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Fiziologia - Physiology  2008.18.3(59)CONTENTS
1. REVIEW - HSP72 and the Immune Response
Carmen Tatu, V Ciocotisan, Gabriela Tanasie, Daniela Puscasiu, RF Tatu, Carmen Bunu................................................................... 4
2. Anthropometric Characteristics and Physical Performance in Young Athletes in Hungary
Ferenc Bóka, László Nagymajtényi, Edit Paulik.................................................................................................................................... 8
3. Olive Leaf Extract Attenuates Ethanol-Induced Gastric Lesions in Rats
Dekanski D, Janicijevic-Hudomal S, Piperski V, Petricevic S, Mitrovic DM......................................................................................... 12
4. Effects of Different Exercise Program on Blood Markers of Oxidative Stress in Young Women
Dragan Radovanovic, Vladimir Jakovljevic, Tatjana Cvetkovic, Aleksandar Ignjatovic, Natasa Veselinovic, Sonja Dondur............... 16
5. Short and Long Term Hypobaric Hypoxia Induces Oxidative Stress in Rats: The Protective Effects of N-Acetylcysteine
Irina Chis, Marius-Ionut Ungureanu, Ramona Simedrea, Monica Maier, Adriana Muresan, Adriana Marton, Nicoleta Decea .......... 20
6. Rest and Postural Tremor in the Power Spectral Structure in Patients with Parkinson’s Disease
Jelena Marić, Suzana Blesić, Sladjan Milanović, Nataša Dragašević, Tihomir Ilić, Vladimir Kostić, Miloš Ljubisavljević................... 23
7. The Correlation between Cardiovascular Risk Factors, Arterial Stiffness and Left Ventricular Diastolic Function in thePatients with Hypertension
L Agoston-Coldea, T Mocan, M Gatfosse, C Bobar, LD Rusu, L Poanta, DL Dumitrascu................................................................... 27
8. SDS-Page Electrophoresis of Urinary Proteins: Diagnostic and Prognostic Value
Kacso Ina, Cristea Anca, Racasan Simona, Spanu Costel, Fedorca Ana, Chindris Adela, Muntean Maria,
Gherman-Caprioara Mirela.................................................................................................................................................................. 34

CUPRINS
1. REVIEW - HSP72 si raspunsul imun
Carmen Tatu, V Ciocotisan, Gabriela Tanasie, Daniela Puscasiu, RF Tatu, Carmen Bunu................................................................... 4
2. Caracteristicile antropometrice si performanta fizica la sportivi tineri in Ungaria
Ferenc Bóka, László Nagymajtényi, Edit Paulik.................................................................................................................................... 8
3. Extractul din frunze de maslin atenueaza leziunile gastrice induse de alcool la sobolani
Dekanski D, Janicijevic-Hudomal S, Piperski V, Petricevic S, Mitrovic DM......................................................................................... 12
4. Efectele diferitelor programe de exercitiu fizic asupra markerilor sanguini ai stressului oxidativ la femeile tinere
Dragan Radovanovic, Vladimir Jakovljevic, Tatjana Cvetkovic, Aleksandar Ignjatovic, Natasa Veselinovic, Sonja Dondur............... 16
5. Hipoxia hipobarica pe termen scurt si lung induce apritia stresului oxidativ la sobolani: efectele protectoare ale n-Acetilcisteinei
Irina Chis, Marius-Ionut Ungureanu, Ramona Simedrea, Monica Maier, Adriana Muresan, Adriana Marton, Nicoleta Decea........... 20
6. Tremorul de repaus si postural in structura spectrala de putere la pacientii cu boala Parkinson
Jelena Marić, Suzana Blesić, Sladjan Milanović, Nataša Dragašević, Tihomir Ilić, Vladimir Kostić, Miloš Ljubisavljević................... 23
7. Corelatiile dintre factorii de risc cardiovascular, scleroza arteriala si functia diastolica a ventriculului stang la pacientii hipertensivi
L Agoston-Coldea, T Mocan, M Gatfosse, C Bobar, LD Rusu, L. Poanta, DL Dumitrascu.................................................................. 27
8. Valoarea diagnostica si prognostica a electroforezei proteinelor urinare in gel de poliacrilamida cu tampon dodecilsulfat desodiu (SDS-Page)
Kacso Ina, Cristea Anca, Racasan Simona, Spanu Costel, Fedorca Ana, Chindris Adela, Muntean Maria,
Gherman-Caprioara Mirela.................................................................................................................................................................. 34

2008.18.3(59)  Fiziologia - Physiology

3

REVIEW
HSP72 AND THE IMMUNE RESPONSECARMEN TATU1, V CIOCOTISAN2, GABRIELA TANASIE1, DANIELA PUSCASIU1, RF TATU1,CARMEN BUNU1
1
Victor Babes University of Medicine and Pharmacy Timisoara, Romania
2
Ricercatore del Dipartimento di Bioingegneria, Politecnico di Milano, Italy

ABSTRACTHeat shock proteins (HSP) are the most phylogenetically conserved proteins present in all prokaryotes and eukaryotes. Intracellular
HSP found within cells serve a cytoprotective role by chaperoning naive, misfolded and/or denatured proteins in response to stressful stimuli by a process known as the stress response. However, stressful stimuli also induce the release of intracellular heat shockproteins into the extracellular milieu and circulation. The extracellular heat shock protein proteins serve a cytostimulatory role byinitiating immune responses designed to fend off microbial infection and destroy neoplastic transformed cells. This review will briefly
cover recent advances into elucidating the mechanism by which stress induces the release of heat shock proteins into the circulation, how it initiates immune responses and suggest the possible biological significance of circulating HSP to the host.
Key words: heat shock proteins, HSP72, immune response, antigen presentation

INTRODUCTION
Heat shock proteins (HSP) are highly conserved proteinsfound in all prokaryotes and eukaryotes. Heat shock proteins
were originally described for their role as chaperones induced
by temperature shock as well as various other kinds of stressincluding environmental (U.V. radiation, heat shock, heavy metals
and amino acids), pathological (bacterial, parasitic infections or
fever, inflammation, malignancy or autoimmunity) or physiologicalstresses (growth factors, cell differentiation, hormonal stimulation or tissue development), that induced a marked increase in
intracellular HSP synthesis known as the stress response (2, 20).
The stress response is designed to enhance the ability of the cellto cope with increasing concentrations of unfolded or denatured
proteins. Of all heat shock proteins, the HSP70 family constitutes
the most conserved and best-studied class. This family consistsof the constitutively expressed HSP70 (Hsc70; 73 kDa), thestress inducible HSP70 (HSP70; 72 kDa), the mitochondrial
HSP70 (HSP75; 75 kDa), and the endoplasmic reticulum HSP70(Grp78; 78 kDa) (2, 3). The primary function ascribed to HSP72
was as an intracellular molecular chaperone of naive, aberrantly
folded, or mutated proteins as well as in cytoprotection following
the kinds of stressful stimuli including environmental (UV radiation, heat shock, heavy metals and amino acids), pathological(viral, bacterial, parasitic infections or fever, inflammation, malignancy or autoimmunity) or physiological stimuli (growth factors,
cell differentiation, hormonal stimulation or tissue development),
induces a marked increase in intracellular HSP72 synthesis (20,21), known as the stress response. HSP72 display a distribution pattern closely paralleling the tissue solute concentration

characteristic of the concentrating kidney (5). Consitent withthis distribution pattern, in one study immunoreactivity to HSP72
was detected in the cortex in individual collecting duct cells
only (22). In the outer medula, all tubules were stained weakly,
whereas in the papilla intense staining of collecting ducts andthe epithelium lining the papilla was noted (25). In another study,
however, in wich a different antibody was used, staining wasapparent in all cortical and medullary tubules after intravenous
injection of vasopressin but not in the saline -injected control.
With few exceptions, changes in medullary solute concentrations
are associated with corresponding changes in inner medullary
HSP72 expression (27). Another study show that accumulationof HSP90 mRNA in response to osmotic stress is unrelated tocellular protein denaturation and that synthesis of HSP90 may beregulated at both the level of transcription and translation (23).
Because, in murine inner medullary collecting duct cells, maximal
HSP72 mRNA expression is already reached at 500 mosmol/kg H2O and because the various stressors (high NaCl and urea
concentrations, low pH) may act in concert to induce HSP72,
the variability in these results may be explained by differencesin the duration and/or extent of changes in medullary solute concentrations in the various studies. Experiments in cultured renal
epithelial cells demonstrate that HSP72 is significantly induced
only when medium osmolality is increased by solutes for which
the cell membrane is poorly permeable (e.g., NaCl, mannitol)
but not by readily permeating solutes (urea, glycerol) (10). Thissuggest that, in the renal inner medulla, the high extracellular
NaCl concentration is a major determinant of the high HSP72
content. The idea that, under specific circumstances, HSP70 may

Received June 2008. Accepted September 2008. Address for correspondence: Dr. Carmen Tatu, Physiology Department, “Victor Babes” University of Medicine anf
Pharmacy Timisoara, Eftimie Murgu Square No. 2A, 300041, Timisoara, e-mail: carmen.tatu@umft.ro4

Fiziologia - Physiology  2008.18.3(59)act as a cytoprotectant has prompted a series of investigations
examining the role and the putative protective effects of HSP72
in acute renal failure (10).
HSP72 INDUCTION AND RELEASE
The gene for HSP72 contains at least two regulatory elements that interact with heat shock transcription factors (HSFs).
Specifically, the induction of HSP72 protein requires HSF1
binding to the heat shock element in the promoter region of the
HSP70 gene (18). Many factors induce transcription and translation of intracellular HSP72 protein, and these vary dependingon the tissue examined. The mechanism of release was studied
by Gallucci and co-workers, who demonstrated that dendritic
cells (DC) are stimulated by endogenous signals received fromstressed, virally infected or necrosis-induced cells, but not by
healthy cells or cells undergoing apoptosis (14). Additional
studies for other laboratories demonstrated that necrotic but not
apoptotic cell death leads to release of HSP’s including gp96,
calreticulin, HSP90 and HSP70 (8). These authors showedthat exposure of DC to necrotic but not apoptotic cells resulted
in maturation of DC. A condition in which necrotic cell deathclearly contributes to the release of HSP72 from cells is after
severe trauma. In a study by Pittet and colleagues a significant
upregulation in circulating serum HSP72 can be measured
in severely traumatized patients as early as 30 minutes after
injury (24). Increased circulating serum HSP72 has also beenmeasured in patients after coronary artery bypass grafting (2).
Importantly, circulating serum HSP72 has been suggested asa marker of myocardial damage, and reported to have a rolein the inflammatory response after acute myocardial infarction(AMI) (12). Other conditions in which elevated levels of circulating serum HSP72 has been demonstrated is in renal disease,
hypertension, atherosclerosis, aging, and sickle cell disease (2).
However, in these conditions although necrosis is proposed as
the mechanism of release, conclusive experimental data is stilllacking. Walsh and coworkers demonstrate that physical exercise
results in the appearance of HSP70 in the circulation prior to any
increase in gene or protein expression in contracting skeletal
muscle (26). Additional studies have confirmed these findings
and recently it was demonstrated that both intensity and durationof exercise influences the concentration of released eHSP72 in
the plasma (3). Psychological stress induced by exposure ofSprague Dawley rats to cats results in the release of HSP72 into
the circulation (13). The human brain is able to release HSP72
into the circulation in response to exercise (3). In an in vitro study,
Guzhova and colleagues demonstrated that HSP70 is released
by glia cells in the absence of necrotic cell death (3, 16). Morerecently, HSP70 has been shown to be released by B cells (11)and peripheral blood mononuclear cells and also under non necrotic conditions. Using conditions will not induce significant cell
death, we showed that IFN-γ and IL-10 induce the active releaseof constitutive seventy-kilo-Dalton heat shock protein, HSC70
from tumors (3). Using conditions that will not induce significant
cell death, Barreto and coworkers showed that IFN-γ and IL-10
induce the active release of constitutively expressed HSP70 also
2008.18.3(59)  Fiziologia - Physiology

designed, Hsc70 or HSP73 from tumors (7). Recently Bauseroand coworkers have begun to elucidate the mechanism of active release of iHSP72 from viable cells (9). It was demonstrated
that certain pro-inflammatory cytokines normally found in highconcentrations within inflammatory foci including IFN-γ, IL-10 but
not the anti-inflammatory cytokine TGF-β1, mediate the active
release of HSP72. It was showed that whereas some eHSP72
could be found as free HSP72, a proportion of eHSP72 wasreleased within exosomes (9). Exosomes are internal vesicles
of multivesicular bodies (MVB) released into the extracellularmilieu upon fusion of multivesicular bodies MVB with the cellsurface. In addition to containing HSP72 (9), exosomes are highly
packed with immunostimulatory mediators including MHC class
I and II and costimulatory molecules (2). Studies by Lancaster& Febbraio recently demonstrate that exosomes provide themajor pathway for secretory vesicular release of HSP72 (19).
However, using methyl-β-cyclodextrin (the cholesterol depleting
agent) to disrupt lipid raft function, these authors were unable toconfirm a role for lipid rafts in stress-induced HSP72 release fromhuman peripheral blood mononuclear cells PBMC. In order toaddress the cellular location of HSP72 after stress, a recent study
demonstrated that newly synthesized HSP72 protein localizes
within the Golgi region of HeLa cells and also concentrates on
the surface of the plasma membrane and in the ruffled zone of
migrating cells (2). Taken together, these studies suggest thatthe active release hypothesis is an important mechanism bywhich HSP72 is released into the circulation. However, studies
remain to be performed that conclusively demonstrate that T cell
responses are primed in response to active release of HSP72
especially in the case of psychological stress or exercise.
BIOLOGICAL SIGNIFICANCE OF RELEASED HSP72
Irrespective of whether HSP72 enters the circulation via an
active or passive release mechanism, what is the role of eHSP72
in circulation? The danger theory postulates that immune activation involves danger/non-danger molecular recognition schemas
and suggests that innate immune cells are activated by danger
signals that are derived from stressed or damaged self-proteins(1). It is now widely accepted that eHSP72 fit this criteria. The
hypothesis is further reinforced by studies howing that circulatingeHSP72 is increased and upregulated in diseased conditions
including renal disease, hypertension, atherosclerosis and sickle
cell disease. However, intriguing questions still remain about therole of increased circulating eHSP72 during psychological stress.
Is it possible that in these situations the circulating eHSP72 ispriming the immune system to real or perceived danger? These
are important questions, the answers to which will provide a keen
insight into numerous psychological and pathophysiologicalconditions. However, extensive studies and concerted efforts
that bring together HSP researchers from such disparate fields
as immunology, molecular biology, cell biology, neurophysiology
and psychology utilizing new breakthrough technologies including the recently deciphered human genome and proteomics are
required before conclusive answers can be given (1).

5

THE CHAPEROKINE ACTIVITY OF HSP72, INITIATIONOF IMMUNE RESPONSES
The term chaperokine describe the unique function of extracellular HSP72 (eHSP72) as both chaperone and cytokine(3). After admixing eHSP70 to APCs, specific signal transduction pathways are activated that result in the stimulation of an
immune response. eHSP72 induces a plethora of immune
responses and the list continues to grow. Asea and coworkers
demonstrated that as early as 2-4 hours post exposure of APC
to exogenous eHSP70, there is significant release of cytokines
including TNF-α, IL-1β, IL-6 and IL-12 (10) and GM-CSF; nitricoxide, a potent apotogenic mediator; chemokines including
MIP-1, MCP-1 and RANTES (3). It was demonstrated that both
peptide-bearing and non peptide-bearing eHSP70 is capable ofinducing pro-inflammatory cytokine production by APCs (3, 4).
eHSP72 induces the DC maturation by augmenting the surface
expression of CD40, CD83, CD86 and MHC class II molecules
on DC and migration of DC and NK cells (15).
HSP 72 AND ANTIGEN PRESENTATION
Once eHSP72 is bound to its specific surface receptor, twointerrelated events occur. First, signal transduction cascades areactivated. Second, the eHSP72 is endocytosed and the chaperoned peptides are presented to the antigen presentation pathway.
Following receptor-mediated endocytosis heat shock proteinscarrying their peptide load is transported via clathrin-coated pits
to endosomes where it colocalizes with both MHCI and MHCII(2). The exact mechanism by which cross-presentation occurs
has not been completely elucidated. However, eHSP72 may
traffic through various intracellular compartments, which resultsin peptide release into the cytoplasm and re-presentation on thecell surface associated with MHC proteins (2). eHSP72 has beenshown to deliver peptides to MHC class I molecules throughthe cross-presentation pathway (6). Following internalizationof eHSP72, chaperoned peptides maybe released into the cytoplasm and processed by the classical antigen representation
pathways. However, cross-presentation may proceed througha recently described alternative route which involves a specialized MHC class I structure known as the ER/phagosome fusioncompartment, which is highly sufficient in inducing antigen
cross-presentation (17).
CONCLUSION
eHSP72 is released via necrotic as would be expected during
non-physiological conditions including trauma. eHSP72 can also
be released into the extracellular milieu within exosomes by a nonclassical protein transport mechanism, requiring intact lipid rafts.
eHSP72 is found in the circulation of healthy individuals, however
elevated levels can be found in various disease conditions and in
response to moderate exercise and acute psychological stress.
eHSP72 binds with high affinity and avidity to specific cells of
the immune system and activates specific signal transduction
cascades depending on which receptor it bind. Finally, eHSP72
stimulates the hosts immune response in an attempt to rid thehost of infection or tumors.
6

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M, Febbraio MA. Exercise increases serum HSP72 in humans. Cell
Stress Chaperones. 2001;6:386–393
27. Yorek M, Dunlap J, Liu W, Lowe L. Normalization of hyperosmoticinduced inositol uptake by renal and endothelial cells is regulated by
NF-kappaB. Am J Physiol Cell Physiol 2000; 287(5): C1011-C1018

HSP72 SI RASPUNSUL IMUNREZUMAT
Proteinele de stres (HSP) reprezinta proteinele cele mai bine conservate din punct de vedere filogenetic, prezente la toate celulele
procariote si eucariote. Proteinele de stress intracelulare au rol protector asupra proteinelor denaturate sau incorect plicaturate careactie de raspuns la diversi stresori. Aceast raspuns este cunoscut sub numele de raspunsul la stres. Stimulii stresori induc deasemenea eliberarea proteinelor de stres in mediul extracelular. Aici HSP contribuie in calitate de citostimulatori, la initierea raspunsului imun in diverse situatii: aparare antimicrobiana, distrugerea celulelor neoplazice. Acest articol incearca sa elucideze cele mai
noi mecanisme, propuse de diversi autori, prin care stresul induce eliberarea de HSP in circulatie, despre cum se initiaza raspunsul
imun si posibile semnificatii biologice ale ale HSP.
Cuvinte cheie: proteinele de stres, HSP72, raspuns imun, prezentarea antigenului

2008.18.3(59)  Fiziologia - Physiology

7

ANTHROPOMETRIC CHARACTERISTICS AND PHYSICALPERFORMANCE IN YOUNG ATHLETES IN HUNGARYFERENC BÓKA, LÁSZLÓ NAGYMAJTÉNYI, EDIT PAULIK
Department of Public Health, University of Szeged Faculty of Medicine, Szeged, Hungary

ABSTRACTThe aim of this study was to investigate the age and sport specific features of anthropometric characteristics and their relationshipwith the training and physical performance parameters in football, water polo and handball players, and kayak-canoeists in Hungary.
Men of 10 to 25 years of age (n = 213) were involved in this cross-sectional study, performed in 2006. The anthropometric measurements included body mass, body height, body fat percent, upper arm girth and shoulder width. Training volume was characterized
by the weekly average time, and physical performance was determined by two simple sport tests (Cooper’s 12 minute run and
Burpee tests).
An age dependent increase was seen in anthropometric parameters and weekly training times. The results of Cooper’s test improved with age in all sport groups, and the Burpee test’s results were rather variable. Correlation between anthropometric data
and training volume was seen only in the older age groups. The results indicated differences between the anthropometric data andphysical performance among male kayak-canoeists, football, water polo and handball players.
The results indicated typical differences in the anthropometric data and physical performance of male kayak-canoeists, football,water polo and handball players.
Key words: body mass index, body fat, sport, training

INTRODUCTION
In the complex biological process called growth, the alteration of body composition is an important feature (9). Numerous
studies were accomplished about effects of usual physical activity
on fitness and anthropometric parameters in subjects of various
age, physical activity, nutritional state etc. (3,4,5). The relationship between physical activity and changes in body compositionin children was described by Stevens et al. (13). In a German
study, made on persons aged between 17 and 26, body weightincreased and fitness decreased in non-obese young adults(6). Rowlands et al. have found a positive relationship between
activity and fitness, and a negative relationship between fatness
and activity (12).
Anthropometric studies on athletes engaged in various sports
indicated a relationship between body composition and the typeof sport (1,7,8,10,14,15,16). Body composition has a significant
effect on athletic performance and, conversely, exercise has thepotential to alter body composition (11), so that the evaluationof body composition in connection with exercise and sport has
emerged as an important field of interest.
In the present study, the age and sport specific features ofanthropometric characteristics and their relationship with thetraining and physical performance was studied in football (soccer), water polo and handball players, and kayak-canoeists in

Hungary. Football, water polo, kayak-canoeist and handball arevery different sports. They are similar in comprising both aerobic
and anaerobic components, but the proportions of the two vary.
Our working hypothesis was that the athletes’ anthropometriccharacteristics and level of physical performance are related totheir age, the kind of sport, and the training volume.
MATERIALS AND METHODS
Men of 10 to 25 years of age (n = 213) were involved in thiscross-sectional study, performed in 2006. Of these, 65 (30.5%)
were 10-13 years old, 73 (34.3%) were 14-17, and 75 (35.2%)
were 18-25 years old. All subjects have had trainings since theirchildhood. According to the types of sport, 55 (25.8%) were football players, 52 (24.4%) were water poloists, 53 (24.9%) were
kayak-canoeists, and another 53 (24.9%) were handball players.
The athletes were selected from the most promising registeredplayers of the given age group in sport clubs of Szeged, andincluded several age-group representative players, as well as
European/World championship and Olympic Game winners
among the adults.
The anthropometric parameters and physical fitness were
measured under standardized conditions in the fitness gym inSzeged, always on Sunday afternoon, at least 2 hours after thelast meal. The anthropometric measurements included bodyReceived May 2008. Accepted August 2008. Address for correspondence: Edit Paulik, Department of Public Health, University of Szeged Faculty of Medicine, Dóm
tér 10, H-6720 Szeged, Hungary, Phone: +36-62-545-119; Fax: +36-62-545-120, e-mail: paulik@puhe.szote.u-szeged.hu

8

Fiziologia - Physiology  2008.18.3(59)mass, body height, body fat percent (BF%), upper arm girthand shoulder width. Body mass was measured to the nearest
0.5 kg, and body height to the nearest 0.5 cm; with the subjects
wearing gym shorts and T-shirts and no shoes. Body massindex (BMI) was calculated from body mass and body height,
and expressed in kg/m2. Body fat percent was determined bybioimpedance analyser (Beurer BF40). Upper arm girth wasmeasured at the mid-point of the arm; and shoulder width,
between the two acromia.
Training volume was characterized by the weekly averagetime (frequency and length of training sessions). Physical performance was determined by two simple sport tests: Cooper’s
12 minute run test (run length in meters) was used for aerobic
capacity, while anaerobic capacity was measured by the Burpee
test performed in two minutes. The Burpee test involves the following practices: standing erect, arms by the side; placing thehands on the floor in front of the feet (squat position); thrusting
the legs back to assume a push up position; squat position;
returning to the starting position.

The data were processed by the Statistical Package forSocial Sciences (SPSS), version 13.0 for Windows. For eachage group and sport, mean and standard deviation of the anthropometric and performance characteristics was calculated.
Comparison of the means by age-groups and sports was done
by non-parametric Kruskal-Wallis test, because the Levene’s
test was significant. Spearman’s rho correlations were used todetermine the degree of association among selected variables.
In all tests, P<0.05 was considered as statistically significant.
The study protocol (No. 11/2006) was approved by the Human Investigation Review Board of the University of Szeged.
Informed written consent was obtained from the participants.
RESULTS
Anthropometric and Physical Performance Characteristics
The results of measurements by age-groups and sport are
shown in Table I. Significant age-group related differences were seenin anthropometric and physical performance parameters except theresults of the Burpee test in football players and water poloists.

Table I – Anthropometric and physical performance parameters in highly trained young athletes

Characteristics
10-13 years old
Body mass index (kg/m2)*
Body fat (%)*
Upper arm girth (cm)*Shoulder width (cm)*Weekly training time (min)*Cooper’s run test (m)*Burpee test (number)*
14-17 years old
Body mass index (kg/m2)*
Body fat (%)*
Upper arm girth (cm)*Shoulder width (cm)*Weekly training time (min)*Cooper’s run test (m)*Burpee test (number)*
18-25 years old
Body mass index (kg/m2)*
Body fat (%)*
Upper arm girth (cm)*Shoulder width (cm)*Weekly training time (min)*Cooper’s run test (m)*Burpee test (number)*

Football players(n=16)
mean
SD
17.06
2.07#
5.31
1.27#
21.87
1.99#
42.09
2.05#
439.6969.01#
2106.51176.89#58.44
9.93(n=19)
mean
SD
19.82
1.68
8.20
3.77
27.00
2.50
47.31
4.76
706.84357.892723.68
409.7352.37
11.75(n=20)
mean
SD
22.11
2.07
15.40
4.09
29.07
2.68
56.30
2.81
1074.00
395.483030.00
389.4758.50
11.70

Handball players(n=16)
mean
SD
20.69
2.12#
7.13
3.70#
25.13
2.33#
47.75
1.88#
378.7578.13#
2090.23222.27#58.13
11.50#(n=18)
mean
SD
23.12
3.37
13.93
6.25
29.75
3.23
57.87
3.18
465.2888.12
2655.56
178.9642.05
5.22(n=19)
mean
SD
24.14
2.21
16.39
2.21
32.63
2.22
61.26
2.64
610.79183.832963.16208.73
52.21
11.87

Significant differences between sports (Kruskall-Wallis test)
Significant differences between age-groups within sports (Kruskall-Wallis test)*

Water poloists(n=16)
mean
SD
21.51
2.69#
9.43
5.80#
23.80
3.01#
47.80
2.48#
326.25
121.76#
1862.50257.88#36.13
8.15(n=15)
mean
SD
23.99
2.41
16.90
4.07
30.63
1.56
55.40
2.56
451.6768.34
2126.67345.3144.00
15.75(n=21)
mean
SD
24.03
1.55
19.42
3.58
32.86
2.61
59.86
5.59
803.09377.512466.67
300.56
42.62
8.65

Kayak-canoeists(n=17)
mean
SD
19.23
2.79#
7.21
3.63#
24.20
2.73#
46.53
3.18#
340.29111.25#2017.65
312.72#44.06
9.49#(n=21)
mean
SD
23.60
2.32
13.69
5.77
30.67
2.23
58.67
1.63
638.57219.442666.67
03.86
49.00
6.79
(n=15)
mean
SD
24.58
1.40
15.18
3.58
33.60
2.03
62.67
8.03
780.00247.383157.33
253.08
60.60
8.09

#

2008.18.3(59)  Fiziologia - Physiology

9Anthropometric data of each age-group were significantly
different by sports with lowest figures among football players,and highest among water poloists or kayak-canoeists.
Weekly training volume increased with age. Within a given
age group, the longest overall training times were seen among
football players, and the shortest, among water poloists andadult handball players.
The results of Cooper’s test improved with age in all sportgroups, with best performance among 18-25 years old kayakcanoeists and football players, and worst among water poloists.
The results of the Burpee test were rather variable. Only the
kayak-canoeists’ results increased with age, water poloists again
had the worst results.
Analysis of Correlations
There were correlation between BMI and other anthropometric parameters (Table II). BMI and BF% was in correlation except
in 10-13 years old football players and 18-25 years old kayakcanoeists. BMI was in strong relationship with upper arm girth
except 10-13 years old football players and 14-17 years old waterpoloists. A strong correlation between BMI and shoulder width
values was in 10-13 years old water poloists, kayak-canoeists
and 14-17 years old handball players (Table II).
Correlation between anthropometric data and training volume
was seen only in the age groups above 13 years. In the 14-17
years old football players’ group, training volume was in strong
relationship with BF% (r=0.869; P<0.001) and with shoulder width(r=0.490; P=0.033). In the 18-25 years old groups, the kayakcanoeists’ upper arm girth (r=0.581; P=0.023), as well as thehandball players’ BF% (r=0.482; P=0.037) and upper arm girth(r=0.473; P=0.041) was in connection with training volume, while
no correlation was found in the water poloists’ group.
Table II – Spearman’s correlation coefficients between body massindex and other anthropometric parametersAge-groups andkinds of sport
10-13 years old
Football players
Handball players
Water poloists
Kayak-canoeists
14-17 years old
Football players
Handball players
Water poloists
Kayak-canoeists
18-25 years old
Football players
Handball players
Water poloists
Kayak-canoeists

Body fat (%) Upper arm Shoulder
girth (cm) width (cm)0.420
0.692**
0.829***
0.849***

0.494
0.745**
0.726**
0.931***

0.163
0.201
0.772***
0.776***

0.869***
0.937***
0.913***
0.798***

0.829***
0.857***
0.397
0.638**

0.647**
0.864***
0.288
0.506*

0.986***
0.887***
0.905***
0.151

0.826***
0.711**
0.502*
0.755**

0.101
0.659**
0.390
0.589** P<0.05, **P<0.01, ***P<0.001

The performance in Cooper’s test was not influenced by
the training volume, while the Burpee test, among the 14-17
10

old football players (r=0.584; P=0.009) and the 18-25 years old
kayak-canoeists (r=0.531; P=0.042) had a correlation with thetraining volume.
When testing the relationship between physical performanceand anthropometric parameters it was found that results of the
Cooper’s test were correlated to BMI and BF%, beside Burpee
test outcome with BMI, BF% and body measures. In the 10-13
years old group, there was no correlation between anthropometric data and physical performance.
Among the 18-25 years old water poloists, negative correlation was found between Cooper’s test results and BMI (r=–0.475;
P=0.030), and between Burpee test performance and shoulder
width (r=–0.579; P=0.006).
Among the 14-17 years old football players, the results of the
Burpee test were in connection with BMI (r=0.478; P=0.039), BF%(r=0.467; P=0.044) and shoulder width (r=0.633; P=0.004). In the
18-25 year old kayak-canoeists’ group, the Burpee test results
were correlated with BMI (r=0.535; P=0.040), upper arm girth(r=0.852; P<0.001) and shoulder width (r=0.632; P=0.012).
DISCUSSION
The observed age-dependent increase of anthropometricparameters might be, in the 10-13 and 14-17 years group, equally
due to growth and training. In the subjects above 18, the major
factors were likely the training volume and the type of sport.
The dissimilar effect of intensive training on the anthropometriccharacteristics in different sports has already been described byother authors (1,8,16).
The choice of sport can, however, itself be influenced bybody constitution: those of thin figure have a preference tofootball while the overweighed children’s choice is probably
water polo.
The high BF% of the water poloists is a sport specific feature,
where fat has probably a thermal insulator role in the water. Water
polo requires much of stamina. Beyond being a heat insulator,
fat tissues actively produce hormones influencing blood level ofsugar and triglycerides, the latter being utilised by the muscles
for energy production (15).
Kayak-canoeists had, determined by the characteristics ofthis sport, the largest muscle mass; but this was nearly free offat (high BMI together with low BF%), which was due partly tothe high physical load and partly to that any overweight would
be unwanted ballast in the boat.
Handball players had a likely good physical state, but in this
sport direct body-to-body fight is frequent so that body mass,represented to a lesser extent by fat and not muscles, is morecrucial. In several surveys, the data of BMI and BF% were only
weakly correlated (14). Body fat measurement by bioimpedance
seemed to be better in describing nutritional state than the standard BMI calculation from body height and weight (3,4).
Compared to the above sports, football players had lower
BMI and BF% data and weaker upper body musculature. Beyond
the influence of physical constitution on the choice of sport, thismight be due to the higher energy consumption during training
work by feet, compared to upper body training, but also to the low
Fiziologia - Physiology  2008.18.3(59)level of physical training of football players in Hungary. Coerver’s
method (2), athletic training of football players, has less weightin Hungary compared to countries with more successful football.
In those, technical development is prioritized in the 5-13 years
old, whereas condition development and physical training is done
mostly in the age groups of 13-14 to 18-20. Beyond increasing
aerobic and anaerobic capacity, this includes the developmentof upper body musculature.
In all sports investigated in the present study, the 10-13 years
olds’ anthropometric data were sport specific without correlation
to training intensity or physical performance (kind of sport chosenby physical constitution).
Among the older, kayak-canoeists had the best, and waterpoloists the worst, condition. The 18-25 years old kayak-canoeists’ very good performance in the 4-beat pushup test was incorrelation with their upper body composition, probably because
this test requires short-term (2-4 min) high anaerobic capacity, acharacteristic of this sport. In the same age group, the results of
the water poloists’ Cooper’s test were in negative correlation with
BMI: more corpulent players had lower aerobic performance.
The results described above indicated typical differencesin the anthropometric data and physical performance of malekayak-canoeists, football, water polo and handball players.
The differences arose partly from the influence of physicalconstitution on the choice of sport, but are later influenced by
the physiological demands of the kind of sport and its specifictraining methods. In practice, the anthropometric examinations
and ongoing performance measurements enable or help thesuggestion and choice of a kind of sport in childhood.
REFERENCES
1. Andreoli A, Melchiorri G, Brozzi M, Di Marco A, Volpe SL, Garofano
P, Di Daniele N. Effect of different sports on body cell mass in highly
trained athletes. Acta Diabetol. 40: S122-S125, 2003
2. Coerver W, Collette R. Soccer fundamentals for players and coaches. Englewood Cliffs, N.J.: Prentice Hall, 1986
3. Dittmar M. Reliability and variability of bioimpedance measures innormal adults: effects of age, gender, and body mass. Am. J. Phys.

Antropol. 122:361-370, 2003
4. Kyle UG, Gremion G, Genton L, Slosman DO, Golay A, Pichard C.
Physical activity and fat-free and fat mass by bioelectrical impedancein 3853 adults. Med. Sci. Sports Exerc. 33(4):576-584, 2001
5. Kyle UG, Morabia A, Schutz Y, Pichard C. Sedentarism affects bodyfat mass index and fat-free mass index in adults aged 18 to 98 years.
Nutrition 20:255-260, 2004
6. Leyk D, Rohde U, Gorges W, Ridder D, Wumderlich M, Dinklage
C, Sievert A, Ruther T, Essfeld D. Physical performance, body weightand BMI of young adults in Germany 2000-2004: results of the physical-fitness-test study. Int. J. Sports Med. 27(8):642-647, 2006
7. McIntyre MC. A comparison of the physiological profiles of elite
Gaelic footballers, hurlers, and soccer players. Br. J. Sports Med.
39:437-439, 2005
8. Melchiorri G, Andreoli A, Candeloro N, De Lorenzo A. Changes
in body composition caused by intense physical training. Clin. Ter.
151:73-76, 2000
9. Pietrobelli A, Tato L. Body composition measurements: from thepast to the future. Acta Paediatr. Suppl. 94(448):8-13, 2005
10. Pucsok J. Effects of increased and reduced physical activity on
body mass index. Háziorvosi Továbbképző Szemle (in Hungarian).
9(6):462-464, 2004
11. Reilly T, Gilbourne D. Science and football: a review of appliedresearch in the football codes. J. Sports Sci. 21(9):693-705, 2003
12. Rowlands AV, Eston RG, Ingledew DK. Relationship between activity levels, aerobic fitness, and body fat in 8- to 10-yr-old children. J.
Appl. Physiol. 86(4):1428-1435, 1999
13. Stevens J, Suchindran C, Ring K, Baggett CD, Jobe JB, Story
M, Thompson J, Going SB, Caballero B. Physical activity as a predictor of body composition in American Indian children. Obes. Res.
12(12):1974-1980, 2004
14. Takada K, Sugita S, Ikeuchi R, Okuda N, Fujinami T. Body composition measurement by electrical bio-impedance method to establish
the effect of daily physical training in adolescents. Med. Prog. Technol.
19(4):187-192, 1993-94
15. Tsekouras YE, Kavoura SA, Campagna A, Kotsis YP, Syntosi
SS, Papzoglou K, Sidossis LS. The antropometrical and physiological characteristics of elite water polo players. Eur. J. Appl. Physiol.
95:35-41, 2005
16. Wittich A, Oliveri MB, Rotemberg E, Mautalen C. Body composition of professional football (soccer) players determined by dual X-ray
absorptiometry. J. Clin. Densitom. 4(1):51-55, 2001

CARACTERISTICILE ANTROPOMETRICE SI PERFORMANTA FIZICA LA
SPORTIVI TINERI IN UNGARIAREZUMAT
Scopul acestui studiu a fost de a investiga varsta si caracteristicile antropometrice specifice sportului si relatia lor cu parametrii deantrenaj si performanta fizica la jucatorii fotbal, polo pe apa, handbal si caiac-canoe in Ungaria.
In studiul crossectional, executat in 2006, au fost involvati barbati de varsta intre 10 si 25 ani (n = 213). Ca parametrii antropometrici,
indexul Masa-Corp, inaltimea, procentajul de grasime, circumferinta bratului si a umerilor au fost considerate. Antrenajul a fost caracterizat prin durata medie de timp al antrenajului saptaminal, performanta fizica a fost determinata prin doua testuri simple: Cooperde 12 minute si testul Burpee.
Parametrii antropometrici si durata saptaminala de antrenaj au aratat o crestere dependenta de varsta. Rezultatele testului Cooper
au aratat o perfectionare cu varsta in toate felurile de sport, pe cand rezultatele testului Burpee au fost variabile. Corelatia dintre
datele antropometrice si volumul de antrenaj a fost demonstrate doar la cei mai varstnici. Rezultatele au indicat diferinte intre datele
antropometrice si perfomanta fizica intre barbati caiac-canoisti, jucatorii fotbal, polo pe apa si handbal.
Rezultatele au indicat diferinte tipice intre datele antropometrice si performanta fizica la barbatii barbati caiac-canoisti, jucatorii
fotbal, polo pe apa si handbal.
Cuvinte cheie: indexul Masa-Corp, procentaj de grasime, antrenaj
2008.18.3(59)  Fiziologia - Physiology

11OLIVE LEAF EXTRACT ATTENUATES ETHANOL-INDUCEDGASTRIC LESIONS IN RATS
DEKANSKI D1, JANICIJEVIC-HUDOMAL S2, PIPERSKI V3, PETRICEVIC S1, MITROVIC DM4
1
R&D Institute, Galenika a.d. Beograd, Serbia
2Medical School Pristina (Kosovska Mitrovica), Serbia
3
Medical Academy, US Medical School, Beograd, Serbia
4
Medical School, Beograd, Serbia

ABSTRACT
Olive leaf extract (OLE) possesses, among other, antioxidative properties, but whether it influences the gastric defense mechanismand gastroprotection against ethanol-induced gastric lesions remains unknown. Previous studies demonstrated that the damaging
action of absolute ethanol could be attributed to the enhancement in the reactive oxygen species (ROS) and the ROS-dependent
increase in lipid peroxidation and inhibition of antioxidative enzyme activity. In this study we investigated the protective effect of
OLE, a natural antioxidant, on gastric mucosal damage induced by absolute ethanol in rats. We applied three different doses of OLE
intragastrically (i.g.) 30 minutes prior to absolute ethanol administration. One hour after i.g. applied ethanol, areas of gastric lesions
were measured by planimetry using ImageJ computer programme. Ulcer index (UI) and percent of inhibition of UI in relation to thecontrol ethanol group were calculated. Furthermore, we compared the effects of applied OLE on gastric mucosal lesions with effectsof i.g. pretreatment of H2 receptor antagonist, ranitidine. Histological evidence of gastric mucosal lesions was also obtained. Absolute ethanol treatment caused severe gastric submucosal haemorrhage, and this finding was confirmed histologicaly. Pretreatment
with OLE (40, 80 and 120 mg/kg), as well as with ranitidine (50 mg/kg), significantly (p < 0.001) attenuated gastric lesions induced
by absolute ethanol. Percents of inhibition in UI were 62%, 82% and 71%, respectively. The protective effect of OLE was similar tothat obtained with ranitidine which inhibited UI for 76%. Our results suggest that OLE exerts a potent gastroprotective activity against
ethanol-induced gastric lesions in rats.
Keywords: olive leaf, gastroprotection, rats

INTRODUCTION
Gastric mucosa plays a role of a barrier that limits exposureof the gastric mucosal cells to numerous injurious luminal agentsand irritants of exogenous and endogenous origin. Pretreatment
with different substances could effectively prevent gastric mucosa
from the development of erosions and ulceration. This action,
called gastro- or cyto-protection is not related to the inhibition ofgastric acid secretion and is known to account for gastroprotection by various irritants.
Interest in the olive leaf and its chemical constituents has
recently been increasing. Its benefits, however, have been
known for centuries, and it has been traditionally used to preventand treat diseases. Olive leaf is used to enhance the immune
system, as an antimicrobial and in heart disease. Folk medicine
uses also include hypertonia, arteriosclerosis, rheumatism, gout,diabetes mellitus, and fever (19). Recently, experimental animalstudies have demonstrated hypoglycemic (2,10), hypotensive(13), antiarrhythmic (25), and vasodilator effects (29), as well as
spasmolytic effect on the intestinal smooth muscle (8). Antimicrobial (5, 9, 16,21), antiviral (15,18), anti-tumor (1,11) andantiinflammatory activity (22) were also reported. The beneficial
properties of olive leaf are further enhanced by the bioavailabilityof its polyphenolic constituents, which are readily absorbed
through the gastrointestinal tract, resulting in significant levelsin the circulation (27,28).
The main constituent of the olive leaves is oleuropeine, oneof iridoide monoterpenes, which is thought to be responsible forpharmacological effects. Furthermore, the olive leaves containtriterpenes including oleanolic and maslinic acid, flavonoides (luteolin, apigenine, rutin), and chalcones (olivin, olivin-diglucoside)(19,17,21). Its chemical content makes olive leaf one of the mostpotent natural antioxidant. Oleuropein has high antioxidant activity in vitro, comparable to a hydrosoluble analog of tocopherol(26) and exibits strong antioxidant protection in oxidative stressduring ishemia-reperfusion i an in vivo experimental model (3).
Moreover, other constituents of olive leaf also exert antioxidative properties, which is experimentaly confirmed (4,7).
A variety of botanical products rich in flavonoids have beenreported to possess anti-ulcer activity (especially from ethnopharmacological studies), and the documented literature has centered primarily on their pharmacological action in experimental
animals (6, 20,30).
Whether olive leaf extract (OLE) influences the gastricdefense mechanism and exhibits gastroprotection against

Received May 2008. Accepted June 2008. Address for correspondence: Dr. Dragana Dekanski, Center for Biomedical Research, R&D Institute, Galenika a.d., Pasterova
str. 2, Beograd, 11000, Serbia, e-mail: ddekan@sezampro.yu

12

Fiziologia - Physiology  2008.18.3(59)experimentaly-induced gastric lesions remains unknown. Previous studies demonstrated that the damaging action of absoluteethanol could be attributed to the enhancement in the reactive
oxygen species (ROS) and the ROS-dependent increase inlipid peroxidation and inhibition of antioxidative enzyme activity(14). Therefore, treatment with potent antioxidant as OLE, could
decrease ethanol-induced gastric mucosal damage.
METHODS
Materials
We used standardized dry olive leaf extract (Olea europaea
L.) purchased from Frutarom Industry Ltd. (Switzerland) whichcontains 16.8 % of oleuropein, previosly determined by HPLC
analysis. For the extrapolation of the dosage from humans torats, we used the metabolic body size or food intake rather thanbody weight as a criterion (23, 24). Hence, 40 mg/kg of olive leaf
extract was administered. A higher doses of 80 and 120 mg/kg
was also given to test for a dose response. Ranitidine tablets wereobtained from Galenika a.d., Serbia. Both OLE and ranitidine wassuspended in distilled water before administration.
Gastric lesions induction and evaluation
This study was approved by the Ethical Committee, Medical School, University of Belgrade, and run in accordance tothe statements of European Union regarding handling of experimental animals. Wistar male rats, weighting between 200
and 220 g were randomly divided into 5 groups (6 rats in eachgroup). The animals were placed in individual metabolic cages.
Before the experiment, they were fasted overnight, but had freeaccess to water.
The first, control group received distilled water intragastricaly(i.g.) 30 minutes prior to 1 ml absolute ethanol administration. We
applied three different doses of OLE on the next three groups,
and finaly, the last group (positive control) received 50 mg/kg ofranitidine, H2 receptor antagonist. One hour after i.g. appliedethanol, animals were sacrificed, abdomen was opened by the
midline incision, the stomach was removed, opened along thegreater curvature, rinsed gently with water and pinned open for
macroscopic examination and for photodocumentation. Areas ofgastric lesions were measured by planimetry using NIH ImageJ
computer programme (12).
Ulcer index (UI) and % of inhibition in UI in relation to thecontrol ethanol group were estimated from formulas:

was done using t-test. Diferences with p<0.01 were consideredas significant.
RESULTS AND DISCUSSION
Effect of OLE suspension on gastric lesions induced byabsolute ethanol
Administration of absolute ethanol to fasted rats resulted insevere gastric damage visible from the outside of the stomach as
thick reddish-black lines. After opening, the gastric lesions were
found in the mucosa and consisted of elongated bands, 1–10mm long, usually parallel to the long axis of the stomach. They
were located mostly in the corpus, the portion of the stomach
secreting acid and pepsin. No visible lesions developed in thenonsecretory part of the stomach.
The effect of absolute ethanol and pretreatment with OLE
applied i.g. in graded concentrations, as well as ranitidine on
the ulcer index is shown on Figure 1. Ethanol caused typicalwidespread gastric lesions on 14.7 % ± 5.5 of total stomach area.
Pretreatment with all three doses of OLE significantly (p < 0.001)
reduced gastric lesions induced by absolute ethanol. The best
inhibition of ulcer index was obtained in group pretreated with
80 mg/kg of OLE, were ethanol caused gastric lesions in only
2.6 % ± 1.4 of total stomach area. The gastroprotective effectof OLE was similar to that achieved by the pretreatment with H2
receptor antagonist, ranitidine, where UI was 3.6% ± 0.8.
Fig. 1. Effect of intragastric pretreatment with OLE applied in gradeddoses ranging from 40 mg/kg up to120 mg/kg and ranitidine (50 mg/kg) on the ulcer index induced by absolute ethanol. Asterisk indicatesstatistical significance of inhibition (P< 0.001), as compared to thecontrol value.

UI = [ulcerated area (mm2)/ total stomach area (mm2)] x100
% inhibition = [1-(UI treatment/UI control)]x100
Histopathological evaluation
Histological evidence of gastric mucosal lesions in all experimental groups was also obtained. Gastric tissue samples
were fixed in 10% buffered formalin and processed for routine
histology. Paraffin sections of 5 μm thickness were cut by rotatory
microtome, stained with haematoxylin and eosin, and examined
microscopically for pathomorphological changes.
Statistical analysis
Results are expressed as means ± SD. Statistical analysis
2008.18.3(59)  Fiziologia - Physiology

Percents of inhibition of UI are shown in Table I. All applied
doses of OLE (40, 80 and 120 mg/kg) significantly inhibited ulcer
index (62%, 82% and 71%, respectively). The protective effectof OLE was similar to that obtained with ranitidine which inhibited UI for 76%. Hence, we confirmed the useful role of gastricantisecretory medication in the prevention of ethanol-related
gastric mucosal damage. The results obtained suggest thatboth antioxidants and antisecretory drugs may be beneficial inpreventing gastric mucosal cell damage induced by ethanol.
13

Table I. Inhibition of ulcer index induced by absolute ethanol
after pretreatment with different doses of OLE and ranitidine. Asterisk indicates statistical significance of inhibition (P< 0.001)

Pretreatment Dose (mg/kg) % of inhibition in UE
OLE
40
62*
OLE
80
82*
OLE
120
71*
Ranitidine
5076*
Effect of OLE suspension on histopathological changes
Histological examination of gastric mucosa showed various
histopathological changes including congestion, haemorrhage,edema, necrosis, inflammatory changes, erosions and ulcers
in ethanol-treated rats, which were in significantly lesser extent
seen in rats pretreated with both OLE and ranitidine.
CONCLUSION
In this study we investigated the protective effect of OLE,
a natural antioxidant, on gastric mucosal damage induced by
topical ulcerogen - absolute ethanol.
The role of oxygen derived free radicals in the generation ofgastric injury is well known. The results obtained indicate that
OLE posseses gastroprotective effect possibly related to its
antioxidative properties. In order to further elucidate the OLE
mechanism of gastroprotective effect, our further investigation
will be focused on determination of gastric mucosal antioxidative enzyme activity.
REFERENCES
1. Abaza L, Talorete TPN, Yamada P, et al. Induction of growth inhibition and diferentiation of human leukemia HL-60 cells by tunisiangerboui olive leaf extract. Biosci Biotechnol Biochem 2007; 71(5):1306-1312
2. Al-Azzawie HF, Alhamdani MS. Hypoglycemic and antioxidant effectof oleuropein in alloxan-diabetic rabbits. Life Sci 2006; 78:1371-1377
3. Andreadou I, Iliodromitis EK, Mikros E, et al. The olive constituent
oleuropein exhibits anti-ischemic, antioxidative, and hypolipidemic effects in anesthetized rabbits. J Nutr 2006; 136: 2213-2219
4. Benavente-Garcia O, Castillo J, Lorente J, et al. Antioxidant activityof phenolics extracted from Olea europea L. leaves. Food Chem 2000;
68(4): 457-462
5. Bisignano G., Tomaino A, Lo Cascio R, et al. On the in vitro antimicrobial activity of oleuropein and hydroxytyrosol. J Pharm Pharmacol
1999; 51: 971-974
6. Borelli F, Izzo AA. The plant kingdom as a source of anti-ulcer remedies. Phytother Res, 2000, 14(8):581-591
7. Briante R, Paturni M, Terenziani S, et al. Olea europaea L. leaf extract and derivatives: antioxidant properties. J Agric Food Chem 2002;
50(17): 4934-4940
8. Fehri B, Mrad S, Aiache JM, et al. Effects of Olea europea L. Extract
on the rat isolated ileum and trachea. Phytotherapy Res 1995; 9(6):435-439
9. Furneri PM, Marino A, Saija A, et al. In vitro antimycoplasmal activityof oleuropein. Int J Antimicrob Agents 2002; 20: 293-296

14

10. Gonzalez M, Zarzuelo A, Gamez MJ, et al. Hypoglycemic activityof olive leaf. Planta Med 1992; 58(6): 513-515
11. Hamdi HK, Castellon R. Oleuropein, a non-toxic olive iridoid, isan anti-tumor agent and cytoskeleton disruptor. Biochem Biophys Res
Commun 2005; 334(3): 769-78
12. ImageJ. Image Processing and Analysis in Java.http://rsb.info.nih.
gov/ij/)
13. Khayyal MT, el-Ghazaly MA, Abdallah DM, et al. Blood pressure lowering effect of an olive leaf extract (Olea europaea) in L-NAME induced
hypertension in rats. Arzneimittelforschung 2002; 52(11): 797-802
14. Kwiecien S, Brzozowski T, Konturek SJ. Effects of reactive oxygen
species action on gastric mucosa in various models of mucosal injury.
J Physiol Pharmacol 2002; 53: 39-50
15. Lee-Huang S, Zhang L, Huang PL, Chang YT. Anti-HIV activity ofolive leaf extract (OLE) and modulation of host cell gene expression by
HIV-1 infection and OLE treatment. Biochem Biophys Res Commun
2003; 307: 1029-1037
16. Markin D, Duek L, Berdicevsky I. In vitro antimicrobial activity ofolive leaves. Mycoses 2003; 46: 132-136
17. Meirinhos J, Silva BM, Valentao P, et al. Analysis and quantificationof flavonoidic compounds from Portuguese olive (Olea europea L.)
leaf cultivars. Nat Prod Res 2005; 68: 189-195
18. Micol V, Caturla N, Perez-Fons L, et al. The olive leaf extract exhibits antiviral activity against viral haemorrhagic septicaemia rhabdovirus (VHSV). Antiviral Res 2005; 66: 129-136
19. PDR for Herbal Medicine. 2nd edition, Fleming T. editor. Montvale,
New Jersey: Medical Economics Company; 2000. p. 556-557
20. Olaleye SB, Farombi EO. Attenuation of indomethacin- and HCl/ethanol-induced oxidative gastric mucosa damage in rats by kolaviron, a natural biflavonoid of Garcinia kola seed. Phytother Res 2006;
20(1):14-20
21. Pereira AP, Ferreira IC, Marcelino F, et al. Phenolic compounds
and antimicrobial activity of olive (Olea europaea L. Cv. Cobrançosa)leaves. Molecules 2007; 12(5):1153-1162
22. Pieroni A Heimler D, Pieters L, et al. In vitro anti-complementary
activity of flavonoids from olive (Olea europaea L.) leaves. Pharmazie
1996; 51(10): 765-768
23. Rucker R, Storms D. Interspecies comparison of micronutrient
requirements: metabolic vs. absolute body size. J Nutr 2002; 132:
2999–3000
24. Rucker RB. Allometric scaling, metabolic body size and interspecies comparisons of basal nutritional requirements. J Anim Physiol
Anim Nutr 2007; 91(3-4): 148-156
25. Somova LI, Shode FO, Mipando M. Cardiotonic and antidysrhythmic effects of oleanolic and ursolic acids, methyl maslinate and uvaol.
Phytomedicine 2004;11:121-129
26. Speroni E, Guerra MC, Minghetti A, et al. Oleuropein evaluated invitro and in vivo as an antioxidant. Phytother Res 1998; 12: 98-100
27. Visioli F, Galli C, Bornet F, et al. Olive oil phenolics are dose-dependently absorbed in humans. FEBS Lett 2000; 468: 159-160
28. Vissers MN, Zock PL, Roodenburg AJC, et al. Olive oil phenols areabsorbed in humans. J Nutr 2002; 132: 409–417
29. Zarzuelo A, Duarte J, Jimenez J, et al. Vasodilator effect of olive
leaf. Planta Med 1991; 57: 417-419
30. Zayachkivska OS, Konturek SJ, Drozdowicz D, et al. Gastroprotective effects of flavonoids in plant extracts. J Physiol Pharmacol
2005; 56(1): 219-231

Fiziologia - Physiology  2008.18.3(59)EXTRACTUL DIN FRUNZE DE MASLIN ATENUEAZA LEZIUNILEGASTRICE INDUSE DE ALCOOL LA SOBOLANIREZUMAT

Extractul din frunze de maslin (OLE) poseda, printre altele, proprietati antioxidante, dar nu se stie inca daca influenteaza mecanismele de aparare gastrica si protectia gastrica in cazul leziunilor produse de alcool la acest nivel. Studii anterioare au demonstrat ca
actiunea distructiva a etanolului pur s-ar putea datora cresterii speciilor reactive ale oxigenului (ROS) si cresterii ROS-dependenta a
peroxidarii lipidice si inhibitiei activitatii enzimatice antioxidative. In acest studio am investigat rolul protector al OLE, un antioxidant
natural, asupra injuriei mucoasei gastrice induse de etanol la sobolani. Am aplicat intragastric (i.g.) trei doze diferite de OLE cu 30
de minute inainte de administrarea etanolului pur. La o ora dupa administrarea i.g. de etanol, s-au masurat ariile leziunilor gastricefolosind planimetria, cu ajutorul programului ImageJ. Am fost calculate indexul ulcerativ (UI) si expresia procentuala a inhibitiei UI in
relatie cu grupul de control caruia i s-a administrat doar etanol. In continuare, am comparat efectele OLE asupra leziunilor mucoasei
gastrice cu efectele pre-tratamentului i.g. cu un antagonist al receptorilor H2, ranitidina. Au fost obtinute de asemenea si dovezile
histologice ale leziunilor mucoasei gastrice. Tratamentu cu etanol absolut a indus hemoragii gastrice submucoase severe, acest
aspect fiind confirmat histologic. Pre-tratamentul cu OLE (40, 80 and 120 mg/kg), precum si cu ranitidina (50 mg/kg), a atenuat
semnificativ (p < 0.001) leziunile gastrice induse de etanol. Procentual, inhibitia UI a fost 62%, 82% si respectiv 71%. Efectele
protectoare ale OLE au fost similare cu cele obtinute folosind pre-tratament cu ranitidina, care a inhibat UI in proportie de 76%.
Rezultatele noastre sugereaza ca OLE exercita o activitate gastroprotectoare puternica impotriva aparitiei leziunilor gastrice indusede etanol la sobolani.
Cuvinte cheie: frunze de maslin, protectie gastrica, sobolani

2008.18.3(59)  Fiziologia - Physiology

15

EFFECTS OF DIFFERENT EXERCISE PROGRAM ON BLOOD
MARKERS OF OXIDATIVE STRESS IN YOUNG WOMENDRAGAN RADOVANOVIC1, VLADIMIR JAKOVLJEVIC2, TATJANA CVETKOVIC3, ALEKSANDARIGNJATOVIC4, NATASA VESELINOVIC1, SONJA DONDUR1
1Faculty of Sport and Physical Education, University of Nis, Serbia
2
Faculty of Medicine, University of Kragujevac, Serbia
3
Faculty of Medicine, University of Nis, Serbia
4
Faculty of Pedagogy Jagodina, University of Kragujevac, Serbia

ABSTRACTIntroduction: Increased aerobic metabolism during exercise is a potential source of oxidative stress. Only a few studies have investigated and compared different aerobic exercise programs and their relations to pro-oxidant and antioxidant activities.
Aim: The aim of this study was to compare biomarkers of oxidative stress: lipid peroxidation, protein oxidation and total antioxidants
in blood, as well as functional variables, before and after 12 weeks of two different exercise programs.
Methods: Blood samples were collected from seven young women who performed pilates training as low-intensity aerobic exercise
and from seven young women who performed tae-bo training as high-intensity aerobic exercise. Samples were analyzed for lipid
peroxidation byproduct malondialdehyde, enzyme catalase, reactive carbonyl derivates, total sulfhydryl groups and serum antioxidant status. The results were statistically evaluated by the by the Wilcoxon Signed Ranks Test and Mann-Whitney U Test.
Results: There were no significant differences in any functional or biomarkers parameters between groups before training programat beginning of study. After 12 weeks CAT increased in both group but increase was significantly higher with pilates training, whereas
TAS increased significantly higher with tae-bo training. Both training programs increased values of MDA, protein carbonyls andsulphydryls, but there were no significant differences between groups.
Conclusion: There was evidence of oxidative stress after both pilates and tae-bo training program. Biomarkers of oxidative stressin blood increased after both 12 weeks training program. Due to different metabolic demands of pilates and tae-bo training, we
can conclude that both training types induced oxidative stress and action effects of O2 is not only mechanism for exercise-induced
oxidative stress.
Key words: oxidative stress, pilates, tae-bo, exercise

INTRODUCTION
It is widely assumed that oxidative stress is detrimental toexercise performance, but there is little experimental evidence tosupport this. The lack of such an effect suggests that exerciseinduced oxidative stress has only minor effects on performancein the short term; long-term effects on health should not be soreadily dismissed however, given the range of diseases thatare associated with enhanced free radical production. On theother hand, the increase in oxidative stress during exercise may
signal an increase in antioxidant defenses that protects against
a wide range of oxidative stresses (5). It is usually stated that
one of the most important source of reactive oxygen species(ROS) during exercise is mitochondrial superoxide production,
via side-reactions of flavin or ubisemiquinoneradicals with oxygen (3,9,14). An alternative mechanism by which exercise may
promote free radical production involves ischemia-reperfusion(13). Muscle damage subsequent to exercise (e.g. in delayed
onset muscle soreness) can cause inflammation and release of
superoxide from the neutrophil NADPH oxidase (11,12).
Increased aerobic metabolism during exercise is a potential
source of oxidative stress. Only few studies have investigatedand compared different aerobic exercise programs and theirrelations to pro-oxidant and antioxidant activities. Only highintensity, or long duration, exercise appears to lead to a large
enough increase in free radical production to overwhelm theantioxidant defenses (11).
The aim of this study was to compare biomarkers of oxidative
stress: lipid peroxidation, protein oxidation and total antioxidants
in blood, as well as functional variables, before and after 12
weeks of two different exercise programs.
METHODS
A total number of fourteen young women participated ininvestigation. None of the participants had suffered from any
illness for at least two weeks before the study. The purposeof the study was explained to the subjects and all gave theirinformed consent. In a preliminary visit body height and weight
were measured using standard techniques (4). Subjects fromboth groups were instructed not to participate in any physicalactivity during the day before the study.
Blood samples were collected from seven women (mean+/SD: age 24.7+/-3.7 years, body height 1.61+/-5.1 m, body weight

Received May 2008. Accepted June 2008. Address for correspondence: Dragan Radovanovic MD, PhD, Faculty of Sport and Physical Education University of Nis,
Carnojevica 10A, 18000 Nis, Serbia; Telephone: +381 18511940 ext.106, e-mail: drdr@bankerinter.net

16

Fiziologia - Physiology  2008.18.3(59)54.9 +/-8.3 kg, body fat 11.1+/-5.2%) who performed pilates
training as low-intensity aerobic exercise and from seven young
women (mean+/-SD: age 26.8+/-4.3 years, body height 1.65+/6.7 m, body weight 58.4 +/-9.8 kg, body fat 15.1+/-6.4%) who
performed tae-bo training as high-intensity aerobic exercise.
Low intensity experimental program consisted of pilates
exercises. After one training of familiarization, subjects exercisedunder the author’s supervision for 3 days per week for a total of
10 weeks. During this period total of 30 training sessions were
performed. Each training lasted 55 min, and consisted from threeparts: worm up, main part of the training and cooling down. The
main part of the training lasted between 35-40 min, and included
between 12-15 pilates exercises using standing, sitting and lying down positions. The supervisor cues subjects through thevarious stretches and movements, offering visualizations andmotivating talks to help them make good postural alignment,
proper breathing, balance, and functional muscle strengthening.
The movements during pilates are performed in the rhythm of theslow music (50 bpm) along with telemetric monitoring of heart
function (Polar, Finland). Heart rate frequency was held underthe 60% of estimated maximal heart rate for age.
High intensity experimental program consisted of tae-bo
exercise. The experimental program was consisted from three
training sessions every week. This made total of 30 training sessions. Each training session lasted one hour. After the warm-up
to properly prepare the muscles and joints for the challenge of

the workout, the main part of the training was consisted from the
combination of tae-bo exercises. The choreographies were madefrom next tae-bo basic moves: jab, hook, cross, upper-cut, kick(front, side and back). Cross train with other forms of exercise
such as walking, jogging, knee up, leg curls, step touch, etc. This
tae-bo exercises program involves boxing gloves and punchingbags. The majority of these classes are driven by fast music(135-155 bpm), although the music in many instances is more
for motivation because the cardio tae-bo choreography is notalways performed to a specific tempo, but intensity in the mainpart of training is adjusting to held hart rate frequency in range
between 60-85% of heart rate. If heart rate frequency reaches
above 85% of estimated maximal heart rate for age subject was
instructed to slow down the intensity of exercising.
Samples were analyzed for lipid peroxidation byproductmalondialdehyde, enzyme catalase, reactive carbonyl derivates,
total sulfhydryl groups and serum antioxidant status. Bloodmarkers of oxidative stress were determined by standardized
spectrophotometry technics (6,7,10).
In order to process the results of the study, the SPSS statistical program for Windows (Release 10.0, Chicago, IL, USA)was used. The results were statistically evaluated by the by the
Wilcoxon Signed Ranks Test and Mann-Whitney U Test.
RESULTSThe results of study are presented in Tables I and II.

Table I. Blood markers of oxidative stress before different exercise programs

Parameters
Malondialdehyde activity - MDA (μM/L)
Catalase - CAT (IU/L)
Total sulfhydryl groups - TSHG (mM/L)
Reactive carbonyl derivates (μM/g protein)
Serum antioxidant activity – AOA (%)

Exercise program
pilatestae-bopilatestae-bopilatestae-bopilatestae-bopilatestae-boMin-max
9.96-28.11
7.72-37.41
1.05-41.47
3.77-50.48
180.36-423.81
177.13-420.58
0.51-1.54
0.41-2.14
25.00-92.70
39.70-98.50

Me (25-75. percentile)
13.22 (10.39-20.77)
13.60 (12.21-19.24)
13.41 (3.56-18.22)
25.14 (7.33-48.60)
281.46 (228.08-326.76)
229.70 (203.16-333.63)
1.20 (0.58-1.28)
1.31 (0.57-1.82)
69.10 (30.90-86.80)
78.20 (60.90-89.60)

Min-max
9.29-64.27
8.57-58.75
16.55-55.51
1.89-29.95
205.44-884.83
200.58-283.89
0.87-2.39
0.35-2.79
17.70-95.60
69.10-97.10

Me (25-75. percentile)
14.42 (10.82-50.72)
13.63 (10.39-22.38)
34.56 (28.49-51.74)*
13.82 (9.64-25.87)
242.64 (221.61-258.82)
219.18 (208.27-248.30)
1.31 (0.99-2.08)
0.91 (0.61-1.85)
64.70 (22.10-83.80)
82.40 (71.35-92.65)*

Table II. Blood markers of oxidative stress after different exercise programs

Parameters
Malondialdehyde activity - MDA (μM/L)
Catalase - CAT (IU/L)
Total sulfhydryl groups - TSHG (mM/L)
Reactive carbonyl derivates (μM/g protein)
Serum antioxidant activity – AOA (%)* Mann-Whitney U Test: p < 0.05
2008.18.3(59)  Fiziologia - Physiology

Exercise program
pilatestae-bopilatestae-bopilatestae-bopilatestae-bopilatestae-bo17

DISCUSSION
Exercise intensity refers to how fast an action is performed,
the power or strength required to achieve an activity, or the effort
put forth by the participant during the activity. Measurement ofheart rate is precise and most often used method to evaluate
intensity in everyday life or to set the level of exercise in physical training.
There is a common acceptance that pilates and other formsof low intensity exercises done slowly and with the breath, will
do far more to improve health status than a vigorous cardiovascular or strength workout (1). They won’t overly raise heart
rates and breathing rates, and won’t overly disturb digestion(parasympathetic exercise) but will reduce stress levels asmuch as possible. Strenuous exercise is characterized by anincreased oxygen consumption and disturbance of intracellular
prooxidant-antioxidant homeostasis (2). Some conditions associated with intense exercise, such as local tissue hypoxia or
elevated tissue temperatures, could also contribute to reactive
oxygen production (8). Since the antioxidant reserve capacity
in most tissues is rather marginal, strenuous physical exercise
characterized by a remarkable increase in oxygen consumption
with concomitant production of ROS presents a challenge to the
antioxidant systems. There is sufficient credible evidence to suggest that exercise is accompanied by an increased generationof free radicals, resulting in a measurable degree of oxidative
modifications to various molecules (15).
In conducted study there were no significant differences inany functional or biomarkers parameters between groups before
training program at beginning of study. After 12 weeks CAT
increased in both group but increase was significantly higher
with pilates training, whereas TAS increased significantly higher
with tae-bo training. Both training programs increased valuesof MDA, protein carbonyls and sulphydryls, but there were no
significant differences between groups. However, t is clear that
ROS play important roles in numerous physiological processes
at rest; however, the detailed physiological functions of ROS in
exercise remain to be elucidated.
CONCLUSION
There was evidence of oxidative stress after both pilates andtae-bo training program. Biomarkers of oxidative stress in bloodincreased after both 12 weeks training program. Due to different metabolic demands of pilates and tae-bo training, we canconclude that both training types induced oxidative stress andaction effects of O2 is not only mechanism for exercise-induced
oxidative stress.

18

REFERENCES
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following exhaustive exercise. Eur J Appl Physiol Occup Physio l
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3. Bejma J, Ji LL. Aging and acute exercise enhance free radical generation in rat skeletal muscle. J Appl Physiol 1999; 87(1) : 465 – 470
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6. Goth L: Serum catalase: reversibly formed charge isoform of erythrocyte catalase. Clin Chem 1991; 37(2): 2043 – 2047
7. Koracevic D, Koracevic G, Djordjevic V, et al. Method for the measurement of antioxidant activity in human fluids. J Clin Pathol 2001;
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8. Koyama K, Kaya M, Ishigaki T, et al. Role of xanthine oxidase indelayed lipid peroxidation in rat liver induced by acute exhausting exercise. Eur J Appl Physiol Occup Physiol 1999; 80(1): 28 - 33
9. Leeuwenburgh C, Hollander J, Leichtweis S, et al. Adaptations ofglutathione antioxidant system to endurance training are tissue andmuscle fiber specific. Am J Physiol 1997; 272: R363 – R369
10. Levine RL, Wiliams JA, Stadtman ER, et al: Carbonil assay for
determination of oxidative modified proteins. Methods Enzymol 1994;
233: 346 - 357
11. Powers SK, Ji LL, Leeuwenburgh C. Exercise training-induced alterations in skeletal muscle antioxidant capacity: a brief review. Med
Sci Sports Exerc 1999; 31(7): 987 - 997
12. Radovanovic D, Rankovic G. Oxidative stress, stress proteins andantioxidants in exercise. Act Med Medianae 2004; 43(4):45-48
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Fiziologia - Physiology  2008.18.3(59)EFECTELE DIFERITELOR PROGRAME DE EXERCITIU FIZIC ASUPRA
MARKERILOR SANGUINI AI STRESSULUI OXIDATIV LA FEMEILE
TINEREREZUMAT
Introducere: Cresterea metabolismului aerob in timpul efortului fizic este o sursa potentiala de stres ovidativ. Doar putine studii au
investigat si comparat diferite programe de exercitii fizice aerobe si relatia acestora cu activitatile pro-oxidante si anti-oxidante.
Scop: Scopul acestui studiu a fost compararea biomarkerilor stresului oxidativ: peroxidarea lipidelor, peroxidarea proteinelor si concetratia sanguina totala a antioxidantilor, precum si variabilele functionale, inainte si dupa 12 saptamani de efectuare a 2 programediferite de exercitiu fizic.
Metode: Au fost recoltate probe de sange de la sapte persoane de sex feminin, tinere, care desfasurau antrenament pilates ca
exercitiu fizic aerobic de intensitate scazuta si sapte femei tinere care desfasurau activitati tae-bo ca exercitiu aerobic de intensitate
crescuta. Analiza probelor a presupus determinarea produsilor metabolici ai peroxidarii lipidice, cum ar fi malondialdehida, catalaza,derivatii reactivi de carbonil, grupurile sulfhidril totale si statusul seric antioxidant. Rezultatele au fost evaluate statistic prin Wilcoxon
Signed Ranks Test si Mann-Whitney U Test.
Rezultate: Nu au existat diferente semnificative intre parametrii functionali si biomarkeri la grupurile studiate, inainte de inceperea
programenlor de antrenament, la inceputul studiului. Dupa 12 saptamani, CAT a crescut in ambele grupuri, dar cresterea a fostsemnificativ mai crescuta in grupul care a efectuat pilates, in timp ce TAS a crescut semnificativ in cursul antrenamentelor tae-bo.
Ambele grupuri de antrenament fizic au prezentat valori crescute ale MDA, protein carbonil si sulfidril, dar nu au existat diferentesemnificative intre grupuri.
Concluzii: Nu exista dovezi ale stresului oxidativ dupa efectuarea programelor pilates si tae-bo. Biomarkerii sanguini ai stresului
oxidativ au crescut dupa 12 saptamani de antrenament. Datorita diferitelor nevoi metabolice in pilates si tae-bo, am putut concluziona ca ambele tipuri de antrenament fizic induc stresul oxidativ, iar efectele actiunii O2 nu sunt singurele mecanisme implicate instresul oxidativ indus de efort.
Cuvinte cheie: stres oxidativ, pilates, tae-bo, efort fizic

2008.18.3(59)  Fiziologia - Physiology

19

SHORT AND LONG TERM HYPOBARIC HYPOXIA INDUCESOXIDATIVE STRESS IN RATS: THE PROTECTIVE EFFECTS
OF N-ACETYLCYSTEINEIRINA CHIS¹, MARIUS-IONUT UNGUREANU², RAMONA SIMEDREA1, MONICA MAIER3,ADRIANA MURESAN¹, PHILIPPE HOANG 4, PATRICIA SUTEU 2, ADRIANA MARTON 2,
NICOLETA DECEA1¹Physiology Department, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj Napoca, Romania²Medicine Student, “Iuliu Hatieganu”University of Medicine and Pharmacy, Cluj Napoca, Romania
3
Northern University, Baia Mare, Romania
4
Centre Tomberg, Bruxelles, Belgium

ABSTRACT
Purpose: The aim of this study was to investigate whether acute and severe hypobaric hypoxia induces the changes of the reactive oxygen species (ROS) and the antioxidant systems. Knowing the efficiency of N-acetylcysteine supplementation in preventing
tissue lesions induced by hypoxic stress, we have also studied if NAC could ameliorate the disfunctionalities generated by the free
radicals caused by hypoxia. Methods: The animals were exposed to hypobaric hypoxia in the barochamber (simulated high altitude
was the equivalent of 5500 meters), for 2 and 14 days. We have followed up 6 groups of Wistar rats: 1st group – control group, which
received a vehicle solution (placebo) ; 2nd group – control group, N-acetylcysteine (NAC, 20 mg/kg); 3rd group – rats exposed tohypobaric hypoxia for 2 days-placebo; 4th group – rats exposed to hypobaric hypoxia for 14 days-placebo; 5th group – rats exposed
to hypobaric hypoxia for 2 days and treated with NAC (20 mg/kg), prior to each exposure; 6th group – rats exposed to hypobaric
hypoxia for 14 days and treated with NAC (20 mg/kg), prior to each exposure. After 14 days, the oxidative parameters in the blood
were determined. Results: The results indicate an increase in the oxidative stress as seen by the increase in free radical production, malondialdehyde (MDA) and carbonylated proteins levels, after the exposure to hypobaric hypoxia. The antioxidant defense
systems such as reduced superoxide dismutase (SOD) and catalase (CAT) levels were significantly decreased after “acute” andlong term hypobaric hypoxia but they significantly increase after the administration of NAC. Conclusions: This study suggests thatthe administration of NAC can be beneficial in attenuating the oxidative stress associated with exposed to high altitude.
Key words: hypobaric hypoxia, oxidative stress, N-acetylcysteine

INTRODUCTION
N-Acetylcystein(NAC), also called Acetylcysteine, is wellknown by its effectiveness in the treatment of acute and chronic
bronchitis and chronic obstructive bronchopneumapthy. Recent
research have brought numerous arguments regarding its rolein different diseases related to oxidative stress.
Nowadays, the implication of reactive species of oxygen(ROS) in a high number of physiological and pathological processes is well-known, some of them having an importance inthe clinical practice, as a causal factor, leading, accompanying
or even aggravating primary lesions.
Acute and chronic exposure to altitude constitutes an oxidative stress, the ROS sources being: mitochondrial respiratory
chain alterations (1), membrane and cytosol alterations, endothelial cell alterations and lipid and protein perturbations. They arecaused by hypobaric hypoxia, ultraviolet radiations and, possibly,
by the temperature variations, dehydration and nutritive deficient.

The oxidative metabolism is altered, and the anaerobic one isactivated. The mitochondria can become targets of the oxidation,
leading to membrane lipid peroxidation (5), protein oxidation,
DNA clivation and, subsequently, deficitary production of ATP (6,7, 8). The purpose of this study was to evaluate whether acuteand severe hypobaric hypoxia induces the changes of the ROS
and the antioxidant systems and also to assess the efficiencyof N-acetylcysteine (NAC) supplementation in preventing tissue
lesions induced by hypoxic stress.
MATERIALS AND METHODS
We have studied 6 groups of 10 Wistar white male rats each,with the body mass between 150-170g.
The animals were exposed to hypobaric hypoxia in thebarochamber (the simulated high altitude was the equivalent of
5500 meters), for 2 days and 14 days.
All studies were conducted in accordance with the National

Received May 2008. Acccepted August 2008. Address for correspondence: Dr. Irina Chis, Physiology Department, “Iuliu Hatieganu” University of Medicine andPharmacy, Cluj Napoca, Romania, e-mail: irinnaus@yahoo.com

20

Fiziologia - Physiology  2008.18.3(59)Institute of Health “Guide for the Care and Use of Laboratory
Animals” and the experiments were carried out with the permission of the Institutional Ethics Committee.
The animals were divided into 6 groups:- 1st group - Control group, which received vehicle solution(physiological serum-placebo)- 2nd group- Control group, which received N-acetylcysteine(NAC, 20 mg/kg);- 3rd group- rats exposed to hypobaric hypoxia for 2 daysplacebo;- 4th group- rats exposed to hypobaric hypoxia for 14 daysplacebo;- 5th group - rats exposed to hypobaric hypoxia for 2 days andtreated with NAC (20 mg/kg), prior to each exposure;- 6th group - rats exposed to hypobaric hypoxia for 14 days
and treated with NAC (20 mg/kg), prior to each exposure.
At the end of the experiment, the rats were fasted overnightand sacrificed by cervical decapitation. Venous blood samples
were collected from the rats’ retroorbital sinus. The serum wasseparated for the estimation of the free radical production: malondialdehyde (MDA) (by Satoh method) (11) and carbonylated
proteins levels (by Reznick method) (9); erythrocytes wereseparated for the estimation of superoxide dismutase (SOD, by
Kakkar method) (4), catalase (CAT, by Sinha method) (12).
RESULTS
Tables I and II show the levels of free radicals: MDA and carbonylated proteins levels in the serum of control and experimental
animals. A significant increased oxidative stress was observed
after the exposure to hypobaric hypoxia. Supplementation withNAC (20 mg/kg body weight) in the hypoxia-exposed group for
2 and 14 days respective shows significant decreased oxidative
stress.
Table I. Effect of N-acetylcysteine (NAC) on MDA, carbonylated
proteins levels in the serum and SOD and CAT in the RBC lysate ofnormal and experimental rats after 2 days the exposure to hypobaric
hypoxia (***p<0.001; **p=0.005; *p<0.05)

Table II. Effect of N-acetylcysteine (NAC) on MDA, carbonylated
proteins levels in the serum and SOD and CAT in the RBC lysate ofnormal and experimental rats after 14 days the exposure to hypobaric
hypoxia (***p<0.001; **p=0.005; *p<0.05)
Parameters

MDA
Carbonyla ted
SODCAT(nmol/ml)
proteins(U/mg Hg) (U/mg Hg)(nmol/mg)
1st Group 2.01± 0.26 0.61± 0.06
3.17±0.11 2.81±0.09
2nd Group 2.03±0.21 0.63± 0.15
3.08±0.18 2.76±0.09
4rd Group 6.97±0.5*** 2.02±0.3*** 1.72±0.3*** 1.79±0.1***
6th Group 4.67±1.85** 1.30±0.1*** 2.97±0.3*** 2.63±0.3***

DISCUSSION
NAC actions by itself extracelularly, and intracelularly it
actions as a precursor of L-cysteine and reduced glutathione(GSH) through a variety of mechanisms, ROS reduction being
one of them (2). These properties are extremely relevant for theskin carcinogenesis induced by UV, due to ROS involvement inthese processes, in conditions in which cutaneous antioxidants
are depleted in the cells exposed to UV (10). NAC is efficientin decreasing their incidence, multiplication and prevents theappearance of scuamous cell carcinoma. In a high number ofstudies there was showed that that NAC acts as an inhibitor of
apoptosis, due to its ability to attenuate the damages launchedby these processes over DNA (2, 3).
SOD protects tissues against oxygen free radicals by catalyzing the removal of superoxide radical (O2•-), which damages themembrane and biological structures.
Catalase has been shown to be responsible for the detoxification of significant amounts of H2O2.
SOD and catalase are the two major scavenging enzymes
that remove the toxic free radicals in vivo. Reduced activities of
SOD and catalase in erythrocytes have been observed after the
exposure to hypobaric hypoxia and this may result in a number ofdeterious effects due to the accumulation of superoxide radicals(O2•-) and hydrogen peroxide.

3.17±0.11 2.81±0.09
3.08±0.18 2.76±0.09
2.67±0.5** 2.41±0.01*
2.96±0.36* 2.73±0.33*

CONCLUSIONS
The data we obtained showed that hypoxic aggression
induces an increased production of lipid peroxides and proteinoxidation. Moreover, our study suggests that NAC is able tosoften the impact of hypobaric hypoxia, based on the fact thatthe markers of ROS production and oxidative damage in rats
were substantially lower in the hypoxic animals treated with NAC
than in the hypoxic animals not treated with NAC.

Tables I and II show the activities of enzymatic antioxidants:
SOD, CAT of control and experimental animals. Activities ofthese enzymes significantly decreased after “acute” and longterm hypobaric hypoxia exposure but they significantly increased
after supplementation with NAC (20 mg/kg body weight) for 2
and 14 days, respectively, of hypoxia exposure.

REFERENCES
1. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC,
Schumacker PT. Mitochondrial reactive oxygen species trigger hypoxia induced transcription. Proc. Natl. Acad. Sci. USA, 1998; 95:
11715-11720
2. De Flora S, Izzotti A, D’Agostini F, Balansky RM. Mechanisms ofN-acetylcysteine in the prevention of DNA damage and cancer, withParameters

MDA(nmol/ml)

Carbonylated
proteins(nmol/mg)
1st Group 2.01± 0.26 0.61± 0.06
2nd Group 2.03±0.21 0.63± 0.15
3rd Group 4.57±1.1*** 1.65±0.12 ***5th Group 3.55±1.4** 0.96±0.20 ***SOD
CAT(U/mg Hg) (U/mg Hg)

2008.18.3(59)  Fiziologia - Physiology

21

special reference to smoking-related end-points. Carcinogenesis
2001; 22: 999- 1013
3. De Flora S, Izzotti A, D’Agostini F,Bagn Asco M, Balansky RM. Antigenotoxic and cancer preventive mechanisms of N-acetyl-L-cysteine.
In Kelloff GJ, Hawk ET and Sigman CC (eds) Cancer Chemoprevention Vol. 1, Promising Cancer Chemopreventive Agents. Humana
Press, Totowa, NJ, 2004; 37- 67
4. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometricassay of superoxide dismutase (SOD). Indian J. Biochem. Biophys.
1984; 21: 130-132
5. Paradies G, Petrosillo G, Pistolese M, Di Venosa N, Serena D, Ruguggiero FM. Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia andreperfusion. Free Radic. Biol. Med. 1999; 27: 42-50
6. Paradies G, Petrosillo G, Pistolese M, Di Venosa N, Serena D,
Ruguggiero FM. Reactive oxygen species affect mitochondrial electron transport complex I activity through oxidative cardiolipin damage.
Gene. 2002; 286: 135-141

7. Petrosillo G, Ruguggiero FM, Pistolese M, PARADIES G. Reactive
oxygen species generated from the mitochondrial electron transport
chain induce cytochrome c dissociation from beef-heart submitochondrial particles via cardiolipin peroxidation. Possible role in the apoptosis. FEBS Lett. 2001; 509: 435-438
8. Rafique R, Schapira AH, Coper JM. Mitochondrial respiratory chain
dysfunction in ageing; influence of vitamin E deficiency. Free Radic.
Res. 2004; 38: 157-165
9. Reznik AZ, Packer L. Oxidative damage to proteins: spectrofotometric method for carbonyl assay. Meth. Enzymol. 1994; 233: 357-363
10. Saliou C, Kitazawa M, McLaughlin L et al. Antioxidants modulate
acute solar ultraviolet radiation-induced NF-kappa-B activation in a human keratinocyte cell line. Free Radic. Biol. Med. 1999; 26: 174- 183
11. Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin. Chim. Acta 1978; 90:
37- 43
12. Sinha KA. Colorimetric assay of catalase. Anal. Biochem. 1972;
47: 389-394

HIPOXIA HIPOBARICA PE TERMEN SCURT SI LUNG INDUCE APRITIASTRESULUI OXIDATIV LA SOBOLANI: EFECTELE PROTECTOARE ALE
N-ACETILCISTEINEIREZUMAT
Scopul acestui studiu a fost sa evidentieze daca hipoxia hipobara acuta si severa induce modificari ale speciilor reactive ale
oxigenului (SRO) si ale sistemelor antioxidante. Cunoscand eficienta suplimentului de N-acetilcisteina in in prevenirea leziunilor
tisulare induse de stresul hipoxic, am studiat de asemenea daca NAC poate ameliora disfunctionalitatile generate de radicalii liberi
ce apar in urma hipoxiei. Metode: Animalele au fost expuse la hipoxie hipobara in barocamera (altitudinea simulate a fost echivalentul a 5500 de metri) timp de 2, respectiv 14 zile. Am urmarit 6 grupuri de sobolani rasa Wistar: grupul 1-grup control, care a primit
solutie vehicul (placebo); grupul 2- grup control- a primit NAC, 20 mg/kg; grupul 3-sobolani expusi la hipoxie hipobara timp de doua
zile- placebo; grupul 4- sobolani expusi la hipoxie hipobara timp de 14 zile-placebo; grupul 5-sobolani expusi la hipoxie hipobara
timp de 2 zile si tratati cu NAC (20 mg/kg), inainte de fiecare expunere; grupul 6- sobolani expusi la hipoxie hipobara timp de 14
zile si tratati cu NAC (20 mg/kg) inainte de fiecare expunere. Dupa 14 zile, parametrii stresului oxidativ au fost dozati din sange.
Rezultate: indica o crestere a stresului oxidativ, marcata de cresterea productiei de radicali liberi, malondialdehida (MDA) si proteine carbonilate, dupa expunerea la hipoxie hipobara. Sistemele de aparare antioxidanta, cum ar fi superoxid-dismutaza (SOD)
redusa si catalaza (CAT), au avut niveluri semnificativ scazute dupa hipoxia acuta si de durata, dar nivelurile acestor parametri au
fost crescute dupa adminitrarea NAC. Concluzii: Acest studiu sugereaza ca administrarea de NAC poate fi benefica pentru atenuarea stresului oxidativ asociat cu expunerea la altitudine.
Cuvinte cheie: hipoxie hipobara, stres oxidativ, N-acetilcisteina

22

Fiziologia - Physiology  2008.18.3(59)REST AND POSTURAL TREMOR IN THE POWER SPECTRALSTRUCTURE IN PATIENTS WITH PARKINSON’S DISEASEJELENA MARIĆ1, SUZANA BLESIĆ1, SLADJAN MILANOVIĆ1, NATAŠA DRAGAŠEVIĆ2,
TIHOMIR ILIĆ3, VLADIMIR KOSTIĆ2, MILOŠ LJUBISAVLJEVIĆ4
1
Institute for Medical Research, Belgrade, Serbia;
2
Institute of Neurology, Clinical Centre of Serbia, Medical School, University of Belgrade, Belgrade, Serbia;
3
Outpatient Neurological Service, Military Medical Academy, Belgrade, Serbia;
4
Faculty of Medicine and Health Sciences, Unite Arab Emirates University, Al Ain, United Arab Emirates

ABSTRACT
It has recently been suggested that frequency harmonics found in power spectra of pathological tremors may contain information on
dynamical properties of tremors, with each finite frequency band (mode) corresponding to a fluctuation period of a single independent source of oscillation. It was thus shown earlier, in the case of Parkinson’s disease (PD) tremor, that the first two modes of oscillation display different patterns of dynamic behaviour. The aim of the present study was to investigate possible difference between
the dynamic oscillatory behaviour of the rest and the postural tremor in PD.
We have studied the time series of hand tremor movements PD by applying the classical Fourier analysis and the detrended fluctuation analysis (DFA) method, to distinguish between their power spectra frequency bands. The analysis of these tremor series
revealed the type of dynamic behaviour that can help differentiate between different modes. The application of DFA analysis helped
us also distinguish between neural mechanisms active in rest and postural tremor in PD patients, as well as discerning difference oftremors in PD, when compared to hand oscillations in healthy subjects. Our findings suggest that pathological tremors in movement
disorders may entail multiple oscillatory circuits that underlie the complexity of their initiation and development.
Keywords: Parkinson’s disease, PD, tremor, power spectra, Fourier analysis

INTRODUCTION
Tremors are typically defined as complex movements, manifested through seemingly rhythmic, oscillatory limb contractions,or contractions of muscle groups, producing oscillatory movements at one or more joints (6). While it is believed that mostof pathological tremors have their origins in the central nervous
system, the pathophysiology of tremor-generating mechanisms
is still an open key question. The power spectra (PwS) of mosttremors usually appear as semi-periodic wave forms, with morethan one typical frequency (see Fig.1b). The recent studies support the assumption that, particularly in tremors of Parkinson’s
disease (PD), multiple sets of neuronal oscillators, rather then asingle one, generate the tremulous activity (1,5,7,9,11).
It has been proposed recently (11) that the presence of multiple oscillatory drives of tremor could be assessed by probing
the difference in dynamic behaviour that is, in values of functions characterizing the dynamic behaviour, of different modes,
extracted from different frequency domains of the power spectraof recorded tremor data series. It was in this way suggested
that the rest tremor in PD most likely originates from multiple
superimposed biological oscillators (11).
The aim of our study was to confirm previous results in thecase of PD tremor (11). Having in mind that the typical tremor(6) in PD patients is the rest tremor, while the other types ofpathological tremors manifest in the existence of postural tremor,
we have investigated both the rest and postural tremor in PD,

and have compared our findings with the normal (physiological) oscillations of the same kind (rest and postural) in healthy
subjects.
The details of DFA method
In order to elucidate the dynamic behaviour of analyzed
tremor data, we applied the detrended fluctuation analysis(DFA), technique that stems from the field of statistical physics,
on a PtP (point-to-point) series of tremor data (10). The DFA
method requires three consecutive steps. In the first step, foreach sequence of T(k), one should calculate the partial sumηy(η) = ∑ T(k) − Tave , where Tave is the average of PtP dura-

1
tions (in k =allcases studied the total number of PtP data for agiven series was of the order of 103). In the next step, the entire
series of y(η) is divided into a set of overlapping segments of thelength n and evaluate the local trend for each segment, whichis the polynomial fit for the segment data (4). The order of thepolynomial fit k specifies the order of the DFA (denoted DFAk).
We used a sliding segment, of size n, which slides over the entirey(η) series, producing thereby a new series of segments yn,i(η).
For the new series, one has to define the so-called detrended

walk yn,d i (η) that is, for a given segment, the difference between
the original series (represented by the partial sums y(η) and the
local trend. Finally, one has to calculate the variance about the
local trend for each segment and determine the average of thesevariances over all segments, which brings about the detrended

Received May 2008. Accepted September 2008. Address for correspondence: Jelena Maric, Institute for Medical Research, PO Box 102, 11129 Belgrade, Serbia,
phone: + 381 64 116 92 77, fax: + 381 11 2643691, e-mail: jelenam@imi.bg.ac.yu

2008.18.3(59)  Fiziologia - Physiology

23fluctuation function:
N − n+1 n

F(n) = 1/(N − n + 1)n ∑ ∑ (yn,d i )2
i =1

η=1

It has been proven that the function F(n) increases withincreasing the segment length n. A linear relationship on a log-log
plot indicates the presence of power-law type scaling behaviourof the analyzed data. Under such conditions, the fluctuations
can be characterized by the scaling exponent η, a slope of theline relating log F(n) to log n. The scaling exponent η characterizes the dynamics of the analyzed series, for it is connected toscaling exponent η of the related PwS, and the exponent η ofthe related autocorrelation function, through the scaling relationa = 1 − g / 2 = (1 + b ) / 2 (12). It may happen, as wasshown in our previous work, that in two neighbouring intervals ofn the function F(n) displays two different power-law behaviours(2,3). The so-called crossover region is then defined by thevalues of n on which the function F(n) changes its behaviour.
MATERIALS AND METHODS
Altogether, 20 patients participated in the study (mean age
62.1 years, range 47-76, mean UPDRS 25.3; see Table 1). The
findings in this patient group were compared with corresponding
data recorded in 20 healthy subjects (mean age 46 years, range
30-72), who had no history of neurological diseases. Patientswere pre-selected from the records of the Institute of Neurology, Clinical Centre of Serbia and Neurological Service of theMilitary Medical Academy in Belgrade. All patients underwent
routine neurological examination that excluded other neurological abnormalities. All subjects gave informed, written consent,
acknowledging that the employed methods had been clearly
explained, and that they understood them prior to the study.
All procedures were approved by the Local Ethics Committee,
and all the precautions were taken to comply with the Helsinki
Declaration of the World Medical Associations (with all current
revisions and amendments).
Tremor movements were recorded by monoaxial accelerometers (EGAX-5, Etran Devices Inc., USA), placed on subject’s
middle finger. All the subjects (patients and healthy controls)
were free of drugs known to influence the tremor for at least
24 hours before the examination. In all the subjects, the tremor
was recorded while comfortably seated in reclined position,with hands at rest (rest condition) and with arms and hands
fully extended (postural condition). Subjects were instructed tokeep their eyes closed during the recordings. Three successive
2-minutes epochs were recorded in all the studied cases. In order
to avoid fatigue, each 2-minute recording session was followed
by a 5-minute break.
Fourier analysis of signals was performed off-line, and thepeak frequencies of the power spectra and the total power of therecords were calculated, in the range between 1 and 25 Hz. We
have analyzed, using the extended DFA method, the first three
PwS modes, in order to access their possible independence,
based on comparison of their dynamic behaviour (8). The total
number of the analyzed data (PtP) of a single mode has beenof the order of 103 data.
24

Fig.1. a) Typical example of recorded hand tremor from apatient with Parkinson’s disease; b) The corresponding power
spectrum of the recorded tremor, shown in a), displays three
distinct frequency modes, used for filtering the original data
RESULTSFor all PD patients studied, functions F(n) of the second
order DFA (denoted DFA2) have been calculated, for the PtPtime series extracted from the regions around three main PwS
modes, while in the healthy controls, since it was impossible todiscriminate different PwS modes, DFA2 curves were calculated
for the area covering law frequencies (from 1Hz to 25Hz) (8).
Table I: Characteristics of the 20 patients with PD, and 20 healthy
controls.

PATIENTS
No. (%)HEALTY SUBJECTS
No. (%)• Sex
Male
11 (55)
12 (60)Female9 (45)
8 (40)
61.47-76
46.30-72
Mean age, range
Handedness
Right
19 (95)19 (90)Left
1 (5)2 (10)• Family historyYes
12 (60)4(20)No
8 (40)
16(80)• Mean disease duration, range 8.2-14• UPDRS
25.3• H&Y		2.5Fiziologia - Physiology  2008.18.3(59)In Fig.2, the results obtained for a patient with Parkinsonian(PD) rest tremor are shown. We present DFA functions on a log10log10 graphs, to measure ηby a linear fit, in the small n regions(within two decades), which was the range for estimating scaling
exponent η. Although there was a notable difference between
the first and the second PwS mode (see Fig.2), the differencein corresponding values of scaling exponents was, dissimilar tofindings of Sapir and colleagues (11), not statistically significant.
However, it is visible from the Fig.2 that the third PD mode
displays markedly different dynamics, in comparison to the firsttwo modes (with p<0.05 for differences of scaling exponents ofthe first and the third region, and second and the third region,
respectively). This was the case in all PD patients studied.

Fig.2. The DFA functions FDFA2(n) of second order, for the series ofpeak-to-peak (PtP) intervals, extracted around the first three modesof the power spectra of hand tremor from patient with Parkinson’s
disease (PD). Straight lines represent the linear least-squares fits to
DFA functions. One can observe markedly different dynamics of thethird harmonic mode, in comparison to the first two PwS modes, in thesmall n region. This is a typical result obtained for PD patients

The results obtained for the low frequency regions of power
spectra of healthy controls group, depicted in Fig. 3, show distinct
difference between the values of the scaling exponent α of therecorded tremor when compared to the corresponding tremorsin PD. All the results obtained from healthy controls in the rest
condition show existence of scaling in the DFA2 curves, with thescaling exponent α=0.34±0.01, and with a visible crossover onhigher scales (more than two orders of magnitude; see Fig. 3),
while the corresponding results for the first (main) mode in PD
case equals to αPD=0.45±0.04, with the visible crossover on lower
scales. In the case of the recorded postural tremor, the results
obtained from healthy controls show existence of scaling in theDFA2 curves, with the scaling exponent α=0.32±0.04, and witha crossover on high scales, while the corresponding results forthe first (main) mode in PD case equals to αPD=0.48±0.01, withthe visible crossover on lower scales. The DFA analysis thus
differentiated between the dynamical behaviour of the rest andpostural tremor in PD patients and healthy controls.

2008.18.3(59)  Fiziologia - Physiology

Fig.3. Comparison of the DFA functions FDFA2(n) for the first(main) modes for a patient with PD and a healthy subject, inrecorded rest tremor condition. Arrows point to the positions ofcrossovers. The calculated values of DFA exponents α are given,
for PD case and a healthy control case

DISCUSSION
We have studied the time series of hand tremor movements
PD by applying the classical Fourier analysis and the detrended
fluctuation analysis (DFA) method, to distinguish between theirpower spectra frequency bands. The analysis of these tremor
series revealed the type of dynamic behaviour that can help
differentiate between different modes.
The application of DFA analysis helped us also distinguishbetween neural mechanisms active in rest and postural tremor
in PD patients, as well as discerning difference of tremors in PD,
when compared to hand oscillations in healthy subjects. Namely,
the rest and the postural tremor recorded in PD patients display
the existence of higher degree of correlations and the existenceof crossover in scaling behaviour on lower scales than the corresponding (rest and postural) oscillations in healthy subjects
that show lesser degree of correlations and have crossovers on
a much higher scales.
Our findings suggest that pathological tremors in movement
disorders may entail multiple oscillatory circuits that underlie
the complexity of their initiation and development. Our findings
also suggest that the existence of pathology in PD tremor may
be connected to higher degree of correlations in corresponding tremor data. In the PD case, the positions of crossovers inbehavior were shifted toward lower scales, in comparison to thedata obtained in healthy subjects, in both rest and postural condition. Generally, the most likely explanation for the appearanceof crossover (2) is that it is the sign of effects of various global
and/or local processes (with longer characteristic time scales)
within the organism. The appearance of these processes onlower scales in PD case could thus be interpreted as anothersign of existent pathology.

25

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7. Gao JB, Tung W-w. Pathological tremors as diffusional processes.
Biol. Cybern. 2002;86:263-270
8. Kantelhardt JW, Koscielny-Bunde E, Rego HHA, et al. Detecting
Long-range Correlations with Detrended Fluctuation Analysis. Physica
A 2001;295:441-454
9. O’Suilleabhain PE, Matsumoto JY. Time-frequency analysis of tremors. Brain 1998;121:2127-2134
10. Peng C-K, Buldyrev SV, Havlin S, et al. Mosaic organization of
DNA nucleotides. Phys. Rev. E 1994;49:1685-1689
11. Sapir N, Karasik R, Havlin S, et al. Detecting scaling in the period
dynamics of multimodal signals: application to Parkinsonian tremor.
Phys. Rev. E 2003;67:031903-8
12. Stanley HE, Buldyrev SV, Goldberger AL, et al. Statistical mechanics in biology: how ubiquitous are long-range correlations? Physica A
1994;205:214-253

TREMORUL DE REPAUS SI POSTURAL IN STRUCTURA SPECTRALA DE
PUTERE LA PACIENTII CU BOALA PARKINSONREZUMAT
Recent a fost sugerat ca frecventa armoniilor din spectrele de putere ale tremorului patologic ar putea contine informatii cu privire la
proprietatile dinamice ale tremorului, fiecare banda de frecventa finita (mod) corespunzand unei perioade de fluctuatie a unei singure surse independente de oscilatie. A fost aratat anterior, in cazul tremorului din boala Parkinson (PD), ca primele doua moduri deoscilatii prezinta patternuri diferite de comportament dinamic. Scopul acestui studiu a fost investigarea posibilei diferente existente
intre comportamentul oscilator dinamic in repaus si tremorul postural in PD.
Am studiat in evolutie miscarile de tremor ale mainii persoanelor cu PD prin aplicarea analizei Fourier clasica si metoda analizei
fluctuatiilor (DFA), pentru a diferentia spectrul de putere in benzile de frecventa. Analiza seriata a relevat tipul comportamentului
dinamic care poate ajuta la diferentierea diferitelor moduri. Aplicarea analizei DFA a ajutat la diferentierea mecanismelor neurale
active in repaus si tremorul postural la pacientii cu PD, precum si la diferentierea diferitelor tipuri de tremor in PD, atunci cand au fostcomparate cu oscilatiile prezente la nivelul mainii unei persoane sanatoase. Rezultatele noastre sugereaza ca tremorul patologic inafectiunile de miscare pot avea diferite circuite oscilatorii care stau la baza complexitatii initierii si dezvoltarii acestora
Cuvinte cheie: boala Parkinson, PD, tremor, spectru de putere, analiza Fourier

26

Fiziologia - Physiology  2008.18.3(59)THE CORRELATION BETWEEN CARDIOVASCULAR RISKFACTORS, ARTERIAL STIFFNESS AND LEFT VENTRICULAR
DIASTOLIC FUNCTION IN THE PATIENTS WITH
HYPERTENSION
L. AGOSTON-COLDEA¹, T. MOCAN², M GATFOSSE³, C. BOBAR¹, LD. RUSU¹, L. POANTA, DL.
DUMITRASCU¹“Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania¹Department of Medical Sciences²Department of Normal Morphology and Functions³Department of Internal Medicine, Coulommiers, France

ABSTRACT
Background and aim: Hypertension accelerates atherosclerosis, collagen synthesis, and arterial smooth muscle hyperplasia andhypertrophy, thereby increasing arterial stiffness. It is well known that left ventricular diastolic function declines in the elderly, especially in patients with hypertension. The study aims determine the relationship between cardiovascular risk factors, arterial stiffness
and left ventricular diastolic dysfunction in patients with hypertension with preserved left ventricular ejection fraction using newly
developed ultrasonic imaging.
Methods: We performed a cross-sectional study including 105 (52 men and 53 women) patients with hypertension, mean age 58.5
(12.4). Left ventricular systolic and diastolic function was evaluated by 2D, M-mode and measuring transmitral flow velocity, mitralannular motion velocity using conventional and tissue Doppler ultrasonic system. Subclinical atherosclerosis also was determinedby measuring the intimae – media thickness and stiffness of the common carotid arteries using 2D-, M-mode and tissue Doppler by
ultrasonography. The dosages of lipids were measured by enzymatic method and the dosages of apolipoproteins were measured
by immunoturbidimetric methods.
Results: The mitral annular motion velocity is correlated with apolipoprotein B (r = 0.64, p<0.05), and LDL-cholesterol (r = 0.54, p =0.05) plasma levels, and apoB/apoA-I ratio (r = 0.58, p<0.05). The diastolic transmitral flow velocity is correlated with hypertension,
without associated with lipids and apolipoproteins. The stiffness of carotid arteries correlated with diastolic mitral annular motion
velocity (r = 0.65, p < 0.05) and with apoB/apoA-I ratio level (r = 0.62, p < 0.05). No significant correlation was obtained between theintimae – media thickness and echocardiographical parameters that define systolic and diastolic function of left ventricle.
Conclusions: These results suggest that the cardiovascular risk factors interact with arterial stiffness and left ventricular relaxationin patients with hypertension. The modern non-invasive imagistic exploration describes a useful method for early tracking of subclinical atherosclerosis.

INTRODUCTION
Hypertension accelerates atherosclerosis, arterial smoothmuscle hyperplasia and hypertrophy, and collagen synthesis,
thereby increasing arterial stiffness. Arterial stiffening is foundin patients with hypertension, but it is not clear whether thearterial disease precedes or is a consequence of sustained
elevated blood pressure (29). Increased stiffness of large arteries commonly occurs with advancing age, thereby leading to thedevelopment of atherosclerosis regardless of the presence ofcoexisting diseases (22). In addition, increased arterial stiffness is
associated with hypertension (26), elevated levels of triglycerides(2), diabetes (36), obesity (25), and may be an early marker forthe development of cardiovascular disease.
As asymptomatic patient with hypertensive left ventricular
hypertrophy and an echocardiography showing a normal ejection fraction and abnormal left ventricle filling can be said tohave diastolic dysfunction (1,7). Diastolic dysfunction refers toan abnormality of diastolic distensibility, filling, or relaxation of
the left ventricle, regardless of whether the ejection fraction isnormal or abnormal and whether the patient is symptomatic or
asymptomatic (7).
Arterial stiffness of hypertension patients has received growing attention during the last decade because of its association
with cardiovascular diseases (3). Interestingly, recent results
demonstrate that arterial stiffness is an independent predictor ofprogression to hypertension in normotensive subjects suggesting that lower arterial elasticity is related to the development ofhypertension (5).
The measurement of pulse wave velocity is generally accepted as the most simple, non-invasive, and reproducible
method indirect to determine arterial stiffness (13). However,the progresses made in last year in noninvasive ultrasonography
concur to assessment of subclinical atherosclerosis by using
tissue Doppler ultrasonography as direct method of simultane-

Received April 2008. Accepted June 2008. Address for correspondence: Dr. Lucia Agoston-Coldea, 2-4 Clinicilor Street, 400006, Cluj-Napoca, Romania, phone:+40264591942; fax: 0264/599817, e-mail: luciacoldea@yahoo.com

2008.18.3(59)  Fiziologia - Physiology

27ous assessment of left ventricular diastolic function as well as
carotid distensibility (8).
The study aims determine the relationship between cardiovascular risk factors, arterial stiffness and left ventricular diastolic
dysfunction in patients with hypertension with preserved left
ventricular ejection fraction using newly developed ultrasonicimaging.
METHODS
Patients and study design
We performed a cross-sectional study with 105 patients witharterial hypertension (52 men and 53 women), hospitalized in the
Department of Internal Medicine, Coulommiers, France between
May 2005 and December 2005. Patients were informed about
the study protocol and written consent was obtained from eachpatient. All the patients were evaluated with the same structuralquiz, the same clinical and paraclinical protocol: medical history,
physical examination, 12-lead electrocardiography, 2D, M-mode
and Doppler echocardiography, 2D, M-mode and Doppler carotid
echography, biochemical analysis.
Essential arterial hypertension was defined in patients asa systolic blood pressure (SBP) >140 mm Hg and/or diastolicblood pressure (DBP) >90 mm Hg or being treated with bloodpressure lowering drugs (14). Only patients with normal restingleft ventricular systolic function (ejection fraction >50% and left
ventricular restrictive filling pattern) and without significant valve
dysfunction were enrolled (12). The patients were divided in two
groups according to the presence or absence of left ventricular
diastolic dysfunction: group 1 – patients without left ventricular
diastolic dysfunction and group 2 – patients with left ventricular
diastolic dysfunction.
Exclusion criteria comprised: 1) any causes of secondary
hypertension; 2) history of cardiovascular disease, defined as
myocardial infarction, coronary heart disease, stroke (includingtransient ischemic attack), congestive heart failure, and peripheral vascular disease; 3) current use of oral hypoglycemic,
antihypertensive, or lipid-lowering drugs; 4) renal failure; and 5)
ejection fraction < 50%.
Blood sampling and biochemical analysis
Venous blood samples were obtained after 12 h of fasting,and samples for lipids, glucose and apolipoproteins, were drawn
without stasis into evacuated glass tubes containing 1/100 volume of 0.5 mmol of ethylenediaminetetraacetic acid/L. Plasma
was obtained by centrifugation at 1500 g for 15 minutes wasmeasured in fresh samples.
All patients had lipids: cholesterol (TC), HDL-cholesterol(HDL-C), LDL-cholesterol (LDL-C), triglycerides (TG) fromplasma, analyzed by enzymatic tests performed by a RocheHitachi 911 analyzer (9). The plasma concentration of apo A-I
and B and Lp(a) were measured by immunoturbidimetry withthe help of the Roche/Hitachi Modular analyzer, using reagentsand standard controls traceable to the International Federationof Clinical Chemistry primary standards (15,16).
Clinical variables
We recorded the presence of several risk factors such as:
28age, sex, family background of coronary heart disease, smoking, hypertension, diabetes mellitus, obesity, dyslipidemia – alldefined in accordance to international rules. Family history ofcoronary heart disease (CHD) was defined by a history of premature coronary artery disease in first-degree relatives (having
occurred in those relatives at age < 55 years for men and < 65years for women). Active smoking was defined as the smoke ofat least one cigarette per day within the previous two months.
The presence of diabetes at baseline was defined as fasting
plasma glucose >110 mg/dL (6.1 mmol/L) or use of oral hypoglycaemia agents or insulin (33). A surrogate marker for obesitycontent is the body mass index (BMI), which is determined byweight (kilograms) divided by height squared (square meters). In
clinical terms, a BMI of 25–29 kg/m² is called overweight; higher
BMI (30 kg/m²) are called obesity. The waist circumference wasmeasured on admission, midway between the last rib and iliac
crest, and the average of 2 measures was recorded (27).
Echocardiography
Patients were studied by conventional and tissue Doppler
echocardiography with HP, Sonos 5500, Philips ultrasound system, using a 2.5 MHz transducer. Recordings were made with asimultaneous superimposed electrocardiography.
Conventional echocardiography 2D, M-mode and Doppler
was used for each patients, tracings from the parasternal longaxis view for measuring septal thickness, left ventricular diameterat end-diastole and end-systole, and posterior wall thickness.
Ejection fraction was derived from Simpson‘s modified single
plane method using the apical 4-chamber view (12).
Comprehensive assessment of left ventricular diastolic function included transmitral pulsed wave Doppler from an apical
4-chamber view. From the transmitral flow, the peck early (E)and late atrial (A) diastolic velocities, E-deceleration time (DT),and isovolumetric relaxation time (IVRT) were successfullyrecorded for all patients. Diastolic function was classified as
normal, impaired relaxation, pseudonormal, or restrictive according to standard diagnostic criteria. Normal diastolic function was
defined by E/A of 0.75–1.5 and a deceleration time of 160–230
ms. Impaired LV relaxation was defined by the combination of E/
A< 0.75 and E wave deceleration time >230 ms (20). No patients
had a restrictive filling pattern.
Tissue Doppler echocardiography was used to left ventricularlongitudinal myocardial wall motion from the apical 4-chamberview (31). Peck systolic myocardial velocity during ejection (Sm),early (Em) and late (Am) diastolic velocities were measured at
the septal and lateral mitral annulus with pulsed wave tissue
Doppler imaging in the apical 4-chamber view. The transducer
was positioned to align the ultrasound beam with longitudinalleft ventricular motion. A sample volume of 2 mm was used withthe frame rate exceeding 100 m/s. The ratio of E to lateral Em
was used to estimate left ventricular filling pressures. Normal
diastolic function was defined by E/Em of <8, and the impaired
left ventricular filling pressure was defined by E/Em of >8 (19).
Carotid Ultrasonography
Ultrasound images were acquired using a 7.5 MHz linear
array transducer and HP, Sonos 5500, Philips ultrasound sysFiziologia - Physiology  2008.18.3(59)tem. Common carotid artery intima – media thickness (IMT) was
determined by high-resolution 2D-mode ultrasound (29). The
transducer was manipulated so that the near and far walls of
the common carotid arteries were parallel to the transducer footprint, and the lumen diameter was maximized in the longitudinalplane. The reference point for measurement of the carotid IMT
was the carotid bulb. On that image, carotid IMT was measured
as the leading edges corresponding to the transition zones
between lumen – intima and media – adventitia over a lengthof 1 cm proximal to the reference point. Maximum IMT were
defined as the greatest measured from 3 contiguous sites at
1-cm intervals.
Posterior wall velocity of common carotid artery in systole(DTI-Vmax m/s) was measured in tissue Doppler ultrasonography for a velocity of 100 mm/s. Sample of 1-2 mm was placed
perpendicular to the posterior wall of common carotid artery.
Stiffness of the common carotid artery was evaluated by
M-mode ultrasonography were used to calculate 2 estimates
the arterial stiffness index β=ln(SBP/DBP)/[(Ds – Dd)/Dd], andthe elastic modulus (EM)=[(SBP-DBP)/(Ds – Dd)]x Dd, where
Ds and Dd are the end-systolic and end-diastolic diameters of
the common carotid artery, respectively (24). The pulse pressure
was evaluated as difference between SBP and DBP.
Statistical analysis
A commercially available statistical program, Statistical Package of Social Sciences (SPSS 6.0, Chicago, Ill) was used. Alldate are presented as the mean and standard deviation (SD).
For univariate analyses were used Chi square or Fisher tests,
according to standard application criteria, to test differences
between qualitative data in the two groups according to thepresence or absence of left ventricular diastolic dysfunction,
respectively. Continuous data were analyzed using Student test
for independent samples.
Pearson’s and when relevant (n<20) additional Spearman’s
correlation was applied and linear regression plot was used toshow relationships. A receiver operating characteristic (ROC)
curve was used to discriminate patients with cardiac diastolic
dysfunction (E/A ratio of <0.75 and E/Em>8) by arterial compliance. The results were considered statistically significant for
p-value < 0.05.
RESULTS
Characteristics of patients
In our study, the diastolic filling pattern was normal in 72(68.6%) of patients with hypertension and the dysfunction pattern (relaxation or pseudonormal) was in 33 (31.4%) patientswith hypertension. There were no significant differences in theclinical characteristics at baseline between the groups (TableI). The mean (SD) age did not differ significantly between the
2 groups. The cardiovascular risk factors – diabetes, obesity,
smoking, dyslipidaemia, familial history of CHD – there were
not significant differences between the 2 groups.

Table I. Characteristics of patient population

Parameters
Group 1
Number patients, n
72 (68.6)
Mean age, years
57.6 (12.1)
Women/Men, n38/34
Systolic blood pressure, mmHg
146.8 (10.7)
Diastolic blood pressure, mmHg
96.3 (8.5)
Pulse pressure, mmHg
55.3 (9.3)
28.4 (5.8)
Body mass index, kg/m2
Family history of CHD, n (%)
14 (19.4)
Obesity, n (%)
15 (20.5)
Diabetes mellitus, n (%)
13 (18.1)
Smoking status, n (%)
21 (29.1)
Total Cholesterol level, mg/dL
180.2 (49.7)
LDL-Cholesterol level, mg/dL
113.4 (46.6)
HDL-Cholesterol level, mg/dL
54.5 (23.1)
Triglycerides level, mg/dL
134.3 (48.2)
Apolipoproteins A-I, g/L
1.3 (0.3)
Apolipoproteins B, g/L
0.8 (0.2)
ApoB/ApoA-I ratio
0.6 (0.2)
Continuous numerical data were expressed as mean (std. dev.); NS, not significant
Echocardiography
There were no significant differences in the M-mode and2-D echocardiography between the 2 groups, regarding systolic
function estimated by ejection fraction and diastolic function estimated by the transmitral flow velocity variables. Left ventricular2008.18.3(59)  Fiziologia - Physiology

Group 233 (31.4)
59.2 (11.4)
18/15
148.9 (11.5)
102.5 (6.9)
67.5 (8.4)
28.9 (5.5)
7 (21.2)
8 (24.2)
7 (21.2)
12 (36.3)
190.6 (42.3)
115.6 (41.2)
45.8 (12.4)
148.4 (42.7)
1.2 (0.2)
0.9 (0.3)
0.7 (0.3)

p-value
<0.05
NS
NS
NS
<0.05
<0.05
NS
NS
NS
NS
NS
NS
NS
<0.05
NS
NS
<0.05
<0.05

parameters by echocardiography in the 2 groups are shown in
Table II. Regarding the diastolic tissue Doppler parameters,
where demonstrated significant differences between the 2groups.
29

Table II. Comparisons of left ventricular parameters between the 2 groups

Parameters
End-diastolic ventricular septal thickness (mm)End-diastolic LV posterior wall thickness (cm)End-diastolic LV diameter (cm)End-systolic LV diameter (cm)LV ejection fraction (%)
Transmitral flow velocity– Peak E velocity (m/s)– Peak A velocity (m/s)– E-DT (ms)– E/A
Mitral annular motion velocity– Peak Sm velocity (m/s)– Peak Em velocity (m/s)– Peak Am velocity (m/s)– E/EmGroup 1
11.2 (1.7)
11.5 (1.6)
46.3 (4.5)
30.1 (2.3)
68.2 (9.1)

Group 2
11.7 (1.9)
11.9 (2.1)
47.4 (5.4)
31.5 (2.7)
67.7 (8.9)

p-value
NS
NS
NS
NS
NS

0.67 (0.2)
0.71 (0.1)
224 (44)
0.9 (0.3)

0.72 (0.1)
0.68 (0.2)
212 (32)
1.18 (0.3)

NS
NS
NS
<0.05

7.8 (1.7)
9.2 (2.9)9.6 (3.8)8.6 (3.3)

8.9 (1.4)
11.4 (3.1)
10.2 (3.9)
6.6 (1.9)

<0.05
<0.05
<0.05
<0.05

Continuous numerical data were expressed as mean (std. dev.); NS, not significant

Peak E velocity, peak early diastolic velocity of transmitral
flow; peak A velocity, peak atrial systolic velocity of transmitral
flow; E-DT, deceleration time from peak to baseline of the early
diastolic transmitral flow velocity; Em, peak early diastolic mitralannular motion velocity; Am, peak atrial systolic mitral annularmotion velocity; Sm, peak systolic mitral annular motion velocity;
E/Em, the ratio of E to Em.

Carotid IMT and arterial stiffness
There were no significant differences in the carotid IMT between the 2 groups. Carotid stiffness parameters and posteriorwall velocity of common carotid artery was significantly lower
in the group 1, whereas there was no significant change in the
carotid diameter between the 2 groups.

Table III. Comparison of carotid ultrasonographic parameters between the 2 groups

Parameters
Intima-media thickness, mm
End-diastolic carotid diameter, mm
End-systolic carotid diameter, mm
Stiffness parameters– Carotid stiffness index β– Elastic modulus, kPa
Posterior wall velocity of common carotid artery, m/s
Correlation between left ventricular diastolic dysfunction
parameters and cardiovascular risk factors
Table IV presents univariate relations assessed with simple
linear regression analysis of left ventricular diastolic dysfunction

Group 10.8 (0.3)
6.5 (1.1)
7.4 (1.4)

Group 20.9 (0.3)
6.8 (1.3)
7.8 (1.2)

p-valueNSNSNS

10.3 (3.7)
124 (32.4)
0.3 (0.2)

11.8 (4.5)
152 (42.5)
0.4 (0.4)

<0.05
<0.05
<0.05

parameters and cardiovascular risk factors. The E/A ratio correlated well the apoB/apoA-I ratio and the E/Em ratio correlated
well the LDL-Cholesterol, apoB and apoB/apoA-I ratio.

Table IV. Correlation coefficients of left ventricular diastolic dysfunction parameters and cardiovascular risk factors
E/A<0.75
E/Em>8Parameters
Rp
R
Total Cholesterol
LDL-Cholesterol
HDL-Cholesterol
Triglycerides
Apolipoprotein A-I
Apolipoprotein B
ApoB/ApoA-I
Intima-media thickness
30

0.199
0.176
0.104
0.027
0.077
0.104
0.490
0.474

0.042
0.073
0.291
0.787
0.435
0.291
0.000
0.000

0.125
0.542
0.014
0.129
0.010
0.641
0.589
0.435

p

0.205
0.032
0.887
0.189
0.917
0.008
0.029
0.047

Fiziologia - Physiology  2008.18.3(59)Correlation between DTI-Vmax, pulse pressure, arterial
stiffness and diastolic dysfunction parameters
Significant correlations were found between posterior wall
velocity of common carotid artery in systole and pulse pressure(r = 0.47, p < 0.05), carotid elastic modulus (r = 0.71, p < 0.05),
and left ventricular diastolic dysfunction parameters – peck early(E) diastolic velocities measured by transmitral flow (r = 0.52, p< 0.05), and peck early (Em) diastolic velocities measured by
the mitral annulus motion (r = 0.67, p < 0.05).

Correlation between carotid stiffness and the presence/
absence of left ventricular diastolic dysfunction
In the ROC curve for discriminating patients with hypertension and cardiac diastolic dysfunction by carotid stiffness, thearea under the curve was 0.71, and the highest discriminating
sensitivity and specificity were 70% and 95% as is shown in
Fig. 2.

3.5
DTI Vmax (m/s)

3
2.5
2
1.5
1
0.5
0
0

50

100

150

200

250

EC (10-3 kPa)

DTI Vmax (m/s)

4.5
4
3.5
3
2.5
2
1.5
1
0.5
0

Fig. 2. Receiver operating characteristic curve between carotid stiffness and the presence/absence of diastolic dysfunction
0

20

40

60

80

100

4

5

PP (mmHg)

3.5

DTi Vmax (m/s)

3
2.5
2
1.5
1
0.5
0
0

1

2

3
Em (m /s)

1.4

DTI Vmax (m/s)

1.2
1
0.8
0.6
0.4
0.2
0
0

0.5

1

1.5

2

E (m /s)Fig. 1. Correlation between carotid DTI-Vmax and pulse pressure, arterial stiffness, left ventricular diastolic dysfunction parameters2008.18.3(59)  Fiziologia - Physiology

DISCUSSIONSIn the present study we assessed conventional echocardiography and tissue Doppler parameters regarding diastolic
function and explored the relationship between arterial stiffness
and cardiovascular risk factors. A method for reliably detecting
the onset of left ventricular systolic and diastolic dysfunction
and subclinical atherosclerosis of carotid arterial in patients withessential hypertension before transition to irreversible damageof the myocardium would be of crucial importance.
Cardiovascular risk factors, including hypertension, abdominal obesity, dyslipidemia, and glucose intolerance (4) contributeto vascular endothelial dysfunction, determining the developmentof atherosclerosis. The atherosclerosis should be evaluatedaccording to the following 2 aspects: atherosis, which reflects
structural changes in the intima and media of vascular walls, andsclerosis, which reflects changes in vascular stiffness or distensibility. The carotid IMT used in the present study is an index ofatherosis, and the stiffness index is an index of sclerosis.
Decreased distensibility of the arterial wall increases SBP
and decreases DBP, leading to increased left ventricular afterload
and impairment of myocardial blood flow because of decreased
coronary perfusion pressure. These abnormalities are reflectedin the decreased left ventricular diastolic function preceding left
ventricular systolic dysfunction (34). The present study - as wellas previous ones - shows that arterial stiffness increases with left
ventricular diastolic dysfunction in hypertensive patients.
31

The relationship between structural and functional changes
in the carotid arteries and left ventricular myocardial function inpatients with cardiovascular risk factors was investigated andfound that left ventricular relaxation is significantly associatedwith carotid stiffness (17,18). In our study, the most significant
correlations were obtained between left ventricular diastolic
dysfunction and apoB/apoA-I ratio.
With pulsed Doppler echocardiography, left ventricular diastolic function can now be evaluated noninvasively by recordingtransmitral flow and mitral annular or left ventricular wall motion
velocities (19). Many studies have indicated that left ventricular
diastolic dysfunction occurs before left ventricular systolic dysfunction in patients with hypertension (21) even in the absenceof apparently cardiovascular disease. Recently, previous studies(6,11) have demonstrated significant univariate relation between
arterial distensibility, mitral inflow propagation velocity, and mitral
E/A in patients with newly diagnosed hypertension. Our study
showed that hypertension increase arterial stiffness and there
is an association between left ventricular diastolic dysfunction
measured by mitral E/A.
The relationship between blood flow derived velocities andregional myocardial wall motion derived velocities, measured bytissue Doppler echocardiography, and expressed as the ratio of
peak early diastolic velocities E/Em, has been shown in several
studies (23,32). In addition, E/Em ratio measured both at thelateral and medial parts of the mitral annulus, has limitations
in discriminating patients with mildly to moderately elevate left
ventricular filing pressures such as hypertension patients (32).
Increased fibrosis in the left ventricular is known to be a part ofthe pathological process in hypertension patients (35). In thepresent study, we demonstrated that hypertension increase arterial stiffness and there is an association between left ventricular
diastolic dysfunction measured by mitral E/Em ratio.
Redfield et al. (28) proposed the criteria of mild cardiac
diastolic dysfunction by Doppler ultrasound examination in thegeneral population. According to their criteria, Yambe et al. (26)showed than the ROC curve that has a brachial-ankle PWV over
1,600 cm/s is a marker of abnormal cardiac diastolic function(E/A ratio of 0.75) (sensitivity = 78% and specificity = 58%). The
present study demonstrated that increased arterial stiffness iscorrelated with the parameters reflecting atherosclerosis but also
to those reflecting cardiac diastolic dysfunction.
In conclusion the tissue Doppler echography is a simple
method for evaluation of arterial stiffness rising and left ventricular relaxation alteration in patients with hypertension recently
tracked. The reliability of the method is similarly or exceeds
the more recently validated markers in diagnosis of studied
pathology, e.g. apoB/apoA-I ratio. The physiological and physiopathological parameters – posterior wall velocity of common
carotid artery in systole, E/Em ratio – have good performancein characterizing of arterial stiffness and left ventricular diastolic
function and can be used for diagnosis and monitoring.
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14. Mancia G, Rosei EA, Cifkova R, et al., European Society of Hypertension-European Society of Cardiology. Guidelines for the management of arterial hypertension, J. Hypertens., 2003, 21, 1011-1053
15. Marcovina SM, Albers JJ, Henderson LO. International Federationof Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B. III. Comparability of apolipoprotein A-I values byuse of international reference material, Clin. Chem., 1993;39:773-781
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by use of International Reference Material, Clin. Chem., 1994;40:586592
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CORELATIILE DINTRE FACTORII DE RISC CARDIOVASCULAR,
SCLEROZA ARTERIALA SI FUNCTIA DIASTOLICA A VENTRICULULUISTANG LA PACIENTII HIPERTENSIVIREZUMAT
Obiective: Hipertensiunea accelereaza ateroscleroza, sinteza de colagen, precum si hiperplazia si hipertrofia musculara neteda arteriala, crescand astfel rigiditatea arteriala. Este bine cunoscut faptul ca functia diastolica ventriculara scade la persoanele in varsta,
mai ales la pacientii cu hipertensiune. Acest studiu isi propune sa determine relatia dintre factorii de risc cardiovascular, rigiditatea
arteriala si disfunctia diastolica la pacientii cu hipertensiune au fractia de ejectie pastrata, folosind imagistica ultrasonografica recent
descoperita.
Metode: Am efectuat un studiu incrucisat care a inclus 105 pacienti (52 barbati si 53 femei) cu hipertensiune, cu varsta medie de58,5 (12,4). Functiile sistolica si diastolica ale ventriculului stang au fost evaluate 2D, M-mode si prin masurarea vitezei fluxului
transmitral, vitezei de miscare a inelului mitral folosind sistemul ultrasonic Doppler conventional si tisular. Ateroscleroza subclinica afost determinata de asemenea prin masurarea grosimii si rigiditatii intima-medie la nivelulu arterei caroide comune, folosind metodeultrasonografie 2D-, M-mode si Doppler tisular. Concentratia de lipide a fost masurata prin metode enzimatice, in timp ce concentratiile apolipoproteinelor au fost determinate prin metode imunoturbidimetrice.
Rezultate: Viteza de miscarea a inelului mitral este corelata cu apolipoproteina B (r = 0,64, p<0,05) si cu nivelurile plasmatice ale
LDL-colesterol (r = 0,54, p = 0,05), precum si cu raportul apoB/apoA-I (r = 0,58, p<0,05). Viteza fluxului diastolic transmitral este
corelata cu hipertensiune, fara asociere cu lipidele si apolipoproteinele. Rigiditatea si ingrosarea arterelor coronare s-a corelat cu
viteza de miscarea a inelului mitral in timpul diastolei (r = 0,65, p < 0,05) si cu raportul apoB/apoA-I (r = 0,62, p < 0,05). Nu au
fost observate corelatii semnificative intre grosimea intima-medie si parametrii ecocardiografici care definesc functia sistolica si
diastolica a ventriculului stang.
Concluzii: Aceste rezultate sugereaza ca factorii de risc cardiovasculari interactioneaza cu rigiditatea vasculara si relaxarea ventriculara la pacientii cu hipertensiune. Explorarea prin metodele imagistice non-invazive moderne este descrisa ca o metoda utila
pentru depistarea precoce a aterosclerozei subclinice.

2008.18.3(59)  Fiziologia - Physiology

33

SDS-PAGE electrophoresis of urinary proteins:
Diagnostic and prognostic value
KACSO INA1, CRISTEA ANCA2, RACASAN SIMONA1, SPANU COSTEL1, FEDORCA ANA1,CHINDRIS ADELA1, MUNTEAN MARIA1, GHERMAN-CAPRIOARA MIRELA1
1
Nephrology and Dialysis Clinic “Mihai Manasia”, Emergency Hospital Cluj-Napoca, Romania 2Immunology Department, University
Hospital, Cluj Napoca, Romania

ABSTRACT
Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis of urinary protein (SDS-PAGE) is a non-invasive method for the studyof proteinuria. Besides its diagnostic usefulness it has recently been shown to have prognostic value in patients with glomerulardiseases. The aim of this study was to evaluate diagnostic and prognostic value of SDS-PAGE in our center. Material and methods:
In 288 patients, in whom SDS-PAGE was performed between 1995 and 2005, we retrospectively studied the link between thepattern of proteinuria (PP) by SDS-PAGE and the diagnosis established by other methods. We then identified patients with glomerular diseases who were followed-up for at least 6 months and studied the prognostic value of certain SDS-PAGE patterns in thisgroup. Results: The PP by SDS-PAGE (glomerular-7%, glomerulo-tubular-65%, tubular 21%, physiologic-7%) correlates with thediagnosis (glomerular diseases-42%, tubulo-interstitial diseases-23%, multiple myeloma-2%, other-33%; p = 0.0001, χ2 57.71). PP
also correlates with quantitative proteinuria (p = 0.0001). In 31 patients with glomerular diseases, at a follow-up of 26 ± 20 months,
we analysed the prognostic value of urinary high molecular weight (HMW ≈150 kda) proteins, very low molecular weight (VLMW<25kda) proteins and polymers of albumin (PA), which are potential risk factors for progression of renal failure. As compared totheir negative counterparts, the HMW positive patients displayed a faster decline in creatinine clearance (CrCl) (1.49±0.21 ml/min/month versus 0.14 ± 0.02 ml/min/month, p = 0.01). The same applied to the VLMW positive vs. VLMW negative patients (1.42 ±0.25 versus 1.15 ± 0.18ml/min/month; p = 0.08), as well as for the PA positive vs. the PA negative patients (1.79 ± 0.24 vs. 0.89
± 0.17ml/min/month; p = 0.04). There was no statistically significant difference between decline in CrCl of patients with or without
low molecular weight (LMW tubular proteins 25-45 kda) proteins in SDS-PAGE. In conclusion SDS-PAGE confirms its value as adiagnostic tool; also its prognostic use in patients with glomerular diseases is to be encouraged, as certain PP are associated witha worse course of disease.
Key words: SDS-PAGE, urinary proteins, very low molecular weight, prognostic, progression – chronic renal failure

BACKGROUND
Proteinuria is an important progression factor of chronic renal
failure as it correlates not only with glomerulosclerosis but alsowith tubulo-interstitial damage, which is an important determinant
in the prognosis of chronic renal failure (11,18). In predicting
the outcome of glomerular diseases, composition of proteinuria
is essential, since excretion of certain urinary proteins carryprognostic significance (19, 1-6, 8). This justifies the qualitative
study of proteinuria in glomerular patients.
First described by Laemmli (16), sodium-dodecyl-sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) of urinary
proteins is a non-invasive method for the qualitative evaluationof proteinuria. SDS-PAGE has been in use in our center since1980 (9). There are some reports about the prognostic value ofsome patterns of urinary SDS-PAGE (11, 17, 18).
The aim of our study was to establish the diagnostic efficiency
of SDS-PAGE on a large cohort of patients in our clinical practiceand to assess the prognostic value of specific components ofproteinuria in patients with glomerular diseases.

PATIENTS AND METHODSWe reviewed the records of patients admitted in our unit
between January 1995 and December 2005 in whom SDS-PAGE
had been performed. Patients whose results were reported
prior to hospital discharge (so that discharge diagnosis could
have been influenced by SDS-PAGE findings) were excluded.
The study cohort consisted of 288 patients; chart records werenoted. Creatinine clearance was estimated according to the
Cockroft formula.
SDS-PAGE was performed following the method initiallydescribed by Laemmli (16). 24 hour urine samples were dialysed,
concentrated and then electrophoresed on 7.5% polyacrylamide
gel slabs (170/120/0.5 mm with 10 protein bands/ slab) in discontinuous buffer. Each sample contained 12 μg proteins. Aftermigration bands were colored with Coomassie Brilliant Blue R
250. Classification was made by visual inspection in comparison
to the migration band of known molecular weight (Amersham
International).
The following types of urinary proteins were identified:

Received July 2008. Accepted September 2008. Address for correspondence: Kacso Ina, Nephrology and Dialysis Clinic “Mihai Manasia”, Emergency Hospital ClujNapoca, Clinicilor 3-5 Street, 400006, Cluj Napoca, Romania, phone/fax 0040 264 592202, phone 0040 741277206, mail: inakacso@yahoo.com

34

Fiziologia - Physiology  2008.18.3(59)proteins ≈ 150 kda (mainly immunoglobulins) – termed highmolecular weight (HMW) proteins; proteins with a molecularweight of
60-72 kda (albumin, transferrin) – termed middle molecularweight (MMW) proteins, proteins with molecular weight between
25 and 45 kda termed low molecular weight (LMW) proteinsand proteins with molecular weight under 25 kda termed very
low molecular weight (VLMW) proteins. These types of proteins
were sometimes associated, creating various patterns. According to data in the literature (3, 4) we classified patients into four
patterns of proteinuria: pure glomerular proteinuria (presence of
HMW and MMW proteins), glomerulo-tubular pattern (presenceof MMW and/or HMW proteins accompanied by LMW and/or
VLMW proteins), physiologic proteinuria – albumin alone andtubular proteinuria (LMW and/or VLMW proteins). Presence of
PA was also recorded.
We then selected patients with glomerular diseases who
had a follow-up of at least 6 months. Patients with improving
renal function (acute renal insufficiency superimposed) or rapidly
deteriorating renal function (decrease in CrCl of more than 50%
in 3 months) were excluded. Cockroft CrCl was calculated on
the first and last visit and the decline in CrCl was divided by the
number of months of follow-up. Correlations between monthly
decline in renal function and various proteinuric patterns werethen studied.
The statistical methods used were χ2 test, t-test, simple
and multiple regression analyses. Data are presented as mean
± SEM.
RESULTS
Diagnostic value of SDS-PAGE
Our group consisted of 288 patients (55% male) with a mean
age of 40.60±15.83 years. They had moderately altered renal
function, significant proteinuria and relatively well controlled
blood pressure (Table I).
Table I – Characteristics of the patientsParameter
Age (years)
Serum Creatinine (mg/dl)
Initial creatinine clearance (ml/min)
Blood Urea (mg/dl)
Hemoglobin (g/dl)
CRP (mg/dl)
Total serum proteins (g/dl)
Serum albumin (%)
Serum cholesterol (mg/dl)
Proteinuria (g/24 h)
Systolic BP (mmHg)
Diastolic BP (mmHg)

Mean ± SEM
48±1
3.36±0.1938±2
99±4
11.6±0.13
1.61±0.17
6.70±0.05
49±1
219±4
3.01±0.13
135±1
84±1

Legend: CRP – C reactive protein, SBP – systolic blood pressure,
DBP – diastolic blood pressure

2008.18.3(59)  Fiziologia - Physiology

120 patients (42%) had glomerular diseases, 69 patients(23%) tubulo-interstitial diseases, 5 patients (2%) had multiple
myeloma and 94 patients (33%) other renal diseases. This lastgroup consisted mainly of patients presenting with relatively
advanced chronic renal insufficiency in which diagnostic certitude
could not be obtained.
SDS-PAGE patterns were as follows: 23 (8%) pure glomerular pattern, 182 (63%) glomerulo-tubular pattern, 60 (21%)
tubular pattern and 23 (8%) physiologic pattern. Among patientswith glomerulo-tubular pattern, 81 (44.5%) had VLMW proteinuria
and 101 (55.5%) had only LMW proteins in their urine.
The diagnosis established by other methods correlated
significantly with SDS-PAGE pattern (p=0. 0001 χ2=57. 71) –Figure 1.

Fig. 1. Correlation between SDS-PAGE pattern and diagnosis
Legend: GD – glomerular diseases; TIN – tubulointerstitial nephritis;
MM – multiple myeloma; other – other nephropathies

As expected, SDS-PAGE pattern also significantly correlatedwith proteinuria (p = 0.0001) - Figure 2.

Fig. 2 - Relation between proteinuria and SDS-PAGE pattern

Prognostic value of SDS-PAGE
We recorded the follow-up of 31 patients (77.4% male) withglomerular renal disease who were followed for at least 6 months(mean 26 ± 10 months). Patients were divided according to their
SDS-PAGE pattern.
First we divided patients according to presence/absenceof HMW proteins in their urine and compared mean monthly
decline in renal function. Patients with HMW proteinuria exhibited a significant, more rapid deterioration in renal function than
35

HMW negative ones (1.49 ± 0.21ml/min/month versus 0.14
± 0.02 ml/min/month, p = 0.01). We compared other factors
known to influence progression of renal failure between the two
above-mentioned groups: patients with HMW proteinuria had asignificantly greater proteinuria. Diastolic BP and CRP were moreelevated in the HMW negative group (Table II).
Patients divided according to presence or absence of LMWproteins in their urine had a similar decline in renal function (1.16± 0.18 ml/min/month versus 1.38 ± 0.25 ml/min/month, NS).
Patients with VLMW proteinuria had a more rapid decline inrenal function than patients without VLMW proteinuria (1.42 ±0.25ml/min/month versus 1.15 ± 0.18ml/min/month, p = 0.08).
No other parameters known to influence progression of renal
disease were different between subgroups except for slightly
different initial renal function (Table II).
We then divided these patients according to the presence orabsence of PA in the SDS-PAGE and again the decline in renal
function was compared. Patients with PA in the urine had a more
rapid progression of renal insufficiency as compared to patients
without PA (1.79 ± 0.24 ml/min/month versus 0.89 ± 0.17 ml/
min/month, p = 0.04). However they also had significantly more
proteinuria and more severe hyperlipidemia (Table II).

Results were similar if only patients with nephrotic syndrome
were analyzed. However, this group was very small (n =13): HMW
positive patients had a more rapid decline in renal function than
HMW negative patients (1.8 ± 0.4 ml/min/month versus 0.16 ±0.05 ml/min/month, p = 0.08), as did VLMW positive patients
versus VLMW negative patients (3.57 ± 0.44ml/min/monthversus 0.84 ± 0.33 ml/min/month, p = 0.01) and PA positive
patients versus PA negative patients (2.23 ± 0.44 ml/min/monthversus 0.40 ± 0.27ml/min/month, p = 0.02). Again there wereno significant differences when we divided patients according
to LMW proteinuria.
DISCUSSION
The main finding in our study was that SDS-PAGE analysis
of proteinuria carries prognostic significance, as certain SDSPAGE patterns (HMW, VLMW proteins and PA) are associatedwith more rapid progression of renal failure.
As already stated, in glomerular diseases, the qualitative
study of proteinuria by SDS-PAGE also has prognostic implications. Presence of HMW proteins excreted in the urine is a direct
marker of the severity of glomerular lesions. In fact it has beenshown that increased urinary excretion of IgG correlates better

Table II – Comparison of patients with glomerular diseases divided according to presence or absence of urinary HMW, VLMW, PA proteins
Glomerular disease n=31

HMW
+

HMW
-

p

VLMW
+

VLMW
-

p

PA
+

PA
-

p

0.14±0.02

0.01

1.42± 0.25

1.15±0.18

0.08

1.79±0.24

0.89±0.17

0.04

60±1

0.002

22±4

27±5

NS

10±1

32±5

0.01

49±1

42±0.5

NS

50±4

43±2

NS

56±3

41±3

0.01

Initial creatinine clearance(ml/min)
54±1

46±1

NS

42±6

63±6

0.05

50±3

53±6

NS

74±1

66±1

NS

43±8

60±7

NS

72±7

57±8

NS

95±2

71±1

NS

102±9

75±8

NS

97±7

69±7

NS

0.45±0.02

2.5±0.05

0.01

0.87±0.14

0.8±0.33

NS

0.60±0.15

1.01±0.35

NS

Hemoglobin (g/dl)

11.34±10.32

12.5±0.64

NS

11.21±1.79

11.9±3.59

NS

11.19±3.5911.70±3.59

NSSerum albumin (%)

48±1

50±1

NS

46±5

49.00±4

NS

49±1

47±1

NS

233±2

236±1

NS

206±9

265±1

NS

272±12215±12

0.02

Proteinuria (g/24h)

4.21±0.08

1.68±0.11

0.05

3.78±0.48

3.80±0.66

NS

5.07±0.61

3.05±0.50

0.04

SBP (mmHg)

139±1

148±1

NS

136±4

146±4

NS

141±4

148±4

NS

85±2

98±0.5

0.02

82±2

92±2

NS

89±3

86±2

NS

Decline in cretinine
1.49± 0.21
clearance(ml/min/month)
Time (months)
18±1
Age (years)

ESR 1 (mm/h)
ESR 2 (mm/2h)
CRP (mg/dl)

Serum cholesterol (mg/dl)

DBP (mmHg)

Legend: HMW – high molecular weight, VLMW – very low molecular weight, PA – polymers of albumin, ESR 1– erythrocyte sedimentation rate at 1 h,
ESR 2– erythrocyte sedimentation rate at 2h, CRP – C reactive protein, SBP – systolic blood pressure, DBP – diastolic blood pressure

36

Fiziologia - Physiology  2008.18.3(59)than 24 hour proteinuria with progression to chronic renal failure(19) with reduced remission and histological changes in patientswith membranous glomerulopathy (2,8) and focal segmental
glomerulosclerosis (6). The same kind of information resultsfrom studies of the predictive value of selectivity of proteinuria invarious glomerular diseases: low selectivity proteinuria correlateswith more prominent tubulo-interstitial changes and accelerated
progression to chronic renal failure, reduced clinical remission
or response to therapy (5, 1).
As information about excretion of different molecules (IgG,
β2 microglobulin, α1 microglobulin) are not routine in our center,
we turned to SDS-PAGE for the characterization of proteinuria.
Available for decades, SDS-PAGE was used for hardly morethan diagnostic purposes, and even in research it tends to beneglected in favour of direct measurements of various proteins.
It is, however, a reliable method and relatively easy to perform,
so we considered it worthwhile to explore its prognostic value. Although our initial population was large enough, since SDS-PAGE
was needed in most cases for diagnostic purposes, the groupof patients with confirmed glomerular diseases and having had
SDS-PAGE is relatively small because diagnosis of glomerulardiseases was in most cases obtained by other methods. HMW
proteins in the urine of glomerular patients should account for
unselective proteinuria and therefore more severe disease. As
expected, presence of urinary HMW proteins identified a groupof patients with more severe disease, greater proteinuria andmore rapid decline in renal function. Other factors of progression (hypertension and inflammation) were worse in the HMW
negative group.
In proteinuric patients with glomerular diseases, filteredproteins will stress the reabsorptive mechanism of the proximal
tubule, eventually inducing tubular lesions and activation ofcytokine and growth factors that produce tubulo-interstitial damage (10). Presence of abnormal quantities of albumin and HMW
proteins in the urine also results in an impaired reabsorption andincrease in the urinary excretion of LMW “tubular” proteins, turning them into a valuable marker for the severity and outcome ofglomerular diseases. In fact β2 microglobulin has been shownto correlate with the progression to chronic renal failure in patients with membranous glomerulopathy (19, 8). Also fractional
excretion of α1 microglobulin was significantly associated withseverity of tubulo-interstitial changes, functional outcome andresponse to therapy in membranous glomerulopathy (2). It also
correlates with tubulo-interstitial damage in patients with focal
segmental glomerulosclerosis (6). It is true that other studies
contested the role of excretion of LMW proteins in predicting
the response to therapy (12).
Using SDS-PAGE for the identification of tubular proteins,several studies have already demonstrated the role of LMW and
VLMW proteins in predicting the renal outcome in glomerulardiseases (17, 21, 3, 15, 13). Bazzi et al. (3) studied 145 patientswith biopsy-proven primary glomerular diseases and showeda correlation between presence of VLMW proteinuria andtubulo-interstitial damage, development of CRF and responseto therapy.
2008.18.3(59)  Fiziologia - Physiology

In our study we obtained a significant relationship between
the presence of VLMW proteins in SDS-PAGE and rapid progression of renal failure in patients with glomerular diseases. Thisconfirms Bazzi et al.’s findings of VLMW proteins as a marker ofworse outcome. Other factors that potentially influence progression of chronic renal failure were compared between patients withand without VLMW proteins in SDS-PAGE and their values weresimilar between the two groups, excluding thereby other causesof worse outcome in the VLMW positive patients.
Presence of PA in the urine of patients with glomerular diseases, first described by Boesken (7), has been suggested to
be a negative prognostic factor for the development of chronic
renal failure (14). Polymerisation of albumin requires certain
conditions to occur, one of which is the presence of a LMW
ultrafiltrable factor (14), so significance of presence of PA and
VLMW in urine could be interconnected. Our study also finds
PA to be a negative marker of accelerated progression of renal
insufficiency in patients with glomerular diseases. However,patients presenting with PA also had other possible confounding
progression factors as they had more severe proteinuria andhigher cholesterol levels, and were also older. This can reflect amore severe underlying disease. Of course, again the relatively
small number of patients and the retrospective nature of thestudy prompt to prudence in interpreting the results.
Although the majority of our patients were biopsied, we were
not able to study the link between different SDS-PAGE patterns
and histological findings since re-examination of biopsy samples
was not feasible.
Proteinuria is the cornerstone of many renal diseases andtherefore an essential diagnostic tool. In the present study weevaluated the value of SDS-PAGE as a non-invasive tool in the
diagnosis of different nephropathies. An unequivocal statisticallysignificant relationship was obtained between SDS-PAGE patternand different diagnostic categories (diagnosis being establishedby other methods, including renal biopsy). As expected SDSPAGE patterns also correlated with proteinuria. These strong
correlations confirm its diagnostic value and suggest that weshould probably prescribe it more frequently with diagnostic
purpose, at least in our local experience.
CONCLUSION
Our study reiterates the clinical usefulness of SDS-PAGE inestablishing the nephrological diagnosis. But most importantly
it confirms the role of certain SDS-PAGE patterns, in particularpresence of HMW and VLMW proteins as well as PA, in predicting accelerated progression of chronic renal failure in patientswith glomerular diseases. In our opinion, given the non-invasive
character of SDS-PAGE and the fact that the method can be
performed without the need of costly material, it should be performed especially in centers where imunonephelometric dosageof urinary proteins is not available. Larger prospective studies are
warranted to confirm the prognostic value of the method; also its
use as a diagnostic tool should be encouraged.

37

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by SDS-PAGE: Clinical significance. Clin Nephrol 1991; 36: 6-11

Valoarea diagnostica si prognostica a ElectroforezEI
PROTEiNELOR URINARE in gel de poliacrilamida cu tampon
dodecilsulfat de sodiu (SDS-PAGE)
REZUMATElectroforeza proteinelor urinare in gel de policarilamida cu tampon dodecilsulfat de sodiu (SDS-PAGE) este o metoda non-invaziva
de evaluare a proteinuriei. Pe langa valentele sale diagnostice s-a demonstrat recent ca detine si o semnificatie prognostica in glomerulopatii. Scopul acestui studiu a fost aprecierea valorii diagnostice si prognostice a metodei in centrul nostru. Pacienti si metoda:
Pe un lot de 288 de pacienti la care s-a efectuat SDS-PAGE intre 1995 si 2005 au fost studiate corelatiile intre tipul proteinuriei si
diagnosticul stabilit prin alte metode. Au fost de asemenea selectati pacienti cu nefropatii glomerulare cu urmarire de minimum 6
luni si a fost evaluata valoarea prognostica a tipurilor proteinuriei la acest grup de pacienti. Rezultate: Tipul proteinuriei conform
SDS-PAGE (glomerulara-7%, glomerulo-tubulara-65%, tubulara-21%, fiziologica-7%) se coreleaza cu diagnosticul (glomerulopatii-42%, nefropatii tubulointerstitiale-23%, mielom multiplu-2%, altele-33%; p = 0,0001, χ2 57,71) si cu proteinuria cantitativa (p =0,0001). La 31 de pacienti cu glomerulopatii si o urmarire medie de 26 ± 20 luni, a fost analizata valoarea prognostica a proteinelorurinare cu greutate moleulara mare (high molecular weight -HMW ≈150 kda), greutate moleculara foarte mica (very low molecularweight -VLMW < 25kda) si polimerilor de albumina (PA), care sunt potential factori de risc ai progresiei insuficientei renale. Pacientii
cu HMW in urina au avut o rata de declin a clearance-ului creatininic mai rapida in comparatie cu cei fara HMW (1,49 ± 0,21 ml/
min/luna versus 0,14 ± 0,02 ml/min/luna, p = 0,01). Aceasta observatie este valabila si pentru pacientii cu VLMW fata de cei fara
fara VLMW (1,42 ± 0,25 versus 1,15 ± 0,18 ml/min/luna; p = 0,08), si pentru pacientii cu PA fata de cei fara PA (1,79 ± 0,24 vs.
0,89 ± 0,17ml/min/luna; p = 0,04). Rapidiatatea declinului functiei renale a fost similara la pacienti cu, respectiv fara, proteinelor cu
greutate moleculara mica (25-45 kda) in urina. In concluzie SDS-PAGE isi confirma valoarea ca mijloc diagnostic; utilizarea sa ca
marker prognostic la pacientii cu nefropatii glomerulare ar trebui incurajata deoarece anumite tipuri proteinurice sunt asociate cu oevolutie nefavorabila.
Cuvinte cheie: SDS-PAGE, proteine urinare, greutate moleculara foarte joasa, prognostic, progresie-insuficienta renala cronica

38

Fiziologia - Physiology  2008.18.3(59)Societatea RomânĆ deûtiinĦe Fiziologice

Colegiul Medicilor din
România

Universitatea de MedicinĆ üi
Farmacie din Craiova

A XXIII-a ConferinĦĆ NaĦionalĆ aSocietĆĦii Române de ûtiinĦe
Fiziologice
Craiova
28-30 mai 2009
"Fiziologia experimentalĆ üi
clinicĆ în contextul medicinei
moderne"Primul AnunĦ
StimaĦi colegi,
Societatea RomânĆ de ûtiinĦe Fiziologice üi Catedra deFiziologie a UniversitĆĦii de MedicinĆ üi Farmacie din Craiova,
vĆ invitĆ sĆ participaĦi la A XXIII-a ConferinĦĆ NaĦionalĆ aSocietĆĦii Române de ûtiinĦe Fiziologice, intitulatĆ "Fiziologia
experimentalĆ üi clinicĆ în contextul medicinei moderne", care seva desfĆüura în Craiova, în perioada 28-30 mai 2009, fiind
onorate de participarea dumneavoastrĆ.
2008.18.3(59)  Fiziologia - Physiology

39Preüedinte de onoare

Ioan HĆulicĆ (Iaüi)
Preüedinte SRSF

Adriana Mureüan (Cluj-Napoca)
Prezidiu de onoare

Valeriu Neütianu (Craiova)
Marius SabĆu (Tg. Mureü)Francisc Schneider (Arad)

Preüedintele Comitetului ûtiinĦific

Maria IancĆu (Craiova)
Comitetul ûtiinĦific:
Carmen Bunu (Timiüoara)
Anca BĆdĆrĆu (Bucureüti)
Petriüor Cismaü (Oradea)
Dan Dobreanu (Tg. Mureü)
Smaranda GoĦia (Timiüoara)
Maria Luiza Flonta (Bucureüti)
Maria Grama (Sibiu)
Simona Gusti (Craiova)
Ileana Ion (ConstanĦa)Rodica Mateescu (Timiüoara)
Georgeta Mihalaü (Timiüoara)
40

Doina Motoc (Arad)
Adriana Mureüan (Cluj-Napoca)
Aurel Nechita (GalaĦi)Virgil PĆunescu (Timiüoara)
Gheorghe Petrescu (Iaüi)
Manuela Pumnea (Sibiu)
Aurel Saulea (ChiüinĆu)Simona SlĆtineanu (Iaüi)
Simona Tache (Cluj-Napoca)
Maria Vrabete (Craiova)
Leon ZĆgrean (Bucureüti)
Fiziologia - Physiology  2008.18.3(59)