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Fiziologia- Physiology 2012.22.4(76)
Fiziologia‑ Physiology 2010supplement

CONTENTS

1. Testing the Discriminant Power of ECG Signal Sonification Procedures...................................................................................................................................................................4
Minodora Andor
2. Pregnancy and Birth Outcome after Maternal Exposure to Ceftriaxone...................................................................................................................................................................9
Simona Şipoş, Daliborca Cristina Vlad, Roxana Popescu, Mirabela Dima, Adina Bucur, Victor Dumitraşcu
3. Optimization of Protocols for Completely Decellularize Equine Cornea.................................................................................................................................................................13
Marius Toma, Radu Lazarovicz, Laura Marusciac, Radu Ivanescu, Florina Bojin, Carmen Panaitescu, Virgil Paunescu
4. Analysis of Synthetised Steroid Dimers Based on the Structure of Pentacyclic Triterpenes...................................................................................................................................19
Alina Heghes, Cristina Trandafirescu, Camelia Sass (Oprean), Simona Ardelean, Florin Borcan
5. Serum Soluble Interleukin-2 Receptor Levels in Chronic Hepatitis C before Interferon Therapy...........................................................................................................................24
Manole Cojocaru, Simona Alexandra Iacob, Eugenia Panaitescu, Diana Gabriela Iacob
6. Efficacy and Toxicological Character of Coriander and Chamomile Extracts..........................................................................................................................................................26
Simona Ardelean, Cristina Trandafirescu, Camelia Oprean (Sass), Alina Heghes, Florin Borcan
7. Oleanolic and Ursolic Acid in Human Skin Cancer – A Preliminary In Vitro Comparative Study..........................................................................................................................30
Camelia Sass, Florina Bojin, Alina Heges, Atena Galuscan, Virgil Paunescu
8. The Relationship between Diabetic Retinopathy and Coronary Artery Disease in Banat County.........................................................................................................................34
N. Niculescu, I. Zolog, M. Muntean, O. Boruga, D. Brie
9. The Pathologic Spectrum of Tonsil Malignant Tumors in Timisoara - Statistic Study.............................................................................................................................................38.
Ramona Maria Ghiran, Stan Cotulbea, Anamaria Vasilica Mag, Delia Horhat

CUPRINS
1. Testarea puterii de discrimiare a semanalelor ECG prin proceduri de sonorizare.....................................................................................................................................................4
Minodora Andor
2. Efectele expunerii materne la Ceftriaxona asupra sarcinii si nasterii .......................................................................................................................................................................9
Simona Şipoş, Daliborca Cristina Vlad, Roxana Popescu, Mirabela Dima, Adina Bucur, Victor Dumitraşcu
3. Optimizarea protocoalelor de decelularizare completa a corneei ecvine..............................................................................................................................................................13
Marius Toma, Radu Lazarovicz, Laura Marusciac, Radu Ivanescu, Florina Bojin, Carmen Panaitescu, Virgil Paunescu
4. Analiza dimerilor steroidici sintetizati pe baza structurii triterpenelor pentaciclice..............................................................................................................................................19
Alina Heghes, Cristina Trandafirescu, Camelia Sass (Oprean), Simona Ardelean, Florin Borcan
5. Nivelurile receptorului solubil al Interleukinei-2 in hepatita cronica C inainte de terapia cu Interferon...............................................................................................................24
Manole Cojocaru, Simona Alexandra Iacob, Eugenia Panaitescu, Diana Gabriela Iacob
6. Eficacitatea si caracterul toxicologic al unor extracte de coriandru si musetel......................................................................................................................................................26
Simona Ardelean, Cristina Trandafirescu, Camelia Oprean (Sass), Alina Heghes, Florin Borcan
7. Acidul oleanolic si ursolic in cancerele cutanate umane – studiu comparativ preliminar in vitro.......................................................................................................................30
Camelia Sass, Florina Bojin, Alina Heges, Atena Galuscan, Virgil Paunescu
8. Relatia dintre retinopatia diabetica si boala coronariana in Regiunea de Vest a Romaniei...................................................................................................................................34
N. Niculescu, I. Zolog, M. Muntean, O. Boruga, D. Brie
9. Spectrul patologic al tumorilor amigdaliene maligne in Timisoara – studiu statistic..........................................................................................................................................38
Ramona Maria Ghiran, Stan Cotulbea, Anamaria Vasilica Mag, Delia Horhat
2012.22.4 (76)  Fiziologia - Physiology

3

TESTING THE DISCRIMINANT POWER OF ECG SIGNALSONIFICATION PROCEDURESMINODORA ANDOR
Semiology Clinic I, University of Medicine and Pharmacy “Victor Babes” Timisoara

ABSTRACTThe purpose of the paper is to develop a protocol for testing the potential improvement in ECG investigations by adding sound tovisual display of ECG signal. As there are numerous procedures for sonification of biological signals, which can be tested on bothnormal and pathological signals, this protocol allows a systematic approach, associated with a quantitative estimation of any of thesonic representations: acoustic continuous or quasicontinuous display, as well as real time, magnified or compressed display. The
analysis of the potential use of various types of tempolenses is also discussed. An application of such a protocol will bring importantdata for further studies on potential use of sonifcation tools in the analysis of various biological signals.
Keywords: ECG, sonification, tempolens, discriminant power.

INTRODUCTION
Sonification is a method using acoustic non verbal convey
for data representation (1). Its potential use for cardiac signals
representation was revealed by the pioneering work of Ballora (2). However, sonification of biological signals is still in its
infancy, studied by just a few research teams. This is due not
only to the absence of appropriate equipment, but also to thelack of a proper semiology, to allow the potential user to retrieve,
recognize and classify a signal - the essential step to bring thismethod within the arsenal of cardiac investigations. This paper
is the first in a set of articles which intend to present a set upof a methodology for a classification of some cardiac signals,
sonified by various methods, starting with a short presentationof these sonification methods.

neighbor points, called “Level A”
ii - with a quasicontinuous representation, which preserve
the discrete form of the signal, producing a sound of a constantfrequency for a duration equal to sampling frequency. (In fact
we can set an arbitrary value for the duration this sound). Thisis called “Level Q”.

SONIFICATION METHODS
A short presentation of sonification methods, as describedby Mihalas et al. (3) is necessary for a better understanding of
the proposed methodology.

c) Musical Transform
An extension of level S, by introducing rhythm and harmony(and timber), would yield a representation which sounds likemusic - this is the “Level M”. This level will not be analyzed inthis paper.

1.1. Sonification levels
There are several ways to transform a parameter of a signal into a parameter of the corresponding sound. It should be
mentioned that most usual techniques record and/or store thesignal in a digital form; hence it has usually a discrete original
representation. One can distinguish three major levels for sonification, briefly presented bellow.
a) Acoustic Transform
This level assumes a direct correspondence between thefrequency of the sound and the amplitude of the signal (linear of
exponential). There are two ways to display the sound:i - with a continuous change of frequency between twob) Sonic Transform
Let us mention here that the frequencies produced by the
acoustic transform belong to a continuous spectrum. If we apply
the procedure of level Q, but associate only frequencies froma discrete spectrum (ex the musical notes), the representation
obtained is called “Level S”. For level S we can add algorithms
for obtaining various durations of the sounds.

1.2. Tempolenses
Various tools developed for enhancing the performances ofa visualization procedure can be transposed also for sonification.
Such a useful tool is the “lens” used for magnifying certain areasof an image. The equivalent transposition which can enhance
the temporal resolution of a sonic representation was called“temporal lens” or “tempolens” (4). The main parameter whichcharacterizes a tempolens is the “magnification” - m, defined as
the ratio between the duration of the display and the original duration of the segment. For m>1 we have a “dilation” of the signal,
while for m<1 the signal is “compressed” (a useful procedure forshortening the display duration of some signals).

Received 24th of October 2012. Accepted 13th of November 2012. Address for correspondence: Minodora Andor, MD, PhD, Semiology Clinic I, University of Medicine and Pharmacy “Victor Babes” Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara, phone/fax: +40256220479, e-mail: andorminodora@gmail.com

4

Fiziologia - Physiology  2012.22.4 (76)The tempolenses can be classified into:
tempolenses with constant magnification
tempolenses with variable magnification (5), which havedifferent magnifications for different segments of the signal.
The preliminary results show a good resolution, without inutile
prolongation of other segments.
1.3. Signals
For this phase of the study we worked with cardiac signals
for humans downloaded from PhysioBank (6), from both healthy
subjects and from patients presenting sleep apnea, congestive
heart failure and arrhythmia. Only one ECG lead was used; withsampling rate of 100 Hz. When it was originally recorded witha different sampling rate, it is resampled. This standardization
will bring a standard processing procedure for all signals. The
ECG signal is imported in MATLAB and filtered (20 Hz low pass).
The sonic representation was generated in MATLAB R2011b.
METHODOLOGICAL SETUP
In order to build a methodology to cover all aspects necessary for introducing a new procedure as an investigation tool, it
is worth to note that it comprises several phases:a) understanding the procedure- in our case it will be represented by collection of demo signals, sonified in various ways.
Our previous work (3) showed that using visualization in parallelwith sonification raises the quality of understanding.
b) testing the discriminant power of various sonification
procedures – the work in this phase is very difficult, taking
into account the very large number of tests and the degree ofsubjectivity of listeners in ranking the procedures; however, thisphase is essential before using the procedures for classification.
c) learning phase – similar to any other method, once established the procedure, step by step, a large collection of signals
will be displayed, both video and audio; the “supervised learning”
procedure will be applied, having for each signal all additional
information about diagnosis, age gender etc.
d) classification assessment – the last phase, when the users will be presented the now signals which have to be classified.
e) the classical analysis (sensitivity/specificity, predictive
values, accuracy and error rate etc.) will be performed, separately
for each class of signal and sonification procedure.
In this paper only the phase about testing of discriminant
power will be presented in detail, without describing the learning
phase and classification assessment. However, the demo phasewill be described, since it precedes the testing phase.
METHODOLOGY FOR UNDERSTANDING THE SONIFICATION PROCEDURE
This phase has two major purposes:a) to understand the sonification levels A and Q (levels S
and M have not been yet introduced in our studies).
b) to understand tempolenses
3.1. Understanding sonification levels
A couple of demo signals have been built (7) to have both2012.22.4 (76)  Fiziologia - Physiology

quick and slow variations.
The A representation is unique; however, for Q representation one can choose various temporal steps (multiplying thesampling period with an integer, q). The same signal can be
reproduced several times, starting with a very short steps (0.02 s
for q=2) when it sounds like A and increasing gradually up to 0.05
s (q=5) and 0.2 s (q=20), when each step is heard separately.
It is worth to note here that, due to the very short duration ofsome events (for instance the R wave of the QRS complex inECG), an increase of the step (q) might yield a “jump” over such
short events (no longer observing Shannon’s sampling theorem).
Hence, we have chosen the maximal value from each set of q
samples. For the preliminary trial we selected q=5.
For S representation, the distinction from level Q with thesame temporal step is less perceivable at quick variations, but
might be perceived at slow variations. We can mention here thatthe distinction will be clearer when the sonification is performedon more channels simultaneously.
3.2. Understanding tempolenses
A. Tempolenses with constant magnification
Let us first remind that for A level we have only one way ofmagnification, which can be easily understood and perceived if,
after a display with normal speed, we can dilate it 2 times (and/or
4 times), then compress it 2x (and/or 4x). Let us now observe thatfor Q and S levels, the tempolens can be applied in two ways:a) method N (no interpolation) each temporal step is magnified “n” times (here “magnified” can be dilated or compressed);
there is no interpolation(or skip) for frequenciesb) method I (interpolation) each time step is split into “n”
subintervals (n>1 for dilation and n < 1 for compression) andthe corresponding interpolated frequencies are computed (forcompression this is equivalent to skipping some values).
The procedure for A and Q levels will start with the original
sound, followed by “4xN” and “4xI” (dilated 4 times, no interpolation, then interpolation).
Note: for the rhythm analysis (RR signals), instead of dilation
the signal will be compressed, and the “Observation sheet” wasadjusted accordingly.
B. Tempolenses with variable magnification
As we showed in a previous paper (4) the interest paid tovarious regions of the sound is different. The tempolens withvariable magnification used by us, in both demo and real signals,
dilates the signal 2x for the first quarter of the duration, preserves
unchanged the next quarter and compresses 2: the rest half.
For understanding this lens the original signal will be firstdisplayed at A level without lens and then with lens, followed by Q
level, both N and I method. For demo signals the S level sounds
similar to Q and will not be displayed separately.
METHODOLOGY FOR TESTING THE DISCRIMINANT
POWER OF VARIOUS SONIFICATION PROCEDURES
The major interest of the study is to test the capacity ofusers to distinguish the difference between signals originating5

from patients with different diseases. Of course, these differences
might be better or less perceivable depending on the sonification procedure chosen the sonification procedure chosen for
representation.
a) Working groups
The study will be performed on three groups of persons: agroup of cardiologists, a group of medical students and a groupof musicians (students). Each person will have a User ID, anda file comprising users relevant data will be built (age, gender,
profession etc)b) Display procedures
Individual setup is prepared for the setups users with two
versions of display: head phones or loud speakers. The lengthof an audio sample will be 10 seconds.
The working protocol comprises more phases:
accommodation phase – the user will be presented successively the demo signals for each of the displaying scheme
to be used
several testing phases, each one dedicated to a pair of two
signals. At the beginning, each pair will have a normal signal
displayed first; however, it can be changed, when the differential
diagnosis comes to be tested.
The screen displayed to a user looks like in figures 1 and2. In Figure 1 the signals were displayed with normal rate, whilein fig.2, the same signals were displayed with a tempolens withmagnification 4x.

Fig.1. Visual display of two signals at normal rate (no tempolens). Left side
- reference signal (normal ECG), right side - tested signal (ECG in sleep
apnea). Upper side - level A, bottom - level Q

The quadrants are allocated standard conditions: the top twocorrespond to the A level and the two at the bottom correspond to
the Q level (for q=5); the value of q can be changed, if necessary.
On the vertical axes, the left side comprises the visual display ofthe signal 1 (the normal ECG in the first set of trials), while theright side displays signal 2.
When the program starts, the first quadrant (up left) is displayed and the corresponding sound is heard (standard duration
is 10 seconds); then, after a pause of 0.5 seconds, the second
quadrant is visualized and heard. The program stops and the user
6

cmyk 6can now estimate the score of his perception. With a click the
program starts the display of the third, then the fourth quadrant,
corresponding to the Q level; the programs stops again andrestarts with a new click.
Fig.2. Visual display of the same two signals with tempolens with magnification 4x. Starting point of magnification for ECG in sleep apnea was t=4

The next test phase displays the same two signals, but withdifferent processing parameters: magnification 4 times without interpolation, then magnification 4 times with interpolation and finally
applying a tempolens with variable magnification (2x : 1x : 1/2x).
The sounds can be heard by accessing the library ofsounds, at the address: <www.medinfo.umft.ro/dim/sonification/
test-andor>
The user can repeat any of the phases. Moreover, when
applying a tempolens, he can choose the starting point (byspecifying the starting time for either reference signal 1, or tested
signal 2, or both). This point will be preserved for all magnifications in that tour. A full round for comparing two signals takes
10-15 minutes, depending on the number of repetitions requiredby the user.
c) Perception scale
The users will be asked to rank the quality of perception of
the differences between the two signals to be compared. As thesignals will be displayed both visual and acoustic, the users will
rank also the differences perceived visually.
The following scale for ranking the differences is proposed(Table I):
Table I. Ranking scale for differences
Class

Score

Description

Not relevant

0

No differences

1

Small differences, difficult to describe2

Not large, but clearly distinguishable

3

Large differences

9

No resemblance/seem to be different
signalsX

Could not hear/see the signal, technicalproblems

Relevant
Other

Fiziologia - Physiology  2012.22.4 (76)A demo with examples for each score is presented before
starting the testing.
d) Observation file
The results of tests will be recorded on an “Observation file”
presented in Annex 1.
Such a file will be filled in for each pair of signals to becompared.AS it can be seen, the users can ask a repetitionof the signals for deciding upon the differences; the number ofdisplays is also recorded (#); a value #>1 represents repetitions.
CONCLUSIONSThe procedures described above allow a solid approach forestimating the real potential of using sonification as a complementary tool in ECG investigations. Introducing such a tool inclinical practice must pass first the full set of tests mentioned
above. This paper refer to the first protocol of studies to be performed before introducing more sophisticated transformations,corresponding to levels S, or even M, where the number ofmappings between the signal parameters and sonic parameters
can increase dramatically.
Let us mention here that such a study is also very usefulfor comparing sonification with visualization (8). There are highexpectations from an integrative representation, combining bothvisualization and sonification, not only for a higher refinement,
but also bringing in supplementary data about the overall physiological condition of a patient (9).
REFERENCES
1. Hermann T, Hunt A, Neuhoff JG eds. The Sonification Handbook.
Berlin: Logos-Verlag, 2011.
2. Ballora M, Pennycook B, Ivanov PCh, Glass L, Goldberger AL. Heart
Rate Sonification: a New Approach to Medical Diagnosis. Leonardo
2004; 37 (1): 41-46.
3. Mihalas GI, Paralescu S, Mirica N, Muntean D, Hancu M, Tudor A,
Andor M. Sonic Representation of Information: Application for Heart
Rate Analysis. Proceedings MIE 2012, Pisa, Italy, www.mie2012.it,
279_CD_SC_Oral_ID403
4. Mihalas GI, Andor M. Tempolenses - A New Tool in Sonic Representation of Medical Data. Application for ECG and Rhythm Analysis.
CD Proceedings of APAMI 2012 Conference, Beijing 23-25 Oct 2012.
5. Mihalas GI, Paralescu S, Andor M, Lighezan D, Mirica N, Muntean D,
Hancu M, Neagu A, Neagu M, Tudor A, Fira-Mladinescu O. Tempolenses
with Variable Magnification for Sonic Representation of Medical Data.
Accepted in: Stud Health Technol Inform. Proceedings of EFMI STC
2013, ref 007, 2012.
6. Goldberger AL, Amaral LAN, Glass L, Hausdorff JM, Ivanov PCh,
Mark RG, Mietus JE, Moody GB, Peng C-K, Stanley HE. PhysioBank,

2012.22.4 (76)  Fiziologia - Physiology

PhysioToolkit and PhysioNet: Components of a New Research Resource
for Complex Physiologic Signals. Circulation 2000; 101(23): e215-e220.
7. Mihalas GI, Paralescu S, Andor M, Muntean D, Mirica N, Lighezan
D, Tudor A, Fira-Mladinescu O. Medical Data Representation by Sonification. Applications for Cardiac Signals. In Stoicu-Tivadar L, Mihalas
GI, Lupse OS, Vida MM, Vernic C (eds) “Innovation and Cooperation”- CD Proceedings 32nd National Conference of Medical Informatics
ROMEDINF 2012, Timisoara: Ed Victor Babes, 204-211.
8. Pauletto S, Hunt A. A comparison of audio and visual analysis ofcomplex time series data sets. ICAD05, 2005: 175-181.
9. Katsis CD, Ganiatsas G, Fotiadis DI. An Integrated Telemedicine
Platform for the Assessment of Affective Physiological States. Diagnostic
Pathology, 2006, doi: 10.1186/1746-1596-1-16.

ANNEX 1. OBSERVATION FILE
I. ESTIMATION OF CLARITY OF AUDIO/VIDEO DISPLAY
User ID …............................................
Date ….................................................
Time ….................................................
Table AI. Accommodation

Accommodation phase
full
short

no

II. COMPARISON OF TWO SIGNALS
Signal 1 ID: ........................... Signal 2 ID: ....................................
Table AII. Discriminant power between 2 signals, by A and Q levels

Type processing
Normal 4X - N 4X - I Var
Method display
A Q A Q A Q A Q
SoundSVisualV
No of displays #Table AII is repeated in the Observation Sheet several times;
one such table is filled in for each pair of signals.
III. REMARKS
Table AIII. Was sonification helpful in perceiving the differences between these two signals?

Not at Not very much A little Clearly I don’t
know
all
yes

3.b. If yes, which was the most relevant procedure? (you canchoose more than one)
3.c. Did you like any of the sounds? Which one?
3.d. Other comments: .................

7

TESTAREA PUTERII DE DISCRIMIARE A SEMANALELOR ECG PRIN
PROCEDURI DE SONORIZAREREZUMAT
Scopul acestui studiu a fost dezvoltarea unui protocol de testare a imbunatatirii investigatiilor ECG prin adaugarea
sunetelor la imaginea grafica a semnalelor ECG. Deoarece exista numeroase proceduri pentru sonorizarea semnalelorbiologice, care pot fi testate atat in cazul semnalelor normale, cat si in cazul celor patologice, acest protocol permite
abordarea sistematica, asociata cu estimarea cantitativa a oricarui tip de reprezentare sonora: prezentarea acustica
continua sau cvasicontinua, precum si cea in timp real, amplificata sau comprimata. Este discutata de asemenea si
analiza potentialelor aplicatii ale tipurilor variate de tempolene. Aplicarea unui astfel de protocol va aduce date importantein studii viitoare legate de utilizarea instrumentelor de sonorizare in analiza diverselor semnale biologice.
Cuvinte cheie: ECG, sonorizare, tempolene, putere discriminanta

8

cmyk 8

Fiziologia - Physiology  2012.22.4 (76)

PREGNANCY AND BIRTH OUTCOME AFTER MATERNALEXPOSURE TO CEFTRIAXONESIMONA ŞIPOŞ1, DALIBORCA CRISTINA VLAD1,2, ROXANA POPESCU2,3, MIRABELA DIMA4,ADINA BUCUR5, VICTOR DUMITRAŞCU1,2
1
Department of Pharmacology, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
2
County Emergency Clinical Hospital Timisoara, Romania
3
Department of Cell and Molecular Biology, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
4
Department of Neonatology, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
5
Department of Public Health, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

ABSTRACT

Objective. To examine the risk of congenital abnormalities, preterm birth, low birth weight and impaired liver function after exposure to ceftriaxone in utero.
Patients and Methods. 55 pregnant patients (20 to 35 years), who redeemed a prescription for ceftriaxone during their pregnancy,
were randomly allocated in three study groups, accordingly to their pregnancy stages: first trimester (n = 23), second trimester (n =22), and respectively third trimester (n = 10). The control group consisted of 55 women who did not redeem any prescriptions until
the end of pregnancy. All adverse drug events were monitored.
Results. In the group of women exposed to ceftriaxone at any time during pregnancy, average birth weight was 3202 g, 3.90%
had a low birth weight, 4.94% had a preterm delivery, and 3.54% gave birth to a child with a minor congenital malformation. The
corresponding data in the control group were similar, respectively: 3328 g, 5.27%, 7.32%, and 4.13 %. 7 patients (12.72%) fromthe first and second study group required two series of intravenous ceftriaxone administrations for recurrent severe pyelonephritis.
Biliary pseudolithiasis has been reported in 8.1% of these cases and reversible cholelithiasis was revealed in 2 cases of newborns
from women who received ceftriaxone therapy in the third trimester of pregnancy
Conclusion. Ceftriaxone is a well tolerated third generation cephalosporine, which can be safely used during pregnancy. Biliary
pseudolithiasis and newborn cholestasis are possible adverse effects due to extensively drug use.
Key words: ceftriaxone, pregnancy, safety, biliary pseudolithiasis, reversible cholestasis.

INTRODUCTION
Cephalosporins are the most widely used class of antibiotics. Based on their spectrum of activity against gram-negative
bacteria, they are classified into four generations. Many of thefirst and generation cephalosporins have been studied extensively in pregnant patients and are considered safe in any stageof pregnancy.
We recently reviewed the current status of antibiotic
therapy for pre-term, pre-labor rupture of membranes (pPROM)
cesarean delivery, newly evolving strategies to enhance theeffectiveness in reducing post-natal infection, and to monitor
the adverse effects on the embryo and fetus. We conducted afull PubMed (January 1976-August 2011) search using the key
words “pregnancy”, “infections”, “antibiotics” and “transplacental
transmission.” The purpose of this analytic review of selectedstudies was to reveal the safety of antibiotic use in pregnancy.
A total of 355 articles were identified and supplemented by abibliographic search (1).
Second and third generation cephalosporins are prescribedtoday to pregnant women, although there are no appropriate
data on their safe use during pregnancy, and particularly during
embryogenesis.

Ceftriaxone is a second generation cephalosporin (Figure 1):

Fig. 1. Chemical structure of ceftriaxone sodium

Ceftriaxone is prescribed as an antibiotic of first choice inpyelonephritis in pregnancy, pneumonia, otitis, sinusitis andmeningitis. It has bactericidal activity against Streptococcus
pneumoniae, Klebsiella pneumoniae, Staphylococcus aureus,
Group A beta haemolytic Streptococcus, and Gram negative
bacteria (2,3).
It is a cephalosporin for parenteral administration. In adults,
the usual dose is 1 to 2 g of ceftriaxone, administered once aday (every 24 hours). In cases of serious infections the dosecan be raised-up to 4g, administered once a day intravenously.
Completely absorbed following parenteral administration, thepeak plasma concentrations (about 80 mg/l) occurs between 2

Received 20th of September 2012. Accepted 10th of October 2012. Address for correspondence: Simona Sipos, Department of Pharmacology, “Victor Babes” Universityof Medicine and Pharmacy Timisoara, Romania, Eftimie Murgu Square No. 2A, RO-300041, e-mail: simonasipos@yahoo.com

2012.22.4 (76)  Fiziologia - Physiology

9and 3 hours after dosing. The pharmacokinetics of ceftriaxone
is non-linear with respect to the dose. The decrease of binding
to plasma proteins explains the non-linearity and leads to anincrease in distribution and elimination of the drug.
Hepato-biliary complication due to use of ceftriaxone were
described in the last ten years considering children and adults,
but not pregnant women (5).
Previous researches have revealed that ceftriaxone has
no effect on reproduction or mutagenic activity; however, only
few studies pointed out that this third generation cephalosporincrosses the placenta and is excreted in human milk at low
concentrations (4).
The aim of the present study was to prospectively evaluate
the safety of ceftriaxone during pregnancy and to evaluate thepotential risks of this third generation cephalosporin on pregnancy outcome, fetus and newborn.
PARTICIPANTS, MATERIALS AND METHODS
Study design and eligibility criteria
Fifty-five eligible pregnant women participated in this randomized, prospective and comparative survey (study groups). The
patients, ranging in age from 20 to 35 years, who redeemed aprescription for ceftriaxone during their pregnancy, were classified in three subgroups, accordingly with their pregnancy stages:
first trimester (n = 23), second trimester (n = 22), and respectively
third trimester (n = 10).
The control group consisted of 55 women who did notredeem any prescriptions from 2 months before pregnancy until
the end of pregnancy (Figure 2).

Fig. 2. Study groups

Exclusion criteria: pregnant women with co-morbidities,
hypersensitivity to ceftriaxone, use of different antibiotics during pregnancy within the past three months. Patients with risk
factors for billiary stasis/sludge or history of reno-urinary lithiasis
were also excluded from the study. This study was performed
between February 2011- March 2012, in accordance with the
ethical standards of the responsible institutional committee onhuman experimentation and with the declaration of Helsinki, as
revised in 2000. All the participants gave informed consent andwere recruited from the patient list of a family healthcare provider
10

from Timisoara, Romania.
MATERIAL AND METHODS
Exposed women from study and control groups were paired
for age, with references being exposed to non-teratogenic antibiotics administered for the same indications. Full clinical details of thestudied patients were obtained from their electronic case notes.
The electronic ICMed system, used by family healthcare
providers, includes the type and amount of the prescribed drug
according to the Anatomical Therapeutical Classification Code(ATC), the defined daily dose, the date of issuing the prescription,and the personal register number of the patient (CPR number)which is assigned to all patients. This electronic registry contains
information on all births; the main variables include maternal age,birth order, gestational age, length and weight at birth, and CPR
number of both mother and child.
The patients received intravenous ceftriaxone therapy, fordifferent bacterial infections, for five to ten days. All adverse drug
events were monitored.
Data on prescriptions for ceftriaxone and outcome wereobtained from population-based registries from the family
healthcare unit were the pregnancies were followed-up. Usinga case–control design, we compared pregnancy outcomes
between women who had been prescribed ceftriaxone during
pregnancy and those who had not, adjusting for available potentially confounding factors.
Statistical analysis
Logistic regression analyses was used in order to estimate
the prevalence of congenital abnormalities, preterm birth(gestational age <37 weeks), and low birth weight (<2500 g)
associated with ceftriaxone exposure, adjusted for maternal age,birth order and smoking. All statistical analyses were performed
using the SPSS software package (version 6.0 for Windows,
SPSS Inc, Chicago, IL.).
RESULTS
We identified 55 women who redeemed a prescription for
ceftriaxone during pregnancy, and 23 of them redeemed theirprescription during the first trimester. The control group consistedof 55 women who did not redeem any prescriptions before
conception until delivery.
In the group of women exposed to ceftriaxone at any time
during pregnancy, average birth weight was 3202 g, 3.90% had
a low birth weight (restricted to full term deliveries > 37 weeks),
4.94% had a preterm delivery, and 3.54% gave birth to a child witha congenital malformation. The corresponding numbers in thecontrol group were 3328 g, 5.27%, 7.32%, and 4.13 %. No casesof perinatal death in the study and control group were revealed.
7 patients (12.72%) from the first and second study groups
required two series of intravenous ceftriaxone administrations
for recurrent severe pyelonephritis. Biliary pseudolithiasis has
been reported in 8.1% of these cases and reversible cholelithiasis
was revealed in 2 cases of newborns from women who received
ceftriaxone therapy in the third trimester of pregnancy (Table I).
Fiziologia - Physiology  2012.22.4 (76)Table I. Characteristics of participants and outcomes instudy and control group
Exposed toCeftriaxone
First
trimester(n=23)
26.5(20-35)

Exposed toCeftriaxone
Secondtrimester(n=22)
26.0(22-31)

Exposed toCeftriaxone
Third
trimester(n=10)
26.3(23-30)26.7(20-35)

No of Prescriptions
Smokers (%)

25
32.28

22
31.63

12
33.52

―
32.83

Birth Weight(mean)
No of Low Birth Weight (%)

3176 g3245 g3185 g3328 g

5.22

3.38

3.11

5.27

No of Preterm Deliveries (%)

6.85

4.22

3.76

7.32

No of malformations (%)

5.42

2.84

2.36

4.13

Ceftriaxone induced biliary
sludge/calculi cases)

―

2

3

―

No of Ceftriaxone
induced cholestasis in the
neonate(cases)

―

―

2

―

Characteristics

Mean ageControls(n=23)DISCUSSION
Although antibiotics are commonly prescribed to pregnant
women, details relating to the effects of many of these drugs
remains poorly understood. If an antibiotic must be prescribed,
it is important to be aware of the effects such drugs can have on
pregnancies, in order to prescribe the most suitable treatment
with the least risk to mother and fetus.
Ceftriaxone is a third generation cephalosporin, commonlyused in general practice for various infections in all age groups,
because of its antimicrobial activity against many gram-positiveand gram-negative organisms. There are controversies regarding “contraindication in pregnancy” induced by ceftriaxone (6).
Pharmacokinetic changes occurring during pregnancy, as
well as fetal pharmacokinetics and passage of the drugs to theunborn are correlated with reproductive and developmental pharmacology. The passage of cephalosporins is different in the threestages of pregnancy. Most studies of drug transfer across thematernal and embryonic/fetal barrier are concerned with the endof pregnancy; but little is known about the transport of substancesin the early phases of pregnancy, in which, morphologically andfunctionally, both the yolk sac and the placenta develop andchange in performance (7). Because of these considerations,
our study involved pregnant women of all three trimesters ofpregnancy, who received treatment with ceftriaxone.
In the third month of pregnancy, the fetal liver is alreadycapable of activating or inactivating chemical substances throughoxidation (8). It is very important that, in the fetal compartment,
the detoxification of drugs and their metabolites takes place at alow level, certainly in the first half of pregnancy. The excretion in
the amniotic fluid explains the accumulation of biological active
substances might take place in the fetal compartment.
Although fetal treatment is still an exception, it is of great interest that in the case of prevention of vertical infections, at the time offunctioning circulation and kidney excretion, antibiotics (penicillins,
2012.22.4 (76)  Fiziologia - Physiology

cephalosporins) concentrate in the fetal compartment (9).
Our prospective and comparative study did not reveal
statistical significant differences among study groups (Table I). In
accordance with other researches we concluded that ceftriaxone
is a safe antibiotic (10). Considering all these, some adverse
effects during ceftriaxone therapy are important to be revealed.
In previous studies we revealed the importance of drug-induced
lithiasis and the composition of ceftriaxone calculi (11). In thepresent study we revealed reversible precipitates in the gallbladders of 2 pregnant women who received ceftriaxone in the second
trimester and 3 in the third trimester; the duration of therapy wasten days, as reported in the literature (12).
Both ceftriaxone exposure cases and their references
had only minor malformations in the newborn, revealed within
the expected baseline risk for the general population. The fiveminor malformations in the ceftriaxone study group were as follows: one cases of undescended testis, one case of metatarsus
adductus, one haemangioma, one minor syndactyly, and one
case of left hallux valgus associated with left preauricular trags(Figures 3 and 4):

Fig. 3. Left halux valgus in a neonate after maternal second trimester exposure to ceftriaxone

The presence of preauricular trags requires an attentive
follow-up of the patient for hearing impairment and especiallyfor hidden abnormalities of the urinary tract.

Fig. 4. Left preauricular traggs in a neonate after maternal third trimester
exposure to ceftriaxone11

In three cases of pregnancies associated with severe
pyelonephritis requiring 3g ceftriaxone/day, administered prior
to delivery, potentially dangerous complications like reversiblecholestasis occured in neonates. All three neonates were bornat term and weighted over 3000 grams. They were icteric,with the liver palpable 3 cm below the right costal margin; nosplenomegaly was associated. Aspartate aminotransferase
ranged between 88 and 120 units, prothrombine time wasprolonged to 36-42 seconds, total bilirubin was 38-44 mg/
dl with direct component of 33-36 mg/dl. After 3 days theicterus subsided and the blood testes normalized. The reversible cholestatic syndrome in the newborn has been widely
discussed in the literature (13-15). Frequently, a reversible
higher level of liver enzymes may prove the previously actionof ceftriaxone (16,17).
The presented study has some limitations. One limitationis the small sample size and the broad confidence limits. The
precise mechanisms of ceftriaxone action during pregnancy will
be clarified by further studies.
CONCLUSION
Ceftriaxone is a well tolerated third generation cephalosporine, which can be safely used during pregnancy. Biliary
pseudolithiasis and newborn cholestasis are possible adverse
effects due to extensively drug use.

REFERENCES
1. Şipoş S, Dima M, Budişan C, Bucur A, Dumitraşcu V. Infections, antibiotics
and pregnancy. Timisoara Medical Journal, 2012; 61(3-4): 225-232.
2. Sanchez-Ramos L, McAlpine KJ, Adair CD, Kaunitz AM, Delke I, Briones
DK. Pyelonephritis in pregnancy: once-a-day ceftriaxone versus multiple doses
opf cefazolin. A randomized, double-blind trial. Am J Obstet Gynecol 1995;

172(1): 129-133.
3. Berkovitch M, Segal-Socher M, Greenberg R. First trimester exposure tocefuroxim. Br J Clin Pharmacol 2000; 50: 161-165.
4. Schaefer C, Peters P, Miller RK. Drugs during Pregnancy and Lactation.
Treatment Options and Risk Assessment. Second Ed., Amsterdam, Boston,Heidelberg, London, New York, Oxford, Paris, San Diego, San Francisco,
Singapore, Sydney, Tokyo, Academic Press, 2007.
5. Ravkin AM. Hepatocellular enzyme elevations in a patient receiving
ceftriaxone. Am J Health Syst Pharm 2005; 62: 2006-10.
6. Briggs GG, Freeman RK, Yaffe SJ. Classification of drugs for teratogenic risk:
an anachronistic way of counseling: a reply to Merlob and Stahl. Birth Defects
Res A 2003; 67: 207-208.
7. Carney EW, Scialli AR, Watson RE. Mechanisms regulating toxicant dispositionof the embryo during early pregnancy: an interspecies comparison. Birth Defects
Res C Embryo Today 2004; 72: 345-360.
8. Juchau MR. Bioactivation in chemical teratogenesis. Ann Rev Pharmacol
Toxicol 2009; 29: 165-187.
9. Knothe GA, Dette A. Antibiotics in pregnancy. Infection 2005; 13: 3-7.
10. Bickford CL, Spencer AP. Biliary sludge and hyperbilirubinemia associatedwith ceftriaxone in an adult: Case report and review of the literature.
Pharmacotherapy 2005; 25, 1389-95.
11. Dumitraşcu V, Matusz AA, Cinca R, Grecu DŞ. Drug-induced calculi.
Identification, structure and composition. Archives of the Balkan Medical Union,
Celsius Publishing House, 40(1), 31-36, 2005, ISSN 0041 6940.
12. Papadapoulou P, Efremidis S, Karyada S, Badouraki M, Karatza E,
Panntelias C. Ceftraxone associated gallbladder pseudolithiasis. Acta Pediatr
88, 1352-1355, 1999.
13. Mulhall A, De Louvois JK. Pharmacokinetics and safety of ceftriaxone inneonate. Eur J Pediatr 1985; 144: 2379-82.
14. Zinberg J, Chernaik R, Coman E, Rosenblatt R, Brandt LJ. Reversible
symptomatic biliary obstruction associated with pseudolithiasis. Am J
Gastroenterol 1991; 86: 1251-54.
15. Czeizel AE, Fladung B, Vargha P. Preterm birth reduction after ceftriaxone
treatment during pregnancy. European J Obstet Gyn Reprod Biol 2004; 116:
157-163.
16. Ravisha MS, Godambe SV. Ceftriaxone induced cholelithiasis in a neonate.
A case report. Ind J Med Sci. 2004; 58: 73-74.
17. Avci YA, Koktener N, Uras F, Catal A, Karadag O, Tetkin H. Nephrolithiasis
associated with ceftriaxone therapz. A prospective study in 51 children. Arch Dis
Child 2004; 89: 1069-1072.

EFECTELE EXPUNERII MATERNE LA CEFTRIAXONA ASUPRA SARCINIISI NASTERIIREZUMAT

Obiectiv. Examinarea riscului de aparitie a anomaliilor congenitale, nasterilor premature, greutate mica la nastere si afectarea functiei
hepatice dupa expunerea in utero la ceftriaxona.
Pacienti si metode. 55 paciente gravide (cu varsta intre 20 si 35 ani), care au primit prescriptie medicala pentru ceftriaxona in timpul
sarcinii, au fost distribuite in trei grupuri de studiu, in functie de stadiul sarcinii: primul trimestru (n = 23), al doilea trimestru (n = 22),
respectiv trimestrul trei (n = 10). Grupul de control a fost format din 55 de femei care nu au avut prescriptie medicala pentru nici un
fel de medicament pana la sfarsitul sarcinii. Au fost monitorizate toate efectele adverse.
Rezultate. In grupul femeilor expuse la ceftriaxona pe parcursul sarcinii, greutatea medie la nastere a fost 3202 g, 3,90% au avut
greutate mica la nastere, 4,94% au avut nasteri premature si 3,54% au dat nastere unui copil cu malformatii congenitale minore. In
grupul de control, datele au fost similare, respectiv: 3328 g, 5,27%, 7,32% si 4,13 %. 7 paciente (12,72%) din primul si al doilea grup
de studiu au necesitat administrarea intravenoasa a ceftriaxonei pentru tratamentul pielonefritei severe recurente. In 8,1% dintre
cazuri a fost identificata pseudolitiaza biliara iar in 2 cazuri de nou-nascuti a fost relevata colelitiaza, in cazul femeilor carora li s-a
administrat ceftriaxona in timpul trimestrului trei de sarcina.
Concluzie. Ceftriaxona este o cefalosporina de generatia a treia care este bine tolerata si poate fi administrata in siguranta in timpul
sarcinii. Pseudolitiaza biliara a nou-nascutului si colestaza sunt efecte adverse posibile, datorate utilizarii abuzive a acestui medicament.
Cuvinte cheie: ceftriaxona, sarcina, siguranta, pseudolitiaza biliara, colestaza reversibila

12

Fiziologia - Physiology  2012.22.4 (76)OPTIMIZATION OF PROTOCOLS FOR COMPLETELYDECELLULARIZE EQUINE CORNEAMARIUS TOMA, RADU LAZAROVICZ, LAURA MARUSCIAC, RADU IVANESCU, FLORINA
BOJIN, CARMEN PANAITESCU, VIRGIL PAUNESCU
Department of Functional Sciences, ”Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT

Cornea can be damaged by various diseases, in which the only treatment is corneal transplantation. A severe shortage of donor
corneas exists worldwide, so alternative solutions are needed. As so, researchers tried to tissue-engineer corneas using autologous
cells cultured on biological scaffolds like decellularized xenograft tissue. The aim of present study was to obtain complete decellularized equine cornea.
Corneas were obtained from 20 normal healthy horses, sacrificed under strict rules and regulation in slaughter houses. Several
protocols that include chemical and mechanical methods were carried out for completely decellularize cornea: hypertonic solutions,detergents, buffers, and alcohol, combined with mechanical agitation or direct stromal perfusion.
DNA extraction and measurement, RT-PCR, DAPI immunofluorescence and HE histochemistry were performed for evaluating effectiveness of our protocols. DNA measurements revealed 91% decellularization in corneas treated with Triton-X, SDS and Tris on
continuous agitation (757 ng/mg), and 96% decellularization in corneas cannulated and perfused with the same chemicals (321 ng/mg), compared to control (8060 ng/mg). The results were confirmed by DAPI nuclear staining, while HE staining revealed integrityof stromal fibers. RT-PCR showed fragmented DNA and expression of GAPDH in all samples.
We succeed 91% decellularization in corneas treated by classical methods which involve cornea placed in detergents-buffer combination on continuous agitation, and 96% in cannulated corneas, data suggesting that our new method could be studied further tosee if the integrity of stromal fibers together with basement membranes maintain as much as possible to physiological conditions.
Key words: equine cornea, decellularization, cannulate, DNA extraction, HE staining

INTRODUCTION
The cornea can be damaged by various diseases that cancause visual impairment and even blindness (1,2). So far, theonly treatment for visually impairing corneal damage or scarring is corneal transplantation. Since the first human corneal
transplant in 1905, corneal transplantation is one of the mostsuccessful forms for tissue transplantation (3-5). Although corneal transplantation is very useful, a severe shortage of donor
corneas exist worldwide (6). In addition, complications suchas infection, graft failure and immune reactions are possible,
and allograft reaction has been reported in patients because ofpenetrating keratoplasty. Hence, alternative solutions for corneal
transplantation are needed to overcome the shortage of corneal
donor organs and the complications resulting from the procedure,
especially rejection and graft failure of the transplanted cornea(7,8). To solve this problem, several researchers have tried totissue-engineer corneas using autologous cells cultured in various extracellular 3D matrices and synthetic polymers (9,10). Both
synthetic and biological matrices have previously been tested forcorneal tissue engineering (11-13), but another new strategy for
preparing a scaffold is the use of decellularized xenograft tissue,
in which the donor cells and antigen molecules are completely
removed to eliminate the host immune reaction (14,15).
The aim of the present study was to obtain complete decellularized equine cornea, including all corneal layers: epithelial

layer, stroma, and endothelial cells. Considering that both outerlayers can be mechanically removed, the main challenge in
obtaining decellularized cornea is represented by the stromalcompartment, populated by independently functioning specialized keratinocytes. We optimized different protocols largely used
for decellularization procedures in order to completely remove
the possible antigenic components from the stromal layer.

Fig. 1. Schematic representation of corneal layers (epithelial cells, Bowman membrane, stroma, Descemet membrane, and endothelial cells) (upper
panel) and histological image of hematoxylin and eosin staining, depicting thesame corneal structure (lower panel)

Received 14th of October 2012. Accepted 14th of November 2012. Address for correspondence: Marius Toma, Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara, phone/fax: +40256 220 479, e-mail: tomamarius_forever@yahoo.com

2012.22.4 (76)  Fiziologia - Physiology

13

MATERIALS AND METHODS
Equine corneas were obtained from 20 normal bread healthy
horses, sacrificed under strict rules and regulation in slaughter
houses. The eyeballs were harvested under sterile conditions andtransported to the laboratory on ice. The surrounding ocular muscles and conjunctivae were removed aseptically and the globes
were washed in sterile medium containing PBS (Dulbecco’s
Phosphate Buffer Saline, Sigma-Aldrich Company, Ayshire,UK) and 2% Pen/Strep (10,000 IU/mL, PromoCell, Heidelberg,
Germany). All animals experiments on comply with European
Convention for the Protection of Vertebrate Animals used forExperimental and Other Scientific Purposes (Strasbourg, France,1986). The University of Medicine and Pharmacy Timisoara Ethical Committee approved the experimental protocol.
1. Equine corneas processing methods
Three methods were used for equine cornea processing before the decellularization protocols were applied. The first method
consisted of harvesting the cornea with a fine sclera limbus formaintaining the shape and physical properties of corneal tissue(Figure 2b.). The second method considered obtaining smaller
fragments of corneal tissue. For this purpose, the equine eyeballs were trephined in the middle of corneal tissue using 6 mm
disposable biopsy punch (Kai Medical, Lohdorfer, Germany),
and the resulting corneal fragments were further submitted todecellularization protocols. This method was used for achieving
better results in penetration of substances which are required forcellular removal from all corneal layers (Figure 2c.).
The third method used in processing the cornea wasadapted from vascularized organs decellularization protocols,in which the organs are perfused with detergent substances.
Large sclera limbus and cornea were harvested from theequine eyeball; the whole fragment was stretched and boundto a 5 cm diameter Petri dish, placed in a vertical position, andcannulated in the upper part, while a small incision was madein the lower part of the cornea (Figure 2d.). The whole construct
was perfused with 1% SDS with a flow of 1 ml/hour for 24 hours.
After detergent wash, the cellular residues were removed using
continuous perfusion with PBS for 12 hours. Corneal deswelling
was performed in 100% glycerol for 6 hours.

Fig.2. Methods for equine corneas processing. a. entire equine eyeball; b.
equine cornea harvested with fine sclera limbus; c. trephined cornea; d. equine
cornea harvested with large sclera tissue, cannulated and vertically perfused14

2. Decellularization of equine corneas
Protocol 1. Detergent decellularization protocol. Corneas were placed in 50 ml centrifuge tubes (Falcon, BD) with
1% sodium dodecyl sulphate (SDS; Sigma-Aldrich Company) atroom temperature on the orbital shaker and continuous agitation for 12 hours. The ratio between SDS and corneal volume
was 25:1. In order to remove the detergents, the corneas were
washed three-five times with PBS, for 2 hours each, at room
temperature. Another washing step included 12 hours placementin 75% ethanol, at room temperature, followed by 3-5 times wash
in PBS, for 1 hour, at room temperature.
Protocol 2. Ethanol decellularization protocol. Corneas
were placed in 50 ml tubes, and 75% ethanol (20:1) was added
at 20oC, for 72 hours, on orbital shaker. Solvent replacement was
performed at 1, 3, 6, 12, 24, 48, and 72 hours. Distilled water
was used for washing, for 12 hours, at room temperature, followed by Trypsin-EDTA 0.05% (Sigma-Aldrich Company) (15:1)
incubation, for 96 hours, at room temperature.
Protocol 3. DNAse and RNAse decellularization protocol.
Corneas were subjected to non-surfactant treatment involving
incubation in 1.5 M NaCl, for 48 hours, at room temperature(change of medium was performed every 24h). This method was
modified in the second step by treating the corneas with DNAse
5 U/ml + RNAse 5U/ml (Sigma-Aldrich Company), for 48 hour, atroom temperature (dilution buffer: 50 mM Tris-HCl, 10 mM MgCl2,
50 μg/ml BSA). The third step consisted of PBS wash, for 72 hours,
at room temperature (medium was changed every 24 hours).
Protocol 4. Aprotinin decellularization protocol. In thisprotocol, corneas were treated with 10 mM Tris buffer + EDTA
0,1% + aprotinin 10 KIU, for 12 hours, at 4o C; 0.3% SDS in TBS,EDTA 0.1%, aprotinin 10 KIU/ml were added for 20 hours, at room
temperature. Next step wash 3 times wash in TBS, followed bytreatment with DNAse I 50 U/ml + 1 U/ml RNAse A (dilution buffer:
50 mM Tris-HCl, 10 mM MgCl2, 50 μg/ml BSA), for 3 hours, at 37o
C with agitation. The final washing step was 3 times in TBS andthe corneas were deswelled for 6 hours in 100% glycerol (Figure3). All chemical substances and reagents were purchased from
Sigma-Aldrich Company, unless otherwise specified.

Fig.3. Aspect of deswelled sectioned equine cornea. After 6h placementin 100% glycerol, the thickness of corneas was reduced to less than 1/3,
compared to swelled corneas, but it was significantly thicker than normal,
freshly isolated ones

Fiziologia - Physiology  2012.22.4 (76)Protocol 5. Osmotic gradient plus detergents. Corneas
were subjected to a stepwise treatment in which we usedcombination of hypotonic and hypertonic buffers, together withdetergents (SDS or Triton X-100). This protocol was adaptedfrom Roy et al. (16). In the first step, corneas were treatedwith hypotonic buffer – 10 mM Tris (pH 8) for 24 hours, at 4oC, on orbital shaker. After 24 hours, the buffer was replacedwith hypertonic solution containing 1% Triton X-100, 1.5 M
KCl, and 10 mM Tris (pH 8). In the last decellularization step,
the hypertonic buffer was replaced with detergent solution
containing 1% SDS and 10 mM Tris (pH 9) for 24 hours, under the same temperature conditions. In order to remove thedetergent, corneas were incubated at 37 oC for 30 minutes
in 1% Triton X-100, and then washed with PBS for 72 hours(change every 24 hours).
Protocol 6. Cannulated corneas, osmotic gradient anddetergents. Our newly developed protocol was a three step protocol, in which we used the previous described method (protocol5) perfused into the cannulated cornea with a flow rate of 1 ml/
hour, for 24 hours. The last step consisted in extensive washof cellular remains using PBS, with the same flow rate, for 12hours. The entire procedure was performed at room temperature
and all chemical substances and reagents were purchasedfrom Sigma-Aldrich Company. Deswelling procedure followed
the same protocol described before, using 100% glycerol for 6hours at room temperature.
3. DNA extraction and PCR evaluation
For DNA extraction we used Fast Tissue-to-PCR Kit(Fermentas, Life Sciences), and followed the manufacturer
instructions. Shortly, DNA was extracted from 5 mg ofequine decellularized cornea, which was mixed by vortexing initially with 100 μl of Tissue Lysis Solution and 10μl ofProteinase K Solution. The mixture was incubated at room
temperature for 10 minutes, and then additional 3 minutes
at 95 oC. At the end of the incubation, not all the tissue wascompletely digested, but this fact did not affect the further
PCR performance. In a next step, 100 μl of Neutralization
Solution T was added to the sample and mix by vortexing.
The neutralized tissue extract was either stored at 4 oC orused immediately in PCR.
The PCR analysis was performed with Tissue Green PCR
Master Mix, which includes a specialized formulation of the efficient DreamTaq DNA polymerase, dNTPs and buffer, optimized
for use with the reagents used for DNA extraction. Loadingdyes and density reagent are also included in the master mix
for direct loading of the PCR product onto an agarose gel. The
primers used for the PCR reaction were the following: G3PDH
F: GA-AGG-TCG-GAG-TCA-ACG-GAT-TTG-GT; G3PDH R:
AT-GTG-GGC-CAT-GAG-GTC-CAC-CAC, with an ampliconsize of 1000 bp. The program followed for DNA amplificationconsisted of subsequent temperature cycles: 50 oC - 30 minutes,
95 oC - 15 minutes, 35x (94 oC - 1 minute, 60 oC - 1 minute, 72
2012.22.4 (76)  Fiziologia - Physiology

C - 1 minute), 72 oC -10 minutes, 4 oC - ∞. The amplification
products were migrated in electrophoresis 2% agarose gel, andvisualized using UV Fluor-S MultiImager (Bio-Rad) and Quantity
One Basic software.
o

4. Histochemical and fluorescence evaluation
Corneas decellularized using different protocols were
cut in small fragments (10 mm3) each and embedded in Killik – cryostat embedding medium (Bio-Optica, Milano, Italy),snap-frozen in liquid nitrogen, and preserved at -80o C until
further processing. 3 μm thick sections were performed using
cryotome, and the slides were stained using haematoxylineosin (DakoCytomation Hematoxylin Mayer’s, Lillie’s modification, Dako) classical labeling, for revealing collagen fibers
organization after decellularization procedures. For showing
the degree of decellularization, we used the fluorescent nuclear staining – 4,6-diamidino-2-phenylindole (DAPI), counted
the remaining nuclei on five different microscopic fields, andperformed comparative statistic analysis between all protocols
using the average value. Microscopy analysis was achieved
on a Nikon Eclipse E800 microscope equipped with adequate
fluorescence filters.
5. Statistic analysis
Statistic analysis was performed using Excell Microsoft Office 2007 (Microsoft Corporation) software. The
central tendencies of the variables were expressed as amean (M), and the dispersion ones as standard deviation(sd). In order to perform the statistic comparisons, „t”Student test and the variance analysis (ANOVA) were used
for continuous variables. Differences were consideredsignificant for p < 0.05.
RESULTSDNA content and integrity
The DNA content analysis revealed different concentrations of DNA remaining in the decellularized samples,
correlated with the complexity and duration of the protocols
used. All DNA samples were expressed as ng of DNA / mgof dried cornea, and calculated in respect with suspension
volume. We used Nanodrop ND-1000 spectrophotometer for
quantification of DNA, and the 260/280 ratio was between
1.2-1.8 in all samples measured (Figure 4). The following were
the average values of DNA amount obtained using different
decellularization protocols (± sd): Protocol 1 – 3466.6 ± 124
ng/mg; Protocol 2 – 4808 ± 203 ng/mg; Protocol 3 – 3111 ±98 ng/mg; Protocol 4 – 2923 ± 87 ng/mg; Protocol 5 – 757± 58 ng/mg; Protocol 6 – 321 ± 35 ng/mg. Control integral
cornea had a significant increased content – 8060.4 ± 109
ng/mg DNA (Figure 5). Based on DNA content, protocols 5
and 6 had the best decellularization rate, estimated to 91%
and 96%, respectively.
15

mented DNA and expression of GAPDH in samples extracted
after decellularization with protocols 1 and 2 (Figure 6b). Samples
extracted after using more complex decellularization protocols(protocols 3 and 4) showed a clear 1000 bp band when migrated
in agarose gel, with no fragmentation, while decellularization
protocols 5 and 6 showed a faded band when amplified for
GAPDH expression. The results are clearly indicating that thedecreased DNA amount is relevant for a good decellularization
protocol, and protocols 5 and 6 seem do have the best outcomes.

Fig.6. Agarose gel electrophoresis migration of DNA. a. control DNA, noamplification; b. protocols 1 and 2 obtained DNA expression of GAPDH(1000 bp ladder); c. protocols 3 and 4 obtained DNA qualitative expression of
GAPDH; d. protocols 5 and 6 obtained DNA amplified for specific expressionof GAPDH

Fig. 4. DNA content measured using Nanodrop ND-1000 spectrophotometerin samples extracted after decellularization of equine cornea using different
protocols. Highly impure DNA content, reflected by the 260/280 ratio which
was lower than 2 in all samples, except for control

Structural integrity of corneal stromal fibers and cell
removalHE staining was used for quantification of cell number,but also for revealing the disposition of stromal fibers within
the equine cornea. The fibers had a parallel arrangement,
both with respect of each other, and the two basal enclosing
membranes (Figure 7a). DAPI staining showed cellular nuclei
in the equine cornea. In normal integral cornea, DAPI staining
showed abundant cellular population within stroma, as well as
the endothelial (mono)layer and epithelial cells placed on basal
membranes (Figure 7b).

Fig.7. Normal integral equine cornea. a. HE staining (magnification 200x);
nuclear DAPI staining (magnification 100x)
Fig. 5. Statistic analysis of DNA content in samples obtained after using all
6 decellularization protocols. Significant decrease is found in all samples,compared with control (p < 0.05), but highly significant decrease in DNA
content was found in cornea samples decellularized using Protocols 5 and 6(p < 0.001)

Control DNA semi-quantitative expression showed integral
pattern of molecular aspect (Figure 6a). RT-PCR showed frag-16

Decellularization protocols used in this study inducedchanges in morphological and structural aspects of equine
cornea, in different degrees, depending on the methods used.
Protocols 1-4 provoked a markedly disarrangement in fibers
structure, while the cellular content was less influenced, thenumber of nuclei presented a small decrease, compared tocontrol (Figures 8 and 9).

Fiziologia - Physiology  2012.22.4 (76)DISCUSSION

Fig. 8. Detergent and ethanol methods for decellularization of equine cornea.
a. Protocol 1, HE staining; b. Protocol 1, DAPI staining; Protocol 2 (c. HE; d.
DAPI) (magnification 200x)

Fig.9. Nuclease and aprotinin decellularization methods. Protocol 3 (a. HE; b.
DAPI). Protocol 4 (c. HE; d. DAPI) (magnification 200x)

When protocols 5 and 6 for decellularization were used,
the number of nuclei had a spectacular decrease, thus showingthat the cells were removed in a greater extent. Regarding thefibers arrangement, protocol 5 seemed to be more aggressiveon overall morphological structure of equine cornea (Figure 10
a and b), while using protocol 6 we were able to maintain fibers
structure in a close to normal disposition (Figure 10 c and d).

We were able to develop and optimize a new protocol for
efficient decellularization of equine cornea, consisting in using
methods and reagents already described in literature (16), but
introducing a revolutionary concept of stretched and cannulated
cornea, which proved to remove 96% of the stromal cells, while
maintaining integrity and fibers arrangement. This protocol could
be applied in cases of bioengineered constructs, which will befurther used in in vivo transplants.
Successful tissue engineering depends on the provision ofa scaffold during the initial stage of reconstruction. A suitablescaffold is readily fabricated and results in a 3D porous structure,
which affects the structure and function of the generated tissue.
Moreover, the proper scaffold not only provides an environment
in which the cells proliferate, integrate and form ECM, but isimportant for metabolic support and waste removal (17,18).
Until now, there have been no synthetic scaffolds that can mimic
the structure of the lamellar stroma, and synthetic and natural
biological materials used as scaffolds for tissue engineeringlack a lot of natural properties, which limit their use for clinicalimplantation (19). Without a proper 3D assembly, cells lack theproper clues for growing into the variety of cells that make up
a particular tissue.
We demonstrated that this tissue can be processed using
a variety of techniques that can maintain the unique ECM structure. In this study, treatments were selected to be compatible
with corneal tissue. We may speculate that these decellularized
corneas should also provide the specific microenvironment forthe stromal and epithelial cells to migrate and repopulate thetissue graft, both in vitro and in vivo.
Acknowledgements
This work was supported by CNCSIS – UEFISCSU, project
number PNII – IDEI 318/2011, and POSDRU 89/1.5/S/60746.
REFERENCES

Fig. 10. Canulated equine cornea, perfused with SDS for 24h. Dehydrated
cornea right after SDS perfusion and stained with HE (a) and DAPI (b). Additional PBS wash for 12 hours, followed by deswelling in 100% glycerol (c.
HE; d. DAPI) (magnification 200x)2012.22.4 (76)  Fiziologia - Physiology

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17

7. Thompson RW Jr, Price MO, Bowers PJ, Price FW Jr. Long-term
graft survival after penetrating keratoplasty. Ophtalmology, 2003; 110:1396-1402.
8. Panda A, Vanathi M, Kumar A, Dash Y, Priya S. Corneal graft rejection. Surv Ophtalmol, 2007; 52: 375-396.
9. Griffith M, Osborne R, Munger R, et al. Functional human corneal
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11. Li F, Carlsson D, Lohmann C, Suuronen E, Vascotto S, et al. Cellular and nerve regeneration within a biosynthetic extracellular matrix
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scaffolds. Cornea, 2003; 22: S81.
13. Chirila TV. An overview of the development of artificial corneas withporous skirts and the use of PHEMA for such an application. Biomaterials, 2001; 22: 3311.

14. Wilshaw SP, Kearney JN, Fisher J, Ingham E. production of anacellular amniotic membrane matrix for use in tissue engineering. Tissue
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15. Oh JY, Kim MK, Lee HJ, Ko JH, Wee WR, Lee JH. Processing
porcine cornea for biomedical applications. Tissue Eng Part C Methods,
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16. Roy S, Silacci P, Stergiopulos N. Biomechanical properties ofdecellularized porcine common carotid arteries. Am J Physiol Heart
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JI, Serrano D, Medialdea S, et al. Construction of a complete rabbit
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OPTIMIZAREA PROTOCOALELOR PENTRU DECELULARIZAREA COMPLETA
A CORNEEI ECVINEREZUMAT

Corneea poate fi alterata in diferite afectiuni oftalmologice, in care singurul tratament este transplantul de cornee. In lumea intreaga
exista un deficit al donatorilor de cornee, astfel incat este nevoie de solutii alternative. Cercetarile actuale sunt indreptate spre obtinerea corneei prin inginerie tisulara, folosind celule autologe, cultivate pe suporturi biologice, cum ar fi xenogrefoane decelularizate.
Scopul acestui studiu a fost acela de a obtine decelularizarea complete a corneei ecvine.
Au fost obtinute 20 de cornee de la cai de rasa comuna, sanatosi, care au fost sacrificati in conditii reglementate de legislatia Romaniei. Au fost utilizate diverse protocoale care folosesc metode chimice si mecanice pentru decelularizarea completa a corneei: solutii
hipertone, detergenti, solutii tampon, alcool, in combinatie cu agitare mecanica sau perfuzie stromala directa.
Pentru evaluarea eficientei de decelularizare a protocoalelor utilizate, a fost efectuata extractia ADN, urmata de RT-PCR si au fostutilizate metode de imunofluorescenta (DAPI) si histochimie (HE). Masurarea continutului de ADN a aratat o decelularizare de 91%
la corneele tratate cu Triton-X, SDS si Tris, cu agitare continua (757 ng/mg) si o decelularizare in proportie de 96% in cazul corneelor canulate si perfuzate cu aceleasi substante chimice (321 ng/mg), comparativ cu corneea control (continut ADN - 8060 ng/mg).
Rezultatele au fost confirmate prin marcarea nucleara cu DAPI, iar colorarea histochimica HE a aratat integritatea fibrelor stromale.
Prin RT-PCR s-a aratat fragmentarea ADN si expresia GAPDH in toate probele analizate.
Am reusit decelularizarea corneei ecvine in proportie de 91% folosind metode clasice, care utilizeaza o combinatie de detergenti sisolutii tampon si agitatie continua, in timp ce prin canularea corneei am obtinut o decelularizare in proportie de 96%, aceste datesugerand ca noua metoda dezvoltata prin acest studiu ar trebui investigata in viitor pentru a arata daca integritatea fibrelor stromale
si membranelor bazale este suficienta pentru mentinerea conditiilor fiziologice ale micromediului celular.
Cuvinte cheie: cornee ecvina, decelularizare, canulare, extractie ADN, marcare HE

18

Fiziologia - Physiology  2012.22.4 (76)ANALYSIS OF SYNTHETISED STEROID DIMERS BASED ON
THE STRUCTURE OF PENTACYCLIC TRITERPENES
ALINA HEGHES1*, CRISTINA TRANDAFIRESCU1, CAMELIA SASS (OPREAN)2, SIMONA ARDELEAN3, FLORIN BORCAN1
1“Victor Babes” University of Medicine and Pharmacy Timisoara, Faculty of Pharmacy, 2nd Eftimie Murgu Sq., Timisoara-300041,
Romania2“Victor Babes” University of Medicine and Pharmacy Timisoara, Faculty of Medicine, 2nd Eftimie Murgu Sq., Timisoara-300041,
Romania3“Vasile Goldis” West University Arad, Faculty of General Medicine, Pharmacy and Dental Medicine, 1st Feleacului Str., Arad-310396,
Romania

ABSTRACTSteroid dimers can serve as architectural components in the chemistry of molecular recognition and may be used as chiral building
blocks to construct artificial receptors. The synthesis of steroid dimers based on pentacyclic triterpenes such as betulin or betulinicacid, was attempted. A simple esterification reaction between betulin and betulinic acid was not satisfactory. The research not shows
better results even in the case of betulinic acid activation with thionyl chloride. 1,4-butanediol was used to create a spacer group andto avoid the steric hindrance and the final product was topical applied on hairless mice skin.
Keywords: steroid, betulin, betulinic acid, esterification, HPLC-MS

INTRODUCTION
The steroids are organic compounds which contain a specific arrangement of four cycloalkane rings which are connectedto each other (1). These substances form a class of biologically
active lipophilic molecules which include steroid hormones, plantsterols (known as phytosterols), cholesterol, and bile acids. The
parent structure, named gonane (also known as the steroid
nucleus), a tetracyclic seventeen carbon steroidal ring system
is known as 1,2-cyclopentano-perhydro-phenanthrene or simply
cyclopentaphenanthrene (Figure 1) (2).

Fig. 1. Gonane skeleton

The four rings of gonane skeleton are lettered A, B, C, andD, and the carbon atoms are numbered beginning in the A ring.
There is well-known that B, C, and D rings of steroids are always
trans-fused, and in most natural steroids, the first two rings arealso trans-fused.
The steroid dimers are representatives of an importantfamily of chemical compounds which are produced by various

marine organisms, and there are also synthesized in laboratories.
These organic compounds possess different pharmacological and biological properties, and can also be used to create“molecular umbrellas” for drug delivery (2). Steroid dimers werefirst observed as synthetic byproducts and then discovered innature (3, 4). The steroid dimers possess interesting micellular,
detergent, and liquid crystal properties (5).
The steroid dimers are linear or cyclic compounds, connected via rings in the steroid nucleus (A–A, B–B, etc.) or throughthe side chains, the latter being commonly used to form bile acids
dimers. There are many kinds of acyclic structures based onbile acids, among which the most interesting are the following:ionophores (6), molecular umbrellas (7), cleft type structures (8),molecular tweezers (9), dendrons (10), gelling agents (11), andinclusion compounds (clathrates) (12).
The steroid dimers represent a class of compounds thathave attracted attention for their rigid and inherently asymmetricarchitecture (13).
A synthesis of 6E-hydroximino steroid homodimers wasreported in the literature (Figure 2) (14). These unusual 6Ehydroximino-3-oxo-4-ene steroids were discovered in the
sponge Cinachyrella alloclada. A rapid and efficient synthesisof homodimers of these cytotoxic compounds was studied. To
6-ketones (X = O) three or four carbon atom linkers with terminal
double bonds were attached in the position 3β. The allylic ether(n = 1) afforded E-homodimer in 82% with small amounts ofthe sterically less favoured Z-isomer. In the case of homoallylic
ether (yield 80%) the ratio E/Z was 1.5:1. After chromatographic
purification, the dimers were converted to the 6E-oximes. How-

Received 15th of October 2012. Accepted 20th of November 2012. Address for correspondence: Alina Heges, Pharmaceutical Tech. Dept., Faculty of Pharmacy, “Victor
Babes” University of Medicine and Pharmacy Timisoara, 2nd E. Murgu Sq. 300041 Timisoara, RO, phone/fax: +40256220479, e-mail: heghes_alina@yahoo.com

2012.22.4 (76)  Fiziologia - Physiology

19ever, homodimers were proved to be not very cytotoxic whencompared with monomer counterparts.

Fig. 2. Synthesis of homodimers

L.P. Valverde and his collaborators have reported a method(15) that involves the modification of the hydroxyl group (C-3from A-ring of pregnenolone) in order to synthesize pregnenolone dimers with and without spacer groups that can be usedas chiral building blocks to construct artificial receptors and asarchitectural components in biomimetic or molecular recognition
chemistry (Figure 3). They reported an easy procedure to synthesize pregnenolone dimer via connection between the two A rings.
The purpose to use of long spacer arm in the pregnenolone-dimer
was to avoid the steric hindrance that the support could havewhen interacting with other biological molecules.

drogene dimers coupled at C3-C3 and C17-C3 and of an androgenprogestogen combination as long-acting hormonal steroids (16).
Cholest-5-en-3-spiro-[6′ α,5′-oxa]-5′ α-cholest-3′-one(2), cholest-5-en-7-spiro-[4′ α,5′-oxa]-5′ α-cholest-7′-one
and 3β-substitutedcholest-5-en-7-spiro-[4′ α,5′-oxa]-3′β-substituted-5′ α-cholestan-7′-ones were synthesized startingfrom cholest-5-en-3-one, cholest-5-en-7-one and 3β-substitutedcholest-5-en-7-ones respectively (17).
The need of new antifungal drugs with high efficacy, low
toxicity and new action mode is well-known. Synthesis andantifungal activity of bile acid derived new steroidal dimers suchas N1,N3-Diethylenetriamine-bis-[cholic acid amide] (Figure 4a)and N1,N3-Diethylenetriamine-bis-[deoxycholic acid amide](Figure 4b) having novel amphiphile topology was described ina US Patent (18).

Fig. 4. Structures of bile acid derived new steroidal dimers

In this study, betulin and betulinic acid were used as raw
materials in an esterification reaction in order to obtain steroid
dimers compounds. 1,4-butanediol was used to create a spacergroup because there were not obtained good results even after
the betulinic acid activation with thionyl chloride.
MATERIALS AND METHODS
Betulin and betulinic acid (Figure 5) were isolated with drycolumn chromatography from external birch bark (Betula Pendula
Roth) as was described in the literature (19, 20) and its IR spectra
were compared with the reference standards. Betulinic acid wascrystallized from chloroform-methanol as shining needles with amelting point 294-296 oC (21). Solvents, thionyl chloride and all theothers substances at analytical reagent grade, were purchased from
Fluka (Germany) and were used without any previous purification.

Fig. 3. Three steps synthesis of pregnenolone-pregnenolone dimer via Ring
A-Ring A connection(1) pyridine/toluene; (2) ethylenediamine dihydrochloride, acetonitrile/water;(3) 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride, acetonitrile/water

H. Kuhl and H.D. Taubert have reported the synthesis of an20

Fig. 5. Structures of betulin and betulinic acid

Fiziologia - Physiology  2012.22.4 (76)Dimerization procedure
As it was already described in the literature in the case of
betulinic acid acyl glucuronide synthesis (22), betulinic acid was
coupled with betulin in the presence of potassium carbonate(K2CO3) in a heterogeneous solution of CH2Cl2 / H2O.
HPLC-MS
Effective analysis was performed by two complementary
methods. It was first performed the screening analysis, looking
for the three compounds that may exist in the sample (betulinicacid, betulin and corresponding dimer). It was run a gradientof 0.4% formic acid in water/methanol and it was record thechromatogram.
Betulinic acid activation
The reaction apparatus consists of a one-neck flask withmagnetic stir bar and reflux condenser. For draining of the evolving gases (HCl and SO2) the reflux condenser is connected to awash bottle filled with an aqueous solution of sodium hydroxide(20%). The reaction flask is charged with 10 ml carbon tetrachloride and 14.50 µl (0.2 mmol) freshly distilled thionyl chloride.
Whilst stirring, 45.67 mg (0.1 mmol) betulinic acid is added in theflask. The reaction mixture is initially slowly heated whilst stirring(as far as possible because of strong formation of gas) up to 50°C, afterwards stirring is continued for 2 additional hours at 80 °C.

were used 4 mice: group FP (2 mice, treated with cream withthe final product), group 0 (2 mice, treated with blank cream).
The applications were done twice a week and the measurements
were done only at the begging and the end of the experimentperiod. The measurements on the mice skin were carried out witha Multiprobe Adapter System (MPA5) from Courage-Khazaka,
Germany.
RESULTSThe obtained chromatogram in the screening analysis ispresented in Figure 7.

Fig.7. The chromatogram of the first reaction products

The molecular mass was the principle of betulinic acid andbetulin identification, after about 1.8 minutes. The two signals at
3.6 and 4.5 minutes correspond to plasticizers from solvent (notfrom analyzed sample). The corresponding mass spectra areshown in Figure 8. There were not identified any compound withexpected molecular weight for a dimer (M = 898, the expectedm / z = 899 or 881 for dehydrated molecule).
The raw materials were quantitative analyzed in order to
see their ratio because in the first stage of analysis it was notrevealed any dimer.
Intens.
x108+MS, 1.7min #356

4
3

Fig. 6. The reaction of betulinic acid activation

425.2

2
1

After the cooling down of the reaction mixture, the reflux
condenser is replaced by a distillation bridge and the excess ofthionyl chloride is removed by distillation (23). The crude acidchloride is for most uses pure enough, so that one can do without
distillation. The product is a colourless, strong refractive liquid,
which crystallizes whilst cooling as a colourless solid product.
The dimerization procedure was repeated using activatedbetulinic acid in reaction with betulin and the products were analyzed by FT-IR spectra using a Jasco FT-IR/410 spectrometer.
In the last step, it was used 1,4-butanediol in order to avoid thesteric hindrance.
Ten weeks old females of hairless mouse SKH1 were usedin this experiment. Creams with and without the final product wereapplied on mice skin for 9 weeks (0.3 mL / application). There2012.22.4 (76)  Fiziologia - Physiology

0x108+MS, 1.7min #368

3
439.2

2

1

0

400

500

600

700

800

900

m/z

Fig. 8. The mass spectra of first reaction products

The absence of any important change in mice skin parameters may be observed on Figure 9.
21

Fig. 9. Differences of mice skin parameters between cream with and without
final product before and after experiement

DISCUSSION
After the first reaction was done, it was weighed a quantityof 6.98 mg product sample, dissolved in acetone with ultrasoundand analyzed the 1:1000 solution with HPLC-MS. The test results
showed that the powder contains about 70% betulin (± 3-4%)
and 32% betulinic acid (± 3-4%). It is unlikely that dimer exist
in this sample given the reactants ratio in the sample provided.
There were used the calibration curves from Figure 10.

Fig. 10. Calibration curves of betulin and betulinic acid

The betulinic acid activation was the second step in orderto obtain the steroid dimer. It was used the procedure describedin the literature (23). After the betulinoyl chloride was purified
and checked by FT-IR spectra, the dimerization procedure wasrepeated.
The reaction products were checked by FT-IR spectra. It
is well-known that organic acids presents a strong absorption
22

between 1700-1725 cm-1 for C=O vibration, a strong and verybroad signal at 2500-3300 cm-1 characteristic for O-H vibration,
and a strong band between 1210-1320 cm-1 for C-O bond. The
O-H bond from alcohols presents signals between 3200-3700
cm-1, depend on the presence of hydrogen bonds. A strong band
between 1735-1750 cm-1 (C=O) and two or more bands between
1000-1300 cm-1 (C-O) are specific for esters (24).
The IR spectra of the products sample did not reveal thepresence of the ester group. Then, 1,4-butanediol was used in
the synthesis in order to avoid the steric hindrance. In this case
the FT-IR spectrum contains the signals specific for esters. The
new product was topical applied on the back skin of hairless micefor its toxicological characterization. There were not observed
important changes of skin parameters (Figure 9).
CONCLUSIONSThe synthesis of symmetric molecules derived from joining
two identical parts has gained importance in last decade. Steroid
dimers may be linear or cyclic compounds which are connected
via rings or through the side chains, the latter being commonlyused to form bile acids dimers. In this experimental study it was
attempted the synthesis of a steroid dimer using pentacyclic
triterpenes such as betulin and betulinic acid as raw materials.
It was done a simple esterification reaction and the products
were analyzed by HPLC-MS; the analysis results show that thisprocedure was not satisfactory. The procedure was then repeatedafter the betulinic acid activation with thionyl chloride, but FT-IR
spectra did also not show the expected results. It seems thatthe steric hindrance prevents the chemical reaction which wasdone to obtain the steroid dimer. It was synthesized an esterwith 1,4-butanediol which not shown any important changes ofmice skin parameters.
REFERENCES
1. Steroid. http://www.britannica.com/EBchecked/topic/565825/
steroid/278048/Steroid-numbering-system-and-nomenclature. Last
accessed: November 5th, 2012.
2. Nahar L, Sarker SD. Steroid Dimers: Chemistry and Applications in
Drug Design and Delivery. Wiley, John & Sons, Chicester (UK), 2012.
3. Fieser LF, Fieser MF. Steroids. Reinhold Publishing Corporation,
New York (USA), 1959.
4. Banerji J, Chatterjee A, Itoh Y, et al. New Steroid Alkaloids from
Chonemorpha-Macrophylla Chonemorpha-Fragrans. Ind. J. Chem.
1973; 11: 1056-57.
5. Li Y, Dias JR: Dimeric and Oligiomeric Steroids. Chem. Rev. 1997;
97: 283-304.
6. Bandyopadhyay P, Janout V, Zhang LH, et al. An Ion Conductor
Derived from Spermine and Cholic Acid. J. Am. Chem. Soc. 2000;
122: 12888-89.
7. Janout V, Lanier M, Regen SL. Molecular Umbrellas. J. Am. Chem.
Soc. 1996; 118: 1573-74.
8. McKenna J, McKenna JM, Thornthwaite DW. Bis-steroids as potential
enzyme models: Perylene solubilisation and dye spectral changes withaqueous solutions of some derivatives of conessine and cholic acid. J.
Chem. Soc., Chem. Commun. 1977; 809-11.
9. Maitra U: Bile-Acids in Asymmetric-Synthesis and Molecular Recogni-

Fiziologia - Physiology  2012.22.4 (76)tion. Curr. Sci. 1996; 71(8): 617-24.
10. Balasubramanian R, Rao P, Maitra U. First bile acid-derived chiraldendritic species with nanometric dimensions. Chem. Commun. 1999;
23: 2353-54.
11. Maitra U, Kumar PV, Chandra N, et al. First donor-acceptor interaction promoted gelation of organic fluids. Chem. Commun. 1999; 7: 595-6.
12. Yoswathananont N, Sada K, Miyata M, et al. Dependence of selective enclathration on types of cholic acid crystals. Org. Biomol. Chem.
2003; 1(1): 210-214.
13. Nahar L, Sarker SD, Turner AB. A review on synthetic and natural
steroid dimers: 1997-2006. Curr. Med. Chem. 2007; 14: 1349-70.
14. Rega M, Jiménez C, Rodríguez J. 6E-Hydroximinosteroid homodimerization by cross-metathesis processes. Steroids 2007; 72: 729 - 735.
15. Valverde LF, Cedillo FD, Tolosa L, et al. Synthesis of PregnenolonePregnenolone Dimer via Ring A-Ring A connection. J. Mex. Chem. Soc.
2006; 50(1): 42 - 45.
16. Kuhl H, Taubert HD. A new class of long-acting hormonal steroid
preparation: synthesis of dimeric androgenes coupled at C3-C3 andC17-C3 and of an androgen-progestogen combination. Steroids 1976;
28(1): 89 - 99.
17. Alam MGS, Siddiqui T. Synthesis of steroidal dimers: Selective
amine catalysed steroidal dimerization. J. Chem. Sci. 2011; 123(4):491-495.
18. Salunke DB, Hazra BG, Pore VS, et al. Bile acid derived steroidal
dimers with novel amphiphilic topology having antifungal activity. Patent
US7235678, 2007.
19. Cinta-Pinzaru S, Dehelean CA, Soica C, et al. Evaluation anddifferentiation of the Betulaceae birch bark species and their bioactive
triterpene content using analytical FT-vibrational spectroscopy and
GC-MS. Chem. Cent. J. 2012; 6(1): 67.
20. Holonec L, Ranga F, Crainic D, et al. Evaluation of Betulin and
Betulinic Acid Content in Birch Bark from Different Forestry Areas ofWestern Carpathians. Not. Bot. Horti. Agrobo. 2012; 40(2): 99-105.
21. Zhao G, Yan W, Cao D. Simultaneous determination of betulin andbetulinic acid in white birch bark using RP-HPLC. J. Pharm. Biomed.
Anal. 2007; 43(3): 959-962.
22. Gauthier C, Legault J, Rondeau S, et al. Synthesis of betulinic acidacyl glucuronide for application in anticancer prodrug monotherapy.
Tetrahedron Lett. 2009; 50: 988-991.
23. NOP Online. http://kriemhild.uft.uni-bremen.de/nop/en-experiment-2013. Last accessed: November 5th, 2012.
24. IR-frequencies. http://www2.ups.edu/faculty/hanson/Spectroscopy/IR/IRfrequencies.html. Last accessed: November 5th,2012.

ANALIZA DIMERILOR STEROIDICI SINTETIZATI PE BAZA STRUCTURIITRITERPENELOR PENTACICLICEREZUMAT

Dimerii steroidici pot servi in calitate de componente arhitecturale in chimia recunoasterii moleculare si pot fi folositi ca blocuri chirale
de constructie a receptorilor artificiali. A fost incercata sinteza dimerilor steroidici pe baza triterpenelor pentaciclice, cum ar fi betulina
sau acidul betulinic. O reactie de esterificare simpla intre betulina si acidul betulinic nu a fost satisfacatoare. Cercetarea nu prezinta
rezultate mai bune chiar si in cazul activarii acidului betulinic cu clorura de tionil. 1,4-butandiol a fost folosit pentru a crea o gruparespatioasa și pentru a evita impiedicarea sterica, iar produsul final a fost aplicat topic pe piele de soarece fara par.
Cuvinte cheie: steroid, betulina, acid betulinic, esterificare, HPLC-MS

2012.22.4 (76)  Fiziologia - Physiology

23

SERUM SOLUBLE INTERLEUKIN-2 RECEPTOR LEVELS IN
CHRONIC HEPATITIS C BEFORE INTERFERON THERAPYMANOLE COJOCARU1, SIMONA ALEXANDRA IACOB2, EUGENIA PANAITESCU2, DIANAGABRIELA IACOB2
1“Titu Maiorescu” University, Faculty of Medicine, Bucharest
2“Carol Davila” University of Medicine and Pharmacy, Bucharest

ABSTRACT
Background: The release of soluble interleukin-2 receptor (sIL-2R) appears to be a characteristic marker of T lymphocyte activation and might be related to immunoregulatory function. Objectives: To determine serum sIL-2R levels in hepatitis C virus (HCV)
infection as compared to normal controls and its association with histological inflammatory indicators. Materials and methods:
Fifteen patients with chronic HCV infection (6 male, 9 female, mean age 47.35±10.78 years) and 14 healthy subjects (6 male, 8
female; mean age 35.00±15.45 years) were included in this study. The diagnosis of the patients with chronic HCV infection wasestablished on the basis of clinical, laboratory, ultrasonographis and histopathologic findings. The healthy subjects had negative
hepatitis serology, normal liver function tests and normal ultrasonographic findings. Serum sIL-2R was measured before interferon(IFN) alpha treatment. Serum levels of sIL-2R were measured by ELISA. Statistical significance was assumed for p<0.05. Results:
Serum sIL-2R levels were significantly higher 809±43.6 pg/mL than those of healthy controls 634.5±32.7 pg/mL, p<0.02 by Student
t test. There were some correlations between serum sIL-2R levels and histological activity score (r=0.482, p<0.01) and serum alanine
aminotransferase levels (r=0.363, p<0.01). Serum sIL-2R was significantly higher in HCV genotype 1 as compared to genotype 3(889±54.8 vs. 756.7±37.9; p<0.02). Conclusion: Serum sIL-2R levels increase due to HCV infection, and the amount of increase
corresponds to the degree of inflammation.
Key words: soluble interleukin-2 receptor, hepatitis C virus infection, pathophysiology

BACKGROUND
The pathogenic mechanisms of chronic HCV infection
remain unclear. Objectives of this study were to assess thepotential role of sIL-2R in chronic HCV infection and to clarify
whether there is a difference in the activation of T lymphocytes
among HCV genotypes.
MATERIAL AND METHODS
Fifteen patients with chronic HCV infection (6 male, 9female, mean age 47.35±10.78 years) and 14 healthy subjects(6 male, 8 female; mean age 35.00±15.45 years) were includedin this study. The diagnosis of the patients with chronic HCV
infection was established on the basis of clinical, laboratory,
ultrasonographis and histopathologic findings. The healthy
subjects had negative hepatitis serology, normal liver function
tests and normal ultrasonographic findings. Serum levels of
sIL-2R were measured by ELISA. The individual samples weredone in duplicate. The Mann–Whitney test was used for groupcomparisons and Fisher’s exact test was used to calculate thedifferences in categorized data. For testing associations, Spearman rank correlation coefficients were calculated. For normally

distributed paired samples, paired t-tests were used. Bonferroni’s
correction was used where appropriate, and corrected p<0.05
were considered significant. Values are given as mean±SD.
Correlation between serum sIL-2R level and the immunohistochemical reading score was analyzed by a simple curve fit
test. Survival curves were plotted with method of Kaplan-Meier.
The statistical difference between groups was compared by the
log-rank test. χ2 analysis showed that the serum sIL-2R levels
correlated well with histological inflammatory indicators.
RESULTS
Serum sIL-2R was measured in 15 patients with chronic
HCV infection before interferon (IFN) alpha treatment. Serum sIL2R levels were significantly higher 809±43.6 pg/mL than thoseof healthy controls 634.5±32.7 pg/mL, p<0.02 by Student t test.
There was some correlation between serum sIL-2R levels andhistological activity index scores (r=0.482, p<0.01) and serum
alanine aminotransferase levels (r=0.363, p<0.01). Serum sIL2R was significantly higher in HCV genotype 1 as compared togenotype 3 (889±54.8 vs. 756.7±37.9; p<0.02).

Received 1th of August 2012. Accepted 11th of September 2012. Address for correspondence: Dr. Manole Cojocaru, Titu Maiorescu University, Faculty of Medicine,
Department of Physiology, Center for Rheumatic Diseases, Bucharest; Thomas Masaryk No. 5 Sector 2, Postal Code 020983, Bucharest, Romania, e-mail:manole.cojocaru@yahoo.com

24

Fiziologia - Physiology  2012.22.4 (76)

severity of chronic liver disease, assessed histologically, and sIL2R levels in selected patients. Clinically it has been reported thatthe level of sIL-2R in serum is a useful marker to follow diseaseactivity. In addition to activated lymphocytes, increased IL-2R gene
expression can be found in chronic HCV infection. Activated T cells
can be an important source of elevated sIL-2R in HCV patients.
This elevated sIL-2R level can in turn act as a prognostic serum
biomarker to assess the aggressiveness of HCV infection (1-4).
Further studies are needed to elucidate the mechanism by which
expression of sIL-2R may be regulated pathophysiologically.
p<0.02
Fig.1. Mean value of the sIL-2R levels in patients with chronic HCV infection
as compared to controls

CONCLUSION
Increased concentrations of sIL-2R, a marker of T lymphocyte activation have been found in chronic HCV infection. HighersIL-2R levels were related to disease progression and genotype.
Conflict of Interest
The authors have declared no conflicts of interest.
Acknowledgement
This paper was supported by CNCSIS-UEFISCSU, PNII,
code 1165/2009.

p<0.02
Fig. 2. Mean value of the sIL-2R levels in HCV genotype 1 as compared togenotype 3

DISCUSSION
In this study we measured sIL-2R levels in serum of patientswith chronic HCV infection. SIL-2R in serum of HCV patients weresignificantly increased compared to those of normal controls.
We also examined, retrospectively, the relationship between the

REFERENCES
1. Rubin LA, Nelson DL. The soluble interleukin-2 receptor: biology,function, and clinical applications. Ann Intern Med. 1990; 113: 619-27.
2. Kawakami Y, Hayashi J, Ueno K, Ohmiya M, Kishihara Y, Yamaji K,
Kashiwagi S. Elevation of serum soluble interleukin-2 receptor levels
in patients with hepatitis C virus infection. Fukuoka Igaku Zasshi 1997;
88(7): 274-82.
3. Sugimura T, Motomura S, Sakai H, Nawata H. Increased serum
soluble IL-2 receptor levels following interferon therapy in patients withchronic hepatitis C. Hepatogastroenterology. 1999; 46(27): 1827-30.
4. Shintaro A, Ryoichi N, Toru M, Takeshi O, Akinari T, Makoto O.
Increased soluble IL-2 receptor levels during interferon and ribavirin
treatment are associated with a good response in genotype 2a/2b
patients with chronic hepatitis C. European J of Gastroenterology &Hepatology. 2008; 20: 373-8.

NIVELURILE RECEPTORULUI SOLUBIL AL INTERLEUKINEI-2 IN HEPATITACRONICA C INAINTE DE TERAPIA CU INTERFERONREZUMAT

Introducere: Se consideră că producerea receptorului solubil al interleukinei 2 (sIL-2R) este un marker caracteristic al activării
limfocitelor T şi ar putea să fie în legătură cu funcţia de imunoreglare. Obiective: Determinarea nivelurilor de sIL-2R în ser în infecţia
cu virusul hepatitei C (HCV) comparativ cu martori sănătoşi şi asocierea cu indicatorii histologici ai inflamaţiei. Material şi metode:
Cincisprezece pacienţi cu infecţie cronică cu HCV (6 bărbaţi, 9 femei, vârsta medie 47,35±10,78 ani) and 14 subiecţi sănătoşi (6bărbaţi, 8 femei; vârsta medie 35,00±15,45 ani) au fost incluşi în studiu. Diagnosticul pacienţilor cu infecţie cu HCV a fost stabilit pebaza examenului clinic, datelor de laborator, de ultrasonografie şi histopatologice. Subiecţii sănătoşi au prezentat serologie negativă
pentru hepatită, testele funcţiei hepatice normale şi datele de ultrasonografie normale. Nivelurile serice de sIL-2R au fost măsurate
folosind ELISA. Semnificaţia statistică a fost stabilită pentru p<0,05. Rezultate: sIL-2R în ser a fost măsurat la 15 pacienţi cu infecţie
cronică cu HCV înaintea tratamentului cu interferon (IFN) alfa. Nivelurile serice de sIL-2R au fost semnificativ mai crescute 809±43,6pg/ml decât cele ale subiecţilor sănătoşi 634±32,7 pg/ml, p<0,02 după testul Student t. Au existat corelaţii între nivelurile serice desIL-2R şi scorul activităţii histologice (r=0,482, p<0,01) şi nivelurile serice de alanin aminotransferază (r=0,363, p<0,01). sIL-2R în sera fost semnificativ mai crescut la pacienţi cu HCV genotip 1 comparativ cu genotip 3 (889±44,8 vs. 756,7±37,9; p<0,02). Concluzie:
Nivelurile serice de sIL-2R cresc din cauza infecţiei cu HCV, iar nivelul corespunde gradului de inflamaţie.
Cuvinte cheie: receptorul solubil al interleukinei 2, infecţia cu virusul hepatitei C, fiziopatologie
2012.22.4 (76)  Fiziologia - Physiology

25

EFFICACY AND TOXICOLOGICAL CHARACTER OF CORIANDER
AND CHAMOMILE EXTRACTSSIMONA ARDELEAN1, CRISTINA TRANDAFIRESCU1, CAMELIA OPREAN (SASS)2, ALINAHEGHES3, FLORIN BORCAN1*
1“Vasile Goldis” West University Arad, Faculty of General Medicine, Pharmacy and Dental Medicine, 1st Feleacului Str., Arad310396, Romania
2“Victor Babes” University of Medicine and Pharmacy Timisoara, Faculty of Medicine, 2nd Eftimie Murgu Sq., Timisoara-300041, Romania
3“Victor Babes” University of Medicine and Pharmacy Timisoara, Faculty of Pharmacy, 2nd Eftimie Murgu Sq., Timisoara-300041, Romania

ABSTRACT

Plants have the ability to synthesize a wide variety of chemical substances that are used to perform important biological functions,and to defend against attack from predators such as insects, fungi and herbivorous mammals. In this study, coriander and chamomile
extracts were obtained using two different pathways (Soxhlet and maceration). The extracts were characterized by density, pH, anddried substance content measurements, by Franz diffusion cell and applications on the mice skin. Generally the results indicate theobtaining of extracts good enough to be used in cosmetics.
Keywords: diffusion cell, TEWL, melanin content, erythema, Stratum corneum

INTRODUCTION
A new stage of plant chemistry (phytochemistry) started
in 1806, when Belgian pharmacist Sertűrner isolated for the
first time morphine from poppy latex. In his experimental study,
it was established for the first time a procedure to obtain theplants’ active principles, so-called the “quintessential” advocatedby his predecessors. In the following centuries, the research
is characterized by large-scale studies on phytochemicals,
and their principles (1, 2). These chemicals as essential oils,
tannins, vitamins, alkaloids, were studied in laboratories andclinics, establishing their therapeutic properties. Scientific studies have also shown that active principles have well-defined
action, and medicinal plants contain a number of substancesthat complement and enhance their action. The active principles
are sometimes scattered throughout the plant, and in this case
it is used together with the root, sometimes they are located inlarge amounts only in leaves, flowers, roots, seeds, fruits andso on; it is harvested and uses only those parts of the plant (3).
Coriander, Coriandrum sativum L. (Apiaceae family), is anannual plant with thin, and less branched roots. It has a cylindrical long stem (about 30-60 cm), finely furrowed, and branchedat the top. The flowers are white, reddish or purplish. It blooms
in June and July (4). Coriander is a culture plant that grows onbrown forest soils. The fruits (Fructus coriander) are collected
in order to be used in August and September (5).

The main component of coriander is the volatile oil withconcentrations between 0.2-1.7% and it depend on the climaticconditions in which the plant grew. Smaller fruits are rich involatile oil. Coriander fruits contain 13-20% fatty oil. There werealso determined protein substances, different acids, mannitol,
tocopherol, coumarins, a mixture of flavonoids, and resins (7).
Coriander oil is a colorless or pale yellow liquid with aromaticodor, characteristic and pleasant, like the rose, and with spicy taste.
It is less dense than water (d = 0.780 to 0.885) and is optically
active. It is soluble in alcohol, ether, chloroform, petroleum ether,fatty oils and is insoluble in water, but it communicates its flavor.
Coriander oil is constituted mostly of linalool and other terpenes(pinene, geraniol, geranyl acetate, borneol, citronellol, camphor).
Coriander fruits possess spasmolytic and bactericidal actions. The
volatile oil is used mainly as a flavoring in perfumery (8).
Chamomile, Chamomilla Matricaria recutita (Asteraceae
family) is an annual herbaceous plant which has a branchedsurface root in soil. The stem is often branched, each branchterminated by an inflorescence. It blooms from May to August(9). Chamomile is a common plant throughout the country, foundin uncultivated places, green land, beside roads, meadows,
healthy soils. The flowers (Flores Chamomillae) are used fortheir content: bitter sesquiterpene substances such as matricine.

Fig. 1. Coriander (6)Fig. 2. Chamomile (10)Received 15th of September 2012. Accepted 20th of October 2012. Address for correspondence: Florin Borcan, Pharmaceutical Chem. Dept., Faculty of Pharmacy,“Victor Babes” University of Medicine and Pharmacy Timisoara, 2nd E. Murgu Sq. 300041 Timisoara, RO, phone/fax: +40256220479, e-mail: fborcan@umft.ro26

Fiziologia - Physiology  2012.22.4 (76)Other substances isolated from chamomile are flavonoids,
represented by flavones and flavonols as aglycones, but mostly
as glycosides. There have been highlighted two coumarins,
phenolic acids, and high fatty acids. Chamomile flowers also
contain mucilage, a phytosterol, a tricyclic alcohol (C15H26O),
a tricyclic hydrocarbon (C15H24), and a keto-alcohol (C15H24O2).
The most important component is the volatile oil which is themost active ingredient of chamomile. It is a blue, dense liquid,with aromatic odor and bitter taste. In contact with air and lightits color changes to green, watery brown (11).
In this study, coriander and chamomile extracts were obtained through two different pathways and there were characterized by density, pH, and dried substance content measurements;
the concentrated extracts were tested by the mice skin model.
MATERIALS AND METHODS
The coriander and chamomile extracts were obtained
through two different pathways: (i) extraction with Soxhlet
apparatus at 78 °C for 2 hours, and (ii) maceration at room
temperature for 7 days.

maintained during the experiments in National Institute of Animal
Health (NIAH) recommended conditions: 12 hours light-darkcycles, food and water ad libidum, temperature 24±2 ºC, humidity
above 55%. The suspensions were applied on mice skin for 9
weeks (0.3 mL / application). There were used 15 mice: group
CoS (3 mice, treated with suspension based on CoS extract),group ChS (3 mice, treated with suspension based on ChS extract), group CoM (3 mice, treated with suspension based on CoM
extract), group ChM (3 mice, treated with suspension based on
ChM extract) and group 0 (3 mice as blank treated with ethanol).
The applications and measurements were done twice a week.
After applications, each determination was performed within
30 minutes. All the measurements on the mice skin were carriedout with a Multiprobe Adapter System (MPA5) from CourageKhazaka, Germany.
RESULTSThe coriander and chamomile extracts present different pH,
density, and dried substance content values depending on thechosen pathway (Table II).

Table I. Samples codes and descriptions

Table II. Samples characterization

Code

Raw materials
Technique

M.U.

Quantity

Code

CoS

Coriander
Ethanol (96%)
Distilled water
Soxhlet

g
ml
ml

5
40
10

CoS

0.94

5.66

3.8

ChS

0.98

6.20

5.6

Chamomile
Ethanol (96%)
Distilled water
Soxhlet

g
ml
ml

5
40
10

CoM

0.92

5.72

4.9

ChM

0.91

6.08

6.1

CoM

Coriander
Ethanol (96%)
Distilled water
Maceration

g
ml
ml

5
40
10

ChM

Chamomile
Ethanol (96%)
Distilled water
Maceration

g
ml
ml

5
40
10

ChS

The obtained extracts were finally concentrated in order toobtain suspensions which can adhere to the mice skin. Thesesuspensions efficacy was evaluated by Franz diffusion cell in
the following conditions: 7 mL phosphate buffer (pH = 7.4) as
acceptor phase; effective diffusion area was 1.767 cm2; rotation
speed was 450 rpm; experiments were done at a temperatureof 37±0.5 °C for 6 hours. Acceptor phase was extracted with anautomatically system and replaced with fresh medium. Quantitative measurements were made with a UV-Vis spectrophotometer
Hewlett Packard model 8453. A synthetic membrane and samples of rat skin were used for the study.
Ten weeks old females of hairless mouse SKH1 were2012.22.4 (76)  Fiziologia - Physiology

Density [g/cm3]

pH

Dried substancecontent [%]Suspensions with different concentrations were used to
draw the calibration curves. Calibration curves were done inorder to evaluate the suspensions efficacy with Franz diffusion
cell (Figure 3).

Fig. 3. Calibration curve used in the case of CoS sample

It was draw the release profile of every obtained suspension. An
example of these is Figure 4.
27

Fig. 4. Release profile of CoS sample

There were not observed any differences about the miceskin macroscopic aspect between the first and the last week ofthe experiment (Figure 5). The evolutions of the skin parameters
are presented in Figure 6.

b

a

a

Fig. 5. Macroscopic aspect of back skin (magnification: 3x) for one mousefrom CoS group in(a) first week; (b) last week of experimentbcFig. 5. Evolution of skin parameters: (a) transepidermal water loss; (b) hydration of Stratum corneum;(c) melanin content; (d) erythemaDISCUSSION
The diffusion capacity of the active principle from theobtained suspensions was studied don synthetic membrane(PORAFIL Membranfilter) and on rat skin (Spraque Dawley). In
all cases, the amount of active principle which diffuses throughthe membrane is high (over 40%), regardless of membraneused. The best results were obtained from samples obtainedby maceration extraction process.
TEWL measurements showed increases in transdermal
water loss less pronounced, which indicates low skin alterations
or substances with low harmful effect. The melanin content
28

Fiziologia - Physiology  2012.22.4 (76)evolutions correspond to an upward trend, which could not becorrelated with the type of suspension. In many studies there
is a slight linear increase melanin content in the skin over skintesting period. Suspensions based on coriander extracts present
lower erythema differences and this trend is probably due tothe chamomile allergic reaction (12). Level of hydration of theStratum corneum had a downward trend in all cases.
CONCLUSIONS
Medicinal plants are used for their medicinal properties.
Only few plants or their phytochemical constituents have been
proven to have medicinal effects by rigorous science or havebeen approved by regulatory agencies. In this research there
were obtained coriander and chamomile extracts using two
different pathways. The extracts were concentrated and theirefficacy was evaluated by Franz diffusion cell and applications
on the mice skin. There were recorded good results of diffusion
through membranes and good results of TEWL and melanincontent. It was observed the well known chamomile allergic
reaction by erythema evolution study.
REFERENCES
1. Chast F. Chapter 1, A History of Drug Discovery: From first steps ofchemistry to achievements in molecular pharmacology. The Practiceof Medicinal Chemistry (Third Edition), 2008.
2. Aggrawal A. History of Toxicology. Encyclopedia of Forensic andLegal Medicine, 2005.
3. Gherman C, Culea M, Cozar O. Comparative analysis of some active principles of herb plants by GC/MS. Talanta 2000; 53(1): 253-262.
4. Coiciu E, Racz G. Plante medicinale si aromatice. vol I, Ed. RPR,
Bucharest, 1962.
5. Femeia stie. http://www.femeiastie.ro/sanatate-dieta/terapii-naturiste-si-alternative/importanta-hormonilor-vegetali. Last accessed on
Dec 7th, 2012.
6. Coriander. http://peonyden.blogspot.ro/2008/11/coriander-compound-umbel-flower.htm. Last accessed on Dec 7th, 2012.
7. Burdock GA, Carabin IG. Safety assessment of coriander (Coriandrum sativum L.) essential oil as a food ingredient. Food and Chemical
Toxicology 2009; 47(1): 22 - 34.
8. Drogheria. http://www.drogheria.ro/Noutati/Galbenele-8-124.html.
Last accessed on Dec 7th, 2012.
9. Krier H. Medicina naturista - Miracolul plantelor. Ed. Helicon,
Timisoara, 1997.
10. Chamomile. http://www.wildcrafted.com.au/Botanicals/Chamomile.
html. Last accessed on Dec 7th, 2012.
11. Mukherjee PK, Ponnusankar S, Pandit S, et al. Botanicals as
medicinal food and their effects on drug metabolizing enzymes. Foodand Chemical Toxicology 2011; 49(12): 3142-53.
12. Petronilho S, Maraschin M, Coimbra MA, et al. In vitro and invivo studies of natural products: A challenge for their valuation. The
case study of chamomile (Matricaria recutita L.). Industrial Crops andProducts 2012; 40: 1-12.

EFICACITATEA SI CARACTERUL TOXICOLOGIC AL UNOR EXTRACTE DECORIANDRU SI MUSETELREZUMAT

Plantele au capacitatea de a sintetiza o mare varietate de substante chimice, care sunt utilizate pentru a efectua diverse functii
biologice importante, si pentru a apara impotriva atacurilor unor pradatori, cum ar fi insectele, ciupercile si mamiferele erbivore. In
acest studiu, extracte de musetel si coriandru au fost obtinute folosind doua cai diferite (Soxhlet si macerare). Extractele au fostcaracterizate prin masuratori de densitate, pH si continut de substanta uscata, cu ajutorul celulei de difuzie Franz si prin aplicatii pepiele de soarece. In general rezultatele indica obtinerea unor extracte destul de bune pentru a fi folosite pentru produse cosmetice.
Cuvinte cheie: celula de difuzie, pierdere de apa transdermica, continut de melanina, eritem, Stratum corneum

2012.22.4 (76)  Fiziologia - Physiology

29

OLEANOLIC AND URSOLIC ACID IN HUMAN SKIN CANCER– A PRELIMINARY IN VITRO COMPARATIVE STUDYCAMELIA SASS1, FLORINA BOJIN1, ALINA HEGES2, ATENA GALUSCAN3, VIRGIL PAUNESCU1
1
Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara
2
Department of Pharmaceutical Technology, “Victor Babes” University of Medicine and Pharmacy Timisoara
3
Faculty of Stomatology, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT

Triterpenic compounds are the subject of many studies because of their multiple activities including anticancer activity. The aim of
this study is to compare the activity of ursolic acid (UA) and oleanolic acid (OA) on A375 human melanoma cells, using the MTT invitro cell viability assay. A375 cells were treated with different concentration of ursolic and oleanoic acid (10, 20, 30, 50, 75 μM) for
24 and 48 hours. UA acted as a cytotoxic compound at concentration of 50 and 75 μM, cell viability decreasing slightly after 24 h
(80% and 97%), and more pronounced after 48 h (47% and 46% respectively) leading to the conclusion that ursolic acid could be
an in vivo anti-proliferative agent on A375 human melanoma cells in a dose- and time-dependent manner. For cells treated with OA,
at 48 h, we observed a very slightly decreasing in cell viability (93-102%) for all the concentration, showing a poor anti-proliferative
effect at the same concentration.
Key words: ursolic acid (UA), oleanolic acid (OA), anti-proliferative, MTT assay

INTRODUCTION
Importance of research skin cancer increases with its
incidence, statistics showing that the number of skin cancers isincreasing every year. In this direction, triterpenic compounds
are the subject of many studies because of their multiple
activities including anticancer activity (1). Oleanoic acid (OA)
and its isomer, ursolic acid (UA) are pentacyclic triterpene
compounds, with 30 carbon atoms (2) which exist in nature
both in free form and as aglycones for saponins triperpenice:oleanane and ursane (3, 4, 5). Their existence is widespread innature, oleanolic acid were isolated from over 120 species ofplants of which the best known are rosemary, oregan, lavender,
Olea Europeae L., Swertia mileensis T., Ligustrum lucidum it.,and Actostaphylos uva-urs, Eriobotrya japonica, Rosmarinus
officinalis, and Glechoma hederaceae (2, 4,5).
The pharmacological activity of oleanolic acid and ursolic
is multiple, related, totaling antitumor activity, antiviral, antiinflammatory, antimicrobial, hepatoprotective, antidiabetic,
gastroprotective and hemolytic (3, 4). Antitumor effects ofursolic and oleanolic acid were observed in various types ofcancers such as breast cancer, melanoma, hepatoma, prostate
carcinoma and leukemia and lymphomas (3, 6). Ursolic acidhas demonstrated cytotoxic effects on P-388 and L-1210
leukemia cells, A-549 human lung carcinoma cell, KB humanepidermoid carcinoma (2, 4). Feng’s study results achieved by
MTT method, show that ursolic acid causes inhibition of B16 cellproliferation with IC50 value at 10 µM (2), which strengthens
the hypothesis that supports anti-proliferative effects of ursolicacid.
Moreover, Es-Saady et al. showed ursolic acid’s effecton G1 cell cicle on B16 melanoma cells (7). Baconasoni etal. examined the effect of ursolic acid on the proliferationand differentiation of A375 human melanoma cells and andshowed that UA inhibits tumor cells proliferation in a doseand time- dependent maner (8). However, the mechanisms
by which these triterpenes act as tumor inhibitors in the skin
may be multiple and complex, some of which are still understudy (9).
The aim of this study is to compare the activity of ursolic
acid and oleanolic acid on A 375 human melanoma cells, using
the MTT cell viability test.
MATERIALS AND METHODS
Preparation of ursolic and oleanolic acid solution
Ursolic and oleanolic acid (Sigma Aldrich Germany)
were dissolved in Dimethyl sulfoxide (DMSO; Sigma-Aldrich,Ayrshire, UK) and stored at 2-8 oC. For all experiments, final
concentration of the tested compounds was prepared bydiluting the stock solution with DMEM.
Cell culture
A375 human melanoma (ATCC, USA) was cultured in
DMEM containing 10% FCS (fetal bovine serum, PromoCell,
Heidelberg, Germany), 1% glutamine, and 1% penicillinstreptomycin (Pen/Strep, 10,000 IU/mL; PromoCell, Heidelberg,
Germany). Cells were cultured in 5 % CO2 atmosphere at 37 o
C.
MTT in Vitro Analysys
The A375 cell line (ATCC, USA) was seeded onto a
96-well microplate and attached to the bottom of the wellovernight. After 24 h, 150 µL of new medium containing
Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco BRL,

Received 16th of September 2012. Accepted 27th of October 2012. Address for correspondence: Camelia Sass, PhD student, Department of Functional Sciences, “Victor
Babes” University of Medicine and Pharmacy Timisoara, 2A Eftimie Murgu Square, RO-300041 Timisoara, phone/fax: +40256490507, e-mail: camelia.sass5@gmail.com

30

Fiziologia - Physiology  2012.22.4 (76)Invitrogen, Carsbad, Ca, USA) and the tested substanceswere added and incubated for 24 h and 48 h respectively;
the medium was supplemented with 10% fetal calf serum(FCS; PromoCell, Heidelberg, Germany) and 1% penicillin/
streptomycin mixture (Pen/Strep, 10,000 IU/mL; PromoCell,
Heidelberg, Germany). Melanoma cells were passaged at
confluence after treatment with 5 mM EDTA; the living cells
were then assayed by the addition of 15 µL of 5mg/mL MTT
solution. The intact mitochondrial reductase converted
and precipitated MTT as blue crystals during a 3-h contact
period. The medium was then removed, and the precipitatedcrystals were dissolved in 150 µL dimethyl sulfoxide(DMSO; Sigma-Aldrich, Ayrshire, UK). Finally, the reduced
MTT was spectrophotometrically analyzed at 570 nm using
a microplate reader; wells with untreated cells were used
as controls. All in vitro experiments were performed on two
microplates with at least four parallel wells. DMSO was used
to prepare stock solutions of the tested substances, and weused DMSO solution as a negative control. The dilution rate
was 1:400 and the concentration of stock solution was 10
mM.

Fig.2.a

RESULTS
Cell morphology

Fig.2. Effect of UA (a) and OA (b) on A375 melanoma cells viability. Cell weretreated with different concentration and cell viability was assesed by MTT
assay at 24 h and 48 h.

Control

UA, 10 µM

UA, 20 µM

UA, 30 µM

UA, 50 µM

UA, 75 µM

Fig.1. A375 cell morphology: control, cells treated with different concentrationof ursolic acid (10, 20, 30, 50, 75 µM)

Figure 1 show the morphology of A375 cell treated with ursolic acid at different concentration and non-treated cells (control).
The morphology of cells treated with oleanolic acid looked likethe morphology of cells treated with ursolic acid.

After 24 h in cells treated with UA, we observed an increase of
proliferarion to 139%, 121%, 102 % (10, 20, 30 μM) compared
to control. For the higher concentration, 50 and 75 μM weobserved a decrease of cell viability percentage to 78%, 98% respectively. For cells treated with OA, at 24 h there all theconcentration (10-75 Μm) showed an increase of proliferation(131%, 107%, 158%, 101%, and 119% respectively). At 48 h,
for cells treated with UA, MTT assay did not show a difference incell proliferation between 10-30 μM, but for 50, 75 μM, it showeda decrease of cell viability from 78% to 47% and from 97% to
46% respectively (Figure 2.a). For cells treated with OA, at 48
h, we observed a slightly decreasing in cell viability (93-102%)
for all the concentration (Figure 2.b).

Cell proliferation assay
Cell proliferation assay was performed using the MTT
assay. MTT is a tetrazolium salt that is cleaved to formazanby the succinate dehydrogenase system which belongs to themitochondrial respiratory chain and is only active in viable cells.
The mitochondrial succinate dehydrogenase reduces the yellow
tetrazolium salt into water-insoluble purple formazan. A375 cells
were treated with different concentration of ursolic and oleanoic
acid (10, 20, 30, 50, 75 μM) for 24 and 48 hours.
2012.22.4 (76)  Fiziologia - Physiology

Fig.3.a31

Fig.3. Comparative effect of ursolic and oleanolic acid at 24 h (a) and48 h (b), assesed by MTT assay.

DISCUSSION
Due to continuous increase in skin cancer frequency, inthe past 10 years the number of melanoma cases augmented
more than any type of cancers (10), researcher’s attention heads
to obtain more effective and less toxic anticancer compounds(11). In this prospect, some of the compounds studied for theiranticancer potential are ursolic and oleanolic acid. Ursolic
acid inhibits growth of tumor cells through multiple functions
including cytotoxicity, induction of apoptosis and prevention ofangiogenesis (12). Our study attempted to discover the activityof ursolic and oleanolic acid and to make a comparison between
their activities on A375 cell line. A375 is a human melanoma
cell line, amelanotic, derived from a 54 year old female withmalignant melanoma.
Using the MTT assay we demonstrated that UA has ananti-proliferative effect on A375 melanoma cells line in a dose
and time-dependent manner. UA acted as a cytotoxic compoundat concentration of 50 and 75 μM, cell viability decreasingslightly after 24 h (80% and 97%), and more pronounced after
48 h (47% and 46% respectively). Lower concentrations did not
induce any cytotoxic activity neither after 24 h, nor after 48 h.
Furthermore concentrations between 10 and 30 μM showed anincrease of MTT reduction, which could lead to the conclusionthat ursolic acid induce the proliferation of A375 melanoma cells.
Other authors reported that MTT could underestimate the antiproliferative effect of substances demonstrating through othermethods such as Trypan blue exclusion method (10) or counting
with hemocytometer (7) that the true number of viable cells could
not correspond with the values obtained by MTT assay. This is
the case of M4Beu melanoma (10), B16 mouse melanoma (7),
MCF7 human breast cancer and HaCaT keratinocyte-derived
cells (10, 13). As we did not use another method to evaluate theeffects of UA on A375 cells, we cannot decide if the MTT values
are the true values or not.
Other results on ursolic acid’s activity on melanoma cancer
cells, such as melanoma B16 murine melanoma and M4Beu
human melanoma report that depending on exposure time,anti-proliferative activity of ursolic acid occurs at concentrations
IC = 10-50 µM (10, 14, 15). However, Manu et al, reported thatafter 48 hours of exposure, concentration between 10-50 µM,
showed a non-toxic activity on B16 F10 murine melanoma cells,
32

but it found to be 76 % and 100% cytotoxic at concentration of
75 µM and 100 µM (16).
Comparison to the ursolic acid, oleanolic acid did not show
a good anti-proliferative activity at the same concentration
neither after 24 h of exposure, nor after 48 h. George et al,
showed using a MTT assay on HaCaT cells, after exposureof 200 μM for 72 hrs, only 26.2% cytotoxicity was observed(11). Oleanolic acid also was observed to reduce metastatisof B16 melanoma cells (17). All taken together could lead tothe conclusion that oleanolic acid’s antiproliferative activity is at
higher concentration than ursolic acid.
CONCLUSION
In conclusion, we may say that ursolic acid could be an
in vivo anti-proliferative agent on A375 human melanoma cells
in a dose- and time-dependent manner. Oleanolic acid show
a poor anti-proliferative effect at the same concentration andfurther investigation with higher concentration are needed.
REFERENCES
1. Muceniece R, Saleniece K, Riekstina U, Krigere1 L, Tirzitis G,
Ancans J. Betulin binds to melanocortin receptors and antagonizes
a-melanocyte stimulating hormone induced cAMP generation inmouse melanoma cells. Cell Biochem Funct. 2007; 25: 591-596.
2. Feng H, Chen W, Zhao Y, Ju X-L. Anti-tumor activity of oleanolic,ursolic and glycyrrhetinic acid. The Open Natural Products Journal.
2009, 2: 48-52.
3. Sun H, Fang W-S, Wang W-Z, Hu C. Structure-activity relationshipsof oleanane-and ursanetype triterpenoids. Botanical Studies, 2006,
47: 339-368.
4. Liu J. Pharmacology of oleanolic acid and ursolic acid. J
Ethnopharmacol. 1995, 49:57-68.
5. Lim SW, Hong SP, Jeong SW, Kim B, Bak H, Ryoo HC, Lee SH,
Ahn SK. Simultaneous effect of ursolic acid and oleanolic acid on
epidermal permeability barrier function and epidermal keratinocyte
differentiation via peroxisome proliferator-activated receptor-α. J
Dermatol. 2007; 34: 625-634.
6. Yim EC, Lee MJ, Lee HK, Umy SJ, Park JS. Antiproliferative andantiviral mechanisms of ursolic acid and dexamethasone in cervicalcarcinoma cell lines. Int J Gynecol Cancer. 2006; 16: 2023-31.
7. Es-Saady D, Simon A, Ollier M, Maurizis JC, Chulia AJ, Delage C.
Inhibitory effect of ursolic acid on B16 proliferation through cell cycle
arrest. Cancer Lett. 1996; 10:193-197.
8. Bonaccorsi I, Altieri F, Sciamanna I, Oricchio E, Grillo C, Contartese
G, Galati EM. Endougenous reverse transcriptase as a mediator of
orsolic acid’s anti-proliferative and differentiating effects in humancancer cell lines. Cancer Lett. 2008; 236:130-139.
9. Zhang P, Li H, Chen D, NI J, Kang Y, Wang S. Oleanolic acidinduces apoptosis in human leukemia cells through caspase activationand poly(ADP-ribose) polymerase cleavage. Acta Biochim Biophys
Sin. 2007; 39(10): 803-809.
10. Hardman PO, Duval RI, Delage C, Simon A. Ursolic acid induces
apoptosis through mitochondrial intrinsic pathway and caspase-3
activation in M4Beu melanoma cells. Int. J. Cancer. 2005; 114: 1-11.
11. George VC, Kumar N, Suresh PK, Kumar RA. Apoptosis-induced
cell death due to oleanolic acid in HaCaT keratinocyte cells - a proofof-principle approach for chemopreventive drug development. Asian
Pacific J Cancer Prev. 2012; 13: 2015-20.

Fiziologia - Physiology  2012.22.4 (76)12. Wang X, Zhang F, Yang L, Mei Y, Long H, Zhang, Zhang J, Suyila
O, Su X. Ursolic acid inhibits proliferation and induces apoptosis ofcancer cells in vitro and in vivo. J Biomed Biotechnol. 2011: 419343.
Es-Saady D, Simon A, Jayat-Vignoles C, Chulia AJ, Delage C.
MCF-7 cell cycle arrested at G1 through ursolic acid, and increased
reduction of tetrazolium salts. Anticancer Res. 1996; 16(1): 481-6.
Pinon A, Limami Y, Micallef L, Cook-Moreau J, Liagre B, Delage C,
Duval RE, Simon A. A novel form of melanoma apoptosis resistance:
Melanogenesis up-regulation in apoptotic B16-F0 cells delays ursolic
acid-triggered cell death. Exp Cell Res. 2011; 317(12): 1669-76.
13. Cha DS, Shin TY, Eun J S, Kim DK, Jeon H. Anti-metastatic

properties of the leaves of Eriobotrya japonica. Arch Pharm Res. 2011;34 (3): 425-436.
14. Manu KA, Kuttan G. Ursolic acid induces apoptosis byactivating p53 and caspase-3 gene expressions and suppressing
NF-κB mediated activation of bcl-2 in B16F-10 melanoma cells. Int
Immunopharmacol. 2008; 8: 974-981.
15. Lucio KA, Rocha G, Monca o-Ribeiro LC, Fernandes J, Takiya
CM, Gattass CR. Oleanolic acid initiates apoptosis in non-small celllung cancer cell lines and reduces metastasis of a B16F10 melanoma
model in vivo. PLoS ONE, 2011; 6(12).

ACIDUL OLEANOLIC SI URSOLIC IN CANCERELE CUTANATE UMANE –STUDIU COMPARATIV PRELIMINAR IN VITROREZUMAT

Compusii triterpenici sunt investigati in multiple studii actuale datorita actiunii acestora in diverse afectiuni, inclusiv activitatea
antitumorala. Scopul acestui studiu a fost acela de a compara activitatea acidului ursolic (UA) si oleanolic (OA) asupra celulelor liniei
de melanom uman A375, folosind testul in vitro de viabilitate MTT. Celulele liniei A375 au fost tratate cu diferite concentratii de acidursolic si oleanolic (10, 20, 30, 50, 75 μM) timp de 24 de ore, respectiv 48 de ore. UA a avut activitate citotoxica la concentratii de 50
si 75 μM, viabilitatea celulara prezentand o scadere usoara dupa 24 de ore (80% si 97%), dar mult mai pronuntata dupa 48 de orede actiune (47%, respectiv 46%), ceea ce a dus la concluzia ca acidul ursolic ar putea fi un agent anti-proliferativ in cazul celulelorde melanom uman A375, actiunea acestui compus fiind dependenta de doza si de timpul de actiune. Pentru celulele tratate cu OA,
la 48 de ore se observa o scadere usoara a viabilitatii celulare (93-102%), pentru toate concentratiile utilizate, ceea ce sugereaza
un efect anti-proliferativ scazut al acestui compus comparativ cu UA, pentru concentratii similare.
Cuvinte cheie: acid ursolic (UA), acid oleanolic (OA), anti-proliferativ, test MTT

2012.22.4 (76)  Fiziologia - Physiology

33

THE RELATIONSHIP BETWEEN DIABETIC RETINOPATHY AND
CORONARY ARTERY DISEASE IN BANAT COUNTY
N. NICULESCU1, I. ZOLOG1, M. MUNTEAN1, O. BORUGA1, D. BRIE2
1
Ophthalmology Clinic, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
2
Cardiovascular Disease Institute, “Victor Babes” University of Medicine and Pharmacy Timisoara, Romania

ABSTRACT

We conducted an observational study to assess the relationship between diabetic retinopathy and coronary artery disease in a
population from Banat County, Romania. Our study included 231 patients (121 men and 110 women) with type 2 diabetes aged
40-65 years who were examined for retinal microvascular changes by retinal photographs in the Ophthalmology Clinic Timisoara
between May 2008 and September 2012. The patients involved in our study were selected from a larger group of 3,400 patients.
The mean follow-up period was 28 ± 12 months. The initial ophthalmologic examination revealed that 109 subjects had some degreeof DR (prevalence rate of ~ 40%), including 87 patients with non-proliferative (NPDR) (prevalence rate of 32%), and 22 patientswith proliferative diabetic retinopathy (PDR), (prevalence rate of 8%). After adjustments for risk factors, diabetic retinopathy was
associated with two times higher incidence of unstable and stable angina - odds ratio =1.95 (95% CI 1.13 - 3.35), and higher incidence of myocardial infarction-odds ratio = 2.36 (95% CI 0.78 - 7.14). The risks of CAD were higher in individuals with proliferative
retinopathy (PDR) - for unstable and stable angina odds ratio =2.86 (95% CI 1.13 - 7.22), for myocardial infarction odds ratio = 4.29
(95% CI 1.1-16.7). Also, in patients with non-proliferative retinopathy (NPDR) was a slightly higher incidence of unstable and stableangina odds ratio=1.76 (95% CI 0.99 - 3.14), and myocardial infarction - odds ratio=1.43 (95% CI 0.44 - 4.59). Our data show thatthe presence of retinopathy in individuals with type 2 diabetes was associated with a twofold higher risk of incident CAD (unstable
and stable angina, myocardial infarction), independent of glycemic levels and cardiovascular risk factors. This association appears
to be graded with retinopathy severity, as in patients with proliferative retinopathy was associated with four time higher incidence of
myocardial infarction and three time higher incidence of stable and unstable angina.
Key words: diabetic retinopathy, coronary artery disease, myocardial infarction, angina, incidence

PURPOSE
We conducted an observational study to asses the relationship between diabetic retinopathy and coronary artery disease
in a population from Banat County, Romania.
INTRODUCTION
Diabetic retinopathy (DR) is a common and devastating
microvascular complication in type 1 and 2 diabetes patients.
The leading cause of mortality in patients with diabetes iscoronary arteries disease (CAD) (1,2). CADs are listed as thecause of death in ≈ 65% of persons with diabetes (3). In diabetic
patients myocardial ischemia due to coronary atherosclerosis
commonly occurs without symptoms. As a result, multi-vessel
atherosclerosis often is present before ischemic symptoms occur and before treatment is instituted. The risk of a myocardial
infarction in diabetic patients with no evidence of CAD matches
that in patients without diabetes who have had a previous myocardial infarction, supporting the idea of diabetes as a coronary
equivalent (4). A delayed recognition of various forms of CAD
undoubtedly worsens the prognosis for survival for many diabetic
patients (5). A number of recent studies showed that diabetes is
an independent risk factor for CAD in both men and women (6,7).
However, it is unclear if the presence of DR, a microvascularcomplication, is an increased risk of CAD.

METHODS
Study population
Our study included 231 patients (121 men and 110 women),
aged 40-65 years who were examined for retinal microvascularchanges by retinal photographs in Ophthalmology Clinic Timisoara between May 2008 and September 2012. The patientswere selected from a larger group of 3,400 patients who visited
the Ophthalmology Clinic Timisoara for eye examination between
May 2008 and September 2012. We selected for inclusion in ourstudy only patients with fasting plasma glucose of ≥ 7.0 mmol/l(126 mg/dl) or more, using diabetic medications, or a physician’s
diagnosis of diabetes. Participants diagnosed with diabetes
were categorized as newly diagnosed diabetes (NDM) patients.
Those with self-reported diabetes and either on current treatment(insulin or oral hypoglycemic medication) or with diabetic glucose
values were categorized as having known diabetes (KDM). In
these patients we assessed hypertension on the basis of systolicand diastolic blood pressure at three consecutive visits (morethan 140 systolic or/and 90 diastolic mmHg, measure in a rest
condition with a mercury sphygmomanometer). The diagnosisof hypertension was confirmed by a cardiologist.
We excluded from our study patients with history of stroke

Received 10th of September 2012. Accepted 20th of November 2012. Address for correspondence: Niculescu N, Ophtalmology Clinic, “Victor Babes” University of Medicine
and Pharmacy Timisoara, 2nd E. Murgu Sq. 300041 Timisoara, RO, phone/fax: +40256220479, e-mail: nico28nov@yahoo.com

34

Fiziologia - Physiology  2012.22.4 (76)or transient ischemic attack; patients with known prior coronary
arteries diseases (history of angina, previous myocardial infarction), significant peripheral vascular disease (previous surgery or
symptoms of intermittent claudication); aortic aneurysm; thyroid
disease; nephrotic syndrome and patients with type 1 diabetes.
In all patients with known or recent diabetes a complete physicalexamination, ECG, and blood pressure was performed, then thepatients were referred for a complete eye examination. Required
data were collected, including: sex, age, disease duration, typeof diabetes, dyslipidemia (based on laboratory findings or useof any lipid-lowering drugs).
Participants were asked to go to a specified laboratory forcollection of blood samples, following a 12-hour fast to identify
the undiagnosed patients. Glycosylated hemoglobin (HbA1c)
level was measured and values less than 7% were considered
as indicators of good glycemic control. Body mass index (BMI)(weight in Kg, divided by height in meters squared) was calculated. The WHO (1977, 1979) classification for BMI was used toestimate the degree of obesity. Complete eye examinations were
performed. Fundus photography performed with a digital camera and full retinal and macular examination conducted by two
experimented operators were carried out. Diabetic retinopathy
is primarily classified into non proliferative DR (NPDR), formerly
termed simple, or background retinopathy (mild, moderate andsevere), and proliferative DR (PDR). We use for classificationof diabetic retinopathy an adaptation of “The International Clinical Diabetic Retinopathy Disease Severity Scale” (8). DR was
defined to include the presence of any of the above lesions.
The presence of coronary arteries diseases was defined aspresence of pectoris angina (with one of the following: specificischemic changes on ECG, positive effort test, positive angiocoronarography) and acute coronary syndrome (ST elevation
myocardial infarction -typical chest pain, specific rise and fallof cardiac enzyme and ST elevation on ECG, unstable anginatypical chest pain, ischemic modification on ECG, without riseof cardiac enzyme). The diagnosis of coronary arteries disease
was made by a cardiologist from Cardiovascular Disease Institute
Timisoara. We performed angiocoronarography in all patientswith CAD.
Statistical analysis
We used the MedCalc Software for calculating the odds
ratio, confidence interval, prevalence proportion. The results forcontinuous variables were given as mean ± SD and for categorical variables as percentage. The limit of P value of statisticalsignificance was considered 0.05.
RESULTSWe included in our study 231 patients (121 men and 110
women) with type 2 diabetes, aged 40-65 years. The meanfollow-up period was 28 ± 12 months. The initial ophthalmologic
examination revealed that 109 subjects had some degree ofDR (prevalence rate of ~ 40%), including 87 patients with nonproliferative (NPDR) (prevalence rate of 32%), and 22 patients2012.22.4 (76)  Fiziologia - Physiology

with proliferative diabetic retinopathy (PDR), (prevalence rate of8%). We detected clinically significant macular edema (CSME)
in 17 patients (6.25%). The clinical characteristics of patients areshown in Table I. Patients with DR (109 patients) were included
in group A. Patients without DR (122 patients) were included ingroup B. The characteristics of patients with DR (group A) andwithout DR (group B) are shown in Table II. The patients with DR
had a significantly longer duration of known diabetes (P < 0.001),
a higher ratio of being on insulin therapy (P < 0.001), higher
serum creatinine levels (P = 0.026). There were no significant
differences with regard to patient age, sex, risk factors for CAD,
and other biochemical parameters (Table II).
Table I. Characteristics of total patients with type 2 diabetes

Total (n = 231)
Clinical characteristics
Age (years)
Sex (Men/Women)
BMI (kg/m 2)
Diabetes
Duration (years)
HbA 1c (%)
Retinopathy (%)
Proliferative retinopathy (%)
Macular edema
Nephropathy (%)
Peripheral neuropathy (%)
Cardiovascular risk factors
Hypertension (%)
Dyslipidemia (%)
Smoking (%)≥2 Cardiovascular risk factors (%)

51 ± 12121/11027.2 ± 6.1
7.4 ± 48.5 ± 2.2
109 (40%)
22 (8.0)17(6.25%)
74(32%)
47 (22%)162 (70%)
150 (65%)
99 (43%)143 (62%)

Table II. Characteristics of patients in group A and B

Characteristics
Age (years)
Sex (Men/Women)
BMI (kg/m 2)
Duration of diabetes (years)
HbA1c (%)
Creatinine (mg/dL)
No. of patientstaking insulin
Total cholesterol(mg/dL)HDL (mg/dL)
LDL (mg/dL)≥2 Cardiovascularrisk factors (%)
Smoking (%)
Hypertension

Group A
(With DR)
109 pts
52±10 y59/56
26.5±510.4±6

Group B
(WithoutDR) 122 pts
53±9 y62/5427.3±66.2±4

P value7.9±1.3
1.13±0.47
33(30%)

8.1±1.7
0.94±0.20
17 (14%)

P=NS
P=0.026
P<0.001

201.4±50.2

213±48.6

P=NS

42.3±9.5
126.5±33.8
69 (63.3%)

40.1±8.7
128±40.2
74 (61%)

P=NS
P=NS
P=NS

47 (43%)75 (69%)52 (42.6%)
87 (71%)

P=NS
P=NS

P=NS
P=NS
P=NS
P < 0.001

35

Over the follow-up period, 101 patients (43.7%) were diagnosed with CAD, 85 patients (36.7%) with stable and unstableangina, and 16 patients (~7%) with myocardial infarction. In group
A there were 59 patients (54%) with CAD, 49 patients (45%)
with stable and unstable angina, and 10 patients (9.17%) withmyocardial infarction. The incidence of CAD was significantly
higher than in group B: 42 patients (34.4%) with CAD, 36 patients(29.5%) with stable and unstable angina, and 6 patients (5%)
with myocardial infarction (p < 0.001) (Table III).
Table III. Incidence of coronary arteries disease

Total patients Group A
231 pts
with DR
109 pts
101 pts-43.7% 59 pts-54%
85 pts-36.7% 49 pts-45%

CAD
Unstableangina, stableanginaMyocardial
16 pts-7%
infarction

Group B
without DR
122 pts
42 pts- 34.4%
36 pts-29.5%

10 pts-17% 6 pts-5%

After adjustments for risk factors, diabetic retinopathy was
associated with two times higher incidence for unstable andstable angina - odds ratio = 1.95 (95% CI 1.13 - 3.35), higher
incidence of myocardial infarction - odds ratio = 2.36 (95% CI 0.78
- 7.14). The risk of CAD was higher in individuals with proliferative
retinopathy (PDR) - for unstable and stable angina - odds ratio =2.86 (95% CI 1.13 - 7.22), for myocardial infarction - odds ratio =4.29 (95% CI 1.1 - 16.7). Also, in patients with non-proliferative
retinopathy (NPDR) was a slightly higher incidence of unstable
and stable angina - odds ratio = 1.76 (95% CI 0.99 - 3.14), andmyocardial infarction - odds ratio = 1.43 (95% CI 0.44 - 4.59).
DISCUSSION
Our study conducted on a sample of patients with type 2
diabetes showed that the prevalence of CAD is significantly
higher in patients with DR compared with those without DR. It is
well know from observational studies that patients with advanced
diabetic retinopathy have a poor life expectancy. A retrospective
review of 128 diabetics followed-up at the Radcliff Infirmary, Oxford, England found the 5-year mortality was 45% for those with
PDR, 8% for those with only microaneurysms, and 8% for those
without any retinopathy (9). Davis and colleagues (10) reported
in a prospective study of 709 patients with type 2 diabetes taking
insulin and followed-up for up to 13 years a 5-year mortality of
44% for those with PDR, 19% for those with moderate NPDR,
and 1% for those with no or minimal retinopathy at baseline.
In the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) based on an 8.5-year follow-up of 1,370
persons with older-onset diabetes mellitus, the age-adjusted andsex-adjusted hazard ratios for CAD mortality were 1.50 (95%
confidence interval, 1.22 - 1.85) in patients with mild NPDR,
1.93 (95% confidence interval, 1.43 - 2.61) in those with moderate NPDR, and 2.07 (95% confidence interval, 1.48 - 2.91) inthose with PDR (11). This study involved patients with type 1
diabetes, while we included in our study only patients with type36

2 diabetes. In the Atherosclerosis Risk in Communities Study
was demonstrated that among patients with type 2 diabetes,
the presence of diabetic retinopathy is associated with a twofold
higher risk of CAD events, and a threefold higher risk of CAD
death, independent of cardiovascular risk factors, diabetes duration and control, and large-vessel atherosclerosis (12). In a study
conducted by Mittinen and colleagues (13) that included 1,059
patients during the 7-year follow-up has been shown that 255(24%) patients developed serious CAD events. In patients withproliferative retinopathy at baseline, the risk of CAD events during
the follow-up was statistically significantly higher compared withpatients without retinopathy changes (odds ratio [OR] 2.31, 95%
CI 1.21 - 4.40). The association between proliferative retinopathy
and CAD events remained significant when other cardiovascularrisk factors were controlled for (13).
In a study of 2,329 type 1 diabetic patients without prior CAD,
the 7-year incidence rate of CAD was 8.0 (per 1,000 personyears) in men and 10.2 in women. Multivariate standardized Cox
proportional hazards models showed that age (hazard ratio 1.5),
albumin excretion rate (1.3 in men and 1.6 in women), waist-tohip ratio (1.3 in men), smoking (1.5 in men), fasting triglycerides(1.3 in women) or HDL cholesterol (0.74 in women), and systolic
BP (1.3 in women) were predictors of CAD (14). In the pooled
analysis of the 17 included studies, the OR for all-cause mortality
and/or CV events of the presence of DR was 2.34 (95% CI 1.96
- 2.80) compared with patients without DR. All but three studies
reported an increased risk for CV events. However, a significant
heterogeneity among the individual estimates was evident whenthe magnitude of the association was evaluated (I2 = 62.7%, P< 0.001). When advanced DR was evaluated (10 reports), theOR for all-cause mortality and/or CV events was 4.22 (95% CI
2.81 - 6.33) compared with patients without retinopathy (I2 =63.0%, P = 0.004). The overall sensitivity of advanced DR for the
combined outcome was 19% (13 - 28), and the specificity was
94% (91 - 96). The positive likelihood ratio was 3.64 (2.52 - 5.26)
and the negative likelihood ratio was 0.84 (0.77 - 0.92) (15).
These meta-analyses and cohort studies showed that thepresence of any degree of DR or advanced DR was associatedwith an increased risk for all-cause mortality and CV events(fatal and nonfatal) in both type 2 and type 1 diabetic patient.
We found similar data in our study. After adjustments for risk
factors, diabetic retinopathy was associated with two time higher
incidence for unstable and stable angina -odds ratio =1.95 (95%
CI 1.13 - 3.35), higher incidence of myocardial infarction-odds
ratio = 2.36 (95% CI 0.78 - 7.14). The risks of CAD were higher
in individuals with proliferative retinopathy (PDR) - for unstable
and stable angina odds ratio =2.86 (95% CI 1.13 - 7.22), formyocardial infarction odds ratio = 4.29 (95% CI 1.1 - 16.7).
Also in patients with non-proliferative retinopathy (NPDR) wasa slightly higher incidence of unstable and stable angina odds
ratio = 1.76 (95% CI 0.99 - 3.14), myocardial infarction- odds
ratio = 1.43 (95% CI 0.44 - 4.59). A recent study of 824 type 2
diabetic patients, using fundoscopic examinations to evaluate
retinopathy, showed a gender difference in the association of
background diabetic retinopathy with CAD death, raising the possibility that microvascular disease might be more important in thedevelopment of CAD in women than in men (16). We didn’t follow
Fiziologia - Physiology  2012.22.4 (76)the gender difference regarding incidence of CAD in our study.
A number of important limitations of this study should be
mentioned. The people in our study were aged between 40-65
years, white Caucasian. Thus, caution should be taken when
extending these findings to other segments of the population older and younger age groups, other ethnic groups, etc. We didn’t
use in our study methods to diagnose silent cardiac ischemia(stress echocardiography, scintigraphy, PET etc.).
CONCLUSION
Our data show that the presence of retinopathy in individuals
with type 2 diabetes was associated with a twofold higher risk ofincident CAD (unstable and stable angina, myocardial infarction)
independent of glycemic levels and cardiovascular risk factors.
This association appears to be graded with retinopathy severity:
in patients with proliferative retinopathy was associated with fourtime higher incident of myocardial infarction and three time higher
incidence of stable and unstable angina.
Our study showed that in diabetic patients evaluation ofdiabetic retinopathy is important because the presence of any
modification is associated with risk of developing coronary
arteries disease. Further studies are necessary to asses thecorrelation with cardiovascular risk factor, diabetic retinopathy
and cardiovascular mortality.
REFERECES
1. Gu K, Cowie CC, Harris MI. Diabetes and decline in heart diseasemortality in US adults. JAMA, 1999; 281: 1291-97.
2. Kannel WB, McGee DL. Diabetes and cardiovascular disease: theFramingham study. JAMA, 1979; 241: 2035-38.
3. Geiss LS, Herman WH, Smith PJ. National Diabetes Data Group. Diabetes in America. Bethesda, Md: National Institutes of Health, National
Institute of Diabetes and Digestive and Kidney Diseases; 1995: 233-257.
4. Haffner SM, Lehto S, Rönnemaa T, et al. Mortality from coronary
heart disease in subjects with type 2 diabetes and in nondiabetic

subjects with and without prior myocardial infarction. N Engl J Med
1998; 339: 229-234.
5. Wingard DL, Barrett-Connor EL, Scheidt-Nave C, McPhillips JB.
Prevalence of cardiovascular and renal complications in older adults
with normal or impaired glucose tolerance or NIDDM: a population-based
study. Diabetes Care. 1993; 16: 1022-25.
6. Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbershatz H,
Kannel WB. Prediction of coronary heart disease using risk factor
categories. Circulation. 1998; 97:1837-47.
7. Wilson PW. Diabetes mellitus and coronary heart disease. Am J
Kidney Dis. 1998; 32:S89-S100.
8. International clinical diabetic retinopathy disease severity scale.
American Academy of Ophthalmology (AAO). Free download from the
ICO: http://bit.ly/wdERl8.
9. Caird FI, Pirie A, Ramsell TG. Diabetes and the Eye, Oxford, England:Blackwell Scientific Publications Inc; 1998.
10. Davis MD, Hiller R, Magli YLM, et al. Prognosis for life in patientswith diabetes: relation to severity of retinopathy. Trans Am Ophthalmol
Soc, 1979; 77: 144-170.
11. Klein R, Klein BE, Moss SE, Cruickshanks KJ. Association of oculardisease and mortality in a diabetic population. Arch Ophthalmol, 1999;
117: 1487-95.
12. Cheung N, Wang JJ, Klein R, Coupep D, Sharrett AR, Wong TY.
Diabetic retinopathy and the risk of coronary heart disease: the Atherosclerosis risk in Communities Study. Diabetes Care, 2007; 30:1742-46.
13. Mittinen H, Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M.
Retinopathy predicts coronary heart disease events in NIDDM patients.
Diabetes Care, 1996; 19: 1445-48.
14. Soedamah-Muthu SS, Chaturvedi N, Toeller M, et al. EURODIAB Prospective Complications Study Group Risk factors forcoronary heart disease in type 1 diabetic patients in Europe: theEURODIAB Prospective Complications Study. Diabetes Care,
2004; 27: 530-537.
15. Diabetic Retinopathy Predicts All-Cause Mortality and Cardiovascular Events in Both Type 1 and 2 Diabetes: Meta-analysis of observational
studies Diabetes Care, 2011; 34: 1238-44.
16. Juutilainen A, Lehto S, Ronnemaa T, Pyorala K, Laakso M: Retinopathy predicts cardiovascular mortality in type 2 diabetic men andwomen. Diabetes Care, 2007; 30: 292-299.

RELATIA DINTRE RETINOPATIA DIABETICA SI BOALA CORONARIANA
IN REGIUNEA DE VEST A ROMANIEIREZUMATAm efectuat un studiu observational pentru a evalua relatia dintre retinopatia diabetica si boala coronariana la nivelul populatiei
din Regiunea de Vest a Romaniei. Studiul a inclus 231 de pacienti (121 barbati si 110 femei) cu diabet tip 2, cu varsta cuprinsa
intre 40-65 ani, care au fost examinati pentru modificari microvasculare retiniene prin tehnici de imagistica retiniana in Clinica de
Oftalmologie, in perioada mai 2008 – septembrie 2012. Pacientii implicati in acest studiu au fost selectati dintr-un grup mai mare,care a cuprins 3400 de pacienti. Perioada medie de urmarire a pacientilor a fost de 28 ± 12 luni. Examinarea oftalmologica initiala aevidentiat 109 pacienti cu un grad de retinopatie diabetica (DR) (prevalenta ~ 40%), dintre care 87 de pacienti cu retinopatie diabetica non-proliferativa (NPDR) (prevalenta 32%) si 22 de pacienti cu retinopatie diabetica proliferativa (PDR) (prevalenta 8%). Dupa
modificarea factorilor de risc, retinopatia diabetica a fost asociata cu incidenta de 2x mai crescuta a anginei instabile si stabile - odds
ratio = 1,95 (95% CI 1,13 - 3,35), precum si cu incidenta mai crescuta a infarctului miocardic - odds ratio = 2,36 (95% CI 0,78 - 7,14).
Riscurile de boala coronariana (CAD) au fost mai crescute la pacientii cu retinopatie proliferativa (PDR) – pentru angina instabila si
stabila, odds ratio = 2,86 (95% CI 1,13 - 7,22), iar pentru infarctul micocardic, odds ratio = 4.29 (95% CI 1.1-16.7). De asemena, lapacientii cu retinopatie non-proliferativa (NPDR) a fost observata o incidenta usor mai crescuta a anginei instabile si stabile, odds
ratio = 1,76 (95% CI 0,99 - 3,14), in timp ce pentru infarctul miocardic - odds ratio = 1,43 (95% CI 0,44 - 4,59). Datele prezentate
releva faptul ca prezenta retinopatiei la indivizii cu diabet zaharat tip 2 a fost asociata cu un risc de doua ori mai crescut de aparitiea CAD (angina instabila si stabila, infarct miocardic), independent de nivelurile glicemice si prezenta factorilor de risc cardiovascular.
Aceasta asociere pare sa fie corelata cu severitatea retinopatiei, deoarece la pacientii cu retinopatie proliferativa incidenta infarctului
miocardic a fost de 4 ori mai crescuta, in timp ce incidenta anginei instabile si stabile a fost crescuta de 3 ori.
Cuvinte cheie: retinopatie diabetica, boala coronariana, infarct miocardic, angina, incidenta
2012.22.4 (76)  Fiziologia - Physiology

37

THE PATHOLOGIC SPECTRUM OF TONSIL MALIGNANT TUMORS IN TIMISOARA - STATISTIC STUDYRAMONA MARIA GHIRAN, STAN COTULBEA, ANAMARIA VASILICA MAG, DELIA HORHATSurgery Department, ENT Discipline, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT

More than 90% of the oropharynx tumors are squamocellular carcinomas. The rest are malignant melanomas, minor
salivary glands tumors, sarcomas, plasma cell tumors, lymphomas and other rare tumors. Metastases in orophayinx
have been also identified.
The retrospective study was performed on a group of 134 patients with tonsil tumors admitted in the ENT Clinic of theClinical City Hospital Timişoara between 2006 and 2010. The variables analyzed were the histopathologic type of thetumor, local extension and lymph node invasion as well as distant metastases. The specimens were fixed in bufferedformalin, sectioned at 5 μm and stained with haematoxyllin and eosin for the histopatologic diagnosis. The majority ofthe tumors studied were squamous cell carcinomas, which was expected, given the increased general incidence of
this type of malignant tumors from this region. The lymphomas were present in 2 female patients, one aged 16, andthe other aged 61. The last type of malignant tumor that we have noticed was lymphoepithelial carcinoma, present in
a 35 years old patent.
Keywords: squamocellular carcinomas, tonsil carcinoma, pathology, radiotherapy
INTRODUCTION
More than 90% of the oropharynx tumors are squamocellular carcinomas. The rest are malignant melanomas,
minor salivary glands tumors, sarcomas, plasma cell tumors,lymphomas and other rare tumors. Metastases in orophayinx
have been also identified (1-4). Lymphoepithelial carcinoma
is more frequent in the tonsil region and in the base of the
tongue. The differential diagnosis between lymphoepithelial carcinoma and squamous cell carcinoma is important,because the first is radiosensitive. Non–Hodgkin lymphoma is
encountered in 5% of the malignant tumors of the tonsil and isa rare occurence in the base of the tongue.
Squamous cell carcinoma is an epithelial malignant tumor
with squamous differentiations such as intercellular bridges andkeratin formation. Its origin is in the cornified squamous epithelium of the oropharynx mucosa or in the respiratory epithelium
with squamous metaplasia (5). In the oropharynx, the most common localizations include the base of the tongue and the tonsils.
Microscopically, squamous cell carcinomas are characterized by invasive growth and squamous differentiations demonstrated by the formation of intercellular bridges, with or without
cornification; and by the formation of keratin pearls. From theimmunohistochemical point of view, squamous cell carcinomas
express epithelial markers, such as cytokeratins and epithelial
membrane antigen (EMA). The cytokeratin expression patternis determined by the differentiation grade of the tumor and the
keratinization degree (6).
The basaloid carcinoma is a poorly differentiated squamouscell carcinoma type composed by basaloid and squamous cells,with a clinically agressive behaviour. It was first described by Wainet al. in 1986 (7). It has a predilection for the aerodigestive tract,
but it was described also in the uterine cervix (8), esophagus (9),lungs (10) and the anal canal (11). The preferential localizationis the hypopharynx (the piriform sinus), but it was described in
the oropharynx as well (12,13) and in the oral cavity (14-16). The
precursor of this tumor type is considered a totipotent primitive
cell localized in the basal layer of the surface epithelium or inseromucous glands (7,16). The lymphoepithelial carcinoma isa poorly differentiated form of the squamous cell carcinomas
or an undifferentiated carcinoma associated with a dense lymphocytic infiltrate in the stroma. From the morphologic point ofview it can not be distinguished from type III WHO nasopharynx
carcinoma (17). It was initially described in the rhynopharynx in1921 by Regaud and Reverchon (18), and independentently by
Schmincke (19). The squamous cell papillary carcinoma is a rarevariant of squamous cell carcinoma initially described by Crissman et al. in 1988 (20). Its main features are a papillary growth
pattern and a good prognosis. In the head and neck region,
it is preferentially localized in the oropharynx, hypopharynx,
larynx and sinonazal tract (20-25). It was found in the skin (26),uterine cervix (27), conjunctiva (28), and thymus. The verucous
carcinoma (Ackerman tumor) is a well differentiated variant ofsquamous cell carcinoma initially described by Ackerman in 1948(29). It is characterized by an exophytic, verucous growth, withslow local invasion which produces extensive destructive lesions
if untreated. It rarely metastasizes. The majority of these tumors(75%) are localized in the oral cavity and 15% in the larynx. In

Received 14th of October 2012. Accepted 5th of November 2012. Address for correspondence: Delia Horhat, MD, PhD, “Victor Babes” University of Medicine andPharmacy Timisoara, Romania, Eftimie Murgu Square No. 2A, RO-300041, e-mail: deliahorhat@yahoo.com

38

Fiziologia - Physiology  2012.22.4 (76)the oral cavity, the most frequently affected are the oral mucosa
and the gum, and in the larynx the vocal folds. It is rare in otherzones of the head and neck, such as nasal cavity, the sinonasal
tract or the rhynopharynx. They were described in the skin, anus,
external genital organs, urinary bladder and esophagus (30).

squamous cells carcinomas, moderately differentiated (G2). The
distribution of the squamous cells carcinomas is represented
in Figure 2.

MATERIAL AND METHODS
The retrospective study was performed on a group of 134
patients with tonsil tumors admitted in the ENT Clinic of theClinical City Hospital Timişoara between 2006 and 2010. The
variables analyzed were the histopathologic type of the tumor,
local extension and lymph node invasion as well as distantmetastases. The specimens were fixed in buffered formalin,
sectioned at 5 μm and stained with haematoxyllin and eosin forthe histopatologic diagnosis.
RESULTS
The histopatologic diagnosis of tonsil tumors
The majority of the tumors studied were squamous cell
carcinomas, which was expected, given the increased generalincidence of this type of malignant tumors from this region.
Squamous cell carcinomas were characterized by the presenceof neoplastic squamouas cells, usually with euchromatic, butmonstruous nuclei, with prominent nucleoli, with marked nuclear
anaplasia and anisonucleolosis. The cytoplasm of the neoplastic
cells was abundant, predominantly eosinophyllic, sometimes witha granular aspect. In some rare cases, a basophyllic material,with basal membrane aspect accumulated around neoplastic
cells. In these cases, the tumors were classified as squamous
cells carcinomas, because other features (the type of tumor cells
and the general microscopic architecture of the tumor) of basal
cells carcinomas were absent. Out of the 134 tumor specimens,
123 were squamous cells carcinomas, mostly moderately differentiated. The histopatologic types of the lesions studied are
represented in Figure 1.

Fig.1. Histopatologic types of the lesions studied

Squamous cells carcinoma
The squamous cells carcinoma was majoritar not only
among the malignant tumors from the study, but from the total of
the specimens. The majority of the tumors were nonkeratinized2012.22.4 (76)  Fiziologia - Physiology

Fig. 2. Squamous cells carcinomas subtypes studied

The differentiation grade of the squamous cell carcinomas
was in the majority of the cases moderate (G2), with tumors withkeratinization and keratozic pearls formation, with squamous
cells with euchromatic nuclei with prominent nucleoli and with
abundant eosinophyllic cytoplasm. The mitotic count on 200x
field was moderate, and no tumor necrosis was noticed.
The well differentiated squamous cell carcinoma was characterized by abundant keratinization.
As mentioned, the moderatly differentiated squamous cell
carcinoma is characterized by moderate formation of keratozic
pearls (keratinocite maturation), and the cells are uniform, someo them with basaloid features. The mitotic count on 200x field is
low, and the tumor cells are grouped.
The poorly differentiated squamous cell carcinoma ischaracterized by marked cellular and nuclear atypia, without
keratinocite maturation and without keratinization, a high mitotic
count and often atypic mitoses. We have noticed this type ofsquamous cell carcinoma in 3 cases.
The nonkeratinized squamous cell carcinoma was present
in more variants, most frequently with cells with indistinct limits, atypic, with a high mitotic count and sometimes with local
necrosis, predominantly comedonecrosis. Often the tumors
were exulcerated and inflamed, with local suprainfection, which
modify the microscopic aspect. The lymph node metastases ofnonkeratinized carcinomas had a particular morphologic pattern,with local necrosis and cystic degeneration.
The mitotic count in this tumor type (squamous cell nonkeratinized carcinomas) was high, with easily visibile mitoses
even at low magnification (medium power field), many of them
being atypic.
Comedonecrosis was frequent in poorly differentiated nonkeratinized squamous cell carcinomas, probably by alteration of
the tumor vascular supply produced by the rapid tumor growthwith insufficient angiogenesis. The tumor growth with high mitotic
index doesn’t seem to be the main mechanism of necrosis, because this phenomenon is noticed also in poorly differentiatednonkeratinized squamous cell carcinomas with low mitotic index.
The rarer variants of nonkeratinized squamous cell
carcinoma were those with foci of umor cell maturation andkeratinization and those involving the deep part of the tonsil
crypts. In his later situation, the tumor cells had indistinct limits,cellular and nuclear anaplasia; euchromatic nuclei with prominent
39

nucleoli and anisonucleolosis, and the tumor cells islands were
infiltrated by lymphocytes, which are normal in this area evenin normal patients.
The nonkeratinized squamous cell carcinomul arising fromthe tonsil crypt epithelium was noticed in a single case.
Another rare variant of nonkeratinized squamous cell carcinoma is that with maturation of the cells from the periphery. The
peripheral cells maturation is proven y their better differentiatedaspect and by keratin production, which accumulates aroundthe islands of tumor cells. We have noticed this type of tumor
in 2 cases.
Almost all of the lymph node metastases had a solid pattern, resembling the primary tumor, with islands of squamous
tumor cells mostly without keratin deposition. In one case we
hav noticed a lymph node metastasis with cystic appearance,
without necrosis or degeneration.
The nondifferentiated squamous cell carcinoma was present in 2 cases, both patients male, one aged 35 and the otheraged 53. It was characterized by large groups of big tumor
cells, with indistinct cellular limits, with abundant eosinophylliccytoplasm and monstruous nuclei, with marked nuclear anaplasia, prominent nucleoli and anisonucleolosis, with high mitotic
count and atypical mitoses. The tumor microvessel density was
also increased, and the tumor stroma tumorală was massively
infiltrated with lymphocytes.
The last rare variant of squamous cell carcinoma was thatwith papillary pattern, noticed in a 59 years old male patient,which presented with a large friabile exophytic tumor, without
necrosis or suprainfection. Microscopically, the tumor is formedby fine loose connective tissue axes, sometimes infiltrate with
inflammatory cells, without massive fibrosis or keratin deposits,
covered by a relatively well differentiated neoplastic epithelium,with medium sized tumor cells, with eosinophyllic cytoplasm andeuchromatic nuclei with prominent nucleoli and anisonucleolosis,
arranged in layers. In the described case, the squamous cell
carcinoma with papillary pattern was associated in variable
proportions with tonsilar epithelium displasia. The displasia wasmoderate, with cellular and nuclear atypia and with disorganizedmaturation of epiteliale cells.
The lymphomas were present in 2 female patients, one
aged 16, and the other aged 61. B cells lymphoma is a medium
sized tumor, of 3/3/1.2 cm, ulcerated. Microscopically, the tumor
was formed of neoplastic lymphocytes, with surface epithelium
ulceration.
The last type of malignant tumor that we have noticed
was lymphoepithelial carcinoma, present in a 35 years oldpatent. It was a small tumor, with a maximum dimension of
0.6 cm, with elastic consistency and formed of large cells,with abundant eosinophyllic cytoplasm, with euchromatic
nuclei with nucleoli with atypia and sometimes of multinucleated cells. The epithelial cells described are intermixed
with small lymphocytes, with phenomena of emperipolesisand peripolesis.
The majority of the malignant tumors studied were squamous cells carcinomas, keratinized or nonkeratinized, of different
40

grades, moderately differentiated, followed by far by lymphomas
and by lymphoepithelial carcinoma, as illustrated in Figure 3.

Fig. 3. The malignant tumors studied. The high frequency of squamous cell
carcinomas is easily noticeable.

DISCUSSION
The1 34 patients with tonsil lesions group is significant for
the analyzed period and for the size of the diagnosis and treatment center where the study was carried on; the majority of thestudies published in the literature are on tens of patients series,
the only large groups studies being on periods longer than 10
years as archival epidemiologic studies. The biggest populational study published so far is the one of Mehta et al (31),which consideres all of the oropharynx and tonsil squamouscell carcinoma from SEER database of the National Cancer
Institute from 1976 to 2006. The central conclusion of this study
is the fact that the tumor differentiation grade has evolved frompoorly differentiated tumors in the ’70s, to well differentiate tumors with longer survival intervals, including an increase of thedisease-free interval. In our study, the majority of the patientswith squamous cell carcinomas had moderatly differentiatedtumors, with G2 grade; a few patients had poorly differentiatedor anaplasic tumors. The well differentiated tumors were evenrarer. The age was also considered as a risk factor in a study
on a large number of patients (5538), establishing the age of
50 as a threshold (32). In this study, the same as in ours, theage was not correlated with the increased incidence of thetonsil carcinoma, even if combined with human papillomavirus
seemed to have a slightly higher role. As for the lymph node metastases, a remarkale fact is that the tonsil tumors may present
with clinically occult metastases, which can evolve in residual
tumors and may compromize the efficiency of the therapy. Most
patients from our study had lymph node metastases, because
most had locally advanced tonsil squamous cell carcinomas
and thus cervical lymph node dissection was necessary due
to the extent of the local lesions. In the case of locally earlystage tumors, the selective dissection of the regional lymph
nodes is important, because it can identify occult lymph nodelesions, which may diminish the efficacy of the treatment, andwhich have usually a bad prognosis, both from the disease-freeinterval and the survival rate (33).
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SPECTRUL PATOLOGIC AL TUMORILOR AMIGDALIENE MALIGNE IN
TIMISOARA – STUDIU STATISTICREZUMATMai mult de 90% dintre tumorile orofaringiene sunt carcinoame scuamocelulare, restul fiind melanoame maligne, tumori ale glandelorsalivare mici, sarcoame, tumori plasmocitare, limfoame sau alte tumori rare. Au fost identificate de asemenea metastaze la nivelul
orofaringelui.
Studiul retrospectiv a fost efectuat pe un grup de 134 de pacienti cu tumori amigdaliene, care au fost internati in Clinica ORL a
Spitalului Municipal Timisoara intre anii 2006-2010. Variabilele analizate in acest studiu au fost: tipul histologic al tumorilor, gradul deextindere locala si invazia nodulilor limfatici, precum si metastazele la distanta. Specimenele au fost fixate in formalina tamponata,sectionate la o grosime de 5 μm si colorate cu hematoxilina si eozina pentru stabilirea diagnosticului histopatologic. Majoritatea
tumorilor studiate au fost carcinoame cu celule scuamoase, ceea ce era de asteptat data fiind incidenta crescuta a acestui tip detumori maligne in aceasta regiune. Limfoamele au fost intalnite doar la 2 paciente cu varsta de 16, respectiv 61 de ani. Un ultim tip
de tumora maligna intalnit a fost carcinomul limfoepitelial, care a fost prezent la un pacient in varsta de 35 de ani.
Cuvinte cheie: carcinom scuamocelular, carcinom amigdalian, patologie, radioterapie

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