ACTA MEDICA BULGARICA

Transcription

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