δ 10% and APTEM_A15 >EXTEM_A15 > Δ 10%)


The childrens’ group didn’t present increased fi brinolytic activity in the fi rst two periods (R1, R2). The percentage of the adults with increased fi brinolytic activity was signifi cantly higher than the percentage of the children in the preanhepatic (R3) and anhepatic period (R4). (adults 41,66% and children 25,00%). The dif-ference between the two groups was insignifi cant during next periods R5 (adults 33,33% and children 25,00%) and R6 (fi g. 2).

Fig. 2. Patients from the corresponding groups with activated fi brinolysis during the perioperative peri-ods R1-R6 (APTEM_CT < EXTEM_CT > Δ 10% and APTEM_A15 >EXTEM_A15 > Δ 10%) The highest percentage of patients with established hyperfi brinolytic activ-

ity from the general group (8,7%) was also determined in the anhepatic period (R4), and decreased to 4,35% during the next R5, R6 periods, with caculated СТ_ АРТЕМ < СТ_ЕХТЕМ > Δ 25% and АРТЕМ_СТ/_ЕХТЕМ CT < 0,75; АРТЕМ_MCF/ЕХТЕМ_MCF > 1,25. 16 % of the patients from the group with heavy co-agulopathy displayed hyperfi brinolytic activity in the anhepatic period R4, while no patient from the moderate coagulopathy group developed hyperfi brinolysis during the perioperative periods (fi g. 3).

Fig. 3. Patients from the corresponding groups with activated hyperfi brinolysis during the perioperative periods R1-R6 ( СТ_ АРТЕМ < СТ_ЕХТЕМ >Δ 25%) and АРТЕМ_ СТ/_ЕХТЕМ CT<0,75),

(АРТЕМ_ A15/ЕХТЕМ_ A15 > 1,25)


In the childrens’ group, the percentage of patients with hyperfi brinolytic activity (12%) was less than the percentage in the adults’ group (13,8%) (fi g. 4).

Fig. 4. Patients from the corresponding groups with activated hyperfi brinolysis during the perioperative periods R1-R6 ( СТ_ АРТЕМ < СТ_ЕХТЕМ > Δ 25%) and АРТЕМ_ СТ/_ЕХТЕМ CT<0,75),

(АРТЕМ_ A15/ЕХТЕМ_ A15 > 1,25)

DISCUSSION

The assessment of the ROTEM® amplitude on the 15th min. (INTEM_A15, ЕXTEM_A15 or FIBTEM_А15) is performed in a fi xed and shorter interval when compared to MCF. The established excellent correlations between MCF and A15 parameters in our study suggest the application of A15 as satisfactory for monitoring the fi brinolitical activity. This is extremely important in cases of urgency with acute bleeding, when the diagnosis is established on the 15th min. before the themogram has reached the maximum clot fi rmness (MCF). Rugeri et al have determined similar correlations in patients with severe trauma [18].

In our study, the fi brinolytic activity under the urgency conditions of liver transplantation was determined through calculating rotation thrombelastometric parameters of APTEM and EXTEM tests, which was in concordance with the investigations of Goerlinger et al. and the Guidelines of the Expert Group [7].

The highest percentage of patients with increased fi brinolytic activity in all groups was detected during the anhepatic period (R4), perhaps due to the reduced biosynthesis of fi brinolytic inhibitors and the absence of the purge out function of the reticuloendotelium system of the liver in this period. The determined in our study most frequent manifestation of activated fi brinolysis during the anhepatic period confi rms the observations of several contemporary authors [2, 5, 12, 15, 16].

The displayed signifi cant difference between the groups with heavy and with moderate coagulopathy in developing hyperfi brinolysis, could be possibly


due to the differences in the hemostatic preoperative status. The heavy preoperative coagulopathy could be regarded as a possible reason for a next-coming manifestation of intraoperative hyperfi brinolysis. The established by us paraoperatively activated hyperfi brinolysis, expressed mostly during the anhepatic period, confi rms the investigations of J. H. Levis [11], Y. G. Kang [10], D. J. Loskutoff [13] who explained the established hyperfi brinolysis with the increased production of tPA from the endothelium cells and lack of clearance in the same period (R4). Less children developed increased fi brinolytic activity in the early R1-R3 transplantation periods, when compared to the adults’ group. A probable reason for this could be the better preoperative general hemostatic status in this group. In childrens’ group, hyperfi brinolysis was unexpectedly established only during the preanhepatic period (R3).

CONCLUSSION

According to the ROTEM® guided hemostatic monitoring, patients with preoperative heavy coagulopathy are more risky to develop hyperfi brinolysis during the preanhepatic and anhepatic periods (R3-R4). This could determine the application of antifi brinolytic premedication as prophylactics for patients with heavy preoperative coagulopathies.

The established excellent correlation between MCF and A15 parameters in our study suggests the application of A15 as satisfactory for the monitoring of fi brinolytical activity.

For determing the increased fi brinolytic activity the following relations between EXTEM and APTEM parameters could be used: APTEM_CT < EXTEM_CT > Δ 10% and APTEM_A15 > EXTEM_A15 > Δ 10%. Hyperfi brinolysis could be defi ned through the relations: СТ_АРТЕМ < СТ_ЕХТЕМ > Δ 25%; АРТЕМ_A15 > ЕХТЕМ_A15 ≥ Δ 20% and АРТЕМ_СТ/_ЕХТЕМ CT < 0,75; АРТЕМ_A15/ЕХТЕМ_A15 > 1,25.

REFERENCESA v i d i a n , M. et al. The effect of aprotinin on thrombelastography with three different activators. – 1 . Anesthesiology, 95, 2001, 1169-1174.B e c h s t e i n , W. O. et P. Neuhaus. Blutungsproblematik in der Leberchirurgie und Lebertrans-2 . plantation. – Chirurg, 71, 2000, 363-368.C a l a t z i s , A. N. et al. Fast and specifi c coagulation monitoring through modifi ed thrombelastog-3 . raphy. – Ann. Hematol., 1996, Suppl 1., P 92.C a l a t z i s , A. N. et al. Thrombelastographic coagulation monitoring during cardiovascular sur-4 . gery with the ROTEG coagulation analyzer. – Cardiovasc. Surg., 119, 2000, 215-226.F i n d l y, J. Y. et S. Retteke. Aprotinin reduces blood cell transfusion in orthotopic liver transplanta-5 . tions. – Liver Transpl., 7, 2001, 808-810.G o e r l i n g e r , K. et al. ROTEM-based management for diagnosis and treatment of acute haemor-6 . rhage during liver transplantation. – Eur. J. Anaesth., 23, 2006, 85.G o e r l i n g e r , K. ROTEM- Erweitertes perioperatives Gerinnungsmanagement. – JAI, 7 . 12, 2005, 53-58.


G r o s s e , H. et L. van Obbereh. Platelet aggregates in small blood vessels and death during liver 8 . transplantation. – Lancet, 338, 1991, 532-534.H i m m e r l e i c h , G. et al. Different approtinin application infl uencing hemostatic changes in 9 . orthotopic liver transplantation. – Transplantation, 53, 1992, 132-136.K a n g , Y. G. et al. Intraoperative changes in blood coagulation and thrombelastographic monitoring 1 0 . in liver transplantation. – Anesth. Analg., 64, 1985, 888-896.L e w i s , J. H. et al. Liver transplantation: intraoperative changes in coagulation factors in 100 fi rst 11 . transplants. – Hepatology, 9, 1989, 710-714.L l a m a s , P. et R. Cabrera. Haemostasis and blood requirements in orthotopic liver transplantation. 1 2 . – Haemostageologica, 83, 1998, 38-46.L o s k u t o f f , D. J. et T. E. Edgington. Synthesis of a fi brinolytic activator and inhibitor by endothelial 1 3 . cells. – Proc. Natl. Acad. Sci. USA, 74, 1977, 3903-3907.M a h l a , E. et al. Thrombelastography for monitoring prolonged hypercoagulability after major 1 4 . abdominal surgery. – Anesth. Analg., 92, 2001, 563-564.P i e t s c h , U. C. et L. Schaffrainietz. Anaesthesiologisches Vorgehen bei OTX. – Ergebnisse einer 1 5 . Umfrage. – AINS, 41, 2006, 21-26.P o r t e , R. J. et I. Q. Molenaar. Aprotinin and transfusion requirements in OLT. EMSALT Study 1 6 . Group. Lancet, 355, 2000, 1289-90.R a m s a y, M., H. Randall et E. Burton. Intravascular thrombosis and thromboembolism during 1 7 . liver transplantation: Antifi brinolitic therapy indicated. – Liver Transplant., 10, 2004, 310-314.R u g e r y, L. et al. Diagnosis of early coagulation abnormalities in trauma patients by rotation 1 8 . thrombelastography. – J. Thromb. Haemost., 5, 2007, 289-295.

Address for correspondence: Assia Stancheva, MD Universtity Hospital "Lozenets" 1 Koziak str. 1407 Sofi a e-mail: [email protected]


UNCOMMON CLINICAL PRESENTATION OF FIBROSARCOMA OF THE THYROID GLAND

IN A PATIENT WITH FATAL OUTCOME: THE ROLE OF IMMUNOHISTOCHEMISTRY FOR CONFIRMATION

OF THE DIAGNOSISJ. Ananiev1, M. Gulubova2, I. Manolova3, G. Tchernev4, V. Ramdan5, V. Velev6 and J. Gerenova7

1,6Department of General and Clinical Pathology, Medical Faculty – Trakia University, Stara Zagora

3Laboratory of Clinical Immunology, University Hospital, Stara Zagora 4Department of Dermatology and Venereology, Medical Faculty, Trakia University,

Stara Zagora5Department of Anesthesiology and Intensive Care Medicine, University Hospital,

Stara Zagora7Department of Internal Medicine, Clinic of Endocrinology, University Hospital, Stara Zagora

Summary. We report a case of a rare thyroid gland tumor – fi brosarcoma, and autopsy fi ndings. A 79-year-old woman presented with a twenty-year history of a thyroid gland enlargement. Since twenty days she had respiratory failure and dull pain in the anterior neck region. Preliminary cytological diagnosis confi rmed undifferentiated thyroid gland tumor. The patient died four days after hospitalization with symptoms of complete respiratory failure, hypoxemia and suspected infl am-mation of the lungs. Autopsy revealed primary neoplasm оf the thyroid gland area, infi ltrated trachea and adjacent tissues; acute tracheitis and lobular pneumonia. Histopathologically, the primary tumor showed proliferation of spindle-shaped tu-mor cells and was ultimately confi rmed by immunohistochemistry as a primary fi b-rosarcoma of the thyroid gland.

Key words: fi brosarcoma, thyroid gland, immunohistochemistry, vimentin

INTRODUCTION

Fibrosarcomas are relatively uncommon tumors and account for 12-19% of soft tissue sarcomas. More than half of all tumors arise in the lower extremities; approximately 10% occur in the head and neck, most com-

Uncommon clinical presentation...20

monly in the sinonasal tract and neck. Moreover fi brosarcoma is exceptionally rare neoplasm of thyroid gland [1]. Depending on differentiation, tumor cells may re-semble mature spindle-shaped fi broblasts, secreting collagen, with rare mitoses. The prognosis as fi brosarcoma greatly depends on the extent of the disease, the size and location of the tumor, presence or absence of metastases and the tumor’s response to therapy.

The data of this type of tumor in literature are scanty and we did not fi nd any thorough publications about thyroid fi brosarcoma in our country.

In this article, we consider the existence of true fi brosarcoma originating in the thyroid gland and confi rm that fi brosarcomas can arise de novo in the thyroid gland.

CASE REPORT

A 79-year-old woman presented with a swelling in her anterior neck. Her med-ical history was unremarkable. On physical examination, a non well-circumscribed, elastic fi rm soft tissue mass was palpated. The mass was attached to the deep tis-sues and was not mobile. Its maximum width was about 66 mm. The skin over the swelling was slightly reddish and showed no signs of either ulceration or necrosis. Laboratory tests revealed no abnormalities.

From fi ne needle aspiration cytology (before exitus letalis) we suspected cells like of an anaplastic – undifferentiated carcinoma or fi brosarcoma of the thyroid (Fig. 1). The neoplastic cells were randomly distributed and dyshesive. Occasional small aggregates occured. The majority of cells in this low-grade tumor are spindle-shaped, with occasional tadpole, stellate, and polygonal forms. Nuclear pleomor-phism and atypia is obviously.

Fig. 1. Fine needle aspiration cytology – the majority of cells are spindle-shaped, with occasional tadpole, stellate, and polygonal forms. Nuclear pleomorphism and atypia are obviously

(Magnifi cation x 400)


Against the background of this neoplasm process, the patient developed hy-postatic pneumonia with fever and enhancement of WBC which increased to 12.6 x 103 ml. The clinicians were unable to make a defi nite diagnosis of the type of tumor, and the patient died four days after admission due to respiratory failure.


Autopsy was performed and after fi xation of the necropsy specimens in 10% formaldehyde solution, tissue samples were taken from different areas and em-bedded in paraffi n. Multiple 3-μm sections were obtained by means of a standard microtome and then stained with haematoxylin and eosin, and by Von Gieson’s mixture.

Immunohistochemistry was performed on de-parafi nized by polimer-peroxi-dase method using commercially available monoclonal antibodies against Cytok-eratin (IR053, DAKO, ready-to-use), desmin (IR606, DAKO, ready-to-use) and Vi-mentin (IR630, DAKO, ready-to-use). Paraffi n sections 5 μm thick were dewaxed in two xyllenes at 56° C for 1 h, and were rehydrated in ethanol. Later, they were washed in 0.1 M phosphate buffered saline (PBS), pH 7.4, incubated in 1.2% hy-drogen peroxide in methanol for 30 min, and rinsed in 0.1 M PBS, pH 7.4, for 15 min. The detection system immunostaining kit used was DAKO LSAB® 2 System, HRP (K0675, DAKO) and DAKO®DAB Chromogen tablets (S3000, DAKO).

Autopsy, histology and immunohistochemistry fi ndingsThe tumor was whitish and with a size of – 58×36×29 mm. The adjacent struc-

tures – trachea, soft tissues, and the skin, were involved (Fig. 2). The tumor cut surface had a fi rm, solid, grey-white to yellow-brown appearance and checkered with areas of necrosis and little or occult hemorrhagies. We didn’t found evident capsule.

Fig. 2. Solitary fi brous tumor of the thyroid gland with infi ltrative growth pattern


The other signifi cant fi nding was in the lungs. Both lungs were large, thick with weighty tumescence. The cut surface demonstrated the typical appearance of a bronchopneumonia with areas of tan-yellow consolidation. These areas con-fl uented in the left basal lobe and on the middle and basal right lobe. The areas of consolidation were fi rmer than the surrounding lung.

Histological examination of the tumor revealed a malignant mesenchymal tu-mor, and primary fi brosarcoma of the thyroid gland was ultimately confi rmed. We found that the tumor was composed of malignant spindle cells and rare pleomorphic cells that vary markedly in size and shape (Fig. 3). It was diffi cult to determine them because of their tendency to be poorly differentiated or anaplastic. Mitotic fi gures and foci of necrosis were observed. The peripheral thyroid parenchyma showed atrophic changes due to the tumor pressure and invasion. The background of fi brosarcoma with abundant collagen fi bers was stained in red by Van Gieson stain (Fig. 4).

Fig. 3. Fibrosarcoma of thyroid gland – atrophic changes due to the pressure of fi brosarcoma. HE stain (Magnifi cation x 200)

Fig. 4. The background of fi brosarcoma with abundant collagen fi bers stain red. Van Gieson stain (Magnifi cation x 100)


Immunohistochemical analysis showed the tumor cells to be strong posi-tive for vimentin, but negative for other markers – desmin and cytokeratin (Fig. 5a-c).

From histology of lung specimens we confi rmed bronchopneumonia. The al-veoli were fi lled with a neutrophilic exudate that corresponded to the areas of con-solidation seen grossly with the bronchopneumonia. We found also diffuse infl am-matory process in trachea responding on acute tracheitis.

A B

C Fig. 5. Fibrosarcoma. Spindle cells exhibit: a) positive reaction for vimentin; b) negative reaction for

cytokeratin; c) negative reaction for desmin. (Magnifi cation x 200)

DISCUSSION

Fibrosarcoma of thyroid gland is uncommon neoplasm observed in middle aged adults, with a slight predominance of females [2, 3]. Solitary fi brous tu-mors of this organ are characteristically immunoreactive for vimentin and some other markers. However, negative reactions were observed for cytokeratin and desmin [4]. It is important that clinical symptoms and radiological signs are not characteristic.


The histological examination of the tumor of our patient revealed medium cel-lular density with a slight polymorphism, abundant intervening collagen fi bers, 4 mi-toses per 10 high power fi elds and foci of necrosis. These features allowed to clas-sify this tumor as moderate differentiated fi brosarcoma. In differential diagnostic plan we discussed some other lesions such as fi bromatosis, malignant spindle cell carcinoma, malignant fi brous histiocytoma and anaplastic carcinoma. Fibromatosis has more collagen fi bers, is less cellular, with less mitoses and shows no necrosis or nuclear overlap or hyperchromasia. Negative immunohistochemical reaction for cytokertain and positive for vimentin of neoplastic cells allowed to rule out spindle cell carcinoma, malignant fi brous histiocytoma, but not convincingly – anaplastic carcinoma.

The clinical data about thyroid fi brosarcoma reported in literature are scarce. Unusual locations make the diagnosis of fi brosarcoma diffi cult, especially when it occurs in the thyroid. Most reports in the literature have demonstrated anaplastic thyroid carcinomas by use of immunohistochemical studies and electron micros-copy, stating that the sarcoma-like tumors of the thyroid gland are in fact of epi-thelial histogenesis, i.e., anaplastic carcinomas [5, 6, 7]. Differential diagnosis with anaplastic carcinomas is very diffi cult due to their similar clinical, histological and immunohistochemical features.

Again, the World Health Organization classifi cation of thyroid tumors indicates that it is very diffi cult or impossible to distinguish some thyroid sarcomas from un-differentiated carcinomas. The recommendation is that primary thyroid sarcoma should only be diagnosed when there is a complete lack of all epithelial differentia-tion and there is defi nite evidence of specifi c fi brosarcomatous differentiation [8], as was the case of our patient.

In conclusion, the case reported here shows a fi brosarcoma of thyroid gland confi rmed by imunohistochemistry, rarely previously reported in the literature. These fi ndings may contribute to broadening of the spectrum of differentiation of this unusual neoplasm.

REFERENCESF i s h e r , C., E. Van den Berg et W. M. Molenaar. Adult fi brosarcoma. – In: Tumors of Soft Tissue 1 . and Bone. Ed. Fletcher, D. M., Unni, K. K., Mertens, F. Lyon, IARC Press, 2002, 100-101.C a m e s e l l e -Te i j e i r o , J. et al. Solitary fi brous tumor of the thyroid. – Am. J. Clin. Pathol., 1994, 2 . № 4, 535-538.R o d r i g u e z , I. et al. Solitary fi brous tumor of the thyroid gland: report of seven cases. – Am. J. 3 . Surg. Pathol., 25, 2001, № 11, 1424-1428.T i t i , S., K. Sycz et M. Umiński. Primary fi brosarcoma of the thyroid gland-a case report. – Pol. J. 4 . Pathol., 58, 2007, № 1, 59-62.


C a r c a n g i u , M. L. et al. Anaplastic thyroid carcinoma:a study of 70 cases. – Am. J. Clin. Pathol., 5 . 83, 1985, 135-158.T h o m p s o n , L. D. et al.. Primary smooth muscle tumors of the thyroid gland. – Cancer, 6 . 79, 1997, № 3, 579-587.N i s h i y a m a , R. H., E. L. Dunn et N. W. Thompson. Anaplastic spindle-cell and giant-cell tumors 7 . of the thyroid gland. – Cancer, 30, 1972, 113-127.H e d i n g e r , C., E. D. Williams et L. H. Sobin. Histologic Typing of Thyroid Tumors. – In:World 8 . Health Organization International Histologic Classifi cation of Tumors. 2nd ed. Berlin, Springer, 1988, 13-15.

Address for correspondence: Georgi Tchernev, Associated Professor Dermatology and Venerology Department of Dermatology and Venereology Trakian University Medical Faculty Armeiska 11, 6000 Stara Zagora Bulgaria

00359 885 588 424 e-mail: [email protected]

Fatty acid composition of fi sh...26

FATTY ACID COMPOSITION OF FISH SPECIES FROM THE BULGARIAN BLACK SEA

M. Stancheva, A. Merdzhanova and L.MakedonskiDepartment of Chemistry, Medical University of Varna

Summary. The total lipids and fatty acid profi le in the edible tissue of two traditionally consumed fi sh species from Bulgarian Black Sea coast – shad and red mullet in two seasons are determined. The fatty acid composition was analy-sed by GC/MS. The total content of omega-3 fatty acids was signifi cantly higher than the total content of omega-6 fatty acids in shad whereas red mullet showed opposite trend. The omega-3/omega-6 FA ratio, an useful indicator for evalua-tion the relative nutritional value of a given fi sh, was within the recommended range for the studied Black Sea fi sh species. Obtained results for FA composi-tion, omega-3/omega-6 and polyunsaturated /saturated fatty acids ratios indicate that these Black Sea fi sh species in both seasons – spring and autumn are good sources of essential fatty acids.

Key words: omega-3, omega-6, fatty acids, GC/MS, fi sh species

INTRODUCTION

Nowadays marine food and especially marine fi sh are an important part of healthy diets. This is because marine fi sh provides us with a large number of nutrients especially omega-3 (ω 3) fatty acids such as ei-

cosapentaenoic (EPA, C 20:5) and docosahexaenoic (DHA, C 22:6) fatty acids. A large body of scientifi c paper shows that fi sh consumption has benefi cial effects on coronary heart disease, growth and development, blood lipids and other health issue [4, 12].

Fish consumption in Bulgaria is lower than recommended by World Health Organization (WHO) and the Food and Agricultural Organization (FAO) (at least 15-20 kg fi sh per capita) [5]. Their is a lack of information on the fatty acid (FA) composition of local fi sh species consumed in Bulgaria. The shad (Alosa pontica) and red mullet (Mullus barbatus ponticus) are traditionally consumed fi sh species


in our country. However, the fat content and FA composition of fi sh lipids are not constant. They vary according to fi sh and its characteristic such as catching place, in response to their habitats as water temperature change, salinity and dietary lip-ids [1, 6, 7, 9, 13]. Some Turkish articles presented information for a proximate composition of the Black Sea species living in South Eastern part of Black Sea but they do not show similar results [6, 9, 13]. Signifi cant differences were also reported in the fat content and FA composition of several marine fi shes during the seasons [15]. The aim of the present study is to determine the fatty acid composition in the edible parts of shad and red mullet. The infl uence of seasonality on total lipids and FA profi le are followed in order to fi nd in which season these species are the best source of essential FA.


SamplingShad was caught from region of Kavarna, the North-Eastern Black Sea,

whereas red mullet was caught from region of Varna (Tracata). They were pur-chased from Varna local fi sh markets during two commercial catching seasons – spring (March-April) and autumn (Octomber-November) of 2009. The biometric and biologic characteristics of analysed Black Sea fi sh species are presented in Table 1.

Table 1. Biometric and biologic characteristics

Fish species Mean total weight

(g) ± SDSpring

Mean total weight

(g) ± SDAutumn

Mean stand. length

(cm) ± SD Spring

Mean stand. length

(cm) ± SD Autumn

Habitat Food habits

Pelagic Demersal Carnivorous

Shad (n=4) 325.00±5.00 315.00±5.20 25.50±2.10 28.50±3.50 + – +

Red mullet (n=12) 35.00±3.00 47.50±4.00 14.50±1.50 16.00±2.00 – + +

n – number of specimens

The edible fi sh tissue was fi lleted with the skin left on and homogenized. The freshly prepared homogenates (5,00 g) were extracted by the method of Blight and Dyer [10] using chloroform/methanol/water in a ratio 2:2:1. After phase separation, the chloroform extracts were evaporated to dry residue and were quantifi ed by weight. Three replicate total lipid analyses were performed gravimetrically and the results were present as g.100g-1 raw tissue.

The dry residue of the chloroform fraction was methylated by base-catalyzed transmethylation using 2M methanolic potassium hydroxide and n-hexane [11]. Af-ter centrifugation, the hexane layer was separated and analysed by GC-MS.


GS-MS conditionGas chromatography analyses were performed by Gas Chromatograph

with autosampler, equipped with Polaris Q MS detector (Thermo Scientific, USA). The capillary column used was a TR-5 MS (Thermo Scientific, USA) universal column 30 m length and 0.25 mm i.d. Helium was used as a carrier gas at flow rate 1 ml/min. Peaks were identified according to retention time based on available fatty acid methyl esters mix standard (37 FAME mix C4-C24, SUPELCO) and mass spectra (ratio m/z). Three replicate GC analyses were performed and the results were expressed in GC area % as mean values ± standard deviation (SD).

Statistical analysisStatistical analysis was performed using Graph Pad Prism 5 program. The

descriptions of the data are given as mean ± Standard Deviation (SD) in Table 2. To assess the statistically signifi cant levels (P >0.05), a one way ANOVA (nonpara-metric test) was employed.

RESULTS AND DISCUSSION

The total lipid (TL) content and FA composition of analysed fi sh species are presented in Table 2. As it can be seen from the above table, red mullet can be classifi ed as moderately oily fi sh in spring season until red mullet (autumn) and shad (over two seasons) are typical fatty species. Our results are similar to other relevant publications describing seasonal changes in TL of pelagic species and in Mediterranean red mullet [10, 15].

A lipid analysis enabled the classifi cation and quantitative determination of FA as well as the sum of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA). FA contents in red mullet fol-lowed a relative pattern with SFA > MUFA > PUFA in both seasons. In contrast to the red mullet, shad (autumn) contained a higher amount of SFA than that of MUFA (e.g. SFA > PUFA> MUFA) whereas the shad (spring) contained a dif-ferent pattern of FA composition as evidenced by the relatively greater MUFA compared to PUFA (e.g. MUFA > SFA > PUFA). Figure 1 presented signifi cant differences obtained when comparing the FA groups in each analysed fi sh spe-cies. These FA groups presented signifi cant differences in their values during both seasons. Those visible FA variations observed in the red mullet in both seasons are in accordance with the data presented by Polat et al. [10] for the Mediterranean red mullet. Fig. 1 shows that the amount of FA groups varies widely among the species and seasons.


Table 2. Total lipids and fatty acid composition in edible fi sh tissue (mean ± SD)

Fatty Acid% of total FA

Red mulletSpring

Red mulletAutumn

ShadSpring

ShadAutumn

TL (g.100-1g r.w) 5.38±0.65 14.69±0.85 13.15±0.70 12.70±0.80C 12:0 0.53±0.02 1.10±0.05 0.05±0.01 0.84±0.02C 14:0 4.27b±0.70 5.85±0.75 2.68b±0.22 3.46±0.65C 15:0 0.02±0.01 0.01±0.01 0.00 1,19±0.25C 16:0 21.43a,b±1.15 30.05b±1.75 26.02a±1.40 27.74a±2.01C 17:0 0.63±0.02 0.31±0.01 0.24±0.01 1,43±0.30C 18:0 6.30±0.050 6.23±0.045 1.54b±0.20 3.02b±0.60C 20:0 0.65±0.02 0.28±0.01 0.10b±0.01 2.47b±0.80C 21:0 0.39±0.01 0.15±0.01 0.02±0.01 0,00C 22:0 0.69±0.02 0.24±0.01 0.06b±0.01 2.52b±0.20C 23:0 0.37±0.01 0.13±0.01 0.02±0.01 0,00C 24:0 0.65±0.02 0.22±0.01 0.05b±0.01 2.87b±0.50Σ SFA 35.44 43.90 30.81 46.03C 14:1 2.06±0.50 1.15±0.40 0.03±0.01 1.32±0.10C 16:1 9.36b±1.02 12.32±1.10 16.82b±0.65 7.72b±1.01C 17:1 0.68±0.05 0.24±0.01 0.29±0.02 1,40±0.30

C 18:1 n9 c 20.9b1±1.50 16.75±1.20 9.35b±0.50 6,76±0.85C 18:1 n9 tr 0.17±0.01 0.06±0.01 1.03a±0.02 1,17a±0.20

C 20:1 2.06±0.40 1.73±0.30 2.45a±0.30 1,89a±0.20C 22:1 n9 1.85b±0.40 0.90±0.05 3.10b±0.30 1,68b±0.15

C 24:1 0.50±0.02 0.22±0.01 1.49±0.02 1,44±0.20Σ MUFA 37.56 39.45 34.56 25.39

C 18:3 ω 6 1.57±0.10 1.01±0.40 0.04±0.01 1,32±0.20C 18:2 ω 6 c 3.07b±0.15 2.14±0.20 5.21b±0.25 4,43±0.25C 18:2 ω 6 t 0.02±0.01 0.01±0.01 0.22±0.01 1,98±0.50C 18:3 ω 3 1.20±0.80 0.63b±0.05 0.41b±0.02 2,11b±0.20C 20:5 ω 3 1.13±0.50 0.68±0.02 0.13b±0.01 1,33b±0.40C 20:4 ω 6 5.11b±0.85 4.41±0.90 0.15b±0.01 2,50±0.20

C 20:2 0.96±0.05 0.34±0.02 0.15±0.01 1,42±0.50C 20:3 ω 3 0.90±0.05 0.45b±0.04 0.07±0.02 1,86b±0.80C 20:3 ω 6 0.96±0.03 0.34±0.01 0.00 1.42±0.20C 22:6 ω 6 7.01b±0.97 4.53±0.65 21.04b±0.30 9,30b±1.05

C 22:2 0.83±0.02 0.43b±0.01 0.06±0.01 1,73b±0.80Σ PUFA 21.40 15.65 28.25 29.60

SFA – saturated FA; MUFA – monounsaturated FA; PUFA – polyunsaturated FA. value without superscript – P=0.01; for value with superscript- a – P=0.001; b – P= 0.0001


0

20

40

60SFAMUFAPUFA

red mulletspring

red mulletautumn

shadspring

shadautumn

% of

total

FA

Fig. 1. Seasonal changes in fatty acid groups in red mullet and shad

In the SFA group, C 16:0 (predominated in all groups), C 18:0 and C 14:0 were found in large quantities. Availability of high levels of this fatty acid supports the results published in many similar studies conducted on seawater fi sh [7, 14]. We found that the increases in total SFA in the autumn in two species were directly related to growth quantities of C 16:0 and C 14:0.

The amounts of unsaturated FA as MUFA vary especially in wild fi sh [7, 15]. The percentage of this FA in the observed Bulgarian Black Sea fi sh during two sea-sons is not similar. The highest total MUFAs value was determined for red mullet (autumn), while the lowest one was for shad (autumn). That was due to the high concentration of C 16:1,C 18:1 ω 9 and C-20:1 ω 9. C16:1 was the main MUFA in the sad, especially in spring season which is in accordance with FA profi le pre-sented for some Mediterranean and Aegean fi sh species as mullet (Mugul cepha-lus) and sardine (Sardinella aurita) [9]. Many studies [1, 6, 7, 13] report that C 18:1 is the main MUFA in seawater fi sh species. Our results for red mullet confi rmed these informations. This fatty acid has exogenous origin and usually refl ects the type of diet of the fi sh. Some Turkish papers report that the highest level of C18:1 (up to 13.00%) is measured in the Black Sea turbot [1, 6, 7, 9, 13]. Some investiga-tions supposed that because of the decline in populations of zooplankton and the increase of phytoplankton mass, as a response to the eutrophication in the Black Sea, the fi sh in that region have low levels of MUFA as C 20:1 and high levels of ω 3 PUFA [14]. Contrary results were obtained in our study where C 20:1 levels were high (for red mullet in both seasons) and low for C 22:1 ω 9 especially in red mullet (autumn). The shad presented high level of C 22:1 ω 9 (spring samples).

The major FA identifi ed as PUFA is DHA. Other important long-chain fatty acids such as linoleic acid (C 18:2 ω6), EPA and arahidonic acid (C 20:4 ω6) were found in signifi cant amounts too. The maximum value of DHA was defi ned in shad (74.50% of total PUFA) and the minimum – in red mullet (48.80 % of total PUFA).


Saglik et al. [11] have analysed ω 3 FA in some Turkish seawater fi sh and reported that DHA occurred in higher amounts. In comparison with our results, signifi cant dif-ferences for EPA and DHA between Black Sea fi shes and presented Mediterranean fi shes were found. In our investigation for all species, EPA levels were lower than those of C 20:4 ω 6 in both seasons.

The obtained results (Table 3) for omega-3/omega-6 ratios for both species varied according to seasons.

Table 3. Total sum of omega-3 and omega-6 content (% of total FA)

Fatty Acids Red mulletSpring

Red mulletAutumn

ShadSpring

ShadAutumn

ω 3 9.34±1.20 5.78±0.90 21.66±1.65 14.60±1.35 ω 6 10.73±1.10 7.64±1.52 5.60±0.94 10.35±1.05

ω 3/ ω 6 0.87 0.75 3.87 1.41

The data indicate that shad was characterized by high level of omega-3 FA series and low levels of omega-6 series while red millet presented reverse version. Nevertheless, we can conclude that these fi sh species are a valuable source of essential fatty acids, especially DHA. The ω 3 / ω 6 ratio has been suggested to be a useful indicator for comparing the relative nutritional value of fi sh. The seasonal changes observed in ω 3 and ω 6 PUFA for both species are presented in Figure 2. The ratio 0.20-3.80 recommended by UK Department of Health [3, 12] would constitute a healthy human diet and our study proved that this ratio for all Black Sea fi sh species is within the recommended level. Due to its diet prior to breeding season shad showed unbalanced omega-3/omega-6 ratio.

r m S r m A s S s A

0

5

10

15

20

25 omega 3omega 6

red mulletspring

red mulletautumn

shadspring

shadautumn

% of t

otal F

A

Fig. 2. Content of omega-3 and omega-6 fatty acids


In Bulgaria, people have a relatively low intake of omega-3 FA compared with omega-6 FA. A moderate to low consumption of fi sh and other seafood (especially fatty fi sh), combined with limited conversion of ALA to EPA and DHA in particular, contributes to a relatively high level of omega-6 fatty acids in the body compared with the marine omega-3 fatty acids. By combining a lower intake of omega-6 fatty acids with a higher intake of omega-3 fatty acids, e.g. through the consumption of seawater fi sh, a relatively signifi cant effect will be achieved [12]. Values for PUFA/SFA ratio greater than 0.45 are recommended [5 ,12]. Our results are in agreement with this requirement showing higher PUFA/SFA ratios for three studied fi sh species (Table 3). The highest PUFA/SFA ratio was observed in the shad (spring), whereas the lowest value was found for red mullet (autumn). The most balanced PUFA/SFA ratio was obtained for the shad.

CONCLUSION

The present study provides useful information about the seasonal variation of total lipid content and fatty acid composition of two Black Sea fi sh species – shad and red mullet:

The total lipids in shad had similar values in both seasons while in red mullet • a threefold increase in quantity in the autumn period was observed.

Seasonal variations were observed in all FA groups as the most signifi cant • were in SFA and PUFA – increasing amounts of SFA at the expense of reduction of PUFAs in the autumn in both species.

The most important omega-3 fatty acids in PUFAs group are DHA and they • were presented with the highest concentration in all species and in all observed seasons. The ω 3/ ω 6 ratio varies in the range of 0.75 up to 3.87. The most bal-anced PUFA/SFA ratio was obtained for the shad.

In conclusion, regarding the lipid contents, the omega-3/omega-6 and PUFA/SFA ratios, we may assume that these Black Sea fi sh species were found to be a valuable source of the essential PUFA for human diet in both seasons.

AcknowledgmentsThe authors would like to thank the National Science Fund, Ministry of

Education and Science of Bulgaria for their fi nancial support (Project DVU 440 / 2008).

REFERENCESB a y i r , A., H. I. Haliloğlu. et N. Sirkecioğlu. Fatty acid composition in some selected marine fi sh 1 . species living in Turkish waters. – J.. Sci. Food Agric., 86, 2006, 163-168.BDS EN ISO 5509. Animal and vegetable fats and oils-preparation of methyl esters of fatty acids. 2. 2000.B l i g h E. et W. J. Dyer. A rapid method of total lipid extraction and purifi cation. – Can. J. Biochem. 3 . Physiol., 37, 1959, 913–917.


C u l y e r , A. Supporting research and development in the NHS: a report to the Minister of Health. 4 . London, HMSO, 1994.FAO/WHO: Scientifi c and ethical challenges in agriculture to meet human needs. http://www.fao.5. org/docrep/003/X8576M/x8576m05.htmG u n e r , S. et al. Proximate composition and selected mineral content of commercially important 6 . fi sh species from the Black Sea. – J. Sci. Food Agric., 78, 1998, 337.I m r e , S. et S. Saglik. Fatty acid composition and cholesterol content of some Turkish fi sh species. 7 . – Turk. J. Chem., 22, 1998, 321-324.M u r r a y, J. et J. R. Burt. The Composition of Fish. Ministry of Technology, Torry Research Station, 8 . 2001, Torry Advisory Note No. 38. http://www.fao.org/wairdocs/tanÖ z o g u l , Y. et F. Özogul. Fatty acid profi le of commercially important fi sh species from the 9 . Mediterranean, Aegean and Black Seas. – Food Chem., 100, 2007, 1634-1638.P o l a t , A., S. Kuzu et G. Özyurt. Fatty acid composition of red mullet: A seasonal differentiation. 1 0 . – J. Muscle Foods, 20, 2009, 70-78.S a g l i k , S. et S. Imre. ω3 fatty acids in some fi sh species from Turkey. – J. Food Sci., 11 . 66, 2001, 210-212.S i m o p o u l o s , A. et L. Cleland (eds). Importance of the Ratio of Omega-6/Omega-3 Essential 1 2 . Fatty Acids: Evolutionary Aspects. Omega-6/Omega-3 Essential Fatty Acid Ratio: The Scientifi c Evidence. – World Rev. Nutr. Diet., 92, 2003, 1-22.Ta n a k o l , R. et al. Fatty acid composition of 19 Species of fi sh from the Black Sea and the 1 3 . Marmara Sea. – Lipids, 34, 1999, 291-294.Z a i t s e v, Y. Recent Change in the Trophic Structure of the Black Sea. – Fish. Ocean., 1 4 . 2, 1992, 180-189.Z l a t a n o s , S. et K. Laskaridis Seasonal variation in the fatty acid composition of three Mediterra-1 5 . nean fi sh – sardine (Sardina pilchardus), anchovy (Engraulis enchrasiholus) and picarel (Spicara smaris). – Food Chem., 103, 2007, 725-728.

Address for correspondence: A. Merdzhanova Department of Chemistry Medical University of Varna 55 Marin Drinov Str. 9002 Varna Bulgaria

035952 650019 e-mail: [email protected]

Rheumatoid arthritis in the general...34

RHEUMATOID ARTHRITIS IN THE GENERAL PRACTITIONER'S RACTICE

V. Paskaleva-Peycheva1, M. Panchovska-Mocheva2 and E. Kavrakov3

1UMBAL “Sveti Ivan Rilski” – Sofi a, Rheumatology Clinic, Medical University – Sofi a2Military Medical Academy – Plovdiv, Clinic of Internal Diseases, Medical University – Plovdiv

3Medical student, MU – Plovdiv

Summary. Rheumatoid arthritis (RA) is a infl ammatory joint disease with a variety of manifestations, decursus and treatment approaches. It is essential to stress the importance of the early proven diagnosis and early treatment which pro-tect from serious complications in the musculoskeletal system and internal organs. This requires some highly specialized help from a rheumatologist. There has to be a good dialogue between general practitioner and rheumatologist who must work together. Such an approach would lead to quick and effi cient treatment of patients with RA.

Key words: rheumatoid arthritis, patients, general practitioner

Rheumatoid arthritis is an autoimmune rheumatic disease with unknown etiology and a complex pathogenesis. It is characterized by the gradual development of chronic erosive arthritis (synoviitis) and appearance of

systemic manifestations – lesions of different internal organs. The frequency of RA is 0,8-1,0%, with male to female ratio of 2-3 : 1. The

disease onset is earlier in women (it starts at the age of 30-50) while in men the onset is later but course is more severe.

This disease is associated with some antigens: HLA-DR3, DR4, DRW52General practitioners (GPs) and RAGPs are the fi rst who get to know about a patient’s problem. Having in mind

that RA happens to be a disease following the patient all his life with its chronic course, the interaction of the physician and the patient is very important.

The best way to manage the disease is to diagnose and treat it early enough. The early referral of patients to specialists in rheumatology may prevent disability and provide a higher quality of life [1].


The American College of Rheumatology developed the following criteria (in 1987) for diagnosing RA:

1) Morning stiffness lasting at least 1 hour2) Arthritis of 3 or more joint areas3) Arthritis of hand joints4) Symmetric arthritis5) Rheumatoid nodules 6) Serum RF7) Radiographic changes typical of RA*The diagnosis is accepted if at least 4 out of 7 criteria are present.**Criteria 1) – 4) have to last at least 6 weeks and to be observed by a physician [2]. Differential Diagnosis: 1. Osteoarthritis: “mechanical” nature of pain, morning stiffness is continuing

less than 30 min., etc.2. Systemic Lupus Erythematosus: characteristic fi ndings in immunological

tests, lack of erosive radiographic changes in impaired joints.3. Gout: a chronic disease more distributed among females; increased uric

acid level, cystose radiographic changes4. Psoriatic arthritis: visible psoriatic lesions, genetic predisposition5. Bechterew’s Disease (Ankylosing spondylitis): HLA – B27, other cases in

the family, sacroileitis, enthesopathia.6. Reactive arthritis: after a short-lasting acute urogenital ot intestinal infection 7. Other types of arthritis Which are the indications for a hospitalization of patients with RA?1. Diagnosis establishment and prognostic evaluation of the disease 2. Selection of a disease-modifying antirheumatic drug (DMARD) for treat-

ment3. Disease activity evaluation4. If infections are developed – for example pneumonia, cholecystitis, septic

arthritis, also in case of complications of RA like amyloidosis or complications of the selected treatment (drug ulcers, toxic drug hepatitis, interstitial nephritis, etc)

5. Surgical treatment (carpal tunnel syndrome, endoprosthesis) 6. Visceral manifestations such as “rheumatoid” lungs, glomerulonephritis, ac-

companying vasculitisThe treatment’s purpose is to:1. Decrease the activity of the disease2. Prevent destruction of joints and deformity, as much as functional impair-

ment3. Reach a remission state4. Improve the overall quality of life5. Increase life expectancy (the mean life expectancy of the population has to

be reached) [3, 4]

Rheumatoid arthritis in the general...36

The behavior of GPs when working with patients with RA has to unify the fol-lowing options:

1. Interdisciplinary treatment – participation of rheumatologist, neurologist, physiotherapist and orthopedist in the treatment plan

2. Avoidance of those factors that can lead to exacerbations – social and living conditions, professional harmful factors

3. Smoking and alcohol intake have to be given up and, in addition, a BMI < 25 has to be achieved and supported

4. A suitable balanced diet rich in Ca, proteins and omega-3-fatty acids must be recommended.

5. Education of the patient; active kinesitherapy6. Physiotherapy in case activity is low or RA is in a stage of remission7. Orthopedic tools – corsettes, walking sticks and other gait facilitating tools8. Sanatorium treatment during periods of remission9. Treatment of accompanying diseases: focal infections, gastroduodenal

problems, thyroid dysfunctions BIOLOGIC AGENTS are used in the treatment schemes of RA in the last

years [5].Biologic agents are protein molecules directed against other molecules, par-

ticipating in the infl ammatory process (i.e.the cytokines). Following groups of biologic agents are directed against the respective target

molecules:1) Remicade, Enbrel, Humira – vs. TNF-α2) Anakinra – vs. IL-13) Tocillizumab – vs. IL-64) Rituximab (Mabthera) – vs. B cellsBiologic agents are indicated in the cases stated below:1. In cases of insuffi cient effect from nonbiologic treatment: methotrexate, hy-

droxychloroquine, lefl unomide, sulfasalazine2. TNF-α inhibitors may be prescribed in the beginning of treatment of newly

diagnosed patients before methotrexate3. Biologic agents combined with methotrexate are prescribed if RA activity is

very high4. Combinations of more than one biologic agent are not recommended5. Treatment contraindications to biologic agents include: bacterial infections,

H.zoster, active and latent TBC, acute or chronic hepatitis B and C6. High-risk patients should be advised to undergo vaccinations against infl u-

enza, pneumococcus infection and HBV GPS AND CONTROL OF PATIENTS WITH RAGPs have the following specifi c tasks concerning patients with RA:1. Arterial blood pressure monitoring due to a tendency to increased values as

a side effect of NSAIDs and corticosteroids


2. Control of drug doses because there is a risk of toxicity and complications3. Due to risk of NSAIDs-induced gastroduodenopathia selective COX-2 in-

hibitors are prescribed (Celebrex, Arcoxia)4. Monitoring of peripheral blood and biochemistry tests – creatinin, amin-

otransferases; endoscopy of the gastric and duodenal mucosa. 5. Risk factors in treatment of patients with RA include: age > 65; cardiovas-

cular diseases; anticoagulant intake; H. pylori infection.6. In cases of allergy towards sulphonamidic drugs, Sulfasalazin and Cele-

coxib should not be used.

CONCLUSION

RA is an infl ammatory joint disease which is characterized by a large variety of manifestations, course and therapeutic approaches. Early diagnosis and early adequate treatment are essential as they prevent disability in these patients. The necessity of providing highly specialized medical help from a rheumatologist de-termines the importance of the collaboration between specialists and GPs. Such an approach would ensure right professional behavior towards patients with RA, higher quality of life and preserved life expectancy.

REFERENCESVa n d e r L i n d e n , M. P. et al. Long-term impact of delay in assessment of patients with early 1 . arthritis. – Arthritis Rheum., 62, 2010, № 12, 3537-3546.S o k o l o v e , J. et V. Strand. Rheumatoid arthritis classifi cation criteria – it’s fi nally time to move 2 . on. – Bull. N. Y. Univ. Hosp. H. Dis., 68, 2010, № 3, 232-238.R o b i n s o n , P. C. et M. J. Taylor. Time to treatment in rheumatoid arthritis: factors associated 3 . with time to treatment initiated and urgent triage assessment of general practitioner. – J. Clin. Rheumatol., 16, 2010, № 6, 267-273.N a r a n j o , A. et al. Cardiovascular disease in patients with rheumatoid arthritis: results from the 4 . QUEST-RA study. – Arthritis Res. Ther., 10, 2008, 1186.A m e r i c a n College of Rheumatology. Recommendations for use of nonbiological and biological 5 . disease – modifying antirheumatic drugs in Rheumatoid arthritis. – Arthritis Rheum., 59, 2008, № 6, 762-784.

Address for correspondence: Assoc. Prof. Veneta Paskaleva-Peycheva, MD Rhematology Clinic UMBAL "Sveti Ivan Rilski" 13 Urvich str. 1612 Sofi a

958-25-53

Immunobiology of endometriosis38

IMMUNOBIOLOGY OF ENDOMETRIOSISN. Manolova, D. Zasheva and M. Stamenova

Department of Immunochemistry, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences

Summary. Endometriosis is a condition in which tissue similar to the lining of the uterus (endometrial cells and glands, which should appear only in the uterus) is found in other parts of the body. This tissue can be implanted itself and grow throughout the abdominal cavity. As a disease, it is a unique clinical and scientifi c challenge. This disease called “enigma wrapped in mystery”, is still etiologic and pathogenetic phenomenon. According to some authors in women with infertility, the rate of genital endometriosis is estimated at 20-55% and 30 to 50% of women with endometriosis are infertile. The disease leads to poor success rate in patients who undergo assisted reproductive technologies. Endometriosis is a benign, chronic, estrogen – dependent gynecological disease accompanied by pelvic pain. Because of the clinical, psychological and social signifi cance of the problem, endometriosis is a widely studied disease. It affects the quality of life of women in their most active age and has serious economic consequences. For accurate diagnosis and proper treatment of endometriosis and prevention it is necessary to perfect knowledge of the mechanisms of emergence and development of pathological process. Basic environment for emergence and development of endometriosis is peritoneal fl uid. Middle of the peritoneal fl uid surrounding the endometrial implants is biochemically dynamic and it becomes a meeting of reproductive and immune systems.

Key words: endometriosis, implantation, infertility, peritoneal fl uid, serum, reproduc-tive system, immune cells, humoral immunity

IMMUNE CELLS AND THEIR FUNCTION IN ENDOMETRIOSIS

I. Cell immunityMonocytes/macrophages1. – peripheral blood monocytes (PBMs) and

macrophages in peritoneal fl uid are major cellular components of the immune sys-tem. Women with endometriosis have an increased circulating monocyte activa-


tion status, which has been found by chemiluminescence [3]. Under disincentives (basal) and stimulated conditions in women with endometriosis, PBMs produced high levels of tumor necrosis factor (TNF-α), interleukin (IL)-6 and IL-8, but not IL-10 compared with monocytes of healthy controls [4]. Macrophages in the perito-neal cavity remove red blood cells, damaged tissue fragments, apoptotic cells and endometrial cells most probably penetrating into the peritoneal cavity through the fallopian tubes. In endometrial peritoneal fl uid, the concentration and the number of macrophages are signifi cantly increased [5]. They are large activated macrophag-es that produce high levels of TNF-α, IL-6 and IL-8 and IL-10 in comparison with macrophages in healthy women [6]. They also produce high levels of smooth-mus-cle-contracting prostaglandins (PGs), such as PGE2 and PGF2α [7]. Endometrial macrophages are also a source of other cytokines, growth factors, adhesion mol-ecules, complement components, hydrolytic enzymes, reaction O2 products stimu-lating eutopic and ectopic endometriosis cell proliferation in vitro and decrease endometriotic-cell apoptose [8]. It is assumed that in healthy women, phagocytic activity of peritoneal macrophages is crucial to perform the elimination of menstrual detritus. This activity is mediated via surface scavenger (cleaning) receptors, which are regulated by different cytokines and growth factors. It has been shown that these scavenger-receptors play a role in cell adhesion and that non-adherentive macrophages do not express type-A scavenger-receptor. Thus the increase in non-adherentive macrophages without scavenger-receptor may contribute to the patho-genesis of endometriosis [9].

NK-cells: 2. NK-cells are large granular lymphocytes that participate in the destruction of abnormal cells derived from viral infections, malignant changes or aging. One of the mechanisms by which NK-cells kill target cells is through an-tibody-dependent cellular cytotoxicity. To this end, NK-cells have receptors that bind immunoglobulin-G (Ig G) and kill then covered with IgG target cells. Another mechanism involving recognition of target cells is through the so-called KAR (killer activating receptors) and KIR (killer inhibitory receptor). If KAR are employed, i.e. are activated, NK-cells have cytotoxic activity, when KIR are vacant, i.e. are inacti-vated, cytotoxic activity is inhibited. Cytotoxic activity of NK-cells can be enhanced by lymphokines such as IL-2, a function known as LAK-activity (lymphokine-acti-vated killer) [5].

Several studies have demonstrated lower NK-cell cytotoxicity against autolo-gous and heterologous endometrial cells in women with endometriosis [10]. This decrease in NK-cell cytotoxicity against autologous endometriotic cells may also refl ected increased resistance of endometrial cells to NK-mediated cytolysis, which was fi rst presented by Oosterlynck et al. [11].

If the NK-cell cytotoxicity is one component of the immune “disposal” system of menstrual detritus, NK-cell defi ciency may facilitate the development of disease. Changes in NK-cell cytotoxicity in endometriosis appear more functional than quan-titative. The percentage of peripheral NK-cells do not change [5].


Several studies demonstrate increased expression of KIRs family of NK-cells in women with endometriosis. These molecules interact with the main complex for tis-sue compatibility class-I (MHC-I) of the potential target cells to block the “killing” abil-ity of NK-cells. Cells with no expression of MHC-I treat waste program activated NK-cell [12, 13]. Studies of Maeda et al. [14] showed that endometriosis has increased the proportion of NK-cells that express KIR2DLI phenotype compared with normal controls. This was demonstrated both in circulating and peritoneal NK-cells.

T-lymphocytes: 3. T-lymphocytes derived from pluripotent stem cells in fetal liver and bone marrow. From these places they travel to the thymus, where complete their development in the two main subpopulations characterized by expression of the glycoprotein CD4 and CD8, functioning as co-receptors for MHC-II class and MHC-I class molecules. CD4 T-lymphocytes can later be turned into subclasses CD4 Th1 and Th2 CD4 cells. Th1 cells enhance differentiation of CD8 cells to killer-cells and activated monocytes/macrophages system to facilitate cell-mediated im-munity. Th2 cells amplify B-cell differentiation to antibody-secreting cells. Th1 and Th2 cells can be distinguished by their cytokine characteristics. Fully activated CD8 T- lymphocytes can eliminate intracellular pathogens by destroying virus-infected cells simultaneously with the activation of monocytes-macrophages system [5].

Quantitative studies provide T-lymphocytes and their subclasses in peripheral blood and peritoneal fl uid in women with endometriosis. Changes were not detect-ed in the total number of T-lymphocytes, and CD4/CD8 ratio in peripheral blood, but peritoneal fl uid observed elevated absolute number of CD4 and CD8 subclasses, and their ratio. In eutopic endometrium, total lymphocytes and CD4/CD8 ratio were similar both in endometriosis and in healthy control patients. In ectopic endometri-um, although the number of T-lymphocytes was increased compared with the pro-liferative and secretory eutopic endometrium, CD4/CD8 ratio remained unchanged [5]. With regard to functional changes in peripheral lymphocytes in women with endometriosis and adenomyosis, there were submitted data in 1980 (Startseva [15]). A little later, using peripheral blood lymphocytes and autologous-Cr-labeled endometriotic target cells in women with endometriosis was found decreased cell lysis to target cells compared with controls and patients with moderate and severe endometriosis [16].

II. Humoral immunityB-lymphocytes: 1. B-cells are precursors of plasma cells which are antibody-

producing cells of the immune system. It is suggested that CD5+ B-cells, which are presented in 10-20% of B-cell population, are responsible for the production of au-toantibodies. The number of CD5+ B-cells is increased in patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and others.

Autoantibodies and location of endometriosis as an autoimmune disease: −The idea for the autoimmune nature of endometriosis was fi rst presented by

Gleicher et al. [17], demonstrating that endometriosis is responsible for most of the criteria to be classifi ed as autoimmune diseases, as presented in Table 1.


Table 1. Common characteristics between autoimmune diseases and endometriosis(Nothnick. Endometriosis and autoimmunity. Fertil Steril 2001)

1. Tissue damage 7. Multiorgan complications

2. Polyclonal B-cell activation 8. Family history

3. T-lymphocyte immunological disorder 9. Potential effects of environmental factors

4. B-lymphocyte immunological disorder 10. Possible genetic basis

5. Association with autoimmune diseases 11. Altered apoptosis

6. Prevalent in women

Like the classic autoimmune disease endometriosis was associated with poly-clonal B-cell activation, fi rst described in 1980 [15]. The disease is associated with frequent abortions and infertility [18] which can be explained by the presence of au-toimmune abnormalities. Further evidence for the role of autoimmunity or autoan-tibodies in infertility, concomitant endometriosis, comes from studies showing that medical treatment with Danazol [19] or gonadotropin-releasing hormone (GnRH)-analogues [20] suppresses levels of autoantibodies associated with endometriosis. To be a disease characterized, however, really as autoimmune in nature, it should be exercised in healthy animals after the implementation of transfer of immuno-globulins from serum or from affected tissues of sick. Until now such studies were not conducted. However, there is uncertainty whether endometriosis is an autoim-mune disease. It has a similar pathophysiology with other autoimmune diseases (rheumatoid arthritis, Crohn’s disease, psoriasis) by: increasing infl ammation, in-creased levels of components of tissue remodeling, altered apoptosis, increased local and/or systemic levels of cytokines [21].

In 1980, a group of Weed and Arguembourg reported for C3 and immunoglob-ulin G (IgG) accumulation in the uterine endometrium in women with endometriosis and lowering total serum complement manifested through antigen-antibody reac-tions in the endometrium [22].

Mathur et al. fi rst described in patients with endometriosis autoantibodies that recognize endometrial antigens [4] (ranging between 34-140 KDa), eventually au-toantigens the immune response [23]. IgG and IgA autoantibodies described by these authors in serum and cervical and vaginal secretions of women are with specifi city against endometrial and ovarian tissues. There are other data from the same research group, indicating that in normal fertile controls and in women with endometriosis, there were observed decreased levels in the circulation and perito-neal fl uid of antibodies against endometrial antigens with different molecular weight. It is believed that this may be a mechanism for clearing the reproductive tract of menstrual detritus. Only against endometrial antigens with molecular weights of 26 KDa and 34 KDa auto-antibodies in endometriosis were identifi ed. There were also antibodies to endometrial transferrin, α2 Heremans-Schmidt glycoprotein (α2-HSG) and carbonic anhydrase in women with endometriois [24]. Antibody response


against carbohydrate epitop (Thomsen-Friedenreich antigen) suggests that the au-toimmune response may have direct contact with the disease process or refl ect an abnormality in glycosylation in endometriosis [25].

During the early stages of endometriosis, the disease is associated with el-evated serum and peritoneal liquid levels of anti-phospholipid antibodies against inositol, kardiolipin, ethanolamine and β2-glycoprotein I, while in patients with stage I-II disease more autoantibodies compared with III-IV stage were observed [26]. Other researchers presented circulating autoantibodies against subcell items (in-cluding antinuclear antibody) or against chemical substances, inseparable from the cellular structures (including anti-DNA or anti-phospholipid antibodies). The high frequency of these autoantibodies was also reported in women with autoim-mune diseases and various forms of reproductive failure, and unexplained infertil-ity and recurrent miscarriage [5]. Gleicher et al. reported that among 31 women with endometriosis, 65% had IgG and 45% – IgM autoantibodies against at least one of the sixteen tested antigens [27]. These fi ndings are most often associated with autoantibodies to phospholipid (especially phosphatidylserine), histones and nucleotides. In support of the theory to unlock the humoral autoimmune response in endometriosis are the data for the high prevalence of thyroid autoimmunity in women with endometriosis as a result of signifi cantly higher serum levels of thy-roid peroxidase-antibody [28, 29] . In infertile patients with the disease, IgG anti-laminin-I antibodies have been found [30]. There were proven also auto-antibodies against oxidative stress-induced antigens, such as malone dialdehyde-modifi ed low density lipoproteins and oxidized LDL (low density lipoprotein) are elevated in the serum of patients with endometriosis [31].

In support of the theory of autoimmune nature of endometriosis, data about apoptotic deregulation process in the disease may be adapted. Breach in apoptosis may trigger an autoimmune process through persistent self-reactive lymphocytes and/or the inclusion of so-called death receptors and/or their ligands in tissue de-struction [32]. Inability of cells to transmit “signal death” or the ability of cells to avoid cell death is associated with other autoimmune diseases. There are studies demonstrating that the endometrial cells in ectopic and eutopic endometrium in women with endometriosis have altered apoptotic mechanisms [33, 34, 35]. Fur-thermore, it is assumed that the change in apoptosis allows retrograde endometrial tissue to “escape” from cell death. The precise mechanisms responsible for these changes of apoptosis in infected women are unknown. It has been proven that this changing the pattern of cell death can be “reversed” by GnRH-analog therapy, sug-gesting that this peptide could play an important role in the reorganization of normal endometrial cells during the menstrual cycle [33].

Growth factors:2. It is known that during the infl ammatory response, mac-rophages release a variety of infl ammatory mediators. Some may be associated with ectopic implantation of endometrial cells, for example: fi broblast-like growth factor (FGF), epidermal growth factor (EGF), transforming growth factor-α (TGF-α),


transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) [30]. It has been proven that these growth factors stimulate proliferation of endome-trial stromal cells in vitro. This implies that they can improve the implantation of endometrial cells [36]. Several studies have reported increasing activity of perito-neal macrophages in endometriosis, which is associated with production of vari-ous growth factors such as thrombocytic growth factor-β (TGF-β) and epidermal growth factor (EGF) [37, 38]. It is also found that levels in serum and peritoneal fl uid of vascular endothelial growth factor (VEGF) are increased in women with endometriosis compared with healthy controls [39]. But other authors argue that there is no correlation between serum levels of VEGF and endometriosis [40]. El-evated levels of the receptor of epidermal growth factor (EGF-R), which is involved in angiogenesis imply an active role of the EGF in the development of disease [41]. Serum insulin-like growth factor-I (IGF-I) in patients with early stage endometriosis and in healthy controls were signifi cantly reduced compared with levels in patients with severe stage of endometriosis, suggesting that IGF-I is an important mediator in the development and/or maintenance of endometriosis or progression to late stage of the disease [42].

Cytokines: 3. Cytokines are proteins or glycoproteins released mainly in the intercellular environment from leukocytes or other cells, exert their effects on cells which have secreted (autocrine action) or on nearby cells (paracrine action). Some of these proteins may circulate or pass through the cavities of the body, thus exert its endocrine action. In some cases, cytokines are found in cell membrane-associ-ated forms, where they act on adjacent cells [43]. Cytokines are key mediators of intercellular communication in the immune system. They act pleyotropic on a vari-ety of target cells, exerting proliferative, cytostatic, or differentiating chemoattrac-tant effect. Cytokines have biological activity at high concentration range and are associated with intracellular signaling and function of secondary mediators through specifi c, high affi nity receptors on target cell membranes [43].

RANTES (Regulated on Activation, Normal T-cell Expressed and Secret-ed): RANTES is a cytokine of β or “C-C” chemokine family. RANTES is hemoat-tractant for monocytes and memory cells. Besides being identifi ed by the secre-tion of chematopoietic cells, this cytokine is also secreted by some epithelial and mesenchymal cells. RANTES may be an important mediator of acute and chronic infl ammation. Numerous potential binding sites for transcription factors regulate the expression of the gene for RANTES. In normal endometrium, RANTES-protein is presented primarily in stromal compartment. In vitro stromal cell cultures synthesize RANTES-mRNA and RANTES-protein, respectively, when they impact with TNF-α and INF-γ, while epithelial cells synthesize neither transcripts nor protein [44]. But there is a signifi cant difference between normal endometrial cell cultures and those derived from endometriotic lesions. Placed under similar conditions, endometrial stromal cell cultures secrete signifi cantly greater amounts of RANTES-protein [45]. It is believed that secretion of RANTES by ectopic implants provides a mechanism


for recovery of peritoneal leukocytes. Expression of RANTES-gene has a high level of regulation in endometrial stromal cells in response to IL-1β (released by mac-rophages) acting pleyotropic. This in turn triggers infl ammation, “loop” in which IL-1β, by activated macrophages, may lead to RANTES production and subsequent monocytic chemotaxis [43]. Figure №1 shows that macrophages occupy a central role in immunobiology of endometriosis.

Fig. 1. Central roll of macrophages in immunobiology of endometriosis. Interactions between endo-metriotic cells and macrophages are mediated by growth factors, cytokines and chemokines leading to

survival of implants instead of their death (Lebovic. Immunobiology of endometriosis. Fertil Steril 2001)

IL-1: This is a cytokine that plays a central role in the regulation of infl ammation and immune responses. Originally recognized as a product of activated monocytes and macrophages, IL-1 is now regarded as an activator of T-lymphocytes and dif-ferentiation of B-lymphocytes. Two receptor agonists, IL-1α and IL-1β, are encoded by different genes, and although both proteins have only 18- 26% amino acid-sim-ilarity, they bind to the same receptors and have similar biological activities. IL-1ra, receptor antagonist of IL-1, is an endogenous inhibitor that blocks binding of IL-1α and IL-1β to IL-I receptor type. IL-1 was isolated from the peritoneal fl uid of patients with endometriosis and its concentration was signifi cantly increased compared to healthy controls [46, 47, 48]. Mori et al. observed increased levels of IL-1β in perito-neal macrophages mRNA in women with “mild” endometriosis, but increased levels of IL-1ra-mRNA in moderate and severe endometriosis. Coordinating activities of these proteins are not suffi ciently studied [49]. The role of IL-1β in strengthening the process of angiogenesis in endometrial lesions is represented by induction of angiogenic factors VEGF and IL-6, which was observed in endometriotic stromal cells but not in normal endometrial stromal-cells [50]. Vigano et al. found that IL-1β increased secretory form of ICAM-1 (sICAM-1) separated from endometrial cells,


but it can intercept, allowing refl uxing endometriotic tissue to avoid destruction in the peritoneal antrum [51].

IL-6: The pleiotropic removable cytokine IL-6 is an important regulator of in-fl ammation and immunity, which serves as a physiological link between endocrine and immune systems. IL-6 also modulates the secretion of other cytokines, in-creasing T-cell activation and B-cell differentiation, inhibits growth of various hu-man cell lines. It is 23-26 KDa, phosphoglycoprotein that exists in multiple iso-forms and is produced by many cell types, including monocytes, macrophages, fi broblasts, endothelial cells, smooth-muscle cells and endometrial epithelial and stromal cells [52, 53]. IL-6 is also produced by several endocrine glands including the pituitary gland and pancreas. Endometrial stromal and epithelial cells produce IL-6 in response to hormones and other immune activators. Endometrial stromal cells release IL-6 protein under the action of IL-1α or IL-1β, TNF-α, TGF and IFN-γ [50, 53]. Since, usually estrogen increases proliferation of endometrial epithelium suggests that estrogen causes proliferation by limiting the synthesis of epithelial cell inhibitors, such as IL-6. Parmakoupis et al. found that IL-6 inhibits proliferation of human endometrial stromal cells, this inhibition depends on the density of cells, suggesting that IL-6 may play a role in epithelial-stromal interactions governing the regulation of normal uterine function [54]. Tabibzadeh et al. suggest that fl uctua-tions of IL-6 during the menstrual cycle show feedback to estrogen action; estrogen concentrations are elevated during the proliferative phase, while the levels of IL-6 are decreased. Serum levels and levels of IL-6 in peritoneal fl uid were signifi cantly elevated in women with endometriosis compared with controls, higher levels of IL-6 were found in women with “chocolate” cysts [55, 56]. But other results reported back that the levels of IL-6 in peritoneal fl uid in patients with endometriosis are decreased [57, 58].

TNF: This are pleyotropic acting cytokines with a range of benefi cial and harmful effects, depending on the quantity produced, their tissue localization, lo-cal activity of TNF-binding proteins and their hormonal and cytokine environment. TNF-α is produced by neutrophils, activated lymphocytes, macrophages, NK-cells, several nonhomeopathic cellular branches, while TNF-β is produced by lympho-cytes. Although these TNF were initially identifi ed by their ability to kill certain cell lines, their main function is in conjunction with IL-1 to initiate a cascade of cytok-ines and other factors associated with infl ammatory responses. TNF have similar biological activities, but regulation of expression and processing is quite different. There is speculation that TNF-α facilitates endometrial cell adhesion to peritoneal mesothelium which provides a potential mechanism for initiation of endometriosis [59]. Other studies found that levels of TNF-α in the serum and peritoneal fl uid were signifi cantly elevated in the early stages of the disease and decreased in severe stages [60], i.e. concentration of TNF-α correlates with the stage of disease [61].

IL-8 and MCP-I: Interleukin-8 is a chemoattractant for neutrophils and a potent angiogenic factor. It is produced by number of cells types, including monocytes, en-


dothelial cells, fi broblasts, mesothelial cells and endometrial stromal cells [62]. Peri-toneal fl uid (PF) of women with endometriosis has been shown to have increased neutrophil chemotactic activity [63]. It has been identifi ed that IL-8 concentrations are higher in the PF of women with endometriosis than in healthy controls.

Monocyte chemoattraktive protein-I (MCP-I) is a potent chemotactic and ac-tivating factor specifi c for monocytes. It signifi cantly increases in the early stages and decreases with severity of disease in serum, while its levels in peritoneal fl uid show improvement in severe stages [60]. Studies provide measurements of se-rum and peritoneal liquid levels of IL-1β, IL-6, IL-8, IL-12, IL-13 and TNF-α at 130 women in an attempt to fi nd non-surgical method for diagnosing disease and to determine the accuracy of this non-invasive method. The survey showed that only measured levels of IL-6 and TNF-α are suffi ciently sensitive and specifi c for the dif-ferentiation of patients with and without endometriosis [63].

Fas-ligand: Fas-ligand or CD95L is a transmembrane-type II protein that be-longs to the family of TNF. It binds with its receptor and induces apoptosis. Fas-ligand receptor interactions play an impotant role in the regulation of the immune system and progression of cancer. Serum levels and the levels of Fas-ligand in peritoneal fl uid were found to be elevated in patients with moderate and severe endometriosis compared with women with minimal form of development or mild disease, and women free of disease [64]. Garcia-Velasco et al. showed that mac-rophage-conditioned media induced Fas-ligand expression by endometrial stromal cells, thus suggesting that peritoneal macrophages in endometriosis might stimu-late a Fas-mediated apoptosis of immune cells as a further means of escape from immune surveillance [65].

Soluble adhesion molecules (sICAM): These molecules can be used as an indicator for endometriosis [66]. Concentration of soluble HLA-I class molecules and sICAM-I was higher in patients with stages I and II disease, suggesting that they possibly take part in the pathogenetic mechanisms of endometriosis [67]. El-evated ICAM-I was found in patients with severe endometriosis, but its sensitivity is not high and the concomitant use of the CA-125 marker will increase the sensitivity and specifi city of detection [68, 69]. Many more soluble adhesion molecules can be used as markers to determine the stage of disease [70]. It is reported that se-rum soluble E-cadherin (sE-cadherin) is secreted throughout the menstrual cycle in women with endometriosis. Patients with III and IV stages of disease had higher serum concentrations of soluble VCAM-I, lower serum concentration of sICAM-I, and no difference in the serum concentration of soluble E-selectin.

Insoluble cell-adhesion molecules: It is believed that the shed endometrium in women affected by endometriosis attaches and spreads in the pelvis by specifi c cell adhesion-receptors [71, 72]. Hence, the adhesion of endometrial cells to the extracellular matrix (ECM) would be expected to play role in the pathogenesis of endometriosis [73]. Various cell adhesion molecules (CAMs) have been investigat-ed for their expression in endometriotic endometrium. Each cell type expresses a


distinct pattern of integrins and other CAMs, including the cadherins, selectins, and members of the immunoglobulin family [74]. The superfamily of cell adhesion pro-teins is believed to be critically involved in most cellular-level processes in higher organisms, including cellular differentiation, motility, attachment, and cell–cell com-munication. The major cell-surface receptors for the ECM are integrins. Integrins are a major class of CAM, so named as “integral” receptors for the ECM. Endo-metrial stroma, epithelium, and vascular cells each display a characteristic pattern of integrin expression. Expression of integrin αvβ3 was signifi cantly decreased in endometriosis tissues [71, 72]. Integrin α3 was absent in all the endometrial tissue of patients with endometriosis, whereas there was no change in integrin α6 and integrin β3 expression in samples from sick and healthy patient [75]. This suggests that integrins are involved in the process of human embryo implantation.

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E i s e r m a n n , J. et al. Tumor necrosis factor in peritoneal fl uid of women undergoing laparo-60. scopic surgery. – Fertil. Steril., 50, 1988, 573-579.A r i c i , A. et al. Regulation of interleukin-8 gene expression in human endometrial cells culture. 61. – Mol. Cell Endocrinol., 94, 1993, 195-204.L e i v a , M. C. et al. Increased chemotactic activity of peritoneal fl uid patients with endometriosis. 62. Am. J. Obstet. Gynecol., 168, 1993, 592-598.B e d a i w y , M. A. et al. Prediction of endometriosis with serum and peritoneal fl uid markers: a 63. prospective controlled trial. – Hum. Reprod., 17, 2002, № 2, 426-431.G a r c i a - V e l a s c o , J. A. et al. Elevated soluble Fas-ligand levels may suggest a role for apop-64. tosis in women with endometriosis. – Fertil. Steril., 78, 2002, № 4, 855-859.G a r c i a - V e l a s c o , J. A. et al. Macrophage derived growth factors modulate Fas-ligand ex-65. pression in cultured endometrial stromal cells: a role in endometriosis. – Mol. Hum. Reprod., 5, 1999, 642-650.D e P l a c i d o , G. et al. Serum concentrations of soluble human leucocyte class I antigens and 66. of the soluble intracellular adhesion molecule-I in endometriosis: relationship with stage and non-pigmented peritoneal lesions. – Hum. Reprod., 13, 1998, № 11, 3206-3210.L e n g , J. et al. Serum levels of soluble intercellular molecule I /sICAM-I/ in endometriosis. – 67. Zhonghua Ji Xue Za Zhi, 82, 2002, № 3, 189-190.S o m i g l i a n a , E. et al. Use a serum-soluble intracellular adhesion molecule-I as a new marker 68. of endometriosis. – Fertil. Steril., 77, 2002, № 5, 1028-1031.F u , C. et J. Lang. Serum soluble E-cedherin level in patients with endometriosis. – Chinese Med. 69. Sci. J., 17, 2002, № 2,121-123.G o l u d a , M., K. Kuliczkowski M. Jedryka. The concentration of exfoliative adhesion molecules 70. (ICAM-I and E-selectin) in serum and peritoneal fl uid of women with endometriosis. – Ginekol. Polska, 68, 1998, № 12, 1175-1178.K o k s , C. A. et al. Adhesion of menstrual endometrium to extracellular matrix: the possible role of 71. integrin alpha(6)beta(I) and laminin interaction. – Mol. Hum. Reprod. 6, 2000, № 2, 170-177.S p u i j b r o e k , M. D. et al. Early endometriosis invades the extracellular matrix. – Fertil. Steril, 72. 58, 1992, № 5, 929-933.S t r e u l i , C., N. Bailey et M. J. Bissell. Control of mammary epithelial differentiation: basement 73. membrane induces tissue-specifi c gene expression in the absence of cell interaction and morpho-logical polarity. – J. Cell Biol., 115, 1991, 1383-1395.B o u d r e a u , N. S. C., Z. Werb et M. Bissel. Suppression of ICE and apoptosis in mammary 74. epithelial cells by extracellular matrix. – Science, 267, 1995, 891-893.S u z u m o r i , N. et al. Expression of secretory leucocyte protease inhibitor in women with endo-75. metriosis. – Fertil. Steril, 72, 1999, № 5, 857-867.

Address for correspondence: Nelly Manolova Department of Immunochemistry Institute of Biology and Immunology of Reproduction 83 Tsarigradsko shosse blvd. 1131 Sofi a, Bulgaria

02 9711395/235 e-mail: [email protected]


ZINC – THE BREAKTHROUGHS. S. Mileva

Department of Infectious Diseases, Epidemiology And Tropical Medicine, Medical University of Varna

Summary. The essentiality of zinc for plants and animals has been estab-lished by science for decades. Nevertheless, its signifi cance for human health has been revealed only at the end of sixties of the XX century. Since then the interest of medical scientists for that element keeps growing. As a consequence in the last 40 years we became witnesses of a dramatic breakthrough in the knowledge of this unique mineral and its applications in medicine. The aim of this review is to ex-plore the current concepts of the zinc signifi cance for human physiology and some pathologic conditions. Special importance is attached to its place in the treatment of infectious diarrhea, because acute diarrhea is a huge part of the infectious pathol-ogy in our country. There is no established practice yet for additional treatment with zinc in the cases of acute diarrhea in Bulgaria.

Key words: zinc, human physiology, diarrhea

Biological functions of zinc. Zinc is an essential element found in ev-ery cell of the microorganisms, plants and animals. The presence of this metal in the biologic systems is ubiquitous despite the fact that it

is not so abundant on earth as a whole. The causes are many, but two of them are fundamental. The fi rst one is the perfection of the regulation of its cell entry, distri-bution and excretion, which made it practically non-toxic. The second one is its ca-pability of stabile and fl exible binding with macromolecules, which allows it to meet the needs of proteins and enzymes with a wide range of biologic functions. That is how zinc participates in the metabolism of proteins, nucleic acids, carbohydrates, lipids, gene transcription control, etc. [54].

Unlike any other metal it enters into the composition of all six enzyme classes: oxidoreductases, transferases, hydrolases, lysases, isomerases and ligases, in-cluding RNA polymerase, carboanhydrase, alkaline phosphatase, and alcohol de-hydrogenase [40]. In addition it infl uences the effect of several signal molecules,

Zinc – the breakthrough52

it plays the role of a neurotransmitter by itself, and according to a new study of Ya-masaki et al. zinc is also an intracellular second messenger [54, 59].

Tissue zinc distribution. The whole zinc content in an adult human is about 1,5-2,5 grams, 83% of it is concentrated in the skeletal muscles and the bones. It is predominantly an intracellular ion (over 95%), with highest concentration in teeth (250 mg/100 g), hair (200 mg/100 g) and prostate (100 mg/100 g). There is no con-ventional tissue depot of zinc that can release or sequestrate quickly the needed quantity according to the diet variations [24].

Less than 0,2% of the whole content of zinc circulates in the plasma, where its concentration is nearly 15μmol/L [9].

Zinc metabolism. Zinc is released as free ions from food during digestion. In the intestinal lumen it connects to endogenously secreted ligands or to exogenous materials. After that it is absorbed in the distal duodenum and proximal jejunum. Zinc is transported through the enterocytes and its entry into the portal circulation is followed by a quick liver uptake. Further it is released in the systematic circula-tion, bound to the albumin in 70%, available for all tissues. Any albumin drop leads to serum zinc depletion. Its concentration also falls in a stress condition (trauma, acute infection) when the elevated levels of cytokines and cortisol stimulate the tis-sue uptake of zinc [24].

Zinc is excreted from the body through the skin, the urinary and gastrointes-tinal systems. Losses through the intestines vary from 7 mmol/24 h (0,5 mg/24 h) to above 45 mmol/24 h (3 mg/24 h) in relation with the intake. Losses through the skin are about 7-10 mmol/24 h (0,5-0,7 mg/24 h) and rise in a high temperature environment, due to an increase in perspiratio insensibilis. The same amount is secreted through the urine and it is rising in starvation and muscle tissue catabolic processes.

That is how the serum concentration depends on the zinc intake, the intensity of tissue absorption, serum albumin levels and excretion process dynamics.

In a case of lessening of the amounts of zinc in the diet the organism falls to a negative zinc balance for a while, until it succeeds to regain it, according to the new conditions. These brave episodes could have discrete consequences, as for example suppression of the immune function [24].

Natural sources, bioavailability of zinc and daily requirements. Zinc as-similation mainly depends on its amount in the food and the presence of substanc-es stimulating or suppressing its absorption.

Several studies in different age groups demonstrated the homeostatic mecha-nisms of positive feedback of zinc absorption. Its assimilation increases by diet restrictions [29].

The strongest inhibitor of zinc absorption is phytic acid (inositol hexaphos-phate) and its salts, known as phytates in the literature. They attach it irreversibly in the intestines. The highest amounts are found in wheat, beans, nuts, less in fruits and vegetables. That is what makes them non-reliant sources of zinc, despite that


the concentration is relatively high in those foods [24]. In many countries rice, corn and vegetables are the main food habit, therefore zinc defi ciency is frequently ob-served even in adequate intake.

The phytate content in a food product can be decreased by enzyme and non-enzyme methods of hydrolysis.

Phytase exrtract from Aspergillus oxyzae or from Aspergillus niger is added to the food in enzyme hydrolysis, but this is an expensive and practically diffi cult method [53]. The more appropriate method is the activation of the endogenous plant phytase or the stimulation of its synthesis de novo, which can be achieved by some practices of food processing, like bread rise, microbial fermentation and germination. In a study in Africa those practices showed a signifi cant reduction of phytate content in some plant-based foods [25, 51].

Phytates in some plant foods can be partially hydrolised nonenzymatically by milling, thermal processing and soaking. Methods of nonenzyme hydrolysis alone are less effective, but the combination of both approaches achieves a signifi cant increase of the zinc bioavailability [25]. It is established that phytates in the food are a mixture of several different molecules, each one suppressing the zinc absorption to a different degree. They are in different proportions in the different foods, so the calculation of the whole phytate content in a product is a rough indicator of the zinc bioavailability.

The highest concentrations of zinc are found in oysters, followed by mam-mals’ and poultry’s meat and organs, fi sh, shellfi sh, in a less extend in eggs, milk and dairy products. As all those foods don’t contain phytates, they seem to be the perfect sources of easy absorbable zinc.

Fibres are also thought to inhibit zinc absorption but several studies showed that this is due to the fact that plants, containing fi bers, contain large quantities of phytates as well. In conclusion, fi bers alone do not exercise a signifi cant infl uence on zinc assimilation.

Until the eighties of ХХ century calcium has been considered an inhibitor of zinc absorption. In accordance of that its quantity in the food was taken into account in the algorithm of Murfy et al. for estimation of the zinc bioavailability [22]. The recent studies found that calcium infl uences the zinc absorption only from foods, rich of phytates. Even, in result of the complex interactions sometimes the supplementation with higher amounts of calcium leads to an increase of the zinc bioavailability. That’s why calcium is already excluded as a predictive factor for the zinc assimilation.

In 1981, Solomons and Jacob found that iron inhibits the zinc absorption in the intestines only in the cases when zinc is in inorganic form and iron is not con-nected in hem (in their study zinc sulfate and iron sulfate) and the proportion iron/zinc is above 1/1. Therefore, a decline in zinc absorption could be expected when it is given as an inorganic compound in patients with iron therapy.

Soya is a well known powerful inhibitor of zinc absorption but it is due to the high amounts of phytates, not to its protein as was considered in the past [32].


It is established that animal proteins increase zinc bioavailability as a whole, except for the milk casein. Nevertheless proteins are not included as a determining factor in the fi nal algorithm of The International Zinc Nutrition Consultative Group (IZiNCG) for estimation of the zinc bioavailability in the diet. The work on that prob-lem has shown that quantities of zinc and phytate and their molar proportion in the food are stabile and adequate indexes of the zinc bioavailability. The phytate/zinc ratio in a food or a diet can be calculated as follows:

mg phytate/660,

mg zinc/65,4

where 660 = phytate molecular weight, 65,4 = zinc molecular weight [24].According to the World Health Organization (WHO) diets with phytate/zinc

molar ratio over 15 are low bioavailability diets with zinc absorption of 15%. Those are cereal-based diets, with very low intake of animal proteins. Over 50% of the energy is acquired from unrefi ned grains and legumes.

Diets with phytate/zinc molar ratio between 5 and 15 are of moderate bio-availability with 30% of zinc absorption. Those are mixed diets and lacto-ovo-vegetarian diets that are not based on unrefi ned cereals or high extraction rate fl ours (over 90%).

Diets with molar ratio under 5 are of high zinc absorption level (50%). The latter are refi ned diets with low amounts of fi bers with animal proteins as a main source of energy [3].

The zinc absorption percents of those three diet types are equal for all people, regardless of sex, age and geographic origin.

In contrast to WHO, IZiNCG divides diets into two categories. The fi rst one is with phytate/zinc molar ratio from 4 to 18 with zinc absorption levels of 26% in men and 34% in women over twenty. Here belong mixed and refi ned vegetarian diets. The second category of diets includes unrefi ned cereal-based diets with molar ra-tio of over 18. The absorption of zinc is calculated to be 18% in men and 25% in women over 20 years of age [24].

Until now nor WHO, neither IZiNCG have found reasons to assume different levels of zinc absorption for the different age groups. Therefore at this stage of knowledge, data for zinc bioavailability in the different diets are applying to children under the age of 18.

Changes of zinc absorption in pregnancy are dependent on the zinc status. In pregnant women with a normal zinc status, there has not been found a signifi cant increase in zinc absorption. In pregnant women with a diet with zinc amounts at the lower limits, the absorption rises signifi cantly. In the lactation period, an increase in zinc absorption has been observed in all cases [15, 47]. According to those studies’ data IZiNCG accepts a level of absorption of 44% in lactating women with mixed and refi ned vegetarian diets (over 19 years) and 40% (between 14 and 18 years).


In lactating women with unrefi ned cereal-based diets, the level of absorption is fi xed at 35% (over 19 years) and 32% (between 14 and 18 years).

In 1996, a committee of the WHO estimated the physiologic requirements for zinc in adults as the sum of the quantities needed for tissue grow and maintenance, metabolism and endogenous losses. The intensity of zinc excretion through the intestines and the kidneys depends on the zinc status, that’s why WHO gives two separate values. First one is the so called “basal requirements” of 1,0 mg/d for men and 0,7 mg/d for women. Those are the amounts of zinc which cover the physi-ologic needs in individuals fully adapted to low levels of zinc consumption. Since these border levels do not leave any reserve in a case of additional drop of the zinc intake, the second value, the “normative physiologic requirements” was assumed to be 1,4 mg/d for men and 1,0 mg/d for women.

After the normative physiologic requirements and the percents of zinc absorp-tion are established for a category of individuals, the requirements for zinc in the diet can be calculated as follows: diet requirements = (normative physiologic re-quirements) / (percents of absorption from the common diet). The WHO committee establishes such a value of the recommended zinc intake for a population, so indi-viduals with consumption of less than 2 SD in comparison with the mean population consumption, to meet their normative physiologic requirements [9].

To establish the children dietary requirements for zinc, the amounts needed for tissue growth and the relatively larger endogenic losses per weight unit, have been considered [3]. According to WHO until the age of 6 months zinc requirements are covered fully by breastmilk with 80% absorption from it, IZiNCG points lower percents – 50% [9, 24].

For the lack of suffi cient studies WHO accepts that zinc requirements of elderly over 60 years do not differ from those assumed for the age 18-60 years. At the end of 2008, a study of Childrens’s Hospital Oakland Research Institute fi nished, with over 6000 healthy persons over 70 included, which defi nitely showed lower levels of zinc absorption in this group, 21% less in men and 17% less in women [14].

IZiNCG revised estimates of zinc dietary average requirements for the differ-ent age groups, sex, pregnancy and lactation periods in accordance with the differ-ent categories of diets can be found as a table in the Food and Nutrition Bulletin, Vol. 25, Number 1, March 2004, Technical Document #1.

Zinc toxicity. Zinc has a low toxicity, in spite of that acute and chronic forms of poisoning were reported.

Acute intoxication occurs after consumption of large amounts of zinc (225-450 mg for adults) and nausea, vomiting, diarrhea, epigastric pain, malaise and fever develop [16].

Chronic overdosing (100-300 mg/daily for adults) leads to lowering of the se-rum copper concentrations due to inhibition of the absorption; anemia; immune disturbances (suppression of the lymphocyte response after phytohemagglutinin stimulation, of the chemotaxis and the bacterial phagocytosis from polymorphonu-


clear leucocytes); decline of the serum HDL [12, 17, 23, 48, 58]. All the reported cases demonstrated full reversibility of the condition, caused by zinc overdosage.

The upper limits of daily zinc intake for the different age groups and sex, as-sumed by expert commissions of WHO, US FNB/IOM and the estimates revised by IZiNCG are published as a table in the Food and Nutrition Bulletin, Vol. 25, Number 1, March 2004, Technical Document #1.

Zinc defi ciency. The multiple biologic functions of zinc determine the wide range of metabolic and physiologic processes, depending on zinc status. Growth, neurobehaviour development, immune competence and reproductive function could be mentioned here.

Stunting is a cardinal symptom of zinc defi ciency, not related to the loss of ap-petite and decreased energy intake, which is demonstrated in studies with animals. Zinc participates in cell division and proliferation by a direct and hormonal regula-tion of DNA and protein synthesis [11].

Zinc infl uences the gene expression through the transcription factor. It con-trols the cell entry in S-phase of the cell cycle and also affects the enzyme thymidin kinase [34].

The axis hypophyseal growth factor – insulin-like growth factor-I (ILGF-І) is zinc dependent. It is established that zinc defi ciency lowers the levels of circulat-ing ILGF-I and alters the postreceptal induction of the cell proliferation [61]. That was established in a trial, involving children with diabetes type I, where the zinc concentrations and the levels of ILGF-I in sera were found to be lower, compared to healthy controls at the same age [6].

A meta-analysis of Brown et al. of 33 randomized control trials about the effect of zinc supplementation on serum zinc concentration and growth found positive ef-fect, markedly in children with low height and weight for age scores [10].

A recent analysis of Ramakrishnan et al. examined 43 studies from the period 1996-2008 of children up to 48 months of age and did not fi nd a positive effect of zinc supplementation on the growth curve [44].

Zinc defi ciency is known to be related to the loss of appetite in the seventies of XX century in animal model experiments. Experiments using mice models revealed that zinc intake increases appetite through a stimulation of the Y neuropeptide and orexin expression in the hypothalamus and through an afferent vagal stimulation [30]. Zinc supplementation increases the appetite in zinc defi ciency populations [24]. A lot of publications already exist about the success of zinc supplementation in the complex therapy of anorexia nervosa [7, 27].

In the second half of the XX century, the relationship between the zinc status and the nervous system development was established in animal experiments. In the central nervous system, zinc is concentrated in the synaptic vesicles of the glutaminergic neurons. Those zinc-containing neurons are concentrated in the ce-rebrum and form a network, connecting brain cortex and limbic structures [20]. The exact function of this network is not clarifi ed yet, but is well known that the


majority of the zinc-containing glutaminergic neurons are sited in the cortex and the amygdale and those ones, which do not contain zinc are mainly subcortical and spinal. According to this, some authors connect the zinc-dependant glutaminergic synapse function with the cognitive and mnemonic operations, characteristic for the cerebrum [20, 37].

In experimental studies with rats, the severe zinc restriction in pregnancy led to fetal neuronal degeneration and diminution of the brain size. The role of zinc as a modulator of the neuronal proliferation and apoptosis was confi rmed in experiments on cell lines as well [1, 35]. The relationship between neurobehavioral function and zinc status in people is established in studies with zinc supplementation in popula-tions with defi ciency. Some of the studies showed a positive relation between the improving of the zinc status and children motor development and neurobehaviour, while others did not. That is why the dependency of the cognitive and motor devel-opment on zinc status in childhood is unconvincing to date [8, 9].

Zinc defi ciency is thought to be related to some disease conditions in preg-nancy, childbirth complications, abnormalities in the fetus development and neo-natal morbidity. Randomized control trials with zinc supplementation in pregnancy demonstrated a relationship between zinc defi ciency and only some of the sus-pected conditions: pregnancy related hypertension, preterm delivery, intrauterine retardation, low birthweight, increased morbidity among the prematurely born in-fants and poor neurobehavioral development. The rest of them were not found to be categorically related to the zinc status (preeclampsia, intra- or postpartum haemorrhage, prolonged labor, placentar abruption, premature rupture of amniotic membrane, ineffi cient uterine contractions, postpartum infections, congenital mal-formations, spontaneous abortions, fetal distress syndrome, neonatal sepsis, low Apgar score and asphyxia) [9]. Recent studies, examining the zinc status of the mothers and their newborn babies did not fi nd a relation between zinc defi ciency and low birth weight [2].

There is no doubt that immune system is most strongly and early affected as zinc status worsens. The immunologic mechanisms of zinc for modulation of the susceptibility to infections have been studied for decades. It’s clear now that it af-fects the immune system at many levels – from the epithelial barrier function to the gene regulation processes in lymphocytes [19, 36]. Zinc plays a crucial role in the proper development and function of the cells, participating in the nonspecifi c im-munity – neutrophils, natural killer cells (NKC) and macrophages. It also regulates T-lymphocyte activation, Тh1 cytokine production, B-lymphocyte immunoglobulin production, mostly of the G class. The zinc dependence of the immune system is founded on its participation in basic cell functions as DNA synthesis, RNA transcrip-tion, cell division and activation. Zinc functions as an antioxidant and membrane stabilizer, its insuffi ciency potentiates cell apoptosis [18].

A long time ago thymus atrophy and lymphopenia became a distinguishing feature of protein-calorie defi ciency, which coexists naturally with zinc defi ciency.


Zinc was found to be an independent factor for the immune system integrity in experimental works using mice models and during the study of acrodermatitis en-teropathica [19].

Acrodermatitis enteropathica is a rare genetic disease with autosomal reces-sive inheritance, due to an error in zinc metabolism resulting in zinc malabsorption. The defective gene is identifi ed as SLC39A4, which encodes transmembrane trans-port protein of zinc Zip4 [31]. In conditions leading to severe zinc defi ciency, symp-toms clinically indistinguishable of the inherited disease have been observed. That acquired variant is rare as well, that is why case reports are published in the peri-odic specialized literature: as a result of alcoholism, parenteral nutrition for a long period, mucoviscidosis, insuffi cient amounts in the breastmilk, especially in preterm born babies, short bowel syndrome after resection, after pancreatoduodenectomia and other surgery interventions, in anorexia nervosa, infl ammatory bowel disease, etc [13, 38, 39, 28, 49, 60, 52, 50]. Acrodermatitis enteropathica was fi rst described in the seventh decade of the last century and has been one of the earliest proves that zinc defi ciency in humans leads to thymus atrophy, lymphopenia, suppressed cell immunity and as a consequence – increased susceptibility to infections. Serum zinc concentrations and urine excretion rates are usually as twice as lower than the normal levels [56]. The most frequent symptoms are periorifi cial dermatitis, inter-mittent diarrhea, alopecia, stunting, weight loss, mood changes.

At the end of the 70-s of the XX century, experiments with healthy adult mice showed that zinc defi ciency for 4 weeks period results in thymus atrophy with sup-pression of the cell mediated immunity, the late anaphylactic reaction and antitumor defense. In the same time, they cleared up that immunity system damage is abso-lutely reversible after zinc status repair, no matter how extensive it was. That was also confi rmed in experiments with people [43].

Animal model experiments, as well as human studies established that subop-timal zinc intake leads to chronic production of glucocorticoids (GС). To date, over 50 publications point out that zinc and protein-calorie defi ciency activate hypothala-mo-hypophyseal-adrenal axis with chronic production of GC [19]. As GC are classic inductors of the apoptosis of T-lymphocytes, B and T precursor cells, it is clear that at least partially the lymphopenia and thymus atrophy in zinc defi ciency are due to those hormonal changes. In the same time, expansion of granulocytes is observed, which could be explained with an inhibition of granulocyte apoptosis by GC [57]. Simultaneously, alteration of the macrophage function and neutrophil chemotaxis takes place [41].

Prasad et al. have found in experiments with volunteers with mild zinc defi -ciency, a signifi cant drop in serum thymulin activity, which modulates T-cells alo-genic cytotoxity, suppressor function and the production of interleukin-2 (IL-2). As zinc status restores, thymulin activity normalizes [41].

Studies using cell lines established that zinc depletion lowers the levels of IL-2 and interferon-gamma (IF-γ) and increases those of tumor necrosis factor-


α(TNF-α), IL-1ß and IL-8. Data show that cytokine levels are changed in result of an altered expression of the coding genes [4]. The functions of NKC depend on IL-2, logically zinc defi ciency decreases their activity and the intake of exogenous zinc stimulates them to produce γ -IF [19, 45].

Experimental studies with healthy volunteers have shown that zinc has antiin-fl ammatory and antioxidant effects as well [26, 42].

All the above mentioned clearly demonstrates the close connection between the immune system integrity and zinc status. This explains the existence of a plenty of publications, describing the benefi cial effect of zinc supplementation in the ther-apy of various infectious diseases (infectious diarrhea, pneumonia, malaria, AIDS, tuberculosis, etc.), especially in patients with some degree of zinc defi ciency.

Zinc in the therapy of diarrheal diseases. Diarrheal syndrome is almost in-evitable in severe zinc defi ciency. The explanation are not only the immune system alterations, but also the reduction of sodium transport and the water bound to it in the intestines, in zinc insuffi ciency conditions [21]. The losses of zinc through the intestinal tract increase in diarrhea, which further leads to worsening of the zinc defi ciency and turns on a vicious circle.

On the other hand, diarrheal diseases are a global health problem, which causes in spite of the modern medicine advances about 2,5 millions of deaths per year. They keep being the second mortality cause in the world childhood population.

The above facts are a reason for the great hopes on the zinc supplementa-tion, applied to the basic treatment for diarrhea, especially in children under 5 years of age. Many trials with zinc supplementation in children with diarrhea have been published, almost all carried out in developing countries.

In the year 1999, Bhutta et al. published the results of an analysis of 10 ran-domized trials on the effect of zinc supplementation on diarrheal episodes in pa-tients younger than 5 years. In 3 of them, zinc was applied for two weeks with a following observation of 2 to 3 months and in the rest 7 trials the supplementation lasted longer (from 2 to 3 months). The conclusion of the authors was that the use of zinc considerably decreases the severity and duration of the diarrheal episodes, not only in the trials with zinc intake for longer periods, but also in the shorter dura-tion supplementation trials [5].

In 2008, Lukacik et al. published the results of a meta-analysis of 22 ran-domized double-blinded trials about the effectiveness and safety of zinc therapy in children with acute and persistent diarrhea. According to that analysis, zinc intake decreases the severity and duration of the disease. More exactly, the median stool frequency in the children who received zinc had been reduced by 18,8 and 12,5%, and the diarrhea duration by 15 and 15,5%, compared to those ones who received placebo in acute and persistent diarrhea, respectively. In 11 of the trials with acute and in 4 with persistent diarrhea, vomiting after the treatment was reported, con-siderably more pronounced in the patients receiving zinc, in comparison to those receiving placebo [33].


Both analyses had included studies carried out in South Asia, South America and Africa. The meta-analysis of Lukacik et al. also took in a randomized control trial from Australia and another one from Turkey [55, 40].

As a result of all the evidences that zinc is effective and safe in acute and persistent diarrhea and is even capable to reduce the diarrhea related childhood mortality, WHO and UNICEF added zinc to their jointly elaborated recommenda-tions for the management of acute diarrhea in 2004 [46].

In spite of the abundance of heterogeneous information about the medical im-portance of zinc, a lot of questions and gaps in knowledge stand still. Here could be mentioned the not fully elucidated interactions between the particular dietary fac-tors, infl uencing zinc absorption from diets, the lack of accessible and exact clinical and biochemical indexes of mild zinc defi ciency, the lack of studies on the effective-ness of zinc supplementation in childhood diarrhea in the developed world.

The review is a part of a scientifi c research project “Assessment of the zinc sta-tus of young infants with acute diarrhea and effi cacy of zinc supplementation therapy”, fi nanced by “Scientifi c research fund”, Ministry of Education, Youth and Science.

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USE OF POSTS IN DENTAL PRACTICEE. Boteva

Department of Conservative DentistryFaculty of Dental Medicine – Sofi a

Summary. Metal posts are often used in the dental practice for non vital teeth .There are different views in the dental literature about the indications for their use. The traditional approach to fi x them always when a cusp or wall is missing, is not commonly used. In most of the cases, posts are fi xed when a large amount of hard tissues is missing. The classical approach is accepted as based mainly on the experience from the preadhesive dental techniques. The aims of the present study are to evaluate the frequency of the complications in front and distal teeth, pins, casted posts and cores or cemented posts. The following parameters were registered: type of the post, length, diameter, complications including empty root canals, periapical lesions, etc. The results have shown that the aesthetic posts are not much used in the dental practice in Bulgaria. The most frequent use of posts is in front teeth and premolars (81.3%). The reason for that can be to avoid complica-tions, more common in molars. Cemented prefabricated metal posts are used most frequently (60.3%), and casted less (8.9%). The study shows that the fi nancial fac-tor plays a central role in the choice of dental posts.

Key words: posts, dentistry

In the last two decades, the use of posts for reconstruction of dental crowns become an routine practice, not only for distal teeth, but for incisors too. There are different visions in the dental literature over this problem. The

traditional approach of replacement of more than one wall or cusp with a post is more or less based on the knowledge of preadhesive dentistry era.

There is a long history back in the use of dental posts. In 1839 and 1848, post crowns (Richmond type) were described and used in restorative dentistry [6]. Other authors defend the vision that not very destroyed teeth are more stable without posts and cores [5, 11]. But even without massive tooth loses MOD cavities them-selves are decreasing the mechanical integrity up to 63% [9]. Fracture resistance

Use of posts in dental practice64


is related to the amount of remaining dentine especially in buccolingual dimension, and to the amount of root canal preparation [10].

In the last 20 years, prefabricated posts become much more popular com-pared with the traditional casted post and cores, which are much more expensive and time consuming [12]. In the last 10 years, aesthetic posts are much more popular for scientifi c research trials and experiments like fi ber, glass-fi ber, ceramic etc.posts. It is not known at all how much all these posts are in use in our dental practices.

In the dental literature, the retentive and mechanical factors of posts are eight [7]: length, diameter, type of post, type of cement and method of cementation [4], dimension of the root canal, its preparation and the place of the particular tooth in the dentition [10].

This method can result in iatrogenic errors and complications leading to teeth extractions or bone operative interventions, different in different quadrants of the mouth.

In our dental literature, this problem is only mentioned in one literature review and only very few of the main problems are looked at, like: length, diameter, and length and diameter of the root canal and the type of the post [1]. Problems like the frequency of use in different groups of teeth [3, 12], and the frequency of iatrogenic errors in dental practice are unknown [2, 3]. Most of the publications are related to fracture resistance and in the last years only to aesthetic posts.

AIMS of the present study are:To register the frequency of iatrogenic errors in the use of posts.1. To defi ne the frequency of use in different groups of teeth.2. To determine the most used types of dental posts. 3.

MATERIALS AND METHODS:

Patients: of the Dental Faculty in Sofi a between 2008-2010 were included in the study. All previous posts and cores were observed on x-rays, three per tooth, n = 1860 total. All endodontically treated teeth were 1291 with 2116 root canals, of them 150 teeth were with 156 posts, two of them fi xed on teeth with periapical pathology.

On dental X-rays of the teeth with posts, there were observed and regis-tered:

T1. ype of the tooth: incisor, premolar, molarType of the post: pins and cemented or casted posts.2. Length i3. n the root canal: less than ½, up to ½ and ½.MD dimension of the root canal: below 1/3, up to 1/3, up to 2/34. Iatrogenic mistakes like posts > 2/3 and perforations5. Posts with periapical lesions – periodontitis6. Posts in empty root canals7.

Exclusion criteria: x-rays with poor quality.

Fig. 1. Teeth with iatrogenic errors with posts from A color atlas and text of endodontics, 1995, second edd., Stock et al.

RESULTS

Of 156 posts on 150 teeth 53 or 35.3% were used for front teeth restorations. For restorations of premolar teeth 69, 46.0% were used and only 23, 15.2% were fi xed on distal teeth. Only few were the posts fi xed of third molars.

According to the type of fi xation 94 were cemented – 60.3% and 48, 30.8% were pins. Only 14, 9.0% were the casted post and cores.

Less than ½ of root canal length were 80 or 51.3%, up to ½ were 49, 31.4% and up to 2/3 were 17or 10.9% of the posts. Less than 1/3 of the MD dimension were 37, 23.7%, up to 1/3 – 38, 24.4% and up to 2/3 were 50 or 32.05%. Over preparation of root canal dentine and sizes over 2/3 of MD dimension were found in 21 teeth or 13.5%, 4 perforations, 2.6% and in 11 cases – 7.0% were cemented in not fi lled root canals.

All groups are shown on table 1.

Table 1. X-ray valuation of posts (3 Ro gr per one post)

LengthType of tooth Type of post МD dimension Errors

> 2/32153 PM

69М23

Cem.94

Pins48

Cast14.

< 1/337

Upto1/338

Upto2/350

< 1/2 29 41 8 53 25 2 35 18 18 8½ 21 20 11 30 12 7 2 16 24 92/3 3 8 4 9 5 3 - 4 8 4



In this study, aesthetic posts were not found.

Fig. 2. Correct and incorrect choices of cemented posts on premolar teeth in two patients

Fig. 3. Incorrect choice of a metal post on a very thin root

Fig. 4. Incorrect choice of a self made small post in a large root

Fig. 5. Correct use of a pin in a distal root of a lower molar

Fig. 6. Correct use of two pins in an upper molar

Fig. 7. Very poor choice and use of a cemented post in upper premolar

Fig. 8. Correct choice of post in one poorly treated uper premolar and perforation of 1 of two posts in tooth 16

Fig. 9. Correct compromice in one short root of a lower tooth

Fig. 10. Poor choice and use of a cemented post in one upper premolar

DISSCUSSION

Two of the posts were fi xed in roots with large periapical lesions, which can be classifi ed as an error. In 11 cases, posts were fi xed in empty root canals and over preparation with damages on the dentine of posts bigger than 2/3 of the MD dimen-sion in 21 patients also can be classifi ed as an profound error. Perforations in 4 cases too. This results in a total of 23.1% were severe iatrogenic errors. Underestimation of non effi ciency of very small and short posts (less than ½ of root length) will be also a mistake. So are 35 – or 22.4%, and 80 – 51.3% of the posts. In 35 patients or 22.4% the posts were both thin and short. All inadequate posts were 45.5% !!!

These data are high and confusing as compared with the data found in the students’ post endodontic restorations where the same percentage is 33.2. We found in this group of patients that posts were used in 11.6% of all endodontically treated teeth and nearly half of them were compromised in the last step of the en-dodontic and restorative treatment.

Nearly half of the mistakes were made in front teeth and premolars, although in these teeth root canals are straight and the visual control is easier. These facts are proving that the working and manipulation technology and the choice of posts are inadequate in many patients.

Very limited use of casted post and cores can be easily explained with their higher price and more diffi cult technology. Some authors have proved the advan-tages of prefabricated posts, compared with casted ones [2, 3]. One of this re-search trials was published in 2010 on 112 teeth, followed up for 10 years [3] and 317 in two trials from 2007 [2].

Most mistakes in our study were found when pins were used – In 6 patients.

CONCLUSIONS:

There are no evidences of routine or even often use of aesthetic posts in 1. the dental practice.



Posts are used mostly in the treatment of incisors and premolars – 81.3%, 2. and much less in molars. Reasons for this are to avoid complications, or in the fi nancially motivated task for more fi xed ceramic restorations.

Cemented posts (60.3%), pins (30.8%) and casted posts (8.9%) are used 3. in very different frequencies. This means that the choice in the practice is based mainly on the lowest possible price.

Iatrogenic errors and complications account for 45.5% which is a bad cer-4. tifi cate for the level of the general dental practice.

It can be extreamlly usefull for patients’ health and better students educa-5. tion all aspects of the use of posts and cors to take a better place in postgraduate education programs, dental materials companies seminars etc. It is highly recom-mended all fi xed posts to be controlled with x-rays.

REFERENCES:M i r o n o v a , J. et R. Vasileva. [Moderm approaches in the usage of posts]. – Dent. Med., 1 . 2, 2008, 137-141. (in Bulgarian)B o l l a , M. et al. Root canal posts for the restoration of root fi lled teeth. – Evid. Based Dent., 2 . 8, 2007, № 2, 42.G o m e s -P o l o , M. et al. A 10 year retrospective study of the survival rate of teeth restored with 3 . metal prefabricated posts versus cast metal posts and cores. – J. Dent., 38, 2010, № 11, 916-920.G o s s , J. M. Radiographic appearance of titanium alloy prefabricated posts cemented with differ-4 . ent luting materials. – J. Prosthet. Dent., 67, 1992, № 5, 632.G u z y, G. E. et J. I. Nicholls. In vitro comparison of intact endodontically treated teeth. – J. Pros-5 . thet. Dent., 42, 1979, № 1, 39-44.H a r r i s , C. A. The Dental Art. Baltimore, Armstrong and Berry, 1839, 305-347.6 . H u , Y. H. et al. Fracture resistance of endodontically treated anterior teeth restored with four post 7 . core systems. – Quint. Int., 34, 2003, № 5, 349-353.M e n t i n k , A. et al. Survival rate and failure characteristics of the all metal post and core restora-8 . tion. – J. Oral Rehabil., 20, 1993, 455-461.R e e h , E. S., H. H. Messer et W. H. Douglas. Reduction in tooth stiffness as a result of endodontic 9 . and restorative procedures. – J. Endodontics, 15, 1989, 512.S t o c k t o n , L. W. Factors affecting retention of post systems: A literature review. – J. Prosthetic 1 0 . Dent., 81, 1999, № 4, 380-384.T r o p e , M., D. O. Maltz et L. Tronstad. Resistance to fracture of restored endodontically treated 11 . teeth. – Endod. Dent. Traumat., 1, 1985, № 3, 108-111.To r b j o r n e r , A., S. Karlsson et P. A. Odmann. Survival rate and failure characteristics for two 1 2 . post designs. – J. Prosthet. Dent., 73, 1995, 439-444.

Address for correspondence: Assoc. Prof. E. Boteva Faculty of Dentisty Medcial University – Sofi a 3 Sv. G. Sofi iski 1431 Sofi a e-mail: [email protected]

USE OF POSTS BY UNDERGRADUATE DENTAL STUDENTS

E. BotevaDepartment of Conservative Dentistry

Faculty of Dental Medicine – Sofi a

Summary. Metal posts are often used in the dental practice for non vital teeth. Teeth with large defects of the clinical crown are often treated during the undergrad-uate students practice. There are different views in the dental literature about the indications for their use. The traditional approach to fi xed them always when a cusp or wall is missing, is not commonly used. In most of the cases, posts are fi xed when a large amount of hard tissues is missing. The classical approach is accepted as based mainly on the experience from the preadhesive dental techniques. The aims of the present study are to evaluate the frequency of: the use of prefabricated metal posts and the complications frequency in front and distal teeth, and the type of the post – pins, casted posts and cores or cemented posts. The following parameters were registered: type of the post, length, diameter, complications including empty root canals, periapical lesions, etc. The results have shown that the aesthetic posts are not much used in the dental practice in Bulgaria. Posts are used in half of the restored teeth. The most frequent use of posts is in front teeth and premolars (88%) and only 11.5% in molars. The reason for that could be to avoid complications, more common in molars. Cemented prefabricated metal posts and pins are used mostly and equally, casted less only in 14.9%. Poor quality of the fi xed posts was observed in 33.2%, and teeth suspected for future root fractures were 12.2% (teeth with over preparation of root dentine). We found essential to focus tutors attention on better choice of the particular post most suitable for the treated tooth and on the control of the preparation technique and x-ray control of the results.

Key words: post and core technique, indications, use

Posts are more and more often used in the dental practice, especially in the students’ halls, where people with the lowest socioeconomic status and severe tooth and dental structure loses are treated. This is valid not

only for distal teeth, but every year the situation is getting worset for front teeth too.

Use of posts by undergraduate...70


The differences in the dental literature on indications and exclusion criteria of posts and cores are well known. Traditionally in the Department of Conservative Den-tistry in Sofi a we are teaching that if there is more than one absent wall or cusp, there should be a post. This view is mostly based on the preadhesive dentistry era.

There is a long history of the use of posts. Back in 1839 and 1848 post crowns (Richmond type) were used for restorative treatments (Harris, Tomes). Some au-thors suggest that if teeth are not very destroyed, they are more stable without posts (Guzy, Trope, Sidoly). Even without large loses of enamel and dentine, MOD cavities are lowering tooth resistance up to 63% (Reeh 1989). The fracture re-sistance is related to the amount of remaining dentine, especially in buco-lingual dimension and to the amount of preparated root canal (Stockton).

On the other hand in the last two decades prefabricated ready made posts are used much more often than casted post and cores. They are cheaper and much faster (Тоrbjorner).

In the dental literature, the retentive and mechanical factors of posts are eight [7]: length, diameter, type of post, type of cement and method of cementation [4], dimension of the root canal, its preparation and the place of the particular tooth in the dentition [10].

All university teachers know that the post and core method can result in iat-rogenic errors and complications leading to teeth extractions or bone operative interventions, different in different quadrants of the mouth.

In our dental literature this problem is only mentioned in one literature review and only very few of the main problems are looked at, like: length, diameter, and length and diameter of the root canal and the type of the post [1]. Problems like the frequency of use in different groups of teeth [3, 12], and the frequency of iatrogenic errors in teaching practice are unknown [2, 3]. Unknown is also the clinical applica-tion of the teaching criteria of their use.

AIMS

To fi nd out the frequency of the use of posts by students in year IV and V 1. and the frequency of complications.

The frequency of use of posts in different groups of teeth.2. What types of posts are in use most and how they are used during stu-3.

dents’ education


Teaching programme: During the preclinical course the practicals include only one pin and one cemented post. In the next 4 clinical semesters, the require-ments are for 5 posts with adhesive restorations and two casted posts and cores restorations.

Participants: patients of 124 dental students from 2008-2010, followed up with x-rays. Total number of endodontically treated teeth was 1291 with 2116 root ca-nals. Our follow up was on 319 teeth with 319 posts, plus 5 fi xed on teeth with periapical lesions. This makes 2.6 of the required 7. Only 47 of these were casted, the rest more than half were not followed up.

X-RAYS: of the treated teeth on small dental radiographs were observed and registered for:

Type of the tooth: incisor, premolar, molar.1. Type of the post: pin, cemented or casted2. Length in the root canal: < 1/2, up to ½, up to 2/33. MD dimension of the root canal < 1/3, up to 1/3 and up to 2/34. Iatrogenic errors > 2/3 and perforations5. Posts on large periapical lesions in 265 treated lesions6. Posts in empty root canals7.

RESULTS

Of all 1291 endodontically treated teeth, 50% were restored with posts and 319 were followed up, according to the protocols. Of these 319, most were used for incisors – 163, 52.5% and premolars – 119, 37.3% and less in distal teeth – 37, 11.5%.

In relation to the method of fi xation, 132 were cemented (42%), 135 (42.9% )were pins and only 47 (14.9%) were casted.

Shorter than ½ of the root canal length were 116 posts, up to ½ were 104 and up to 2/3 – 93 posts. Less than 1/3 of the MD dimension were 45 posts, up to 1/3 125 and up to 2/3 125 too. Over preparation of dentine in the root canal and posts bigger than 2/3 were found in 30 patients and only in one case there was a perforation. In nine cases in empty root canals posts were found to be fixed.

In table 1 all groups are shown:

Table 1. X-ray valuation of the posts, 3 Ro gr per post

Lenght

Type of tooth Type of post MD dimension Errors > 2/3

30I163 PM119

М37

Cem132.

Pins135

Cast47.

< 1/345

Upto1/3125

Upto2/3125

< 1/2 60 43 13 60 42 16 35 45 27 10½ 55 35 14 44 48 13 4 50 47 52/3 48 35 10 28 45 18 6 30 51 15



A B Fig. 1, a, b. Upper canine after endodontic treatment – a, and after correct post cementation – b

A B Fig. 2, a and b. Upper incisor after endodontic treatment – a, and after correct pin fi xation – b

Fig. 3. Short post on two examined radiographs of an upper incisor

DISSCUSSION

Five posts were fi xed in cases with large periapical lesions – 1.3%. In this treatment decisions this fact cannot be classifi ed as a mistake, because it was undertaken only in patients which needed fi nishing of the treatment related to their personal plans and on their own responsibility.

In nine endo- treatments, unfortunately posts were fi xed in empty root ca-nals – 2.8%. This is not a high rate but as it is a big mistake we found this rate a bad symptom. Dentine over preparation, which is another big mistake was found in 30 patients, or in 9.4%. And if the perforation was a unique case, the very bad mistakes were 12.2%. Underestimation of very thin (14.1%) and very short – 116 (36.4%) posts is also not in favor of good dental treatment. The combination of these two is poor treatment and it was found in 35 or 11%. This accounts for 33,2% of all not quite accurate posts.

This fact is very unpleasant looking into the students results and achieve-ments in clinical endodontics – mean marks for 124 students, 5.06 of 6. If they use in 50% of the treatments posts and 33.2% are not good, it means that 17-18% of very good endodontic treatments are blamed by posts.

Another confusing fact is that 60 or 36.8% of the mistakes are in incisors and 43 or 36.1% are in premolars. Rate of 72.9% mistakes of all mistakes in the front area, where the visual control is easy and manipulation and technology much bet-ter, means that the choice and use of posts is not very well controlled by the as-sistant professors and tutors.

Less are the faults in the use of posts in molars – 13 or 35.1%. The explana-tion of this can be that both students and tutors are more careful in the distal area and that they underestimate the dangers in the anterior area.

CONCLUSIONS:

The use of prefabricated metal posts in endodontic treatments of dental 1. students is regular and up to 50% of the treated teeth.

The most frequent use of posts is in incisors and premolars – 88% and 2. much less in molars – 11.5%.

Pins and posts are used equally and only 14.9% of the cases are 3. treated with casted post and cores.

For better results in the students education and for better patients treat-4. ment outcomes it would be usefull in students preclinical practical teaching more posts and pins to be included on different groups of teeth.

More attention by tutors during the clinical practicals on the choice 5. and use of posts and pins is essential.

REFERENCESM i r o n o v a , J. et R. Vasileva. Modern approaches in the use of radicular posts. – Dent. Med. 1 . (Sofi a), 2, 2008, 137-141. (in Bulgarian)B o l l a , M. et al. Root canal posts for the restoration of root fi lled teeth. – Evid. Based Dent., 2 . 8, 2007, № 2, 42.G o m e s -P o l o , M. et al. A 10 year retrospective study of the survival rate of teeth restored with 3 . metal prefabricated posts versus cast metal posts and cores. – J. Dent., 38, 2010, № 11, 916-920.



G o s s , J. M. Radiographic appearance of titanium alloy prefabricated posts cemented with differ-4 . ent luting materials. – J. Prosthet. Dent., 67, 1992, № 5, 632.G u z y, G. E. et J. I. Nicholls. In vitro comparison of intact endodontically treated teeth. – J. Pros-5 . thet. Dent., 42, 1979, № 1, 39-44.H a r r i s , C. A. The Dental Art. Baltimore, Armstrong and Berry, 1839, 305-347.6 . H u , Y. H. et al. Fracture resistance of endodontically treated anterior teeth restored with four post 7 . core systems. – Quint. Int., 34, 2003, № 5, 349-353.M e n t i n k , A. et al. Survival rate and failure characteristics of the all metal post and core restora-8 . tion. – J. Oral Rehabil., 20, 1993, 455-461.R e e h , E. S., H. H. Messer et W. H. Douglas. Reduction in tooth stiffness as a result of endodontic 9 . and restorative procedures. – J. Endodontics, 15, 1989, 512.S t o c k t o n , L. W. Factors affecting retention of post systems: A literature review. – J. Prosthetic 1 0 . Dent., 81, 1999, № 4, 380-384.T r o p e , M., D. O. Maltz et L. Tronstad. Resistance to fracture of restored endodontically treated 11 . teeth. – Endod. Dent. Traumat., 1, 1985, № 3, 108-111.To r b j o r n e r , A., S. Karlsson et P. A. Odmann. Survival rate and failure characteristics for two 1 2 . post designs. – J. Prosthet. Dent., 73, 1995, 439-444.

The reference list is equal with "Use of posts in dental practice".

Address for correspondence: Assoc. Prof. E. Boteva Faculty of Dentisty Medical University – Sofi a 3 Sv. G. Sofi iski 1431 Sofi a e-mail: [email protected]

DENTIGEROUS CYSTS: A CLINICAL STUDY OF 146 CASES

Petia F. PechalovaDepartment of Maxillofacial Surgery, Faculty of Dental Medicine,

Medical University – Plovdiv

Summary. Dentigerous cysts are epithelial cysts related to the development of tooth germ. The present study was aimed at providing a clinical and epidemio-logical analysis of patients with dentigerous cysts. The study was conducted over a ten-year period (1998-2007). Data were obtained from records of 146 patients. The mean age of patients was 22.63 ± 1.56. The ratio of patients at the age of up to 20 towards patients over 21 was 1.98:1. The ratio male: female was 1.12:1. Mandible was involved by dentigerous cysts 2.65 times more frequently than maxilla. Denti-gerous cysts developed most commonly around mandible premolars (36.9%). The most common reason for diagnosis was the presence of asymptomatic swelling of the jaw bone (62.3%). The ratio between the extracted (110) and preserved tooth germs (104) was 1.06:1. It is concluded that dentigerous cysts were more common in younger patients and males.

Key words: dentigerous cysts, maxilla, mandible

INTRODUCTION

Cysts are one of the major causes of bone loss in jaws [1]. In 1974, Kramer defi ned the jaw cyst as a “pathological cavity fully or partially lined with epithelium, having fl uid, semi-fl uid or gaseous contents, not

created by the accumulation of pus” [2]. Literature data for the second place in incidence of dentigerous cysts compared to other odontogenic cysts of the jaws [3-5] were confi rmed by our earlier investigation as well [6]. Dentigerous cysts are epithelial cysts related to the development of tooth germ. They involve partially or completely the crown of an unerupted tooth and come into contact with it in the area of cement-enamel junction. The radiographic appearance is a unilocular shadow in contact with an unerupted tooth with a pronounced sclerotic band, except in case

Dentigerous cysts: a clinical study of 146 cases76


of infl ammation [7]. Radiographically dentigerous cysts are visualized centrally, lat-erally and circumpherentially. The differentiation between a small dentigerous cyst and a voluminous dental follicle is diffi cult, except in cases when the radiological shadow is more than 3-4 mm wide [8].

Prevailing opinion in the literature is that the dentigerous cyst may be either of extrafollicular or of intrafollicular origin, as the second type may develop due to accumulation of fl uid between the reduced enamel epithelium and the enamel, and also in the enamel organ. Another theory for the origin discusses the possibility that during the eruption, the crown of a permanent tooth gets into a radicular cyst formed around the apex of its deciduous predecessor [9]. Also, the opinion that in-fl ammatory changes in the apical region of the deciduous tooth – predecessor may be responsible for the development of dentigerous cysts around the corresponding permanent teeth can be found [10].

The aim of this study was to present clinical and epidemiological analysis of patients with dentigerous cysts treated at the Clinic for Maxillofacial Surgery at Uni-versity Hospital, Plovdiv, Bulgaria.

MATERIAL AND METHODS

The hospital records of 10,986 patients treated from January 1998 to De-cember 2007 in the Clinic for Maxillofacial Surgery at University Hospital, Plov-div, Bulgaria were examined. The study involved all 146 patients with defi nitive pathological diagnosis “dentigerous cyst” of upper and lower jaw during the 10-year period. The following variables were analysed: age, gender, location, adjacent teeth and behaviour towards them, reasons for diagnosis, type of an-esthesia, surgical approach, bone cavity obturation, length of hospital stay. As a level of signifi cance P < 0.05 was accepted. Data processing was performed with software SPSS 17.0.

RESULTS

The mean age of 146 patients with dentigerous cysts was 22.63 ± 1.56. The youngest patient was 4 years old and the oldest patient was 81 years old. The distribution of dentigerous cysts by age groups is presented in Figure 1. The ratio of patients at the age of up to 20 towards patients over the age of 21 was 1.98: 1.

The ratio male: female was 1.12: 1. Mandible was involved by dentigerous cysts 2.65 times more frequently than the maxilla - the study found 106 cysts (72.6%) in the mandible: 40 cysts (27.4%) in the maxilla. The most common loca-tion was the mandibular body, followed by the mandibular angle, the ramus and the mentum in the ratio 7.42: 2.5: 1.42: 1. In the maxilla, dentigerous cysts developing

in the alveolar ridge had the highest proportion, followed by cysts in the maxillary sinus and destroying the hard palate (25: 14: 1). The examined 146 dentigerous cysts developed around 214 teeth. Dentigerous cysts developed most commonly around the premolars (36.9%) and molars (23.4%) of the mandible, and most rarely – around the maxillary incisors (1.4%) (Figure 2). The distribution of dentigerous cysts in both jaws for different age groups around the groups of teeth is presented in Figures 3 and 4. The one-way analysis of variance (One-way ANOVA) reveals a statistically signifi cant difference (P < 0.05) in different age groups in the incidence of involvement of:

1) Premolars:The second premolars of the mandible bilaterally (P<0.0001)The fi rst right premolars of the maxilla (P<0.0001) The fi rst left premolars of the maxilla (Р=0.001) The second right maxillary premolars (P=0.037)2) Molars:The fi rst right molars of the maxilla (Р=0.001) The second left molars of the mandible (P=0.002)The left mandibular wisdom teeth (P=0.025) 3) Incisors and canines:The second left incisors of the maxilla (P=0.022)The right mandibular canines (Р=0.032)

41

56

15

6

10 9

4 41

0

10

20

30

40

50

60Number

up to 10 11–20 21–30 31–40 40–50 51–60 61–70 71-80 over 81

Age groups

Fig. 1. Distribution of dentigerous cysts by age groups



Maxilla incisors

3

18MandibleIncisors

Maxilla canines

20

15Mandiblecanines

Maxilla premolars

12

79Mandiblepremolars

Maxillamolars

17

50Mandible

molars

Num

ber o

f tee

th

Fig. 2. Distribution of dentigerous cysts in the maxilla and mandible by involved groups of teeth

0

1

2

3

4

5

6

7

8Number

up to 10 11-20 21-30 31-40 41-50 51-60 61-70 71- 80 over 81 Age groups

incisors

canines

premolars

molars

Fig. 3. Distribution of groups of teeth in the maxilla involved by dentigerous cysts by age groups

0

5

10

15

20

25

30

35Number


incisors

canines

premolars

molars

Fig. 4. Distribution of groups of teeth in the mandible involved by dentigerous cysts by age groups

The study found that the ratio between the extracted during the surgery (110) and preserved tooth germs (104) was 1.06: 1.

The reasons for diagnosing dentigerous cysts, type of anesthesia used, surgi-cal approach and data on bone cavity obturation are summarized in Table 1.

The average duration of hospital stay of patients with dentigerous cysts was 6.01± 0.319 days. The shortest stay was 1 day, and the longest – 22 days. The distribution of hospital stay by age groups is presented in Figure 5.

5.225.89

5.07

6.67

5.8

7.67

11.75

7.25 7.0

0

2

4

6

8

10

12Days


Fig. 5. Average hospital stay (days) in patients with dentigerous cysts by age groups



Table 1. Distribution of reasons for diagnosing, type of anesthesia used, surgical approach, data on bone cavity obturation in 146 dentigerous cysts

Reasons for diagnosing Type of anesthesia Surgical approach Bone cavity obturation

casual X-ray

painless mass suppuration local general intraoral extraoral without

obturation

temporary gauze fi lling

bone substitution

material

Number 21 91 34 21 125 143 3 81 55 10

% 14.4 62.3 23.3 14.4 85.6 97.9 2.1 55.5 37.7 6.8

DISCUSSION

Our study found that male gender is more often affected by dentigerous cysts, which is consistent with the data of Tortorici et al. [4] about 61.1% of dentigerous cysts in males, Meningaud et al. [11] about the ratio of male: female patients with dentigerous cysts of 108: 46, Koseoglu et al. [12] about the ratio between both genders in patients with dentigerous cysts 7: 6 in favour of males. Prevailing of the male gender in patients with dentigerous cysts is announced also by Mosqueda-Taylor et al. [1] - 56.9% and Ochsenius et al. [3] – 63%.

Data from our study show that the mean age of patients with dentigerous cysts was 22.63 ± 1.56. They are in accordance with the fi ndings of Daley et al. [13] and Shear and Speight [2] - dentigerous cysts are the most common type of cysts in the fi rst decade of life, increase in the second and reach a peak during the third decade. But Ochsenius et al. [3] found peak in the incidence of dentigerous cysts in the second decade. Other authors report that dentigerous cysts are more common during the fi rst two decades [4, 11].

Our study found that 66.4% of dentigerous cysts were diagnosed in patients at the age of up to 20. These data coincide with those published by Nakamura et al. [14] who examined 259 dentigerous cysts and found that 60% of them developed in patients at the age of up to 20.

In our series, dentigerous cysts developed most commonly around the pre-molars of the mandible (36.9%), mandibular molars (23.4%) and maxillary canines (9.3%). The data are in accordance with those of Varinauskas et al. [15], which show that dentigerous cysts develop most commonly around the mandibular premolars. There is discrepancy with the data of Ochsenius et al. [3] about the development of dentigerous cysts most often around the mandibular molars (30.6%), followed by maxillary incisors and canines (28.2%), while dentigerous cysts around the man-dibular premolars were observed by them rarely (11.9%). Shear and Speight [2]

consider that dentigerous cysts are most common around the mandibular wisdom teeth and maxillary canines.

CONCLUSIONS

The ratio of patients at the age of up to 20 towards patients over the age of 21 was 1.98: 1. The male gender was affected more frequently (1.12 males: 1 female). Mandible was involved 2.65 times more frequently than the maxilla (106 mandibu-lar cysts: 40 maxillary cysts). Dentigerous cysts developed most commonly around premolars of the mandible (36.9%). There was a statistically signifi cant difference (P<0.05) in the incidence of involvement of groups of teeth in patients from different age groups. The most common reason for diagnosing of a dentigerous cyst was the presence of asymptomatic swelling of the jaw bone (62.3%). Preferred method of analgesia in patients with dentigerous cysts was intubation anesthesia (85.6%). The surgical approach for their removal was predominantly intraoral (97.9%). The average duration of hospital stay was 6.01 ± 0.319 days.

REFERENCESM o s q u e d a -Ta y l o r , A., M. E. Irigoyen-Camacho et M. A. Diaz-Franco. Odontogenic cysts. 1 . Analysis of 856 cases. – Med. Oral Patol. Oral Cir. Bucal., 7, 2002, 89-96.S h e a r , M. et P. M. Speight. Cysts of the oral and maxillofacial regions. 4th ed. Oxford, UK, Black-2 . well Munksgaard, 2007, 1-171.O c h s e n i u s , G. et al. Odontogenic cysts: analysis of 2 944 cases in Chile. – Med. Oral Patol. 3 . Oral Cir. Bucal., 12, 2007, 85-91.To r t o r i c i , S. et al. Prevalence and distribution of odontogenic cysts in Sicily: 1986-2005. – J. 4 . Oral Sci., 50, 2008, 15-18.G r o s s m a n n , S. M. et al. Demographic profi le of odontogenic and selected nonodontogenic 5 . cysts in Brazilian population. – Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod., 104, 2007, 35-41.P e c h a l o v a , P. et A. Bakardjiev. Cysts of the jaws: a clinical study of 621 cases. – Acta Stoma-6 . tol. Croat., 43, 2009, 215-224.N e v i l l e , B. W. et al. Oral & Maxillofacial Pathology. Philadelphia, USA, W. B. Saunders Co, 7 . 2002, 107-136; 589-601.D a l e y , T. D. et G. P. Wysocki. The small dentigerous cysts. A diagnostic dilemma. – Oral Surg. 8 . Oral Med. Oral Pathol. Oral Radiol. Endod., 79, 1995, 77-81.W o o d , R. E. et al. Radicular cysts of primary teeth mimicking premolar dentigerous cyst: report 9 . of three cases. – J. Dent. Children, 55, 1988, 288-290.S h i b a t a , Y. et al. Radiographic examination of dentigerous cysts in the transitional dentition. – 1 0 . Dentomaxillofac. Radiol., 33, 2004, 17-20.M e n i n g a u d , J. P. et al. Odontogenic cysts: a clinical study of 695 cases. – J. Oral Sci., 11 . 48, 2006, 59-62.



K o s e o g l u , B. G., B. Atalay et M. A. Erdem. Odontogenic cysts: a clinical study of 90 cases. – J. 1 2 . Oral Sci., 46, 2004, 253-257.D a l e y , T. D., G. P. Wysocki et G. A. Pringle. Relative incidence of odontogenic tumors and oral 1 3 . and jaw cysts in a Canadian population. – Oral Surg. Oral Med. Oral Pathol., 77, 1994, 276-280.N a k a m u r a , T. et al. A study of cysts in the oral region. Cysts of the jaw. – J. Nihon Univ. Sch. 1 4 . Dent., 37, 1995, 33-40.Va r i n a u s k a s , V., A. Gervickas et O. Kavoliuniene. Analysis of odontogenic cysts of the jaws. 1 5 . – Medicina (Kaunas), 42, 2006, 201-207.

Address for correspondence: Petia F. Pechalova Department of Maxillofacial Surgery, Faculty of Dental Medicine, Medical University, № 66 “Peshtersko shose” Str. 4000 Plovdiv, Bulgaria

0898 468 498 e-mail: [email protected]

PHARMACOECONOMICS OF RARE DISEASES THERAPY – AN EXAMPLE OF PHENYLKETONURIA

S. Desancic1, A. Stoimenova2, A. Savova2, M. Manova2, V. Petkova2 and G. Petrova2

1University of Belgrade, Faculty of Pharmacy, Belgrade, Serbia2Medical University, Department of Social pharmacy, Faculty of Pharmacy – Sofi a, Bulgaria

Summary. Due to the limited number of patients and lack of market interest towards the development of medicines by pharmaceutical companies, the ther-apy of rare diseases is usually very expensive. Because of that the pharmaco-economic assessment of the rare disease therapy is commonly unfavourable. In this study, the standards and cost of therapy of phenylketonuria (PKU) in Bulgaria and Serbia are analysed. The number of patients was gathered from the offi cial databases of National Health Insurance Fund of Serbia and Ministry of Health in Bulgaria. The costs of low protein diet for patients with PKU were calculated. The lists of reimbursed medicinal products for rare diseases were reviewed. The preliminary results showed that the average cost per patient in Serbia is 528 EUR per month and 494 Euro in Bulgaria. In both countries, the dietetic foods for patients under 18 years are reimbursed. The prices of dietetic foods vary in wide range. No other therapy is provided to the patients with PKU. Our results show that in both countries the patients with phenylketonuria did not have an access to modern medicine therapy.

Key words: rare diseases, phenylketonuria, cost of therapy, orphan medicines, Bul-garia, Serbia

INTRODUCTION

Treatment of rare diseases is usually expensive due to the limited num-ber of patients and lack of interest by pharmaceutical companies for the development of medicines. Therefore, pharmacoeconomic evaluation

of the therapy has unfavourable ratio between the input costs and clinical results. On the other hand, patients with rare diseases must be provided with health care of the same quality as the patients with common diseases [11].

Pharmacoeconomics of rare disease...84


Phenylketonuria is a rare genetic disorder associated with increased concen-trations of phenylalanine due to mutation of the gene responsible for the degrada-tion of phenyl-alanine hydroxylase [3, 4, 5].

The aim of this study is to analyse the current therapy of phenylketonuria and its cost in Bulgaria and Serbia.


The interview with the national medical consultants and representatives of as-sociations of patients was performed to analyse the current therapeutic approaches.

The number of patients was determined from the database of the Institute for Health Insurance in Serbia and specialized pediatric clinics in Belgrade and Sofi a. Analysed were also lists of reimbursed medicines in both countries to determine the reimbursed medicinal products, used for the treatment of phenylketonuria.

The average weight of individuals by 1 year, between 1-5 years and over 5 years and average daily required amount of dietary foods were used to calculate the necessary monthly amount of food, according to the opinion of national consul-tants. These values were averaged over the entire treated population.

Costs of low protein diet were calculated based on clarifi ed dietary standards, prescribed quantities of special food stuff in different age groups, patients’ weight, and prices of the dietetic foods. Prices were taken from the respective lists of medi-cines, which are reimbursed by the national health insurance funds and are recal-culated into euros at the exchange rate for 2009 (1 EUR= 95 DIN = 0.9545 BGN).

The perspective of this analysis was that of the health insurance fund for one year period.

RESULTS

History of diagnosis of phenylketonuria (PKU)The fi rst evidence for the diagnosis of PKU dates from 1934 when Asbjorn

Feling examined the urine of two sick children. He found a lack of proteins and glu-cose, specifi c smell, and with the addition of ferric chloride urine turned green. After a detailed analysis, he found an increased concentration of fenilpirograpes acid. Initially Dr. Feling called the disease “phenylpyruvate oligophrenia” [14].

In 1953, prof. Bikel initiated the treatment of fi rst diagnosed patient with pro-tein hydrolysed and low protein diet.

In 1963, thanks to the discovery of Robert Gatrij, it was created a diagnostic test for early detection of PKU based on microbiological and bacterial inhibition of the blood sample.

In 1969, Kaufman discovered the structure of the enzyme responsible for deg-radation of phenylalanine to tyrosine, namely phenylalanine hydroxylase [3-6].

EpidemiologyAccording to the defi nition of the World Health Organization (WHO), rare dis-

eases are those with prevalence less than 1 per 10,000 inhabitants. Every year 750 new cases of PKU are diagnosed in Europe [12]. The frequency of disease in litera-ture varies by region, even within a country [9] as shown in Table 1. The population with PKU is increasing from north to south and from west to east.

Table 1. Frequency of PKU in Europe

Frequency of PKU Country1 in < 5000 residents Slovenia, Turkey13

1 in 5-10 000 residents Austria, Belarus, Bosnia and Herzegovina, Latvia, Lithuania, Poland, Portugal, Russia, Scotland, Slovakia, Spain, Ukraine

1 in 10-15 000 residents Croatia, Czech Republic, Denmark, Estonia, Madagascar, Netherlands, Norway, Serbia, Sweden, Wales, USA15

1 in 15-25 000 residents Belgium (Wallonia), France1 in >25 000 residents Flanders region of Belgium, Bulgaria, Greece

Differences in genetics and geographic prevalence of the disease determine the differences between the numbers of patients, as well as between the treatment policies in the countries.

For the Balkan countries and their neighbours the number of patients and ac-cess to the reimbursement is presented in Table 2 [1, 2, 6, 12].

Table 2. Patients with PKU and access to reimbursed treatment in Balkan countries

Bulgaria Serbia Croatia Slovenia Slovakia Czechia Turkey

Population 7.6 mill. 9.5 mill. 4.5 mill. 2 mill. 5.5 mill. 10 mill. 71 mill.

Screening at birth Yes Yes Yes Yes Yes Yes Yes

Frequency 1 >25 000 1:10 000-15 000

1:10 000- 15 000

1 < 5000 1:5 000-10 000

1:10 000-15 000

1 < 5000

Patients 47 50 180 120 700 111 4 000

Reimbursement 80-100% 100% 90-100% 100% 20-100% 100% 100%

The high absolute number of patients with PKU in Turkey, Slovenia and Bel-gium is determined not only by population but also by the fact that in these countries there are marriages between distant or close relatives [6, 8, 12]. The combination of related genetic material increases the risk of genetic mutations and prevalence of PKU.



All counties are screening the new born children in order to benefi t from the earlier diagnosis and to minimise the consequences from the disease develop-ment. High concentrations of phenylalanine in childhood lead to nervous system damage, mental retardation, convulsions, trouble with memory and behaviour, poor concentration and low IQ.

In all the countries, the dietetic foods are reimbursed nearly 100%, except in Slovakia.

Standards for treatment of PKU in Bulgaria and SerbiaAnalysis of lists of reimbursed medicines and medical foods, as well as in-

terviews with national consultants and patient organizations, revealed that in both studied countries standard treatment of PKU is the low protein diet. Diet is con-ducted with dietary foods for special medical purposes containing synthetic protein supplements. It is regularly adapted to the child’s age and the concentration of phenylalanine in the blood [7, 16].

Strict maintenance of the amount of phenylalanine in the blood within the acceptable values is essential for the normal development of the nervous sys-tem and mental maturity of children, especially during the fi rst fi ve years. Stan-dards for diagnostics and current therapeutic behaviour are presented in Table 3 [1, 2, 10, 11].

Table 3. Standards for diagnostics and therapy and reimbursement of patients with PKU

Diagnostics standards Bulgaria SerbiaScreening + (100 % obligatory) + (100 % obligatory)

Genotypization Since 2-3 years Since 2009

Therapy – dietFrom 1 to 18 years 80-100 % reimbursed 100% reimbursed

>18 years not reimbursed not reimbursed

Treatment costsIn both countries, the health insurance funds reimburse the cost of dietary

foods between 80-100% till the aged less than 18 years. Complying with an average weight by age and necessary average daily

quantities of the medicinal foods, according to the opinion of the national con-sultants, we assumed that every patient gets reimbursed in average 5 packs of dietetic food with full strength of nutrient content per month, and 7 packs of low protein fl oury mixture monthly. Based on that assumption and on the prices de-clared in the positive lists of medicines, we calculated the average annual costs which are shown in Table 4.

Table 4. Average diet cost per patient and for all registered patients

Country Average costs per patient per year (EUR)

Patients Total cost for all patients per year(EUR)

Serbia 6 000 50 300 000

Bulgaria 5 930 47 278 710

Since dietary foods are manufactured by international companies, their prices are close among the countries in the region and diet cost is similar.

Interviewed representatives of patient organizations in Bulgaria consider that allocated resources are insuffi cient, and patients need more funds to maintain their health.

Currently in both countries there are no medicinal products available for the treatment of PKU, although some can be found on the website of the European Medicines Agency as centrally authorized for sale in EU [5].

CONCLUSIONS

The study showed that both countries have national policies to diagnose and support treatment of patients with PKU, but the policies cover only patients less than 18 years of age.

Patients with PKU in Bulgaria and Serbia are treated mainly with diet and do not have access to new medicines authorized for the treatment of their disease in Europe. Further analyses should be done for the possible reasons and policy measured to improve the rare diseases patients’ access to modern therapy.

REFERENCESM i n i s t r y of health. Registiss. Register of prices of medicaments. http://www.mh.government.bg 1 . (accessed 20.05.2010)T h e N a t i o n a l health insurance fund. http://www.nhif.bg (accessed 20.05.2010). (in Bulgarian)2 . A r n o l d , G. L. et al. Prevalence of stimulant use for attention dysfunction in children with 3 . phenylketonuria. – J. Inherit. Metab. Dis., 27, 2004, № 2,137-143.E u r o p e a n Society for Phenylketonuria and Allied disorders treated as phenylketonuria. About 4 . PKU, 2008, http://www.espku.org/3.0/index.php?option=com_ content&task=view&id=1&Itemid=2 (accessed May 2010)E u r o p e a n Organisation for Rare diseases, Eurordis survey on orphan drugs availability in Europe, 5 . 2007. http://www.eurordis.org/IMG/pdf/2007ODsurvey-eurordis.pdf (accessed May 2010)G e n e t i c s Home Reference. Tetrahydrobiopterin defi ciency,.http://www.ghr.nlm.nig.gov/conditio6 . n=tetrahyrobiopterindefi ciency (accessed May 2010)G i o v a n n i n i , M. et al. Phenylketonuria: dietary and therapeutic challenges. – J. Inherit. Metab. 7 . Dis., 30, 2007, 145-152.L a n d o l t , M. A. et al. Quality of life and psychologic adjustment in children and adolescents with 8 . early treated phenylketonuria can be normal. – J. Pediatr., 140, 2002, 516-521.



L o e b e r , J. G. Neonatal screening in Europe; the situation in 2004. – J. Inherit. Metab. Dis., 9 . 30, 2007, № 4, 430-438.M e d i c i n e N e t . Com. Hyperphenylalaninemia, 2005. http://www.medicinenet.com/1 0 . hyperphenylalaninemia/ article.htm (accessed May 2010)N a t i o n a l Institutes of Health, Phenylketonuria: screening and management, 2000. http://www.11 . consensus.nih.gov/2000/2000Phenylketonuria113html.htm (accessed May 2010)O r p h a n e t , Prevalence of rare diseases: a bibliographic survey, 2008. http://www.orpha.net/1 2 . orphacom/cahiers/docs/GB/Prevalence_of_rare_diseases.pdf (accessed May 2010)D u r s u n , A. et al. Mutation analysis in Turkish patients with hereditary fructose intolerance. – J. 1 3 . Inherit. Metab. Dis., 24, 2001, № 5, 523-526.P K U news.org. The discovery of PKU by Dr. Asbjorn Folling: Norway, 1934.2000. http://www.1 4 . pkunews.org/about/history.htm (accessed May 2010)U S , National Institutes of Health. Consensus Development Conference Statement: phenylketonu-1 5 . ria: screening and management, October 16-18, 2000. – Pediatrics, 108, 2001, № 4, 972-982.Wa l t e r , J. H. et al. How practical are recommendations for dietary control in phenylketonuria? – 1 6 . Lancet, 360, 2002, 55-57.

Address for correspondence: Assena Stoimenova, M. Sc. Pharm., Ph.D. Department of social pharmacy Faculty of Pharmacy, Medical University – Sofi a 2 Str. Dunav 1000 Sofi a, e-mail: [email protected]

CONTINUING PROFESSIONAL EDUCATION OF BULGARIAN PHARMACISTS 2007-2009

A. Stoimenova1,2, G. Petrova1,2, I. Nikolov2, M. Manova1, G. Draganov1,2, Z. Ivanova 2, D. Dimitrov2, G. Mihova2 and A. Savova1

1Department of Social Pharmacy, Faculty of Pharmacy, Medical University – Sofi a2Quality Committee at the Bulgarian Pharmaceutical Union

Summary. Continuing professional education is very important for the health professionals to maintain a high level of knowledge and for the improvement of their professional practice, patient outcomes and generally the social health. In 2007, a system for continuing education of pharmacists in Bulgaria was established, follow-ing the formation of Bulgarian Pharmaceutical Union. The aim of the current study is to analyse the system for the continuing professional education, preferred top-ics and number of courses accredited for the Bulgarian pharmacists for the period 2007-2009. The system for classifi cation of the postgraduate education and num-ber of accredited educational events were analysed. The retrospective analysis of the approved topics of educational events was performed during the observed pe-riod and systematized according to type of event, topics covered, geographic area of performance and event initiators and organizers. The established accreditation system contains 4 categories of continuing education programmes. 93 educational forms were accredited and performed for the studied period (n=26 in 2007, n=44 in 2008, n=23 in 2009). The most often organised forms are educational meetings (n=24 out of 26 forms in 2007; n=41 out of 44 forms in 2008; n=22 out of 23 forms in 2009). In general, the educational system is positively accepted by the pharma-cists in the country. The lectures and short term events remain the most favourite format of continuing education for Bulgarian pharmacists although possibility for on-line training is foreseen in the near future. We recommend greater involvement of academia in continuing professional education of pharmacists.

Key words: pharmacists, continuing professional education, educational meetings, Bulgaria

INTRODUCTION

Pharmacists play a vital role in helping doctors and patients to select an appropriate therapy and in monitoring of side effects in patients with various diseases [1-5]. With the educational background pharmacists

Continuing professional education...90


have, they are reliable source of very important information regarding the use of medicines and prevention of some diseases. Different care programs in com-munity and hospital pharmacies are introduced to improve treatment and adher-ence to prescribed medicines. [6-20]. Continuing professional development is very important for the pharmacists to maintain a high level of knowledge, to improve their professional practice and to serve better the patients and generally the social health. The continuous professional development prepares the pharmacists for the requirements of the changed role of pharmacists in the society [7, 8]. Substantial part of the continuous professional development is the continuing education. Dif-ferent approaches to continuous professional education ranging from lectures to peer-mentoring work shops and web tools are developed throughout the last 20 years [9-20].

11 years after the adoption of new Law on medicinal products and pharma-cies in human medicine in 1995 [21], in 2006, a Law on professional association of pharmacists was endorsed [22] and Bulgarian Pharmaceutical Union (BPhU) was established. Thus the obligation set by the Health Law [23] towards the profession-als associations to develop a continuing education started to be implemented. In June 2007, the Bulgarian Pharmaceutical Union accepted the system for continu-ing education of pharmacists, created by the Quality Commission of BPhU [24]. The fi rst educational cycle started in June 2007.

The aim of the current study is to analyse the system for the continuing profes-sional education, preferred topics and number of courses accredited for the Bulgar-ian pharmacists from the Quality Commission of BPhU for the period 2007-2009.

The main study questions are:Does the system provide contemporary topics as educational events?1. Which are the most often organised educational forms and by whom? 2. What is the general evaluation of t3. he educational events?


This study is a retrospective database analysis. The offi cial register of the BPhU was considered a source of information for the approved continuing educa-tion programmes during the studied period (June 2007 – Dec 2009).

The system for classifi cation of the postgraduate education was analysed and number of accredited educational events per type of classifi cation form were sys-tematized.

The retrospective analysis of the approved topics of educational events was performed during the observed period and systematized according to the type of event, geographic area of performance and topics covered. The information con-cerning the accredited forms of continuing education of pharmacists was extracted from the offi cial protocols issued by the Quality Commission of BPhU.

For the assessment of the general evaluation, the comments and questions shared on the offi cial internet page of the BPhU by the pharmacists in the corre-sponding internet section were reviewed.


I. Analysis of the system of continuing professional education of phar-macists in Bulgaria

In 2007 after the formation of Bulgarian Pharmaceutical Union (BPhU), the Qual-ity Commission prepared the Rules for continuing education of pharmacists, based on the Law on professional association of pharmacists endorsed [22]= A list of topics of interest for continuing education, rules for its approval and accreditation were cre-ated. Later in June 2007, a system of continuing education of pharmacists was ap-proved by the Managing Board of the BPhU and started to be implemented. The fi rst accredited event was the fi rst Bulgarian Pharmacy Days held in June 2007.

The aim of the developed system for continuing education was to prepare the pharmacists for requirements of Good Pharmaceutical Practice including improve-ment of their communication skills.

The continuing education system comprises 4 main categories of educational events presented on Table 1.

Table 1. Structure of continuing education system for pharmacists

Category Subcategory Type of event CreditsA A1 Participation in seminars and qualifi cation courses 1 credit/hour

A2 Participation in congresses and symposia 1 credit/hourA3 Participation in the BPhU seminars 1 credit/hour

B B1 Publication in pharmaceutical press 5 creditsB2 Publication in pharmaceutical scientifi c journals 7 creditsB3 Case study presentation 7 credits

C C1 Long term specialisations in university events Up to 40 creditsC2 Long term specialisations according to Health law 5 to 20 creditsC3 Master degree in the health or related fi elds 6 to 30 creditsC4 PhD degree education and defending 30 to 60 credits

D D1 Tutoring pharmacy students in pharmacy 0.5 credits/student

The system provides credits for different types of forms of continuing educa-tion including seminars, symposia, lectures, conferences, workshops, as well as forms for self education or university specialisations or other forms of professional self-development. The variety of the categories offers to the pharmacists in Bul-garia various options to upgrade their knowledge despite of their function and job description.



A uniform credit system for assessment of the short-term and long-term forms for continuing education was developed and the highest estimation received the long-term educational programs implemented jointly with academia. The credit sys-tem allows differentiation of the educational activities and objective evaluation of all pharmacists’ initiatives.

All forms in category A need to be accredited by the Quality Commission of BPhU, as well as some of the long term educational programs. For the other forms of education or carrier development the pharmacists need to apply individually in front the BPhU to receive the credits relevant to the event. Every pharmacist should gather 30 credits per year, but not less than 20/year and 90 credits in total for the whole cycle of 3 years.

The rules for continuing education are hosted on the internet page of the Bulgarian Pharmaceutical Union permanently [24]. Every accredited event is also publicly announced on the web page with the place of event, topics, lectures and number of credits. There is also a dedicated space for questions and comments. Till the end of 2009 no negative comments have been received concerning the system and provided topics and educational system in general.

II. Analysis of accredited continuing education events of Bulgarian phar-macists for the period 2007-2009

In general 93 educational forms were accredited and performed for the studied period (n = 26 in 2007, n = 44 in 2008, n = 23 in 2009) (Table 2). Ten proposals for educational meetings have not received accreditation by the Quality Commission because they failed to meet the established requirements (Table 2). In 2007, distri-bution of educational events according to the category was as follows: category A1 (n =7); category A2 (n=2), category A3 (n=11) and category C1 (n=6). In 2008 cate-gories were presented as follows: A1 (n =2); category A2 (n=3), category A3 (n=31) and category C1 (n=7). In 2009, the preferred forms were A1 (n=1), A 2 (n=2), A 3 (n=18), C1 (n=1) and one individual training accredited by the Quality Commission which was not categorized. Individual applications for tutoring pharmacy students and publications were not included in the analysis. The most wanted category for the studied period was category A3 – seminars, organized with the help and under control of BPhU. These seminars consisted of 3 lectures: one for socially important diseases, a lecture for normal physiological conditions (i.e. food supplements use, pregnancy, breastfeeding, oral hygiene etc.) and pharmacy practice specifi c topics such as communication skills, marketing, etc. Unlike A3 seminars, A1 seminars could consist of only one lecture.

The topics were proposed either by academia or by the industry due to fast development of the concerned sector and intensive marketing authorization of re-lated medicinal products. The accredited forms were performed in different formats ranging from several hours seminars to 4-modules programme (each module con-sisting of 6 academic hours and performed during the weekend).

Table 2. Main topics included in continuing education of Bulgarian pharmacists in 2007-2009

2007 2008 2009

Topic N Organizer Topic N Organizer Topic N Organizer Flue preven-tion

7 Wholesaler jointly with the manufac-turer

Allergy 8 Wholesaler Antibacterial medicines

9 Manufacturer

Periodontal diseases

5 Manufacturer Pharmacy practice

7 Wholesaler Probiotics 7 Wholesaler jointly with the manu-facturer

Asthma 5 Wholesaler BACTERIAL conjunctivitis

6 Wholesaler jointly with the manufacturer

Eye infections 3 Wholesaler jointly with the manu-facturer

Homeopathy 3 Manufacturer jointly with Medical universities

Hypertension 3 Wholesaler jointly with the manufacturer

Hospital phar-macy

1 Hospital phar-macy association jointly with a manufacturer

Food supple-ments

3 Wholesaler, Manufacturer and Pharmaceutical Faculty, Sofi a

Overweight 3 Manufacturer Logistics 1 Individual training provided by train-ing organization

Other 3 BPhU; Training & consultancy orga-nization.

Communication skills

2 Wholesaler jointly with the manufacturer

Homeopathy 1 Manufacturer jointly with Medical universi-ties

Cosmetics 2 Training organi-zation

Homotoxicology 1 Regional phar-maceutical asso-ciation jointly with manufacturer

Breast cancer 2 Wholesaler jointly with the manufacturer

Food supple-ments

2 Wholesaler, manufac-turer and Pharmaceutical Faculty, Sofi a

Other 9 BPhU; training organiza-tions; Hospital pharmacists association

Total Y2007 26 Total Y2008 44 Total Y2009 23

In all the years, the disease oriented education prevails as presented in Table 2. A variety of acute and chronic diseases were included in the postgraduate courses like fl ue prevention, periodontal diseases, asthma, food supplements and homeopa-



thy. According to the rules stated by the Quality Commission, the lectures included general information about the diseases, their treatment and information about par-ticular medicinal products. A questionnaire for assessment of level of understanding was distributed at the end of every educational event. Several long term courses were organized for general health topics like homeopathy, food supplement legislation and classifi cation, pharmacy practice, hospital pharmacy, and logistics. (Table 2).

The most popular form of continuing education for the pharmacists in Bulgaria during the studied period were events included in the category A, like educational meetings (Table 3) (n = 24 out 26 forms in 2007; n = 41 out of 44 forms in 2008; n = 22 out of 23 forms in 2009). The educational meetings include seminars, sym-posia, lectures, conferences, workshops etc. Bulgarian Pharmacy Days was also recognized as a form of continuing education as it is the biggest forum gathering the pharmacists from all parts of Bulgaria and the programme includes lectures from academia representatives as well as pharmacists from other countries. 4 of the seminars in 2007, respectively 6 in 2008 and 2 in 2009 were organized and performed together with Medical universities.

Table 3. Forms of continuing education of Bulgarian pharmacists in 2007-2009

2007 2008 2009Form N Form N Form N

Educational meetings 24 Educational meetings 41 Educational meetings 22

Bulgarian Pharmacy Days 1 Bulgarian Pharmacy Days 1 Individual training 1

Internal education 1 Conference 1Internal education 1

Total Y2007 26 Total Y2008 44 Total Y2009 23

The analysis by geographic area covered revealed that despite the increased number of educational meetings in 2008, 24 of them were performed in the fi rst three biggest cities including the capital (Table 4). It was also evident that the other big cities, which possess universities have been also preferred.

Table 4. Continuing education of Bulgarian pharmacists in 2007-2009 by place of performance

2007 2008 2009Town N Town N Topic NSofi a 7 Sofi a 16 Sofi a 7Plovdiv 6 Plovdiv 5 Varna 3Varna 3 Varna 3 Plovdiv 1Other, but university towns 10 University towns 13 University towns 4

Other 7 Other 7Total Y2007 26 Total Y2008 44 Total Y2009 23

The comment and questions section of the BPhU internet page revealed that there were no negative evaluations or comments during the whole period con-cerning the education system. Although it is not systematically performed because of the expiry of some comments, the collected information permits to summarise some general perceptions. The lectures were recognised as most appreciated for-mat of continuing education and the practical application of theoretical knowledge was identifi ed as need. Wish for better coverage of smaller regional associations and rural areas was expressed.

DISCUSSION

Analysis of the continuing educational system for the period 2007-2009 showed that lots of contemporary health and practical topics were covered. We can assume that the education fi ts the pharmacists’ needs in terms of theoretical and practical material covered. The decrease in the number of educational forms during the third year of the studied period could be due to the fact that most of pharmacists have already fulfi led the requirement for credits or that there is not a suffi cient inter-est among the organizing institutions. Due to the fact that no data are available for the number of pharmacists covered we can not clarify if there is a relation among the decreasing number of educational forms and number of pharmacists. Further studies should be done to clarify the reasons.

It is logical that the most often organized educational forms are 1-2 days seminars and workshops because they are found very convenient for the phar-macies. These forms of education are mainly covering professional working in community pharmacies, and weekends appear to be the most convenient days. The preference towards the bigger towns is also logical from the point of view of organizers as these locations have universities, bigger hospitals and signifi cant number of pharmacies, but further efforts should be done to cover the pharma-cists from smaller rural areas who do not have access to educational events unless they attend the meetings in bigger cities. The new distance learning edu-cational program which is in implementation phase proposes some solutions in this respect.

Our study shows that the role of the professional unions in the continuing education of the pharmacists in Bulgaria is very important. Despite of some organi-zational problems and needs of improvement, the continuing education system of Bulgarian pharmacists is a positive fi rst step in the development of their continuous professional development according to the current standards.

Our study possesses some limitations as missing information about the num-ber of pharmacists covered by the continuing education during the whole studied period and all the events. This is due to the fact that it was not measured during all educational events, as well as due to some internal organizational problems.



CONCLUSIONS

The continuing educational system of the BPhU is well accepted and fi ts the basic needs of the pharmacists serving the need for updating their professional knowledge and skills.

The lectures and short term events remain the most favourite format of con-tinuing education for Bulgarian pharmacists although possibility for on-line training is foreseen in the near future.

We recommend greater involvement of academia in continuing professional education of pharmacists not only as inclusion of academia representatives as lecturers, but also in the organization of the process for increasing the credibility of the events.

REFERENCESN o r d e e n , J. D. et A. J. Smith. Pharmacist’s role in acute decompensated hearth failure man-1. agement. – US Pharm., 35, 2010, № 2, HS8-HS19.B e n k o , R. et al. The participation of pharmacist in antibiotic related activities of Hungarian hos-2. pitals and intensive care units. – Acta Pharm. Hung., 79, 2009, № 2, 57-62.W o r t m a n , S. B. Medication reconciliation in a community, nonteaching hospital. – Am. J. 3. Health-Syst. Pharm., 65, 2008, № 21, 2047-2054.G i l m a r t i n , C. Pharmacist’s role in managing anemia in patients with chronic kidney disease: 4. Potential clinical and economic benefi ts. – Am. J. Health-Syst. Pharm., 64, 2007, № 13, Suppl., S15-S22.H e b r a n t , J. Role of pharmacist in promoting a “healthy” use of medicaments. – Int. J. Health 5. Educ., 17, 1974, № 1, 24-31.C a r d e n a s , V. J. et al. Introduction and improvement of a care program for elderly on multiple 6. medication in a primary care area. – Rev. Calidad Asist., 24, 2009, № 1, 24-31.A u s t i n , Z. et M. H. H. Ensom. Education of pharmacists in Canada. – Am. J. Pharm. Educ., 7. 72, 2008, № 6, 128.K n a p p , K. K., M. D. Ray MD et S. Feldman. Education and training of pharmacists: comments 8. on sustaining continuous improvement. – JAPhA, 48, 2008, № 4, 544-549.F a u s , M. J. et al. A new pharmaceutical care tool from Dader Program. – Pharm. Care Esp., 9 . 10, 2008, № 3, 137-141.A u s t i n , Z. et al. Peer-mentoring workshop for continuous professional development. 1 0 . – Am. J. Pharm. Educ., 70, 2006, № 5, 117.S r u m o v a , M. [Continuous education of pharmacists in Czech Republic.] – Farmac. Obzor, 11 . 74, 2005, № 11, 288-295. (in Chekh)A u s t i n , Z., A. Marini et B. Desroches. Use of a learning portfolio for continuous profession-1 2 . al development: A study of pharmacists in Ontario (Canada). – Pharm. Educ., 5, 2005, № 3-4, 175-181.H e n n - M e n e t r e , S. et al. Continuous education programme in hospital pharmacies for manag-1 3 . ing cytotoxic drug preparation: The Oncolor’s network experience. – Bull. Cancer, 90, 2003, № 10, 910-916.C a l o p , N. et al. The effect of continuous education on the professional practice of French com-1 4 . munity pharmacists. – Pharm. Educ., 2, 2002, № 4,1 185-190.

J a m e s , D. A framework for assessing the continuous professional development needs of com-1 5 . munity pharmacists. – Pharm. Educ., 2, 2002, № 2, 63-68.S i n c l a i r , H. K. et al. Training pharmacists and pharmacy assistants in the stage-of-change 1 6 . model of smoking cessation: A randomised controlled trial in Scotland. – Tobacco control, 7, 1998, № 3, 253-261.D r i e s e n , A. et al. Motivation and views related to continuing education. – Pharm. World Sci., 1 7 . 27, 2005, № 6, 447-452.F a r h a n , F. A review of pharmacy continuing professional development. – Pharm. J., 1 8 . 267, 2001, № 7171, 613-615.M o t t r a m , D. R. et al. Pharmacists’ engagement in continuing education and attitudes towards 1 9 . CPD. – Pharm. J., 269, 2002, 618-622.D e a n , B. et al.CPD implementation in a large NHS trust. – Hosp. Pharmacist, 2 0 . 8, 2001, 105-108.N a t i o n a l peoples Assembly. Law on medicines and pharmacies in human medicine. – State 2 1 . Gazette, № 36/18.Apr.1995.N a t i o n a l peoples Assembly. Law on professional association of pharmacists. – State Gazette, 2 2 . № 75/12.Sep.2006.N a t i o n a l peoples Assembly. Health Law. – State Gazette, № 70/10. Aug. 2004.2 3 . R u l e s for the implementation of the continuing professional education of pharmacists. http:// 2 4 . www.bphu.eu. Assessed 08.August.2010.

Address for correspondence: Assena Stoimenova Department of Social Pharmacy Faculty of Pharmacy Medical University – Sofi a 2, Dunav Str. 1000 Sofi a Bulgaria e-mail: [email protected]



BIOLOGICALLY ACTIVE COMPOSITION AND HEALTH IMPACT OF ALLIUM CEPA

S. Tsanova-SavovaDepartment of Chemistry, Faculty of Pharmacy, Medical University – Sofi a

Summary. The aim of this study is to present contemporary information for the biologically active compounds in Allium cepa and correspondingly their health benefi ts, as well as to present analytical HPLC results for the content of the main antioxidants in Allium cepa – the fl avonols quercetin and kaempferol, in different types of Bulgarian onion, and to submit data about onion consumption on national and international level. The potent antioxidant activity of the biologically active com-pounds in onion is an important factor for the prevention against different degenera-tive diseases like cardiovascular disease, cancer, diabetes. Results for content of the fl avonols quercetin and kaempferol in different types of Bulgarian onions are presented. The red onion contains highest amount of quercetin – average value 452.5 mg/kg and a maximum value found – 832.2 mg/kg. It was determined that the green part of spring onion contains relatively equal values of quercetin (30.2 – 61.1 mg/kg) and kaempferol (27.4 – 52.3 mg/kg). This results characterize spring onion as very important dietary source of kaempferol in Bulgarian healthy diet emphasiz-ing their role in development of different preventive diets.

Key words: Allium cepa, onion, health impact, quercetin, kaempferol

INTRODUCTION

The onion family (Allium) comprises more than 500 species, thus mak-ing it diffi cult to trace their origin. A candidate for the predecessor of the culture type Allium cepa is not only one, but fi ve wild plant species from

Central Asia. The onion bulbs were used as food for thousands of years. During the centuries, the onions were esteemed not only for their culinary use and gustatory qualities, but also for their healing properties. It is known that the Allium cepa repre-sentatives are rich source of biologically active substances like fl avonoids, namely quercetin; sulfi des; isothiocyanates; the microelement chrome, etc. Anti-infl amma-

tory properties of onions and their preventive role against cardiovascular diseases, diabetes and other contemporaneous degenerative diseases are well known. Re-cently research articles on the biologically active compounds in foods increase like avalanche, requiring presentation of the summary of general information.

The aim of this study is to present a contemporary information for the biologi-cally active compounds in Allium cepa and correspondingly their health benefi ts, as well as to present analytical results for the content of the main antioxidants in Allium cepa – the fl avonols quercetin and kaempferol, in different types of Bulgarian onion, and to submit data about onion consumption on national and international level.


Plant materialIn the present study, three different types of Allium cepa were analysed as

follows: 7 samples white onion, 5 samples red onion, and 3 samples spring onion, divided to hole plant, white and green part. In order to obtain representative sam-ples, the sampling plan was conducted during two years. Each analysed sample of Allium cepa comprises composite of tree individual samples, purchased from three different places in one day. The individual samples were not less than one kilogram (BDS ISO 874:1996) [1]. From the composite sample by random selection an aver-age sample was made, and non-edible parts were removed. The average sample was freeze-dried and stored at 4°C in a hermetically sealed vacuum container. Right before the analysis, the lyophilized average sample was grinded to fi ne pow-der, sieved through a 0.5 mm pore size, and homogenized.

MethodsIn the present study, the extraction and hydrolysis of the fl avonols from the

plant material was performed with 1.2 M HCl in 50% methanol, refl uxing lyophilized sample for 2 h at 90°C.

HPLC analysis. For the quantitative determination of quercetin and kaemp-ferol the internal standard morine was used. The chromatographic separation was performed by using Hewlett Packard liquid chromatograph, HP pump 1050; ther-mostat: HP 1100; UV detector: HP 1050; and data handling software ChemStation (Agilent Technology); Alltima (100 × 4.6 mm i.d., 3 μm) C18 analytical column, con-nected to pre-column Alltima (4 × 4.6 mm i.d., 3 μm) C18, Alltech Association Inc. An isocratic elution with 28% acetonitrile in 2% acetic acid was applied, with a fl ow rate of 0.9 ml/min, resulting to a working pressure of 11.5-12.0 MPa. For determina-tion of the selected fl avonoids a fi xed UV detection at 365 nm was used.

The method used for determination of fl avonols is characterized by a limit of detection 1 mg/kg fresh weight for quercetin and 2 mg/kg fresh weight for kaemp-ferol, and by very good repeatability (RSD < 5%) [2].

Biologically active composition...100



Onion – health benefi tsOnions contain quercetin – a potent antioxidant from the group of fl avonoids

[3]. The antioxidants are compounds helping the delay of the oxidative damages in cells [4]. A large number of articles have shown that quercetin may act as scav-enger of free radicals, and as inhibitor of the low-density lipoprotein oxidation (im-portant step in development of atherosclerosis and coronary diseases). Quercetin also may regenerate the activity of other potent antioxidant – vitamin E, and may inactivate the transitional metal ions, catalysts of oxidative processes in the body by chelating [5]. In the literature, there is great number of data for the physiological role of the fl avonoids. In addition to the antioxidant activity, their vasodilatation, anti-carcinogenic, anti-infl ammatory, antibacterial, immunostimulation, antiallergenic ac-tivity were described, as well as their antivirus activity against HIV, herpes simplex, infl uenza, rhinovirus, and their ability to act as inhibitors of platelet aggregation [5, 6]. The fl avonoids have specifi c infl uence upon the activity of different enzymatic systems like cytochrome P450, phospholipase A2, lipoxygenase, etc. [7].

Recent studies of Briggs et all. show that the onion consumption inhibits the blood platelet aggregation in vivo [8, 9]. Onion extract inhibits the enzymes cycloox-ygenase and 12-lipoxygenase (in thrombocytes), taking part in the arahidonic acid metabolism. Via this biochemistry pathway, the active compounds in onion give rise to inhibition of thromboxane synthesis and play a positive role on blood coagulation function, i.e. have a preventive effect against thrombus formation and consequently against development of cardiovascular diseases [10, 11, 12].

The anti-infl ammatory properties of onion are well studied [13]. It is determined that it alleviates the pain in osteoarthritis and rheumatic arthritis, has a benefi cial ef-fect in asthma and common infl uenza. The anti-infl ammatory activity of Allium cepa is due to vitamin C, quercetin, and other biologically active compounds like isothio-cyanates, which have synergic antioxidant activity. Therefore in the popular medi-cine of almost all countries in the world, the onion consumption is recommended.

The regular intake of onions, for instance two or three times per week is as-sociated with a considerable decrease of the risk for colon cancer development [14, 15, 16]. As already was mentioned, onion is rich source of quercetin. It can suppress the tumor growth in animals, and protect the colon cells from the harmful effect of some known carcinogens.

Furthermore the onions are rich dietary source of other biologically active compounds, like organic acids, disulfi des, three sulfi des, etc. Allium cepa is a good source of vitamin C, vitamin B6, potassium, dietary fi bers, folates, calcium, iron. The onions are pour source of sodium and do not contain fats. Onion is a very reach source of the microelement chromium, important factor of insulin mechanism of action and for glucose tolerance [17]. Clinical research on diabetic patients has shown that chromium can reduce the blood glucose level, improve glucose toler-ance, as well as reduce cholesterol and triglycerides level. In many developed

countries, the chromium defi ciency is widespread, since the chromium level sharp-ly lowers with consumption of refi ned sugar and white fl our products. It is important to know that one glass of row onion may assure about 20% of the recommended daily intake of this important microelement.

Important study conducted by University of Bern in 2005 reports that one gram of onion added to the food of rats inhibits signifi cantly (p < 0.05) bone resorption as assessed by the urinary excretion of tritium released from bone of 9-week-old rats prelabeled with tritiated tetracycline from weeks 1 to 6. It was determined that during the above conditions the mineral density increased by 17%, which is by 13% higher in comparison with the control group animals. This result suggests that high onion consumption has a potential to reduce the risk for development of osteopo-rosis. In this study, a new compound in onion was isolated, L-glutamyl-trans-S-1-propenyl-L-cysteine sulfoxide, inhibitor of osteoclast activity. The study suggests that Allium cepa may play an important role for menopausal women with a high risk for osteoporosis development [18].

Flavonol content in Bulgarian onionOn table 1, analytical results for the quercetin and kaempferol content in dif-

ferent types Bulgarian onions are presented.Three types of Allium cepa representatives were analysed: white, red and

spring onion. In order to study the fl avonoids’ distribution in spring onion the green and white part of the plant were analysed separately.

Table 1. Quercetin and kaempferol content in Bulgarian onions

Quercetin Kaempferol

mg/kg fresh weight

Samples n Average value Min Max Average

value Min Max

White onion 7 204.1 91.9 292.8. n.d.

Red onion 5 452.5 264.7 832.2 n.d.

Spring onion (hole) 5 103.2 75.6 158.4 14.6 2.0 27.1

Spring onion, (green part) 5 45.4 30.2 61.1 45.4 27.4 52.3

Spring onion, (white part) 5 179.4 110.2 230.5 < 2 0.7 2.8

n – number of samples analysed, Min – minimum value, Max – maximum value, n.d. – not detected

The results show that the red onion contains higher level of quercetin – average value of 452.5 mg/kg, and maximal value – 832.2 mg/kg. The present results are simi-lar to literature data. The data for quercetin content of Bulgarian white onion are lower than those reported by Hertog et al., 1992; Crozier et al., 1997; Justesen et al., 1998 [19, 20, 21], but higher than results of Bilyk et al., 1984; Mattila et al., 2000, Price et al., 1995 [22, 23, 24]. The data for fl avonols in spring onion are fare more scattered in



comparison with those for white and red type of Allium cepa. Only Justesen et al., 1998 reported data for fl avonoids content in spring onion samples [20]. Due to the lack of literature data for quercetin and kaempferol in spring onion, the present results enrich the knowledge of the composition of this plant variety. It was determined that the green part of spring onion contains relatively equal values of quercetin (30.2 – 61.1 mg/kg) and kaempferol (27.4 – 52.3 mg/kg). This result characterizes the green part of spring onion as very important dietary source of kaempferol in Bulgarian healthy diet since its distribution in plant kingdom is very restricted. In the white part of spring onion, high amounts of quercetin are concentrated (110.2 – 230.5 mg/kg), while kaempferol was detected in only two of the samples analysed, but in very low levels (maximum value 2.8 mg/kg). The analysis of whole spring onion shows that the average values for quer-cetin and kaempferol have a medium position among the above-mentioned data.

On the basis of the analysis of the recent scientifi c information for the health benefi ts of onion, its large part in healthy diet is recommended. The onion is the third vegetable consumed in Bulgaria, after potatoes and tomatoes – about 5 kg per capita per year. According to FAO statistics for onion production and consumption for 2007, the highest intake is in Libya – 28 kg per capita per year. In Europe, the highest amount of onion per year is consumed in Balkan and Iberia peninsulas, as follows: Romania – 16.7 kg, Greece – 15 kg, Croatia – 8.9 kg, Spain – 20.1 kg, Portugal – 14.3 kg per capita. In some other European countries, the average onion consump-tion is very low, for instance in Norway and Belgium it is only 1.3 kg per capita [25].

CONCLUSION

On the basis of the recent scientifi c data for physiological importance of on-ions’ compounds, the traditional involvement of onion in national diet is very appre-ciable. Expanding the spectrum of Allium cepa participation in other diets, it can be mentioned that onion defi nitely takes a central part in the Balkan diet, which along with the Mediterranean diet are considered as examples of healthy nutrition.

REFERENCESB u l g a r i a n State Standard ISO 874. Fresh fruits and vegetables. Collecting samples. 1986.1 . Ts a n o v a , S. [Methods of determination of content of fl avonols miricitin, quercetin and kaemp-2 . ferol in foods with high – performance liquid chromatography. – In: Collection of methods of the National Centre of Hygiene, Medical Ecology and Nutrition, Vol. IV. Part: Chemical Content of Foods], 2004, 91-94. (in Bulgarian)H e r t o g , M. G .L., P. C. H. Hollman et M. B. Katan. Content of potentially anticarcinogenic fl a-3 . vonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. – J. Agric. Food Chem., 40, 1992, 2379-2383.K a n c h e v a , V. Аntioxidants – dependence between structure and activity. Antioxidants – 4 . prevention and healthy aging. Sofi a, Simolpress, 2010, 56-73. (in Bulgarian)R i c e - E v a n s , C. A., N. J. Miller et G. Paganga. Structure-antioxidant activity relationships of 5 . fl avonoids and phenolic acids. Review article. – Free Radical Biol. Med. 20, 1996, 933-956.K a t i a r , S. K. et al. Green tea polyphenols: DNA photodamage and photoimmunology. – J. Pho-6 . tochem. Photobiol. B., 65, 2001, 109-114.

H o d e k , P., P. Trefi l et M. Stibirova. Flavonoids-potent and versatile biologically active compounds 7 . interacting with cytochromes P450. – Chem.-Biol. Interactions, 139, 2002, 1-21.B r i g g s , W. H., et al. Differential inhibition of human platelet aggregation by selected Allium thio-8 . sulfi nates. – J. Agric. Food Chem., 48, 2000, № 11, 5731-5735.B r i g g s , W. H., et al. Administration of raw onion inhibits platelet-mediated thrombosis in dogs. – 9 . J. Nutr., 131, 2001, № 10, 2619-2622.M i l l e r , A. I. Antioxidant fl avonoids: structure, function and clinical usage. – Alt. Med. Rev., 1 0 . 1, 1996, № 2, 103-111.X i e , M. L., Q. Lu, et Z. L. Gu. Effect of quercetin on platelet aggregation induced by oxyradicals. 11 . – Zhongguo Yao Li Xue Bao, 17, 1996, № 4, 334-336.A k i b a , S. et al. Involvement of lipoxygenase pathway in docosapentaenoic acid-induced inhibi-1 2 . tion of platelet aggregation. – Biol. Pharm. Bull., 23, 2000, № 11, 1293-1297.W i l s o n , E. A. et B. Demmig-Adams. Antioxidant, anti-infl ammatory, and antimicrobial properties 1 3 . of garlic and onions. – Nutr. Food Sci., 37, 2007, № 3, 178-183.D o r a n t , Е. et al. Consumption of onions and a reduced risk of stomach carcinoma. – Gastroen-1 4 . terology, 110, 1996, 12-20.K y l e , J. et al. Dietary fl avonoid intake and colorectal cancer: a case- control study. –Br. J. Nutr., 1 5 . 103, 2010, 429-436. D o r a n t E., P. A. van den Brandt et R. A. Goldbohm. A prospective cohort study on the relation-16. ship between onion and leek consumption, garlic supplement use and the risk of colorectal carci-noma in The Netherlands. – Carcinogenesis, 17, 1996, № 3, 477-484.W e t l i , H. A. et al. A gamma-glutamyl peptide isolated from onion (Allium cepa L.) by bioassay-17. guided fractionation inhibits resorption activity of osteoclasts. – J. Agric. Food Chem., 53, 2005, № 9, 3408-3414.B l o o d sugar lowering effects of оnion. http://www.shvoong.com/medicine-and-health/1772295-1 8 . blood-sugar-lowering-effects-onion/ Published: February 25, 2008 Updated: October 15, 2010.H e r t o g , M. G. L., P. C. H. Hollman et M. B. Katan. Content of potentially anticarcinogenic fl a-1 9 . vonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. – J. Agric. Food Chem., 40, 1992, 2379-2383.C r o z i e r , A. et al. Quantitative analysis of the fl avonoid content of commercial tomatoes, onions, 2 0 . lettuce, and celery. – J. Agric. Food Chem., 45, 1997, 590-595. J u s t e s e n , U., P. Knutsen et T. Leth. Quantitative analysis of fl avonols, fl avones and fl avonones 2 1 . in fruits, vegetables and beverages by high-performance liquid chromatography with photo-diode array and mass spectrometric detection. – J. Chrom. A, 799, 1998, № 1-2, 101-110.B i l y k , A., P. L. Cooper et G. M., Sapers. Varietal differences in distribution of quercetin and 2 2 . kaempferol in onion (Allium cepa L.) tissues. – J. Agric. Food Chem., 32, 1994, 274-276.M a t t i l a , P., J. Astola et J. Kumpulainen. Determination of fl avonoids in plant material by HPLC 2 3 . with diode-array and electro-array detections. – J. Agric. Food Chem., 48, 2000, 5834-5841.P r i c e , K. R., J. R. Bacon et M. J. C. Rhodes. Effect of storage and domestic processing on the 2 4 . content and composition of fl avonol glucosides in onion (Allium cepa). – J. Agric. Food Chem., 45, 1997, 938-942.F o o d Balance Sheet. http://faostat.fao.org2 5 .

Address for correspondence: Silvia Tsanova-Savova Department of Chemistry Faculty of Pharmacy Medical University – Sofi a Dunav 2 Str. 1000 Sofi a



CARDIOVASCULAR FUNCTIONAL DIAGNOSTICS METHODS

R. Nikolova1 and S. Danev2

1National Center of Public Health Protection – Sofi a2Medeia Inc. – Sofi a

Summary. Effective functional diagnostics is a priority of EU strategy on pre-vention of health status. Effective non-invasive functional diagnostic cardiovascu-lar methods are methods for examination of central nervous system and autono-mous nervous system functions through analyses of heart rate variability, blood pressure variability, and barorefl ex sensitivity. Cardiovascular functional diagnos-tic methods might enhance our understanding of physiological and pathophysi-ological mechanisms of mental work load and stress, etiopathogenesis of cardio-vascular diseases.

Key words: functional diagnostics; heart rate variability; blood pressure variability; barorefl ex sensitivity; cardiovascular diseases

World Health Organization (WHO) indicates that one of the most impor-tant health topics is an early diagnosis and prevention of morbidity and mortality of cardiovascular diseases (CVD). Effective functional

diagnostics is a priority of EU strategy on prevention of health status. Populations of low- and middle-income countries of the EU are exposed to a greater extent to morbidity and mortality of CVD, and have less access to an effective functional di-agnostics [1]. Until 2030, the mortality from CVD will reach 23.6 millions. In low- and middle-income countries of EU, individuals are exposed to a greater extent to the infl uence of cardiovascular risk factors, and have a low access to CVD prevention [2]. Next trend of EU public health policy is on health prevention through research studies. The accent is on transformation of research studies into clinical programs, elaboration of new intervention programs and therapies, effective functional diag-nostic methods, stimulation of preventive strategies, medical technologies, and ef-fective health systems.

Effective non-invasive functional diagnostic cardiovascular methods are methods for examination of central nervous system (CNS) and autonomous ner-

vous system (ANS) functions through analyses of heart rate variability (HRV), blood pressure variability (BPV), and barorefl ex sensitivity.

Cumulative infl uence and effect of stressogenic social, economic, profession-al, aging, and environmental factors affect neural-regulated cardiovascular function. The infl uence of these stressors changes the functional state and activity of CNS and ANS. HRV, BPV and barorefl ex sensitivity are functional non-invasive methods for investigation of ANS and CNS regulated cardiovascular function [3-12]. Cardio-vascular function might be studied through beat-to-beat analysis of heart rate and blood pressure monitoring.

Since the time of the earliest measurements of heart rate and arterial blood pressure, it was observed that signals of cardiovascular origin though almost periodi-cal were characterized by slight cycle-by-cycle variations in both amplitude and time domain. Discrete series describing these oscillations (either as a function of cardiac cycles or equivalently as time fl uctuations obtained from these variables by means of interpolation techniques) are generally referred to as variability signals. Technological progress in the fi eld of data collection and analysis of heart rate and arterial blood pressure variability has led to more sophisticated approaches to rhythmic circulatory phenomena and to their more frequent investigation by power spectral analysis.

Power spectral analysis of cardiovascular parameters is a promising non-invasive method for assessment of the ANS and CNS regulated cardiovascular function. Determination of neural control is of particular clinical interest since diag-nosis, therapy and prognosis of CVD might rely on the balance between sympa-thetic and parasympathetic activity. Based on experimental and clinical studies, the general concept is that very low-, low-, and mid-frequency oscillations (0.00-0.15 Hz) refl ect sympathetic and parasympathetic activities whereas respiration-related high-frequency oscillations (0.2-0.4 Hz) refl ect the parasympathetic tone [9]. Power spectrum refl ects the amplitude of heart rate and blood pressure fl uctuations pres-ent at different oscillation frequencies. Spectral variability measures are defi ned as the total spectral energy in a considered frequency band, i.e. the integral of the spectral density function in the relative frequency area.

The power spectrum consists of frequency bands ranging from 0 to 0.5 Hz, and can be classifi ed into four bands: the ultra low frequency band (ULF), the very low frequency band (VLF), the low frequency band (LF), and the high frequency band (HF) [10, 12, 13].

Variable Units Description Frequency rangeTotal power ms2 variance of all NN intervals < 0.4 HzULF ms2 ultra low frequency < 0.003 HzVLF ms2 very low frequency < 0.003-0.04 HzLF ms2 low frequency power 0.04-0.15 HzHF ms2 high frequency power 0.15-0.4 HzLF/HF ratio ratio of low-high frequency power

Cardiovascular functional diagnostic methods106


Short-term spectral recordings (5 to 10 minutes) are characterized by the VLF, HF and LF components, while long-term recordings include a ULF component in addition to the three others. The table above shows the most used frequency do-main parameters. The spectral components are evaluated in terms of frequency (Hertz) and amplitude which is assessed by the area (or power spectral density) of each component. Thus, squared units are used for the absolute values expressed in ms squared (ms2). The total power of cardiointervals (RR interval) variability is the total variance and corresponds to the sum of the four spectral bands, LF, HF, ULF and VLF.

The HF component is generally defi ned as a marker of vagal modulation. This component is respiration-mediated and thus determined by the frequency of breathing.

The LF component is modulated by both the sympathetic and parasympa-thetic nervous systems. In this sense, its interpretation is more controversial. Some scientists consider LF power, particularly when expressed in normalised units, as a measure of sympathetic modulations; others interpret it as a combination of sympathetic and parasympathetic activity. The consensus is that it refl ects a mix-ture of both autonomic inputs. In practical terms, an increase of the LF compo-nent (tilt, mental and/or physical stress, sympathomimetic pharmacologic agents) has been generally considered a consequence of sympathetic activity. Conversely, β-adrenergic blockade resulted in reduction of the LF power. However, in some conditions associated with sympathetic overexcitation, for example in patients with advanced CHF, the LF component was found to be drastically diminished, refl ect-ing thereby the decreased responsiveness of the sinus node to neural inputs.

The LF/HF ratio refl ects the global sympatho-vagal balance and can be used as a measure of this balance. With an average normal adult in resting conditions, the ratio is generally between 1 and 2.

ULF and VLF are spectral components with very low oscillations. The ULF com-ponent might refl ect circadian and neuroendocrine rhythms and the VLF component long period rhythms. The VLF component has been found to be a major determinant of physical activity and was proposed as a marker of sympathetic activity .

Research teams evaluating the effect of mental work load on ANS activity and clinical utility of HRV consider following spectral measures of HRV: spectral powers of RR intervals in the low-frequency, mid-frequency and high-frequency bands [9, 14].

Low-frequency band is limited between 0.02 and 0.06 Hz. Peripheral vascu-lar resistance exhibits intrinsic oscillations with a low-frequency. These oscillations might be infl uenced by thermal skin stimulation and are thought to arise from ther-moregulatory peripheral blood fl ow adjustments. The fl uctuations in peripheral vas-cular resistance are associated with fl uctuations with the same frequency in blood pressure and heart rate and are mediated by sympathetic nervous system [15];

Mid-frequency band is the band between 0.07-0.14 Hz. The 10-second rhythm in heart rate originates from self-oscillation in the vasomotor part of the barorefl ex

loop. These intrinsic oscillations result from the negative feedback in the barore-fl ex and are accompanied by synchronous fl uctuations in blood pressure (Mayer waves) [16]. The frequency of the oscillations is determined by the time delay of the system. They are augmented when sympathetic tone is increased and they decrease with sympathetic or parasympathetic blockade.

High frequency band is the area between 0.15-0.4 Hz. The inspiratory inhibi-tion of the vagal tone causes heart rate fl uctuations with a frequency equal to re-spiratory rate. The inspiratory inhibition is evoked primarily by control irradiation of impulses from the medullary respiratory to the cardiovascular center. Variations in HF band can mainly be attributed to respiratory sinus arrhythmia (RSA). RSA can be abolished by atropine or vagotomy and is parasympathetically mediated.

HRV and BPV are increasingly used to examine disease-related changes in the autonomic tone or the effects of therapeutic interventions [17, 18]. Hyperten-sion remains one of the most signifi cant cardiovascular risk factors – in 70% the cause for the excess of mortality in hypertensive individuals is mild hypertension. Signifi cant topic in diagnosis and therapy of essential arterial hypertension is deter-mination of autonomic cardiovascular control through the study of HRV and BPV. Short-term heart rate and blood pressure variability recordings registered in labora-tory supine rest conditions show that essential hypertension is characterized by a greater low-frequency power and a smaller high-frequency power of RR intervals [19]. Similar results were reported in the studies of Julius and Johnson, 1985 [20] and Folkow, 1982 [21]. These studies reveal that in essential hypertension car-diac sympathetic tone is increased and cardiac vagal tone and modulation are decreased. HRV and BPV can be used to assess the effects of antihypertensive drugs on cardiac autonomic tone. Antihypertensive drugs have signifi cant effect on autonomic tone. Effect direction and magnitude might be estimated by frequency-domain measures of HRV and BPV. The link between the sympathetic nervous system activity assessed by HRV, BPV and barorefl ex sensitivity and the hyperten-sive state might be supported by the clinical evidence showing the effi cacy of drug therapy interfering with sympathetic control.

Barorefl ex sensitivity is determined by computing the modulus (gain) of the transfer function between changes in systolic blood pressure (input) and heart rate interval (output).

Barorefl ex sensitivity might compute by the gain (mmHg/ms) in barorefl ex us-ing trigonometric regressive spectral analysis (TRSA) [22]. TRSA is a method for mapping of beat-to-beat recorded autonomic cardiovascular measures: HRV, and systolic and diastolic BPV. TRSA examines barorefl ex sensitivity by computation of cross correlations R>0.7 between corresponding pairs of frequencies (frHR ≈ frBP) in very-low-, low- and high-frequency bands of variances of HRV and BPV [23].

Research interest in barorefl ex sensitivity (BRS) as an indicator of cardiac autonomic control has grown in recent years [24]. Available studies in cardiovas-cular psychophysiology elucidate decreasing of BRS in different cardiovascular



diseases, including arterial hypertension, coronary artery disease, and congestive heart failure [25]. Low BRS was associated with increased blood pressure levels in arterial hypertension [26]. Reduced arterial compliance and increased sympa-thetic activity have also been suggested to be responsible for the decreased BRS in hypertension [27, 28]. There is also evidence that impaired barorefl ex regulation is not only a consequence of hypertension but also may contribute to the develop-ment of hypertension [29, 30]. Barorefl ex impairment is considered a major causal mechanism because of the important role of the barorefl ex in reducing blood pres-sure and enhancing heart rate oscillations [31].

The involvement of baroreceptor refl ex control in regulating cardiovascular reactions to mental stress tests has also been explored. The simultaneous increase in systolic blood pressure and heart rate typically observed in response to mental stress test implies the disruption or overriding of short-term barorefl ex regulation. Cardiac barorefl ex sensitivity is suppressed during mental stress tests [32]. Time-domain and frequency-domain analyses for computer evaluation of the arterial barorefl ex have shown that the sensitivity of the baroreceptor-heart rate refl ex is much lower in essential hypertensive than in normotensive subjects for each hour of the 24 hours thereby confi rming previous conclusions obtained by studying the barorefl ex with laboratory techniques [33].

In summary, it can be stated that the main function of baroreceptor refl ex-es (barorefl exes) is to buffer acute changes of blood pressure. Impairment of the barorefl exes (e.g. by denervation of the sino-aortic afferents) results in a hyperre-activity to external stimuli. Human studies show that the sensitivity of the barorefl ex is diminished during reactivity tasks such as dynamic and isometric exercise, and during mental arithmetic tasks. Individuals with decreased barorefl ex sensitivity re-veale increased blood pressure lability. Barorefl ex sensitivity is inversely related to overall blood pressure variability (which depends primarily on the lowest-fre-quency powers) and directly related to overall heart rate variability. This change implies that barorefl ex infl uences are not limited to fast parasympathetically me-diated blood pressure and heart rate fl uctuations but extend to the low and very low frequency fl uctuations, considered sympathetically and parasympathetically mediated. Barorefl ex sensitivity is infl uenced by both divisions of the autonomic nervous system: sympathetic and parasympathetic activity. During mental stress blood pressure and heart rate elevations are associated with suppression of car-diac barorefl ex sensitivity.

HRV, BPV and barorefl ex sensitivity are cardiovascular functional diagnostic methods which have considerable potential to assess the function of the ANS and CNS in healthy individuals to assess the effect of mental work load on functional state, and to study the etiopathogenesis of diseases in patients with cardiovas-cular and non-cardiovascular disorders. Functional diagnostic methods might en-hance our understanding of physiological and pathophysiological mechanisms of mental work load and stress, etiopathogenesis of cardiovascular diseases, and

the mechanism of action of medications. Identifi cation of individuals at risk for subsequent morbidity and non-risk groups necessitate future prospective studies to determine the sensitivity, specifi city and predictive values of HRV, BPV and barorefl ex sensitivity.

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R ü d i g e r , H., L. Klinghammer et K. Scheuch. The trigonometric regressive spectral analysis – a 2 2 . method for mapping of beat-to-beat recorded cardiovascular parameters on to frequency domain in comparison with Fourier transformation. – Comp. Meth. Progr. Biomed., 58, 1999, 1-15.R ü d i g e r , H. et al. Spectral analysis of peripheral blood pressure and heart rate in laboratory 2 3 . under mental load and rest in persons with and without mild hypertension. – 9th European Meeting on Hypertension. Milan, 11-15 June 1999.M u l d e r , G. et al. A psychophysiological approach to working conditions. – In: R. Backs, W. 2 4 . Boucsin (Eds.) Engineering Psychophysiology. Mahwah, New Jersey, Lawrence Erlbaum Associ-ates Publ., 2000, 139-159.E c k b e r g , D. et P. Sleight. Human Barorefl ex in Health and Disease. Oxford, UK, Clarendon, 2 5 . 1992.K i n g w e l l , B. et al. Arterial compliance may infl uence barorefl ex function in athletes and hyper-2 6 . tensives. – Am. J. Physiol., 268, 1995, H411-H418.L a g e , S. et al. Relationship of arterial compliance to barorefl ex function in hypertensive patients. 2 7 . – Am. J. Physiol., 265, 1993, H232-H237.M a t s u k a w a , T. et al. Reduced barorefl ex changes in muscle sympathetic nerve activity during 2 8 . blood pressure elevation in essential hypertension. – J. Hypertens., 9, 1991, 537-542.P a r m e r, R., J. Cervenka et R. Stone. Barorefl ex sensitivity and heredity in essential hyperten-2 9 . sion. – Circulation, 85, 1992, 497-503.L o w, P. Clinical Autonomic Disorders. 2nd ed. Philadelphia, Lippincott-Raven Publishers, 1997.3 0 . S c h e u c h , K. et H. Schroder. Mensch unter Belastung: Stress als ein Humanwissenschaftliches 3 1 . Integrationskonzept. Berlin, Deutscher Verlag der Wissenschaften, 1990.Vo g e l e , C. et A. Steptoe. Emotional coping and tonic blood pressure as determinants of cardio-3 2 . vascular responses to mental stress. – J. Hypertens., 10, 1992, 1079-1087.S m i t h , H., P. Sleight et G. Pickering. Refl ex regulation of arterial pressure during sleep in man. 3 3 . – Circul. Res., 24, 1969, 109-121.

MENTAL WORK CAPACITY AND RELIABILITY AMONG NUCLEAR POWER PLANT OPERATORS

A. Agovska and R. Kirkova Department of Hygiene, Medical Ecology and Nutrition, Medical University – Sofi a

Summary. Examination is performed on the mental work capacity of the operators in NPP – Kozloduy, and its importance for the control of the operative staff’s reliability. Psycho-physiological studies on 50 operators were performed. By computer-based psycho-physiometer (PPM) the following examinations were car-ried out: Simple sensorymotor reaction (SSMR), Visual-motor reaction of moving object (VMRMO), Landolt’s test, Dynamic and static tremor-reaction as well as the dynamic of their changes in the real work time. At the same time, there were mea-sured the heart rate (HR) and the skin resistance (SR). The operators kept high level of mental work capacity during the whole shift, also an indicator for high reli-ability. In all tests at the end of the shift, the HR was slightly raised, an indicator for a higher “physiological price” of the fulfi lment. The SR was constant, which shows emotional steadiness in the examined operators. The evaluation of operator’s be-havioral reliability is closely related to the evaluation of their mental work capacity. The proposed PPM-system could be used for periodical control of the work capac-ity and the functional state of the operators and is suitable for work in the services of occupational medicine.

Key words: nuclear operators, reliability, mental work capacity

INTRODUCTION

The work of the operative staff in the Power Plants (PP) is exclusively cognitive with expressed mental stress, high responsibility, necessity of taking non- alternative decisions in conditions of shortage of time.

Therefore, the problems related to the reliability of the human team in management of PP are of vital importance for ensuring a safe work with no accidents [1, 2, 3, 4, 5]. The term “human reliability” is usually defi ned as a probability that a person will correctly perform some system-required activity during a given time period (if time is a limiting factor) without performing any extraneous activity that can degrade the

Mental work capacity and reliability...112


system [6]. Short and complete defi nition of human reliability is adequacy, timeli-ness and underlying [7]. Here are comprised: type of higher nervous activity, in-tellect and memory (lasting, brief and operative); the characteristics of attention (scope, concentration, stability, distribution, commutation, mobility of the psychic processes), velocity of the sensory-motor reactions; functional status, psychoso-matic health; emotional stability/non-stability, self control; reactions during stress and frustration, motivation, etc [8, 9, 10].

Index of importance for evaluating the human reliability in PP are the changes in the mental work capacity during work shift [11, 12, 13, 14].

On the one part, the behavioral reliability depends on the experience and knowledge of the individual and personal resource, of the psychosomatic health, etc. qualities and requirements, which are evaluated on professional choice level [7, 15].

On the other part, the operative staff reliability is affected by the development of the stress reactions related to the work activity, the rate of nervous tension, motional stability/instability and the approach of fatigue, which can lead to taking inadequate and wrong decision [16, 17, 18].

In the present work, there are given data of indices on the mental work capaci-ty of operators in NPP – Kozloduy, and its importance for the control of the reliability of the operative staff. The given results are part of the complete study “Assessment of Kozloduy Nuclear Power Plant Operator’s Work Load and Health Risk”.

SUBJECTS AND METHODS

Psychophysiological studies are performed in the Command Desks of the management of 5th and 6th block in the NPP – Kozloduy, during working shifts (at the beginning and the end of work during day shift 8-16 hrs). Fifty persons of the operation staff were examined.

A computer-based psycho-physiological testing-system (Psycho-Physio-Me-ter – PPM) is used. By the latter quantitative indices can be received, objectively characterizing the state of the central nervous system (CNS) and some functional indices – heart rate (HR) and skin resistance (SR).

The examined person receives stimuli from the computer screen by prelimi-nary entered instruction on the same screen. The response is by functional key-board – divided into separate sectors, responding to the individual tests and acti-vated one by one according to the test performed. The following examinations were carried out.

1. Simple sensorymotor reaction (SSMR). 30 sound stimuli with frequency 1000 Hz are handed over in pseudo-accidental way in previously given interval of 0,50 – 2,50 sес. In response to the sound stimuli the person has to press one but-ton as quickly as possible. The time of the person’s reaction is measured from the moment of giving the signal (sound) to the moment of touching the sensory button.

On the basis of 30 given stimuli the average time of reaction (in sec) is recorded. This is the test by which the total level of the agitated processes (activation level) and the psychomotor activity’s changes are studied.

2. Visual-motor reaction of moving object (VMRMO). On the screen static marker (reper – green hexagon) is represented, which is disposed on the perimeter of the basic circumference of accidently generated from the program place. On the outside circumference, there is represented a moving marker – white hexagon of the same size. The movement of the hexagon starts from position zero to the right or left direction in pseudo-accidental order at preliminary given velocity of the movement 0.40 m/sec. Every new movement of the white hexagon from position zero is registered as separate stimulus – 30 stimuli were set. The person has to perform a quick motor reaction (press the button) when the both repers fall in line. The test serves to evaluate the psychomotor activity, the concentration and distri-bution of the attention, possibility to take fast decision. The number of correct and wrong answers is recorded in left and right movement.

3. Landolt’s test – On the screen, “Landolt’s circle” is displayed – with cut out sectors in one of the eight possible directions. The person has to press the button from the functional keyboard, where the respective circle is displayed. The number of wrong and correct answers is recorded as well as the average time of reaction. By this test the short-time memory, reaction time in the condition of choice, i.e. the speed of coding the relevant information is evaluated.

4. Static and Dynamic tremor-reaction (tremometry). To evaluate the static tremor-reaction (STR), the person takes a “pen” with sharp nib and strives to hold the nib of the pen in the 5 mm-opening, without touching its walls. The number of touches, the average time of touches and the coeffi cient of steadiness are record-ed. The latter represents the relation of total time for touchings to the total time of the investigation multiplied by hundred (in %). When examining the dynamic trem-or-reaction (DTR), the examined person has to take over a “pen” along a labyrinth (one meter) without touching its walls. The time of passing through the labyrinth (in sec), the number of touchings, the average time of touchings and the coeffi cient of steadiness (%) are measured. Both data for STR and DTR give information on the basic quality of attention – steadiness, visual-motor coordination, as well as on the will-power qualities of the examined person and the emotional status.

5. Heart rate (HR) and skin resistance (SR). Before starting the examinations and during performance of the separate neuropsychic tasks, the pulse (beats/min) and skin resistance (kΏ) are registered. The average values and variety of both in-dices are taken into consideration. This gives information about the effort by which the different neuropsychic tasks are performed and emotional status.

The results were statistically processed by SPSS-variation, alternative and correlation analysis, Wilcoxon Matched Pairs analysis. Reliability of the change р < 0.05.



RESULTS

Таble 1. Results of the neuro-behavioral tests

Indices Beginning of the shift End of the shift Beginning/Endх- Sx x Sx р

Simple sensory-motor reaction (SSMR)Reaction time (sec) 0,250* 0,037 0,228* 0,035 p<0,05*Heart rate (beats/min) 84,50 11,76 87,01 10,87 p>0,05Steadiness Coeffi cient of Pulse (%) 5,27 2,18 5,73 1,35 p>0,05Skin resistance (kΏ) 419,63 217,97 413,48 125,53 p>0,05

Visual-motor reaction of moving object (VMRMO).Number of correct answers– in left– in right

10,8010,20

3,794,10

13,0013,85

3,122,85

p<0,05*p<0,05*

Number of incorrect answers– in left– in right

4,304,55

2,002,14

1,280,75

1,851,30

p<0,05*p<0,05*

Heart rate (beats/min) 87,70 12,54 89,00 11,42 p>0,05Steadiness Coeffi cient of Pulse (%) 5,40 2,30 7,27 2,30 p>0,05Skin resistance (kΏ) 329,61 112,97 363,48 150,32 p>0,05

Landolt’s TestNumber of correct answers 27,94 4,11 29,86 3,62 p>0,05Number of wrong answers 1,27 1,16 1,34 0,90 p>0,05Average Reaction time (sec) 1,56 0,26 1,32 0,16 p<0,02*Heart rate (beats/min) 97,60 13,53 104,70 10,12 p<0,05*Skin resistance (kΏ) 337,40 120,70 432,60 170,32 p<0,05*

Static tremor-reaction (tremometry)Number of touchings 2,66 1,97 2,00 1,43 p>0,05Average time of touchings (sec) 0,051 0,006 0,036 0,003 p>0,05Coeffi cient of steadiness (%) 0,323 0,047 0,122 0,080 p=0,02*Heart rate (beats/min) 83,80 9,31 87,00 12,50 p>0,05Skin resistance (kΏ) 376,40 102,70 432,60 107,32 p>0,05

Dynamic tremor-reaction (tremometry)Time for fulfi lment (sec) 36,56 14,78 37,00 12,10 p>0,05Number of touchings 99,80 25,1 81,20 23,40 p<0,05*Average time of touchings (sec) 0,035 0,006 0,030 0,007 p>0,05Coeffi cient of steadiness (%) 8,86 3,73 6,56 4,90 p>0,05Heart rate (beats/min) 86,70 13,10 90,70 13,20 p>0,05Skin resistance (kΏ) 371,40 120,70 398,51 170,32 p>0,05

x – average value of the result; Sx – standard deviation; * – statistically reliable difference

The results point out to reliable time decrease of the SSMR at the end of work in comparison with the beginning, an indicator for improved attention. The heart rate inreliability increases to the end of the shift. The skin resistance shows uncon-siderable variations.

At the end of the work shift, the VMRMO improves in both directions (in left and in right), the number of correct answers reliably increases, while that of wrong answers reliably decreases - index for good mobility of the basic nervous process-es at the beginning and at the end of work. In the HR and SR values, a tendency of increase with no statistical reliability is established.

Table 1 shows that in Landolt’s test the number of the correct answers at the end of work slightly increases, at unchanged time for fi nding the button, corre-sponding to the displayed fi gure. The HR and SR reliable increase when perform-ing the test at the end of the work day in comparison with the beginning.

The STR render an improvement at the end of the work shift – there is a tendency of decrease in the number of touches and reliable improvement of the coeffi cient of steadiness. At the performance of this test, the HR and SR are higher towards the end of the work day.

By the task of evaluating the DTR, a slight increase in the total time for fulfi l-ment of the task is registered, combined with reliable decrease in the number of touches.

The data given in tables 1 point out that the HR and SR values are different during the carrying out the separate nervous-behavioral tests.

For the HR the diffi culty of the tests could be classifi ed in the following way: 1) Landolt’s test – 99.5 beats/min; 2) DTR – 93.3 beats/min; 3) VMRMO – 90.4 beats/min; 4) SSMR – 85.7 beats/min; 5) STR – 85.1 beats/min.

Landolt test’s HR is reliably higher than the HR in the remaining 4 tests (Wilcoxon Matched Pairs p < 0.05). Reliably higher is the HR during the VM-RMO in comparison with the HR for studying the time of SSMR (p < 0.05). HR in dynamic tremometry is reliably higher in comparison with the HR in the static tremometry (р < 0.05).

On Figure 1, the results of the daily dynamic of the HR’s changes are given. The results surveyed in dynamic point out to higher HR at the end of the work

day in comparison with the beginning in all used tests (fi g. 1). Best expressed is the pulse increase in the fulfi lment of the Landolt’s test (p < 0.05), followed by the tests static and dynamic tremometry (p > 0.05), SSMR and VMRMO (p > 0.05). These data to a great extent correspond and confi rm the mentioned above rating in diffi culty by the used tests in the study. The SR in daily dynamic shows no clear changes.

The comparison between the separate tests points out that the SR is highest in time during the fulfi lment of SSMR (421.9 kΏ) which, according to the diffi culty, is on one of the last places and on the lowest in time for completion of the task for VMRMO (329,2 kΏ) and the Landolt’s test (337.4 kΏ), which in diffi culty take the fi rst two places. It is accepted that SR values to a great extent confi rm the rating in diffi culty, mentioned above, met in the performance of the tasks.



0 20 40 60 80 100 120

1

2

3

4

5

Beginning of the shiftЕnd of the shift

1-SSMR; 2-VMRMO; 3-Landolt’s test; 4-STR; 5-DTR

Fig. 1. Heart rate (p/min) dynamics for different neurobehavioral tests

As far as HR and SR are affected by emotional stress, there is a negative dependence between them – at high emotional stress the HR increases, while the SR decreases. The data given show such dependence during the performance of the DTR (KK = -43; p = 0.06) and during the time of performance of the VMRMO (K = -51; p = 0,028). During the examination of the Landolt’s test and the STR, a tendency of negative dependence is observed.

DISCUSSION

The data of the used tests carried out show that the operative staff keeps good abilities for the completion of complex neuro-sensorial tasks during a whole work day.

The fulfi lment of the SSMR task is very simple, but the changes in absolute values are good integral index for the level of the general excited processes and the processes of attention. In this case, they show an increase of the excited pro-cesses in the central nervous system in the end of the shift. On the background of the raised excitability at the end of work the neuropsychic capacity for work, evaluated after the other used behavioural methods remains unchanged and even improved [19].

The number of correct and wrong answers in VMRMO to the left and to the right in both examinations (in the beginning and in the end of the shift) is index for good mobility of the basic nervous processes, good concentration and distribution of the attention during the work time.

The results from Landolt’s test (the correct answers at the end of work slightly increases without change in the reaction time) besides index for quick codifi cation of the relevant information are index for fast and accurately short-term (operat-ing) memory, good concentration and distribution of the attention, possibility for

maintaining the active attention and the differential ability during the work shift. It is important that the short-term memory is more sensitive to fatigue and more often correlated with the parameters of the cardiovascular system (CVS) than attention [20]. Confi rmation of this is that Landolt’s test is fulfi lled with highest HR in the be-ginning and the end of the shift.

Tremor-reaction is good indicator for mental load’s infl uence and expresses the emotional state. Study of Haider E et al. [22] shows that tremor-reactions with informatory-mental stress can be interpreted by considering performance. Reac-tions with informatory-emotional stress are task specifi c when tremor is inter-indi-vidually normalized.

The good results in the static tremor-reaction also confi rm the general ten-dency for keeping on high level the neuropsychic work capacity during the whole work day.

In the dynamic tremor-reaction, at the end of the shift, there is registered a reliable decrease in number of touches (i.e. improving the accuracy of work) com-bined with slight increase in time for performing the test (i.e. worsen the velocity). The alteration of the correlation “velocity/accuracy” is an index for a change in the “strategy” of the examined person while accomplishing this task. The change in the “strategy” is a mechanism for maintaining the neuropsychic (mental) working capacity [20], but it refl ects on CVS parameters – the data from the pulse show a tendency of increase at the end of the shift. Because of this mechanism the mental work capacity shows to be less dependent on the fatigue as compared to the CVS activity [23].

As the neurobehavioural tests used raise demands for velocity and accu-racy on behalf of the operating memory and the motor response, good steadi-ness and distribution of the attention and engagement the emotional will-power sphere, the heart rate to a higher degree refl ects the diffi culty with which the person accomplished the task – the so called “physiological price” for good answer [21, 24].

At the end of the work, the HR in fulfi llment of all tests is insignifi cantly higher in comparison with the beginning of the day, i.e. at the end of the shift the neurobe-havior tasks are performed with higher “physiological price” – a stronger tension is needed on behalf of the nervous system for keeping the initial level of work capac-ity, which refl ects on CVS [20, 24].

The SR expresses the emotional stress during the performing of a given task – the calmer a person is, the higher is the SR. On the other part, the emotional instability strongly decreases the reliability of the human team in the system “man-management” of the NPP. The results point out that during work the emotional state of the examined operators is kept on con stant level – indicator for this are the SR values, where there are no differences during the work shift. This emotional steadi-ness is an index for high reliability of the operative staff in NPP Kozloduy.



The high level of mental (nervous-psychic) work capacity in the operators of NPP Kozloduy we connect with their high individual resources and strong motiva-tion. These individuals have strong feeling for responsibility, work control, high la-bour morality, refl ecting the effect of the strict professional selection [7]. Most of the examined operators are satisfi ed with their work – their knowledge and experience are used completely. They are sure in their activities during work, they have the possibility to advance in hierarchy, nice payment. Of importance is also the young age of the operators – average age 33,3 ± 2,5 years [25].

The work satisfaction, the high personal resources and strong motivation are factors for powerful reserve to counteract to the stress [8, 26]. The operators with high levels of psychic stability revealed that individuals with high emotional stability, good self-control, low frustration and sthenic reactivity type demonstrate higher resistance to psychologic stress at work. Opposite personal features lower resistance to stress and result in transfer of stress at work into family relation-ships sphere that itself could become a stressor [8]. These factors are of impor-tance in maintaining high level of mental work capacity and decrease the state of fatigue [26].

It could be generalized that the examined operators are with preserved abili-ties for the fulfi lment of complex psycho-sensorial tasks and with high behavioral reliability for ensuring safe work without accidents.

The following nervous-behavioral tasks are objectively identifying the opera-tor’s mental work capacity – good criteria for evaluating the reliability of the human team in the operative staff in NPP. For that reason we consider that the evaluation of operator’s behavioral reliability is closely related to the evaluation of their mental (nervous-psychic) work capacity.

The system of psycho-physiological examinations used for this study reports the basic nervouspsychic processes and the “physiological price” – the effort thanks to which the tasks are fulfi led. The proposed psycho-physiological test-system could be used in periodical control of the health and functional state of the operators and is suitable in the work of the services related to occupational medicine.

REFERENCES G r o z d a n o v i c , M. Usage of human reliability quantifi cation methods. – Int. J. Occup. Saf. Er-1 . gon, 11, 2005, № 2, 153-159.G o r e , B. Human performance cognitive-behavioral modeling: a benefi t for occupational safety. – 2 . Int. J. Occup. Saf. Ergon, 8, 2002, № 3, 339-351. R e v u e l t a , R. Operational experience feedback in the World Association of Nuclear Operators 3 . (WANO). – J. Hazard Mater., 111, 2004, № 1-3, 67-71.S u k s i , S. Methods and practices used in incident analysis in the Finnish nuclear power industry. 4 . – J. Hazard Mater., 111, 2004, № 1-3, 73-79.

Ta k a h a s h i , M. et T. Tanigava. Modifying effects of perceived adaptation to shift work on health, 5 . wellbeing and alertness on the job among nuclear power plant operators. – Ind. Health, 43, 2005, № 3, 93-118.D o u g h e r t y, E. Human reliability analysis – Where shouldst thou turn? – Reliability Engineering 6 . and System Safety, 29, 1990, № 1, 283-299.Yo n k o v a A. [Psychofi siological and psychological criteria for professional selection of operators 7 . in nuclear power plants.] – Hyg. Public Health, 1, 1993, № 4, 6-20. (in Bulgarian).B o n d a r e v, I. et I. Kozlovskaia. [Individual, social and psychologic factors of resistance to stress 8 . among operators and dispatchers in power plant.] – Med. Tr. Prom. Ekol., 8, 2001, № 2, 15-19. (in Russian).H i r s c h b e r g , S. Human Reliability Analysis in probabilistic safety assessment for nuclear power 9 . plants. – CSNI Tech. Opin. Papers, 6, 2004, № 2, 18-25.S o r o k i n a , N. et al. [The effect of motivational and emotional aspects of work on the structure of 1 0 . operator’s errors.] – Aviakosm. Ekolog. Med., 27, 2003, № 1, 23-26. (in Russian)B o b k o , N. [Diurnal changes in the effectiveness of mental activity of the operators during shift-11 . type work.] – Fiziol. Zh., 6, 1989, № 2, 83-87. (in Russian)B o b k o , N. [Computer monitoring of the effectiveness of the operator’s adaptation to shift work.] 1 2 . – Med. Tr. Prom. Ekol., 9, 1999, № 1, 17-20. (in Russian)K a r p e n k o , A. [Problem of controlling current mental capacity in operators.] – Gig. Tr. Prof. 1 3 . Zabol., 1, 2002, № 3, 12-14. (in Russian)K o r o b e i n i k o v, H. et N. Kharkovliuk. [The characteristics of autonomic regulation in persons 1 4 . with different levels of mental work capacity.] – Fiziol. Zh., 46, 2000, № 1, 82-88. (in Russian)G e r a s i m o v, A. [Psychophysiologic criteria in predicting the working effi ciency of power plant 1 5 . operators at their admission to the training center.] – Fiziol. Cheloveka, 19, 1993, № 4, 77-88. (in Russian)B a s o v, V. et al. [Psychological services in the professional training of the NES (nuclear energy 1 6 . station) operators.] – Med. Tr. Prom. Ekol., 13, 2004, № 3, 17-20.B o b k o , N., A. Karpenko et V. Chermyuk. [Combined infl uence of work tension, fatigue and cir-1 7 . cadian rhythms on the effectiveness of the operator’s mental performance. – Fiziol. Zh, 44, 2008, № 1, 35-42. (in Russian)R a u , R. Psycho-physiological assessment of human reliability in a simulated complex system. – 1 8 . Biol. Psychol., 42, 1996, № 4, 287-300.L a s k o v a , I. V. et E. E. Tret’yakova. [Characteristics of neurological status and the electroen-1 9 . cephalogram in nuclear power station control operators.] – Neurosci. Behav. Physiol., 40, 2010, № 4, 457-460. (in Russian)L a s k o v a , I. V. et E. E. Tret’yakova. [Infl uence of working shift on nervous system function in 2 0 . operators of control unit in nuclear power station.] – Neurosci. Behav. Physiol., 3, 2009, № 4, 32-36.B o b k o , N. [Effect of time of day and fatigue on psycho-physiological functions of an operator.] – 2 1 . Fiziol. Zh.., 49, 2003, № 5, 89-96. (in Russian)H a i d e r , E. et al. Tremor reaction to mental and emotional stress. – Int. Arch. Occup. Environ. 2 2 . Health, 53, 1983, № 2, 175-179.Ta k e m u r a , Y., S. Kikuchi et Y. Inaba. Does psychological stress improve physical performance? 2 3 . – Tohoku J. Exp. Med., 187, 1999, № 2, 111-120.



B o b k o , N. A. [Effect of stress on the cardiovascular system function in operators performing 2 4 . predominantly mental work at the different times of the day and week days]. – Fiziol. Cheloveka, 33, 2007, № 4, 55-62. (in Russian)R e i d , K. et D. Dawson. Comparing performance on a simulated 12 hour shift rotation in young 2 5 . and older subjects. – Occup. Environ. Med., 58, 2001, № 1, 58-62.B o d n a r , E., G. Zarakovskii et L. Chainova. [Motivation as a factor in the development of an op-2 6 . erator’s functional state of tension.] – Fiziol Cheloveka, 25, 1999, № 2, 1-78. (in Russian)

Address for correspondence: Assoc. Prof. A. Agovska, MD, PhD Department of Hygiene Medical Ecology and Nutrition Medical University 15 Akad. Iv. Geschov bulv. 1431 Sofi a Bulgaria

(359 2) 9152103; 0888 775284 (359 2) 851 08 37

e-mail: [email protected]

PSYCHOACTIVE SUBSTANCES IN DIFFERENT CULTURES AND RELIGIOUS PRACTICES

J. Radenkova¹, E. Saeva² and V. Saev²1Toxicology Clinic, Emergency Medicine Institute “N. I. P irogov” – Sofi a

2University “La Sapienza” – Rome

Summary. Many drugs are used for their mood and perception change ef-fects, including those with accepted uses in medicine and psychiatry. There is ar-chaeological evidence for the use of psychoactive substances dating back at least 10 000 years, as well as historical data for cultural purposes in the last 5000 years. Some psychoactives, particularly hallucinogens, have been used for religious pur-poses since prehistoric times. Examples of traditional entheogens include: kykeon, ambrosia, iboga, soma, peyote, ayahuasca. Other traditional entheogens include cannabis, ethanol, ergine, psilocybe mushrooms, opium. Although entheogens are taboo and most of them are offi cially banned in Christian and Islamic societies, their ubiquity and importance in terms of different spiritual traditions of other cultures is unquestioned.

Key words: psychoactive substances, drugs, entheogens, hallucinogens

Many drugs are used for their mood and perception change effects, in-cluding those with accepted uses in medicine and psychiatry. Classes of drugs frequently used recreationally include: stimulants, hallucino-

gens, hypnotics, analgesics [16]. There is archaeological evidence for the use of psychoactive substances dating back at least 10 000 years, as well as historical data for cultural purposes in the last 5000 years [14]. Some psychoactives, particu-larly hallucinogens, have been used for religious purposes since prehistoric times.

There is speculation that hallucinogenic mushrooms and cacti, heavily infl u-enced the major religions of India, Americas, Middle East and Europe, including Christianity [9, 17]. Entheogen in the narrow sense, is the psychoactive substance used in psychotherapy, religious, spiritual or shamanic context. The term is derived from two words of ancient Greek, (entheos) and (genesthai). The literal meaning of the word entheogen is “that which causes God to be within an individual”. Histori-

Psychoactive substances in different...122


cally, entheogens are mostly derived from plant sources and were used in various traditional religious contexts.

Examples of traditional entheogens include: kykeon, ambrosia, iboga, soma, peyote, ayahuasca. Other traditional entheogens include cannabis, ethanol, ergine, psilocybe mushrooms, opium [9, 22, 23].

Kykeon is Greek and drinks that are used in the Eleusinian mysteries - the opening ceremony of the cult of Demeter and Persephone. Kykeon is mentioned in the texts of Homer “Iliad”, and is described as consisting of barley, water, herbs and ground goat cheese. In an attempt to solve the mystery of how many people over a period of two millennia could constantly try revelations, during the climax of the ceremony of the Eleusinian Mysteries, it is postulated that the barley used in the Eleusinian kykeon is parasitized by ergot and that the psychoactive properties of fungi lead to strong experience mentioned by participants in Eleusis [15].

Ergot – Claviceps purpurea is a parasitic fungus that occurs in some grass-es and cereals, mostly rye. In the class of infected rye grains instead appear purple cells in the form of horn, hence the name of the fungus. Ergot contains psychoac-tive alkaloids (ergotamine) and in larger doses is toxic to humans (ergotism). Since then it gets styptic drug that is used in medicine.

While doing experiments with ergot, looking styptic, the Swiss chemist Albert Hofmann discovered the psychoactive properties of the alkaloid ergotamine [10] and shortly thereafter synthesized LSD.

Albert Hofmann, with Uosan and Rukh, substantiate the hypothesis that the sacred drink in ancient Eleusinian Mysteries (kyukeon) contained barley or rye in-fected with ergo, which had caused hallucinations in dedicate to Mystery.

In the Middle Ages, there are known outbreaks among humans and animals caused by bread made with ergot-infected grain. They are called “Fire of St. An-thony” or “Holy Fire (ignis sacer)” – now called ergotism.

Delphic oracle was founded eight centuries BC and is the most popular and renowned in antiquity holy place in the Mediterranean. This is best documented religious site of the classical Greek world [11]. Pythia is the name of any prophet-priestess of the god Apollo in his sanctuary at Delphi, located at the foot of Mount Parnassus. When giving predictions Pythia sat on the tripod of Apollo in the temple, chewed bay leaves, fell into a trance, sent by Apollo, and in this state gave pre-dictions of who came to inquire of God. Since speech was incomprehensible and unrelated, her words were interpreted by special priests.

There is a hypothesis that the prophet-priestess of the god Apollo used hallu-cinogenic substances, most likely plant which produced the visions – cannabis, opium or daydream. According to another hypothesis, a tripod was ground fi ssure from which emerged intoxicating gases – nitrous oxide, or ethylene that induced a state of trance.

Among the most famous ancient writers who speak for the sanctuary of Apollo at Delphi are Pindar, Herodotus, Aeschylus, Sophocles, Euripides, Plato, Aristotle, Diodorus, Strabo, Pausanias, Plutarch, Livy, Ovid, Luke, Julian, Justin [11].

Soma or Haoma was a ritual drink of importance among the early Indo-Irani-ans, and later and greater Persian Vedic cultures [2]. The active ingredient of Soma is presumed by some to be ephedrine, an alkaloid with stimulant and (somewhat debatable) entheogenic properties derived from the soma plant, identifi ed as Ephe-dra pachyclada). However, there are also arguments to suggest that Soma could have also been Syrian Rue, Cannabis, Belladonna or some combination of any of the above plants. In the Vedas, Soma is portrayed as sacred and as a god. The god, the drink and the plant refer to the same entity. Two holy drinks exist: Soma for the immortal soul and Amrita for the immortal body. In this aspect, Amrita is similar to the Greek ambrosia; both are what the gods drink, and what made them deities. Indra and Agni are portrayed as consuming Soma in copious quantities. The con-sumption of Soma by human beings, as attested in Vedic ritual, probably results from the belief that it bestows divine qualities on them [20, 21].

Entheogens have played a key role in the spiritual practices of American cul-ture for millennia [5, 6]. Not one of the cacti of the New World has psychoactive properties, but arguably the most famous among them is Lophophora williamsii [18]. This cactus, called by locals meskal or peyote (more accurate peyotl) semi deserts grows from central Mexico to the Rio Grande River. There is archaeologi-cal evidence that it was known to the locals at least before 8000 and because of its particular properties it has for centuries played an important role in religious beliefs and rituals. Since 1620 this plant was condemned by the Inquisition as contrary to the purity and integrity of the holy Catholic faith.

However, there is a large group of people who continue to use peyote in a tra-ditional manner. Over the 19-th century, its cult spread among the Indian tribes north of the Rio Grande and soon conquered the Great Plains purposes. This cult united traditional shamanism to Christianity, according to Indian Christian God is the same with the Great Spirit who created the world and left a piece of peyote in its power, and Jesus is the man who gave the people this plant. Peyote was used in Saturday’s gathering in traditional tents, where the Indians under its infl uence prayed and sang in the morning ceremony which ended with a common breakfast. Core values of this religion are brotherly love, care, self-denial and plenty of alcohol.

In 1918, its followers were united to resist persecution and founded the Ameri-can Church Indigenous (Native American Church), which today has around 250,000 followers, i.e. one third of all Indians in the U.S. The organization has managed to resist the attempts use of peyote to be outlawed and by a 1970 act of Congress it was offi cially legalized as part of the ritual.

Peyote quickly became known in Europe and many scientists and intellectu-als were captivated by its ability to immerse the individual in another reality. Peyote opened the doors of perception – that is the title of Aldous Huxley’s book dedicated to his experiences under the infl uence of mescaline.

Along with the psychological research the effective start of peyote was ac-tively sought. In 1900 it was already known that this is an alkaloid called mescaline – colorless oil, soluble in water, alcohol and chloroform.



The structure of mescaline was established with certainty only after Shpet in 1919 managed to synthesize it in a chemical laboratory. It has been isolated from peyote and other alkaloids, e.g. lofoforin which has strychnine similar properties. Al-though to a lesser extent, they also contribute to the general effect of peyote. As of 1925 all that could be known about peyote and mescaline was known; today mesca-line and LSD have become standard for hallucinogenic substances [26, 31].

Indigenous peoples of South America used a wide variety of entheogens. Prominent example is ayahuasca [7]. Dimethyltryptamine containing ayahuas-ca is used primarily as a religious sacrament. Those whose usage of ayahuasca is performed in non-traditional contexts often align with the philosophy and cos-mologies, associated with ayahuasca shamanism, as practiced among indigenous peoples such as the Peruvian Amazon Urarina. Ayahuaska is referred to as nectar of the gods. It has strong hallucinogenic properties, but is signifi cantly softer than pure DMT, mainly due to lower concentration. “Spiritual experiences” are also char-acteristic of ayauaska.

Iboga tree is the main pillar of the religion practiced Bwiti in West Central Africa, mainly Gabon, Cameroon and Congo, which use alkaloid containing plant roots in a number of ceremonies [5, 7].

Iboga is taken in large doses by initiates when entering the religion, and is regularly eaten in smaller doses associated with rituals and tribal dances, usually performed at night. Bwitists were subject to persecution by Catholic missionaries and today are totally opposed to the growing religious movement of Bwiti. Leon M’ba, before becoming the fi rst president of Gabon since 1960, protected religion Bwiti and use of iboga in French colonial courts. Council of Ministers of the Republic of Gabon announced Tabernanthe iboga that is a national treasure on June 6, 2000.

At lower doses Iboga has a stimulant effect and is used to maintain vigilance in hunting. The best-known entheogen using culture of Africa is Bwitists, using de-tergent based peel Iboga (Tabernanthe iboga) [7].

One of the most widely used entheogens is cannabis which was used in China, Europe and India, and in some cases for thousands of years. It also ap-peared as part of religions and cultures, such as movement Rastafari, of Sadhus of Hinduism, the Scythians, Sufi Islam, and others [3].

Cannabis has an ancient history of ritual use as an aid to cross and is tradition-ally used in religious context throughout the Old World. Herodotus wrote about the early ceremonial practices by the Scythians, who are considered to have occurred from 5-th to 2-nd century BC. Early Greek history and modern archaeology indicate that the peoples of Central Asia have been using cannabis 2500 years ago [3, 14].

Cannabis or ganja is associated with the worship of Hindu deity Shiva, who is popular as he considered that plant hemp. Bhang to Shiva offers images, espe-cially the festival Shivratri. This practice is particularly witnessed in the temples of Benares, Baidynath and Tarakeswar [20, 21].

Early Christians used cannabis oil for medicinal purposes and as part of bap-tism to confi rm the forgiveness of sins and “right of passage” into the Kingdom of Heaven [1]. According to the Living Torah, cannabis was an ingredient of holy anointing oil mentioned in various sacred Hebrew texts [7, 12]. The herb of interest is best known as kaneh-bosm (Hebrew). This is mentioned several times in the Old Testament as a bartering material, incense, and a component of the holy anointing oil used by the high priest of the temple [12].

In ancient Germanic culture cannabis is linked to the German love goddess Freya. The collection of works is associated with an erotic high festival. It was be-lieved that Freya lived as a fertile force in the female fl owers of the plant and the ingestion made one affected by this divine force.

Ethanol is a powerful psychoactive substance and one of the oldest recre-ational drugs. Alcohol is oldest and still most common substance that causes re-laxation, excitement or anger. Archaeological evidence suggests that the vine was domesticated by man with corn around 6000 BC and that all civilizations of the ancient world – Egypt, Greece, Rome, knew alcohol – mainly beer and wine [13].

When the Indo-Europeans reached the world of the Caucasus and the Ae-gean, they encountered wine, the entheogen of Dionysus, who brought it with him from his birthplace in the mythical Nysa, when he returned to claim his Olympian birthright.

Dionysus is the god of wine and sexual orgies. He was accompanied by maenads and satires - people with goat feet. Their leader is Pan, the god of fl ocks.

The Nordic nations also had their own alcoholic beverages. Germans and Slavs drank mead – “medovina”, steppe peoples - fermented milk – “koumiss” and Chinese people drank brewed from fermented rice.

Islam strictly forbids alcohol, but despite the bad treatment of it, namely the Arab alchemists were the fi rst to receive it in pure form. They believed that distilla-tion reached the soul of intoxicating drinks.

Alcohol has gained popularity among the masses only in the 16th century, when it became cheaper and pubs everywhere started springing up.

The indigenous peoples of Siberia (from which the term shaman has been attached) used fl y mushroom Bride (Bride muscaria) as an entheogen. Ancient inebriant Soma, mentioned frequently in the Vedas, seems to be consistent with the effects of an entheogen. The active ingredient in Soma is supposed by some to be ephedrine, an alkaloid with stimulant and (less controversial) entheogenic proper-ties arising from the Soma plant, identifi ed as pachyclada Ephedra [2]. However, there are arguments to show that Soma may have been Syrian Rue, cannabis, Belladonna, or any combination of the above plants.

The muscimol containing in Amanita mushroom has been used for ritual pur-poses in prehistoric Europe. Sponge itself ingested in minimum quantities, in most cases is not fatal, but serious poisoning is common when mistaken for the bride,



if these quantities are large. Some people had consumed it in certain quantities to get hallucination – in the role of a substance causing visions it is especially known for the rite of the Vikings and Siberian shamans. There are assumptions that it was sacred mushroom in early Christianity. Similarly fl ying sponge is dedicated to Odin, the god of ecstasy and henbane stood under the dominion of the thunder god – Thor in Germanic mythology – and Jupiter among the Romans [28].

Mushrooms have a particular role in the mythology of almost all nations thought that they are children of thunder or being reborn phallus of the dead men, in all cases the magical powers of mushrooms – good or bad – are not questioned. If the religions of ancient mushrooms were overshadowed by other plants in Sibe-ria, their use until recently was preserved in its original form. All European explorers turned their attention to the role of exclusive in shamanic rituals fl yagaric. To fall into a trance, shamans drank a drink made from fl y agaric; furthermore, fl y agaric used ordinary people, apparently to experience unusual feeling. Before alcohol was brought by the Europeans, the sponge was popular entertainment among cer-tain peoples in Northeast Asia.

Many authors believe that the popular in medieval Scandinavia berserk owes its fame to fl yagaric. Berserk means man-bear, so they called the people who suf-fered particular state with a drink made from fl y agaric. The drink is usually used before battle and by those soldiers who get into a rampage and fury, causing the enemy holy terror. After the battle they took several days for recovery [25, 30].

Hallucinogenic Psilocybe was known to the aboriginal Mexicans as teo-nanácatl (“divine mushroom”) and became served at the coronation of Montezuma II in 1502. After the Spanish conquest of the Americas, the use of hallucinogenic plants and fungi, and other pre-Christian traditions, were forcibly suppressed and driven underground [5, 8].

The seeds of several tropical vines have long been used by Indians in Mexi-co for ritual purposes. All of these vines belong to the family Convolvulaceae.

The active substance is contained in the seeds, they are ground, soaked in water, water fi lter and drink, but the godless that do not hold the ritual, simply chewed.

The group of natural products belong deliriants used since the dawn of hu-man civilization as a healing and “magic” tools (belladonna, mandragora, datura, reverie). Later many chemical compounds were synthesized in their likeness, some of which are among the most active known psychoactive substances [4, 29].

Drugs containing deliriants of this kind have a long history of use. Aztecs used them for rituals in the Temple of the Sun. Shamans of the Peruvian Indians practiced sacred datura for dedication to youth in the mysteries of the spirit world. Prophet of the temple of Apollo at Delphi used small doses datura, before making their predictions. Homer mentions datura in their epic Odyssey. The priests of the ancient Thracians on our lands used deliriants in his ceremonies. Seeds and leaves of Datura were added to ganja (cannabis) in India to obtain additional mental health

effects of smoking. In India, trance then drugged with delirianti was part of cult ritual human sacrifi ces in honor of the goddess Kali.

Deliriants are associated with Western history, especially in the period 15th - 17th century. This is the era of witches and their fanatical persecutors of Holy Church. Witchcraft can be seen as a continuation of ancient pagan traditions, res-urrected in the form of a peculiar social and religious rebellion. Which is drugged by deliriants of “magic” herbs (belladonna, mandragora, datura) specially prepared in various forms – ointments, powders, potions? It is known that the active alkaloids – atropine, hioscyamin, scopolamine, can penetrate the skin and affect the body.

From ancient times Mandragora is considered an aphrodisiac. This property was noted in the Old Testament, which describes how after its impact Rachel and Leah became pregnant and had children.

Fame and a means exciting amorous desires, has been sung by many ancient and medieval authors. Mandragora is often mentioned by Shakespeare, the play is another example of Mandragora Niccolo Machiavelli.

Such fame had another plants of the same family that grow in Central and Northern Europe. Among them, most familiar are Scopolia carniolica, Arch of the door (Atropa belladonna), Datura Stramonium and Day dream (Hyosciamus niger). The name of the plant – “Day dream”, speaks well for its psychoactive properties.

The name of the Bella donna (beautiful woman) came from glory and vehicles for embellishment as due to its impact pupils wide by open and eyes look very shiny and black. Its other name by which it is known in this country is crazy ticket – hardly need to explain why. Linking all these plants with the release of sexual forces make them a natural component of ointments, used by witches. Many medieval women who experienced the impact of the plants remained with the fi rm conviction that they actually fl ew and communicated with spirits. There was a widespread belief that the ointments of the witches could turn people into an animal, usually a wolf, this conversion was called by medieval scholars likantropiya.

In many places in Europe, including Bulgaria, North America and Asia, there are drugs with properties of deliriant – Datura Stramonium. Intoxication cause contained in the plant alkaloids – atropine, 1-hioscyamin, scopolamine. There are known about 15-20 species Datura with suffi cient content of alkaloids to induce strong psychoactive effects in the world.

In the books by Carlos Castaneda, there is a detailed description of the use of such ointments obtained from Indian datura (Datura inoxia). Don Juan, Castane-da’s teacher, not an isolated case – wizards and priests of many nations often use them to get into a condition suitable for communication with the spirit world.

The priests of the ancient Thracians also have used similar means to reach divine madness.

Drink from datura was used in India by followers of Kali, who fall into trance and expressed tribute to the goddess in the usual way for their sect, killing many people. Although rare, some of these plants are used to induce a kind of pleasure;



normally used is datura as the most widespread. The report of the Indian hemp Commission says that cannabis is sometimes mixed after with leaves of datura and an Indian smoking mixture that person falls into a special kind of delirium, in which “throws his clothes and saw horrible things, like snakes, scorpions and so etc.” Datura leaves smoked some Arab tribes [4].

Cultivation of Opium poppies for food, anesthesia, and ritual purposes dates back to at least the Neolithic (New Stone) Age. In Sumerian, Assyrian, Egyptian, Indian, Minoan, Greek, Latin, Persian and Arab Empires each made widespread use of opium, which is the most powerful form of pain relief then available, allowing ancient surgeons to perform prolonged surgical procedures.

Opium is mentioned in the most important medical texts of the ancient world, including the Ebers Papyrus and the writings of Dioskorid, Galen and Avicenna.

Widespread medical use of unprocessed opium continued through the Ameri-can Civil War before giving way to morphine and its successors, which can be injected at a precisely controlled dosage [19, 27].

CONCLUSION

Entheogens have been used in ritualized context for thousands of years, their religious signifi cance is well established in anthropological and contemporary evi-dence. Entheogens have been used in various ways, including as part of the es-tablished religions, secularly for personal spiritual development as a tool (or “plant teachers”) to enhance the mind, secularly as recreational drugs, and for medical and therapeutic purposes. The use of entheogens in human culture and tradition is nearly ubiquitous throughout recorded history.

Although entheogens are taboo and most of them are offi cially banned in Christian and Islamic societies, their ubiquity and importance in terms of different spiritual traditions of other cultures is unquestioned.

REFERENCESB a r n e t t , R. E. The Presumption of Liberty and the Public Interest: Medical Marijuana and Fun-1 . damental Rights. 2007.B o t a n y of Haoma. – In: Encyclopedia Iranica. N. Y., Columbia University, 2007.2 . H o f f m a n , M., C. Ruck et B. Staples. Conjuring eden: art and the entheogenic. vision of paradise. 3 . – Entheos, 2001, № 1, 13-50.C e l d r á n , J. et C. Ruck daturas for the virgin. – Entheos, 4 . 1, 2002, № 2, 49-74. D í a z , J. L. Ethnopharmacology of sacred psychoactive plants used by the Indians of Mexico. – 5 . Ann. Rev. Pharmacol. Toxicol., 17, 1977, 647-675.E l -S e e d i , H. R. et al. Prehistoric peyote use: alkaloid analysis and radiocarbon dating of ar-6 . chaeological specimens of Lophophora from Texas. – J. Ethnopharmacol., 101, 2005, № 1-3, 238-242.F e r n a n d e z , J. W. An Ethnography of the Religious Imagination in Africa, Princeton, Princeton 7 . University Press, 1982, 731 p.

G r i f f i t h s , R. R. et al. Psilocybin can occasion mystical-type experiences having substantial and 8 . sustained personal meaning and spiritual signifi cance. – J. Psychopharmacol., 187, 2006, 268.H a l l , A. Entheogens and the origins of religion. – Psychoactive drug, 2007, Nerdshit.com9 . H o f m a n n , A., Use of LSD in Psychiatry. – In: LSD, My Problem Child. Chapter 4. New York, 10. McGraw-Hill Book Company, 1980.R o a c h , J. Delphic oracle’s lips may have been loosened by gas vapors. 11 . – Nat. Geographic News, 2001-08-14. K a p l a n , A. The Living Torah. New York, 1981, 442.1 2 . M e d l i n e Plus. Medical Encyclopedia: Alcohol Use. 1 3 . M e r l i n , M. D. Archaeological Evidence for the tradition of psychoactive plant use in the Old 1 4 . world. – Economic Botany, 57, 2003, № 3, 295–323.“ M i x i n g the Kykeon”, ELEUSIS. – J. Psychoactive Plants Comp., New Series 1 5 . 4, 2000.N e u r o s c i e n c e of PAS use and dependence. Geneva, WHO, 2007.1 6 . P a h n k e , W. N. Drugs and mysticism. – Int. J. Parapsychol., 1 7 . 8, 1966, № 2, 295-313.P r e h i s t o r i c peyote use: alkaloid analysis and radiocarbon dating of archaeological specimens 1 8 . of Lophophora from Texas. – J. Ethnopharmacol., 101, 2005, № 1-3, 238–242.R a m D a s s : Longtime Spiritual Leader, Opponent of the “War on Drugs”. 2004-03-08. www.1 9 . drogpolicy.orgS o c i a l and Religious Customs. – In: Report of the Indian Hemp Drugs Commission. Chapter IX. 2 0 . Simla, India, Government Central Printing House, 1894. R i c h t e r -U s h a n a s , E. The Indus script and the 2 1 . Ṛig-Veda. Bremen, 2001.R o b e r t s , T. B. Chemical Input, Religious Output – Entheogens. (Chapter 10) – In: Where God 2 2 . and Science Meet: Vol. 3: The Psychology of Religious Experience. Westport, CT, Praeger/Green-wood, 2006.R o b e r t s , T. B. (ed.). Psychoactive Sacramentals: Essays on Entheogens and Religion. San 2 3 . Francisco, Council on Spiritual Practices, 2001.S a m o r i n i , G. Traditional use of psychoactive mushrooms in Ivory Coast. – Eleusis, 1995, 2 4 . № 1, 22-27.S a m o r i n i , G. The mushroom-trees in christian art. – Eleusis, 1998, № 1, 87-108.2 5 . S t a f f o r d , P. Psychedelics. Oakland, California, Ronin Publishing, 2003.2 6 . U N World Drug Report 2007 – Afghanistan.2 7 . Ve t u l a n i , J. Psychoactive substances in the past and presence. – Pol.J. Pharmacol., 2 8 . 53, 2001, № 3, 201-214.V i z i , S. Drugs of Abuse – The Myth of Creativity and the Reality of Destruction. Cambridge, Cam-2 9 . bridge University Press, 2007, 241-256.Wa s s o n , R. G. The Wondrous Mushroom: Mycolatry in Mesoamerica. (1980). New York, 3 0 . McGraw-Hill Book Co., 2007.W i n k e l m a n , M. J. et B. R. Thomas (editors). Psychedelic Medicine: New Evidence for Halluci-3 1 . nogens as Treatments. Westport, CT, Praeger/Greenwood, 2007.

Address for correspondence: Julia Radenkova – Saeva, MD, PhD, Toxicology Clinic, Emergency Hospital “N. I. Pirogov” Sofi a е-mail: [email protected]



ANGIONEUROTIC EDEMA CAUSED BY DENTAL MATERIALS: A CASE REPORT

S. Dermendjiev1 and Z. Stoyneva2

1Occupational Diseases and Toxicology Department, Medical University of Plovdiv, Bulgaria2Clinic of Occupational Diseases, University Hospital Sv. Ivan Rilsky, Sofi a, Bulgaria

Summary. The synthetic polymers are high-weight molecular organic sub-stances which are formed from one compound (monomer) or by several compounds through chemical processes of polymerisation or polycondensation. Acrylamide, for example, is used in dentistry to harden compositions with high adhesion. Methyl methacrylate (MMK) is the main monomer in obtaining acrylic. It is widely used in households and in different industries – production of organic glasses, lacquers, resins, displays. MMK is often used as a substance in the preparation of various composites, used in dentistry, dental fi llings, etc. MMK can be a health risk factor, both in terms of acute, often occupational exposure and prolonged effects on the human body. While acute exposure to high concentrations MMK produces clinical manifestations of intoxication, which clinical picture and course are well known, the adverse health effects arising from prolonged contact with this substance are very diverse, involve different organs and systems, and some are still poorly studied. It is known for example that MMK, like other polymeric substances and plastics exhib-its sensitizing effect in contact with skin and mucous membranes. After exposure to methyl methacrylate, cases of contact dermatitis are described. Is it possible, however, MMK, and substances in which it participates, to create another type of allergic reactions? Responses to this question can provide the case we present.

Key words: Quinke’s edema, etiology, methyl methacrylate

INTRODUCTION

Synthetic polymers are high-weight molecular organic substances formed by one compound (monomer) or by several monomers through chemical processes polymerisation or polycondensation. Acrylamide,

for example, is used in dentistry to harden compositions with high adhesion. Methyl methacrylate (MMK) is the main monomer in obtaining acrylic. It is widely used in

households and in different industries – production of organic glasses, lacquers, resins, displays. MMK is often used as a substance in the preparation of various composites, used in dentistry, dental fi llings, etc. MMK can be a health risk factor, both in terms of acute, often occupational exposure and chronic effects on the hu-man body [1-5]. While acute exposure to high concentrations of MMK produces clinical manifestations of intoxication with well known manifestations and course, the adverse health effects arising from prolonged contact with this substance are very diverse, involve different organs and systems, and some are still poorly stud-ied [6-8]. It is known, for example, that MMK, like other polymeric substances and plastics, exhibits sensitizing effect in contact with skin and mucous membranes. After exposure to methyl methacrylate, cases of contact dermatitis are described [9]. Is it possible, however, MMK, and substances in which it participates, to create another type of allergic reactions? Responses to this question can provide the case we present.

AIM

To study the possible adverse allergic effects, caused by contact with compos-ite materials (methyl methacrylate, evicrol, etc.), used for dental fi llings and other dental procedures.


We present a case report of a 52-years-old man, admitted to the Department of occupational diseases and toxicology of University hospital “Sv. Georgi” (Plovdiv, Bulgaria) from 25.01.2010 till 29.01.2010. The diagnosis at discharge was agreed as angioneurotic edema.

GENERAL AND SPECIFIC HISTORY OF THE PATIENT

The man was admitted for the fi rst time to the department, in order to diagnose health problems with allergic/toxic-allergic etiology. According to anamnesis and documentary data from 2002, the patient was with recurrent edema and rashes of the body and face, which etiology remained unclear, so far. The fi rst acute allergic incident manifested during his hospitalization in the Cardiology Clinic of Plovdiv Uni-versity Hospital, where he got urticarial itchy rash during a course with fi brinolysis. During 2004 – 2006 he was hospitalized 4 times in the Clinic of Dermatology and Venereology (Alexandrovska University Hospital, Sofi a) because of urticaria and Quinke’s edema. Different trigger factors were suspected then, such as food, nuts, sweets, drugs, etc. After repeated treatment courses with systemic corticosteroids and H1-blockers, the symptoms gradually resolved. He received therapeutic cours-es for long periods of time, including antihistamines (Lorano, Fenistil, Clemastin, Allergosan) and H2-blockers. Since 2 days he took again H1-blocker (Cetirizin) in a

Angioneurotic edema caused...132


dose of 1 pill (10 mg) because of a new relapse of angioedema with predilection to the left facial half and around the oral angle. History and medical records showed variable localization of the edema, including genitals, extremities, but mainly af-fecting the face, sometimes migratory, often involving the eyelids, and accidentally half of the tongue. Occasional voice changes were also reported. Commonly, the angioedema was accompanied by itchy urticarial rash. The patient presented a set of photos of himself with massive facial angioedema.

Dental status, assessed by the patient’s dentist: Reports established the presence of a gold crown, amalgam fi llings, containing silver, copper, zinc and mer-cury in a certain ratio, as well as 2 white composite fi llings, most likely from evicrol, containing methyl methacrylate. In the medical documentation, skin allergic tests with different drugs were carried out, with borderline values for Concor, Aspirin, Tenox, Crestor, Piracetam.

Family history: since 2001 his daughter had experienced several episodes of urticarial rash. His uncle was known as “allergic”, too.

Treatment: At the time for an associated heart disease, the patient was treat-ed with a daily combination of the following medicines: Trombex (clopidogrel) – a pill (75 mg) in the morning; Aspirin protect – a pill (100 mg) in the evening; Betaloc ZOK – a pill (100 mg) in the evening; Tenox (amlodipine) – 2 x 1 pill (5 mg); Torvac-ard (atorvastatin) – 1 pill (20 mg) in the evening.

It is well known that most of the above drugs have got side effects similar to the patient’s symptoms, especially some of them as Aspirin, Clopidogrel, Torvac-ard, Beta-blockers.

Patient’s total occupational service was 41 years, as a military person. At the time of hospitalization the patient was retired. He presented a copy of expert deci-sion by the territory expert medical committee, certifying the following diseases:

Coronary artery disease. Incurred acute myocardial infarction of the lower, rear, lateral, basal part of the right chamber of the heart in chronifi ed stage; Trifur-cating coronary artery disease. Condition after stent placement and PTCA* of LAD** because of a high class angina. Congestive Heart failure NYHA-FC*** II. Arterial hypertension stage 3, cerebral-cardiac form. Dyslipidemia. Polyallergy. Diabetes mellitus type 2, mild without complications. Cerebrovascular disease. Condition after ischemic stroke of vertebrobasilar system without neurological defi cit.

Clinical examination established: a satisfactory general condition; clear consciousness, adequate, afebrile; mental status, speech, mood, motor and sen-sory system – normal; pink skin with brownish pigmented patches on the back; well expressed hairs of the beard and chest; moderate swelling of the left facial half in the area of the oral angle (see Fig.1a, b); exanthema was not found; peripheral lymph nodes were not enlarged.

* PTCA – percutaneous transluminal coronary angioplasty ** LAD – Left Anterior Descending coronary artery *** NYHA-FC – New York Heart Association functional class


Fig. 1a, b. Quinke’s edema

Respiratory system: symmetrical breast halves; hypersonic percussion tone; bilaterally weakened vesicular breathing over the lung bases, bronchial spasm and wheezing.

Cardiac system: rhythmic heart rate; pulse 72 b/min; deaf heart tones; blood pressure 140/95 mm Hg.

Abdomen – soft and painless on palpation. Liver – at the rib arc. Spleen is not discovered on palpation. Succusio renalis negative bilaterally.

Bone and muscle system – properly developed.Laboratory tests were carried out in accordance with the diagnostic algo-

rithm in clinical pathway 291 of the National health insurance fund: Hematological tests: 1. ESR – 4 mm, HB – 155 g/l, Er – 5.02 T/l, Leu – 8.33

G/l, Hct – 0.459, MCV – 91.4, PLT – 296 G/l, Differential leukocyte count: Sg – 74.7%, Mo – 5.4%, Ly – 17.5%, Eo – 1.9%, Ba – 0.5%.

Biochemical tests: 2. Chol – 3.7 mmol/l, Trigly – 0.80 mmol/l, Gluc – 7.0 mmol/l, T Prot 77 g/l, Creat – 89 mkmol/l, Urea – 4.1 mmol/l, T Bilirub – 13.4 mkmol/l, ASAT – 30, ALAT – 29, CK – 163, LDH – 399 U/l.

Immunoassays: 3. Level of general IgE – 101.23 IU/ml (ref. up to 100) −IgG – 39.875 (increased, ref. up to 18.37 g/l) −IgA – 0.381 −IgM – 1.029 −C3 – 1.034 g/l (normal) −C4 – 0.536 g / l. (increased) −C1 – esterase i − nhibitor – 230 mg/l (normal)


ECG4. – sinus rhythm, data for incurred lower MI, ST – T criteria for left ven-tricular hypertrophy.

Gas analyses of mixed venous blood:5. pH – 7.420, PaO2 – 72.8 mmHg, PaSO2 – 39.9 mmHg, BE + 1.4 HbO2 – 95.6%.

Treatment regimen: injectable steroid (Methylprednisolone) 40 mg. i.m., Chloropyramine hydrochloride 1 ampule (2 ml) i.m. daily, H1-blocker (Deslorata-din), 2 x 1 pill (5 mg), H2 – blocker (Famotidine) – 2 x 1 pill (40 mg)

Outcome of hospitalization: The patient was discharged in a good general condition, asymptomatic.

CASE DISCUSSION

We present a typical case of angioneurotic edema, which has a prolonged course with frequent relapses. Having in mind the very broad differential diagnosis of angioedema, in this particular case, mainly two types of factors should be ex-cluded:

The role of used medications to treat concomitant cardiac problems, for 1. which angioedema has been described as relatively common side effect. The fact that the swelling often recur after withdrawal of the aforementioned drugs, practi-cally excludes their leading role as an etiological factor in the presented case.

Hereditary angioedema (HAE) is another possible diagnosis, to be exclud-2. ed. It is known that the “gold standard” in the diagnosis of HAE includes a combi-nation of familial predisposition, typical clinical picture with solid type of swellings which are more often painful than itchy and laboratory diagnostic tests, of which the greatest diagnostic value is determination of C4. In this case, the level of C4 is increased, while C1-esterase inhibitor is normal.

Possible functional C1-esterase inhibitor defi ciency cannot be ruled out, it 3. is plausible. In our case, qualitative study of this factor has not been investigated due to lack of such possibility of the immunological laboratory.

The probability of angioneurotic edema due to a combination of factors with 4. different mechanisms is very high. The history of intolerance to certain foods, drugs, some of which are known to operate in nonimmmune mechanisms, as well as the lo-cal irritative and sensitizing effects of the mentioned synthetic polymeric substances, applied in this patient during dental procedures should be considered.

CONCUSIONS

The spectrum of allergic symptoms after contact with composite substanc-1. es containing evicrol, methyl methacrylate, etc. can be very diverse, as described by other authors [4, 6, 9], too.

Notwithstanding the broad differential diagnosis of angioneurotic edema, 2. suspected as a factor that triggers allergic symptoms, the sensitizing effect of syn-


thetic polymers upon the buccal mucosa should be considered as well. This state-ment is confi rmed also by many research studies, done recently [5, 6, 9].

Although the fact that angioneurotic edema is a systemic allergic reaction 3. which unfolds largely as of fi rst type (IgE-mediated hypersensitivity, fast type), there should be taken into account other nonimmune factors and mechanisms, that may cause similar symptoms.

The good knowledge of the factors and mechanisms of allergic reactions 4. is a guarantee for the effective treatment to the existing problems in the presented patient.

REFERENCESD e v a l , R. et al. Natural rubber latex allergy. – Indian J. Dermatol. Venereol. Leprol.,1 . 74, 2008, № 4, 304-310.A n t ó n Gironés, M. et al. Immediate allergic reactions by polyethylene glycol 4000: two cases. – 2 . Allergol. Immunopathol. (Madr), 36, 2008, № 2, 110-112.G o o p t u , B. et D. A. Lomas. Conformational pathology of the serpins: themes, variations, and 3 . therapeutic strategies. – Annu. Rev. Biochem., 78, 2009,147-176.A s s a l , C. et P. Y. Watson. Angioedema as a hypersensitivity reaction to polyethylene glycol oral 4 . electrolyte solution. – Gastrointest. Endosc., 64, 2006, № 2, 294-295.Ta y l o r , J. S. et E. Erkek. Latex allergy: diagnosis and management. – Dermatol. Ther., 5 . 17, 2004, № 4, 289-301.K i n d , F., K. Scherer et A. J. Bircher. Allergic contact stomatitis to cinnamon in chewing gum mis-6 . taken as facial angioedema. – Allergy, 65, 2010, № 2, 276-277.K i e f f e r , C., B. Cribier et D. Lipsker. Neutrophilic urticarial dermatosis: a variant of neutrophilic 7 . urticaria strongly associated with systemic disease. Report of 9 new cases and review of the lit-erature. – Medicine (Baltimore), 88, 2009, № 1, 23-31.I n c o r v a i a , C. et al. Allergy and the skin. – Clin. Exp. Immunol., 8 . 153, 2008, Suppl. 1, 27-29.B e a u d o u i n , E. et al. Allergies in orthodontics. – Eur. Ann. Allergy Clin. Immunol., 9 . 35, 2003, № 9, 344-351.

Address for correspondence: Svetlan Dermendjiev, MD Occupational diseases and toxicology department Medical University of Plovdiv 15a Vasil Aprilov blvd. 4000 Plovdiv, Bulgaria e-mail: [email protected]


A CASE REPORT OF A LARGE SUBSEROUS FIBROID AND INCOMPLETE ABORTION IN A JUVENILE PATIENTP. Madzharov¹, P. Panchev¹, E. Kovachev², S. Ivanov², R. Minkov¹, A. Tsonev², A. Abbud²

and I. Bakardzhiev³1Obstetrics and Gynecology Hospital – Varna, Bulgaria

2Department of Obstetrics and Gynecology, Medical University – Varna, Bulgaria3Medical College – Varna, Bulgaria

Summary. We report a case of an 18-year-old patient with a large abdominal tumor, originating from the uterus and with positive β-hCG, without ultrasound in-dications for vital intrauterine or ectopic pregnancy. After the endometrial abrasion, which showed signs of incomplete abortion, we performed a laparotomy, where we found a large subserosal fi broid. Myomectomia was performed and the fi nal histopathological report revealed a benign fi broleiomyoma with focal coagulation and hemorrhagic necroses.

Key words: fi broid, incomplete abortion, uterus

INTRODUCTION

Uterine fi bromyomas are the most common tumors of the female genital tract and are classifi ed as submucosal, intramural, or subserosal. Myomas are usually associated with abnormal vaginal bleeding, lower abdominal pain,

threatening abortion, recurrent miscarriage, intrauterine growth retardation, abruptio placentae, preterm labor, infertility [1, 5]. Fibroids are typically found in the middle and late reproductive age and extremely rare before 20 years of age [4]. Some fi broids may interfere with pregnancy and possible complications may occur [3]. Symptoms caused by uterine fi broids are the most common indication for hysterectomy, especially in women who have accomplished their reproductive functions.

CASE PRESENTATION

An 18-year-old nulliparous woman was hospitalized in the gynecological department with lower abdominal pain, scarce vaginal bleeding, amenorrhea continuing

A case report of a large subserous...138

for 2 months and abdominal tumor. Pelvic examination revealed the presence of a large tumor mass, measuring over 20 cm in diameter. The ultrasound examination demonstrated a large fi broid like tumor with heterogeneous texture (fi g. 1) as well as uterus septus or uterus bicornis (fi g. 2). Laboratory tests showed low hemoglobin level of 94 g/L; hematocrit-27,6%; MCV-78,2 fL; MCH-26,6 pg; RDW-CV-12.9%; WBC-9.0 x 109/L. Ca 125-57,8 U/ml. The pregnancy test was positive. With these clinical fi ndings we performed an endometrial abrasion, with histological report: decidual reaction and amniotic parts. After the intensive reanimation, we performed an exploratory laparotomy with lower longitudinal incision. We found an enormous fi broid like tumor, 30cm in diameter originating from the uteral fundus on a wide basis and the adnexa were intact (fi g. 3 and fi g. 4). The fi broid was clamped, ligated under direct vision, resected and the uterine wall was restored with single stitches on 2 levels. When bleeding persisted, electrocoagulation was performed. The fi broid was sent for frozen section and the result was a benign leiomyoma. The postoperative period was uneventful and the patient was discharged from the hospital sixth days after the surgery.

The fi nal histopathological report revealed a benign fi broleiomyoma with focal coagulation and hemorrhagic necroses. Venous thrombosis and congestion with interstitial hemorrhage are responsible for the red (carneous) degeneration.

Fig. 1. Large subserosal uterine fi broid-

ultrasonographic imageFig. 2. Uterus bicornis – ultrasonographic image

Fig. 3. Large fi broid, originating from fundus Fig. 4. Large fi broid – 30 cm in diameter


DISCUSSION

We summarized our own and the foreign experience in diagnosis and treatment of large uterine fi broids in young women with preserving the reproductive function of the patient [6]. While a bimanual examination typically can identify the presence of larger fi broids, ultrasonography is the standard tool to evaluate the uterus for fi broids. Also magnetic resonance imaging (MRI) can be used to defi ne the depiction of the size and location of the fi broids within the uterus [2].

CONCLUSION

We report a case of a large fast growing uterine fi broid in a young 18-year-old patient, incomplete abortion, vaginal bleeding and anemia. Our patient had not visited routine gynecological and ultrasound examinations and the reason was the enormous tumor size (2000 g).

Imaging modalities cannot clearly distinguish between the benign uterine leiomyoma and the malignant leiomyosarcoma or even ovarian carcinoma.

Myomectomy for subserosal leiomyomas can be accomplished by open or laparoscopic approaches.

REFERENCESB o y n t o n -J a r r e t t , R. et al. A prospective study of hypertension and risk of uterine leiomyomata. 1 . – Am. J. Epidemiol., 161, 2005, № 7, 628.http://en.wikipedia.org/wiki/Digital_object_identifi erH o d g e , J. et C. Morton. Genetic heterogeneity among uterine leiomyomata: insights into 2 . malignant progression. – Human Mol. Gen., 16, 2007, № 1, R7-13.D e c h e r n e y, A. et L. Nathan. Current Obstetric&Gynecologic Diagnosis&Treatment. 9th ed. N. 3 . Y., Lange Medical Books/McGraw-Hill, 2003, 693-699.G o t o , A. et al. Usefulness of Gd-DTPA contrast-enhanced dynamic MRI and serum determination 4 . of LDH and its isozymes in the differential diagnosis of leiomyosarcoma from degenerated leiomyoma of the uterus. – Int. J. Gynecol. Cancer, 12, 2002, № 4, 354-361.N e i g e r , R. et al. Pregnancy-related changes in the size of uterine leiomyomas. – J. Reprod. 5 . Med., 51, 2006, № 9, 671-674.O k o l o , S. Incidence, aetiology and epidemiology of uterine fi broids. – Best Pract.Res. Clin. 6 . Obstet.Gynaecol., 22, 2008, № 4, 571-588.R e i n , M. S. Advances in uterine leiomyoma research: the progesterone hypothesis. – Environ. 7 . Health Perspectives, 108, 2000, Suppl. 5, 791-793.Wa l l a c h , E. E. et N. F. Vlahos. Uterine myomas: an overview of development, clinical features, 8 . and management. – Obstet. Gynecol., 104, 2004, 393-406.

Address for correspondence: Atanas Tsonev, MD Tzar Osvoboditel Str.150 9000 Varna, Bulgaria

00359895551955 e-mail:[email protected]

C O N T E N T S

H. Georgiev, B. Matev, N. Dimitrov and P. Georgiev. Osteoclastoma of metacarpal bones ................... 3A. Stancheva, L. Spassov and K. Tzatchev. Hemostatic monitoring of the perioperative

fi brinolytic activity during liver transplantation .................................................................................. 9J. Ananiev, M. Gulubova, I. Manolova, G. Tchernev, V. Ramdan, V. Velev and J. Gerenova.

Uncommon clinical presentation of fi brosarcoma of the thyroid gland in a patient with fatal outcome: the role of immunohistochemistry for confi rmation of the diagnosis ............... 19

M. Stancheva, A. Merdzhanova and L. Makedonski. Fatty acid composition of fi sh species from the bulgarian Black Sea ........................................................................................................ 26

V. Paskaleva-Peycheva, M. Panchovska-Mocheva and E. Kavrakov. Rheumatoid arthritis in the general practitioner's practice .............................................................................................. 34

N. Manolova, D. Zasheva and M. Stamenova. Immunobiology of endometriosis ................................ 38S. S. Mileva. Zinc – the breakthrough .................................................................................................. 51E. Boteva. Use of posts in dental practice ............................................................................................ 64E. Boteva. Use of posts by undergraduate dental students ................................................................. 70P. Pechalova. Dentigerous cysts: a clinical study of 146 cases ........................................................... 76S. Desancic, A. Stoimenova, A. Savova, M. Manova, V. Petkova and G. Petrova.

Pharmacoeconomics of rare diseases therapy – an example of phenylketonuria ......................... 84A. Stoimenova, G. Petrova, I. Nikolov, M. Manova, G. Draganov, Z. Ivanova,

D. Dimitrov, G. Mihova and A. Savova. Continuing professional education of Bulgarian pharmacists 2007-2009 ............................................................................................. 90

S. Tsanova-Savova. Biologically active composition and health impact of Allium cepa ....................... 99R. Nikolova and S. Danev. Cardiovascular functional diagnostic methods ........................................ 105A. Agovska and R. Kirkova. Mental work capacity and reliability among nuclear

power plant operators ..................................................................................................................112J. Radenkova, E. Saeva and V. Saev. Psychoactive substances in different cultures

and religious practices ................................................................................................................. 122S. Dermendjiev and Z. Stoyneva. Angioneurotic edema caused by dental materials:

a case report ................................................................................................................................ 131P. Madzharov, P. Panchev, E. Kovachev, S. Ivanov, R. Minkov, A. Tsonev,

A. Abbud and I. Bakardzhiev. A case report of a large subserous fi broid and incomplete abortion in a juvenile patient ........................................................................................................ 137

ACTA MEDICA BULGARICA 1/2011Editor in Chief: Prof. V. Mitev, MD, Ph. D., DScScientifi c editor: Prof. W. Bossnev, MD, Ph. D., DScEditor of the English text: B. Stancheva, MDArt editor and computer design: D. AlexandrovaOrganizing secretary: M. DimitrovaPublisher’s sheets: 8.3Printer’s sheets: 6.25Format: 70 x 100/16Issued by the Central Medical Library

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