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CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERIOANA SISKACARMEN TATUMIHAI NECHIFORSORIN RIGA
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Fiziologia- Physiology 2011.21.4(72)
Fiziologia- Physiology 2010supplement

CONTENTS
1. Controversies Related to Cell Cultures Obtained from Various Tissular Samples
Florina Bojin, Oana Gavriliuc, Valentin Ordodi, Mirabela Cristea, Simona Anghel, Daniela Crisnic, Daciana Nistor, Carmen Tatu, Gabriela Tanasie, Carmen Panaitescu, Virgil Paunescu ........................4
2. Comparative Analysis of Bone Marrow-Derived Mesenchymal Stem Cells and Immortalized Mesenchymal Cell Line
Marusciac Laura, Panaitescu Carmen, Paunescu Virgil .............................................................................................................................................................................................................................. 10
3. Prevalence of Obesity in Patients with Various Changes of Glycemic Balance and Thyroid Diseases
Adriana Gherbon, Lavinia Noveanu, Georgeta Mihalas . ........................................................................................................................................................................................................................... 15
4. Cardiovascular Manifestations in Autoimmune Rheumatic Diseases
Manole Cojocaru, Inimioara Mihaela Cojocaru, Violeta Sapira .................................................................................................................................................................................................................. 20
5. The Assessment of ERG Differences between Red and White Stimuli
Alexandru D, Catalin B, Georgescu M, Georgescu D, Iancau Maria . ........................................................................................................................................................................................................... 24
6. Strategy to Predict Potential for a “Regular Donor Career” in First Time Blood Donors
Alina Mirella Dobrota, Ileana Ion, Lavinia Voineagu . ................................................................................................................................................................................................................................ 29
7. The Use of Keratinocyte and Fibroblast Cultures in Treatment of Burns and Chronic Wounds
Simona Vermesan, Tiberiu Bratu, Virgil Paunescu . .................................................................................................................................................................................................................................... 36
8. Markers of Oxidative Stress in Chronic Renal Failure
Lavinia Voineagu, Cecilia Adumitresi, Ileana Ion, Alina Dobrota, Victoria Badea, Liliana Tuta . ................................................................................................................................................................ 40

CUPRINS
1. Controverse legate de culturile celulare obtinute din diferite surse tisulare
Florina Bojin, Oana Gavriliuc, Valentin Ordodi, Mirabela Cristea, Simona Anghel, Daniela Crisnic, Daciana Nistor, Carmen Tatu, Gabriela Tanasie, Carmen Panaitescu, Virgil Paunescu ........................4
2. Analiza comparativa a celulelor stem mezenchimale obtinute din maduva osoasa hematogena si a liniei de celule stem mezenchimale imortalizate
Marusciac Laura, Panaitescu Carmen, Paunescu Virgil .............................................................................................................................................................................................................................. 10
3. Prevalenta obezitatii la pacientii cu variatii ale echilibrului glicemic si afectiuni tiroidiene
Adriana Gherbon, Lavinia Noveanu, Georgeta Mihalas . ........................................................................................................................................................................................................................... 15
4. Manifestări cardiovasculare in bolile reumatice autoimune
Manole Cojocaru, Inimioara Mihaela Cojocaru, Violeta Sapira .................................................................................................................................................................................................................. 20
5. Evaluarea diferentelor ERG prin stimulare cu lumina rosie, monocromatica si cu lumina alba
Alexandru D, Catalin B, Georgescu M, Georgescu D, Iancau Maria . ........................................................................................................................................................................................................... 24
6. Strategie pentru estimarea potentialului donatorilor de sange initiali pentru o cariera de “donator cu donari regulate”
Alina Mirella Dobrota, Ileana Ion, Lavinia Voineagu . ................................................................................................................................................................................................................................ 29
7. Folosirea culturilor autologe de keratinocite si fibroblaste in tratamentul arsurilor si plagilor cronice
Simona Vermesan, Tiberiu Bratu, Virgil Paunescu . .................................................................................................................................................................................................................................... 36
8. Markeri ai stresului oxidativ in insuficienta renala cronica
Lavinia Voineagu, Cecilia Adumitresi, Ileana Ion, Alina Dobrota, Victoria Badea, Liliana Tuta . ................................................................................................................................................................ 40
2011.21.4 (72)  Fiziologia - Physiology

3

CONTROVERSIES RELATED TO CELL CULTURES OBTAINEDFROM VARIOUS TISSULAR SAMPLESFLORINA BOJIN, OANA GAVRILIUC, VALENTIN ORDODI, MIRABELA CRISTEA,SIMONA ANGHEL, DANIELA CRISNIC, DACIANA NISTOR, CARMEN TATU,GABRIELA TANASIE, CARMEN PANAITESCU, VIRGIL PAUNESCU
Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT

Mesenchymal stem cells (MSCs) are nonhematopoietic stromal cells that are capable of differentiating into, and contribute to theregeneration of, mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose. MSCs are identified by theexpression of many molecules including CD105 (SH2) and CD73 (SH3/4) and are negative for the hematopoietic markers CD34,
CD45, and CD14.
We isolated two cellular populations of MSCs, from bone marrow (BM), and umbilical cord (UC), and we assessed them comparatively
for presence of phenotypical markers, trilineage potential and morphological characteristics. Although, both BM-MSCs and UC-MSCspresented similar phenotypical pattern and morphological characteristics (in optic microscopy), UC-MSCs failed to differentiate into
adipocytes, osteoblasts and chondrocytes, thus suggesting their poor developed or modified functionality. A more detailed analysis
on morphologic appearance (electron microscopy) demonstrated presence of intercellular junctions - desmosomes - which could
account for their behavior. Whether the UC-isolated cells acquired this phenotype, or the isolation method failed to separate only
mesenchymal cells, is still to be determined. However, the “stemness” characteristics should relay more on function than on presenceof phenotypical markers, since stem cell types are considered for clinical applications.
Key words: BM-MSCs, UC-MSCs, differentiation, function

INTRODUCTION
Stroma is in most of the cases seen as a connecting “device”
for the specific structures of an organ. Usually, people perceive
interstitial cells as being mainly fibroblasts and great confusion
still exists amongst cell biologists and other specialists interested
in regenerative medicine regarding the in vivo identity of humanbone marrow (BM) mesenchymal stem cells (MSCs). Contrary toviews of many scientists, methods for the robust identification andpurification of BM-MSCs are now well established. Human BMMSCs represent a phenotypically homogeneous cell populationthat share an identical phenotype with marrow adventitial reticular
cells, which are stromal cells similar in nature to pericytes. Whenan extensive panel of markers is used to characterize BM-MSCs,
it appears that the diverse MSC markers described in different
laboratories are expressed on the same cell population. Rare cell
phenotypical analysis and in vitro colony forming unit-fibroblast(CFU-F) assays produce no compelling evidence that BM-MSCs
circulate in healthy man. Furthermore, although investigators
speak of a number of specific MSC markers, a true marker of
MSC ‘stemness’ and multipotentiality has not yet been definedsince culture-expanded MSCs may lose some of these markers,
but remain multipotential. This knowledge provides a platform
for understanding MSCs in vivo leading to novel approaches fortherapy development, including in situ tissue engineering.
The concept of a mesenchymal stem cell (MSC) arose fromthe work of Friedenstein and colleagues four decades ago (1).
They noted that upon plastic adherence of bone marrow (BM)cells, a rare cell population developed into colony forming units
that were fibroblastic (CFU-F) (2). Following in vitro culture expansion, clonal cultures derived from individual CFU-Fs could
be introduced into diffusion chambers in experimental models
where the formation of bone, cartilage and stromal elements
was observed (3,4).
The interest in MSCs increased greatly almost a decade ago
with the reporting of novel markers for culture-expanded MSCs
including CD73 and CD105 and the development of robust in
vitro assays of MSC tripotentiality (5). Some investigators suggested that these findings were erroneously celebrated by thescientific community and media as the happy outcome of an
extraordinary hunt for MSCs (6). Indeed, the studies in question
described the same culture-expanded CFU-F population thatwent back as far as Friedenstein’s work, and the identity of theunknown ancestral cell remained enigmatic. The only firm clue
to the in vivo identity of BM MSCs came from the work of Simmon’s group (7) who showed that an antibody Stro-1 could be
used to enrich CFU-Fs approximately 100-fold; however, their
purification was still not feasible.
The ongoing confusion in the MSC field has been contributed
to by the assumption that any marker expressed on culture-expanded MSCs was also likely to be present in vivo. Consequently,
independent laboratories have begun to use different markers
of expanded MSCs to search for MSCs in vivo (8, 9). This has
resulted in the perception that these in vivo MSCs were a heterogeneous cell population, and could be distinct from Stro-1þ stromal

Received August 10th 2011. Accepted September 5th 2011. Address for correspondence: Florina Bojin, MD, PhD, Physiology Department, “Victor Babes” University of
Medicine and Pharmacy Timisoara, Spl. Tudor Vladimirescu no. 14A, phone/fax: +40256490507, e-mail: florinabojin@umft.ro4

Fiziologia - Physiology  2011.21.4 (72)

cells and progenitors. Indeed, the confusion to the in vivo identityof the BM-MSC has lead to difficulty with terminology whereby the
MSC acronym continues to signify both MSCs and marrow stromal
stem cells (5, 6, 10). In addition to the identification of MSCs basedon their morphologic or phenotypic characteristics, a further way
to identify supposed MSC populations is by their capacity to be
induced to differentiate into bone, fat, and cartilage in vitro.
Based on our studies of in vitro MSCs and related literature, the purpose of this article is to reconcile these apparent
contradictions and to discuss their implications for further use
in clinical applications.
MATERIALS AND METHODS
Cell isolation and culture
Unprocessed bone marrow (10 ml) obtained from 10 human adult subjects free of hematological disorders was used
for isolation of mesenchymal stem cells (MSCs). Bone marrow
was placed in culture plates, and the fibroblastic-like, plastic
adherent fraction, was isolated following multiple passages andused in our experiments. The BM-MSCs were further cultured
and expanded in alpha-minimum essential medium (MEM;
Gibco BRL, Invitrogen, Carlsbad, CA, USA), supplemented with
10% fetal calf serum (FCS; PromoCell, Heidelberg, Germany)and 2% Penicillin/Streptomycin mixture (Pen/Strep, 10,000 IU/
ml; PromoCell), by incubation at 37oC in 5% CO2 atmosphere.
Medium replacement was performed every third days and whenreaching 80-90% confluence, the cells were passed using 0.25%
Trypsin-EDTA solution (Sigma Aldrich Company, Ayrshire, UK)followed by centrifugation (10 minutes, 300g) and replated in
T75 culture flasks at a density of 10,000 cells/cm2.
Human umbilical cords (n = 10) were collected from full-term
births with informed consent of the mother after either Caesarean
section or normal vaginal delivery and stored at 4 oC up to 12h insterile physiological saline prior to processing. Following disinfection in 75% ethanol for 30 s, the umbilical cord was rinsed several
times with PBS-Buffer (Sigma Aldrich Company). The cord blood
was drained and clots flushed from the vessels. The umbilical cord
was dissected into cubes of approximately 1cm3 and the vessels
were stripped manually from these cord segments. This umbilicalcord tissue was then diced into pieces of about 0.2 cm and treatedwith an enzyme cocktail for 3 h at 37 oC. The enzyme cocktail
consisted of 4mg/ml BSA, 4mg/ml Collagenase (Sigma AldrichCompany), 1mg/ml Hyaluronidase, and 0.1mg/ml Trypsin-Inhibitor(all substances were purchased from Sigma Aldrich Company).
The dissociated mesenchymal cell solution was diluted with PBS(1:10), pelleted twice by low speed centrifugation (300 x g for 10min) and suspended in fresh media. The UC-MSCs were counted
under the microscope with the aid of a hemocytometer and were
subsequently used for cell cultures.
Osteogenic, chondrogenic and adipogenic differentiation experiments
The trilineage potential of BM-MSC and UC-MSC to differentiate into adipogenic, osteogenic and chondrogenic lineages
2011.21.4 (72)  Fiziologia - Physiology

was assessed at different passage levels, starting with passage
2 for each cellular type. Cells were seeded in 4-well Lab-Tek
glass chamber slides (Nunc, Rochester, NY, USA) at a cellulardensity of 10,000 cells/cm2 in standard growth medium until
they reached confluence, being then stimulated to differentiate
under appropriate medium conditions. Nonhematopoietic stem
cell medium for generation of osteoblasts, chondrocytes andadipocytes (Miltenyi Biotec, Bergisch Gladbach, Germany) wasused, supplemented with 1% Penicillin/Streptomycin.
Flow-cytometry
BM-MSCs and UC-MSCs in culture reaching 80-90%
confluence were detached using 0.25% Trypsin-EDTA (Sigma
Aldrich Company), washed two times with PBS, resuspended
in 100 μl PBS at a concentration of 105 cells/ml and incubatedin the dark at room temperature for 30 minutes with mouse antihuman fluorochrome-conjugated antibody at a dilution specified
in manufacturer’s protocol. Cells were then washed twice with
1 ml Cell Wash Solution (BD Biosciences, San Jose, CA, USA)each and resuspended in 500 μl of the same solution for further
analysis on a four color capable FACSCalibur (Becton-Dickinson)
flow-cytometer. Conjugated antibodies utilized included PE-conjugated CD14 (BD Pharmingen™), CD117 (BD Pharmingen™),
α-SMA (BD Biosciences), CD29, CXCR4, Nestin, VEGF-R1(Flt-1), VEGF-R2 (Kdr), E-Cadherin, TGF-β RII, TGF-β RIII
(R&D Systems) as well as FITC-conjugated CD34, CD44, CD45,
CD73, CD90, CD106, HLA-DR (BD Pharmingen™), Cytokeratin(R&D Systems) and APC-conjugated CD31 (BD Pharmingen™).
Acquisition and data analyses were performed using CellQuest
Pro software (BD).
Immunohistochemical analysis
Immunohistochemistry was performed for BM-MSCs and
UC-MSCs. Cells prepared for these analyses were grown in
4-well glass chamber slides, and 3-5 days from plating medium
was removed, cells were washed, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 and theninvestigated for expression of the proteins of interest, using forlabeling the following antibodies: monoclonal mouse anti-swine
Vimentin (clone V9), monoclonal anti-human endoglin, CD105(clone SN6h), monoclonal mouse anti-human cytokeratin (clone
MNF116). All primary antibodies were provided by DakoCytomation (Glostrup, Denmark) and tested for human specificity andcross-reactivity. Staining protocol continued with secondary
biotinylated antibody binding, substrate addition, and hematoxylin
counterstaining of the nuclei (LSAB2 System-HRP, Dako) following the manufacturer procedures.
BM-MSCs and UC-MSCs differentiation experiments towards adipocytes, chondrocytes and osteoblasts was assessedusing anti-mFABP4, anti-hAggrecan, and anti-hOsteocalcin,
primary antibodies, respectively antibodies from the Human
Mesenchymal Stem Cell Functional Identification Kit (R&D Systems), while the visualization system was LSAB2 System-HRP(Dako). Microscopy analysis was performed on a Nikon Eclipse
E800 microscope.
5Electron microscopy
Scanning electron microscopy (SEM) was performed
for identification of morphological changes between BM and
UC-derived MSCs. Cells were cultured at cellular density of
10,000 cells/cm2 in 24-well format cell culture inserts (BD).
After 24 hours cells were pre-fixed for 1 hour with 2.5%
buffered glutaraldehyde (in PBS), rinsed three times in PBS,
and the 0.4 μm pore-sized membranes were detached fromthe culture inserts. For better image quality, cells fixed on themembranes were sputter-coated with platinum-palladium andexamined with a FEI Quanta 3D FEG electron microscope(FEI Company, Eindhoven, The Netherlands) generating digitalelectron micrographs.
Transmission electron microscopy (TEM) was used tocompare MSCs’ ultrastructural characteristics. For TEM analysis,
cells were spined down and immediately fixed with 4% bufferedglutaraldehyde. They were then postfixed with 1% OsO4 in 0.1M
cacodylate buffer, included in agar, ethanol dehydrated and thenembedded in Epon 812 at 60ºC for 48 hours. The ultrathin sections were cut using a diamond knife and double stained withuranyl acetate and lead citrate. Ultrathin sections were examinedusing a Morgagni 286 TEM (FEI Company, Eindhoven, Nederlands) at 60 kV. Digital electron micrographs were recorded witha MegaView III CCD using iTEM-SIS software (Olympus, Soft
Imaging System GmbH, Germany).
RESULTS
Microscopy of BM-MSCs and UC-MSCs
As revealed by light microscopy, both MSC types share asimilar morphology, while UC-MSCs being smaller and more
confluent (Figure 1A). However, several notable differences havebeen observed in transmission electron microscopy between thetwo cell types. Hence, BM-MSCs have nuclei exhibiting discrete
indentations, numerous mitochondria, reduced endoplasmic
reticulum, lysosomes, packages of intermediate filaments andshort and rare cytoplasmic elongations. On average, their diameter is 15-25 µm. On the other hand, UC-MSCs have nuclei withlobulated morphology (large indentations), occupying more thanhalf cellular diameter, few mitochondria, and highly developed endoplasmic reticulum with dilated cisterns. Their average diameteris 10-15 µm (Figure 1B). Both cellular types exhibited numerous
thin cytoplasmic elongations and intermediate filaments. Detailed
and enlarged view of UC-MSCs revealed presence of intercellularjunctions – desmosomes (Figure 1C).

A

6

B

C
Fig. 1. Morphologic characteristics of isolated stem cells. A. BM-MSCs and
UC-MSCs in SEM; B. TEM revealing ultrastructure of stem cells; C. Presenceof desmosomes connecting two individual UC-MSCs

Immunophenotype of MSCs
Flow cytometry analysis has revealed many phenotypical similarities between the BM and UC-derived MSCs. Bothcellular types expressed all the cell surface markers generally
used to characterize the mesenchymal stem cells, like CD44,
CD90, CD73, CD29, etc., while being essentially negative for
CD34 and CD45 expression (Figure 2). CD29, CD90, and CD73
expression was increased for BM-derived MSCs compared toUC-derived MSCs, being almost double when considering CD73
expression pattern.
No cytokeratin and E-cad expression was detected by either
flow-cytometry or IHC.
As revealed by IHC, both BM-MSCs and UC-MSCs strongly
expressed vimentin (Figures 3 and 4), the staining being locatedwithin the cytoplasm and perinuclear. Vimentin is a defining
cytoskeletal protein found in connective tissues, and all primitive cell types express vimentin but in most non-mesenchymal
cells it is replaced by other intermediate filament proteins during
differentiation.
Regarding the proliferation rate, we could demonstrate that
UC-MSCs have an increased proliferation rate, as presented
by Ki67 expression; almost 80% of the UC-MSCs expressed
this marker, the passage number of both cellular types being
the same. VEGF expression was laking in both cellular types,
even though the BM-MSCs subpopulations are known for theirpro-angiogenic potential.
Another stem cell marker was assessed – CD117 (c-kit, stem
cell factor receptor) and its expression was increased on both
BM and UC-derived MSCs, thus demonstrating the “stemness”
of the considered cellular populations.
Fiziologia - Physiology  2011.21.4 (72)Fig. 4. UC-MSCs express vimentin and c-kit in similar pattern with BM-MSCs,
being negative for VEGF and cytokeratin; Ki67 is present in 80% of cells,
indicative of highly proliferative processes (Magnification 400x)

Plasticity of BM-MSCs and UC-MSCs
Adipogenesis was markedly induced in BM-MSCs after
exposure to specific culture medium (50% of cells presented lipid
drops within the cytoplasm), while differentiation of UC-MSCs
towards the adipocytic lineage was not achieved, although thecells presented a morphological change prerequisite to such a
process. Visualization of MSCs-differentiated adipocytes was
performed using primary mouse anti-human FABP4 antibody.
Upon induction of both BM and UC-MSCs to differentiate towards
osteoblasts and chondrocytes, we could not see the UC-derived
cells differentiating, although the BM-derived cells presented
these abilities (data not shown) (11).

Fig. 2. Expression of characteristic surface markers on BM and UC-MSCs.
A. BM-MSCs positive expression of CD29, CD90, and CD73; B. UC-MSCs
follow the same expression pattern when compared to BM-MSCs

Fig. 3. BM-MSCs present expression of Vimentin and intracytoplasmic c-kit(CD117). Proliferation marker Ki67 is expressed in low amount, and cells arenegative for cytokeratin (Magnification 400x)

2011.21.4 (72)  Fiziologia - Physiology

DISCUSSION
Overall, we demonstrated that even though different sourcesof MSCs are used, the characteristic markers and morphology
converge towards the same pattern. When we talk about theheterogeneity of isolated cells, there is a great debate regarding which of the tissular sources can give rise to a more pure
population. However, when a more detailed morphological andfunctional analysis was performed, we identified structures – desmosomes – which impaired the function of UC-MSCs. So that,the debate about which are the characteristics markers of stem
cells will longer continue, due to the fact that not only the structure
give the “stemness”, but also the function of cells we want to
identify as stem cells. To conclude, in the attempt to isolate stem
cells, we should carefully look to the morphology, characteristic
markers, and function of cells we want to identify.
There is still a widely held perception that BM-MSCsrepresent a phenotypically heterogeneous population of cells.
There are a number of reasons for this. First, as far back as thepioneering work of Friedenstein et al. (12), it has been recognizedthat not all CFU-Fs were highly proliferative and multipotential.
Second, many different groups have used a limited number ofdiverse phenotypic markers to identify in vivo MSCs/CFU-Fs
using magnetic enrichment or flow cytometry (8, 9, 13–17).
Taking a synthesis of these functional and phenotypic data toa logical (but not necessarily correct conclusion) has led to the7impression that MSCs were both functionally and phenotypicallyheterogeneous.
To clarify this, we used multiparameter flow cytometry andcross-tested different MSC markers and purification methods,
including plastic adherence for their selectivity and specificity
for in vivo BM-MSCs (14, 18). We found that all these methods
identified a phenotypically identical rare cell population that was
distinct from BM hematopoietic cells by their very low CD45
expression and a larger cell size.
Nevertheless contrary to some currently propagated views(19, 20), a phenotypically distinct, in vivo BM- MSC population
has now been identified. Importantly, a striking consensus regarding the morphology of fresh MSCs is emerging, regardlessof the method of isolation used. They appear as large cells thathave prominent nucleoli and bleb-like projections, which extend
further as MSCs adhere-this is different from spindle-shaped
morphology of typical cultured MSCs (14, 15, 21). Based onfunctional assays, the presence of MSCs in extra skeletal locations including synovium, fat and even placental tissue andumbilical cord has been firmly established. Identifying the MSC
population from the much larger stromal fraction will be a more
formidable challenge compared with MSC identification in the
marrow. There has been a common opinion that CD73, CD105,
CD90 and CD44 are highly specific for MSCs, and hence candiscriminate multipotential cells from the more mundane tissue
resident fibroblasts. More recently, however, several studies
showed that these markers were ubiquitously expressed onstromal cells from many locations as well as on skin fibroblasts(22–24), and at best they only inform an investigator that thephenotyped cells are non-hematopoietic and stromal in origin.
Despite their functional heterogeneity, MSC populations
obtained from most tissues commonly express a number ofsurface receptors including CD29, CD44, CD49a-f, CD51, CD73,
CD105, CD106, CD166, and Stro1 and lack expression of definitive hematopoietic lineage markers including CD11b, CD14, andCD45. Recent studies have shown cells that express the aforementioned surface markers and are capable of differentiating into
connective tissue cell types can be enriched from peripheral andumbilical cord blood by selection for CD133 and from bone marrow by selection for stage-specific embryonic antigen (SSEA)-1,
SSEA-4, or the nerve growth factor receptor CD271.
In the BM, where the overwhelming majority of cells arehematopoietic, these markers may indeed be useful, but in connective tissues, where most of the cells are fibroblastic, their utility
for the isolation of resident MSCs will be limited and a search for
new, more specific markers, if they indeed exist, is needed. For
the isolation of MSCs from post-partum tissues, such as placenta,
an embryonic stem cell marker SSEA-4 was found to be useful
and, more recently, it was successfully applied for the isolationof MSCs from adult BM. Another important issue to bear in mind
is the stability of putative MSC markers in culture. Despite theloss of certain markers following passaging (7, 14) and the gainof others (18), MSC cultures remain multipotential, indicatingthat these markers are unlikely to be reflective of the MSC’strue ‘stem cell’ nature or its multipotentiality. More likely, many8

markers present on MSCs in vivo may be induced by the BM
microenvironment or be reflective of some other MSC function invivo that is lost upon plastic adherence and exposure to culture
media. At this stage, it would appear that the heterogeneity in
the MSC proliferative and differentiation capacities, first notedby Friedenstein et al. (1) cannot be explained on the basis of
known surface markers alone.
However, it is important to realize that no single isolation
method is regarded as a standard in the field. Therefore, thevaried approaches used to culture-expand and select for MSCs
make it difficult to directly compare experimental results. Moreover, some isolation schemes introduce epigenetic and genetic
changes in cells that may dramatically affect their plasticity andtherapeutic utility. Finally, human MSCs exhibit some variation in
their pattern of expressed genes among different donor preparations using the same isolation protocols, and larger variations assparse cultures become confluent and are expanded by serial
passage and approach senescence (23). These subtleties havebeen overlooked in several publications in which high density
and confluent human MSC cultures were assumed to consist of
homogeneous cell populations.
ACKNOWLEDGMENTS
This work was supported by CNCSIS-UEFISCSU, project
number PNII-IDEI 1748/2008 and by the Sectorial Operational
Programme for Human Resources Development, financed fromthe European Social Fund, FSE POSDRU/89/1.5/S/60746.
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2. Friedenstein AJ, Latzinik NV, Gorskaya YF, Luria EA, Moskvina
IL. Bone-marrow stromal colony formation requires stimulation byhematopoietic-cells. Bone Mineral 1992; 18: 199-213.
3. Castro-Malaspina H, Gay RE, Resnick G et al. Characterisation ofhuman bone marrow fibroblast colony-forming cells (CFU-F) and theirprogeny. Blood 1980; 56: 289-301.
4. Civin CI, Trischmann T, Kadan NS et al. Highly purified CD34-positive
cells reconstitute hematopoiesis. J Clin Oncol 1996; 14: 2224-33.
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adult human mesenchymal stem cells. Science 1999; 284: 143-7.
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7. Simmons PJ, Torokstorb B. Identification of stromal cell precursors
in human bone-marrow by a novel monoclonal-antibody, Stro-1. Blood
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8. Majumdar MK, Thiede MA, Haynesworth SE, Bruder SP, Gerson
SL. Human marrow-derived mesenchymal stem cells (MSCs) express
hematopoietic cytokines and support long-term hematopoiesis whendifferentiated toward stromal and osteogenic lineages. J Hematother
Stem Cell Res 2000; 9: 841-8.
9. Boiret N, Rapatel C, Veyrat-Masson R et al. Characterization ofnonexpanded mesenchymal progenitor cells from normal adult humanbone marrow. Exp Hematol 2005; 33: 219-25.
10. Prockop
�����������������������������������������������������������������
DJ. Marrow stromal cells as steam cells for nonhematopoietic tissues. Science 1997; 276: 71-4.
11. Paunescu V, Bojin F, Tatu CA, Gavriliuc OI, et al. Tumour-associatedFiziologia - Physiology  2011.21.4 (72)

fibroblasts and mesenchymal stem cells: more similarities than differences. J Cell Mol Med., 2011; 15(3):635-646.
12. Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone-marrow
osteogenic stem cells - In vitro cultivation and transplantation in diffusion
chambers. Cell Tissue Kinet 1987; 20: 263-72.
13. Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C, Deliliers GL.
Isolation of bone marrow mesenchymal stem cells by anti-nerve growth
factor receptor antibodies. Exp Hematol 2002; 30: 783-91.
14. Jones EA, Kinsey SE, English A et al. Isolation and characterizationof bone marrow multipotential mesenchymal progenitor cells. Arthritis
Rheum 2002; 46: 3349-60.
15. Gronthos S, Zannettino ACW, Hay SJ et al. Molecular and cellularcharacterisation of highly purified stromal stem cells derived from humanbone marrow. J Cell Sci 2003; 116: 1827-35.
16. Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal
stem cells in human bone marrow and dental pulp. J Bone Mineral Res
2003; 18: 696-704.
17. Deschaseaux F, Gindraux F, Saadi R, Obert L, Chalmers D, Herve P.
Direct selection of human bone marrow mesenchymal stem cells using
an anti-CD49a antibody reveals their CD45(med,low) phenotype. Br J
Haematol 2003; 122: 506-17.
18. Jones EA, English A, Kinsey SE et al. Optimization of a flow

cytometry-based protocol for detection and phenotypic characterizationof multipotent mesenchymal stromal cells from human bone marrow.
Cytometry Part B: Clin Cytometry 2006; 70B: 391-9.
19. Tuan R. Stemming cartilage degeneration: Adult mesenchymal stem
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20. Kolf C, Cho E, Tuan R. Mesenchymal stromal cells. Biology ofadult mesenchymal stem cells: regulation of niche, self–renewal anddifferentiation. Arthritis Res 2007; 9: 204-304.
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Novel markers for the prospective isolation of human MSC. Ann NY
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23. Ishii M, Koike C, Igarashi A et al. Molecular markers distinguish bonemarrow mesenchymal stem cells from fibroblasts. Biochem Biophys Res
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24. Jones EA, English A, Henshaw K et al. Enumeration and phenotypiccharacterization of synovial fluid multipotential mesenchymal progenitor
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50: 817-27.

CONTROVERSE LEGATE DE CULTURILE CELULARE OBTINUTE DINDIFERITE SURSE TISULAREREZUMAT

Celulele stem mezenchimale (MSC) sunt celule stromale non-hematopoietice, care sunt capabile sa se diferentieze si sa contribuie
la regenerarea tesuturilor mezenchimale, cum ar fi tesutul osos, cartilaj, muschi, ligamente, tendoane si tesut adipos. MSC sunt
identificate prin expresia moleculelor de suprafata, cum ar fi CD105 (SH2) si CD73 (SH3/4), fiind negative pentru markerii celulelor
hematopoietice CD34, CD45 si CD14.
In prezentul studiu am izolat doua populatii celulare, din maduva osoasa hematogena (BM) si cordonul ombilical (UC) si am investigat
comparativ prezenta markerilor fenotipici, potentialul de diferentiere spre trei linii celulare (trilineage) si caracteristicile morfologice.
Cu toate ca atat BM-MSC, cat si UC-MSC au prezentat un profil fenotipic similar si caracteristici morfologice comune (in microscopie
optica), nu am reusit diferentierea UC-MSC spre adipocite, condrocite si osteoblaste, ceea ce sugereaza functionalitatea scazuta
sau modificata a acestora. Analiza detaliata a aspectului morfologic (microscopie electronica) a demonstrat prezenta jonctiunilor
intercelulare de tipul desmozomilor, care ar putea explica partial comportamentul in vitro al acestor celule. Trebuie investigat dacacelulele stromale izolate din cordonul ombilical au dobandit acest fenotip in conditii de cultivare in vitro, sau daca metoda utilizata
pentru izolarea acestora nu a fost cea potrivita. Totusi, caracteristicile “stemness” ar trebui sa fie bazate mai mult pe functia celulara
si mai putin pe caracteristicile fenotipice celulare, deoarece aceste celule sunt candidate potentiale pentru terapiile regenerative si
aplicatii clinice.
Cuvinte cheie: BM-MSCs, UC-MSCs, diferentiere, functie

2011.21.4 (72)  Fiziologia - Physiology

9

COMPARATIVE ANALYSIS OF BONE MARROW-DERIVEDMESENCHYMAL STEM CELLS AND IMMORTALIZEDMESENCHYMAL CELL LINE
MARUSCIAC LAURA, PANAITESCU CARMEN, PAUNESCU VIRGIL
Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT
Human mesenchymal stem cells (hMSCs) were first identified when observing groups of cells that developed into fibroblastic colony
forming cells (CFU-F). Since then, the potential clinical applications of MSCs have increased interest in this field. However, identification of these cells seems to be a challenge. Several identification criteria have been proposed, in order to establish homogenous
hMSCs for use in research.
The aim of this study was to characterize two populations of MSCs – one derived from human bone marrow, and an immortalized
hMSC line –, in order to determine whether they are equivalent for use in further research. We have determined the expression of
CD11b, CD29, CD34, CD45, CD73, CD90, CD105, CD117, and vimentin in the two populations. They both showed similar expression for all the analyzed markers. We concluded that the immortalized cell line has the same characteristics as the freshly isolated
hMSCs, and therefore can be used in further research.
Key words: hMSCs, phenotypical markers, characterization, flowcytometry

INTRODUCTION
Mesenchymal stem cells (MSCs) are multipotent, nonhematopoietic stromal cells that can differentiate into a multitude
of mesenchymal tissues, such as muscle, bone, cartilage, andadipose tissue [1]. They are usually isolated from bone marrow,
where they constitute about 0.01% of the total cell population[2]. However, they have been found in numerous other adulttissues: brain, spleen, liver, kidney, lung, muscle, thymus, pancreas, as well as in large (aorta, vena cava) and small bloodvessels (kidney glomeruli) [3]. By definition, MSCs have to be
negative for the following surface markers: CD11b or CD14,
CD34, CD45, CD79a or CD19, HLA-DR, which distinguishes
them from hematopoietic cells, and they express several surface markers, among which CD73, CD90 and CD105. There
are also two functional criteria that define MSCs: adherence toplastic when maintained in standard culture conditions using
tissue culture flasks, and multipotent differentiation potential (toosteoblasts, adipocytes, and chondroblasts) under standard invitro differentiating conditions [4] (see Table I).

Table I. Criteria to identify MSCs related to phenotype, adherence andplasticity
Phenotype

Positive (>95%)
CD73
CD90
CD105

Negative (<2%)
CD11b or CD14
CD19 or CD79a
CD34
CD45
HLA-DR

Adherence to plastic
In vitro differentiation to osteoblasts, adipocytes, and chondroblasts

Integrins have been known to play a key role in cell adhesion
and migration [5]. One of the integrins involved in cell interactions
is CD29, or integrin β1. The CD29 molecule is a 130 kDa single
chain type I glycoprotein that is expressed in a heterodimericcomplex with one of six distinct α subunits, comprising the verylate activation antigen (VLA) subfamily of adhesion receptors [6].
It is one of the essential surface molecules expressed on human
MSC from bone marrow and other sources. The ligand for hMSC
CD29 is the vascular cell adhesion molecule 1 (VCAM-1) [7].
Vimentin is an intermediate filament protein widely applied

Received 23rd of September 2011. Accepted 15th October 2011. Address for correspondence: Laura Marusciac, MD PhD student, Department of Functional Sciences, “Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara, phone/fax:+40256220479; e-mail: laura.marusciac@gmail.com

10

Fiziologia - Physiology  2011.21.4 (72)

as a mesenchymal indicator [8]. It is functionally involved inmaintaining the structure of mesenchymal cells [9]. In addition
to serving as a marker in the epithelial to mesenchymal transition, it plays a versatile role in cancer cell motility [10]. In normal
tissue injuries, vimentin-deficient mice suffer from delayed
wound healing due to the failure of mesenchymal contraction at
the wound site [11] and impairment of fibroblast migration [12].
Vimentin seems to be related to the activation of mesenchymal
cells, but little is known about the relationship between vimentin
expression and normal cell activation [13].
CD117, also known as the mast/stem cell growth factor receptor (SCFR), proto-oncogene c-Kit or tyrosine-protein kinase Kit, isa protein that in humans is encoded by the KIT gene. It functions
as a cytokine receptor, and signalling through CD117 has beenshown to play a role in cell survival, proliferation, and differentiation[14]. CD117 has been used to identify and characterize different
types as stem and progenitor cells, including hematopoietic stem
cells [15] and mesenchymal stem cells [16, 17].
The aim of this study was to characterize human mesenchymal stem cells in regard to surface markers, and to provide
a comparison between the characteristics of isolated hMSCs
and immortalized hMSCs.
MATERIAL AND METHODS
1. Isolation and culture of human MSCs
Human MSCs were obtained from the iliac crest bonemarrow of healthy male donors, with ages between 18 and40 years. The donors had been previously evaluated for thepresence of hepatitis B surface antigens (HbSAg), hepatitis C
antibodies (HCV Ab), human immunodeficiency virus antibodies(HIV Ab), and cytomegalovirus antibodies. All samples of bonemarrow were collected after informed consent was obtained inaccordance with the guidelines on the use of human subjects,
and approval by the ethics committee. 10-20 ml of bone marrowwas collected from each donor in heparin-coated tubes.
The bone marrow was filtered through 100 μm sieves (BDFalcon, San Jose, CA, USA), diluted with Phosphate Buffer Solution (PBS, Invitrogen, Carlsbad, CA, USA) in a 1:1 ratio, and thencollected into sterile 50 ml Falcon tubes (BD Falcon). Then Biocoll
density gradient (Biochrome AG, Germany), with a density of 1,077
g/ml was added carefully at the bottom of the tube, underneaththe bone marrow, in a 1:2 ratio. The tubes were then centrifuged
for 30 minutes, at 1800 rpms, at room temperature.
The mononuclear cell layer (“buffy coat”) was then carefullycollected in new sterile 50 ml Falcon tubes, and diluted with
Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) in a 1:1
ratio. The tubes were then centrifuged at 2000 rpms, at room
temperature. After centrifugation, the supernatant was discarded
and the cells were resuspended in 1 ml DMEM and counted. The
mononuclear cells were then cultured in T75 culture flasks (BDFalcon), at a density of 1x105 cells/cm2, at 37 °C, in a humidified
atmosphere that contained 5% CO2. The culture medium that wasused contained DMEM supplemented with 20% fetal calf serum2011.21.4 (72)  Fiziologia - Physiology(FCS, Invitrogen), 1% penicillin/streptomycin mixture (Pen/Strep,
10,000 IU/ml; PromoCell, Heidelberg, Germany). The culture
medium was changed to remove the remaining nonadherent cells
24 hours after the initial plating. Thereafter, the culture medium
was replaced twice per week.
The cells were processed no longer than 4 hours after bonemarrow harvesting. In parallel, human MSC line was obtained
from Vitro BioPharma (Native Human MSC, CO, USA).
2. Immunophenotypical characterization of hMSCs
For flowcytometry, human MSCs were labeled with conjugated antibodies against several human proteins to analyze the cellsurface expression of typical MSC antigens, as well as the absenceof antigen expression for other CD molecules, considered negative for mesenchymal stem cells. The antibodies were conjugatedwith Allophycocyanin (APC), Fluorescein isothiocyanate (FITC), or
Phycoerythrin (PE) as follows: CD29-PE, CD73-PE, CD90-APC,
CD105-FITC, CD117-APC, CD11b-APC, CD34-PE, CD45-APC.
All antibodies were purchased from BD Pharmingen.
Human MSCs from passages 2-5 were used when theyreached a confluence of 70-80%. They were trypsinized, counted
and then resuspended in 1 ml staining buffer, containing 0.5%
bovine albumin serum (BSA), 2 mM EDTA, pH=7.2, and 0.05%
azide. For each monoclonal antibody, 1x105 cells were put into
Eppendorf tubes, staining buffer was added up to 500 µl, and then
2 µl of monoclonal antibody were added to each tube. The tubes
were incubated in the dark, at 4 °C, for 20 minutes. The cells werethen washed twice with 1000 µl of staining buffer, and centrifugedat 300g, for 5 minutes, at 4 °C. The cells were then resuspended
in 200 µl of staining buffer and, for each antibody, 1x104 labeledcells were analyzed using a flow cytometer. Data acquisition was
performed using a FACSCanto II flow cytometer (Becton Dickinson,
Franklin Lakes, NJ, USA), and the data was analyzed by FlowJo
software, version 7.6 (Flowjo, Ashland, Oregon, USA).
For immunofluorescence assays, human MSCs were labeledwith antibodies against CD117 (c-kit) and vimentin. For CD117,
the primary antibody consisted of a polyclonal rabbit anti-humanantibody (clone A4502, Dako, Glostrup, Denmark) and for vimentinof a monoclonal mouse anti-swine antibody (clone V9, Dako).
The secondary antibody consisted of an anti-rabbit antibody for
CD117, and an anti-mouse antibody for vimentin. Both secondary
antibodies were conjugated with Alexa Fluor 488. The antibodies
were diluted with PBS in a ratio of 1:300 before use.
Human MSCs from passage 2 were cultured in cover slip
slides until they reached a confluence of 80-90%. After discarding
the growth medium, the cells were washed with PBS, and thenfixed with 4% formaldehyde, for 8 minutes, at 4 °C. The cells werethen washed with PBS for 5 minutes, and the primary antibody
was added. The slides were then incubated for 24 hours, at 4
°C, in the dark. The cells were then washed twice with PBS, thefluorescent secondary antibody was added, and the slides wereincubated for 1 hour, at room temperature, in the dark. The nuclei
were then counterstained for 1 minute, using 4,6’-diamidino-2phenylindole (DAPI, 1mg/ml; Sigma-Aldrich Company, Ayrshire,UK), diluted with PBS, in a ration of 1:5000. The cells were then11

washed twice with PBS and left to dry. The slides were mounted
using cover slip (ESCO microscope cover glass, Erie ScientificCompany, Portsmouth, N.H., USA) and fluorescence mounting
medium was then added (ProLong® Gold anti-fade reagent,
Invitrogen Molecular Probes™). The slides were analyzed using
a fluorescence microscope (Nikon Eclipse E800).
For immunohistochemistry assays, human MSCs were labeledwith antibodies against vimentin (Dako) and CD29 (R&D Systems,
Minneapolis, MN, USA). Human MSCs from passage 2 were cultured in Nunc plates (Thermo Fisher Scientific Inc., Hennigsdorf,
Germany) until they reached a confluence of 80-90%. They werethen trypsinized and 500 μl cellular suspension were cytospun andcytospin slides were obtained by 6 minutes centrifugation at 600
rpm in Shandon Cytospin 4 (Thermo Fisher Scientific). Slides were
air-dried for 10 minutes and then used for immunocytochemistry
procedure. The cells were then fixed with 4% formaldehyde, for
8 minutes, at 4 °C. The cells were then washed with PBS for 5
minutes, and the primary antibody was added. The cells were thenincubated on an orbital shaker, at 200 rpm, for 30 minutes, at room
temperature. The cells were then washed again with PBS, and thesecondary antibody was added.
For staining for vimentin, the Dako EnVision+ System-HRP
kit for use with mouse antibodies (Dako) was used. The cells
were incubated with 1 drop of Labelled Polymer-HRP Anti-Mousesecondary antibody, on the orbital shaker, at 200 rpm, for 30
minutes, at room temperature. The substrate was prepared, using
500 µl of DAB+ substrate buffer and 1 drop of DAB+ Chromogen.
The slides were then washed again with PBS, and 50 µl of substrate were added, followed by incubation on the orbital shaker,
at 200 rpm, for 10-20 minutes, at room temperature, dependingon the intensity of the staining.
For staining for CD29, the Cell & Tissue Staining Kit, HRPAEC system (R&D Systems) was used. The cells were incubatedwith 1 drop of Biotinylated Secondary Antibody, on the orbital
shaker, at 200 rpm, for 30 minutes, at room temperature. The
slides were washed with PBS, and then the cells were incubatedwith 1 drop of HSS-HRP on the orbital shaker, at 200 rpm, for
30 minutes, at room temperature. The substrated was prepared,
using 500 µl of Chromogen buffer and 1 drop of AEC Chromogen.
After washing with PBS, the cells were incubated with 50 µl of
substrate, the orbital shaker, at 200 rpm, for 10-20 minutes, atroom temperature, depending on the intensity of the staining.
For both vimentin and CD29 staining, the slides were thenwashed with running tap water, and stained for 5 minutes with
50 µl of hematoxylin solution (Hematoxylin, Mayer’s Lillie’s
Modification, Dako, Glostrup, Denmark) diluted with tap waterin a ration of 1:5. After another washing with running tap water,
one drop of mounting media was added to the slides, and thecover slips were put into position.
RESULTS AND DISCUSSION
Isolation and cultivation
The success rate for isolating bone marrow MSCs was 100%
(8 out of 8 donors). The average donor age was 26.375±7.76
years old. The average quantity of bone marrow collected from
12each donor was 16 ± 3 ml. The average number of mononuclear
cells isolated from the donors was 85x106±22.8 x106.
Optical microscopy
Both types of human MSCs – isolated from the bonemarrow of donors, and derived from cell lines – had a typicalmorphology, exhibiting a fibroblast-like, and spindle shape,
characterized by a small cell body, with few long, thin cellprocesses. The cell body contained a large, round nucleus.
The cells adhered to the flask. When reaching confluence, they
exhibited a “whirl” arrangement in culture. Immortalized MSCs
from cell lines had a more elongated cell body, with longer cellprocesses (Figure 1).

Fig. 1. A. Isolated MSCs, passage 2, 40% confluence; B. MSCs cell line, passage 7, 40% confluence. Magnification 100X

Immunophenotypical characterization of isolated MSCs
Bone marrow-derived MSCs presented a configurationof positive characteristic markers, including CD73, CD90, andCD105. Negative CD molecules were CD11b, CD34, and CD45
(Figure 2). CD90 positive population is divided in two subpopulation, suggesting that within the heterogenous MSCs some of
the cells are more mature than others, CD90 being a marker ofcellular immaturity.
Fiziologia - Physiology  2011.21.4 (72)

normal cell phenotype [18]. It has also been shown to have a
critical role in pancreatic cancer progression and metastasis,by inhibiting pancreatic cancer cell adhesion, proliferation andmigration [21].
These observations highlight the importance of investigating
the role of adhesion molecules in the appropriate context.

Fig. 2. Flowcytometric characterization of bone marrow-isolated MSCs. MSCs
cellular line expressed similar proportion of characteristic markers.

MSCs and cell line-obtained MSCs immune staining
Both bone-marrow derived human MSCs and the MSC line
expressed of vimentin (Figure 3). The expression was not inabundance, due to the fact that the cells were not permeabilized
beforehand.
CD117 was positive for both bone marrow-derived andMSCs cell line, but in a small amount of cells (Figure 4). For
flowcytometric procedure, cells were not permeabilized, so that
detection level could not exceed the cell surface. Given the
bipolar structure of this marker (stem cell factor receptor, c-kit),
we expected to find an increased expression of CD117 within thecellular cytoplasm, using other phenotypical analyses.
Immunohistochemistry showed an abundance of CD29
marker on the cell surface, in both isolated hMSCs and hMSC line
(Figure 5, A and B). This result is confirmed by the flowcytometricresults, where the majority of the cell population is positive forCD29 (Figure 5, C and D).
Integrin receptors have major importance in cell signaling,
one of their functions being the modulation of the activity andexpression of intracellular proteins and signaling factor, including kinases and scaffold proteins [18]. The assembly of protein
complexes determines the activation of downstream signaling
pathways, some of which overlap with pathways mediated by
growth factors; therefore, integrin signaling is required for adhesion-dependent survival, growth, and migration of cells [19].
There are 18 different α and 8 different β subunits, which
can combine in various ways. The largest subgroup is formedby the β1 subunit, whose members bind to different extracellularmatrix molecules, such as collagen, laminin, fibronectin, but alsointeract with cellular receptors like VCAM-1 [20].
Integrin β1 has an important role in cell invasion, especiallyin a 3D matrigel. Inhibition of integrin β1can restore the ability ofsome tumor cells to form acinar structures, which represents anindicator of reduced tumorigenicity, as well as a return to more
2011.21.4 (72)  Fiziologia - Physiology

A
AA

B
BB

Fig. 3. Comparative expression of cytoskeleton protein Vimentin in isolated MSCs (A) and hMSC line (BFig.
3.
expressionofThecytoskeleton
proteinVimentinin isolatedofFig.
cells
areComparative
positiveexpression
forthis marker.
cell nuclei
arestainedwith
DAPI
and appear
blue-whiteundComparative
expression
cytoskeleton
proteinVimentininisolated
MSCs
(A)
hMSC
(B
Fig.
3. 3.
Comparative
ofofcytoskeleton
proteinVimentinisolated(A)andandhMSClineline
MSCs(A) positive
and
hMSC
line
(B).
NoteThethatcellmajoritycells
are inpositiveforMSCs
this appear
Magnification
400x.
cells
thismarker.
marker.
nucleiofare
arestained
with
of of
cells
arearepositive
forforthis
The cellnucleistainedwith DAPIDAPI andand appearblue-white
blue-whiteunu
marker. The
cell nuclei are stained with DAPI and appear blue-white under
Magnification
400x.
Magnification
400x.

fluorescent light. Magnification 400x.

A
A
A
C
C
C

B
B
B
D
D
D

Fig. 4. CD117 expression on both types of MSCs - isolated hMSCs (A) and hMSC line (B) - shows abun
Fig.
expression
on. bothMSCsisolated- isolated
hMSCs
(A)
and
(B)
- shows abun
withincellularcytoplasm
Magnification
400x.
Flowcytometryshows
thathMSC
only
small
percentageof t
Fig.4.the4.CD117
CD117
expression
on types
both oftypes
of -MSCshMSCs(A) a line
within thecellular cytoplasm. Magnification
400x. Flowcytometry shows that only a small percentage of(4.56%,
respectively
12.73%) (Cand D).
and
hMSC
line
(B)
shows
abundanceof
thismarker
withinthecellularFig.
4. CD117
expression
on bothtypes(4.56%,
respectively
12.73%)(C andD).of MSCs - isolated hMSCs (A) and hMSC line (B) - shows ab
withinthe cellularcytoplasm . Magnification
400x. Flowcytometry
only a small percentage ocytoplasm. Magnification
400x. Flowcytometryshows thatshows
only thata small(4.56%,
respectively
12.73%)
D).
percentageof thecells (Careand
CD117+(4.56%, respectively 12.73%) (Cand D).

A
A

B
B

A

B

13

1 integrins in human endometrial stromal and decidual cells. J Clin
Endocrinol Metab. 1996; ;81(4): 1533-40.
7. Margadant C, Monsuur HN, Norman JC, et al. Mechanisms of integrin
activation and trafficking. Curr Opin Cell Biol. 2011; 23(5): 607-14.
8. P M, S H, R M, et al. Adult mesenchymal stem cells and cell surface
characterization - a systematic review of the literature. Open Orthop J.
2011; 5(Suppl 2): 253-60.
9. Mendez MG, Kojima S, Goldman RD. Vimentin induces changes inCDcell shape, motility, and adhesion during the epithelial to mesenchymaltransition. FASEB J. 2010; 24(6): 1838-51.
Fig. 5. CD29 expression on MSCs shows abundance of this marker in both
10. Li B, Zheng YW, Sano Y, et al. Evidence for mesenchymal-epithelial
isolated hMSC (A) and MSC line (B). Please note the whirl-like arrangementtransition associated with mouse hepatic stem cell differentiation. PLoS
cells.
Magnification
100x.
Thisabundance
result is confirmedby theFig.of5.theCD29
expression
on MSCsshowsof this marker
in flowcytometricboth isolated hMSC (A) andMSC
line (B).
Pleasee17092.
note
One.
2011;
11;6(2):
results(C and D).100x. This result is confirmed by the flowcytometric results (C and D).
the whirl-like arrangement of the cells.
Magnification
11. Eckes B, Colucci-Guyon E, Smola H, et al. Impaired wound healing
in embryonic and adult mice lacking vimentin. J Cell Sci. 2000; 113 (Pt
CONCLUSIONCONCLUSION
13): 2455-62.
Ourconfirm
results confirm
studies,whichshow expression
Our results
previousprevious
studies, whichshow
expressionof vimentin, CD117,
CD29
on human
12. and
Eckes
B, Dogic
D, Colucci-Guyon E, et al. Impaired mechanicalof vimentin,stem
CD117,
andCD29
on humanhMSC
mesenchymal
cells.
mesenchymal
cells.
The
immortalized
line stains stemtrue forall these markers,
seems toandbe acontractile capacity in vimentin-deficient fibrostability,and
migration
good
for usehMSC
in further
Thecandidate
immortalized
line experiments.
stains true for all these markers, andblasts. J Cell Sci. 1998; 111 (Pt 13): 1897-907.
seems
to
be
a
good
candidatefor use in further experiments.
13. Shirahata A, Sakata M, Sakuraba K, et al. Vimentin methylation as a marker
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of beta 1 integrins in humanstromalfluid stem cells are capable of modulating thelectedendometrial
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Chamberlain
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J, Ashton Metab.
B, Middleton
J. Concise
decidual cells.G,J Clin
1996; ;81(4):1533-40.review:
immune response. PLoS One. 2011; 6(10): e26535.
7. mesenchymal
Margadant C, Monsuur
HN, their
NormanJC, et al. Mechanisms
of integrin
trafficking.
Currstem cells:
phenotype,
differentiation
capacity,activation and18. 18.
Schooley
AM,Opin
Andrews NM, Zhao H, et al. β1 integrin is required
Cell Biol. 2011;features,
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Silva Meirelles
L, Chagastelles
PC, Nardi
NB. Mesenchymal stem
19. 19. Scales TM, Parsons M. Spatial and temporal regulation of
9. Mendez MG, Kojima S, Goldman RD. Vimentin induces changes in cell shape, motility, and adhesion during
cells reside in virtually all post-natal organs and tissues. J Cell Sci.
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2006; 119(Pt 11): 2204-13.
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10. Li B, Zheng YW, Sano Y, et al. Evidence for mesenchymal-epithelial transition associatedwith mouse hepatic
4.stemDominici
M, Le Blanc PLoSK, Mueller
I, et al.
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TheImpaired
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24(3): 403-11.
ImhofB,BA,
Aurrand-LionsM. Adhesion
mechanisms
regulating
themigration 21.
12.5.Eckes
Dogic
D, Colucci-Guyon
E, et al. Impaired
mechanicalstability,
and21.
contractile
capacityin Cao HS, Burton DW, et al. Knockdown of theGrzesiak
JJ, Tranvimentin-deficientfibroblasts.
J CellImmunol.
Sci. 1998;2004;
111 (Pt
13):432-44.
1897-907.
migrationof monocytes.
Nat Rev4(6):β(1) integrin subunit reduces primary tumor growth and inhibits pancre13.6.Shirahata
A,S,Sakata
M, Sakuraba
K, et al.
Vimentinmethylation
as a marker for advanced colorectal
Shiokawa
Yoshimura
Y, Nagamatsu
S, etal. Expressionof beta
atic cancer metastasis. Int J Cancer. 2011 Dec 15; 129(12): 2905-15.

carcinoma. Anticancer Res. 2009; 29(1): 279-81.
14. Ashman LK. The biology of stem cell factor and its receptor C-kit. Int J Biochem Cell Biol. 1999; 31(10):1037-51.
ANALIZA COMPARATIVA A CELULELOR STEMMEZENCHIMALE
OBTINUTE DIN MADUVA OSOASA HEMATOGENA SI A LINIEI DE
CELULE STEM MEZENCHIMALE IMORTALIZATEREZUMAT
Celulele stem mezenchimale umane au fost identificate pentru prima oară în momentul în care s-au observat populaţii celulare care
au suferit o transformare în unităţi formatoare de colonii fibroblastice (CFU-F). Din acel moment, potenţialele aplicaţii clinice ale
MSC-urilor au menţinut un interes crescut pentru acest domeniu. Cu toate acestea, identificarea MSC-urilor pare să reprezinte o
provocare. Au fost propuse mai multe criterii de identificare, pentru a stabili uzul unor populaţii omogene de MSC-uri în studii.
Scopul acestui studio a fost de a characteriza două populaţii de MSC-uri – una izolată din măduvă hematopoietică, iar cealaltă fiind
constituită dintr-o linie imortalizată de MSC-uri – pentru a determina dacă acestea sunt echivalente în ceea ce priveşte uzul lor în
cercetări ulterioare. Am determinat expresia CD11b, CD29, CD34, CD45, CD73, CD90, CD105, CD117, şi vimentină în cele douăpopulaţii. Acestea au prezentat expresie similară pentru markerii analizaţi. Concluzia studiului este că linia celulară imortalizată
prezintă aceleaşi caracteristici ca MSC-urile proaspăt izolate, şi astfel pot fi folosite în cercetări ulterioare.
Cuvinte cheie: hMSC, markeri fenotipici, caracterizare, flowcitometrie

14

Fiziologia - Physiology  2011.21.4 (72)PREVALENCE OF OBESITY IN PATIENTS WITH VARIOUS
CHANGES OF GLYCEMIC BALANCE AND THYROID DISEASESADRIANA GHERBON, LAVINIA NOVEANU, GEORGETA MIHALAŞ
Department of Physiology, UMF „V. Babeş”, Timişoara

ABSTRACT
Obesity is a disease commonly associated with type 2 diabetes and thyroid disease with hypothyroidism. The purpose of this study
was to determine the prevalence of obesity in patients with various changes of glycemic balance and thyroid diseases. General lot
studied was represented by 733 cases, aged 7-79 years.
The study group was subdivided by age criterion in two groups: children and adult group. They used clinical, imaging, biochemical,
hormonal, and immunological parameters.
Keywords: diabetes, thyroid disease, and obesity

INTRODUCTION
WHO recognizes obesity as a disease of epidemic proportions in many countries. Its prevalence is higher from a year toanother. Obesity also became a problem in children and young
people, recent data suggest that 8% of them are obese (2).
It seems that it occurs as a consequence of decreased daily
physical activity and increased calorie intake, especially intakeof fast food products.
The WHO MONICA study conducted in Europe in 39 countries on subjects aged 35-64 years reported a prevalence ofobesity of 10-20% for men and 15-20% for women. Overweight
were more common among men; obesity and overweight rate
was over 50% in Europe (9).
In our country, the prevalence of obesity is about 17% in rural
areas and 20% in urban areas. It is more common in women.
The children range varies between 5-10%.
The Urziceni study showed that overweight in adult population is 22.8%, and obesity is 25.2%, accounting 48% of cases.
The higher prevalence of obesity is in the U.S.: 67% of men
are obese and 27.5% overweight, while 62% of women are obeseand 27.5% overweight (5).
Obesity is also responsible for the increase of type 2
diabetes, 90% of patients with type 2 diabetes are obese. The
risk for type 2 diabetes increases with increasing BMI (risk is40-80 times higher for a BMI>40 kg/m2 to a BMI<21 kg/m2).

Also, overweight (BMI>25 kg/m2) occurs in 64% cases in men
and 74% in women (10).
Although obesity is a risk factor for developing diabetes,
only 50% of obese develops diabetes (10).
MATERIAL AND METHOD
Investigated population
The study included adult subjects with diabetes mellitus,
which in time present thyroid disease, or adult subjects withthyroid disease that subsequently present diabetes mellitus.
The study group comprised 733 cases, aged 7-79 years.
Subjects were divided as follows:-group of children that included 83 children and adolescents
aged 7–17 years (14.57±2.25 years), with a ratio F/M of 5.9/1.
-group of adults that included 650 adults aged 18–79 years(52.03±12.46 years), with a ratio F/M of 9.48/1.
Methods of investigation
Methods of investigation were the clinical data-history,
present status, and imaging-thyroid ultrasound, biochemistrycarbohydrate metabolism parameters: fasting bloods glucose,urine glucose, glycosylated hemoglobin and thyroid hormones
investigations and some immunological parameters.

Received September 15th, 2011. Accepted October 20th, 2011. Address for correspondence:���������������������������������������������������������������������������
Adriana Gherbon, MD, PhD, Department of Physiology, ����������������������
“Victor Babe����������
s���������
” University of Medicine and Pharmacy Timisoara, Romania, Eftimie Murgu Square No. 2A, 300041, Timisoara, phone/fax: +40256220479; e-mail: adrianagherbon@yahoo.com

2011.21.4 (72)  Fiziologia - Physiology

15

Glucose determination was performed by enzymatic
techniques with glucose oxidase. Were considered normal
fasting blood glucose between 70-110 mg%, diabetes mellitusfasting blood glucose values above 126 mg%, impaired glucose
tolerance-fasting blood glucose values between 110-126 mg%
and the oral glucose tolerance test (OGTT) at 2 h between
140-200 mg% and fasting impaired glucose tolerance-fastingblood glucose values between 110-126 mg% and OGTT at 2h under 140 mg%.
Determination of glycosylated hemoglobin (HbA1c) was
achieved through the DiaStat program for glycosylated hemoglobin HbA1c that measures the ratio of glycated hemoglobin tototal HbA.
Determination of serum levels of TSH, free fraction ofserum level of triiodothyronine (FT3), free fraction of thyroxin(FT4) were ARCHITECT quantitative method, which is an immunologic determination by chemiluminescence’s with small
Chemilumnescent Micro particle Immunoassay (CMIA). The
following values were considered normal: TSH=0.465 to 4.68
mIU/ml, FT3=3.69-10.4 pmol/l, FT4=10 to 28.2 pmol/l.
Immunological parameters were represented by some markers of thyroid autoimmunity-antiperoxydase (antiTPO) and antithyroglobulin (antiTG) antibodies (AB). To determine the serum
titers of antiTPO AB AxSYM antiTPO kit was used, the method is
enzyme immunoassay with micro particles, Meia (Micro particle
Enzyme Immunoassay). It was considered normal: antiTPO AB<35 IU/ml. To determine the serum titers of antiTG AB AxSYM
antiTG kit was used, the method is enzyme immunoassay withmicro particles, Meia (Micro particle Enzyme Immunoassay). It
was considered normal: antiTG AB <55 IU/ml.
Thyroid ultrasound performed in all cases is a non-invasive
method of exploration that allows measurement of thyroid volume, thyroid study report with cervical anatomical structures andthyroid parenchyma changes.
The appearance of normal thyroid parenchyma is characterized by a high intensity echogenic, homogeneous, easily distinguishable from the neck muscles which look hypoecogenic.
Inflammatory and autoimmune processes are hypoecogenic. The degree of thyroid hypoecogenity was assessed as:
discreet +, moderate + + and marked + + +.
In autoimmune thyroid disease is found hipoecogenity ofthyroid parenchyma.
Graves’ disease appears: thyroid volume generally increased and hipoecogenity with different intensities with variablehomogeneity.
Chronic autoimmune thyroiditis appears: hipoecogenity
generally uneven and normal or increased thyroid volume.
The appreciation of overweight and obesity in children andadolescents was based on BMI, whose value was correlatedwith age and sex (Table I). According to BMI, the weight of achild older than 2 years is estimated as follows: underweight at
a BMI under percentile 5, overweight at a BMI equal to or above
the 85th percentile, but under the 95th percentile and obese at a
BMI above the 95th percentile.
16

Table I. Definition of child overweight and obesity based on BMI (kg/m2) (6, 3)
Age(years)

2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
12.5
13.0
13.5
14.0
14.5
15.0
15.5
16.0
16.5
17.0
17.5
≥ 18

The value of BMI
which estimatedoverweight (85thpercentile)
Boys
Girls
18.41
18.02
18.13
17.76
17.89
17.56
17.69
17.40
17.55
17.28
17.47
17.19
17.42
17.15
17.45
17.20
17.55
17.34
17.71
17.53
17.92
17.53
18.16
18.03
18.44
18.35
18.76
18.69
19.10
19.07
19.46
19.45
19.84
19.86
20.20
20.29
20.55
20.74
20.89
21.20
21.22
21.68
21.56
22.14
21.91
22.58
22.27
22.98
22.62
23.34
22.96
23.66
23.29
23.94
23.60
24.17
23.90
24.37
24.19
24.54
24.46
24.70
24.73
24.85
25
25

The value of BMI
which estimated obesity (95th percentile)
Boys
20.09
19.80
19.57
19.39
19.29
19.26
19.30
19.47
19.78
20.23
20.63
21.09
21.60
22.17
22.77
23.39
24.00
24.57
25.10
25.58
26.02
26.43
26.84
27.25
27.63
27.98
28.30
28.60
28.88
29.14
29.41
29.70
30

Girls
19.81
19.55
19.36
19.23
19.15
19.12
19.17
19.34
19.65
20.08
20.51
21.01
21.57
22.18
22.81
23.46
24.11
24.77
25.42
26.05
26.67
27.24
27.76
28.20
28.57
28.87
29.11
29.29
29.43
29.56
29.69
29.84
30

In the case of an adult, depending on height and weight
it was determinate body mass index classified by WHO andInternational Obesity Task Force as follows (Table II).
Table II. Weight status classification after WHO and Obesity Task Force (7)Classification
Underweight
NormalOverweightObese- type I- type II- type III

BMI (kg/m2)< 18.518.5 -24.9
25 – 29.9≥ 30
30 – 34.9
35 – 39.9≥ 40

RESULTS AND DISCUSSION
The children’s group included 83 subjects, aged 7–17
years (Table III). All studied children had type 1 diabetes.
Fiziologia - Physiology  2011.21.4 (72)AgeCases numbern%Female
n
%

Male
n
%

2
32
49

2
22
47

10
2

0-4 years
5-9 years
10–14 years
15–17 years

2.4
38.56
59.04

100
68.75
95.92

31.25
4.08

Depending on height and weight was determined body massindex (BMI). Its medium value was 20.98±3.19 kg/m2, with aminimum of 14.64 kg/m2 and a maximum of 30.22 kg/m2.
According to BMI, the weight status of children study grouphas been established (Table IV).
Table IV. Distribution of children and adolescents group in relation to theobesity type

Weight status
Underweight
NormalOverweight
Obesity- type I- type II- type III

Total(n = 83)
n
%
18
21.68
58
69.87
6
7.22
1
1.2
1
1.2
-

Female(n = 71)
n
%
9
50
55
94.82
6
100
1
100
1
100
-

Male
(n = 12)
n
%
9
50
3
5.18
-

According to BMI the majority of subjects were normal(69.87%). A percentage of 21.68% were underweight and 7.22%
overweight. Only 1.2% had obesity, which is type I.
It is noted that all overweight and obese individuals werefemale. The obesity type was “apple shape”, characterized byan increased WHR.
We could not specify the patients’ weight at onset diabetes,
before the therapy with insulin. Weight gain could occur as aresult of insulin therapy or following installation of puberty.
Various studies have shown conflicting results on this issue.
A study in Portugal showed that intensive insulin therapy in patients with type 1 diabetes improves metabolic control, but increases
the prevalence of obesity and overweight, leading to an increased
risk of cardiovascular disease (1). Another study conducted in Poland
showed that intensive insulin treatment of diabetes type 1 at puberty
is not a factor for the development of obesity (4).
Between glucose and BMI showed a direct correlation, with a very

Weight status
Under weight
NormalOverweightObesity-type I
-type II
-type IIIDM type 1 (%)
T
F
M
100
7.7
3.34

low intensity (r=0.15, p<0.001). Between HbA1c and BMI was also showna direct correlation, with a very low intensity (r=0.08, p<0.001).
Adults group included 650 people, young adults and elderly
adults, aged between 17 and 79 years (Table V). The study
included adult subjects with diabetes mellitus, which in timepresent thyroid disease, or adult subjects with thyroid disease
that subsequently present diabetes mellitus.
Table V. Distribution of adults group by age and sex
Age
18–19 years
20–29 years
30–39 years
40–49 years
50–59 years
60–69 years
70–79 years

Cases number
n
%

n

11
29
48
168
219
118
57

10
27
43
141
209
112
46

1.7
4.46
7.38
25.84
33.7
18.15
8.77

Female
%
90.9
93.1
89.58
83.93
95.43
94.91
80.7

n

Male
%

1
2
5
27
10
6
11

9.1
6.9
10.42
16.07
4.57
5.09
19.3

Adult group was subdivided in function of the type at
glycemic balance in 4 subgroups (Fig. 1):
 group with type 1 diabetes represented by 60 cases(9.23%)
 group with type 2 diabetes accounted for 290 cases(44.61%)
 group with impaired glucose tolerance (IGT) accounted
for 183 cases (28.15%)
 group with fasting impaired glucose tolerance (IFG)
accounted for 117 cases (18%)
50%

Case prevalence

Table III. Distribution of children and adolescents group by age and sex

44.61%

40%
28.15%

30%

18%

20%
10%

9.23%

0%
DM type 1

DM type 2

IGT

IFG

Fig. 1. Distribution of cases by type of changes in glycemic balance

In table VI are shown the results about weight status atpatients with DM and various changes in glycemic balance.
Table VI. The weight status at patients with DM and various changes inglycemic balance.

DM type 2 (%)
TF
M

IGT (%)TF

M

IFG (%)
T
F
M

1.67
43.34
50

92.3
96.66

12.41
28.62

77.77
85.54

22.23
14.46

0.54
9.28
34.97

100
1.7
100
76.47 23.53 29.91 80
2096.87 3.12 26.49 93.55 6.45

5
3.34
1.67
-

66.66 33.34 58.96
50
50
54.97
100
28.65
16.37

89.47
90.43
83.67
96.43

10.53
9.57
16.33
3.57

55.19
50.49
22.77
26.73

96.04
94.11
95.65
100

2011.21.4 (72)  Fiziologia - Physiology

3.96 41.88
5.88 67.34
4.35 20.4
5.12

100
100
100
100

17

In the case of the studied group with type 1 diabetes, themedium value of BMI was 25.1±3.71 kg/m2, with a minimum of
18.22 kg/m2 and a maximum of 35.08 kg/m2.
According to BMI, 50% of patients are overweight. The
percentage of normal weight was 43.34% and the percentage ofunderweight was 1.67% cases. Obesity was present in 5% cases.
It is noted that obesity and overweight was more encounter in
56.36% women and 40% men (p=0.48, X2=0.5). The obesity
type was “apple shape”, characterized by a WHR>0.85. The
prevalence of obesity in type 1 diabetes is very rare. It is notknown the weight at onset of diabetes.
It is possible that patients with type 1 diabetes become
obese due to insulin therapy. Increased fat mass increases
insulin resistance. This occurs due to decreased number of
insulin receptors in adipose tissue and other tissues. A numberof studies show that adipose tissue is higher in the elderly. If aperson has the same body weight at 65 years to 25 years in thisperiod it is doubled fat mass (8).
In the case of the studied group with type 2 diabetes, themedium value of BMI was 31.94±6,53 kg/m2, with a minimumof 20 kg/m2 and a maximum of 59.11 kg/m2. According to BMI
was establish the weight status of the studied subjects with DM
type 2 (Tab.VI).
According to BMI, 58.96% of patients are obese. Normal
weight subjects were 12.41%. It was not found the presenceof underweight subjects. Overweight were present in 28.62%
of cases.
Regarding to obesity type, 54.97% had obesity type I,
28.65% obesity type II and 16.37% obesity type III.
In the type 2 diabetes group, obesity and overweightencounter in 88.88% women and 78.94% men (p=0.08, X2=3).
Obesity type which predominated was “apple shape” characterized by a WHR>0.85 (1.18% “pear shape” and 98.82% “apple
shape”, p<0.001, X2 = 484.28).
In the case of the studied group with IGT, the medium valueof BMI was 31.89±6.72 kg/m2, with a minimum of 16 kg/m2 anda maximum of 55.63 kg/m2. The results about weight status inpatients with IGT are shown in Tab.VI.
According to BMI, 55.19% of patients are obese. Normal
weight subjects were 9.28% of cases, and underweight subjects
were 0.54% cases. Overweight were present in 34.97% of cases.
Regarding the obesity type, 50.49% had obesity type I, 22.77%
obesity type II and 26.73% obesity type III.
In the IGT group, obesity and overweight encounter in
91.9% women and 60% men (p = 0.0009, X2=10.85). Obesity
type which predominated was “apple shape” characterized bya WHR>0.85 (3.03% “pear shape” and 76.97% “apple shape”,
p<0.001, X2 = 291.21).
In the case of the studied group with IFG, the medium valueof BMI was 28.8±5.96 kg/m2, with a minimum of 17.64 kg/m2 anda maximum of 50.6 kg/m2. According to BMI was establish theweight status of subjects with IFG (Tab.VI).
According to BMI, 41.88% of patients were obese. Normal
weight subjects were 29.91% of cases, and underweight were1.7% cases. Overweight were present in a proportion of 26.49%
18

of cases. Regarding the obesity type, 67.34% had obesity typeI, 20.4% obesity type II and 5.12% obesity type III.
In the IFG group, obesity and overweight encounter in
72.22% women and 22.22% men (p=0.0019, X2=9.61%). Obesity
type was “apple shape” characterized by a WHR>0.85.
The role of obesity as a risk factor for type 2 diabetes has
been shown by many prospective studies that included Caucasian populations in Norway, Sweden, Israel and the U.S.,
Mexicans in Texas, U.S., and Pima Indians (5).
In another two studies conducted in Israel and at the Pima
Indians, it was found that the duration and degree of obesity arerisk factors for type 2 diabetes. In the Pima Indian group, therisk of type 2 diabetes is two-fold in individuals with a history ofobesity for 10 years and over this period compared to those witha history of less than 5 years (5).
Central distribution of intra-abdominal fat is a major risk factor for type 2 diabetes independent of obesity degree, hypothesis
demonstrated by prospective studies conducted on populations
from Sweden, Japan and Pima Indians.
In the Pima Indians group, 5-year longitudinal studies haveshown the association of abnormal glucose tolerance transition
with weight gain, decreased insulin secretion and action. Nochanges were observed in hepatic glucose production. Emphasizing excess weight and defect of synthesis/action of insulin
and increased hepatic glucose production is characteristic ofabnormal glucose tolerance progression to diabetes (5).
Between BMI and blood glucose in all types of changes
in glycemic balance of the studied group was a direct correlation, very weak (r=0.048, p<0.001 for the group with type 1
diabetes, r=0.19, p<0.001 for the group with type 2 diabetes,
r=0.13, p<0.001 for the IGT group and r=0.09, p<0.001 for the
IFG group). Also, between BMI and HbA1c was found a direct
correlation, very weak (r=0.36, p<0.001 for the group with
type 1 diabetes and r=0.18, p<0.001 for the group with type 2
diabetes).
Significant differences between patients with differentchanges in glycemic control about obesity prevalence are show
in table VII.
Table VII. Significant differences between patients with different changes inglycemic control about obesity prevalence

DM type I
DM type 2
IGT
IFG
DM
childrenDMtype I
p<0.001
X2=35.77
p<0.001
X2=37.03
p=0.07
X2=3.07p<0.001
X2=37.48

DM type2
p<0.001
X2=35.77
-

IGT

IFG

p<0.001p=0.07
X2=37.03 X2=3.07
p=0.39p<0.001
X2=0.74 X2=20.09
p=0.39p<0.001
X2=0.74
X2=22.63
p<0.001p<0.001
X2=20.09 X2=22.63
p<0.001p<0.001 p<0.001
X2=192.42 X2=166.92 X2=70.98

DMchildrenp<0.001
X2=37.48
p<0.001
X2=192.42
p<0.001
X2=166.92
p<0.001
X2=70.98-

Depending on glycemic balance, obesity and overweight
predominate in people with type 2 diabetes and IGT to thosewith type 1 diabetes. There were no significant differences
Fiziologia - Physiology  2011.21.4 (72)

between DM type 1 and IFG. Also, obesity and overweight
predominate in people with type 2 diabetes to those with IFG,
but was no significant differences between DM type 2 and IGT.
Also, between IGT and IFG, obesity and overweight predominate
in people with IGT.
In function of age, there were significant differences between
DM type 1 at children and different changes in glycemic control
at adult (obesity and overweight prevailed at adults).
CONCLUSIONS
In patients with various changes in glycemic balance andthyroid disorders, obesity is more common in women in case of
IGT and IFG. In case of DM type 1 and DM type 2, there was no
significance difference between men and women.
Depending on glycemic balance, it predominates in people with
type 2 diabetes and IGT to those with type 1 diabetes, in people with
type 2 diabetes to those with IFG, and to those with IGT vs. IFG.
In function of age, there was a net prevalence of obesity andoverweight at adult with different changes in glycemic control vs.
children with DM type 1.
Depending on obesity type, the predominant type was“apple shape”, its predominance shown an increased risk ofcardiovascular morbidity-mortality, especially for atherosclerotic
cardiovascular disease.
Association of thyroid disease, which in time can evolve withhypothyroidism, is an additional risk factor for atherosclerotic
cardiovascular disease.

That’s why it is indicated the screening of thyroid diseases
in patients with various changes in glycemic balance in order
to diagnose these conditions early and to institute appropriate
treatment.
REFERENCES

1. Arcanjo CL, Piccirillo LJ, Machado Ido V, Andrade CR Jr, Clemente
EL, Gomes Mde B. Lipid profile and anthropometrical evaluation in type1 diabetes. Arq Bras Endocrinol Metabol, 2005; 49 (6): 951-8.
2. Bundred P, Kitchiner D, Buchan I. Prevalence of overweight andobese children between 1989 and 1998: population based series ofcross sectional studies. BMJ, 2001; 322: 326-8.
3. Cole TJ. Establishing a standard definition for child overweight
and obesity worldwide: international survey. Br Med J, 2000; 320:
1240-43.
4. Noczynska A, Filipowski H, Wasikowa R. Is overweight in patientswith type 1 diabetes in the puberty a problem ? Endokrynol Diabetol
Chor Przemiany Materii Wieku Rozw, 2003; 9 (2): 89-92.
5. Pencea C, Ionescu-Târgovişte C. Obezitatea. În: Tratat de diabet
Paulescu, Editura Academiei Române, 2004: 701-725.
6. Popa I, Brega D, Drăgan M, Alexa A, Raica M. Obezitatea copiluluişi ţesutul adipos. Editura Mirton, 2001, pag. 24-42
7. Şerban V, Babeş AP. Clinică Medicală. Teorie şi practică. Editura deVest, 1999; 1: 11-149.
8. Şerban V, Vlad A, Sima A. Diabetul zaharat al vârstnicului, Editura
Brumar, 2003 9. Uwaifo������������������������������������������������������
GI, Arioglu E. Obesity. In Endocrinology (electronic book), 2002.
9. Wilding JPH. Obesity and nutritional factors in the pathogenesis oftype 2 diabetes mellitus. In: Textbook of diabetes. 3rd ed., Blackwell
Science Ltd, 2003: 21.1-21.16.

PREVALENTA OBEZITATII LA PACIENTII CU VARIATII ALE ECHILIBRULUIGLICEMIC SI AFECTIUNI TIROIDIENEREZUMAT
Obezitatea este o afecţiune frecvent asociată diabetului zaharat tip 2, precum şi unor afecţiuni tiroidiene însoţite de hipotiroidie.
Scopul acestui studiu a fost de a determina prevalenţa obezităţii la pacienţii cu diferite modificări ale echilibrului glicemic şi afecţiuni
tiroidiene. Lotul general studiat a fost reprezentat de 733 cazuri, cu vârste cuprinse între 7-79 ani.
Lotul studiat a fost subîmpărţit după criteriul vârstei în 2 loturi: lotul de copii şi lotul de adulţi. S-au folosit parametrii clinici, imagistici,
biochimici, hormonali, imunologici.
Cuvinte cheie: diabet zaharat, afecţiuni tiroidiene, obezitate2011.21.4 (72)  Fiziologia - Physiology

19

CARDIOVASCULAR MANIFESTATIONS IN AUTOIMMUNERHEUMATIC DISEASES
M. COJOCARU1, INIMIOARA MIHAELA COJOCARU2, VIOLETA ŞAPIRA3
1”Titu Maiorescu” University, Faculty of Medicine, Department of Physiology, Center for Rheumatic Diseases, Bucharest
2“Carol Davila” University of Medicine and Pharmacy, Department of Neurology, Colentina Clinical Hospital, Bucharest
3“Ovidius” University, Faculty of Dental Medicine and Faculty of General Medicine, Department of Pathophysiology, Constanţa

ABSTRACT
Cardiovascular manifestations in autoimmune rheumatic diseases (ARD) have been infrequently recognized and mainly neglectedin the medical community. Cardiac involvement may represent the initial manifestation of an ARD. Cardiac manifestations range
from unusual to prominent and from mild to dramatic. Although ARD affects all cardiac structures, pericarditis is the most common
manifestation. Early detection of cardiac abnormalities may have important therapeutic or prognostic implications. Aggressive antiinflammatory therapy might additionally reduce clinical manifestations of cardiovascular disease. We review the cardiovascular
manifestations of more common ARD in the light of the reported literature.
Keywords: cardiovascular manifestations, autoimmune rheumatic diseases

INTRODUCTION
In the last several decades, the increased prevalenceof cardiovascular manifestation in patients with autoimmune
rheumatic diseases (ARD) was observed (1). Heart disease in
ARD develops through several pathophysiological mechanisms
including myocardial inflammation and/or fibrosis, infiltration by
granulomatous tissue, vasculitis, thrombus formation, of accelerated coronary atherosclerosis. Beyond this, pulmonary arterial
hypertension leads to significant cardiac dysfunction and rapid
progression of heart failure. Whilst it is likely that chronic systemic
inflammation promotes accelerated atherosclerosis in thesepatients (2,3). Cardiac manifestations range from unusual toprominent and from mild to dramatic. In particular, coronary heartdisease seems to be associated with inflammatory rheumaticconditions (4,5). Autoantibodies can damage all heart structures(Figure 1) (3). Autoantibodies may interact with target tissue or
induce downstream mechanisms leading to cardiac lesions (6).
Rhythm and conduction disorders are not only a frequent clinicalmanifestation of cardiovascular involvement in ARD but also a
possible cause of sudden death. The conduction disorders are
more frequent in ARD than the cardiac arrhythmias. Cardiovascular autonomic dysfunction was revealed in the majorityof autoimmune patients (7). Another difficulty is to differentiate
cardiovascular effects of drugs from ARD involvement. Basic

research in imaging and rheumatology might provide a better
insight into ARD pathogenesis and might guide development oftargeted disease-specific therapies (8,9).

Fig. 1. Involvement of the heart structures in autoimmune rheumatic diseases (3)Rheumatoid arthritis
Cardiovascular features in rheumatoid arthritis (RA) arecommon, including pericarditis, cardiomyopathy/myocarditis, cardiac amyloidosis, coronary vasculitis, arrythmia, valve diseases
and, most importantly, congestive heart failure and ischaemic

Received October 5th, 2011. Accepted November 20th, 2011. Address for correspondence: Manole Cojocaru, MD, PhD, ���������������������������������������������
”Titu Maiorescu” University, Faculty of Medicine, Department of Physiology, Center for Rheumatic Diseases, Bucharest, Thomas Masaryk No. 5 Street, Sector 2, RO-020983, Bucharest, Romania, phone/fax:+4021.324.30.13, e-mail: manole.cojocaru@yahoo.com

20

Fiziologia - Physiology  2011.21.4 (72)heart disease (10-13). Chronic inflammation is primarily responsible for accelerated atherosclerosis in RA (14-16). Clinicallysignificant valvular disease attributable to RA appears to be uncommon (17). Fibrinous pericarditis may be detected at autopsy
in RA patients but is generally not of clinical relevance. Arterial
stiffness is increased in RA. It is related to disease duration(18). Rheumatoid arthritis is associated with an increased risk ofcongestive heart failure. The prevalence of left ventricular systolic
dysfunction was to be higher in patients with RA (19). Coronary
arteritis and fibrinous pericarditis may be detected at autopsy in
RA patients. Important for clinical practice is the positive effect of
methotrexate therapy on the reduction of cardiovascular mortality
and the contraindication for TNF-α antagonists in patients withsevere heart failure (20). In patients with RA, a major cause of
sudden cardiac death is atherosclerotic coronary artery disease,leading to acute coronary syndrome and ventricular arrhythmias(21). In RA, infiltration of the atrioventricular node can cause
right bundle branch block. AV block is rare in RA, and is usuallycomplete. In RA patients, conduction abnormalities may regress
when the underlying disease is controlled (22,23).
Systemic lupus erythematosus
The premature development of atherosclerotic coronary
artery disease has been documented in autopsy. Arterial stiffness is increased in SLE and is related to disease
duration and to circulating levels of C-reactive protein andinterleukin-6. It is associated with active disease, including
myositis and serositis. SLE-related factors associated withclinical manifestations of coronary artery disease include older
age at diagnosis, longer duration of SLE, higher damage
score, longer duration of steroid therapy, and higher levels ofoxidized low-density lipoprotein cholesterol and homocysteine.
Valvular disease associated with Libman-Sacks lesions, serositis resulting in pericardial disease, and venous and arterial
thromboses associated with the presence of antiphospholipid
antibodies are well-established cardiovascular manifestationsof SLE (24). Myocarditis is rarely diagnosed clinically and valvular nodules have been described in the majority of patients
at autopsy. The premature development of atherosclerotic
coronary artery disease has been documented in autopsy(25). Pericardial disease, as a manifestation of serositis, is adiagnostic feature of SLE. Pericardial effusions occur mostcommonly in the setting of active disease (flares) (26). In SLE,
sinus tachycardia, atrial fibrillation and atrial ectopic beats are
the major cardiac arrhythmias (27).
Systemic sclerosis
The most prominent cardiovascular abnormalities associated with systemic sclerosis (SSc) are microvascular perfusion
abnormalities of the ventricular myocardium resulting in ischemia,
fibrosis, systolic dysfunction, and conduction disease (28). Myocardial disease in SSc may be related to associated pulmonary or
renal involvement or to hypertension (29). The severity of theselesions was unrelated concomitant pulmonary and systemic
hypertension, Raynaud’s phenomenon, and renal disease but
2011.21.4 (72)  Fiziologia - Physiology

was related to the presence of congestive heart failure, conduction abnormalities, ventricular arrhythmias, and cardiovasculardeath (30). The large artery stiffening parallels that describedabove in RA and SLE. Stiffening of the conduit arteries develops
as a consequence of inflammation and/or microvascular disease(31,32). Pulmonary arterial hypertension is a major issue in ARD,
especially SSc significantly affecting quality of life and prognosisof the patients. The most frequent cardiac rhythm disturbancesin SSc are premature ventricular contractions, often appearing
as monomorphic, single ventricular contractions, or rarely as
bigeminy, trigeminy or pairs (33). Conduction disturbances in
SSc are due to fibrosis of sinoatrial node, presenting as abnormal
ECG, bundle and fascicular blocks (34).
Dermatomyositis and polymyositis.
Cardiac involvement is now well recognized as a clinicallyimportant manifestation in patients with polymyositis (PM) or dermatomyositis (DM), although its actual frequency is still uncertain.
Despite the fact that clinical manifestations are relatively rare,
asymptomatic cardiovascular features are frequently reported
in patients with PM and DM and are predominated by conduction abnormalities and arrhythmias (35). The most frequently
reported clinically overt manifestations are congestive heart
failure, conduction abnormalities, that may lead to complete
heart block, and coronary artery disease. Cardiac arrhythmias(including conduction disturbances and abnormal systolic timeintervals) can occur but are often asymptomatic. These patients
can manifest with AV blocks and ventricular or supraventriculartachyarrhythmias. Subclinical manifestations, characterizedby conduction abnormalities and arrhythmias are frequently
observed. In DM and PM, heart involvement may have fatal
consequences, despite clinically overt involvement of the heart
is seldom evident (36).
Psoriatic Arthritis
Premature atherosclerosis has been recognized as animportant co-morbidity in patients with psoriatic arthritis (PA).
Patients with PA had higher prevalences of both cardiovasculardisease risk factors, and ischemic heart disease, peripheral vascular disease, and congestive heart failure. The risk of incident
myocardial infarction was increased (37).
Ankylosing spondylitis
Patients with ankylosing spondylitis (AS) may develop
cardiovascular manifestations ranging from asymptomatic forms
to life threatening conditions. Heart disease is a well recognizedcomplication of AS (38). The spectrum of disease is wide andincludes mitral valve disease, cardiomyopathy, and pericarditis.
The most common cardiac lesions in patients with AS are aortic
regurgitation and conduction abnormalities. Conduction disease
may develop as a consequence of postinflammatory scarring of
myocardial tissue (39). First-degree atrioventricular block is mostcommon; however, higher-grade atrioventricular block and rightand left bundle-branch block have also been reported. Heartblock appears to occur more frequently in HLA-B27-positive21

individuals, even in the absence of clinical manifestations of
AS. Diastolic filling abnormalities have been described in AS.
Aortitis of the ascending aorta may lead to distortion of the aortic
ring, causing aortic regurgitation (40). Fibrosis of the conduction
system may result in various degrees of atrioventricular block,including complete heart block. Aortic aneurysmal dilation is arecognized rare association with AS, and bicuspid aortic valve or
coronary artery disease is only occasionally present. Decreased
aortic elasticity with impaired endothelial function could be
responsible for the development of the aortic complication in
AS patients (41).
CONCLUSION
Cardiovascular manifestations are known to occur in patients with ARD often in subclinical form. Cardiovascular morbidity and mortality have been found to be increased in association
with many of the ARD. A thorough history for cardiac symptoms
is very important to prevent any future major cardiac event.
Rheumatologists should carefully consider, both in late and inearly phases of the disease, the occurrence of cardiovascular
manifestations in patients with ARD.
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Prevalence, disease manifestations, and treatment of psoriatic arthritis
in Western Norway. J Rheumatol, 2005; 32: 1918-22.
38. Caliskan M, Erdogan D, Gullu H, Yilmaz S, Gursoy Y, Yildirir A,
Yucel E, Muderrisoglu H. Impaired coronary microvascular and left
ventricular diastolic functions in patients with ankylosing spondylitis.
Atherosclerosis, 2008; 196: 306-12.
39. Divecha H, Sattar N, Rumley A, Cherry L, Lowe GD, Sturrock R. Cardiovascular risk parameters in men with ankylosing
spondylitis in comparison with non-inflammatory control subjects:
relevance of systemic inflammation. Clin Sci (Lond), 2005; 109:
171-6.
40. Brunner F, Kunz A, Weber U, Kissling R. Ankylosing spondylitis andheart abnormalities: do cardiac conduction disorders, valve regurgitation and diastolic dysfunction occur more often in male patients withdiagnosed ankylosing spondylitis for over 15 years than in the normal
population? Clin Rheumatol, 2006; 25(1): 24-29.
41. Yuan S-M. Cardiovascular involvement of ankylosing spondylitis:
Report of three cases. Vascular, 2009; 17: 342-54.

MANIFESTĂRI CARDIOVASCULARE ÎN BOLILE REUMATICE AUTOIMUNEREZUMAT
Manifestările cardiovasculare în bolile reumatice autoimmune nu sunt frecvente şi de obicei sunt subdiagnosticate. Afectarea
cardiacă poate să reprezinte manifestarea iniţială a unei boli reumatice autoimune. Manifestările cardiace variază de la neobişnuite
la evidente şi de la uşoare la dramatice. Deşi bolile reumatice autoimmune afectează toate structurile cardiace, pericardita estecea mai frecventă manifestare. Evidenţierea precoce a tulburărilor cardiace este importantă pentru terapie sau prognostic. Terapia
antiinflamatorie agresivă reduce manifestările clinice ale bolii cardiovasculare. Autorii prezintă manifestările cardiovasculare mai
frecvente ale bolilor reumatismale autoimmune în lumina datelor din literatură.
Cuvinte cheie: manifestări cardiovasculare, boli reumatice autoimmune

2011.21.4 (72)  Fiziologia - Physiology

23

THE ASSESSMENT OF ERG DIFFERENCES BETWEEN RED
AND WHITE STIMULIALEXANDRU D.1, CĂTĂLIN B.2, GEORGESCU M.2, GEORGESCU D.1, IANCĂU MARIA2
1
Department of Medical Informatics, University of Medicine and Pharmacy of Craiova, Faculty of Medicine
2
Department of Physiology, University of Medicine and Pharmacy of Craiova, Faculty of Medicine

ABSTRACTIn the study featured in this article we investigated the differences between the series of amplitudes andlatencies of ERG waves recorded to flash stimuli, produced by different light, red (monochromatic) or white,using increasing intensities, from -1 to 2 log with steps of 0.5 log, plus the 1.7 log intensity (50 cd·s/m2). We
obtained significantly different amplitudes for the “a” waves only for flash intensities over 1.5 log cd·s/m2 andfor the “b” waves only for the maximum amplitude of the series, measured at 0.5 log cd·s/m2. Analyzing thelatencies, we found highly significant statistical differences for all the intensities used.
Keywords: photopic ERG, red light, white light, amplitude, latency

IntroduCTION
Although the cellular structure of the retina is known in detailfor over 100 years, the interactions between different cell types
and the roles of their subtypes are still far from being completely
characterized. The information they generate and the way thisinformation is processed at the retina level are topics more actual
than ever, because of the improvement of research methods,
due to the development of information technology and that ofbio-signal recording techniques.
Image generation has been highly studied and recent discoveries regarding the image processing that begins in the retina
or the cellular pathways identified at this level are arguments that
this area is in constant development.
Still, basic aspects of light perception are not well documented in the literature, such as the general behavior of the retina
in response to selective color stimuli, in normal conditions.
As a result of this observation, we conducted an electroretinography study, meant to highlight the differences between
the ERG parameters obtained by stimulating the retina withmonochromatic (red) and white light. The study consisted of
two tests:- in the first test, we recorded Flash ERG in photopic conditions, in response to a series of 8 increasing light intensities,
stimulation being done with white light, using a strobe lamp;- in the second one, we recorded Flash ERG in photopic
conditions, in response to a series of 8 increasing light intensities, stimulation being done with red light (660nm), using LED
goggles.
In both tests, the eyes were light adapted and pupils were
not dilated, to recreate normal daytime conditions. Minimum
intensity was -1 log cd·s/m2 (0.1 cd·s/m2), which allowed us toobtain both ”a” and ”b” ERG waves for red light stimuli.
MATERIALS AND METHOD
We performed these studies according to the ethical andmoral principles of the Helsinki Declaration of Human Rights.
The most important factors taken into account were the wellbeing and safety of subjects. Before testing, each subject wasgiven a detailed presentation of the purpose and the conductof the experiment, clearly specifying what data will be recorded
and how it will be used. All subjects have agreed to voluntary
participation.
At the time of the experiments no subject was sufferingfrom eye disorders, acute or chronic. Volunteers have made acommitment to come to experiments rested, without consuming
coffee, alcoholic beverages or any substances affecting attention
and reaction time, for at least 12 hours before the experiment,
for the results to be scientific relevant.
In both tests, subjects were prepared for the recordings
as follows:
1. to reduce the discomfort of the recording electrode, thesubject was given a drop of lidocaine clorhydrate (XILINE) 4%
solution in the lower conjunctival sac
2. to prevent irritation and infection of the conjunctiva,
the subject was given a drop of tobramycin + dexamethasone(TOBRADEX) solution in the lower fornix
3. we attached the required electrodes for recording the
ERG:- the recording electrode, HK-loop type, previously wettedwith physiological serum to not irritate the conjunctiva, wasintroduced into the lower conjunctival sac of the right eye andfixed on the lower eyelid with medical adhesive tape

Received October 20th, 2011. Accepted November 3rd, 2011. Address for correspondence: Teaching assistant Dragoş Alexandru, MD, PhD, University of Medicine andPharmacy of Craiova, Faculty of Medicine, Department of Medical Informatics, 2 Petru Rareş Street, 200349 , Craiova, Romania, e-mail: dragosado@yahoo.com

24

Fiziologia - Physiology  2011.21.4 (72)- the reference electrode, self adhesive, was set to theipsilateral external cantus- the grounding electrode, self-adhesive, was placed on
the forehead
4. we proceeded with light adaptation, for 10 minutes
5. we applied the stimuli, each repeated 10 times, thus doing
the recording of ERG responses. The end result was achieved
through the coherent averaging of the 10 samples.
On the Flash ERG recordings many waves are described;
we were interested in the first negative wave, denoted “a” and in
the first positive wave, denoted “b”. Parameters used to describe
them were the amplitude and latency.
For this, we marked their extreme points that are theminimum of the “a” wave and the maximum of the “b” wave.
We measured the amplitude of “a” as difference between the
average voltage measured before applying the stimulus andthe peak of the wave, and the amplitude of “b” as difference
between the „b” and the “a” peaks („trough to peak” amplitude).
Latencies of the “a” and “b” waves were reported as the difference between the time from the stimulus onset and the time
when the waves peak.
For stimulation and data recording we used the Neuropack
M1 MEB-9100 system (Nihon Kohden Corp., Tokyo, Japan). Red
light stimuli were obtained with special LED goggles - LS-102J,
part of the system (Nihon-Kohden Corp, Tokyo, Japan), emitting
a narrow band of frequencies, with peak wavelength of 660nm,
and white light stimuli were produced with a TSD122b strobe
lamp (Biopac Systems Inc., Goleta, CA, USA).
Table I. Characteristics of the used stimuli

Luminance(log cd·s/m2)

Luminance(cd·s/m2)

Inter
StimuliInterval
(s)

-1

0.1

10

-0.5

0.3

10

0

1

10

0.5

3

10

1

10

15

1.5

30

15

1.7

50

15

2

100

15

The recording electrodes were HK-loop type („HawlinaKonec loop”)(1). This electrode consists of a thin silver wire,
shaped to form a loop that is bent and inserted into the lower
conjunctival sac. The wire is electrically insulated with Teflon,
except for three windows at the top of the loop, as opposed to theelectrode connector. Electrical contact with conjunctiva is madeby the not insulated, exposed portion of the wire. Its advantages
are stability similar to surface electrodes and sensitivity comparable to corneal contact electrodes, foil or wire type (DTL).
2011.21.4 (72)  Fiziologia - Physiology

AB
Fig.1. HK loop electrodes (A) and their fixation in the conjunctival fornix, forrecordings (B)The reference electrode was self-adhesive, Ag-AgCl type,with foam, model PG10S (FIAB SpA, Florence, Italy), while thegrounding one was also Ag-AgCl type, model VivoMed Solid Gel(Servoprav GmbH, Wesel, Germany).
Data processing and computing of classical statistical indicators (arithmetic mean, standard deviation, coefficient of variation),
as well as tests for comparing averages (Student) were performed using Microsoft Excel 2003 (Microsoft Corp., Redmond,
WA, USA). To compute data normality tests (Shapiro-Wilks andAnderson-Darling) and nonparametric tests for comparing averages (Mann-Whitney-Wilcoxon), we used the XLSTAT add-on(Addinsoft SARL, Paris, France).
In the first test we performed ERG recordings on 31 volunteers (9 men and 22 women), using white light stimulation,
under photopic conditions, in response to a series of flashes withincreasing intensities, between -1 and 2 log, from 0.5 to 0.5 log,
plus the 1.7 log intensity (50 cd·s/m2).
For the second test of the study, 20 ERG recordings weremade (for 6 men and 14 women, 11 persons from the first experiment missing), in response to a series of red light flash stimuli,using the same intensities as above.

Men,
9,
29.03%
Women,
22,
70.97%

Fig.2.Gender distribution in the first test

25

reduced to 4 and 2 μV.
As expected, for the “b” wave recorded on the light-adapted
eye, when the main contributors to the ERG are the cone cells,
we noticed the “photopic hill” phenomenon: the amplitudes of
the “b” waves increase up to a stimulus intensity of 0.5log (3cd), then begin to decline, reaching about half the peak value
for the final step of 2 log.

Men,
6,30.00%
Women,
14,
70.00%"a" white light"b" white light

Mean amplitude (μV)

80

60

40

20

0

Fig.3.Gender distribution in the second test

-1

-0.5

0

0.5

1

1.5

1.7

2

2

Stimulus intensity (log cd·s/m )Fig.5. Comparison between the values of “a” and “b” waves obtainedwith white light

Mean ± std.dev.

30
22.26

25

22.35

20
15
10
5

For the second set of tests, with red light, the “a” wave amplitudes have been increasing, too, but consistently lower than
the responses recorded in the first test, for the same stimulus
intensities.
For the “b” wave amplitudes, obtained in response to red
light stimuli, we encountered the “photopic hill” phenomenon, too,but here the values seem more systematic and their evolution
can be approximated by a parabolic shaped curve.
"a" red light0Type of stimuli used
Fig.4. Comparison of average age of volunteers

In this study we found no significant differences between
males and females, thus we performed the analysis on theentire group.
This study involved volunteers aged between 19 and 31
years. The average age for the test subjects in the first test was
22.26±3.29 (C.V.=14.76%), and for the second test 22.35±3.47(C.V.=15.52%). Comparing the two means, using the Student
test, we obtained p=0.924, higher than 0.05, so the difference
between the average age of the original subjects and the remaining subjects is not statistically significant.
RESULTSIn the first experiment, conducted with white light, we noticed
a continuous growth for the amplitudes of the “a” wave, but withdifferent rates of change: between -1 and 0.5 log it increasedby 4-5μV from one step to the next, between 0.5 and 1.5 - with
about 10μV and for the last two intensities the increase was
26

Mean amplitude (μV)

Red Light

60

40

20

0
-1

-0.5

0

0.5

1

1.5

1.7

2

2

Stimulus intensity (log cd·s/m )Fig.6. Comparison between the values of “a” and “b” waves obtained with red
light

60

"a" white light"b" white light"a" red light"b" red light

50

Mean latency (ms)White light"b" red light

80

40
30
20
10
0
-1

-0.5

0

0.5

1

1.5

1.7

2

2

Stimulus intensity (log cd·s/m )Fig.7. Comparison of mean wave latencies

Fiziologia - Physiology  2011.21.4 (72)Analyzing the evolution of the latencies of the “a” and “b”
waves, we found highly significant statistical differences for all
the intensities used (Mann-Whitney p<0.001), but we also noticed
a trend of parallel variation in the recorded values.
Calculating the differences between the latencies of the “a”
waves recorded in the two tests, respectively between the latencies of the “b” waves, we observed only little variation, close to
3 ms for the “a” waves and to 6 ms for the “b” waves. Therefore,
it is possible that the significant differences we found between
latencies to be due to the different conditions and equipmentused, and not to reflect an objective difference between whiteand red light stimulation.
Table II. Values and ratios of the measured amplitudes

Intensity„a” white„a” reda white /a red
p Student„b” white„b” redb white /b red
p Student

-1
4.13
4.08
1.01
0.923
8.74
8.23
1.06
0.459

-0.5
8.25
8.17
1.01
0.906
20.42
28.88
0.71
0.000

0
13.88
12.38
1.12
0.138
49.00
49.51
0.99
0.881

the time to peak, both for “a” and “b” waves, was significantly
lower than the implicit time for red light (Table III). Because insome studies cited (6,7) we have not encountered this situation
and because the differences were relatively constant (2.83±0.45
ms for the “a” waves – C.V. equal to 15.83%, and 6.33±0.79
ms for the “b” waves – C.V. equal to 12.56%), we suspect aninterference due to the different equipment used for stimulation,a strobe lamp for white light stimuli and LED goggles for red
light stimuli.
In regard to a comparative analysis of the absolute values of​​wave amplitudes recorded, with data from other studies (2,3,4,5),
the method does not seem viable because of large variations
due to different ERG stimulation and recording systems, and,
0.5
18.35
19.55
0.94
0.429
73.49
61.85
1.19
0.031

1
28.58
24.75
1.15
0.088
65.33
59.75
1.09
0.227

1.5
36.75
28.88
1.27
0.002
52.18
51.27
1.02
0.804

1.7
40.83
30.53
1.34
0.000
42.26
45.38
0.93
0.418

2
42.47
33.00
1.29
0.000
38.38
37.13
1.03
0.719

Std.dev.
0.45
0.79

C.V.(%)
15.83%
12.56%

Table III. Differences between the pairs of recorded latencies

Int.
Dif.a
Dif.b

-1
3.69
7.28

-0.5
2.98
6.88

0
2.37
6.98

0.5
2.96
6.69

1
2.80
5.52

1.5
2.46
6.57

DISCUSSIONSIt should be noted that the study was performed on light
adapted eyes, but without dilating the pupils, as is usually done,
because we were interested in recording normal retinal activity,
comparable to everyday situations. The range of used intensities
also contributes to this end, the lower value being close to thelimit for perceiving a color scene, while the upper one is equivalent to the light produced by a standard 60-75W bulb.
The multiple interactions between retinal cells, that occur
mainly for the photopic sight, cause a highly variable behavior of
the series of amplitudes recorded in this study. For example, forstimuli implying high intensities, the amplitudes of the “a” waves
equalize or even surpass the amplitudes of the “b” waves, andthe amplitudes of the “b” waves are not significantly higher for
white light tan for red light, except for the maximum values ofthe series, obtained for 0.5 log cd·s/m2, for the other intensities
the amplitudes’ ratio being close to 1. The only thing proven,corresponding to what we expected to happen, is that “a” waves
produced with white light, at high intensities, are significantly
larger “a” waves obtained with red light (Table II). Amplitude
values ​​measured in these experiments have not been edifying,showing statistically significant differences only for few intensities
and mainly for “a” waves, although the results were almost always
higher for white light than those recorded for red light.
Recorded latencies have shown that, for white light stimuli,2011.21.4 (72)  Fiziologia - Physiology

1.7
3.06
5.40

2
2.36
5.30

Mean
2.83
6.33

last but not least, the characteristics of the population that the
subjects are part of. For these reasons we turned to the use of
the ratios between the amplitudes of “b” and “a” waves, fromthe same experiment. Most articles that refer to series of ERG
responses, describe only general behavior trends or the functions
used to estimate the series of values recorded, exemplifying only
the results for the maximum amplitude, so a detailed comparison
is not possible.
In two studies that examine various aspects of ERG obtained with white and monochromatic light stimuli (6,7), we foundresults consistent with those from our study. Stimulus intensity,
for which results are reported, is the one corresponding to the
maximum amplitude of the “b” waves series.
Table IV. Comparison of the „b”/„a” amplitude ratios for the maximal
responses

bmax/aIntensity

White light

Red light

Present study
Rufiange 2005
Sustar 2009
Sustar 2009

0.5 log
0.5 log
0.4 log
0.4 log

4
3.49
3.75
3.41

3.16
3.1
3.37
2.77

Unfortunately, a thorough numerical comparison of the
behavior of the latencies with data from literature is not possible,
most authors preferring to ignore the details concerning the mea27

sured times or showing them only as charts, from which we canonly see the general trend of evolution. That kind of informationis also useful, because the data from our research are congruent
with ideas found in similar studies (5,6,7).
CONCLUSIONS
1. By measuring the amplitudes of the ERG waves obtainedwith white and red light stimuli we obtained significantly different
results for the amplitudes of the “a” waves only for stimuli of morethan 1.5 log cd·s/m2 and for the “b” waves only for the maximum
amplitude of the series, measured at 0.5 log cd·s/m2 - in all thesecases, the amplitude of the waves recorded with white light was
higher than for red light.
2. Stimulation with white light systematically produced
significantly lower latencies than the red light, for “a” waves, aswell as for “b” waves (p<0.001). Still, further investigations should
be made to rule out any influences due to the equipment used
for producing the stimuli.

REFERENCES

1. Hawlina M, Konec B. New noncorneal HK-loop electrodefor clinical electroretinography. Doc Ophthalmol., 1992; 81(2):253-259.
2. Robson JG, Saszik SM, Ahmed J, Frishman LJ. Rod and cone
contributions to the a-wave of the electroretinogram of the macaque. J
Physiol., 2003; 547.2:509-530.
3. Euler T, Masland RH. Light-evoked responses of bipolar cells in a
mammalian retina. J. Neurophysiol., 2000; 83:1817-29.
4. Hamilton R, Bees MA, Chaplin CA, McCulloch DL. The luminance–
response function of the human photopic electroretinogram: A mathematical model. Vision Research, 2007; 47:2968-72.
5. Al Abdlseaed A, McTaggart Y, Ramage T, Hamilton R, McCulloch
DL. Light- and dark-adapted electroretinograms (ERGs) and ocularpigmentation: comparison of brown- and blue-eyed cohorts. Doc Ophthalmol., 2010; 121:135-46.
6. Rufiange M, Dumont M, Lachapelle P. Modulation of the humanphotopic ERG luminance-response function with the use of chromatic
stimuli. Vision Research, 2005; 45, 2321-30.
7. Sustar M, Cvenkel B, Brecelj J. The effect of broadband andmonochromatic stimuli on the photopic negative response of the electroretinogram in normal subjects and in open-angle glaucoma patients.
Doc Ophthalmol., 2009; 118: 167-77.

EVALUAREA DIFERENŢELOR ERG PRIN STIMULARE CU LUMINĂ ROŞIE,MONOCROMATICĂ ŞI CU LUMINĂ ALBĂRezumatÎn cercetările descrise în articolul de faţă am investigat ce diferenţe există între seriile de amplitudini şi latenţe ale undelor ERG
înregistrate pentru stimuli de tip flash, produşi cu lumină diferită, roşie (monocromatică) sau albă, folosind intensităţi crescătoare,între -1 şi 2 log, din 0.5 în 0.5 log, plus intensitatea de 1.7 log (50 cd·s/m2). Am obţinut amplitudini diferite semnificativ pentru undele„a” doar pentru stimulii de peste 1.5 log cd·s/m2, iar pentru undele „b” doar în cazul amplitudinii maxime a seriei, măsurată la 0.5 log
cd·s/m2. Analizând evoluţiile latenţelor, am identificat diferenţe înalt semnificative statistic pentru toate intensităţile folosite.
Cuvinte cheie: ERG fotopică, lumină roşie, lumină albă, amplitudini, latenţe

28

Fiziologia - Physiology  2011.21.4 (72)STRATEGY TO PREDICT POTENTIAL FOR A “REGULAR
DONOR CAREER” IN FIRST TIME BLOOD DONORSALINA MIRELLA DOBROTA1, ILEANA ION2, LAVINIA VOINEAGU2
1Regional Blood Transfusion Centre of Constanta
2
Faculty of Medicine, University of Medicine and Pharmacy “Ovidius”, Constanta

ABSTRACTIron deficiency anemia is one of the most frequent reasons for blood donors’ deferral. We propose a strategy to identify first time
donors at risk to subsequent deferral and methods to keep them as donors.
141 first time donors, eligible according to the standards, were recruited (96 men and 45 women).Complete cell blood count, serum
iron, total iron binding capacity, transferrin saturation index and ferritin were measured. Data were analyzed to identify donors’ profileand prevalence of iron deficiency without anemia.
Overall, the results showed that prevalence of iron deficiency without anemia, with or without modified erythrocyte indices (expression of iron deficient erythropoiesis) estimated on the circulating iron markers (serum iron, TIBC, SI) and ferritin were found in 11.45% of males and 26 % of women tested.
Combined screening with both hemoglobin, required by the standard, and iron status markers with the occasion of first donation very
effectively predicts first time donors at risk of subsequent deferral. This strategy allows the physician to personalize the donation
schedule according to the prospective donor characteristics and provide him with appropriate counseling.
Key words: blood donor, iron imbalance, strategy, blood supply

INTRODUCTION
Ensuring an appropriate blood supply for a community is themain goal for any blood establishment. This may be achieved
through 3 actions: sustained promotion of voluntary non-remunerated blood donation and recruitment of new blood donors,
retention of first time and occasional donors and rational use of
the available blood and blood components by clinicians (1).
It is admitted by all professionals involved in the transfusion field that regular blood donors, giving blood several times
per year according to the European and national regulations,represent the safest blood resource: they are already informed
and educated on the donation process, know the importanceof honest and accurate filling of the questionnaire, understandthe value of self exclusion and, moreover, are regularly tested
for transfusion transmissible diseases. Therefore, people giving
blood are kindly advised to return on a regular basis to donate;part of them do become regular donors, with a variable donation career, going up to 30-40 years of blood donations or even
more.
Even though many people in the general eligible population
are aware of the need for blood to treat thousands of patients, nomore then 2% of the total population gives blood in Romania. The
situation is similar in Constanta County. This reduced participation to saving others’ life makes blood establishments dependon committed donors in order to have a proper blood supply andmeet urgent needs. Hence, committed donors are admitted to
donate for the maximum number of allowed donations per year,

as frequently as every 8 weeks. This approach is in compliancewith the European and national regulation: women may donate
whole blood maximum 4 times per year, while men 5 times per
year; the minimal interval between two whole blood donations
being of 8 weeks (2-4).
The eligibility of prospective candidates for donation isevaluated through a selection procedure, meant to identify any
risk that might affect either the donor, or the future receiver ofthe blood component resulted from that donation. Along with
the questionnaire, medical interview and clinical evaluation, apre-donation check of hemoglobin is performed, to prevent bloodcollection from those with anemia or values under the standard:
minimum 12.5 g/dl for women and 13.5 g/dl for men. Hemoglobin
is tested as an indirect marker for iron status evaluation.
In spite of the safe procedure for donor selection andblood collection, the risk of adverse reactions related to blood
donation still exists, although most of them are rare and mild
(5). Iron deficiency and iron deficiency anemia are presented
by medical literature as the most common secondary effect ofblood donation, especially if it is done regularly and frequently(6-10). This hasn’t led so far to a common European approach
for an evidence-based revised selection procedure.
We consider that blood donation by itself shouldn’t be declared a priori responsible for iron deficiency or iron deficiency
anemia occurring in a blood donor, as long as only a variable
percentage of regular or occasional donors, mostly women, aredeferred for lower hemoglobin level, while the majority of regular

Received August 6th, 2011. Accepted November 26th, 2011. Address for correspondence: Alina Mirela Dobrota, MD, Regional Blood Transfusion Centre of Constanta,
Romania, Nicolae Iorga Street No. 85, Constanta, phone/fax: +40341170930, e-mail: alina_mirella@yahoo.com

2011.21.4 (72)  Fiziologia - Physiology

29donors develop a long donation career, without any adverse or
secondary effect. Given this evidence, we consider that irondeficiency might appear in a regular donor if additional physiological or pathological factors contribute to, or have previously
caused, an undiagnosed iron status imbalance. High donation
frequency, even though within the legal limits, may also lead toiron status impairment if periodical health evaluations are not
performed.
The present paper presents part of the results obtained in a
broader study conducted in the frame of doctoral studies.
We planned to evaluate the iron status in first time blood
donors, for the following reasons:a. as a mean to foresee the donor’s potential for a regulardonation careerb. as a proactive measure to identify the risk of secondaryiron deficiency anemia if repeated donations are allowed without
protective measuresc. to ensure retention of first time donors, by increasing thequality of medical care provided
MATERIAL AND METHODS
A number of 141 first time eligible donors, 96 men (68, 09%) and 49 women (31, 91%) were randomly included in the
study, based on the incoming order, unless incompliance withthe 2 sets of criteria was identified. They were selected out of theprospective donors presented for the first time, on a voluntarybasis, to the Regional Blood Transfusion Centre of Constanta
County. The selection procedure included two steps:a) setting-up their eligibility as donor according to thestandardb) setting-up their eligibility for the study, by checking thecompliance with the established preconditions:- informed consent for being included in the study, after
information provided by the physician- exclusion criteria:
 history of anemia or iron deficiency,
 digestive, genital diseases or other physiological or
pathological conditions that might influence iron status, for thelast two years vegetarians, vegans, special diets, chronic alcohol users
 recent menses
 recent���������������������������������������������������������
or ongoing treatment that might interfere iron metabolism or laboratory tests values
All the 141 donors were tested for:a. blood pressure, radial pulse
b. hemoglobin and erythrocyte indices (MCV, MCH, MCHC,
RDW), by complete cell blood count performed with hematological analyzer NIHON. Samples were taken during the selection
procedure, in the pre-donation phase.
c. iron serum level, total iron binding transferrin capacity(TIBC), transferrin saturation index (SI) and ferritin were tested
on blood samples drawn up at the beginning of donation, in addition to the samples necessary for routine testing.
Iron serum level and total iron binding transferrin capacity
were measured on automatic biochemical analyzer VITROS
30

250.Transferrin saturation index was calculated from serumiron divided by TIBC value. Ferritin was measured by a chemiluminescence method, on IMMULITE analyzer. All the 141 firsttime donors were declared eligible and finalized the whole blood
donation; a volume of 450ml + 30 ml for testing was collectedfrom each person.
RESULTSThe 141 first time donors included in the study were representative for the panel of first time donors addressing the Regional Blood Transfusion Centre of Constanta: 2/3 men and 1/3
women, aged between 18-61 years old. Average age was 30.67±
9.4 in men and 32.11± 10.5 in women. Distribution per age isrepresented in Figure 1. Distribution based on living environment:
75.18% (106) urban and 24.82% (35) rural area. Distribution per
sex and living environment is shown in Figure 2.

Fig. 1. Donors’ distribution per age

Fig. 2. Distribution per gender and area

During the selection procedure, a medical interview andclinical exam were performed. Cardiovascular parameters –blood pressure and peripheral pulse – were measured, to checkcompliance with the standards (2, 4). All subjects presented
compliant values. Systolic pressure presented average values of
128 ± 12 mmHg in men and 119± 15 mmHg in women. Diastolicpressure presented average values of 73± 7 mmHg in men and
67± 8mmHg in women. A slight difference was observed between
subjects living in urban and rural environment, with higher meanvalues in the latter, both in men and women. 12.5% of men and
11% of women presented hypertension, with values between 140
and 180 mmHg, without other cardiac diseases; therefore all of
Fiziologia - Physiology  2011.21.4 (72)them were admitted to give blood. Peripheral pulse registeredvalues in the range of 62-97/ minute in men (75± 8/min) and
62-98 / minute in women (69± 7/ min). No differences between
urban and rural area were noticed.
Hemoglobin check is mandatory as part of the selection
procedure. In our institution, we have chosen since years ago
to get the hemoglobin value by performing complete cell bloodcount. All the 141 donors presented hemoglobin values compliant
with the standards (12.5-16.5 g/dl for women and 13.5-17.5 g/
dl for men), as they had been selected as eligible for donation.
Values registered for men ranged from 13.5 to 17 g/dl (mean15.2± 0.76g/dl), while women presented hemoglobin values in
the range 12.5-14.9 g/dl (mean 13.38±0.54 g/dl); no differenceswere noticed between urban and rural living environment, asdeclared by donors. The reference range used for hematocrit
is 35-46% in women and 39-50% in men. Values registered formen ranged from 33.60% to 50.20% (mean 43.09± 2.87%), while
women included in the study presented values between 29.30%
and 49.50% (mean 38.32±3.17%).
The next steps of the study envisaged the evaluation ofiron status going beyond the standard requirements, by testing
the additional markers (MCV, MCH, MCHC, RDW, serum iron,
TIBC, SI, ferritin), as previously explained. 24 / 141 (17 %) donors presented one or more markers modified, 12/96 (12.5%)
in men and 12/45 (26.67 %) in women. 82% of them came fromthe urban living environment. For both genders, prevalence ofmodified markers was higher in urban than in rural area (men:12.68% versus 8%; women: 28.57% versus 20%).

The age distribution of donors presenting one or more
modified parameters is presented in Figure 4.

Fig. 4. Distribution of pathological values in males (A) and women (B)Complete cell blood count presents the advantage ofadditional information provided, derived from the erythrocytes
indices. Compliance of MCV, MCH, MCHC and RDW with reference values was analyzed. Values range, mean and SD in male,respectively women are presented in Tables I and II.
Table I. Descriptive statistics for erythrocyte indexes in males

Min

Max

MCV

Total
Mean
SD
Count
96
88.324 3.957

76.60099.300

MCH
MCHC
RDW

96
96
96

27.30032.00011.40033.00036.00014.200

Variable

30.47534.83312.785

1.348
0.890
0.618

Table II. Descriptive statistics for erythrocyte indexes in women

SD

Min

Max

MCV

Total
Mean
Count
45
87.451

6.180

71.400100.000

MCH
MCHC
RDW

45
45
45

1.551
1.158
0.762

27.20031.60011.50032.60036.00014.900

VariableFig. 3. Prevalence of iron imbalance per gender and environment. A- female;
B-male2011.21.4 (72)  Fiziologia - Physiology

29.61834.31113.093

No differences among donors living in urban versus rural
areas were found. VEM showed lower values in 1.04% of men
and 13.33% of women (Figure 5).

31Descriptive statistic analysis for iron status markers tested
is presented in Tables III and IV.
Table III. Descriptive statistics for males

Variable

Total
Count

Mean

SD

Min

Max

Iron

96

104.46

30.23

48.00

168.00TIBC

96351.6953.91

256.00487.00ferritin

96

127.2

93.9

27.2

433.0

SI

96

0.30380.10070.10000.5500

Table IV. Descriptive statistics for womenFig. 5. Prevalence of lower MCV values in first time donors (A-males; Bwomen)

None of the first time donors presented modified values for
MCH. Modified values for MCHC (Figure 6) and RDW (Figure 7)
were found only in female group.

Variable

Total
Count

Mean

SD

Iron

45

90.56

33.76

29.00

149.00TIBC

45390.0758.94

263.00506.00ferritin

45

34.89

18.79

9.06

69.50

SI

45

0.24200.1032 0.0600

Min

Max

0.4500A certain number of donors, higher in women than in men,showed modified values for iron markers, outside the reference
range, suggestive for iron imbalance (Figures 8, 9, 10).

Fig. 6. MCHC in womenFig. 7. RDW in women

32

Fig. 8. Prevalence of low serum iron level in men (A) and women (B)Fiziologia - Physiology  2011.21.4 (72)

deficiency stages, after the stores had been emptied, before
the appearance of anemia. In order to identify an eventual
earlier stage of imbalance, such as depletion of iron stores, withnormal circulating iron, serum ferritin was tested. Values range,mean and standard deviation for men, respectively women arepresented in Table III and IV. To identify depletion and early
deficiency stages, 2 different cut-off were set up: iron deficiency
was considered for values under 15 ng/ml (with or without modified circulating iron), while iron depletion for values falling in therange 15-20 ng/ml in women and 15-30 ng/ml in men, with normal
values for serum iron, TIBC or SI.
Prevalence of iron depletion and iron deficiency in men,
respectively in women, estimated on ferritin values is presented
in Figure 11.

Fig. 9. Prevalence of increased TIBC in men (A) and women (B)Fig. 11. Iron stores status in men (A) and women (B)Fig. 10. Saturation index (SI) in men (A) and women (B)Serum iron, TIBC and SI are considered screening tests forevaluation of iron status; taken together, they provide informationon the status of circulating iron. Its level is decreased in iron2011.21.4 (72)  Fiziologia - Physiology

Overall, the results showed that prevalence of iron deficiency without anemia, with or without modified erythrocyte
indices (expression of iron deficient erythropoiesis) estimatedon the circulating iron markers (cumulative results for specificprofile of serum iron, TIBC, SI) and ferritin was 11.45 % in males
and 26 % in women tested. Ferritin testing provided additional
cases of earlier stages of iron imbalance: iron stores depletion,
included in the figure. No differences between urban and ruralarea have been noticed.

33

DISCUSSION
All 141 subjects included in the study were eligible first time
donors, selected according to the standards and study preconditions set up. It would had been expected that all these personspresent normal values for the evaluated biological markers, aslong as they all fitted with criteria, declared no relevant history or
behavior at risk for an imbalanced iron status and a good healthappearance was identified by the responsible physician. Still,
various abnormalities were found in 17 % of cases. The rangeof modifications went from isolated modified erythrocyte indices(MCV- 1 man) to different associations of modified markers, outof the considered reference range. Clear profiles for different
stages of iron imbalance, from iron stores depletion to irondeficiency, with or without signs of iron deficient erythropoiesis,
were found.
Thus, the study showed the insensitivity of hemoglobin as
iron status marker. Eligible donors identified with different stagesof iron imbalance, might evolve to iron deficiency anemia even
without any other future donations because of their status, without
influence of the first donation. Moreover, they would reach a lower
hemoglobin level soon, considering the iron loss – approximately
200 – 250 mg – caused by each donation, if they came backto give blood, without any previous iron supplementation. This
category of first time donors is not appropriate for giving blood
regularly, as frequently as each 8 weeks. Without testing ironstatus with the occasion of first donation, they would believe thatblood donation is the cause of anemia reached, and maybe they
will never come back to give blood. This might affect the adherence of general population to regular or repeat blood donation
programs. Combined screening with both hemoglobin, requiredby the standard, and iron status markers very effectively predicts
first time donors at risk of subsequent deferral. This approach
may support the donor management strategy, identifying those
who might benefit from iron supplementation, increased donationintervals and changing from whole blood donation to apheresis;
all these measures are meant to keep the recruited first time
donors and prevent the anemia. Thus, the combined testing,performed in the frame of donor selection procedure, allow thephysician to personalize the donation schedule according to
the prospective donor characteristics. It is up to the institution

34

management to decide which iron status markers to use, basedon the available technical and financial resources. Cost-efficiency
is also a constraint. Appropriate information on healthy nutrition,
life style and attitude after donation, with regards to diet andpaying attention to any symptoms and signs potentially related
to blood donation should be provided by the physician in charge
with donors’ surveillance.
The responsibility to ensure donor well-being while facing
continue pressure to prevent shortage of the appropriate blood
supply has to be kept as priority by all the professionals involvedin donor selection.
REFERENCES

1. Directive 2002/98/EC of The European Parliament and of The Councilof January 2003 setting standards of quality and safety for the collection,
testing, processing, storage and distribution of human blood and blood
components and amending Directive 2001/83/EC. Official Journal of theEuropean Union, L 33, 8.2.2003: 30-40.
2. Commission Directive 2004/33/EC of 22 March 2004 implementing
Directive 2002/98/EC of the European Parliament and of the Council as
regards certain technical requirements for blood and blood components.
Official Journal of the European Union, L 91, 30.03.2004: 25-39.
3. European Directorate for the Quality of Medicines & HealthCare,Council of Europe - Guide to the Preparation, Use and Quality Assuranceof Blood Components, 16th edition 2010: 85-102, 199-212.
4. Ministry of Health - Order number 1193 of 7 July 2007 endorsing
regulations regarding information to be provided to blood donors,
information to be provided by blood donors with the occasion of each
donation and eligibility criteria for blood donation. Official Journal no.
540 of 8 August 2007.
5. Klein HG, Anstee DJ. Blood Transfusion in Clinical Medicine- edition
11th, 2005: 1-12.
6. Famulener LW. Anemia in professional donors: a study in a newphase of public health. The American Journal of Public Health, (N Y)
1922 May; 12(5): 376-377.
7. Boulton F. Donor selection. Towards evidence-based donor selection.
Transfusion Today, 2007; 71: 4-5.
8. De Sousa G. Ferro e dadores- ABO, 36 Out/Dez 2008: 5-11.
9. Simon LT, Gerry PJ, Hooper EM. Iron stores in blood donors. JAMA,
1981; 245(20): 22-29.
10. Cable RG, Glynn SA, Kiss JE, Mast AE, Steele WR, Murphy EL,
Wright DJ, Sacher RA, Gottschall JL, Tobler L, Simon TL. Iron deficiency
in blood donors: the REDS-II Donor Iron Status Evaluation (RISE) study.
Transfusion, 2011; Oct 24. doi: 10.1111/j.1537-2995.2011.03401.x.

Fiziologia - Physiology  2011.21.4 (72)STRATEGIE PENTRU ESTIMAREA POTENŢIALULUI DONATORILOR
DE SÂNGE INIŢIALI PENTRU O CARIERĂ DE „DONATOR CU DONĂRIREGULATE”
RezumatAnemia feriprivă este unul dintre cele mai frecvente motive pentru excluderea de la donare a donatorilor de sânge.
Propunem o strategie pentru a identifica donatorii iniţiali cu risc de excludere la prezentări ulterioare şi metode de a-ipăstra ca donatori eligibili.
Au fost recrutaţi 141 donatori iniţiali, eligibili în conformitate cu standardele, (96 bărbaţi şi 45 femei). Au fost efectuate următoarele teste: hemoleucogramă, fierul seric (Fe), capacitatea totală de legare a fierului (CTLF), coeficientul
de saturare a transferinei (CS) şi feritina. Datele au fost analizate pentru a identifica profilul donatorilor şi prevalenţa
stadiilor deficitului de fier, fără anemie.
În ansamblu, rezultatele au arătat că prevalenţa deficitului de fier, fără anemie, cu sau fără indici eritrocitari
modificaţi (expresie a eritropoezei cu deficit de fier), estimată pe baza markerilor de evaluare a fierului circulant (Fe, CTLF, CS) şi a fierului de depozit (feritina) a fost de 11,45% la bărbaţi şi 26% la femeile testate.
Screening-ul donatorilor iniţiali prin asocierea hemoglobinei, impusă de standard, cu markeri de evaluare a statusului
fierului , la prima prezentare, poate prezice în mod eficient care dintre donatorii iniţiali admişi prezintă un risc de a fi
exclus temporar la prezentări ulterioare, din cauza unei valori neconforme a hemoglobinei, cu sau fără anemie. Această
strategie permite medicului să personalizeze programul de donare în funcţie de caracteristicile potenţialului donator şi
să-i furnizeze consiliere adecvată.
Cuvinte cheie: donator de sange, deficit de fier, strategie, rezerva de componente sanguine

2011.21.4 (72)  Fiziologia - Physiology

35

THE USE OF KERATINOCYTE AND FIBROBLAST CULTURESIN THE TREATMENT OF BURNS AND CHRONIC WOUNDSSIMONA VERMESAN1, TIBERIU BRATU1, VIRGIL PAUNESCU2
1
Department of Plastic Surgery, “Victor Babes” University of Medicine and Pharmacy Timisoara
2
Department of Physiology-Immunology, “Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACT
Tissue-engineered skin substitutes have emerged over the past 20 years to one of the most carefully studied and advanced methodsof treatment of large skin defects. This article shows a general view of the full structure of a dermic substitute. While acellular biologic
constructs are discussed, the authors focus on products that contain autologous or allogenic cells. The efforts of the manufacturersof biomaterial to reduce the production costs in the future could establish this method a the one of choice in the treatment of largeskin defects, instead of the partial or total thickness skin grafts that were used until now. These create donor site defects with thepotential developing of scarring and infection and cannot be applied in cases with very large defects, like extensive burns, where
no donor sites are available. This article compares several cell-culture protocols, some aspects of tissue-engineering, as well as a
discussion about the advantages and disadvantages of autologous or allogenic skin cultures. Additional treatment options, like UV-C
irradiation and negative pressure dressings are also discussed.
Key words: autologous keratinocyte cultures; autologous fibroblast cultures; artificial derm; chondroitin-6-phosphate

INTRODUCTION
Development of new techniques for fibroblasts culture wasestablished long before discovery of method for keratinocytes
culture, which was performed by Rheinwald and Green in 1975(1). This method requires presence of immortalized murine 3T3
fibroblasts, which will ensure proliferation of human keratinocytes.
Fibroblasts are mesenchymal stem cells which can becultured in laboratory conditions and play a significant role inepithelial-mesenchymal interactions, de to secretion of numerous growth factors and cytokines with direct effect on epithelial
proliferation, differentiation and formation of extracellular matrix.
These cellular cultures have several clinical applications, suchas in treatment of chronic decubital ulcers, venous ulcers, burns,
coverage of post-traumatic tegumentary defects, as well asother clinical application in the field of dermatology and plastic
surgery (2).
Fibroblasts represent heterogenous cellular population ofmesenchymal origin, found in several tissues. Fibroblasts fromdifferent anatomical sites have similar morphology, but PCA
studies demonstrated that fibroblasts from different regions havetheir own specific gene expression profiles and characteristic
phenotypes, synthesizing extracellular matrix (ECM) proteinsand cytokines in a region specific manner (3).
Dermal fibroblasts are endowed with multiple functions,mainly synthesizing and storage of ECM compounds, but also
involved in proliferation and cellular migration, as a response to

chemotactic, mitogen and modulatory chemokines. Among the
fibroblasts functions, autocrine and paracrine interactions are ofincreased importance (4).
METHODS FOR OBTAINING SKIN FIBROBLASTSSkin samples can be harvested from the patients, after
signing the informed consent. Dermal and epidermal layers
are separated from the adipose and subcutaneous tissue andplaced in a sterile test tube containing Phosphate Buffer Saline(PBS; Sigma-Aldrich Company, Ayrshire, UK). Samples aretransferred immediately into sterile culture flasks for processing
under sterile conditions.
Dermal fibroblasts can be obtained after enzymatic digestion
using Collagenase IV (from Clostridium histolyticum; SigmaAldrich Company) or using the explant method. The latest method
can be successfully used for obtaining cellular cultures from small
skin biopsies. Usually, the culture media is Dulbecco’s Modified
Eagle’s Medium (DMEM; Gibco BRL, Invitrogen, Carlsbad, CA,USA), supplemented with fetal calf serum (FCS; PromoCell,
Heidelberg, Germany) and 1% Penicillin/Streptomycin mixture(Pen/Strep, 10,000 IU/ml; PromoCell, Heidelberg, Germany)(Figure 1).
Growth parameters and fibroblasts characteristics in culture
will be influenced by passage number, donor’s age, fibroblasts
subtype (reticular or papillar dermis) and the anatomical siteof skin sample harvesting. When comparing the fibroblasts
characteristics of older donor to a younger one, these cells tend

Received October 20th, 2011. Accepted November 3rd, 2011. Address for correspondence: Simona Vermesan, MD, PhD student, Department of Plastic Surgery, “Victor
Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu Square No. 2A, RO-300041, Timisoara, phone/fax: +40256220479, e-mail: simover1@yahoo.com

36

Fiziologia - Physiology  2011.21.4 (72)to have a slower migration, reach earlier the senescence andhave a prolonged doubling time of cellular population. Moreover,fibroblasts obtained from an older donor are less responsive to
growth factors added in vitro culture conditions, such as platelet
derived growth factor (PDGF), epidermal growth factor (EGF),
dexamethasone, insulin, and transferrin.

Fig. 1. Skin fibroblasts obtained using the enzymatic digestion method (A)and explant method (B). Magnification 100x.

Other factors influencing fibroblasts behavior in culture
include vitamins, such as vitamin C, and antioxidants, such ascoenzyme Q10. By adding 100µmol/L of vitamin C, the effecton fibroblasts will be that they can produce twice as much collagen than the control fibroblasts (cultured without this vitamin),a response which is independent of donor’s age when obtaining
the fibroblasts. However, coenzyme Q10 has an increased contribution to wounds healing, by augmenting cellular proliferationand fibroblasts mobility.
Another protocol stipulates implant of fibroblasts culture on
collagen scaffolds. Irradiation of collagen scaffolds with 254 nm
wave length UV light (short wave length, named UV C) enables
adherence of fibroblasts on collagen matrix. This radiation typewas also used as anti-bacterial agent, as well as sterilization
method, and more recently in treatment of chronic wounds withslow healing. Absorption peak for nuclei acids is reached at 254
nm wave length. Used in higher doses, this radiation type can
induce cellular destruction at the DNA level. It was demonstrated
that UV C stimulates in vitro production of transforming growth
factor (TGF-α), as well as number of receptors for epidermal
growth factor.
UV C irradiation increased the level of released fibronectin.
2011.21.4 (72)  Fiziologia - Physiology

Fibronectin is a multifunctional adhesion protein, mediating
cellular adhesion, migration and regulating cellular growth andgene expression. Cellular culture on tridimensional collagenscaffolds provides to the fibroblasts new properties, different
from the monolayer cell culture. Using this new technique, cellschange their shape, from the elongated, spindle-like shape toan irregular, with many elongations and multiple spatial orientation one. Conclusion emerging from this new procedure of UV
C irradiation is that it can accelerate fibroblasts integration andthus healing of chronic wounds.
Additional method of improving the vascular bed is represented by use of negative pressure dressing, VAC type. Someof the dermal lesions can be very difficult to treat, despite thealternative skin grafts, local skin or free microvascular transfers.
Some of the patients are not suited for these procedures. For
instance, in case of chronic wounds, injured vascular bed andinappropriate granulation make these procedures unusable.
Argenta et al. (9) have described a method of wound healing
assisted by a vacuum pump, which is based on using a spongeover the lesion, on top of which one can apply negative controlledpressure on the sponge, and consequently on the wound. Thismethod favors granulation, lesion contraction and decreases
bacterial contamination of the wound
ELEMENTS OF BIOENGINEERING
It was demonstrated that fibroblasts secrete a multitude ofsoluble factors which diffuse within the superjacent epidermis,
thus influencing the keratinocytes in paracrine method. Thus,
keratinocytes expanded in monolayer culture are able to produce
only a thin epidermal layer, and 2 weeks after, the apoptotic process will destroy most of the cells. Dermal fibroblasts promoted
a better development of keratinocytes from all epidermal layers,thus contributing to keratinocytes proliferation. It was demonstrated that air-fluid interface is optimal for fibroblasts culture,
because it is similar to skin microenvironment in vivo.
Fibroblasts can release cytokines and growth factors resulting in autocrine and paracrine effects. Autocrine activity includes
transforming growth factor (TGF-β), which in turn induces synthesis and secretion of growth factor for conjunctive tissue. Thisis contributing to collagen synthesis and fibroblasts proliferation.
Keratinocytes are specifically influenced in a paracrine mannerby fibroblasts secretion of keratinocytes growth factor (KGF),
interleukin-6 (IL-6), granulocytes and macrophages colony
stimulating factor (GM-CSF), and fibroblasts growth factor 10(FGF-10). As a result, keratinocytes can synthesize interleukin-1(IL-1) and a peptide related to parathyroid hormone, which inits turn, stimulates fibroblasts to produce KGF, so that a double
paracrine loop is formed, which supports epidermal formationand facilitate wound healing.
Fibroblasts density is very important, but in culture condition,
optimal seeding density remains to be established. Fibroblasts
have only a partial contribution to basal membrane formation,by producing collagen types IV and VII, induced by TGF-β, andlaminin-5, but also through cytokines secretion, which stimulates37keratinocytes to produce components of basal membrane.
Neovascularization and lymphangiogenesis are also important processes for maintaining normal homeostasis of the skin.
Members of vascular endothelial growth factor family (VEGF),
such as VEGF-A, B, C, and D are produced by normal humanfibroblasts and are important for regulation of endothelial cells
proliferation, acting on specific receptors. It is well known that
VEGF-A is involved in activation of resident endothelial cells
and progenitor endothelial cells capable of vasculogenesis
process. VEGF-B is less mitogenic for endothelial cells, while
VEGF-C and -D have receptor specificity, binding to VEGF-R2
which is mediating angiogenesis and VEGF-R3, influencing
lymphangiogenesis. A recent study analyzed dermal fibroblasts
culture, overexpressing VEGF-C, together with human dermic
microvascular endothelial cells. The result was that endothelial
cells were activated and had and increased expression of matrix
metalloproteinase 1 (MMP-1), which allowed the cells to digest
surrounding collagen, invade the supportive gel, and form tubularconnections between cells, similar to capillaries, suggesting theirability for neoangiogenesis.
Another important aspect which might be considered intreatment of chronic wounds using fibroblasts culture is that
applying local heat can induce acceleration of integration andhealing process. Wound microenvironment, comprising the exudate generated by wound can have both beneficial, as well as
inhibitory effects on wound healing process. It was demonstrated
that the fluid surrounding acute wound favored in vitro proliferation of fibroblasts, while the fluid resulting during a chronic wound,aminly venous ulcers, had inhibitory effects on wound healing
process. Locally applied heat induces vasodilation, increases
local blood flow and tissue oxygenation. Thus, this method canbe useful for diminishing inhibitory activity of fluid secreted by achronic, atonic wound. Up to present, it is not elucidated whichof these factors is responsible of inhibitory activity exerted bychronic exudate, but an hypothesis suggest that the proteases
can be involved, thus explaining the beneficial effect of local
heat, which induces denaturation of these enzymes.
CONCLUSION AND DISCUSSION
Fibroblasts used in cell cultures can be autologous or allogenic. The advantages of autologous fibroblasts are that there
are no risks of developing graft versus host disease or intercurrent infections. Also, use of autologous fibroblasts results in better
therapeutic outcomes, such as dermal reconstruction and more
esthetic scar, when compared to use of allogenic fibroblasts.
However, allogenic fibroblasts present the advantage of time
factor. While the autologous fibroblasts culture requires a lot oftime for establishing the culture, and expanding the cells, theallogenic fibroblasts can be cryopreserved and are readily available. They can also be used as biological dressing, with the roleof optimization of healing process until the permanent grafting.
Nevertheless, permanent grafting requires use of autologous
fibroblasts (5-8).
The immunological impact of allogenic fibroblasts was alsostudied using the PCR method. Even when using skin substitutes
38

composed of keratinocytes and allogenic fibroblasts, foreign cells
were not detectable 6 weeks after the grafting. Starting fromthese studies, a new hypothesis emerged, according to which
allogeneic cells are replaced by host cells. Clinical studies results
showed that there is no graft versus host disease in these cases,
explained by many mechanisms. Neither induction of antibodies
specific for class I HLA antigens expressed on allogenic cells
was observed, nor proliferation of T cells in patients exposed tosuch antigens. Dermal fibroblasts lack class II HLA antigens,required for antigen presenting to immune cells. Moreover, it wasobserved that the in vitro-cultured fibroblasts and keratinocytes
gradually lose their ability as antigen presenting cells (similar toLangerhans cells), along cellular passages, and they will not bepresent in cultured skin substitutes (9).
Presently, cell culture-based therapies have a provenbenefic role on faster and better epithelization, in accelerating
healing of skin defects and closure of atonic wounds. However,potential improvement of technical skills can increase in the
future the effectiveness of this therapeutic procedure (10-12).
For instance, improvement of cellular preservation techniques
can increase the cellular life span, as well as possible duration ofstorage. In case of cryopreserved products, simplifying the tawing
and washing techniques would make these cells more available
for use. Moreover, studies need to be conducted concerning the
optimal application regimen of these therapies, including numberof useful application, and time interval between two consecutive
procedures. Application field of these cellular therapies can beconsiderably enlarged if it is demonstrated that they can improvecertain aspects of wounds, such as cosmetic, scar thickness,recurrence, etc (13). Even the studies regarding the mix allogenic
keratinocytes culture deserve much consideration because they
seem to be a more rapid and cheap alternative for autologous
keratinocytes (14-16).
In the future, a marked development of present techniques
for obtaining the cellular cultures can be achieved. However, we
think that use of these methods in the entire world, and especiallyin our country, should be increased, due to the fact that theyshould not be regarded as the last alternative, after patients
suffered for a long time of pain and non-closure of wounds. It
is strongly recommended to establish methods for identificationof patients which could benefit most of traditional or modern
therapeutic approaches.
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keratinocytes: formation of keratinizing colonies from single cells. Cell
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2. Pianigiani E, Cherubini Di Simplicio F, Ierardi F. Dermal Allograft Plus
Autologous Epidermal Graft: Evaluation of Two Different Techniques in
Chronic, Nonhealing Leg Ulcer. Wounds 2004; 16(3):91-96.
3. Eisenbud D, Huang NF, Luke S, Silberklang M. Skin Substitutes
and Wound Healing: Current Status and Challenges. Wounds 2004;
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4. Hansbrough JF, Franco ES. Skin replacements. Clin Plast Surg
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5. Greenleaf G, Hansbrough JF. Current trends in the use of allograft

Fiziologia - Physiology  2011.21.4 (72)skin for patients with burns and reflections on the future of skin banking
in the United States. J Burn Care Rahabil 1994; 14: 428-31.
6. Bravo D, Rigley TH, Gibran N, et al. Effect of storage and preservation methods on viability in transplantable human skin allografts. Burns
2000; 26: 367-78.
7. Flores F, Eaglstein WA, Kirsner RS. Hydroxyurea-induced leg ulcers
treated with Apligraf. Ann Int Med 2000; 132: 417-18.
8. Martin LK, Kirsner RS. Ulcers caused by bullous morphoea treatedwith tissue-engineered skin. Int J Dermatol 2003; 42: 402-4.
9. Wong SL, Defranzo AJ, Morykwas MJ, Argenta LC. Loxoscelism andnegative pressure wound therapy (vacuum-assisted closure): a clinicalcase series. Am Surg. 2009;75(11):1128-31.
10. Streit M, Bohlen LM, Braathen LR. Ulcerative�������������������������������
sarcoidosis successfully treated with Apligraf. Dermatology 2001; 202:367-70.
11. Gath HJ, Hell B, Zarrinbal R, et al. Regeneration of intraoral defects
after tumour resection with a bioengineered human dermal replacement(Dermagraft). Plast Reconstruct Surg 2002; 109: 889-93.
12. Gohari S, Gambla C, Healey M, et al. Evaluation of tissue-engineered skin (human skin substitute) and secondary intention healing
in the treatment of full thickness wounds after Mohs micrographic or
excisional surgery. Dermatol Surg 2002; 28:1107-14.
13. Oka H. Changes in quantity of fibronectin from human skin fibroblasts
with cellular aging. Ann Plast Surg 1985; 14: 248-57.
14. Gerstein AD, Phillips TJ, Rogers GS, et al. Wound healing andaging. Dermatol Clin 1993; 11: 749-57.
15. Krejci-Papa NC, Hoang A, Hansbrough JF. Fibroblast sheets enable
epithelialization of wounds that do not support keratinocyte migration.
Tissue Eng 1999; 5: 555-62.
Marston WA, Hanft J, Norwood P, Pollak R. The efficacy and safetyof Dermagraft in improving the healing of chronic diabetic foot ulcers:
Results of a prospective randomized trial. Diabetes Care 2003;
26:1701-5.

FOLOSIREA CULTURILOR AUTOLOGE DE KERATINOCITE SI FIBROBLASTEIN TRATAMENTUL ARSURILOR SI PLAGILOR CRONICE
RezumatSubstituentii dermici obtinuti prin bioinginerie au evoluat mult in ultimii 20 de ani si au devenit una dintre cele intens studiate si avansate
metode de tratament ale unor defecte mari de substanta la nivelul pielii. In cadrul acestui articol se face o prezentare generala astructurii complete a unui substituent dermic, sunt discutate si avantajele substituentilor dermici acelulari, dar accentul este pus perealizarea culturilor autologe de fibroblaste si keratinocite. Eforturile producătorilor de biomateriale de a reduce in viitor costurile
de productie ale acestora ar putea implementa aceasta metoda drept cea de electie in tratamentul defectelor mari de substanta, in
detrimentul grefelor de piele liberă sau despicata. Acestea din urma implică si crearea unui defect la zona donatoare, cresc riscul
de infectie si nu pot fi aplicate in cazurile cu defecte foarte mari, ca si arsurile extinse, unde nu mai exista zone donatoare. Acest
articol prezinta in mod comparativ mai multe protocoale de obtinere a culturilor celulare, anumite elemente de bioinginerie, cat si odiscutie asupra avantajelor si dezanvantajelor de culturi autologe sau alogene. Sunt prezentate de asemenea si modalitati aditionale
benefice de tratament, precum iradierea cu lumina UV C sau bandajele cu presiune negativa.
Cuvinte cheie: culturi de keratinocite autologe, cuturi de fibroblaste dermice autologe, substituent dermic, condroitin-6-sulfat

2011.21.4 (72)  Fiziologia - Physiology

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MARKERS OF OXIDATIVE STRESS IN CHRONIC RENAL FAILURE
LAVINIA VOINEAGU1, CECILIA ADUMITRESI1, ILEANA ION1, ALINA DOBROTĂ2, VICTORIABADEA3, LILIANA TUTĂ1
1
Faculty of Medicine - ”Ovidius” University, Constanta, Romania
2
Regional Center of Blood Transfusion Constanta, Romania
3
Faculty of Dental Medicine - ”Ovidius” University, Constanta, Romania

ABSTRACT
Oxidative stress is defined as an imbalance of oxidant and antioxidant mechanisms and is generally thought to contribute to thedevelopment of many pathological conditions such as hypertension, heart diseases, Parkinson’s disease and even cancer. There aredifferent markers which can be used to assess oxidative stress. The reduced glutathione (GSH), a major antioxidant, the determinationof the GSH/GSSG ratio and the quantification of GSSG are useful indicators of oxidative stress in cells and tissues but more stablemolecules like F2-isoprostane and 8OHdeoxiguanosine (8OHdG) might present some advantages in assessing this condition
In our study we have chosen the renal pathology to show the changes in oxidative markers concentrations on a group of 52 patientswith chronic renal failure from Constanta County Hospital. We tried to establish whether there is a significant correlation between theconcentrations of reduced glutathione (GSH), its oxidized form (GSSG) and GSH/GSSG ratio) and, on the other side, F2-isoprostaneand 8OhdG concentrations. Tests were performed from whole blood and urine, using ELISA kits of reagents.
A significant difference between patients with renal failure and healthy controls was found in the levels of GSH, GSSG, and GSH/GSSG
ratio. Compared with controls, the patients with renal insufficiency had a significantly higher level of urinary 8OHdG and isoprostane.
Increased concentrations suggest an excess of free radicals and oxidant injury. A significant correlation was found between GSH,
GSSG, GSH/GSSG ratio, isoprostane and 8OHdG.
Key words: oxidative stress, chronic renal failure, F2-isoprostane, 8OHdeoxiguanosine.

INTRODUCTION
The broad definition of reactive oxygen species (ROS) includes oxygen-containing species that are all capable of reactingwith proteins and lipids to produce abnormal cellular responses.
ROS include molecules such as superoxide, hydroxyl radical,
H2O2, peroxynitrite and nitric oxide. Oxidative stress is defined
as an imbalance of oxidant and antioxidant mechanisms andis generally thought to contribute to the development of many
pathological conditions such as hypertension, chronic renal
failure, heart disease, Parkinson’s disease and even cancer.
There are several key enzymes and cytokines in vascularand tubular cells within the kidney that produce increased
amounts of reactive oxygen species (NADPH oxidase, xanthineoxidase, cyclo-oxygenase, lipoxygenase). Renal sources for
ROS are activated macrophages, vascular cells, and various
glomerular cells. ROS may affect cells of the host organism,
especially at sites of inflammation, in addition to playing a rolein the defense system against other agents. This effect plays arole in a variety of renal diseases such as glomerulonephritis andtubulointerstitial nephritis, which can contribute to proteinuria andother conditions. ROS are also thought to contribute to the pathogenesis of ischemia reperfusion injury in the kidney. This suggests that the kidney may be particularly susceptible to oxidative
stress. Additional uremia-related metabolic aberrations such as

intravenous iron exposure, biocompatibility changes associatedwith dialysis, and hyper homocystinemia may also contribute toincreased oxidative stress. Renal anemia is another contributor
to oxidative stress in patients with chronic renal failure.
Reduced glutathione (γ-glutamyl-cysteine-glycine) has been
identified as the prominent, highly effective radical-eliminating
system. When this is inhibited, there is significant delay in theelimination of free radicals, illustrating a defect in the antioxidant forces. GSH is a major antioxidant that provides reducing
equivalents for the glutathione peroxidase (GPx). In the GPx
catalyzed reaction, the formation of a disulfide bond between two
GSH molecules gives rise to oxidized glutathione (GSSG). Whencells are exposed to increased levels of oxidative stress, GSSG
will accumulate and the ratio of GSH to GSSG will decrease.
Therefore, the determination of the GSH/GSSG ratio and thequantification of GSSG are useful indicators of oxidative stressin cells and tissues.
Evaluation of oxidative stress in a clinical setting is difficult
because free radicals have very short half-lives (in seconds). In
contrast, there are actual, more stable marker molecules thathave longer half-lives, ranging from hours to weeks, which canbe used to assess oxidative stress.
For example, F2-isoprostane, which is a nonenzymatic, free
radical-catalyzed isomer of cyclooxygenase-derived enzymatic

Received August 15th, 2011. Accepted September 5th, 2011. Address for correspondence: Lavinia Voineagu, University ”Ovidius”, Constanta, Faculty of Medicine,
Universitătii Street, C2, Constanta 900527, phone/fax: +40241-672899, e-mail: lvoineagu@yahoo.com

40

Fiziologia - Physiology  2011.21.4 (72)

products of arachidonic acid, can be found in both urine andplasma. It is a stable end product of arachidonic acid oxidationand can be measured with high sensitivity and specificity. Isoprostane increased level was associated with hepatorenal syndrome, rheumatoid arthritis, atherogenesis and carcinogenesis.
Several of these compounds possess potent biological activity, as
evidenced mainly through their renal vasoconstrictive effects, andhave short half-lives. Both human and experimental studies haveindicated associations of isoprostanes and severe inflammatory
conditions, ischemia-reperfusion, diabetes and atherosclerosis.
Bioactive 15-isoprostane F2t are continuously formed in various
tissues and large amounts of these potent compounds are foundunmetabolized in their free acid form in the urine in normal basal
conditions with a wide inter-individual variation.
8-OH-dG (8-hydroxy-2-deoxy Guanosine) is produced by
the oxidative damage of DNA by reactive oxygen and nitrogenspecies. Although more than 20 base lesions have been identified, only a fraction of these have received appreciable study,
most notably 8-oxo-2’deoxyguanosine. In complex samples suchas plasma, cell lysates, and tissues, 8-OH-dG can exist as either
the free nucleoside or incorporated in DNA. Once the blood
enters the kidney, free 8-OH-dG is readily filtered into the urine,
while larger DNA fragments remain in the bloodstream. Its levels
are increased by smocking and excessive physical exercise
MATERIALS AND METHODS
The analyses were conducted in two groups of individuals:
Group of healthy controls – HC (n = 40) recruited from among
the patients with biochemical parameters in normal range andwithout any history of renal disease (age: 56.56 ± 4.95 years).
This control group comprises individuals who have not been
exposed to the main exogenous factors of ROS production.
The individuals in the control group were not on any prescribed
medications (including anti-oxidants), no subject was an active
smoker, the individuals did not participate in regular intense
exercise, and all had moderate exposure to the sun; Group ofpatients with chronic renal failure – CRF (n = 52) were recruitedfrom the Constanta County Hospital, (age: 65.57 ± 9.36 years).
The CRF group comprised a variety of disease etiologies, andwith glomerular filtration rate between 30 and 89 ml/min/1.73
m2. All participants gave fully informed consent to participationin the study.
Samples we have used are whole blood and urine. Blood
was obtained by venous puncture and collected in EDTA tubes
for GSH and GSSG measurement. Urine was collected as thefirst morning void. It was obtained for 8-OH-dG and isoprostane
detection.
Freezing and thawing will lyse red blood cells and maximize
the concentration of GSSG in the sample. Blood samples thathave been frozen without prior treatment with the scavenger are
not ideal for GSSG assay, although the scavenger can be added
to frozen samples before they thaw. This will result in a reductionof GSSG and an increase in GSH in the sample. GSH and GSSG
are relatively stable in intact ”resting” cells for up 24 hr at 4°C. The
stability of ”elevated” GSSG in intact red blood cell has not been
2011.21.4 (72)  Fiziologia - Physiology

determined. It is recommended that blood samples are treatedwith scavenger as soon as possible and frozen immediately.
Upon disruption of the cell, GSH is rapidly oxidized.
Quantitative and sensitive detection of oxidative stressmarkers have been carried out using ELISA both for blood andurine sample. ELISA conventional method is excellent in light of
its sensitivity and great convenience. It is a competitive assaythat can be used for the quantification of isoprostane in urine. The
plates are coated with specific polyclonal antibodies. The tracer
consists in an enzyme linked with the isoprostane. The analytes
in the samples are competes with tracer for binding to antibodies
coated on the microplate. The activity of the enzyme results incolor development when substrate is added, with intensity of
the color inversely proportional to the amount of unconjugatedanalyte in the samples.
Specific ELISA kits were used for measuring serum levelsof isoprostane, according to the manufacturer’s protocol. Eachsample was tested in duplicate. Regarding the reproducibility,
we measured isoprostane levels more than twice in sevenserum samples; as a result, the mean percentage coefficient ofvariation was 3.6%.
Using the SOCR program we perform statistical analysis of
the data, searching for correlations between the GSH, GSSG,
GSH/GSSG ratio and isoprostane, 8OHdG. A p value less than
0.05 was considered to indicate statistical significance.
RESULTS AND DISCUSSIONS
A significant difference between patients with renal failureand healthy controls was found in the levels of GSH, GSSG,
and GSH/GSSG ratio. The renal patients had significantly lower
levels of GSH (p<0.001) (Figure 1). GSH is an important lowmolecular-weight antioxidant and, as such, low values would
indicate oxidative stress. Healthy people are protected against
free radicals by several defense mechanisms. Reduced GSH
is the most important intracellular scavengers of free radicals.
GSH serves as a reductant in oxidation reactions resulting in
the formation of GSSG. High values of GSSG therefore indicate
oxidative stress (p<0.04) (Figure 2). A decrease in GSH/GSSG
ratio (p<0.001) was taken as indicative of oxidative damage. The
GSH/GSSG ratio is considered one of the best ways to determine
the antioxidant capacity of cells, and any decreases suggest astrong oxidant effect (Figure 3).

Fig. 1. Average level, standard deviation, and standard error of GSH for
healthy control and patients with chronic renal failure41isoprostane levels correlated with low GSH/GSSG ratio andhigh GSSG suggests that excessive oxidative stress is related
to vascular damage in CRF.
It has already been shown that patients with diabetes mellitus and renal insufficiency undergo oxidative stress, as indicatedby decreased plasma superoxide dismutase, increased serum
8-OHdG or comet assay parameters [9]. For 8OHdG we founda negatively correlation between GSH/GSSG ratio and 8OHdG(r = -0.75) (Figure 4). In this study, we confirmed that blood
8-OHdG in CRF patients was significantly higher than in generalsubjects. These findings suggest that circulating 8-OHdG mightreflect oxidative stress in renal tissue.
Fig. 2. Average level, standard deviation, and standard error of GSSG for
healthy control and patients with chronic renal failure

Fig. 4. Linear regression curve of GSH versus isoprostaneFig. 3. Average level, standard deviation, and standard error of GSH/GSSG
ratio for healthy control and patients with chronic renal failure

Compared with controls, the patients with renal insufficiency
had a significantly higher level of urinary 8-OH-dG (p<0.002) andisoprostane (p<0.001). Increased F2-Isoprostanes concentration suggests an excess of free radicals and oxidant injury.
Remarkably, 90% (51/52) of patients with CRF exhibited elevated
8-isoprostane levels and isoprostane, while none of the healthy
individuals showed increased levels. Thus, almost all patients
exhibited elevated levels of markers that could discriminate
patients from normal controls.
A significant negatively correlation was found between
GSH and isoprostane for controls (r = -0.67) and patients with
CRF too (r = -0.85). In addition, positive correlation were foundbetween GSSG and isoprostane (r = 0.75). We found a significant
negatively correlation between GSH/GSSG ratio and isoprostane(-0.78) (Figure 5). These may be attributable to the inflammatory
process, but vascular endothelial dysfunction is a very important
event in CRF, and oxidative stress induced by vasospasm is themost characteristic sign that reflects this dysfunction. Ischaemia
and reperfusion injury during renal disease can generate reactive
oxygen species that may result in vascular endothelial damage.
In addition, isoprostane itself is a potent vasoconstrictor, hasplatelet pro-aggregate functions and stimulates endothelial
cells to bind monocytes, which may promote vascular obliteration, inflammation and spasm. Therefore, the finding that high42

Fig. 5. Linear regression curve of GSH/GSSg versus 8OhdG

CONCLUSIONS
1. Patients with chronic renal failure have diminished response to oxidative stress due, probably, to a decrease in theantioxidant capacity or to a continued consumption of GSH inoxidative processes; the mechanisms underlying this decrease,
however, are not well established.
2. A significant difference between patients with renal failureand healthy controls was found in the levels of GSSG, and GSH/
GSSG ratio.
3. Patients with renal insufficiency had a significantly higher
level of urinary 8-OH-dG.
4. Patients with chronic renal failure have an increased
isoprostane concentration which suggests an excess of free
Fiziologia - Physiology  2011.21.4 (72)radicals and oxidant injury.
5. A progressive reduction of GSH/GSSG ratio and a
progressive increase in lipid peroxidation products such as
isoprostane provide a strong evidence of increasing oxidative
stress in chronic renal failure.
6. For 8OHdG we found a negatively correlation between
GSH/GSSG ratio and 8OhdG (r = -0.75) (Figure 4).
7. Isoprostane is authentic biomarkers of lipid peroxidationand can be used as potential in vivo indicators of oxidant stressin renal failure.
8. The urinary excretion of DNA repair product 8OHdG has
been proposed as a noninvasive biomarker as oxidative DNA
damage in humans in vivo.
9. A larger number of biomarkers bring more information on
the extent of tissular damage in oxidative stress.

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MARKERI AI STRESULUI OXIDATIV IN INSUFICIENTA RENALA CRONICA
RezumatStresul oxidativ, consecinţă a dezechilibrului între intensitatea acţiunii factorilor oxidanţi si cea a mecanismelor protective antoixidante,s-a dovedit a fi implicat în patogeneza şi evoluţia unui număr semnificativ de afecţiuni cum ar fi hipertensiunea, bolile cardio-vasculare,boala Parkinson, cancerul, etc. Există un număr considerabil de markeri destinaţi studiului stresului oxidativ. Măsurarea concentraţiei
glutationului redus (GSH), unul dintre cei mai importanţi apărători intracelulari impotriva acţiunii speciilor reactive ale oxigenului,cuantificarea formei sale oxidate (GSSG), determinarea raportului GSH/GSSG reprezintă printre markerii cei mai utilizaţi în acest
scop. Au fost descrise însă şi molecule mai stabile, cum ar fi F2-isoprostanul si 8OHdeoxiguanozina (8OHdG) ce pot aduce o serie
de avantaje în aprecierea intensităţii acţunii factorilor oxidanţi.
Studiul nostru a fost efectuat pe un lot alcătuit din pacienţi aflaţi în insuficienţă relală cronică, internaţi în secţia de nefrologie a
Spitalului Clinic Judetean Constanta. Am urmărit să stabilim dacă există o corelatie între evoluţia concentraţiei markerilor de stresoxidativ reprezentaţi prin: GSH, GSSG, raport GSH/GSSG, F2-isoprostan, respectiv 8OHdG. Testele s-au realizat din sânge integral
si urină, folosind truse de reactivi pentru metode ELISA.
Am găsit diferente semnificative între pacientii cu insuficientă renală cronică si lotul de control privind concentraţiile GSH, GSSG siraportul GSH/GSSG. Pacientii cu insuficientă renală cronică au prezentat nivele de 8OHdG si isoprostan semnificativ mai ridicate
decât cele ale lotului de control. Concentratiile crescute ale acestora sugerează existenţa unui exces de radicali liberi si atac oxidativ
şi/sau o diminuare semnificativă a puterii de apărare antioxidantă. Valorile GSH, GSSG si ale raportului GSH/GSSG s-au corelat cu
cele ale F2-isoprostanului si ale 8OHdG.
Cuvinte cheie: stres oxidativ, insuficientă renală cronică, F2-isoprostan, 8OHdeoxiguanozină

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