FIZIOLOGIA
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Fiziologia - Physiology • 2015.25.4(88)FIZIOLOGIA

physiology
CONTENTS
Role of PNPLA3 I148M Variant in Chronic Liver Disease .............................................................................. 5
Enache EL, Minca DM, Enache LS

Incidence and Antibiotic Susceptibility of Microorganisms Isolated From Female Genital
Tract From Outpatients ................................................................................................................................ 11
Simon LM, Pepelea L, Junie LM, Licker M

Etiology, Frequency and Antimicrobial Resistance of Enterobacteriaceae Infectionsin an Intensive Care Unit ........................................................................................................................... 18
Axente C, Baditoiu L, Muntean D, Horhat F, Bedreag O, Hogea E, Moldovan R, Licker M

Binge Drinking Reality in Relation to Aspects of Sexual Behavior among Students ............................... 23
Bagiu I, Tuţă-Sas I, Băcean Miloicov C, Vlaicu B, Bagiu R

Furin-Cleavable Insulin Expressed in Human Mesenchymal Stem Cells as Diabetes Therapy ............... 28
Gavriliuc OI, Bojin FM, Paunescu V

The Involvement of Enterococcus Spp. Strains in the Etiology of Infections in Hospitalized Patients ...... 35
Pepelea LS, Pandrea SL, Simon L, Matroş L, Şerban OI, Berinde L, Junie LM, Ciontea MI, Chiorean E,
Muntean D, Licker M

The Role of BMI and Co-Morbidities in Hospitalization Length Stay in Surgical Repair
of Incisional Hernia ....................................................................................................................................... 42
Noditi G, Lazar C, Mateescu T, Brebu D, Lazar F, Duta C

GABAergic Potentiation of Reduced Bowel Movements Induced with Zinc Sulphate by Baclofen ........ 47
Rufus Ojo Akomolafe, Olusoji Adeola Adalumo, Kayode Dominion Samuel Bamitale

Biodistribution of Fe3O4/Citric Acid Nanoparticles in a Murine Model ....................................................... 54
Trincu NF, Balșeanu TA, Ungureanu BS, Pirici I, Buteica SA, Săftoiu A, Neamțu J

Body Composition Assessment in Patients with Chronic Obstructive Pulmonary Disease .................... 59
Voica SA, Oancea C, Crisan AF, Tudorache E, Fira-Mladinescu O, Tudorache V

The Effects of Two Lipid-Lowering Drugs on Mitochondrial Respiration In H9C2 Cell Line .................... 66
Scurtu I, Popescu R, Privistirescu A, Sturza A, Pavel IZ, Verdeș D, Duicu OM, Muntean DM

Pulmonary Embolism: To Be or Not To Be ................................................................................................... 70Crişan S, Nicola AR, Valcovici M, Matei O, Lazar MA, Gaşpar M, Petrescu L, Luca CT

Fiziologia - Physiology • 2015.25.4(88)3CUPRINS
Rolul polimorfismului PNPLA3 I148M în bolile cronice hepatice ................................................................. 5
Enache EL, Minca DM, Enache LS

Incidența și sensibilitatea la antibiotice a microorganismelor izolate de la nivelul tractului
genital feminin la paciente din ambulator ................................................................................................... 11
Simon LM, Pepelea L, Junie LM, Licker M

Etiologia, frecvența și rezistența la antibiotice a enterobacteriilor din infecții ale pacienților
într-o secție de terapie intensive ................................................................................................................. 18
Axente C, Baditoiu L, Muntean D, Horhat F, Bedreag O, Hogea E, Moldovan R, Licker M

Abuzul de alcool în relație cu aspectele comportamentului sexual în rândul studenților ........................ 23
Bagiu I, Tuţă-Sas I, Băcean Miloicov C, Vlaicu B, Bagiu ROpțiuni terapeutice în diabet: insulina clivabilă de furină și exprimată
în celulele stem mezenchimale .................................................................................................................... 28
Gavriliuc OI, Bojin FM, Paunescu V

Implicarea tulpinilor de Enterococcus spp. în etiologia infecţiilor la pacienţi spitalizaţi ......................... 35
Pepelea LS, Pandrea SL, Simon L, Matroş L, Şerban OI, Berinde L, Junie LM, Ciontea MI, Chiorean E,
Muntean D, Licker M

Rolul IMC si al co-morbiditatilor asupra duratei de spitalizare in cazul repararii chirurgicale
a eventratiilor postoperatorii ........................................................................................................................ 42
Noditi G, Lazar C, Mateescu T, Brebu D, Lazar F, Duta C

Potențarea GABAergică a reducerii mișcărilor intestinale cu sulfat de zinc de către Baclofen .............. 47
Rufus Ojo Akomolafe, Olusoji Adeola Adalumo, Kayode Dominion Samuel Bamitale

Biodistribuția nanoparticulelor de Fe3O4/acid citric într-un model murin .................................................. 54
Trincu NF, Balșeanu TA, Ungureanu BS, Pirici I, Buteica SA, Săftoiu A, Neamțu J

Măsurarea compoziției corporale la pacienții cu BPOC .............................................................................. 59
Voica SA, Oancea C, Crisan AF, Tudorache E, Fira-Mladinescu O, Tudorache V

Efectele a două medicamente hipolopemiante asupra respirației mitocondriale
la nivelul liniei celulare H9C2 ....................................................................................................................... 66
Scurtu I, Popescu R, Privistirescu A, Sturza A, Pavel IZ, Verdeș D, Duicu OM, Muntean DM

Embolismul pulmonar: A fi sau a nu fi .......................................................................................................... 70Crişan S, Nicola AR, Valcovici M, Matei O, Lazar MA, Gaşpar M, Petrescu L, Luca CT

4

Fiziologia - Physiology • 2015.25.4(88)ROLE OF PNPLA3 I148M VARIANT IN CHRONICLIVER DISEASEELENA L. ENACHE1, DELIA M. MINCA2, LIVIU S. ENACHE3*
1University of Medicine and Pharmacy Tirgu Mures, Romania

2Emergency Clinical County Hospital “Pius Brinzeu” Timisoara, Romania
3Emergency Clinical Hospital Tirgu Mures, Romania

ABSTRACTPatatin-like phospholipase domain containing 3 (PNPLA3) is an enzyme preferentially expressed in the liver, with dual
anabolic and catabolic roles in lipid metabolism. A single nucleotide polymorphism in PNPLA3 gene (rs738409 C>G),
leading to the substitution of isoleucine with methionine in position 148 of the protein, was recently described as a key
determinant of liver fat content. Variant I148M was associated with the development of the full spectrum of liver injury
related to lipid accumulation, from simple steatosis to steatohepatitis. Subsequent studies established a strong link
between PNPLA3 I148M variant and liver fibrosis progression towards advanced stages, cirrhosis development, clinicaloutcome in chronic hepatitis C, and hepatic carcinogenesis. PNPLA3 rs738409 polymorphism represents a valuablegenetic predictor and a potential therapeutic target in liver disease. In this review, we briefly summarize the main
hypotheses on the cellular functions of PNPLA3 and the role of its I148M variant as a risk factor in the progression ofmajor forms of liver disease.
Keywords: Patatin-like phospholipase domain containing 3 (PNPLA3), liver disease, fibrosis, fatty liver disease,hepatocellular carcinoma, genetic risk factor

INTRODUCTION
Patatin-like phospholipase domain containing 3(PNPLA3), also known as adiponutrin, is a member of thepatatin-like phospholipase family. It shares a common
domain with patatin, a major protein of potato with
non-specific lipid acyl hydrolase activity [1].
Although its in vivo function is still subject to debate, asingle variant in PNPLA3 (rs738409) was recently
associated with liver fat content in individuals of various
ethnicities [1]. This variant soon emerged as a key
genetic determinant of advanced injury in various
etiologies of liver disease, such as chronic viral hepatitis,
alcoholic or non-alcoholic liver disease, and hepatocellular
carcinoma (HCC).
In this review, we briefly summarize the main
hypotheses on the cellular functions of PNPLA3 and therole of its sequence variation as a risk factor in the
progression of major forms of liver disease.
Role of PNPLA3 in lipid metabolism
In humans, the PNPLA3 gene is localized on the longarm of chromosome 22 and encodes for a transmembrane
polypeptide chain of 481 amino acids. Among the 9 patatin

domain-containing proteins encoded by our genome, thestructure of PNPLA3 is most closely related to that of thesecond member of the family (PNPLA2), which is thetriacylglycerol hydrolase of the adipose tissue [2]. Earlyattempts to characterize its biochemical function showedthat recombinant PNPLA3 protein expressed in Sf9 cells
had predominantly acyl glycerol hydrolase activity [2]. The
enzyme was active on the three major classes ofglycerolipids(triacylglycerol,
diacylglycerol,andmonoacylglycerol), with a preference for oleic acid as thefatty acid moiety. The non-synonymous C>G polymorphismrs738409 leads to the substitution of an isoleucine with amethionine residue in codon 148. The longer side chain ofmethionine at this position impairs substrate access to thecatalytic serine of the enzyme's active site, leading to asignificant decrease of glycerolipid hydrolase activity of theprotein [2]. These results were supported by anindependent study employing a different expression system
for the PNPLA3 [3]. The loss of function of the variant
protein implied by these results may explain the higher liver
fat content observed in carriers of the I148M form. However,in vivo studies failed to support the loss-of-function theory,
as mice deficient in PNPLA3 did not develop fatty liverdisease or metabolic syndrome [4,5].

Received 12th of September 2015. Accepted 10th of October 2015. Address for correspondence: Liviu S. Enache, Emergency
Clinical Hospital Tirgu Mures, Medical Analyses Laboratory, 3rd Internal Medicine Clinic, 35, Revolutiei st., Tirgu Mures,Romania;
e-mail: enachesliviu@yahoo.com

Fiziologia - Physiology • 2015.25.4(88)5Another group of researchers described an anabolic
function of PNPLA3, which was found to convert
lysophosphatidic acid into phosphatidic acid and thus
promote cellular lipid synthesis [6]. The lysophosphatidic
acid acyl transferase (LPAAT) activity of the I148M variantof PNPLA3 was higher than that of the wild-type protein,both in vitro and in vivo. Conversely, adiponutrin-deficient
mice showed decreased hepatic LPAAT activity, but normal
triglyceride (TG) hydrolase function. This gain-of-function
theory is further supported by the observation that chronic
overexpression of PNPLA3 I148M in mouse liver results indevelopment of hepatic steatosis [7]. In this model, thevariant enzyme seems to exert three effects on liver TG
metabolism: increased synthesis of fatty acids and TG,
reduced TG hydrolysis, and relative depletion of TG
long-chain polyunsaturated fatty acids.
PNPLA3 expression regulation suggests that the
enzyme has a more important role in lipid anabolism
than in catabolic processes [8]. PNPLA3 levels arehighly controlled by nutritional stimuli [9]. This regulation
is modulated via transcription factors such as liver X
receptor (LXR) and sterol response element binding
factor 1 (SREBF1) [10]. During fasting, the low levels of
SREBF1 entrain a decrease in PNPLA3 transcription
and an efficient degradation of the protein, resulting invery low levels of PNPLA3 in the liver. When glicemia
increases, insulin induces SREBF1 which, in turn,upregulates PNPLA3 expression and increases fatty
acid biosynthesis. Thus, PNPLA3 is induced when theliver synthesizes TG and includes them into lipid droplets[10]. Using an animal model, it was also shown thatPNPLA3 regulation in the adipose tissue mirrors that in
the liver [9].
A unifying hypothesis on PNPLA3 function wasrecently proposed [8]. Accordingly, adiponutrin may play
dual roles, in both the biosynthesis and hydrolysis of lipids,
depending on various factors related to the nutritional
environment, such as sources of lipids, or to possible
interactions with as yet unidentified cofactors.
PNPLA3 and liver steatosis
PNPLA3 polymorphism rs738409 was first identified ina genome-wide association study as an independent
factor associated with liver fat content [1]. Interestingly, it
was not linked with major changes in biochemical markers
of glucose or lipid metabolism. No association wasobserved between PNPLA3 allele G and BMI, fasting
serum glucose, insulin or homeostatic model assessmentof insulin resistance (HOMA-IR). It was also not
associated with lipid profile markers such as plasma
triglycerides, total cholesterol, high-density-lipoproteincholesterol or low-density-lipoprotein cholesterol.
Several subsequent studies assessed the associationof PNPLA3 polymorphism with liver fat accumulation in its
various forms of increasing severity: from simple steatosis6

to NAFLD and NASH. Data from the meta-analysis
performed by Sookoian and Pirola [11] provides
suggestive evidence that rs738409 is a strong modifier of
NAFLD natural history. Among individuals without
steatosis of secondary causes, including alcohol abuse,chronic hepatitis B or C, total parenteral nutrition, and useof steatosis-inducing drugs, carriers of GG homozygote
genotype had a liver fat content with about 70% higher
than CC homozygotes. GG homozygotes also had morethan three-fold greater risk of higher necro-inflammatory
scores and fibrosis when compared with CC genotype
individuals. NASH was also more frequent in patients with
GG genotype. rs738409 polymorphism seems to provide
an additive genetic risk for NAFLD development.
Interestingly, the meta-analysis revealed a negative
association between the effect of PNPLA3 polymorphismon liver fat content and male gender. This implies thatPNPLA3 I148M variant more effectively increases the riskof NAFLD in females than in males. It is possible that themodulation of lipogenic genes such as SREBP1c by
estrogens be responsible for this sexual dimorphism [11].
A more recent meta-analysis by Xu et al. [12] did not
find a significant association between the rs738409
polymorphism and simple steatosis, but showed the same
link between PNPLA3 and the risk of NAFLD and NASH.
PNPLA3 and HCV infection
Liver steatosis accompanies CHC in up to two thirds ofcases [13] and is favored by a series of factors including
alcohol consumption, overweight/ obesity, hyperglycemia,
diabetes and viral genotype 3 [14]. The presence ofsteatosis contributes to histological progression in CHC
and is independently associated with advanced fibrosis[13]. Liver steatosis is also an independent negative
predictor for response to interferon-based antiviral therapy
in CHC [15,16].
There is increasing evidence that cellular lipids, andparticularly lipid droplets (LDs), play a key role in the
production of infectious HCV particles [17]. The
recruitment of HCV replication complexes induces theaposition of LDs and endoplasmic reticulum-derived
membranes, generating a lipid membranous complex
wherein virion assembly can proceed [18].
Impairment of HCV core protein binding to LDs
sharply decreases the formation of viral replication
complexes and the production of infectious virions [19]. It
is still not clear whether the cytosolic LDs are recruited tothe endoplasmic reticulum where virus assembly takes
place or if they serve themselves as a platform for the
initiation of assembly [20]. However, a fraction of HCV
particles circulating in vivo in infected patients areassociated with triglycerides and ApoB, and theirformation seems to be linked to the process of VLDL
assembly [20-22]. These less dense particles also exhibit
higher infectivity [23].
Fiziologia - Physiology • 2015.25.4(88)PNPLA3 is a protein tightly associated with LDs andcontributes to their formation [24,25]. The I148M form
contributes to intracellular lipid accumulation andincreases LDs size compared with the wild-type protein[26], a phenomenon compatible with the observation thattransgenic mice overexpressing PNPLA3 I148M develp a
NAFLD-like phenotype [24]. Triglyceride accumulation
may promote HCV replication [27]. On the other hand,
PNPLA3 influences VLDL secretion from hepatocytes,
both in humans and in vitro, and the I148M motation
results in a loss-of-function [25]. Thus, carriers of PNPLA3
allele G have a lower VLDL secretion than CC
homozygotes for the same amount of liver fat and it was
proposed that the PNPLA3 variant may reduce lipidationof nascent VLDL [25]. In this context, we might expect
PNPLA3 I148M to negatively affect secretion of lipidated,
more infectious, HCV particles.
It is not clear so far whether PNPLA3 or its variant
I148M affect HCV life cycle. It is however well documented
that PNPLA3 I148M influences the clinical course ofchronic hepatitis C.
Besides the decrease of TG hydrolase and increased
LPAAT activities, impairment of TG export fromhepatocytes is another mechanism by which PNPLA3
I148M variant promotes intracellular fat accumulation [25].
In vivo, genotype rs738409 GG of PNPLA3 contributes as
an independent risk factor for the development of liver
steatosis and advanced injury in CHC patients of various
ethnic origins. Thus, in Caucasian CHC patients from
Belgium, Germany and France, adiponutrin genotype GG
was associated with steatosis, fibrosis stage, and fibrosis
progression [28]. In a Swiss cohort, the same genetic
marker was linked with a high risk of steatosis in patients
infected with HCV genotype non-3 [29]. In Italian CHC
patients, rs738409 GG was a risk factor for steatosis,
fibrosis stage, cirrhosis and poor response to antiviraltreatment and HCC development [30]. PNPLA3 genotype
was also associated with the presence of steatosis,
severe necro-inflammation and advanced fibrosis inJapanese HCV carriers [31].
Further on, PNPLA3 genotype seems to interact with
IL-28B genotype, a well-known predictor of response toantiviral therapy [32], in the development of CHC-related
hepatic steatosis. In this regard, PNPLA3 allele G favoredsteatosis in patients carrying IL-28B genotypes CT or TT,
but not CC [33]. Valenti et al. showed that PNPLA3 GG
patients infected with HCV genotype non-3, also carrying
IL-28B CC genotype, were protected against steatosis[34].
Despite the strong association of PNPLA3 I148M
variant with processes that favor HCV-related diseaseprogression, such as lipid biosynthesis, lipid droplet
formation and liver steatosis, it has a negligible influenceon viral clearance achievement following antiviraltreatment in unselected CHC patients [35,36]. However,
PNPLA3 GG may negatively affect sustained viral
Fiziologia - Physiology • 2015.25.4(88)response rates in difficult-to-cure CHC patients infectedwith viral genotype 1 or 4 and presenting advanced
pretreatment fibrosis [36].
PNPLA3 genotype GG is an important risk factor for
CHC disease progression. A suggestive association is theincreasing prevalence of this genotype among patientswith increasing disease severity: 4% in CHC without
steatosis, 17% in CHC with steatosis, and 24% in HCC(30). PNPLA3 allele G is linked with a higher risk of clinicaldeterioration, defined as the development of ascites,
hepatic encephalopathy, variceal hemorrhage, HCC, orliver-related death in patients with CHC-related cirrhosis[37]. The association remains significant even in age, sex,
and race-adjusted analysis.
PNPLA3 and liver fibrosis
The effect of rs738409 genotype on liver fibrosis
severity was assessed in a recent meta-analysis
summarizing 24 studies and 9915 patients [38]. The
included studies were mostly performed in Europe and in
the US. There was no significant heterogeneity amongstudies. Advanced fibrosis was significantly associatedwith PNPLA3 genotype, in the dominant as well as in therecessive genetic models. In the recessive model, which is
the most commonly used, the GG carrier status was morestrongly associated with advanced fibrosis than CG and
GG genotypes. The effect of PNPLA3 genotype on fibrosis
stage was similar among studies conducted in the US,
Europe and elsewhere. GG genotype had also similareffect on fibrosis in patients with various etiologies of liverdisease, including NAFLD, ALD and CHC [38]. The same
effect of rs738409 was observed in Asian populations. In
Japanese patients with CHC, the rs738409 GG genotype
was associated with advanced fibrosis, as well as with thepresence of steatosis and severe necroinflammatory
activity [31].
The I148M variant of PNPLA3 was also associatedwith increased risk of cirrhosis in patients with underlying
liver disease, genotype GG carriers having a more than
three-fold risk of cirrhosis than CC individuals [39].
Another meta-analysis underlined a clear association of
rs738409 G allele with alcoholic liver cirrhosis, in an
additive genetic model for this variant [40].
Interestingly, PNPLA3 genotype seems to interact withage at infection in determining fibrosis progression in CHC
patients [41]. Thus, PNPLA3 GG genotype increased
fibrosis progression rates in individuals infected at older
age. Additionally, PNPLA3 impact on fibrosis progression
was more pronounced in patients at higher risk of alteredhepatic lipid metabolism, such as overweight patients,
with grade 2-3 steatosis and infected with HCV genotype 3[41]. A stronger effect of PNPLA3 genotype on CHC
severity in the presence of additional metabolic
disturbances is also supported by the observation that GG
genotype diabetic patients had an almost 9-fold risk
7

increase for the presence of advanced fibrosis comparedwith non-diabetics [42].
It is known that hepatic stellate cells (HSCs) play amajor role in the development of liver fibrosis [43]. A novel
link between PNPLA3 variant, HSCs and the genetic
predisposition to chronic liver disease was recently
proposed by Pirazzi et al. [44]. The authors showed thatPNPLA3 is highly expressed in human HSCs. PNPLA3
expression in primary HSCs was regulated by retinol
availability and insulin. Thus, PNPLA3 was downregulated
when retinol was abundant, whereas retinol deficiency
resulted in an increased expression of the protein.
Moreover, PNPLA3 was able to hydrolyze retinyl palmitate
and allow the extracellular release of retinol. The retinyl
esterase activity of PNPLA3 was restricted to HSCs andnot observable in hepatocytes. The I148M variant of the
enzyme lacked the retinyl-esterase activity and promoted
retinol retention within HSCs, in parralel with decreased
circulating levels of retinol and retinol binding protein 4.
This in turn may interact with chronic liver injury [44], sinceloss of retinyl esters-containing LDs was suggested todiminish hepatic fibrogenesis and carcinogenesis [45].
PNPLA3 and risk of hepatocellular carcinoma
HCC is the fifth most frequent form of cancer and thethird cause of cancer-related death worldwide [46]. In
Europe and the United States, the rise of HCC incidence
was mainly related to CHC cases.
In a meta-analysis including nine independent studies,
2937 patients were assessed with respect to the risk ofdeveloping HCC related to their rs738409 genotype [38].
With the exception of one study conducted in Japan, all
the others were performed in Europe, with Caucasianpatients. Depending on the genetic model, there was asignificant risk of HCC development associated with
rs738409 alleles. In the recessive model, GG genotype
carriers were significantly more likely to develop HCC than
CG and CC carriers. PNPLA3 was also independently
associated with the risk of HCC in a subset of 6 studies
including patients with liver cirrhosis. In the dominantmodel, CG and GG patients had higher risk of HCC
development than CC patients. These results suggest thatthe role of PNPLA3 variant in liver pathology is not limited
to development of steatosis and NAFLD, but it also
promotes fibrosis progression towards advanced stagesand HCC development. Interestingly, the association of
PNPLA3 genotype with HCC was not observed in thesubgroup analysis including HCV-related cirrhosis
patients [38]. Similar results were provided by a separate
meta-analysis with individual patient data including 2503
participants with cirrhosis, which found a tight association
between rs738409 and overall HCC [47]. The strength ofassociation between allele G and HCC was high in ALD
patients and less evident in CHC-related cirrhosis. In a
French and Belgian cohort of cirrhotic patients, rs738409
8

genotypes did not influence the risk of HCC development
or death in HCV-infected individuals, whereas genotype
GG independently predicted the risk of HCC in
ALD-related cirrhosis [48].
In a different cohort from UK, the risk of HCC
development conferred by PNPLA3 rs738409 GG
genotype in NAFLD patients was 5 times higher than CC
genotype in participants with the same underlying liverdisease, and 12 times higher than CC genotype in thegeneral population [49].
PNPLA3 genotype GG predicts HCC occurrence at an
earlier stage in ALD and NAFLD and is a negative
predictor for patient survival, being associated with morediffuse HCC at presentation [50].
It
wassuggested
that,
when
hepatic
microenvironment is altered by steatosis/ steatohepatitis,the liver may be predisposed to cancer even in theabsence of cirrhosis. Several mechanisms have been
proposed to explain the procarcinogenic effect of the
I148M variant [51]: intensified lipogenesis and availabilityof fatty acids as energy source for rapidly proliferating
cells, altered adipokine secretion influencing
microinflammation and insulin resistance, low-level
hepatic inflammation promoting release of tumor necrosis
factor alpha and interleukin-6, lipotoxicity altering
intracellular signaling, mitochondrial damage and oxiative
stress due to lipid peroxidation.
CONCLUSION
In conclusion, adiponutrin is a new key determinantof liver disease progression. Future studies are required toestablish the cellular roles of PNPLA3 and themechanisms driving its involvement in liver pathology.
Hopefully, further understanding of the pathophysiologicalrole of PNPLA3 and its variant in the development liver
steatosis, fibrosis and carcinogenesis, will enable thediscovery of new therapeutic targets. Also, genetic testingof PNPLA3 polymorphism may be included in diagnostic
protocols meant to identify individuals at risk of aggressive
forms of liver disease and to allow a better personalizationof clinical monitoring and treatment of these patients.
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ROLUL POLIMORFISMULUI PNPLA3 I148M IN BOLILE CRONICE HEPATICEREZUMATPNPLA3 este o enzima exprimata preferential la nivelul ficatului, avand rol atat in anabolismul, cat si incatabolismul lipidelor. Un polimorfism punctiform la nivelul genei PNPLA3 (rs738409 C>G), care are drept
consecinta substitutia isoleucinei cu metionina in pozitia 148 a lantului polipeptidic, a fost descris recent drept un
determinant esential al continutului hepatic de lipide. Varianta I148M a fost asociata cu dezvoltarea intregului
spectru de afectare hepatica asociata acumularii de lipide, de la steatoza simpla la steatohepatita. Studii
ulterioare au evidentiat o asociere stransa dintre varianta PNPLA3 I148M si progresia fibrozei hepatice catrestadii avansate, dezvoltarea cirozei, evolutia clinica in hepatita cronica de tip C si carcinogeneza hepatica.
Polimorfismul PNPLA3 rs738409 reprezinta un factor genetic predictiv important si o potentiala tinta terapeutica
in patologia hepatica. In acest review, vom trece in revista principalele ipoteze actuale asupra functiilor celulare
ale PNPLA3 si rolul variantei I148M ca factor de risc in progresia bolilor hepatice cronice.
Cuvinte-cheie: PNPLA3, afectiune hepatica, fibroza, steatoza, cancer hepatic, factor de risc genetic

10

Fiziologia - Physiology • 2015.25.4(88)INCIDENCE AND ANTIBIOTIC SUSCEPTIBILITY
OF MICROORGANISMS ISOLATED FROM FEMALE
GENITAL TRACT FROM OUTPATIENTSLAURA M. SIMON1, LIA PEPELEA1, LIA M. JUNIE1, MONICA LICKER2
1Microbiology Department, UMF ’’Iuliu Haţieganu”, Cluj-Napoca, Romania.
2 “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania.

ABSTRACTThe aim of the study was to determine the etiology of genital infections and antibiotic sensitivity of strains of Mycoplasma
hominis (M. hominis) and Ureaplasma urealyticum (U. urealyticum) isolated from outpatients. Matherials and Methods:
The study included 130 women with clinical diagnosis of genital infection, aged between 18 and 67 years. Data werecollected from the medical records of patients who presented to laboratory Intermed Service Lab Cluj-Napoca during theperiod February- July 2015. We used A.F. GENITAL SYSTEM, a 24-well system containing dehydrated biochemical andantibiotic substrates for presumptive identification and susceptibility testing of bacteria from genital secretions. Results:
The distribution percentage of the infection of the lower genital tract was: 61% vaginosis (79 patients), 9.48% candidosis(12 patients), candidosis + vaginosis 18.9% (25 patients). The distribution of Gram-positive bacteria isolated was: E.
faecalis- 83%, S. agalactiae- 15% and S. aureus -2%. E. coli was the most frequently isolated Gram-negative bacteria,
followed by G. vaginalis - 43.28%, N. gonorrhoeae - 4.47%, Pseudomonas aeruginosa 2.98% and Proteus spp. 1.5%.
We diagnosed 37 patients (28.45%) with candidosis. M. hominis and U. urealyticum were isolated from 36,92% of
patients. Conclusions: Age group 21-30 presented most genital infections. From the total number of genital infections,bacterial vaginosis had the highest prevalence. E. faecalis was the most frequently isolated Gram-positive bacteria andE. coli the most commonly isolated Gram-negative bacteria. U. urealyticum was the most commonly isolated bacteria
from the infections with Mycoplasma spp and U. urealyticum. All M. hominis strains were sensitive to Tetracycline,
Pefloxacin, Ofloxacin, Doxycycline, Minocycline and Clindamycin.
Keywords: female genital tract, outpatients, Mycoplasma hominis, Ureaplasma urealyticum

INTRODUCTION
In recent years the incidence of local or systemic
complications of asymptomatic female genital tract
infections increased significantly [3,4,8,9]. The possibilityof simultaneously contamination with two or moresexually transmitted microorganisms and the increased
percent of chronicization requires an early diagnosis anda specific treatement [11].
The increased frequency of genital infections withobstetric and gynecologic complications and antibiotic
resistance of isolated bacteria was the motivation of thisstudy.
The aim of the study was to determine the etiology ofgenital infections and antibiotic sensitivity of strains ofMycoplasma hominis (M. hominis) and Ureaplasma
urealyticum (U. urealyticum) isolated from outpatients.

The study had the following objectives: identificationof bacterial agents, antibiotic susceptibility testing of M.
hominis strains and U. urealyticum and the resultsinterpretation by comparing them with data from theliterature.

MATERIALS AND METHODS
To achieve the objectives an observational,
retrospective study on a representative sample wascarried.
The study included 130 women with clinical diagnosis
of genital infection, aged between 18 and 67 years. Data
were collected from the medical records of patients who
presented to laboratory Intermed Service Lab
Cluj-Napoca during the period February- July 2015.

Received 15th of September 2015. Accepted 20th of October 2015. Address for correspondence: Laura M Simon, Microbiology
Department, UMF ’’Iuliu Haţieganu” Cluj-Napoca, 6th Pasteur Street, RO-400349, Cluj-Napoca, Romania; phone/fax:+40264597257, e-mail: lauramihaelasimon@yahoo.com

Fiziologia - Physiology • 2015.25.4(88)11The method used was A.F. GENITAL SYSTEM, a24-well system containing dehydrated biochemical andantibiotic substrates for presumptive identification andsusceptibility testing of bacteria from genital secretions.
Collection of pathological products. The
pathological products were vaginal or cervix secretions
collected with a steril synthetic fibre swab. We used
Stuart and Amies mediums incorporated into sterile tubes
attached to sterile fiber swabs. The samples were sent
for inoculation in the A.F. GENITAL SYSTEM
immediately after they were taken.
A.F. GENITAL SYSTEM allows the detection,
semi-quantitative count, presumptive identification andsusceptibility test of M. hominis and U. urealyticum, thedetection and presumptive identification of the
microorganisms most frequently isolated from vaginal andurethral swabs and seminal fluid, such as: Escherichia coli,
Proteus spp./Providencia spp., Pseudomonas spp.,
Gardnerella vaginalis, Staphylococcus aureus (S. aureus),Enterococcus
faecalis,
Neisseriagonorrhoeae,
Streptococcus agalactiae (Group B) (S. agalactiae) andCandida spp.The tests were interpreted after color
changing in different wells and microscopic examination.
The presence of Candida spp. was determined by
microscope (40x) examination of a drop of culture liquidassessing the presence of chlamydospores and hyphae.
The presence of S. aureus was confirmed by the
coagulase test. The presence of S. agalactiae wasconfirmed with agglutination test STREPTO B latex KIT.
Every batch of A.F. GENITAL SYSTEM was
subjected to quality control using the following
microorganisms as referrence: M. hominis ATCC 23114,
U. urealyticum ATCC 27618, Gardnerella vaginalis ATCC
14018, S. aureus ATCC 25923,
Streptococcus
faecalis
ATCC 19433, C. albicans ATCC 10231,
Escherichia coli ATCC 5922, Proteus mirabilis ATCC
25933, Neisseria gonorrhoeae ATCC 19424, S.
agalactiae ATCC 13813, Ps. aeruginosa ATCC 27853.

Fig. 1. M. hominis negative, U. urealyticum negative, Trichomonas
vaginalis negative, Escherichia coli negative, Proteus spp. negative,
Pseudomonas spp. negative, Gardnerella vaginalis negative, S.
aureus negative, Enterococcus faecalis negative, Neisseria
gonorrhoeae negative, S.agalactiae negative and Candida spp
pozitive.
12

Fig. 2. M. hominis negative, U. urealyticum negative,
Trichomonas vaginalis negative, Escherichia coli negative,
Proteus spp. negative, Pseudomonas spp. negative, Gardnerellavaginalis pozitive, S. aureus negative, Enterococcus faecalis
negative, Neisseria gonorrhoeae negative, S. agalactiae andCandida spp. negative.

Statistical analysis
The data collected were placed in an Excel databasewhich was then processed to obtain statistical results.
The database was created as spreadsheets using
Microsoft Office 2013, depending on variables pursued.
Individual tables were created for each variable that wasmonitored. Based on the tables the current graphics weregenerated.
The following statistical softwares: Epi Info™, G*Power
Win_3.1.9.2 and R for Windows 3.2.0 were used. The
sample size and statistical parameters: Se, Sp, PPV, NPV,P were computed using the mentioned tools.
The size of representative sample was determined by
the prevalence of genital tract infections in the general
population.
The null hypothesis of the study: no association
between genital tract infections and isolated bacteria.
Alternative hypothesis of the study: there isassociation between genital tract infections and isolated
bacteria.
Factors that may invalidate the results were poor
inoculum standardization, inadequate materials, A.F.
GENITAL SYSTEM expired, improper temperatures andincubation times.
Data obtained by using statistical tools had thefollowing percentage distributions: by patients group age,for isolated Gram-positive bacteria, for isolated
Gram-negative bacteria, for C. albicans strains, for M.
hominis and U. urealyticum strains, for M. hominis
sensitivity to antibiotics, for U. urealyticum sensitivity toantibiotics, the most frequent bacteria associated with U.
urealyticum, the most frequent bacteria associated with
M. hominis, the most frequent bacteria associated with E.
faecalis.
Fiziologia - Physiology • 2015.25.4(88)RESULTS
Based on the data collected Table I was compiled:
Table I. Contingency Table
Test result
Test +
Test Total

Infection present
114
2
116

No infection
2
12
14

The distribution percentage of the infection of thelower genital tract as shown in Figure 4 was: 61%
vaginosis (79 patients), 9.48% candidosis (12 patients),
candidosis + vaginosis 18.9% (25 patients), andno-infection- 10.34% (14 patients).

Total
116
14
130

Diagnostic test performanceHi-square test was applied and p < 0.005 was
obtained, rejecting the null hypothesis and accepting thealternative hypothesis. The test is very highly significant
since p-value < 0.001 (with 95% confidence interval).
The following statistical parameters were calculated:
1. Sensitivity (Se) = 0.983; is the proportion ofpatients correctly identified as positive subjects
2. The specificity (Sp) = 0.857; is the proportion ofurged subjects correctly identified as negative
3. Prevalence (P) = 0.89; the number of cases of
illness at a given time relative to total
population.
4. Positive predictive value (PPV) = 0.983; It is theprobability of a subject with a positive test to
have the disease.
5. The negative predictive value (NPV) = 0.857; isthe probability of a subject with a negative test
to not have the disease.
Due to Se and Sp values the test is highly sensitiveand highly specific. Values are close to 100% which
implies a high diagnostic quality.
Age patients included in the study was the age groupas shown in Figure 3.
From the 130 patients included in the study, 8% (11patients) were aged between 18-20 years, 58% (75patients) were aged between 21-30 years, 21% (27patients) were aged between 31-40 years and 13% (17patients) were aged over 40 years.

Fig. 3. Percentage distribution of patients by age

Fiziologia - Physiology • 2015.25.4(88)Fig. 4. The percentage distribution of lower genital tract
infections

The distribution of Gram-positive bacteria isolated
was: E. faecalis- 83%, S. agalactiae- 15% and S. aureus-2% (Figure 5).

Fig. 5. The percentage distribution of isolated
Gram-positive bacteria

E. coli (37 strains) was the most frequently isolated at
47.77% Gram-negative bacteria, G. vaginalis had apercentage of 43.28% (34 strains), N. gonorrhoeae had ashare of 4.47% (8 strains), Pseudomonas aeruginosa-2.98% (7 strains) and Proteus spp. -1.5% (6 strains)(Figure 6).

Fig. 6. The percentage distribution of Gram-negative
bacteria (Legend: Ec=E. coli, Gv=G. vaginalis, Ng=N.
gonorrhoeae, Psa=P. aeruginosa, P=Proteus)13

We diagnosed 37 patients (28.45%) with candidosis(Figure 7).

A percentage of 73.68% (31 strains) of U.
Urealyticum (42 strains) were sensitive to Tetracycline,
Pefloxacin, Doxycycline, Clindamycin, 76.31% (32strains) were susceptible to Ofloxacin, and Minocycline,
65.78% (28 strains) were sensitive to Clarithromycin and55.26% (23 strains) were susceptible to Josamycin andErythromycin (Figure 10).

Fig. 7. Percentage distribution of strains of C. albicans

From 130 patients, M. hominis and U. urealyticum
were isolated from 48 patients (36,92%), as shown in
Figure 8. We isolated U. urealyticum from 42 patients(32.3%), M. hominis from 3 patients (2,3%). The
combination of the two bacteria occurred at 3 patients(2,3%).

Fig. 10. Antibiotic susceptibility of U. urealyticum strains(Legend: T=Tetracycline, Pef=Pefloxacin, D=Doxycycline,
Cm=Clindamycin, Mc=Minocycline, Ofl=Ofloxacin,
Crm=Clarithromycin, Jos= Josamycin)

In the co-infection with M. hominis and U.
urealyticum, 50% of strains were susceptible toTetracycline, Pefloxacin, Doxycycline, Erythromycin,
Minocycline, Josamycin, Clindamycin and all strains weresusceptible to Ofloxacin and Clarithromycin (Figure 11).

Fig. 8. Percentage distribution of M. hominis andU. urealyticum strains

All strains of M. hominis (3 strains) were sensitive to
Tetracycline,
Pefloxacin,
Ofloxacin,
Doxycycline,
Minocycline, Clindamycin and 2 strains (66.66%) weresusceptible to Erythromycin, Clarithromycin and Josamycin(Figure 9).
Fig. 11. Graphical representation of antibiotic susceptibility of
M. hominis and U. urealyticum strains (Legend: T=Tetracycline,
Pef=Pefloxacin, D=Doxycycline, E=Erythromycin,
C=Clindamycin, Jos=Josamycin, Mc=Minocycline,
Ofl=Ofloxacin, Crm=Clarithromycin)

Fig. 9. Antibiotic susceptibility of M. hominis strains (Legend:
T=Tetracycline, Pef=Pefloxacin, Ofl=Ofloxacin, D=Doxycycline,
Mc=Minocycline, Cm=Clindamycin, E=Erythromycin,
Crm=Clarithromycin, Jos= Josamycin)

14

In Figure 12 is shown the percentage associationof U. urealyticum strains with other bacteria. 7 strainsof U. urealyticum (15%) were associated with G.
vaginalis, 24 strains (60.5%) have been associatedwith E. faecalis, 4 strains (6%) with S. agalactiae, anda percentage of 18.5% (9 strains) have been
associated with C. albicans.

Fiziologia - Physiology • 2015.25.4(88)Fig. 12. Percentage distribution of U. urealyticum strains
associated with other microorganisms

M. hominis strains have been associated in a
proportion of 50% with G. vaginalis, 25% with E. faecalisand 25% were associated with C. albicans (Figure 13).

Fig. 13. Percentage distribution of M. hominis strains
associated with other microorganisms

From 68 isolated strains of E. faecalis, 16 wereassociated with G. vaginalis (23.52%), 14 with C.
albicans (20.58%), 23 strains with E. coli (33.82%), and
15 strains (22.05%) weren’t associated with any bacteria(Figure 14).

Fig. 14. Percentage distribution of E. faecalis strains associatedwith other microorganisms

DISCUSSION
A study in Brazil on 100 sexually active women
watching the prevalence and risk factors for bacterial
vaginosis and other vulvovaginitis in sexually active
Fiziologia - Physiology • 2015.25.4(88)women showed that the prevalence of bacterial vaginosis
was 20%, the prevalence of candidosis was 22% andtheir coinfection was 2% [7].
In our study, out of 130 women aged between 18 andover 40 years, the prevalence of bacterial vaginosis was
61.02%, the prevalence of candidosis was 9.4% and theprevalence of bacterial vaginosis associated withcandidosis was 18.90%.
A survey conducted in China highlighted theprevalence of genital tract infections with M. hominis andU. urealyticum for women in outpatient. From a total of6051 women a percentage of 31% accounted for
infections with Ureaplasma and 0.7% was the infection
with
Mycoplasma [13].
Atefeh M. et al conducted a study in 2013 in Iran to
diagnose genital infections with Mycoplasma in infertile
women. Out of 104 patients, 39 (37.5%) were diagnosed
with U. urealyticum, 3 patients (2.9%) with M. hominis
and the coinfection was 3.8% [1].
In a study conducted in Poland on a sample of 161
women (60 fertile and 101 infertile), the results revealed
a number of 9 infertile women (9%) and 5 fertile females(8%) infected with U. urealyticum. The presence of M.
hominis has been identified only in four infertile women ina proportion of 4%.
In our study on 130 women aged between 18 andover 40 years, the prevalence of U. urealyticum and M.
hominis was 36,92%, also higher for U. urealyticum.
Another study that revealed U. urealyticum
sensitivity, which was 94.6% for: Doxycycline,
Tetracycline, Minocycline; 69.2% for: Azithromycin,
Erythromycin, Clarithromycin, Roxithromycin. M. hominis
was 100% sensitive to Doxycycline, Tetracycline,
Minocycline and 95.6% to Azithromycin, Erythromycin,
Clarithromycin, Roxithromycin [13].
In our study antibiotic sensitivity of U. urealyticum
was: 73.68% to Tetracycline, Pefloxacin, Doxycycline,
Clindamycin, 76.31% to Ofloxacin, and Minocycline,
65.78% to Clarithromycin and 55.26% to Josamycin andErythromycin. All strains of M. hominis strains weresensitive to Tetracycline, Pefloxacin, Ofloxacin,
Doxycycline, Minocycline, Clindamycin and 2 strains(66.66%)
were susceptible
to Erythromycin,
Clarithromycin and Josamycin.
In previous studies in Hungary between 2008 - 2011
on a sample of 4466 patients aged 21 - 60 years showed
the incidence and sensitivity of genital mycoplasmas. U.
urealyticum incidence was 8.5% and 0.91% for M.
hominis. U. urealyticum was sensitive to Tetracycline(95.9%), Doxycycline (97.32%) and Azithromycin(85.79%) and resistant to Erythromycin (81.23%),
Clindamycin (75.06%), Ofloxacin (25.2%). M. hominis
was sensitive to Clindamycin, Ofloxacin, Doxycycline(95%) and Tetracycline (82.9%) [10].
Studies available have shown the prevalence ofsexually transmitted infections in 296 women aged15

16-49. N. gonorrhoeae infection prevalence was 5.1%
(15 women) [6].
Out of 130 cases examined, our results highlighted
that N. gonorrhoeae was isolated in 4.47% of cases.
In Portland, out of 215 women surveyed, 147(68%) showed no infection, 41 (19.1%) were infectedwith candidosis, and 27 (12.6%) were diagnosed with
infectious vaginitis. 6 patients (22.8%) diagnosed with
infectious vaginitis had S. agalactiae as etiologic agent[5].
Our results revealed the candidosis prevalence with
9.4% and S. agalactiae prevalence from isolated
Gram-positive bacteria was 15%.
Earlier work identified that from 200 women withsymptoms of lower genital infection, bacterial vaginosis
prevalence was 51.5%, and G. vaginalis was isolated in
8.7% of cases of vaginosis [2].
Our results showed prevalence of bacterial vaginosis
at 61.02% and 43.28% prevalence of G. vaginalis fromisolated Gram-negative bacteria.
Another study showed the prevalence of
microorganisms in the vaginal secretion of fertile andinfertile women. 952 patients were included over a periodof 2 years who have carried out tests on the vaginal
discharge for: G. vaginalis, T. vaginalis, Candida, S.
agalactiae, M. hominis, U. urealyticum and C.
trachomatis. The most common organisms identified in
fertile women was 12.1% Candida, 26.6% G. vaginalis
and S. agalactiae at 9.2% [12].
The most common microorganisms isolated from ourstudy were: 26.15% G. vaginalis, 28.45% Candida and S.
agalactiae at 12.76%.

CONCLUSIONS
‐
‐
‐
‐

‐
‐
‐

16

A.F GENITAL SYSTEM test has a high diagnostic
quality and is highly sensitive and highly specificdue to Se amd Sp being close to 100%;
Age group 21-30 presented most genital infections;
From the total number of genital infections, bacterial
vaginosis had the highest prevalence;Enterococcus faecalis was the most frequently
isolated Gram-positive bacteria and Escherichia coli
the most commonly isolated Gram-negative
bacteria;
U. urealyticum was the most commonly isolated
bacteria in the infections with Mycoplasma spp andU. urealyticum;
All M. hominis strains were sensitive to Tetracycline,
Pefloxacin, Ofloxacin, Doxycycline, Minocycline andClindamycin;
The incidence of infections wih M. hominis and U.
urealyticum coincides with the incidence reported in
the literature;

‐

‐

‐

‐

The asymptomatic pathology determined by
Mycoplasmas
causeslong-term
healthconsequences, pose serious issues for individualand couple health;
It is necessary to develop guidelines for diagnosis
and treatment of non-specific genital infections
guided by their incidence in our population, as well
the current profile of sensitivity to antibiotics;
This study, through the given limits, draws attention
to some etiological, diagnostic, treatment andprevention aspects of non-specific genital infections
in Romanian female population;
Developing an effective prevention program incompliance with the rules of sexual health andscreening actions are particularly important.

REFERENCES
1. Atefeh M, Fariba F, Reza M, Rashid R. Detection of genital
mycoplasmal infections among infertile females by multiplex
PCR. IJM, 2014; 398-403.
2. Baruah F, Sharma A, Das C, Hazarika N, Hussain J. Role ofGardnerella vaginalis as an etiological agent of bacterial
vaginosis. IJM, 2014; 6(6): 409-414.
3. Carter J, Beck D, Williams H, Foster J, Dozier G. GA-Based
Selection of Vaginal Microbiome Features Associated with
Bacterial Vaginosis. Genet Evol Comput Conf. 2014; 2014: 265-8.
4. Djukić S, Opavski N, Mijač V, Ranin L. Current knowledge ofbacterial vaginosis. Srp Arh Celok Lek. 2011; 139(5-6): 402-8.
5. Leclair M, Hart A, Goetsch M, Carpentier H, Jensen J. Group B
streptococcus: prevalence in a non-obstetric population. NIH
Public Access, 2010; 14(3): 162-6.
6. Marks M, Kako H, Butches R, Lauri B, Puiahi E, Pitakaka R, etal. Prevalence of sexually transmitted infections in female clinic
attendees in Honiara, Solomon Islands. BMJ Open, 2015; 5:1.
7. Moreira R, Machado S, Silva B, Pimentel R, Ferreira T,
Leoni F, et al. Prevalence and risk factors for bacterial
vaginosis and other vulvovaginitis in a population of sexuallyactive adolescent from Salvador, Bahia, Brazil. Hindawi
Publishing Corporation, 2012: 1-6.
8. Nelson DB, Bellamy S, Nachamkin I, Ruffin A, Allen-Taylor
L, Friedenberg FK. Characteristics and pregnancy outcomes ofpregnant women asymptomatic for bacterial vaginosis. Matern
Child Health J 2008; 12(2): 216-2.
9. Ness RB, Hillier SL, Kip KE, Soper DE, Stamm CA,
McGregor JA, Bass DC, Sweet RL, Rice P, Richter HE.
Bacterial vaginosis and risk of pelvic inflammatory disease.
Obstet Gynecol. 2004; 104(4): 761-769.
10. Ponyai K, Mihalik N, Ostorhazi E, Farkas B, Parducz L,
Marschalko M, et al. Incidence and antibiotic susceptibility ofgenital mycoplasmas in sexually active individuals in Hungary.
Clin Microbiol Infect Dis, 2013; 32(11): 1423-6.
11. Spandorfer SD, Neuer A, Giraldo PC, Rosenwaks Z,
Witkin SS. Relationship of abnormal vaginal flora,
proinflammatory cytokines and idiopathic infertility in women
undergoing IVF. J Reprod Med. 2001; 46(9): 806-810.
12. Sujit R, Klausner J, Krupp K, Reingold A, Madhivanan P.
Epidemiologi features of vulvovaginal candidiasis among

Fiziologia - Physiology • 2015.25.4(88)reproductive-age women in India. Infect Dis Obstet Gynecol,
2012: 1-8.
13. Wang QY, Li RH, Zheng LQ, Shang XH. Prevalence andantimicrobial susceptibility of Ureaplasma urealyticum andMycoplasma hominis in female outpatients, 2009-2013.
Microbiol Immunol Infect., 2014; S1684-1182(14)00118-2.

INCIDENTA SI SENSIBILITATEA LA ANTIBIOTICE A MICROORGANISMELORIZOLATE DE LA NIVELUL TRACTULUI GENITAL FEMININ LA PACIENTE DINAMBULATORREZUMAT
Scopul acestui studiu a fost de a determina etiologia infectiilor genitale si sensibilitate la antibiotice a tulpinilor de
Mycoplasma hominis (M. hominis) si Ureaplasma urealyticum (U. urealyticum) izolate de la paciente din
ambulator. Material si Metoda: Am luat in studiu 130 femei avind diagnostic clinic de infectii genitale, cu varsta
cuprinsa intre 18 si 67 ani. Datele s-au colectat din buletinele de analiza ale pacientelor care s-au prezentat laIntermed Service Lab Cluj-Napoca în perioada februarie – iulie 2015. Am utilizat A.F GENITAL SYSTEM, care
reprezintă un sistem de 24 de godeuri conținând substraturi biochimice deshidratate și antibiotice, cu scopul
identificării prezumtive și testării sensibilității bacteriilor izolate din secrețiile genitale. Rezultate: Repartiția
procentuală a infecțiilor de tract genital inferior a fost: 61% vaginoză (79 paciente), 9.48% candidoză (12paciente), candidoză + vaginoză 18.9% (25 paciente). Repartiția procentuală a bacteriilor Gram-pozitive a fost: E.
faecalis - 83%, S. agalactiae 15%, iar S. aureus- 2%. E. coli a fost izolată la 47.77% din paciente (37 tulpini), G.
vaginalis la 43.28% (34 tulpini), N. gonorrhoeae la 4.47% (8 tulpini), Pseudomonas aeruginosa la 2.98% (7tulpini) iar Proteus spp. la 1.5% (6 tulpini). 37 de paciente (28.45%) au fost diagnosticate cu candidoză. M.
hominis și U. urealyticum au fost izolate la 36,92% din paciente. Concluzii: Grupa de vârstă 21-30 ani a prezentat
cele mai multe infecții genitale. Din totalul infecțiilor genitale, vaginoza bacteriană a avut prevalența cea mai
mare. E. faecalis a fost cel mai frecvent izolata bacterie Gram-pozitiva iar E. coli a fost cel mai frecvent
izolata bacterie Gram-negativa. Dintre tulpinile de M. hominis și U. urealyticum, cea mai frecvent izolata a fost U.
urealyticum. Toate tulpinile de M. hominis au fost sensibile la Tetraciclină, Pefloxacină, Ofloxacină, Doxiciclină,
Minociclină, Clindamicină.
Cuvinte cheie: tractului genital feminin, ambulator, Mycoplasma hominis, Ureaplasma urealyticum

Fiziologia - Physiology • 2015.25.4(88)17ETIOLOGY, FREQUENCY AND ANTIMICROBIAL RESISTANCE
OF ENTEROBACTERIACEAE INFECTIONS IN AN INTENSIVECARE UNIT
AXENTE CARMEN1, BADITOIU LUMINITA 1,2, MUNTEAN DELIA1,
HORHAT FLORIN 1,3, BEDREAG OVIDIU 1,3, HOGEA ELENA 1,3,
MOLDOVAN ROXANA1,2, LICKER MONICA 1,3
1Timisoara “Victor Babes” University of Medicine and Pharmacy
2Timisoara Regional Centre for Public Health

3Timisoara Emergency Clinical County Hospital

ABSTRACTInfections produced by Gram-negative bacilli continue to be of key importance in the ICU, as they are considered
important determinants of hospital mortality, prolonged hospitalization and increased health-care costs as compared to
infections caused by nonresistant bacteria.
We investigated antimicrobial resistance and bacterial distribution of Enterobacteriaceae isolated from the ICU of theTimisoara Emergency Clinical County Hospital (TECCH), Romania. The purpose of this study is to provide up-to-date
information on the frequency, microbial etiology, and susceptibilities of Enterobacteriaceae infections, with the goal of
improving their management.
The study group was represented by patients who received antibiotic treatment in the ICU and from whom
enterobacteria were isolated in the collected specimens. From 1 January 2012 to 31 December 2013 a total of 1,605
patients were included in our study group. Our study revealed that 43.08% of the infections in the ICU, are produced byEnterobacteriaceae, and the 3 most frequently isolated agents of this family are Klebsiella pneumoniae, Proteus
mirabilis and Escherichia coli.
We recorded low percents of beta-lactam sensitive enterobacteria. K. pneumoniae and P. mirabilis strains were more
frequently classified in the ESBL phenotype, while in E. coli strains HPAZA was the most frequently identified
phenotype. E. coli strains were more sensitive to aminoglycosides as compared to K. pneumoniae and P. mirabilis.
Although differences regarding the antimicrobial resistance patterns of K. pneumoniae, P. mirabilis and E.coli infections
have been identified, high resistance to most commonly used antibiotics has been found.

INTRODUCTION
Thanks to the progress in medical care and theimprovement in patient treatment over the last decade,effective treatment is now possible for many Intensive
Care Unit (ICU) patients who previously could have been
lost [1,2]. Antibiotics have contributed greatly to
improvements in health, but, in addition to this, theirirrational use led to the emergence of antimicrobial
resistant microorganisms [3-5]Antimicrobial resistant microorganisms mainly
develop in ICU’s, and this seems due to massive
antibiotic use [1,3]. It is alarming that a limited number ofantimicrobials retain some potency against theseincreasingly resistant pathogens and novel therapies
currently in the developmental pipeline appear to be
years away from the wide scale clinical use [6-8].
Infections produced by Gram-negative bacilli (i.e.
Escherichia coli, Klebsiella pneumoniae etc.) continue tobe of key importance in the ICU, as they are considered
important determinants of hospital mortality, prolonged
hospitalization and increased health-care costs ascompared to infections caused by nonresistant bacteria[6-9].
We investigated antimicrobial resistance andbacterial distribution of Enterobacteriaceae isolated fromthe ICU of the Timisoara Emergency Clinical County
Hospital (TECCH), Romania.

Received 14th of September 2015. Accepted 24th of October 2015. Address for correspondence: Licker Monica, Microbiology
Department, UMF ’’Victor Babes” Timisoara, Eftimie Murgu Sq. 2A, RO-300041, Timisoara, Romania; phone: +40 722 890 774,
e-mail: lickermonica@yahoo.com

18

Fiziologia - Physiology • 2015.25.4(88)The purpose of this study is to provide up-to-date
information on the frequency, microbial etiology, andsusceptibilities of Enterobacteriaceae infections, with thegoal of improving their management.

MATERIALS AND METHODS
Data were collected in the ICU of the Timisoara
Emergency Clinical County Hospital (TECCH), Romania.
The study data were recorded for 24 months, between
01.01.2012 and 31.12.2013, for all ICU admitted patients
who received antibiotic treatment.
For each patient we recorded: age, gender,
admission diagnosis, underlying surgical pathology,
presence of invasive procedures, infection sites, isolated
germs, susceptibility pattern, type, dose and length ofantibiotic therapy, days of ICU stay and ICU status after
discharge. Microbiology results were obtained from theMicrobiology Laboratory of the hospital.
Cultures were performed based on the clinical statusof the patient. All patients with clinical signs of infection
received empirical antimicrobial therapy. After culture
results were available, antibiotic treatments were either
maintained or adapted according to the results ofstandard sensitivity testing.
All isolated microbial strains were identified using theVitek 2 Compact automated system (bioMérieux).
The anitimcrobial sensitivity of germs isolated incollected specimens was determined by measuring theMIC (minimal inhibitory concentration), with further
classification into resistance phenotypes by use of the
Vitek 2 Compact system, based on the CLSI standard(Clinical Laboratory and Standards Institute Inc.).
Copy strains - defined as an isolate with the same
susceptibility pattern throughout a 1-month period in thesame patient – were excluded.
Institutional review board approval was obtained
before study initiation.
Statistical analysis methodology
The statistical analysis of the database was
performed using the SPSS version 10.0. Continuous
numerical variables characterized by mean value andstandard deviation were tested regarding the type of data
distribution using the Kolmogorov-Smirnov test.
Numerical variables with normal distribution werecompared using the unpaired t test, those with
non-Gaussian distribution were compared using the
nonparametric Mann-Whitney test, and dichotomic
variables characterized by percent values werecompared using the chi-squared test with Fisher
correction. All statistical tests were calculated with twoends, and the p value for statistical significance was set
at values ≤ 0.05.
Fiziologia - Physiology • 2015.25.4(88)RESULTS
The study group is represented by patients who
received antibiotic treatment in the ICU and from whom
enterobacteria were isolated in the collected specimens.
From 1 January 2012 to 31 December 2013 a total of
1,605 patients were included in our study group.
The mean patient age was 60.23, 42.39% of patientswere female, and 57.61% were male. The mean length of
ICU stay was 17.32 days and the mean length ofantibiotic therapy was 12.21 days.
The strains were cultured from bronchial aspirate(238 isolates, 41.90%), urine (111 isolates, 19.54%),
blood (67 isolates, 11.79%), wound secretion (55 isolates,
9.68%), catheters (40 isolates, 7.04%), peritoneal fluid(19 isolates, 3.34%), pus (17 isolates, 2.99%), CSF (6isolates, 1.05%), pleural fluid (5 isolates, 0.88%), nasal
exudate (3 isolates, 0.53%), conjunctival secretion (2isolates, 0.35%), sputum (2 isolates, 0.35%), faeces (1isolate, 0.17%), urethral secretion (1 isolate, 0.17%) andvaginal secretion (1 isolate, 0.17%). Enterobacteria wereisolated in 568 biological specimens.

Fig. 1. Distribution of bacterial load depending on the type of
biological specimen

As shown in Figure 1, the majority of infections weremonomicrobial but in certain samples collected fromurethral and vaginal infection sites (with a very low
prevalence in the present study) two microorganisms
were isolated in the same specimen.

Fig. 2. Incidence of circulating Enterobacteriaceae species in the ICU
during the period between 01.01.2012-31.12.201319

During the studied period, our study revealed that 43.08%of the infections in the intensive care unit, are produced byEnterobacteriaceae, and the 3 most frequently isolated agentsof this family are Klebsiella pneumoniae, Proteus mirabilis andEscherichia coli (Figure 2).

Fig. 3. Distribution of Enterobacteriaceae species in biological
specimens

Enterobacteriaceae species isolated in our study arevariably distributed in biological specimens as shown in
Figure 3. Thus, the most frequently involved in lower
respiratory tract infections are: K. pneumoniae, P.
mirabilis, E. coli, Proteus spp, P. stuartii; in urinary tract
infections the most frequently involved are: E. coli, K.
pneumoniae, P. mirabilis, Proteus spp., Enterobactercolacae, Serratia marcescens; in blood stream infections
K. pneumoniae, P. mirabilis, E. coli, P. stuartii wereisolated in higher percents.
Infections were defined according to the International
Sepsis Forum Consensus Conference on Definitions ofInfections in the Intensive Care Unit (10). The organism
associated with infection was defined by the isolation of
the germ in a biological material in the presence of signs
and symptoms of infection.

Moreover, ICU mortality of the study group was very
high (61.70%), and distribution in hospital departments isshown in the above figure, as is the evolution of cases
upon ICU discharge, also depending on the originating
hospital department.

Fig. 5. Distribution of the most prevalent strains according to
the resistance phenotype
Legend: ESBL - extended spectrum beta-lactamase, CAZA cephalosporinase, CHN - high level cephalosporinase, LPAZA low level penicilinase, HPAZA - high level penicilinase, SXT –sulfamethoxazole and trimethoprim

We recorded low percentage of beta-lactam sensitiveenterobacteria. K. pneumoniae and P. mirabilis strains
were more frequently classified in the ESBL phenotype,while in E. coli strains HPAZA was the most frequently
identified phenotype.
E. coli strains were moresensitive to aminoglycosides as compared to K.
pneumoniae and P. mirabilis. Fluoroquinolone sensitivity
was similar for E. coli and P. mirabilis, a lower percent
being recorded in K. pneumoniae strains. Regarding
trimethoprim-sulfamethoxazole, sensitive strains of E.coli
and K.pneumoniae exceeded the number of resistantstrains. Tetracycline sensitivity was maintained in most
tested enterobacteria.

DISCUSSION

Fig. 4. Fatality rates in infectious with Enterobacteriaceae
depending on originating hospital department

20

Searching for the means to understand, control, andprevent the emergence and spread of infections andantimicrobial resistance has become a public healthpriority. The prevention, control, and treatment of ICU
infections demand thorough knowledge of the infection
incidence and infection site rates, the occurrence rates oforganisms and their antimicrobial resistance profiles [11].
Since each hospital unit has a distinct bacteriological
profile and antibiotic resistance pattern, knowledge ofthese differences is critical for planning effective therapy
and reducing infection-related costs, morbidity, andmortality [11,12].

Fiziologia - Physiology • 2015.25.4(88)A large European single-day point prevalence studyof
nosocomial
ICU
infections,
reported
Enterobacteriaceae as most common (34.4%), with
Pseudomonas second most common (28.7%) [12].
Resistance in Gram negative bacilli, including
Escherichia coli, Klebsiella spp, is of great concern,
because a number of reports have documented
mechanisms whereby these microorganisms havebecome resistant to all available antibacterial agentsused in therapy [13,14].
Enteriobacteriaceae are the most common
Gram-negative isolates in laboratories. They can beisolated from numerous sites, the majority from: urine,
blood, peritoneal cavity, respiratory tract but also fromother sites like cerebrospinal fluid, synovial fluid,
abscesses [15,16].
This ICU is also a surgical ICU, and in this type of
department the use of antibiotics not only for therapy but
also for prophylaxis contributes to an increase in total
antibiotic consumption, but also to the selective pressure
by antibiotics, and to the pharmaceutical budget [15,17].
The objective of the present prospective
observational study was the identification and analysis of
the demographic, clinical, and microbiologicalcharacteristics of the patients who receive antimicrobial
treatment and from whom microorganisms of theEnterobacteriaceae family have been isolated during
their hospitalization in ICU of our hospital. In particular,
we evaluated the occurrence rates of the pathogens andtheir susceptibility to antibiotics.
The top three specimen types collected in thepresent study, similarly to several studies in the literature,[11,12] were sputum, urine and blood. It is emphasizedthat Gram negative bacilli are mainly responsible forpneumonia, urinary tract infection and septicaemia in thestudied ICU.
The microbiological profile of infections included K.
pneumoniae, P. mirabilis, E. coli, Proteus spp,
Providencia stuartii, Enterobacter cloacae and Serratia
marcescens as the most frequently isolated species;
similar results have also been found in other studies[11,18]. K. pneumoniae was the most common, while P.
mirabilis and E. coli were second and third mostfrequently identified pathogens in our study population,
respectively.
Although differences regarding the antimicrobial
resistance patterns of K. pneumoniae, P. mirabilis andE.coli infections have been identified, high resistance tomost commonly used antibiotics has been found inseveral studies [11,16].
E.
coli
and
Klebsiellacanproduceextended-spectrum beta-lactamases (ESBLs). ESBLs
are widely prevalent in Latin America, with rates as highas 32.6% for K. pneumoniae and 11.8% for E. coli in the
ICU [11,19]. The present study showed a prevalence of
ESBLs-production ranging from 56.72%-49.70% in
Fiziologia - Physiology • 2015.25.4(88)Klebsiella pneumoniae and Proteus mirabilis. The highprevalence of ESBLs-producing isolates described in thisstudy was probably due to the large amount of
third-generation cephalosporins consumed by ICU
patients.
Significant differences with regard to infection rates,
the rates of occurrence of organisms, infection sites
incidence, and antimicrobial resistance profiles havebeen identified between different countries, between
centres in the same country, and even between thedepartments of a hospital [11]. There is also a big
difference in antimicrobial resistance rates between
different European countries, as shown by the European
Antimicrobial Surveillance System (EARRS) [5].
In conclusion, infections are a very important problem
in our ICU, associated with high incidence, mortality, andmulti-drug resistance of the responsible microorganisms.
Eradication requires implementation of rigorous infection
control measures, prudent antibiotic use, and effective
antimicrobial therapy. Recognition of clinical andmicrobiological characteristics of these patients isessential for prevention and treatment purposes.

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Pharmacoeconomic study of antibiotics used in the treatment oflower respiratory tract infections in ICU patients: A case study inan Egyptian hospital. Bulletin of Faculty of Pharmacy, Cairo
University 2014; 52: 135-144.
7. Sedláková MH, Urbánek K, Vojtová V, Suchánková H,
Imwensi P, Kolář M. Antibiotic consumption and its influence on
the resistance in Enterobacteriaceae. BMC Research Notes
2014; 7: 454.
8. Villegas MV, Briceno DF, Ruiz SJ, Furtado GH, Nicolau DP.
Assessing the pharmacodynamics profile of intravenous
antibiotics against prevalent Gram-negative organisms
collected in Colombia. Braz J Infect Dis 2011; 15(5): 413-419.

21

9. Jing Sun, Li-Fen Hu, Min Wang, Wei Shi, Xi-Hai Xu, Jun
Cheng, et al. Clinical investigation for infections caused byEnterobacteriaceae in intensive care unit of Anhui, China. BrazJ Infect Dis. 2012; 16(1):109-110.
10. Calandra T, Cohen J. The international sepsis forum
consensus conference on definitions of infection in the intensive
care unit. Crit Care Med. 2005; 33(7): 1538-48.
11. Markogiannakis H, Pachylaki N, Samara E, Kalderi M,
Minettou M, Toutouza M, Toutouzas KG, Theodorou D,
Katsatagakis S. Infections in a surgical intensive care unit of auniversity hospital in Greece. Int J Infect Dis, 2009; 13:
145-153.
12. Sligl W, Taylor G, Brindley PG. Five years of nosocomial
Gram-negative bacteremia in a general intensive care unit:
epidemiology, antimicrobial susceptibility patterns, andoutcomes. Int J Infect Dis, 2006; 10: 320-325.
13. Xu J, Zhang W, Chen Q, Zhou Q. Surveillance ofAntimicrobial Resistence and Bacterial Distribution of Gram
Negative Bacilli Isolated from Intensive Care Unit in the First
Bethune Hospital in 2009. Procedia Environmental Sciences,
2012; 12: 1375-1378.
14. Anago E, Ayi-Fanou L, Akpovi CD, Hounkpe WB,
Agassounon-Djikpo Tchibozo M, Bankole HS, et al. Antibiotic
resistance and genotype of beta-lactamase producing
Escherichia coli in nosocomial infections in Cotonou, Benin.
Ann Clin Microbiol Antimicrob. 2015; 14:5.

15. Bennett JE, Dolin R, Baser MJ, et al. Mandell, Douglas,
and Bennett’s Principles and Practice of Infectious Diseases 8 thEd., 2015. ISBN: 978-1-4557-4801-3.
16. Wollheim C, Guerra IM F, Conte VD, Hoffman SP,
Schreiner FJ, Delamare APL, et al. Nosocomial and community
infections due to class A extended-spectrum b-lactamase(ESBlA)-producing Escherichia coli and Klebsiella spp. insouthern Brazil. Braz J Infect Dis, 2011; 15(2):138-143.
17. Meyer E, Schwab F, Pollitt A, Bettolo W,
Schroeren-Boersch B, Traumann M. Impact of a Change in
Antibiotic Prophylaxis on Total Antibiotic Use in a Surgical
Intensive Care Unit. Infection 2010; 38:19-24.
18. Osthoff M, McGuinness SL, Wagen AZ, Eisen DP. Urinarytract
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79-83.
19. Villegas MV, Correa A, Perez F, Miranda MC, Zuluaga T,
Quinn JP. Colombian Nosocomial Resistance Study Group.
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2004; 49: 217-22.

ETIOLOGIA, FRECVENTA SI REZISTENTA LA ANTIBIOTICEA ENTEROBACTERIILOR DIN INFECTII ALE PACIENTILOR INTR-O SECTIE DETERAPIE INTENSIVAREZUMAT
Infecţiile produse de bacilii Gram-negativi sunt frecvente în secţiile de terapie intensivă (TI), fiind considerate
importanti factori de risc în cresterea mortalitatii, spitalizarea prelungită, dar şi în creşterea costurilor, în
comparaţie cu infecţiile produse de bacterii non-rezistente.
In cadrul acestui studiu am investigat rezistenţa microbiană şi distribuţia bacteriilor aparţinând familiei
Enterobacteriaceae, în secţia de TI a Spitalului Judeţean din Timişoara (SCJUT), România. Scopul acestui studiu
a fost determinarea frecvenţei, etiologiei microbiene şi a sensibilităţii la antibiotice a enterobacteriilor identificate,in vederea îmbunătăţirii managementului acestor infecţii.
Grupul de studiu a fost reprezentat de pacienţii care au primit tratament antibiotic pe durata spitalizării în secţia
de TI şi cărora le-au fost izolate enterobacterii. Au fost incluşi în studiu 1,605 pacineţi internaţi pe perioada 1
Ianuarie 2012 - 31 Decembrie 2013.
S-a observant că 43,08% dintre infecţii au fost produse de bacterii aparţinând familiei Enterobacteriaceae, iarcele mai frecvent izolate bacterii aparţinând acestei familii au fost: Klebsiella pneumoniae, Proteus mirabilis şi
Escherichia coli.
S-au identificat procente scăzute ale sensibilităţii enterobacteriilor la beta-lactamine. Tulpinile de K. pneumoniaeşi P. mirabilis au apartinut cel mai frecvent fenotipului BLSE, iar cele de E. coli fenotipului HPAZA. Tulpinile de E.
coli au fost mai sensibile la aminoglicozide decât cele de K. pneumoniae şi P. mirabilis. Deşi există diferenţe între
fenotipurile de rezistenţa ale celor mai izolate trei tipuri de enterobacterii (K. pneumoniae, P. mirabilis şi E.coli ) ingeneral se observă un grad mare de rezistenţă la antibioticele cel mai frecvent utilizate.

22

Fiziologia - Physiology • 2015.25.4(88)BINGE DRINKING REALITY IN RELATION TO ASPECTS
OF SEXUAL BEHAVIOUR AMONG STUDENTS
BAGIU I, TUŢĂ-SAS I, BĂCEAN MILOICOV C, VLAICU B, BAGIU R
University of Medicine and Pharmacy "Victor Babeș" Timișoara

ABSTRACTThe study aims was to investigate some aspects of sexual behavior associated with binge drinking among students
from Timis County, Romania. We applied a transversal population study to the representative sample of 2076 students,
62.49% girls and 37.51% boys with a mean age of 21.09 years with SD 1.48. The boys practice binge drinking
significantly more often than girls, the difference is small to medium: prevalence of 22.1% for boys and 8.7% girls, 1-2
times in the last month. Girls forced into sexual relation practiced binge drinking significantly more often compared tothose who were not forced. The frequency of practicing binge drinking is significantly higher in students who have sex
with partners of one night. The frequency of practicing binge drinking is significantly higher in students who consumed
alcohol intercourse average size of the gap is small to medium in boys and girls.
Keywords: students, binge drinking, sexual behavior

INTRODUCTION

MATERIAL AND METHOD

Alcohol is one of the most commonly used
substances [1]. The quantities of alcohol consumed andthe health problems due to the alcohol abuse are specificto each age group. Characteristic for the young adults is
the increased alcohol consumption in one occasion binge drinking, intoxication, drunk driving and assault [2].
Compared with adults, young people consume lessalcohol, but per occasion, quantities consumed by young
adults exceed those consumed by adults [3].
Alcohol plays an important role in sexual risk
behavior, including unwanted, unintended andunprotected sexual activity, also sex with multiple
partners. All of these behaviors increase the chance of
unwanted pregnancies and contacting sexuallytransmitted diseases including HIV infection [4]. In
pregnant women, alcohol consumption leads to thetermination of the evolving pregnancy, miscarriage,
physical and / or mental fetal malformations [2].
In adolescents and young people, alcohol
consumption increases the risk of inadequate physicaland sexual assault [5], and lifetime alcohol abuse.
We proposed a study on manifestations of sexual
behavior (sexual assault, sex with one night partners,
alcohol associated intercourse) associated with binge
drinking among students.

The representative sample of students in the study
totaled 2076 students from higher education institutions
from Timiș county, 62.49% (1296) girls and 37.51% (778)boys. The average age was 21.09 years with SD 1.48,
ages ranging from 18-25 years old.
The method used was the transversal population
study based on the use of CORT Questionnaire 2004 on
health risk behaviors in adolescents and young people.
The questionnaire was validated by the Ethics
Committee of the University of Medicine and Pharmacy"Victor Babeș" Timişoara.
The study was conducted with the written approval of
the higher education establishments from Timiş.
Inclusion of young people in the study carried out
only after their expressed consent of each participant in
the study, with respect for all the individual rights.
The processing and interpretation of data was doneby using modern statistical methods and advanced
medical PASW 18 software (SPSS 18) 2010. The valueof statistical significance was set at p <0.05, except incases where the Bonferroni correction was applied, theacceptable threshold level was stated in the text. For
ordinal data comparisons we used Mann-Whitney andKruskal test-Wallis. Chi-square test was used for ordinal
data / nominal.

Received 15th of September 2015. Accepted 20th of October 2015. Address for correspondence: Bagiu Iulia, MD, UMF “Victor
Babes” Timisoara, Eftimie Murgu Sq. 2A, RO-300041, Timisoara, Romania; phone/fax: +40256-490412; +40256-492101; e-mail:bagiuradu@yahoo.com

Fiziologia - Physiology • 2015.25.4(88)23RESULTS
1. Binge drinking
By practicing binge-drinking or increased
consumption of more than 5 servings of alcohol on one
occasion, we have: for boys group 11.6% (89) havepracticed binge drinking more than 3 times in the lastmonth, 22.1% (170) have practiced 1-2 times, and 66.3%(510) did not practice binge drinking in the last month;
2.5% for the group of girls (32) have practiced binge
drinking more than 3 times in the last month, 8.7% (112)
have practiced 1-2 times, and 88.8% (1145) have notpracticed binge drinking last month. The frequency ofpracticing binge drinking is significantly higher in boys
than girls, U = 381 799, z = -12.63, p <0.001, r = 0.27,
the size difference is considered small to medium
according to the Cohen's criteria (Figure 1).

Fig. 1. Percentage distribution of students according tofrequency of binge drinking practice last month, by genderFig. 2. Percentage distribution of students according to whether
a situation when they had forced sex and frequency of binge
drinking practicing during the past month among boys

Fig. 3. Percentage distribution of students according to whether
a situation when they had forced sex and frequency of binge
drinking practicing during the past month among girls

3. Sex with one night partner
2. The situation when they had forced intercourse
A percentage of 3.0% (23) of the boys and 4.8% (62)of girls admit that they were obliged to have sexualintercourse against their will. The prevalence of forced
sexual relations is higher among girls compared to boys,
U = 484409.5, z = -2.01, p = 0.044, r = 0.044, the size
difference is very small.
The prevalence of binge drinking in boys who wereforced to have sex against their will was 40.9% (9) andthose who did not report this incident was 33.6% (249).
We could not find a relationship between the frequency of
binge drinking and the claim that they had been forced tohave sexual relations, p = 0.324 (Figure 2).
The prevalence of binge drinking among girls who
were forced to have sex against their will was 24.2% (15)and those who did not report this incident was 10.6%(129). The girls who were forced to have sex against theirwill, practiced more often binge drinking, compared withthose who were not forced, U = 32 718, z = -3.23, p =0.001, r = 0.09, the difference is small in size (Figure 3).

24

35.1% (264) of boys and 7.9% (101) of girls practiced
sex with one night partners. Compared to girls, boys
practiced 6.3 times more common, χ2 (1) = 237.03, p<0.001.
In the group of boys, 49.0% (128) of those who havesex with a one night partner, practiced binge drinking and26.3% (128) of those who did not practiced sex with onenight partners practiced binge drinking. The frequency ofpracticing binge drinking is significantly higher in boys
who have sex with one night partners, U = 48 961, z =-5.22, p <0.001, r = 0.15, the size difference is small(Figure 4).
In the group of girls, 26.7% (27) of those who havesex with a one night partner, practiced binge drinking,and 9.8% (115) of those who did not practiced sex with
one night partners practiced binge drinking . The
frequency of practicing binge drinking is significantly
higher in girls who have sex with one night partners, U =48 511, z = -6.32, p <0.001, r = 0.23, the size differenceis small (Figure 5).

Fiziologia - Physiology • 2015.25.4(88)Fig. 4. Percentage distribution of students according practicing
sex with one night partners and frequency of binge drinking
practicing during the past month among boys

Fig. 5. Percentage distribution of students according practicing
sex with one night partners and frequency of binge drinking
practicing during the past month among girls

4. The use of alcohol associated with sex
A percentage of 29.1% (219) of boys and 15.7% (200)of the girls use alcohol during sex, boys 2.21 times more
often than girls, χ2 (1) = 52.20, p <0.001.
In the group of boys, 62.6% (136) of those whopractice sex associated with alcohol consumption
practice binge drinking, and 22.6% (120) of those who do
not practice associating alcohol and sex, practice binge
drinking. The frequency of practicing binge drinking issignificantly higher in boys who consumed alcohol during
intercourse, U = 33,293, z = -10.79, p <0.001, r = 0.39,
the size difference is medium (Figure 6).
In the group of girls, 28.5% (57) of those whopractice sex associated with alcohol consumption
practice binge drinking, and 7.9% (85) of those who do
not practice associating alcohol and sex, practice binge
drinking. The frequency of practicing binge drinking issignificantly higher in girls who consumed alcohol during
intercourse, U = 84923.5, z = -8.51, p <0.001, r = 0.23,
the difference is small to medium size (Figure 7).

Fiziologia - Physiology • 2015.25.4(88)Fig. 6. Percentage distribution of students according
to the association of the use of alcohol and sexual intercourseand frequency of binge drinking practicing during the last monthamong boys

Fig. 7. Percentage distribution of students according to theassociation of the use of alcohol and sexual intercourse andfrequency of binge drinking practicing during the last monthamong girls

DISCUSSION
In a study by Boyd and colleagues [6], it was shownthat the students witch are practicing binge drinking
were associated with sexual risk behaviors and sexualassault.
Given the sensitive nature of the subject, coercion orsexual activity is hard to investigate. Studies have shown
the different ways of evaluating the forced sexual activity.
In some cases compulsion was defining in any sexual
activity that was not consensual. In other cases it was
heavily defined as rape or with men that trade money
and/or gifts for sex [7-10].
Other authors [11] found the association between
promiscuous unprotected sexual experiences anddrinking, especially binge drinking practice and the early
onset of alcohol consumption.

25

In multiple studies, young people reported early onsetof sexual activity, multiple sexual partners, one night
sexual partners, the use of psychoactive substances andsexual abuse during childhood. A relatively small
percentage said that they used condom during the last
sexual intercourse. Unprotected sex was related to no
behavioral intentions to use condoms, pregnancy, a casual
partner and people who practiced anal sex [12,13].
Alcohol consumption occurs in association with sexual
behavior in a social and cultural variety. Where young
people use alcohol before they engage in sexual relations
occur behaviors risk such as unprotected sex [14,15].
Young people are less likely to adopt safe procedures forthe sexual behavior when are drunk. The perception that
alcohol has an uninhibited effect propels some people toconsume alcohol and then to engage in behaviors that
normally would not attend. Alcohol should be recognized
as a risk factor in the transmission of HIV and othersexually transmitted infections. The synergy between
sexual behavior and alcohol consumption multiplies
enormously the negative potential consequences of thetwo separate behaviors [16,17].

CONCLUSIONSThe prevalence of binge drinking practicing 1-2 times in
the last month is 22.1% for boys and 8.7% girls, and morethan 3 times in the last month is 11.6% for boys and 2.5%
girls. The boys practice binge drinking significantly more
often than girls, the difference is small to medium size.
A percentage of 3.0% of boys and 4.8% of girls admitthat they were obliged to have sexual intercourse against
their will. The prevalence of forced sexual relations ishigher among girls compared to boys, the size differenceis very small. Girls forced into sexual activity practiced
binge drinking significantly more often compared to those
who were not forced.
35.1% of boys and 7.9% of girls practiced sex with a
one night partner, boys practicing 6.3 times more
frequent than girls. In the group of boys, 49.0% of those
who have sex with one night partners practice binge
drinking. In the group of girls, 26.7% of those who havesex with one night partners practice binge drinking. The
frequency of practicing binge drinking is significantly
higher in students who have sex with one night partners,
the difference size is small.
A percentage of 29.1% of boys and 15.7% of girls
practice the use of alcohol during sex, boys 2.21 times more
frequently than girls. The prevalence of binge drinking
between students who practice sexual activity associatedwith alcohol is 62.6% for boys and 28.5% girls. The
frequency of practicing binge drinking is significantly higher
in students who consumed alcohol during intercourse, thedifference size is medium for boys and small for girls.

26

REFERENCES
1. Department of Health and Human Services (US). The
Surgeon General's Call to Action to Prevent and reduce
underage drinking. DHHS, Office of the Surgeon General (US).
2007. Available from: URL: http://www.surgeongeneral.gov
2. Newbury-Birch D, Gilvarry E, McArdle P, et al. Impact of
Alcohol Consumption on Young People: A Review of Reviews,
Institute of Health and Society at Newcastle University, 2008.
3. Substance Abuse and Mental Health Services
Administration (US). Results from the 2005 National Survey on
Drug Use and Health: National Findings. Rockville (MD):SAMHSA, Office of Applied Studies (US); 2006 NSDUH Series
H-30, DHHS Publication No. SMA 06-4194.
4. Currie C, et al. Eds. Social Determinants of Health andwell-being among young people. Health Behaviour in
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No. 6).
5. Hingson R, Heeren T, Winter M, Wechsler H. Magnitude ofalcohol-related mortality and morbidity among US college
students ages 18-24: changes from 1998 to 2001. Annu Rev
Public Health 2005; 26: 259-79.
6. Marmorstein NO. Longitudinal Associations between
Depressive Symptoms and Problems Alcohol: Through Early
Adolescence Early Adulthood. Alcohol Clin Exp Res. 2009;33(1):49-59.
7. NACO, The National Behavioural Surveillance Surveys(Female Sex Workers and Their Clients, MSM & HDI). New
Delhi, Ministry of Health and Family Welfare, 2002.
8. Burton DL. Were adolescent sex offenders with sexual
behavior problems children? Sex Abuse, 2000; 12: 37-48.
9. Burton D. An examination of social cognitive theory withdifferences among sexually aggressive, physically aggressive
and nonaggressive children in state care. Violence and Victims,
1999; 14 (2): 161-178.
10. Friedrich WN. Psychological assessment of SEXUALLY
abused children and Their Families. Thousand Oaks, CA: Sage
Publications, 2002.
11. Clark D. The natural history of adolescent alcohol use
Disorders. Addiction. 2004; 99 (Suppl 2): 5-22.
12. Koniak-Griffin D, Lesser J, Uman G, Nyamathi A. Teenpregnancy, motherhood, and unprotected sexual activity. Res
Nurs Health 2003; 26 (1): 4-19
13. Williams PG, Holmbeck GN, Greenley RN. Adolescenthealth psychology. J Consult Clin Psychol, 2002; 70(3):828-842.
14. Achenbach TM. Manual for the Child Behavior Checklist /4-18 and 1991 Profile. Burlington: University of Vermont.
15. Saxena S. Alcohol Problems and Responses: Challenges
for India. Journal of Substance Use, 2000; 5: 62-70.
16. World Health Organization, Global status report on alcohol.
Geneva 1999.
17. World Health Organization, The Rapid Assessment andResponse Guide on Substance Use and Sexual Risk
psychoactive Behaviour, Geneva, 2000.

Fiziologia - Physiology • 2015.25.4(88)ABUZUL DE ALCOOL ÎN RELAȚIE CU ASPECTELECOMPORTAMENTULUI SEXUAL ÎN RÂNDUL STUDENȚILORREZUMAT
Acest studiu își propune investigarea unor aspecte ale comportamentului sexual asociate cu abuzul de alcool în
rândul studenților din județul Timiș, Romania. Am aplicat un studiu populațional transversal unui lot reprezentativ
de 2076 studenți, 62,49% fete și 37,51% băieți, cu vârsta medie de 21,09 ani și SD 1,48. Persoanele de sex
masculin prezintă episoade de abuz de alcool semnificativ mai frecvent decât cele de sex feminin, diferența fiindmică spre medie: prevalența 22,1% pentru băieți și 8,7% pentru fete, de 1-2 ori în ultima lună. Persoanele de sex
feminin forțate să aibă relații sexuale abuzează de alcool mai frecvent decât cele care nu au fost forțate.
Frecvența episoadelor de abuz de alcool este semnificativ mai crescută la studenții care au relații sexuale
ocazionale. Frecvența episoadelor de abuz de alcool este mai mare în rândul studenților care consumă alcool în
timpul contactului sexual, diferența dintre băieți și fete fiind mică spre medie.
Cuvinte cheie: studenți, abuzul de alcool, comportament sexual

Fiziologia - Physiology • 2015.25.4(88)27

FURIN-CLEAVABLE INSULIN EXPRESSED IN HUMANMESENCHYMAL STEM CELLS AS DIABETES THERAPYOANA I GAVRILIUC1, FLORINA M BOJIN1,2, VIRGIL PAUNESCU1,2
1Department of Immunology, „Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania
2 County Clinical Hospital „Pius Brinzeu” Timisoara, Romania

ABSTRACT
Despite recent progress in the chemically-induced transdifferentiation of human and rat MSC into insulin-producing cells,
the efficiency and clinical aplicability of factor-based conversions is still questionable. In contrast, genetically-modified
hMSCs, capable of long-term, controlled secretion of biologically-active mature insulin, represent a promising therapeuticalsolution for the treatment of diabetes. In the present study, we explored the posibility of generating plasmid vectors
carrying mutant construct human proinsulin construct, where the PC1/3 and PC2 cleavage sites had been altered bysite-directed mutagenesis to form furin-cleavable sites and which can be successfully transcribed correctly and at very highlevels, following the in vitro non-viral transfection into human mesenchymal stem cells.
Keywords: furin-cleavable insulin; mesenchymal stem cells

INTRODUCTION
Currently recognized as a major public healthconcern of both highly industrialized and developing
countries, diabetes, a metabolic disorder affecting morethan 220 million people worldwide, warrants a definitive
cure. Exogenous insulin supply, the current treatment, is
not fully capable of achieving tight control of glucose
regulation [1], while pancreatic islet transplantation is nota viable solution due to the shortage of donors [2]. As
Type 1 diabetes (T1D) is caused by the selective
autoimmune destruction of the highly specialized
insulin-secreting β-cells, it follows logically that thedisease is a good candidate for gene therapy to correct
insulin defficiency.
The possibility of ectopic insulin expression by genetransfer in a non-β-cell has been explored since 1983 [3],but further experiments have shown that simple
replacement of insulin expression is not likely to be
useful as a therapy for T1D unless it contains a system toregulate insulin levels in response to minute variations ofblood glucose. Considering the complex machinery ofstimulus-secretion coupling in β-cells, a successful genetherapy for diabetes should deliver in the target cells,
separately or in combination, at least two key elements: aregulatory system responsive to glucose concentrations,and appropriate processing of proinsulin into mature,
biologically active insulin [4]. Correct proinsulin
processing in non-β cells has been overcome by

expressing a modified single-chain insulin [5], lacking
C-peptide, which has a higher biological activity thanproinsulin, but still only about 30% of that of thenormally-occurring insulin, and does not require any
enzymatic cleaving. A different approach focused onengineering a mutated version of the proinsulin gene, inwhich the aminoacid cleavage sites recognized by
β-cell-specific proteases PC1/3 and PC2 have been
replaced with the tetrabasic sites recognized by the more
ubiquitary endoprotease furin [6].
The introduction of furin consensus sequences at the
B-chain/C- peptide and C-peptide/A-chain junctions
increased the processing of proinsulin to mature,
biologically active insulin [7-10]; however, the productionof furin-cleavable proinsulin was shown to be sensitive tothe aminoacid substitutions used [11].
Ideally, both the cause and the consequence of T1Dshould be addressed [12] in a combined therapy pairing
β-cell replacement by genetic engineering of insulin
secreting cells with protection of the newly transplanted
β-cells from the recurrence of islet-specific autoimmune
attack. Mesenchymal stem cells (MSCs), which generate
minimal immune reactivity and have anti-inflammatory[13] and immunomodulatory [14-16] effects, havebecome favourites in recently initiated or on-going clinical
trials involving the use of stem cells for the treatment ofdiabetes mellitus, matching the more comprehensive
interest in the potential of MSCs to alleviate abnormal
immune responses in graft-versus-host-disease [17],Received 10th of September 2015. Accepted 15th of October 2015. Address for correspondence: Oana Gavriliuc, PhD,
Department of Immunology, “Victor Babeș” University of Medicine and Pharmacy of Timişoara, 14 Tudor Vladimirescu st., 300173
Timisoara, RO, Tel/Fax: +40-256-490507; e-mail: gavriliuc.oana@umft.ro28

Fiziologia - Physiology • 2015.25.4(88)solid organ transplantation [18] and Crohn’s disease,among others. The precise therapeutic mechanism
through which MSCs would be able to treat T1D, either
by release of regulatory cytokines such as TGF-β orIL-10 or by cell-cycle inhibition of lymphocyte proliferation[19], has yet to be conclusively determined.
In the present study, we explored the posibility ofgenerating plasmid vectors carrying mutant constructhuman proinsulin construct, where the PC1/3 and PC2
cleavage sites had been altered by site-directedmutagenesis to form furin-cleavable sites and which canbe successfully transcribed correctly and at very highlevels, following the in vitro non-viral transfection into
human mesenchymal stem cells.

MATERIALS AND METHODS
Isolation and culture of hMSC
Human MSCs were isolated by adherence to plastic,from bone marrow aspirates obtained by sternal punction
from healthy donors. Bone marrow samples wereobtained after informed consent elaborated under anapproved protocol, according to the World Medical
Association Declaration of Helsinki. Prior to thetransfection experiments, all hMSC samples weremaintained in culture and expanded for 5-7 passages inalpha-minimum essential medium (α-MEM; Gibco BRL,
Life Technologies, 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
37°C in 5% CO2 atmosphere. Medium replacement was
performed every third day and, upon reaching 80% to
90% confluence, the cells were subcultured using 0.25%
Trypsin-EDTA solution (Sigma, St. Louis, MO, USA)
followed by centrifugation (10 minutes, 300xg) and were
replated in T75 culture flasks at a density of 10,000
cells/cm2 to ensure optimal proliferation.

TTGTACAAGAAAGCTGGGTCCTAGTTGCAGTAGTTCTCCAGCTGGTAGAG. The proinsulin CDS was further
subcloned by GatewayTM LR recombination into thedestination vectors pcDNA-DEST53 and pcDNA-DEST47(Life Technologies), which contain a neomycin selection
marker and a N-terminal or C-terminal, respectively,
Cycle3 GFP gene. The expression clones, DEST53-hIns
and
DEST47-hIns,
weretransformed
intosupercompetent OneShot OmniMAX 2 T1 E.coli andisolated with the PureLink HQ Mini Plasmid Purification
Kit (both from Life Technologies).
Mutant proinsulin construction
We analysed six furin cleavage sites with the neuralprediction network ProP 1.0 [20] and chose to reconstruct
the sites resulting in the highest probability of cleavage
after the R56 and R89 aminoacids of proinsulin. The
furin-cleavable consensus sequences were generatedusing QuikChange II Site-directed Mutagenesis Kit (Agilent
Genomics) and the plasmid expression clone
DEST-53-hIns as substrate. The mutagenesis sense
primers were as follows: for the B-chain/C-peptide junction
5'-GAACGAGGCTTCTTCTACACACCCAGGAGCAAGCGGGAGGCAGAGG-3',
and
for
the
C-peptide/A-chain
5'-TGGAGGGG
TCCCGGCAGAAGCGTGG. Mutagenesis was performedin two steps, following the manufacturer's instructions.
First, a single-mutant insulin was generated, in a plasmid
vector, by PCR amplification using the first set ofmutagenesis primers. The DpnI-treated plasmid wastransformed into supercompetent bacteria. The
single-mutant insulin plasmid was isolated and subjected
to another PCR amplification, using the second set ofprimers. The resulting double-mutant proinsulin 2M in theexpression vector DEST53-2M was isolated after
transformation into supercompetent OneShot OmniMAX
2 T1 bacteria and was back-cloned into an entry
Gateway vector, which, subsequently served forgenerating the expression vector DEST47-2M, containing
a double-mutant furin-cleavable proinsulin.

Expression plasmids
Total RNA was isolated from human pancreas using
the Absolutely RNA Miniprep Kit (Agilent Genomics, La
Jolla, CA, USA) and its concentration was determined on
the ND-1000 spectrophotometer (NanoDrop Technologies,
Wilmington, DE, USA). Proinsulin coding sequence (CDS)
was cloned by RT-PCR (OneStep RT-PCR Kit; Qiagen,
Valencia, CA, USA) and GatewayTM BP recombination
into the pDONR/Zeo vector (Life Technologies) using
primers designed to introduce an attB1 and a Kozak
sequence at the 5' end and an attB2 and stop-codon at the
3' end: forward 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGCCCTGTGGATGCGC-3',
reverse5'-GGGGACCACT
Fiziologia - Physiology • 2015.25.4(88)DNA sequencing of plasmid vectors
The transgene presence and correct orientation in the
DEST53-hIns and DEST47-hIns expression clones, as wellas in the plasmids carrying the single- and double-mutant
insulin constructs, was confirmed by capillaryelectrophoresis DNA sequencing with the ABI 3130 Genetic
Analyzer using the BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems, Foster City, CA, USA).
The primers were purchased from Life Technologies. Prior tosequencing, the PCR products were purified byethanol/EDTA precipitation and denatured with HiDi
Formamide (Applied Biosystems). Raw data was processedwith SeqAnalysis 5.3 software (Applied Biosystems), while29

contig assembly was perfomed with the DNA Workbench
program (CLC Bio, Aarhus, Denmark).
Lipofection of hMSC

RESULTS

Prior to the transfection, hMSCs were permitted toreach 80-90% confluence, and the complete culture
medium was changed to medium without antibiotics 30
minutes before lipofection. The expression plasmids
DEST53-hIns and DEST53-2M were purified attransfection grade from competent E.coli OneShot
OmniMAX 2 T1 using the S.N.A.P. MidiPrep Kit anddiluted in serum-free OptiMEM. Lipofectamine 2000 was
mixed and diluted at the appropriate amount inserum-free OptiMEM. The diluted DNA was mixed with
Lipofectamine 2000 in a ratio of 1:2.5, and theliposomal-DNA complexes formed after a 20- minuteincubation at room temperature were added to the hMSC.
We used as a positive control for assessing transfection
rate the pmaxGFP plasmid (Amaxa Lonza). The
transfection medium was changed to complete growth
medium after 16-20 hours and the transgene expression
was evaluated at different time points. All transfection
reagents were purchased from Invitrogen.

Human mesenchymal stem cells ubicuitary express
the proconvertase enzyme furin, which can successfully
replace the pancreatic endoproteases in processing
proinsulin to the active, mature hormone. We analyzed
six different batches of MSCs isolated in our laboratory
and compared the expression of furin in MSCs with thatof human embryonic stem cell (hESC) and other celllines available. We also performed a metaprofile analysis
on a public microarray database, Genevestigator V3 [21],to confirm that furin is constantly expressed in MSCs,
allowing the mutant insulin construct to be cleaved intarget cells.

qRT-PCR assay
We isolated total RNA from hMSC by means of the
GenElute Mammalian Total RNA Miniprep Kit (Sigma)
and determined its concentration with the ND-1000
spectrophotometer. Sample purity, as indicated by the
260/280 absorbance ratio, was 1.9-2.02. We used 1 ug
total RNA for every 20 ul reverse transcription reaction
performed with the AccuScript High Fidelity 1st Strand
cDNA Synthesis Kit (Stratagene Agilent Technologies).
cDNA samples were analyzed by quantitative real-time
PCR, using the LightCycler 480 SYBR Green I Master(Roche, Florence, SC, USA). HPRT1 was chosen as asuitable reference gene. The primers for Gen-Ins, 2M-Ins
and N-Ins were designed using the open-access program
Primer3. The 2M-Ins primers were chosen to anneal only
to the mutant insulin, the N-Ins only to the wild-type
insulin, while Gen-Ins can amplify both variants. Furin
primer sequences were chosen from qPrimerDepot and
HPRT1 primer sequences from Harvard Primer Bank
databases. We performed a relative basic quantitation
based on the ΔΔCt method with the LightCycler480
Software (Roche).
Statistical analysis
All statistical tests and values were calculated with
GraphPad Prism 4 (GraphPad Software, San Diego, CA,
USA). Data were expressed as mean ± s.e.m. Groups werecompared using one-way ANOVA for normally-distributed
data or the Mann-Whitney U test for nonparametric values.
P<0.05 was considered to be significant.

30

Furin expression in mesenchymal stem cells

Construction of insulin plasmids
The wild-type insulin plasmid DEST53-hIns was
obtained by subcloning the 333 bp human preproinsulin
CDS, including the stop-codon, into the pcDNA-DEST53
destination vector. As described in the Methods section,
we isolated 1740 ng/uL total undegraded RNA from 39 mg
human pancreas. By PCR amplification of human insulin
CDS with attB-containing primers, we obtained a 397 bp
product, corresponding to the insulin CDS and theprimer-generated Gateway recombination segments. This
product was further subcloned, by recombination reactions
into destination vectors, capable of inducing transgene
expression in eukaryotic cells. The enzymatic
recombination reactions, followed by amplifi- cation of thevectors by transforming supercompetent bacteria, werehighly efficient and resulted in numerous plasmid-carrying
colonies on selective agar plates. We chose 5 colonies forplasmid isolation with the PureLink HQ MiniPrep kit anddownstream sequencing. Finally, pending analysis ofsequencing data, one of the plasmids was isolated with atransfection-grade midiprep kit. Sequencing results
documented a synonimous mutation C306G, with thesame aminoacid, isoleucine, being transcribed at position
102. Otherwise, the reading frame for GFP was the sameto that of insulin and we concluded that the proteins can be
expressed simultaneously following transfection of theDEST53-hIns vector.
To ensure proinsulin processing in nonendocrine cells,
we engineered a double mutant proinsulin (2M), in which
the PC1/3 and PC2 cleavage sites were altered bysite-directed mutagenesis to form consensus tetrabasic
cleavage sites, containing the motif R-X-[K/R]-R,
recognized by the subtilisin/kexin-like enzyme furin
expressed in hMSC (Fig. 1). According to the ProP 1.0
neural prediction network, replacing proinsulin aminoacids
53-55 with Arg-Ser-Lys residues and L89 with Arg results
in a 0.728 probability of cleavage of the mutant proinsulin
after R56 and 0.751 after R89, respectively.
Fiziologia - Physiology • 2015.25.4(88)expression, in non- neuroendocrine cells. qPCR analysisof relative insulin mRNA expression showed a 105 times
more intense transcription of the transgene in thetransfected cells comparative to that in the INS-1
transgenic insulinoma cell line, which was used as apositive calibrator. The presented data was derived fromfive experiments, performed in duplicates, at different
time points and we verified insulin expression withseveral primer pairs, designed to bind to different
complementary regions within the preproinsulin sequence.
The insulin mRNA detected in the transfected hMSC wastranscribed entirely from our plasmid vectors as theendogenous insulin primers designed to bind targets
outside the preproinsulin CDS cloned in the DEST47/53
plasmids detected only negligeable quantities, similar in
the controls and transfected cells (Fig. 3).
Fig. 1 Mutant insulin cleavage sites. The dibasic sequences
Arg55-Arg56, where the B chain is cleaved from the C-peptide,and Lys88-Arg89, where the C-peptide is separated from the
A-chain from human insulin were replaced by consensus aminoacid sequences recognized by furin (shown in red).

We also analyzed several other tetrabasic motifs bothfor furin and a more general proconvertase cleavage
probability, as well as the energy cost of mismatches
when constructing the cleavage sites by in vitro
mutagenesis (QuikChange Primer Design Program,
Agilent Genomics). DNA sequencing confirmed thesuccessful mutagenesis of insulin clevage sites.
Non-viral gene transfer in hMSC
We achieved a 40-50% rate of transfection with
90-100% hMSC cell viability following the lipofection of thecontrol plasmid pmaxGFP, at a ratio of DNA: Lipofectamine
2000 of 1: 2.5, as documented by fluorescence microscopy
24 hours from transfection (Fig. 2).

Fig. 2 Rate of transfection of hMSC with pmaxGFP. The
transfection rate is about 50%, 24 hours from lipofection, whilecell viability is 100%. Optic and fluorescence microscopy, 100x.

Insulin transcription following lipofection of hMSC
We transfected by lipofection hMSC at passages 5-7
with the expression vectors DEST53-hIns/2M and
DEST47-hIns/2M to assess the potential for insulin
Fiziologia - Physiology • 2015.25.4(88)Fig. 3 A Relative mRNA expression of different proinsulin
variants, detected by real-time qPCR. Endogenous insulin ispresent at almost undetectable levels and does not account for
insulin expression in the transfected cells. mRNA level ofexpresson for mutant 2M-insulin is similar to that of thewild-type insulin. B We designed and tested on the plasmid
vectors spe- cific primer pairs that allow us to differentiate
between the 2M-insulin and the native insulin. Gen-Ins primers
amplify both the normal and the mutan transgenes, while the2M and the hIns primers amplify only the particular sequencesof the engineered and the normal insulin.

Interestingly, the level of expression of mutant
proinsulin mRNA was similar to that of the native
proinsulin, when transfected into the same batch of
MSCs, using the same plasmid backbone. This is an
important observation because previous studies reported
reduction of more than 70% of furin-cleavable insulin
expression [9,11,22].
There was no significant difference in mRNA
expression when hMSCs were transfected with either the
GFP-insulin fusion protein construct or with the proinsulin
alone (Fig. 4A). On the other hand, the level of insulintranscription after lipofection varied significantly (p<0.0003)
between individual batches of hMSC (Fig. 4B). The MSCs
were isolated from patients of different clinicalbackgrounds and age groups. The transfection was
performed in identical experimental settings (DNA quantity,
31

DNA:Lipofectamine 2000 ratio) for all samples, and mRNA
expression was assayed 24 hours post-transfection.

Fig. 4. A. Relative mRNA expression of native and mutant
proinsulin, respectively, determined by real-time qPCR.
Following lipofection of either the DEST53 or DEST47 vectors,
which differ in their capacity to generate GFP-fusion constructs,
the expression of the two in- suliant variants does not varysignificantly ( p >0.05, N=4 for each vector). B. Proinsulin
mRNA levels after lipofection of hMSC samples derived fromdifferent patients were heterogenous.

DISCUSSION
Despite progress in the chemically-induced
transdifferentiation of human and rat MSC [23-25] into
insulin-producing cells or the even more momentous
conversion of embryonic stem cells into insulin-secreting
cells reported by Douglas Melton and his team [26],genetically-modified hMSCs, capable of long-term,
controlled secretion of biologically-active mature insulin,
still represent a promising therapeutical solution for thetreatment of diabetes mellitus [27].
The current strategies for gene therapy in diabetes
with the objective of correcting the underlying metabolic
disfunction and restoring normal glucose levels involve
the transfer of genes to express different variants ofinsulin [28,29], transcription factors closely related topancreatic β-cell ontogeny [30,31] or proteins that control
glucose metabolism [32]. In this experimental study, we
reported the cloning of human preproinsulin CDS into a
GatewayTM non-viral expression vector in view of astraighforward and efficient approach to obtaining aclinically-relevant number of insulin-producing cells fromhMSC.
As hMSC do not express the pancreatic-specificendopeptidases PC1/3 and PC2, which convert
proinsulin into the mature, active hormone, we modified
the proinsulin proconvertase recognition sequences by insitu mutagenesis. The tetrabasic furin-cleavable motifs
RSKR and RQKR replaced the wild-type proinsulin
cleavage sites in the mutant proinsulin 2M, to ensure thecorrect processing of proinsulin, a key requirement of any

32

long-term therapeutic solution for diabetes. To the best of
our knowledege, the 2M proinsulin we constructed, with
Arg-Ser-Lys-Arg : Glu-Ala (B:C cleavage site) and
Arg-Gln-Lys-Arg : Gly- Ile (C:A cleavage site), has notbeen previously reported in the scientific literature,
although similar furin-recognition sites have also beenreconstructed. The motifs were chosen due to the highprobability to be cleaved by the ubiquitary enzyme furin,
following a bioinformatics analysis with the ProP 1.0
prediction server [20]. They are also very likely to becleaved by other furin-like proconvertases, as predictedby the same program, which can ensure the posibility of
expression in various cell types and tissues. The
presence of furin was documented in the hMSC used inour experiments, and we confirmed that it is constantly
expressed at least at medium levels, sufficient for an
efficient processing, by screening a free functional
genomics microarray database (Genevestigator V3, [21]).
We then proceeded to cloning the 2M proinsulin in the
DEST53-2M and DEST-47-2M non-viral plasmid vectors.
Transgene expression from the plasmid vectors
DEST53-hIns/2M and DEST47-hIns/ 2M was controlled
by the CMV constitutive promoter. The CMV promoter isfrequently employed in gene transfer experiments due toits high efficiency in most cells [33] and we encountered
few literature mentions of transcriptional inactivation due
to promoter sequence methylation [34]. The next stepwould be to replace the CMV promoter with an
endogenous, glucose-sensitive promoter, such as the
EGFR1 (early growth response-1) element identified byChen et al. [28], induced by rising glucose levels, in theabsence of any protein synthesis, which also activates
several genes invoved in glucose homeostasis [35]. The
DEST53 plasmid vector controls the expression of afusion GFP-insulin construct, with the GFP located at the
N terminus of the protein. As we cloned the insulin CDS
with a stop-codon, this would prevent the fusion of insulin
with GFP at the C terminus, thus generating only thetransgenic insulin. The insulin mRNA expression directedfrom either vector did not differ significantly. We have yet
to discern whether the N-terminal GFP will interfere withprotein folding or signal protein cleavage.
The advantages of using a non-viral transfection
method by lipofection include the relative ease and safetyof the procedure, with high levels of transgene
expression in less than 24 hours from transfection in
40-50% of the cells, and very high cell viability. As theexpression plasmids also contain an eukaryotic antibiotic
resistance gene, we can also select only the transfected
cells.
Several research groups testing a series ofgenetically engineered human and rat insulins reported adecrease by almost 90% of furin-cleavable proinsulin
expression following transfection [9,22,36,37]. Hay et al.
attributed this observation to an instability of proinsulin
structure caused by the basic amino acids (arginine andFiziologia - Physiology • 2015.25.4(88)lysine) present in the furin consensus sites [11].
Surprinsingly, the lipofection of DEST53-2M expression
plasmid into hMSC resulted in a level of insulintranscription similar to the wild-type insulin.
In conclusion, our data demonstrate that humanmesenchymal stem cells transfected with wild-type and amutant furin-cleavable human proinsulin were capable ofexpressing insulin and the mutant proinsulin wastranscribed at a level comparable to wild-type insulin.
Subsequently, we will further analyze the processing,secretion kinetics and physiological effect of the newly
engineered proinsulin.

ACKNOWLEDGEMENTS
This work was supported by the PNI-II postdoctoral grantno. 31/2013 awarded to OIG.

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OPȚIUNI TERAPEUTICE ÎN DIABET: INSULINA CLIVABILĂ
DE FURINA ȘI EXPRIMATĂ ÎN CELULE STEM MEZENCHIMALEREZUMATÎn pofida unor progrese recente în domeniul transdiferențierii chimice a celulelor mezenchimale umane sau deșobolan în celule secretoare de insulină, eficeința și aplicabilitatea clinică a acestor metode este chestionabilă. În
contrast, celule mezenchimale umane modificate genetic, capabile de secreție pe termen lung, controlabilă, ainsulinei mature, activă biologic, ar putea reprezenta o soluție terapeutică promițătoare pentru tratarea diabetului.
În acest studiu, am explorat posibilitatea de a obține vectori genetici de tip plasmid pe care să conțină unconstruct de proinsulină umană, în care situsurile de clivare ale enzimelor PC1/3 și PC2 au fost modificate
genetic prin mutageneză în situsuri de clivare penmtru furină. Am urmarit ca această transgenă pentru insulină
sa poată fi transcrisa corect și la un nivel înalt de expresie prin transferul non-viral în celule stem mezenchimale
umane.
Cuvinte-cheie: insulină, furina, celule stem mezenchimale

34

Fiziologia - Physiology • 2015.25.4(88)THE INVOLVEMENT OF ENTEROCOCCUS SPP.
STRAINS IN THE AETIOLOGY OF INFECTIONSIN HOSPITALIZED PATIENTSLIA S. PEPELEA1, STANCA L. PANDREA1,2, LAURA SIMON1,
LUMINIŢA MATROŞ1, OANA I. ŞERBAN1, LAVINIA BERINDE1,
LIA M. JUNIE1, MONICA I. CIONTEA2, ERICA CHIOREAN2,
DELIA MUNTEAN3, MONICA LICKER3
1Department of Microbiology, “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania
2Department of Microbiology, Central Laboratory, Regional Institute of Gastroenterology and Hepatology “Prof. Dr. O. Fodor”,

Cluj-Napoca, Romania
3“Victor Babeş” University of Medicine and Pharmacy, Timişoara, Romania

ABSTRACTThe present study was motivated by the low rate of morbidity due to enterococcal infections reported at a national level
and associated with a high risk of such infections in hospitals. The aims of the present study are to: i) Specify theaetiology of infections diagnosed in a Cluj university hospital from 2010 to 2012; ii) Investigate the associations
between enterococci and other microorganisms involved in the aetiology of infections in the above-mentioned hospital;iii) Study the distribution by various medical services of infections caused by Enterococcus spp.
Material and method. During the period January 2010 – December 2012, of the 878 bacterial strains isolated fromdifferent pathological products collected from hospitalized patients, 137 were identified by the Vitek 2 Compact
automatic system as being species of the genus Enterococcus.
Results. The species most frequently isolated in hospitalized patients was E. faecalis (64.23%), followed by E. faecium(27.73%). 8.04 % were other Enterococcus species encountered only in the hospital environment: E. durans (2.19%),
E. casseliflavus (2.19%), E. gallinarium (2.19%), and E. avium (1.45%). E.faecalis was most frequently isolated fromurine samples (39.77%), followed by CVC (14.8%), pus from fistula/drainage tube, and wound secretions and puncture
fluids (9.1%). E. faecium was most frequently isolated from pus from fistula/drainage tube (34.21%), followed by woundsecretions (23.7%), puncture fluids (18.42%), and respiratory secretions (13.15%).
Conclusions. The specification of the aetiology of enterococcal infections in hospitalized patients, the distribution ofthese infections by medical service, and the incidence of species and bacterial associations have all proved theimportance of knowledge on enterococci.
Key words: Enterococci, infections, aetiology, hospital, patients.

INTRODUCTIONEnterococci, although they are part of the intestinal
flora commensals, may become nosocomial pathogens.
In the past three decades, a constant growth in thenumber of infections caused by species of the genus
Enterococcus has been reported, a phenomenon
probably caused by a growth in the population thatshows risk factors (chronic inflammations, surgery,
intestinal neoplasia), but also by the emergence ofenterococci strains with multiple resistance to antibiotics,
especially Vancomycin [1].

In hospitals, enterococci infections are one of themost important therapeutic challenges, due to theirintrinsic resistance, on the one hand, and the constant
growth of acquired resistance, on the other hand.
In this era of increased resistance of microorganisms
to antibiotics, it is mandatory to give due consideration to
the judicious use of antibiotics, which is certainly also
helpful in stopping the emergence of resistant andvirulent enterococci strains.

Received 24th of October 2015. Accepted 25th of November 2015. Address for correspondence: Lia S. Pepelea, Microbiology
Department, UMF ’’Iuliu Haţieganu” Cluj-Napoca, 6th Pasteur Street, RO-400349, Cluj-Napoca, Romania; phone: +40750774432;
fax: +40264597257, e-mail: liapepelea@yahoo.com

Fiziologia - Physiology • 2015.25.4(88)35

MATERIAL AND METHOD
The present study was conducted in a prospective
manner, in that the Enterococcus spp. strains involved in
the aetiology of infections in hospitalized patients wereobserved, by ward, during the period January 2010 –December 2012.
The hospitalized patients were from:‐ the Intensive Care ward (54 beds)‐ 2 General Surgery wards (130 beds)‐ the Orthopaedics ward (60 beds)‐ the Gastroenterology ward (50 beds)‐ the Neonatal ward (40 beds)‐ the Obstetrics and Gynaecology wards (85 beds)
In the hospital, data from the Clinical Laboratory’s
Microbiology Compartment was used. In order to collectdata from the hospital, we used the standard surveillancedata sheet, presented in the annex of Health Ministry
Order no. 10/2005.
For each year studied (2010, 2011, and 2012), data
was collected both about the enterococci strains thatproved to be a unique etiological agent and theenterococci strains isolated in association with othermicroorganisms.
The samples were normally collected from patients by
specialized hospital staff, on account of the fact that theobservance of sample-collecting and transportation rules
for pathological products intended for bacteriologicalanalysis influences the different stages and the accuracy ofthe diagnosis.
Urine, blood, and cephalorachidian fluid samples
were collected, as well as samples from purulent
collections, bronchial aspirate, sputum, and central
venous catheters. The collected samples were sown on
Columbia gelose supplemented with sheep blood (5%),
and then incubated at 37C for 24 hrs.
In order to isolate the enterococci, we also used theBile Esculin Azide Agar (Sanimed) medium, CPS(bioMérieux), Trypticase Soy Agar with 5% sheep blood(bioMérieux), and the Uriselect (BioRad) chromogenic
medium [2].
The identification of bacteria belonging to the genus
Enterococcus was done based on culture andbiochemical characters.
The observance of the culture characters involved
the presence of the following types of colony:‐ on gelose-blood, type S, small, white-grey,
non-haemolytic colonies - E. faecalis, beta-haemolyticcolonies - E durans, or alpha-haemolytic colonies;‐ on the Bile Esculin Azide Agar medium, theenterococci develop white-grey colonies with ablack halo;‐ by wiping with a wad the 24 hour culture obtainedon the Trypticase Soy Agar medium and 5% sheep
blood, one can notice the medium’s yellow
colouring- E. casseliflavus, E. mundtii, E. sulfureus;
36

on the chromogenic CPS medium, the enterococcicolonies are green and small.
In the hospital, the isolated enterococci cultures wereidentified via the Vitek 2 Compact automatic system(bioMérieux). The Vitek 2 cards for the identification ofGram-positive cocci are standardized products that
contain miniature biochemical tests.
The Enterococcus species identified by VITEK 2 GP:
E.faecalis, E.faecium, E.avium, E. casseliflavus,
E.cecorum, E.columbae, E.durans, E.gallinarum, E.hirae,
E. raffinosus, E. Saccharolyticus.
‐

RESULTS
During the study, 878 bacterial strains isolated fromdifferent pathological products from hospitalized patientswere processed in the hospital laboratory.
Table I. The distribution of the strains isolated during the study,
by pathological product collected from hospitalized patients
Pathologicalproduct

2012

2011

2010

No.

%

Urine

49

44

42

135

15.37

Puncture
fluids

11

16

14

41

4.66

102

96

96

294

33.53

Pus from
fistula/drainag
e tube

16

17

23

56

6.37

Wound
secretions

46

39

54

139

15.83

Centralvenouscatheter

16

19

23

58

6.60

Blood

36

35

33

104

11.84

Other
products

15

19

17

51

5.80

Total

291

285

302

878

100

Respiratory
secretions(bronchialaspirate/trach
eal secretion)

Total

The isolated bacterial strains came from: respiratory
secretions, 294 (33.53%), wound secretions, 139(15.83%), urine, 135 (15.37%), blood, 104 (11.84%),
central venous catheter, 58 (6.60%), pus from
fistula/drainage tube, 56 (6.37%), puncture fluid, 41(4.66%), and other products, 51 (5.80%).
Of the 878 bacterial strains isolated, 137 strains(15.6%) belonged to the genus Enterococcus; their origin
from different pathological products is shown in Table II.

Fiziologia - Physiology • 2015.25.4(88)Table II. The distribution of Enterococcus strains isolated in thehospital, from various pathological products
Pathologicalproduct

2012

2011

2010

Total

No. ofstrains

No. ofstrains

No. ofstrains

No.

%

Urine

14

13

13

40

29.19

0.993

Puncture
fluids

7

6

7

20

14.59

0.954

Respiratory
secretions(bronchialaspirate/trachealsecretion)

4

5

4

13

9.48

0.900

8

9

8

25

18.24

0.929

Wound
secretions

6

5

6

17

12.48

0.946

Centralvascularcatheter

5

4

4

13

9.48

0.952

Blood

1

1

1

3

2.18

0.999

Bile

1

1

1

3

2.18

0.999

Cervicalsecretion

1

1

1

3

2.18

0.999

47

45

45

137

100

/

Pus from
fistula/drainage
tube

Total

application of the central venous catheter (9.48%), andrespiratory infections (9.48%).

P

It can be noticed that during the three-year study
period, no statistically-significant differences wererecorded in the incidence of enterococci strains isolated
in hospital.
The main types of enterococcal infections diagnosedin the hospital over the three-year study period areshown in Table III.

Fig. 1. Distribution by clinical ward of UTIs caused by enterococci

Fig. 2. Distribution by clinical ward of post-operative infections
caused by enterococci

Table III. The distribution of infections caused by enterococci
Infections caused

Number%Urinary tract infections

40

29.19

Respiratory infections

13

9.48

Post-operative infections

42

30.72

Bacteraemias/septicaemias

3

2.18

Gynaecological infections

3

2.18

Peritonitis

20

14.59

Cholecystitis

3

2.18

Infections caused by the applicationof the catheter

13

9.48

Total

137

100

The most frequent infections are post-operative
infections (30.72%), followed by urinary tract infections(29.19%), peritonitis (14.59%), infections caused by the
Fiziologia - Physiology • 2015.25.4(88)Fig. 3. Distribution by species of the enterococci strains
isolated in the hospital wards

As Figure 3 indicates, the species most frequently
isolated was E. faecalis (64.23%), followed by E. faecium(27.73%).
Other enterococci species encountered in thehospital environment isolated in a percentage of 8.04%
were: E. durans (2.19%), E. casseliflavus (2.19%), E.
gallinarium (2.19%), and E. avium (1.47%).

37

Table IV. Distribution of E. faecalis strains isolated in the hospital
2012

2011

2010

Pathological productNo. ofstrains

No. ofstrains

No. ofstrains

Total
No.

%

Urine

11

12

12

35

39.77

Puncture fluids

3

2

3

8

9.1

Respiratory secretions(bronchialaspirate/
tracheal secretion)

2

2

2

6

6.81

Pus from
fistula/drainage tube
Wound secretions

3

2

4

9

10.22

2

2

4

8

9.1

Central venous catheter

5

4

4

13

14.8

Blood

1

1

1

3

3.40

Bile

1

1

1

3

3.40

Cervical secretion
Total

1
29

1
27

1
32

3
88

3.40
100

Table VI indicates a higher statistically-significant
incidence of E. faecium in pus and wound secretions,while E faecalis is, in a statistically-significant manner,
more present in urine and on central venous catheters.
In the numerous pathological products collected fromthe hospitalized patients, the enterococci were isolated
alongside other bacterial strains. In the following graph,
we show the microbial associations involved in the
aetiology of the infections comprised in the present study.

E.faecalis was most frequently isolated from urine
samples (39.77%), followed by CVC (14.8%), pus from
fistula/drainage tube (10.22%), and wound secretions
and puncture fluids (9.1%).
Table V. Distribution of the E. faecium strains isolated in the hospital
Pathological product

2012

2011

2010

Urine

No. ofstrains2

No. ofstrains1

No. ofstrains1

No. ofstrains
4

Total

10.52

Puncture fluids

3

2

2

7

18.42

Respiratory secretions(bronchialaspirate/tracheal
secretion)
Pus from
fistula/drainage tube

1

2

2

5

13.15

4

5

4

13

34.21

Wound secretions

4

3

2

9

23.7

13

13

12

38

100

%

Total

E. faecium was most frequently isolated from pus
from fistula/drainage tube (34.21%), followed by woundsecretions (23.7%), puncture fluids (18.42%), andrespiratory secretions (13.15%).
Table VI. Comparative presentation of the E. faecalis andE. faecium strains isolated in hospital from diverse
pathological products

Urine

E.
faecalis(n=88)
35

E.
faecium(n=38)
4

0.001

Puncture fluids

8

7

0.146

Respiratory secretions(bronchial aspirate/tracheal
secretion)

6

5

0.304

Pus from fistula/drainage tube
Wound secretions

9
8

13
9

0.001
0.027

Central venous catheter

13

0

0.009

Blood

3

0

0.553

Bile

3

0

0.553

Cervical secretion

3

0

0.553

Pathological product38pFig. 4. Distribution of the microbial associations of the isolated
Enterococcus spp. strains

As Figure 4 indicates, the enterococci were more
frequently isolated in association with Acinetobacterbaumannii, Klebsiella pneumoniae, Pseudomonas
aeruginosa, and E. coli.

DISCUSSION
The aim of the present study was to evaluate theinvolvement of the Enterococcus spp. strains in the
aetiology of certain infections diagnosed in hospitalized
patients and in outpatients.
According to the results presented above, we isolated
and identified a total number of 137 Enterococcus spp.
strains involved in the aetiology of certain infections in
hospitalized patients, which represent 15.6% of the total
germs isolated during the period in question.
By comparing the isolation of enterococci fromvarious pathological products, in the hospital we noted ahigher extremely statistically-significant prevalence in pus,
wound secretions (p<0,001), and puncture fluids(p<0,001). The most frequently isolated enterococcus
species were E. faecalis and E. faecium, which isconsistent with the data provided by the specialty
literature [3].
On the whole, E. faecalis was represented by 88
strains (64.23%). E. faecium totalled 38 strains (27.73%).
By comparing the distribution by pathological product,
we noted that the E. faecalis strains were better
represented as compared to the E. faecium strains inurine (p<0.001), pus (p<0.001), wound secretions
Fiziologia - Physiology • 2015.25.4(88)(p=0.027), and central venous catheter secretions(p=0.009).
Other Enterococcus species included in this study
were E. gallinarium, E. durans, and E. casseliflavus, with 3
strains each (1.36%), and E. avium, with 2 strains (0.91%).
The Enterococcus species are part of the normal
intestinal flora, enterococci being considered as low
virulence germs. However, affecting the local intestinal
defence mechanisms, usually by surgery, neoplasms, orchronic intestinal inflammations, causes invasive
enterococcal infections, such as septicaemia, peritonitis,cholecystitis, and endocarditis. Due to the high incidence ofthe risk factors mentioned above, in recent years we havewitnessed a constant rise in enterococcal infections [4].
In this study, in the case of hospitalized patients, theenterococci were more frequently involved in the
aetiology of post-operative infections (30.72%), followed
by urinary tract infections (29.19%), and peritonitis(14.59%).
Post-operative infections, irrespective of theirlocalization, were more frequently mixed, being causedby associations of enterococci with Gram-negative bacilli,
especially Acinetobacter baumannii and Klebsiellapneumoniae.
We did not notice any statistically-significant
differences between the Anaesthesia and Intensive Care,and the Surgery wards, which are frequently faced withsuch infections (p=0,006), whose number is, nonetheless,larger in the surgery ward.
Although enterococci are frequently isolated frompatients with intra-abdominal infections, their role inthese infections is controversial.
In six studies conducted on patients with mixed
abdominal infections, caused by enterococci and
Gram-negative bacteria, antibiotics were administered that
had no effect on the enterococci; it was observed that, in
this case, the abdominal infections had a favourable
evolution. Similar studies, conducted on outpatients who
had mixed intra-abdominal infections, demonstrated afavourable evolution after the surgical treatment, in theabsence of enterococcal antibiotherapy. Also, it wasobserved that the enterococci do not cause
intra-abdominal infections when they are injected
intraperitoneally into laboratory animals, except if they areassociated with other, more virulent, microorganisms [5].
On the other hand, other researchers havedemonstrated that the presence of enterococci cancause an increased occurrence rate of post-surgery
infections and increased mortality. A study conducted on
330 patients indicated the isolation of enterococci inintra-abdominal collections as the cause for treatment
failure [5,6]. Enterococci can also be involved in skininfections, soft tissue infections, or wound infections.
Usually, these infections are mixed, the role of theenterococci in their aetiology being not fully known. Most
frequently, enterococci are isolated from sore and ulcer
Fiziologia - Physiology • 2015.25.4(88)infections of the diabetic foot [5]. In this study, the 17
Enterococcus strands come from wound infections.
The 2 species, E. faecalis and E. faecium, were
almost identically represented, by 8 and 9 strands,
respectively, which were associated with P. aeruginosa (5cases), and Acinetobacter baumannii (4 cases).
In the case of hospitalized patients, surgical wound
infections were included in the category of post-operative
infections, and they were more frequently diagnosed in
the Anaesthesia and Intensive Care, and Surgery wards.
Urinary tract infections ranked second in the case of
hospitalized patients.
During the three-year period that the study spanned,
we did not observe any statistically-significant differences,from one year to the next, in the incidence of UTI in thehospital.
As far as aetiology is concerned, E. faecalis wasmore present, and in a much more statistically-significant
manner, as compared to the other Enterococcus species(p<0,001).
In the case of the hospitalized patients diagnosed
with UTI, the enterococci were relatively frequently
associated with enterobacteria, especially with wide
spectrum beta-lactamase-secreting E.coli strains.
Although enterococcal UTI more frequently affect theurinary bladder, the ground on which they occur iscompletely different. Thus, in the general population, women
without anomalies of the urinary tract or urologicalinterventions have UTI more often. Enterococci, like other
Gran-positive cocci, are more frequently associated withnosocomial urinary infections in patients with obstructive
uropathy or after endourologic procedures [7-11].
It is considered that approximately 10% of
hospitalized patients are catheterized, and of these,
20-25% develop UTI, the incidence of the infection being
directly proportional to the length of catheterization. Aftera unique bladder catheterization, UTI is present in 1% of
outpatients and 10% in hospitalized patients, it being
more frequent in the elderly, in immunodepressed
patients, and in those having urological diseases. Urinary
infections are significantly more frequent in patients withlong catheterization. In the first two weeks ofcatheterization, the relation between the onset of UTIs
and catheterization is linear, 50% of patients developing
UTI after 15 days of catheterization, and almost all
patients after one month. According to Ferrar, all patientsdevelop bacteriuria 4 days after catheterization, under
the conditions of an open urinary drainage system, while
the use of a closed drainage system is associated withbacteriuria 10 days later [12-16].
The data cumulated from 463 hospitals in the USA
indicates enterococci as the third cause for UTIs
associated with catheterization, the most isolated being
the E. faecium and E. faecalis species [17].
In the specialty literature, UTIs caused byenterococci are associated with the antibiotic treatment in39

previous cases, and often it’s a case of recurring UTIs,
diagnosed more frequently in the case of men over 50
years of age, with a urological pathology. Thoughrelatively rarely, the enterococci can nevertheless cause
pyelonephritis or renal abscess, accompanied bybacteraemia [18].
Ranking fourth in order of frequency, in the hospital,
were respiratory infections and central venous
catheter-related infections.
Respiratory infections are not frequently caused byenterococci, but ortotracheal intubation, assisted breathing,prolonged dorsal decubitus, and antibiotherapy, especiallywith cephalosporins, are favouring factors in the case of
hospitalized patients. In our study we have diagnosed such
infections in a number of 13 patients coming from thehospital’s Anaesthesia and Intensive Care, and Surgery
wards, which participated in our study. The majority of
respiratory infections were mixed, 3 being caused by E.
faecalis and Acinetobacter baumannii, 3 by E. faecium
associated with Acinetobacter baumannii, and 3 werecaused by E. faecalis in association with Pseudomonas
aeruginosa, Klebsiella pneumoniae or Acinetobacterbaumannii. By comparing the distribution of the E. faecalis
and E. faecium strains involved in the aetiology of respiratory
infections, we did not obtain any statistically-significant
differences (p=0,304) during our study.
Of the enterococci, only E. faecalis was responsible
for central venous catheter infections. As far as the
distribution by ward of CVC infections is concerned, they
were more present, in a statistically-significant manner, in
the Anaesthesia and Intensive Care ward than they werein the Gastroenterology ward (p=0,030); this difference
was no longer present when we compared theAnaesthesia and Intensive Care ward with the Surgery
ward (p=0,620).
The bacteraemias caused by enterococci, althoughfrequently reported in the specialty literature, were
extremely rare in this study, in that only 3 such infections
were identified: 2 in the Anaesthesia and Intensive Careward and one in the Surgery ward. E. faecalis was
isolated in all 3 cases of bacteraemia.
Both the bacteraemias and CVC infections caused byenterococci frequently recognize as their source of infection
the gastrointestinal or genitourinary tracts of inpatients or
outpatients. In hospital, other sources of infection can be thevascular or urinary catheters and the wounds, including
burns. Approximately half the cases of enterococcal
bacteraemias occur in immunocompromised patients and in
those who are administered antibiotics [5].
If, in the case of outpatients, enterococcal infections
are less severe, the infections diagnosed in hospital, due
to the ground on which they occur, raise the length of
hospitalization and the cost of treatment, and sometimes
worsen the state of the patients. Under these conditions,
it is necessary to strictly observe certain procedures forfighting and controlling infections.
40

The general control of infections, which also include
enterococcal infections, involves isolating the patients
who are likely to transmit the germs to other patients ormedical staff, and the use of a “physical barrier” [19].

CONCLUSIONSThe specification of the aetiology of enterococcal
infections in hospitalized patients, the distribution of these
infections by different medical services, the incidence ofspecies and of bacterial associations have all demonstrated
the importance of knowledge on enterococci.
The specification of the frequency and aetiology ofthe different types of enterococcal infections (urinary
infections, post-operative infections, respiratory infections,bacteraemias/septicaemias, gynaecological infections,
peritonitis, cholecystitis, and CVC infections) is genuinely
helpful in the prevention and control of these infections,especially of those caused by VRE strains.

REFERENCES
1. Hunt CP. The emergence of enterococci as a cause of
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2. Buiuc D, Neguţ M. Clinical Microbiology Treatise. Editura
Medicală Publishing House, Bucharest, 1999.
3. CA Arias, BE Murray. Enterococcus Species, Streptococcus
Gallolyticus Group, and Leuconostoc Species. In Mandell,
Douglas, and Bennett's Principles and Practice of Infectious
Diseases. Eighth Edition, 2015. Volume 2. Elsevier Saunders.
4. Popescu GA, Pistol A, Șerban R. Report on the Use of
Antibiotics, Bacterial Resistance, and Nosocomial Infections in
Romania, in 2012.
5. Gorbach SL. Intraabdominal infections. Clin Infect Dis. 1993; 17:961-965.
6. Burnett RJ, Haverstock DC, Dellinger EP, et al. Definition of theRole of Enterococcus in Intraabdominal Infection: Analysis of a
Prospective Randomized Trial. Surgery 1995; 118:716-721;
discussion 721-723.
7. Ciocâlteu A. Clinical Nephrology Infomedica Publishing House,
Bucharest, 2000.
8. Fowler JE Jr, Stamey TA. Studies of Introital Colonization in
Women with Recurrent Infections. VII. The Role of Bacterial
Adherence. J Urol. 1977; 117: 472-6.
9. Hovelius B, Mardh P. Staphylococcus Saprophyticus as aCommon Cause of Urinary Tract Infections. Infect Dis. Review,
1984; 6: 328-37.
10. Jordan PA, Jravani A, Richard GA, et al. Urinary Tract
Infection Caused by Staphylococcus Saprophyticus. J Infect Dis.
1980; 142: 510-5.
11. Mims CA, Playfair JHL, Roitt IM, Wakelin D, Williams R.
Medical Microbiology. Mosby-Year Book Europe Limited, 1993.
12. Farrar EW Jr. Infections of Urinary Tract. Med Clin North Am.
1983; 67(1): 187.
13. Gluhovschi G, Drăgan I, Gluhovschi L, et al. Urinary Tract
Infection in Medical Practice. Helicon Publishing House,Timişoara, Romania, 1992.

Fiziologia - Physiology • 2015.25.4(88)14. Batt MD, Galaviz CJ. Urinary Tract Infections. APIC 12: 1-3.
15. Naber KG, Bergman B, Bishop MC, et al. Guidelines on
Urinary and Male Genital Tract Infections. 2005. European
Association of Urology.
16. Stamm WE. Bacteriuria in Women with Long-Term Catheters:
Paired Comparison of the Indwelling and Replacement Catheter.
J Infect Dis. 1984; 157: 199-202.
17. Hidron AI, Edwards JR, Patel J, et al. NHSN Annual Update:
Antimicrobial-ResistantPathogens
AssociatedwithHealthcare-Associated Infections: Annual summary of datareported to the National Healthcare Safety Network at theCenters for Disease Control and Prevention, 2006-2007. InfectControl Hosp Epidemiol. 2008; 29: 996-1011.
18. Murray BE. The Life and Times of the Enterococcus. Clin
Microbiol Rev.1990; 3: 46-65.
19. Nicolle LE. Infection Control Programmes to Contain
Antimicrobial Resistance, World Health Organization 2001: 9-15.

IMPLICAREA TULPINILOR DE ENTEROCOCCUS SPP.
ÎN ETIOLOGIA INFECŢIILOR LA PACIENŢI SPITALIZAŢIREZUMAT
Obiective. Prezentul studiu a fost motivat de morbiditatea scăzută prin infecții enterococice raportată la nivelnațional, asociată riscului crescut al unor astfel de infecții în spitale. Cercetarea de față și-a propus: i) Precizarea
etiologiei infecțiilor diagnosticate în cadrul unui spital universitar
clujean în perioada 2010-2012; ii)
Investigarea asociațiilor dintre enterococi și alte microrganisme implicate în etiologia infecțiilor în spitalulmenționat; iii) Studierea repartiției infecțiilor determinate de Enterococcus spp. pe diferite servicii medicale.
Material şi metodă. In perioada ianuarie 2010- decembrie 2012 din cele 878 de tulpini bacteriene izolate dindiferite produse patologice provenite de la pacienti spitalizaţi, 137au fost identificate cu sistemul automat Vitek2
Compact ca fiind specii din genul Enterococcus.
Rezultate. Specia cel mai frecvent izolată la pacienţii spitalizaţi a fost E. faecalis (64.23%) urmată de E. faecium(27.73%). Alte specii de enterococi întâlnite doar în mediul spitalicesc s-au izolat în procent de 8.04 %: E.
durans (2.19%), E. casseliflavus (2.19%), E. gallinarium (2.19%), E. avium (1.47%). E.faecalis a fost cel mai
frecvent izolat din prelevatele de urină (39.77%), urmat de CVC (14.8%), puroi fistulă/tub de dren (10.22%),secreţiile de plagă şi lichidele de puncţie (9.1%). E. faecium a fost cel mai frecvent izolat din puroi fistulă/tub dedren (34.21%), urmat de secreţiile de plagă (23.7%), lichidele de puncţie (18.42%) şi secreţiile respiratorii(13.15%).
Concluzii. Precizarea etiologiei infecţiilor enterococice la pacienţii spitalizaţi, repartiţia acestor infecţii pe diferite
servicii medicale, incidenţa speciilor şi a asocierilor bacteriene a demonstrat importanţa cunoaşterii
enterococilor.
Cuvinte cheie: Enterococi, infecţii, etiologie, spital, pacienţi.

Fiziologia - Physiology • 2015.25.4(88)41THE ROLE OF BMI AND CO-MORBIDITIES INHOSPITALIZATION LENGTH STAY IN SURGICALREPAIR OF INCISIONAL HERNIANODITI GEORGE, LAZAR CAIUS, MATEESCU TUDOR, BREBU DAN,LAZAR FULGER, DUTA CIPRIAN“Victor Babes” University of Medicine and Pharmacy Timisoara

ABSTRACTIntroduction. BMI as risck factor and co-morbidities evaluated pre-operative can predict the hospitalization lenght stay
in incizional hernia repair. We conducted a 18 months retrospective observational study on incizional hernia repair in
our Clinics after registering to the EuraHS platform in order to evaluate the patients demographic characteristics (age,sex, BMI), co-morbidities, and surgical outcomes (early and late complications, total length of hospital stay).
Materials and methods. We have established the target group of 126 consecutive patients with incizional hernia repair.
We collected data about demographics, comorbidities, and surgical outcomes (length of hospital stay, incidence ofearly and late complications). Data are presented as mean and range. Correlation coefficient r (Pearson), 95%
confidence interval (p<0.05) with have been assessed for each analyzed variable.
Results. The age average of the group was 62.7 y. Sex ratio showed females preponderance (76%) when comparedwith males (24%). The midline localization with all the possibilities as describes on the Eura HS site has been seen in
most of the cases (78.6%), followed by right iliac fossa localization (9.5%). None of the patients had a 4 SOC score.
Score SOC 3 was the most encountered. Out of 126 cases, 54 presented BMI < 29.99; 72 had BMI > 29.99 and 7
more than 40. We have seen that BMI and SOC score are very strong statistically correlated with correlation coefficientr (Pearson) = 0.36, and p = 0.0001. The length of total hospital stay has registered values between 2 and 38 days.
The BMI and total hospital stay are strongly correlated: correlation coeficient r (Pearson) = 0.27, and p = 0.002.
Between SOC score and total hospital stay, there have been no statistical correlation (r = 0.14, and p = 0.12 (>0.05). Intraoperative we reported 2 cases of iatrogen bowel lesions. In terms of surgical complications we had 9 cases(7.14%) of acute or early post-operative complications (4 cases of hemorrhage, 3 cases of bowel obstruction and 2
cases of hematoma) and 5 (3.96%) late post-operative complications (3 hernia recurrence and 2 parietal abcesses) as
per EuraHS classification, in patients with BMI >30.
Conclusions. BMI has a good correlation with SOC score and total hospital stay. Appropiate definitions andclassifications as provided by EuraHS platform could enable the evaluation of patients prior and after abdominal
wall surgery in a unitary manner and thus studies and meta-analysis of this pathology could be improved.
Key words: SOC score, BMI, hospital stay, surgical outcomes

INTRODUCTION
Incisional hernia is a common complication of
abdominal surgery. In a clinical review, Sanders [1]summarized that the reported incidence after a midline
laparoscopy ranges from 3% to 20% and it is double if
the index operation is complicated by wound infection [2].
About 50% of incisional hernias are detected within one
year of surgery, but they can occur several years
afterwards, with a subsequent risk of 2% a year [2, 3].
Millions of abdominal incisions are performed each year
worldwide, so incisional hernias are a major problem,
both in terms of morbidity and socioeconomic cost [1].

Every incizional hernia repair is a challenge for thesurgeon, with different factors related to the patient andhernia type that potentially could influence thepostoperative outcomes.
Despite considerable improvements in prosthetics
used for hernia surgery, the incidence of incision hernias
and the recurrence rates after repair remain high.
Arguably, no other benign disease has seen so little
improvement in terms of surgical outcome [1]. For
predicting the development of early (less than six months
after surgery) incisional hernia models have been
published recently [4]. Van Ramshorst and colleagues [5]identified the major independent risk factors as age, sex,

Received 10th of November 2015. Accepted 5th of December 2015. Address for correspondence: Noditi George, MD, Surgery
Clinics II, Emergency Clinical County Hospital Timisoara; Iosif Bulbuca Street No.10, RO-300736, Timisoara, Romania; phone:+40735311639; e-mail: george.noditi@gmail.com

42

Fiziologia - Physiology • 2015.25.4(88)chronic pulmonary disease, ascites, jaundice, anemia,
and emergency surgery, type of surgery, postoperative
coughing, and wound infection.
Consequently the length of hospital stay is a valuable
indicator of patient care needs before and after surgery.
The main objective of this study is to identify the role of
BMI and co-morbidities variables that significantly predictthe length of hospital stay. Another objective is toimplement definitions and classifications proposed by theinternational online platform for registration and outcome
measurement EuraHS (European Registry for Abdominal
Wall HerniaS) in defining certain variables in order tocollect data in a standardized manner [6].

MATERIALS AND METHODSElectronic medical records of all consecutive patientsfor whom an alloplastic substitution in an open repair
procedure for abdominal wall defects was performed
from July 2012 until December 2013 (18 months) at ourSecond Surgery Clinic of Timisoara Emergency County
Hospital were retrospectively reviewed. For each patienta set of data have been recorded in order to evaluate
their status pre-, intra-, and postoperative.
We have used definitions and classifications postedon the international online platform for registration andoutcome measurement EuraHS (European Registry for
Abdominal Wall HerniaS) for hernia class, SOC score forco-morbidities (Table I), early and late postoperativecomplications (Table II).
Table I. SOC score evaluation [6]We have used histograms to report the number ofobservations/frequency of data of certain figures or
belonging to a certain interval. Statistic parameters havebeen calculated as mean values, and standard deviation,using chi-square analysis. Correlation coefficient r(Pearson), 95% confidence interval (p<0.05) with havebeen assessed for each analyzed variable.

RESULTS
During the above mentioned period, a total number of
134 abdominal wall hernia repairs have been performed.
Because of lack of information about BMI and other data,
8 patients have been excluded from the present study.
Where excluded the patients for whom a laparoscopic
approach has been performed.
During pre-operative evaluation a set of variables
have been used: age, sex, BMI, hernia class and SOC
score as defined by EuraHS. For postoperative outcomes
we have used EuraHS classification of surgicalcomplications.
The age average of the group was 62.7 y. Sex ratio
showed females preponderance (76%) when comparedwith males (24%).
Considering class hernia distribution the midline
localization with all the possibilities as describes on the
EuraHS site has been seen in most of the cases (78.6%),
followed by right iliac fossa localization (9.5%).
While evaluating co-morbidities we have noticed thatnone of the patients had a 4 SOC score. Score SOC 3
was the most encountered as it is showed in Figure 1.

Severity of co-morbidity score
SOC score
SOC
score

Definition0

No co-morbidities

1

Asymptomatic, no medical consultation needed in last
12 months2

Stable disease, intermittent therapy and medical consultationneeded ≤4x/year3

Stable disease, continuous therapy with regular medical
consultation >4x/year

4

Progressive disease, with changing or intensified therapy andfrequent medical consultation >12x/year

Table II. EuraHS classification of surgical complications [6]Fig. 1. SOC score based repartition
Intra-operativecomplications

Are complications occurring during the time of thepatients’ arrival in the operating room and thepatient leaving the operating room

“Acute” or “early”
post-operativecomplications

Are complications occurring during the
hospitalisation or within 30 days postoperatively

Late post-operativecomplications

Are complications related to the hernia repair
occurring after discharge and more than 30 days
postoperatively

Fiziologia - Physiology • 2015.25.4(88)Since weight is a risk factor for hernia formation we
have calculated the BMI for each patient. Out of 126
cases, 54 presented BMI < 29.99; 72 had BMI > 29.99
and 7 more than 40. The total repartition had a normal
theoretical Gaussian distribution as it is illustrated in
Figure 2.

43

Fig. 2. BMI distribution

We have analyzed the BMI and SOC score behavior.
We have concluded that the two variables are very strong
statistically correlated with correlation coefficient r(Pearson) = 0.36, and p = 0.0001 (Figure 3).

Fig. 3. Correlation between BMI and SOC score

The length of total hospital stay has registered values
between 2 and 38 days. The correlation between BMI andthe length of hospital stay is presented in the graph bellow(Figure 4). The BMI and total hospital stay are strongly
correlated and the statistic calculated parameters are:correlation coeficient r (Pearson) = 0.27, and p = 0.002.
Between SOC score and total hospital stay, there
have been no statistical correlation (r = 0.14, and p =0.12 (> 0.05). While intra-operative evaluation we noticed
that no complications have occurred.
In terms of surgical complications we had 9 cases(7.14%) of acute or early post-operative complications (4cases of hemorrhage and 3 cases of bowel obstructionand 2 cases of hematoma) and 5 cases (3.96%) of latepost-operative complications (3 hernia recurrence and 2
parietal abcesses) as per EuraHS classification.

44

Fig. 4. Correlation between BMI and the length of hospital stay

DISCUSSION
Surgical hernia repairs is one of the most common
operative procedures and also the most commun elective
procedures performed by general surgeons since it
occurs in about 11% of laparotomies [7].
Preoperative appreciation of patient characteristics
may help choosing the type of procedure repair and predict
for surgical outcomes. Several investigators haveattempted to identify risk factors for postoperative woundmorbidity in patients undergoing abdominal wallreconstruction [8, 9, 10, 11]. A lot of important risk factors
for predicting surgical site occurrence (SSO) wereidentified. Patients with obesity, a smoking history, diabetes,and chronic obstructive pulmonary disease had a16% riskof developing a complication as SSO [10]. Obesity has
long been considered a risk factor for the development ofprimary and incisional ventral hernias [12].
We conducted a 18 months retrospective
observational study on incizional hernia repair in our
Clinics after registering to the EuraHS platform in order toevaluate the patients demographic characteristics (age,sex, BMI), comorbidities, and some surgical outcomes(early and late complications, total length of hospitalstay). The group consisted in 126 patients with the age
average of 62.7 y. Sex ratio showed females
preponderance (76%) when compared with males (24%).
More than two third of patient had a midline localizationof abdominal wall defect (78.6%), followed by right iliac
fossa localization (9.5%). Since we could notice that 72
patients from the group had BMI > 29.99 and 7 morethan 40 we have looked for the statistical correlation of
BMI with SOC score and total hospital stay. In both cases
we observed a strong statistic correlation in terms ofcorrelation ceoficient (r, Pearson) with p<0.05. In terms ofsurgical outcomes we had 9 cases (7.14%) of acute or
early post-operative complications (4 cases ofhemorrhage and 3 cases of bowel obstruction and 2
Fiziologia - Physiology • 2015.25.4(88)cases of hematoma) and 5 cases (3.96%) of latepost-operative complications (3 hernia recurrence and 2
parietal abcesses) as per EuraHS classification, inpatients with BMI >30.
This study has several limitations. The complex
nature of our referral practice has enabled us to have alarger experience with comorbid patients undergoing
ventral hernia repair reporting to EuraHS platform andlonger follow-up, in order to evaluate the surgicaloutcomes on a larger scale.

CONCLUSIONSIn our study, we could notice that BMI as risk factor
showed a strong statistical correlation with SOC score
and with total hospital stay.
In terms of surgical complications as defined by
EuraHS we had 7.14% of cases with acute or earlypost-operative complications and 3.96% of cases withlate post-operative complications, in patients with
BMI >30.
Appropriate definitions and classifications asprovided by EuraHS platform could enable the evaluationof patients prior and after abdominal wall surgery in aunitary manner and thus studies and meta-analysis of
this pathology could be improved.

REFERENCES
1. Sanders DL, Kingsnorth AN. The modern management ofincisional hernias. BMJ 2012; 344: e2843.
2. Mudge M, Hughes LE. Incisional hernia: a 10 year

prospective study of incidence and attitudes. Br J Surg, 1985;
72: 70-1.
3. Bucknall TE, Cox PJ, Ellis H. Burst abdomen and incisional
hernia: a prospective study of 1129 major laparotomies. BMJ
1982; 284: 931-3.
4. Veljkovic R, Protic M, Gluhovic A, Potic Z, Milosevic Z,
Stojadinovic A. Prospective clinical trial of factors predicting theearly development of incisional hernia after midline laparotomy.
J Am Coll Surg 2010; 210: 210-9.
5. Van Ramshorst GH, Nieuwenhuizen J, Hop WC, Arends P,
Boom J, Jeekel J, et al. Abdominal wound dehiscence in adults:
development and validation of a risk model. World J Surg 2010;34: 20-7.
6. Muysoms F, Campanelli G, Champault GG, DeBeaux AC,
Dietz UA, Jeekel J, et al. EuraHS: the development of aninternational online platform for registration and outcome
measurement of ventral abdominal wall hernia repair. Hernia.
2012; 16(3): 239-50.
7. SPERANZINI MB, DEUTSCH CR. Giant incisional hernias.
Arq Bras Cir Dig 2010; 23(4): 280-286.
8. Dunne JR, Malone DL, Tracy JK, Napolitano LM.
Abdominal wall hernias: risk factors for infection and resource
utilization. J Surg Res 2003; 111: 78-84.
9. Finan KR, Vick CC, Kiefe CI, Neumayer L, Hawn MT.
Predictors of wound infection in ventral hernia repair. Am J Surg
2005; 190: 676-81.
10. Krpata DM, Blatnik JA, Novitsky YW, Rosen MJ. Evaluationof high-risk, comorbid patients undergoing open ventral hernia
repair with synthetic mesh. Surgery, 2013; 153(1): 120-5.
11. Grevious MA, de la Torre JI, Souza JM, Steinberg JP,
Wilhelmi BJ, Dumanian GA, Hasan JS. Abdominal wallreconstruction treatment and management. Medscape, 2012;
emedicine.medscape.com/article/1297226.
12. Novitsky YW, Cobb WS, Kercher KW, Matthews BD, SingRF, Heniford BT. Laparoscopic ventral hernia repair in obese
patients: a new standard of care. Arch Surg. 2006; 141(1):57-61.

ROLUL IMC ȘI AL CO-MORBIDITĂȚILOR ASUPRA DURATEI DE SPITALIZAREÎN CAZUL REPARĂRII CHIRURGICALE A EVENTRAȚIILOR POSTOPERATORIIREZUMAT
Introducere. Evaluarea preoperatorie a IMC ca factor de risc si a co-morbiditatilor pot previziona durata despitalizare in cazul repararii eventratiei peretelui abdominal. Am efectuat un studiu retrospectiv, despre repararea
eventratiilor postoperatorii in Clinica noastra pe o durata de 18 luni, dupa inregistrarea la platforma online
EuraHS cu scopul de a evalua caracteristicile demografice ale pacientilor (varsta, sex, IMC), comorbiditatile si
rezultatele postoperatorii (complicatii precoce si tardive si durata totala de spitalizare) .
Material si metoda. A fost selectionat retrospectiv un grup de 126 pacienti operati pentru eventratie. S-au colectat
date despre caracteristicile demografice ale pacientilor (varsta, sex, IMC), comorbiditatile si rezultatele
postoperatorii (complicatii precoce si tardive, durata totala de spitalizare). Datele sunt prezentate ca valori
medii si procentuale. Coeficientul de corelatie (Pearson), si limita de confidenta 95% (p<0.05) a fost apreciata
pentru fiecare variabila.

Fiziologia - Physiology • 2015.25.4(88)45Rezultate. Varsta medie a grupului a fost de 62.7 ani cu preponderenta sexului feminin (76%) comparativ cu
sexul masculin (24%). Localizarea eventratiei pe linia mediana, cu toate posibilitatile descrise de EuraHS a fost
intalnita in majoritatea cazurilor (78.6%), urmata de localizarea in fosa iliaca dreapta (9.5%). Niciunul dintre
pacienti a prezentat scorul SOC 4. Majoritatea au avut scorul SOC calculat in valoare de 3. Dintre cei 126
pacienti, 54 au prezentat BMI < 29.99; 72 au avut BMI > 29.99 si 7 mai mult de 40. S-a observat o puternica
corelatie statistica intre BMI si scorul SOC cu coeficient de corelatie r (Pearson) = 0.36, si p = 0.0001. durata
de spitalizare a avut valori cuprinse intre 2 si 38 zile. Deasemenea intre BMI si durata de spitalizare s-a observat
o puternica corelatie statistica cu coeficient de corelatie r (Pearson) = 0.27 si p = 0.002. Intre scor SOC si
durata de spitalizare nu s-a observat nicio corelatie(r = 0.14 si p = 0.12 (> 0.05). In timpul actului operator s-au
semnalat 2 cazuri de leziuni intestinale iatrogene. In ceea ce priveste rata complicatiilor postoperatorii s-au
semnalat 9 cazuri (7.14%) de complicatii precoce (4 cazuri de hemoragie, 3 cazuri de ocluzie si 2 cazuri dehematom) si 5 (3.96%)complicatii tardive (3 cazuri de recidiva si 2 cazuri de abces parietal), conform clasificarii
EuraHS, la pacienti cu IMC >30.
Concluzii. IMC are o corelatie puternica cu durata de spitalizare. Definitii si clasificari adecvate, asa cum suntdisponibile online prin inregistrare la platforma EuraHS ofera o evaluare pre si postoperatorie a pacientilor
intr-o maniera unitara si astfel creste calitatea studiilor si a meta-analizelor acestei patologii.
Key words: SOC score, BMI, hospital stay, surgical outcomes
Cuvinte cheie: scor SOC, IMC, durata de spitalizare, rezultate chirurgicale

46

Fiziologia - Physiology • 2015.25.4(88)GABAERGIC POTENTIATION OF REDUCED BOWEL
MOVEMENTS INDUCED WITH ZINC SULPHATE BY BACLOFEN
RUFUS OJO AKOMOLAFE§1, OLUSOJI ADEOLA ADALUMO1 AND
KAYODE DOMINION SAMUEL BAMITALE2
1Department of Physiological Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria
2Department of Medical Pharmacology and Therapeutics, Obafemi Awolowo University, Ile-Ife, Nigeria

ABSTRACTThis study evaluated the influence of orally administered zinc sulphate on bowel movement of rats by the faecal output
and gastrointestinal transit time in the rats. The effects of zinc sulphate on feeding as well as water intake were also
determined. The dose of zinc sulphate that yielded the maximal effect was administered alongside baclofen toinvestigate the role of GABA on bowel propulsion. All the three doses of zinc sulphate (1 mgkg -1, 5 mgkg-1 and 10
mgkg-1) produced significant reduction (p < 0.05) in total faecal pellets produced in eight hours of study (12.00 ± 0.93,
9.17 ± 0.31 and 3.17 ± 0.17) respectively, compared with the control group (15.67 ± 1.38). Also, the three doses ofzinc sulphate produced dose-dependent significant reduction (p < 0.05) in the faecal mass (1.78 ± 0.17, 2.50 ± 0.22
and 1.07 ± 0.10) respectively when compared with control group (3.39 ± 0.25).The total transit time of zinc sulphate(10 mgkg-1) treated group (593.8 ± 2.84 min) increased significantly (p < 0.0001, t = 26.98) compared with the control
group (251.2 ± 5.48 minutes). Zinc sulphate (5 mgkg-1 and 10 mgkg-1) significantly (p < 0.05) reduced food intake(10.83 ± 0.54 and 12.50 ± 0.51) respectively while (1 mgkg-1) produced increase (p < 0.05) in food intake (22.50 ± 0.56)
when compared with the control group (18.33 ± 0.67). Administration of zinc sulphate and zinc + baclofen significantly(p < 0.001, f = 63.9) reduced faecal pellet output (4.80 ± 0.74 and 1.60 ± 0.25) respectively when compared with
control (10.2 ± 0.74). The study concluded that zinc sulphate reduced bowel movement in rats stimulating γaminobutyric acid (GABAB) receptors.
Keywords: Zinc sulphate, Bowel, GABAB, Baclofen and Transit Time

INTRODUCTION
Bowel movements (Motility) in the gastrointestinal
tract is an involuntary and self-generated movement of
the entire gut which accounts for the propulsion, mixing,
and reservoir functions necessary for the orderly
processing of ingested food and the elimination of waste
products. Propulsion is the coordinated motion ofingested foods, liquids, gut secretions, and sloughedcells from the mucosa through the gastrointestinal tract. It
propels the food from the stomach into the small intestine
and along the small intestine, with appropriate timing for
efficient digestion and absorption. Peristaltic waves
propel undigested material into the large intestine andeliminate waste through defecation [1].
Trituration, the crushing and grinding of ingested foodby the stomach, decreases particle size, increasing thesurface area for action by digestive enzymes in the small
intestine. Mixing movements mix pancreatic, biliary, andintestinal secretions with nutrients in the small intestineand bring products of digestion into contact with theabsorptive surfaces of the mucosa. Reservoir functions
are performed by the stomach and colon. The body of thestomach stores ingested food and exerts steadymechanical forces that are important determinants ofgastric emptying. The colon holds material during thetime required for the absorption of water and stores theresidual material until defecation is convenient. Each of
the specialized organs along the digestive tract exhibits avariety of motility patterns. These patterns differ
depending on factors such as time after a meal, awake orsleeping state, and the presence of disease. Motorpatterns that accomplish propulsion in the esophagus,
small and large intestines are derived from a basic
peristaltic reflex circuit in the ENS [1,2].
The musculature of the gastrointestinal tract is for themost part smooth muscle. Electrical slow waves and action
potentials are the principal sources of electrical activity inthe gastrointestinal musculature. Gut smooth muscles
have properties of a functional electrical syncytium. A

Received September 10th 2015. Accepted October 12th 2015. Address for correspondence: Rufus Ojo Akomolafe, Department of
Physiological Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria; phone: +2348034393396; e-mail:rufakom@yahoo.co.uk

Fiziologia - Physiology • 2015.25.4(88)47hierarchy of neural integrative centers in the central
nervous system (CNS) and peripheral nervous system(PNS) regulates and controls the functioning of the gut.
The gastrointestinal tract is innervated by the sympathetic,
parasympathetic, and enteric divisions of the autonomic
nervous system (ANS). Vagus nerves carry afferent
sensory information to the brain and parasympathetic
autonomic efferent signals to the digestive tract.
Splanchnic nerves convey sensory information to thespinal cord and sympathetic autonomic efferent signals tothe digestive tract. The enteric nervous system (ENS)
functions as a mini-brain within the gut [1-5].
Existence of GABA neurons in enteric ganglia ofmany species has been described, including human androdents. In the rat and human intestine, enteric
GABAergic neurons have at least three distinguishable
components viz; neurochemical populations and twomorphological cell types (Krantis, 2000). Enteric
GABAergic ﬁbers are present in great quantity, branchingextensively all around the ganglionated andnon-ganglionated enteric nerve interconnection of the gut
wall [6]. In addition to GABAergic nerve ﬁbers, themucosa of the rodent and human bowel have GABA in
endocrine-like cells in the gastric antrum over the wholedistance to the distal colon within them [6].
There is paucity of evidence on the role of GABA
receptors in the alteration of gastrointestinal motility byzinc salts, hence this study.

MATERIALS AND METHODS
Animal Care and Management
Twenty-four adult male Wistar rats weighing 120 –180 g were obtained from the Animal House, College of
Health Sciences, Obafemi Awolowo University, Ile-Ife.
The rats were kept in plastic rat cages for two weeks toacclimatize in the Animal Holding, Department of
Anatomy and Cell Biology, where they were given accessto feed (Caps Feed, Osogbo) and water ad-libitum. Later,
the animals were housed in modified cages in which foodand water consumption as well as faecal pellet output
can be assessed.
Chemicals and Drugs
Zinc sulphate was obtained from May and Baker
Laboratory Chemicals Limited, Dagenham, England.
Grade-S.L.R; CAS 7446-20-0 and Baclofen tablets waspurchased from MA Halder TEVA UK. Limited 83915 T.
For the experiments the both drugs were dissolved in
distilled water for oral administration.
Experimental Design
The rats were randomly divided into four groups
consisting six rats each. Group 1 which served as thecontrol, was given distilled water while groups 2, 3 and 448

received 1, 5 and 10 mg/kg/day oral administration ofzinc sulphate respectively.
Test on Gastrointestinal Motility (Charcoal Meal)
In Rats
Gastrointestinal transit time in the rats wasdetermined using the method of [7]. The rats weredeprived of food for three hours but had free access towater. The dose of zinc sulphate (i.e. 10 mgkg-1) that
produced the most significant change in faecal pellet
output was administered to rats of groups 2, 3 and 4.
After 90 min, 0.3ml of an aqueous suspension of 5%
charcoal in 10% water was administered to each animal
orally. Sixty minutes later (time 150 min), the rats were
given free access to food. They were observed at 20 min
intervals until the first appearance of charcoal in theirfaecal pellet. Charcoal was observed on the faeces
under normal light, using a hand lens to identify the blackspots. The transit time was based on time for charcoal tobe eliminated from the gastrointestinal tract [7].
Test on GABAβ Receptor: GABAB receptor agonist(baclofen) was administered orally at 1 mg/kg 15 minutes
before administration of zinc sulphate.
Test to determine Faecal Pellet Output and WeightCollection of Faecal pellets followed the method of [8]with slight modifications. Faecal pellets were counted at
two-hour intervals for eight hours following zinc salt
administrations. The faecal droppings were collected on
the absorbent paper placed beneath the rat perforated
cages. The weight of the air-dried pellets were
determined after 24-hours.
Determination of food and water intake: Food andwater intake was determined during the experiment as
described by [9].
Statistical Analysis: Statistical analyses werecarried out using Graphpad Prism 5.0 version software.
Data are presented as mean ± SEM. Analysis of
Variance (ANOVA) was performed followed by Newman
Keuls’ post-hoc test. A value of p < 0.05 was considered
statistically significant.

RESULTS
Pre-treatment with zinc sulphate (5 mgkg-1 and 10
mgkg-1) significantly reduced (p < 0.05) food intake(10.83 ± 0.54 and 12.50 ± 0.51) respectively while (1mgkg-1) produced increase (p < 0.05) in food intake(22.50 ± 0.56) when compared with the control group(18.33 ± 0.67) (p < 0.05) (Figure 1).

Fiziologia - Physiology • 2015.25.4(88)(p < 0.05) in the total faecal weight (1.78 ± 0.17, 2.50 ±0.22 and 1.07 ± 0.10) respectively when compared with
control group (3.39 ± 0.25). Of the three doses of zincsulphate, 10 mgkg-1 produced the highest reduction in
faecal weight in 8 hours (1.07 ± 0.10) while 5 mgkg-1
produced the least in 8 hours (2.50 ± 0.22) (Figure 4).

Fig. 1. Effect of Zinc Sulphate Administration on Feeding:Pre-treatment with zinc sulphate (5 mgkg-1 and 10 mgkg-1)
significantly reduced (p < 0.05) food intake (10.83 ± 0.54 and12.50 ± 0.51) respectively while (1 mgkg-1) produced increase(p < 0.05) in food intake (22.50 ± 0.56) when compared with thecontrol group (18.33 ± 0.67) (p < 0.05).

Pre-treatment with zinc sulphate (1 mgkg-1, 5 mgkg-1
and 10 mgkg-1) produced a dose-dependent reduction (p< 0.05) in water intake (17.50 ± 0.66, 17.89 ± 0.61 and
16.74 ± 1.11) respectively compared with the control
group (21.42 ± 0.93) (Figure 2).

Fig. 4. Effect of Zinc Sulphate Administration on the Weightof Faecal Pellets, * = significantly different from control (p< 0.0001).

The total transit time of zinc sulphate (10 mgkg-1)
treated group (593.8 ± 2.84 min) increased significantly(p < 0.0001, t = 26.98) compared with the control group(251.2 ± 5.48 min) (Figure 5).

Fig. 2. Effect of Zinc Sulphate Administration on Water
Intake: Pre-treatment with zinc sulphate (1 mgkg-1, 5 mgkg-1
and 10 mgkg-1) produced a dose-dependent reduction (p < 0.05)
in water intake (17.50 ± 0.66, 17.89 ± 0.61 and 16.74 ± 1.11)
respectively compared with the control group (21.42 ± 0.93).

The three doses of zinc sulphate (1 mgkg -1, 5
mgkg-1 and 10 mgkg-1) produced a significant dose
dependent reduction (p < 0.05) in the number of total
faecal pellets (12.00 ± 0.93, 9.17 ± 0.31 and 3.17 ±0.17) respectively when compared with control group(15.67 ± 1.38) (Figure 3).

Fig. 5. G.I Transit Time Induced by Zinc Sulphate, * =significantly different from control (p < 0.0001). The totaltransit time of zinc sulphate (10 mgkg-1) treated group (593.8 ±2.84 min) increased significantly (p < 0.0001, t = 26.98)
compared with the control group (251.2 ± 5.48 min).

Zinc sulphate and zinc + baclofen significantly (p <0.001, f = 63.9) reduced faecal pellet output (4.80 ± 0.74
and 1.60 ± 0.25) respectively when compared with
control (10.2 ± 0.74) (Figure 6).

Fig. 3. Effect of Zinc Sulphate Administration on Number of
Faecal Pellets. The three doses of zinc sulphate (1 mgkg-1, 5
mgkg-1 and 10 mgkg-1) produced a significant dose dependent
reduction (p < 0.05) in the number of total faecal pellets (12.00± 0.93, 9.17 ± 0.31 and 3.17 ± 0.17) respectively whencompared with control group (15.67 ± 1.38).

All the three doses of zinc sulphate (1 mgkg-1, 5
mgkg-1 and 10 mgkg-1) produced a significant reduction
Fiziologia - Physiology • 2015.25.4(88)Fig. 6. Baclofen Potentiation of Zinc Induced Reduction in
Frequency of Defecation. * = significantly different from control;§ = significantly different from ZnSO4 only; β = significantly
different from baclofen only (p < 0.0001, f = 63.9).

49

Zinc sulphate and zinc + baclofen significantly (p <0.001, f = 63.9) reduced faecal pellet output (4.80 ± 0.74
and 1.60 ± 0.25) respectively when compared with
control (10.2 ± 0.74).

DISCUSSIONS
Zinc Sulphate Induced Alterations in Food Intake.
In a report by [10], the messenger RNA (mRNA)
expression of neuropeptide Y (NPY) and orexin in the
hypothalamus significantly increased after three hours oforal administration of zinc which was not observed in thegroup given zinc sulphate intraperitoneally and theincreased food consumption was inhibited by vagotomy.
The route of administration used in this current study isthe same as that of [10]. Also, the dose reported by theauthors to have stimulated food intake (19 mmol/kg (3.0mg/kg) of zinc sulphate (ZnSO4) in saline) also
corresponds to the low dose of (ZnSO4 1 mg/kg) that
increased food consumption in this current study. The
only difference being the solvent. In this study, distilledwater was used while Ohinata et al., used saline. The
mRNA expressions of hypothalamic peptides, such as
orexin and NPY, increased after oral administration ofzinc causing increased food intake.
Ghrelin is a potent orexigenic factor that is
synthesized and released mainly from the oxyntic cells of
the stomach, it is also released from duodenum, ileum,caecum and colon [11,12]. It is possible that at a low
dose of zinc sulphate, ghrelin is released from the oxyntic
cells which then increased food intake. This is because
peripheral and centrally administered ghrelin have beenshown to increase food intake in rodents [13,14] throughthe arcuate nucleus of the hypothalamus by increasingc-fos gene expression in the ARC Neuro-peptide Y(NPY)-synthesizing neurons [15].
Further, the anorexigenic effects of the zinc sulphate
tested can be attributed to the peptide-YY secreted
majorly from the L-cells of the distal gastrointestinal tract,
particularly the ileum, colon and rectum [16,17]. This
peptide PYY exists in two major forms: PYY1–36 and
PYY3–36, the latter is the peripherally active anorectic
signaling peptide. The observed anorectic effect of zincsalt at high doses therefore may be by directly releasing
peptide PYY from the distal part of the gut. Peripheral
administration of PYY3–36 to rodents has been shown to
inhibit food intake in rodents [18,19].
Another weight of evidence explaining the effect ofzinc on appetite is by indirect release of Pancreatic
Poly-peptide (PP-Peptide). It is synthesized from thecells at the periphery of the islets of the endocrine
pancreas, and to a lesser extent in the exocrine pancreas,colon and rectum. Studies have identified Zn2+ as a G
Protein-Coupled Receptor 39 activating factor (GPR39)[20-22] and GPR39 is expressed in the endocrine

50

pancreas [23]. Moreover, in the endocrine pancreas Zn2+functions as a chemical messenger co-stored and released[23] suggesting a correlation between Pancreatic
Poly-peptide (PP-peptide) and Zn2+. Peripheral treatment
with PP-Peptide was shown to reduce food intake [24,25]
just as higher dose of zinc in this study, also normal-weight
human volunteers given an infusion of PP-Peptide
demonstrated decreased appetite, and a 25% reduction infood intake over the following 24 h [26].
Although circulating PP is unable to cross the
blood–brain barrier, it may exert its anorectic effect on the
ARC via the area postrema [27]. This effect may occur
via the Y5 receptor as there is no response in Y5
receptor-knockout mice, although the anorectic effect is
not reduced by Y5 receptor antisense oligonucleotides[28]. Following the peripheral administration of PP, theexpression of hypothalamic NPY and orexin mRNA issignificantly reduced [25]. PP may also exert someanorectic action via the vagal pathway to the brainstem,as vagotomy seems to reduce its efficacy [25].
Zinc Sulphate Induced Effect on Water Intake,
Faecal Pellet Output and Transit Time.
The effect of zinc sulphate on water intake in thisstudy is in agreement with a similar finding on the effectof zinc acetate on water intake. The probablemechanisms responsible for the observation were also
extensively discussed in the report [9]. Zinc sulphatefrom previous studies have been shown to have an
inhibitory effect on faecal pellet output. The observationfrom this experiment is also related with the findings of[9]. The detailed possible mechanisms by which zinccauses the reduction were as well discussed.
Zinc sulphate significantly increased the total transit
time in the rats used for this experiment. The increased
total transit time points to a reduction in propulsion
velocity through the entire bowel of the experimental
animals. Other details on the effect of zinc salts on
gastrointestinal transit time has also been discussed in aprevious report [9].
GABAergic Potentiation of Reduced Bowel
Movements Induced by Baclofen
Baclofen significantly reduced the frequency ofdefecation as observed in this experiment. It exerted an
additive effect when given together with zinc sulphateproducing further reduction in faecal pellet output.
Gamma Amino-butyric Acid (GABA) exerts both
stimulatory and inhibitory inﬂuence over enteric neuronalsystem, this is contingent upon the GABA receptor
subtypes excited: stimulation is via GABAA receptors andinhibition is via GABAB receptors. GABA is implicated in
the neural and endocrine/paracrine regulation of diverse
functions within the gut. GABA can either stimulate (via
GABAA receptors) or inhibit (via GABAB receptors)
intrinsic cholinergic motor neurons that are responsible
Fiziologia - Physiology • 2015.25.4(88)for the ascending excitation limb of the peristaltic reﬂex.
Thus perturbation of enteric GABA transmission could
potentially contribute to the cholinergic hyper-contractionof the gut [6].
Within the mucosa, GABAergic nerve ﬁbers branchextensively within the small nerve plexus located beneaththe base of the crypts, which serve as the interface forthe innervation of the mucosa [6]. This allows baclofen toexert its inhibitory influence on bowel motility as shownby the result of the current study.
In addition to the neural localization, GABA is
synthesized, stored, and secreted by mucosal
endocrine-like cells throughout the rat antrum andintestine [29,30]. The mucosa, therefore, may well be
under the inﬂuence of GABA released from local neurons[31] as well as GABA secreted from endocrine cells. In
the rodent gastric antrum, GABAergic endocrine-like cells
resolve into subpopulations of cells colocalizing either
gastrin (G cells) or somatostain (D cells). Baclofen may
therefore modulate G cells’ secretion by exerting
inhibitory influence on the secretion of gastrin known tostimulate gastric motility on one hand while stimulating
the secretion of somatostatin (D cells) from the gastricantrum. Baclofen may likewise stimulate the secretion of
Gastric Inhibitory Peptide (GIP) by the K cells found in
the antrum of the stomach to further inhibit gastric motility
leading to reduction in faecal output.
In the rat, GABAergic neurons account for >5–8% of
the total number of myenteric neurons in the large
intestine, baclofen through its action on GABAB receptors
is believed to trigger the release of somatostatin from D
cells found in the pancreas and the small intestine to
inhibit gastric motility. In the rodent intestine, GABAergic
endocrine-like cells display a morphology similar to
D-type endocrine cells [30]. GABA released from thesecells into the local circulation or interstitial space may act
as a classic gastrointestinal hormone or a local paracrine
or autocrine factor. Baclofen may further stimulate thesecretion of Gastric Inhibitory Peptide (GIP) from the Kcells which are found in the jejunum and duodenum toalso inhibit gastric motility.
Micro-dialysis sampling of myenteric plexus
neurotransmitter release established a substantial
inhibitory effect of the GABAB receptor agonist (baclofen)
on dog’s intestinal acetylcholine (ACh) release. This was
sensitive to GABAB receptor antagonism [32]. It has been
discovered that baclofen, a GABAB receptor agonist
inhibit dog’s intestinal Ach release. GABA antagonist wasreported to reverse this inhibitory effect of baclofen on
ACh release [32]. It is evident from various findings thatGABAB receptors exert an inhibitory effect on release ofACh, without any significant effect on inhibitory
neurotransmitter release or synthesis [33], this may
partially explain the results as obtained in rats from thisstudy. Further, in rat intestine, a neural circuit comprising
GABA-, somatostatin-, and enkephalin-containing
Fiziologia - Physiology • 2015.25.4(88)interneurons was suggested to be crucial for the
regulation of descending relaxation [6].
Furthermore, GABA receptors was reported tomodulate release of 5-Hydroxytryptamine (5-HT) fromendocrine cells by Nakajima and colleagues [34].
GABAB receptors have also been shown to regulate therelease of enterochromaffin cell-derived serotonin fromguinea-pig small intestine, the action of baclofen from theresult of this study can simply be thought to be by way ofstimulating the release of 5-HT which binds to 5HT3
receptor while inhibiting 5HT4. The consequence of whichis the slowing down of gastrokinetic activities, reductionin the rate of gastric emptying due to reduction inperistalsis of jejunum and duodenum.
In addition to GABA receptors modulating 5-HT
release from enterochromafﬁn cells, it has also beenreported to modulate histamine release from mast cells,
gastric mucous secretion [35], prostaglandins released
from interstitial cells [36], and mucosal electrolyte
transport [31,37].
GABAergic myenteric interneurons in the human andrat colon fall into three neurochemically distinct,
non-overlapping subpopulations: 1) neurons withsomatostatin, which account for ~40% of the GABA
population; 2) neurons with enkephalin, which accountfor ~10 % of the GABA population; and 3) neurons with
NADPH diaphorase-related nitric oxide synthase (NOS)
activity, which account for ~20% of the GABA populationand neither somatostatin- nor enkephalin-positive cells
show nitric oxide synthase (NOS) activity [5]. The
reduction in bowel movement in this study may be aconsequence of direct excitation of NOS activity bybaclofen at enteric synapses. This is because, within the
submucosa and mucosa, GABAergic cells were seen to
co-localize either somatostatin or NOS, but not both [6].
Myenteric GABA neurons co-localizing enkephalin
may represent inhibitory innervation of the muscularis,
which modulates cholinergic mediated contractions[38,39], whereby the release of acetylcholine and othertransmitters is attenuated [40]. This is one of the possible
mechanisms by which baclofen reduced bowel motility as
observed in the current study.
GABA and its receptor-mediated effects on bowel
motility seem to be dependent on an intact ENS as
isolated rat smooth muscle cells are unresponsive toaddition of GABA. Moreover, independent of innervationby the central nervous system, peripheral
GABAB receptor activation induces TTX- andatropine-sensitive gastric contractility in vitro [41],suggesting that baclofen locally increases gastric tone
through activation of intrinsic cholinergic neurons.
The propulsive velocity of a dilated balloon along
rabbit colonic preparations was significantly reduced by
GABAB receptor activation with baclofen in a study by
Tonini and colleagues [42]. This is in concert with thefindings from our current study. Further, baclofen had a51

minor inhibitory effect on colonic longitudinal muscle tone
but a more significant inhibitory effect on TTX- andhyoscine-sensitive electrically stimulated responses,
suggesting that the inhibitory effects of the
GABAB agonist on colonic activity in the rabbit isdependent on cholinergic neurotransmission [42].

CONCLUSION
It is evident that baclofen acting through GABAB
receptors reduces bowel movements through many
concerted mechanisms, modulating the activities of many
endogenous neurotransmitters within the gut. We
conclude that zinc sulphate induced reduction of
propulsive movement within the gut is mediated by
GABAB receptor stimulation. This observation opens newopportunities for understanding bowel function in healthand disease.

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53

BIODISTRIBUTION OF FE3O4/CITRIC ACID NANOPARTICLESIN A MURINE MODELNICU FLORIAN TRINCU1, TUDOR-ADRIAN BALȘEANU2,BOGDAN SILVIU UNGUREANU3, IONICA PIRICI4,SANDRA ALICE BUTEICA1, ADRIAN SĂFTOIU3, JOHNY NEAMȚU1
1Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, Romania
2Department of Functional Disciplines, University of Medicine and Pharmacy of Craiova, Romania
3Research Center of Gastroenterology and Hepatology, Gastroenterology Department, University of Medicine and Pharmacy

of Craiova, Romania
4Research Center for Microscopic Morphology and Immunology, University of Medicine and Pharmacy of Craiova, Romania

ABSTRACTIron oxide magnetic nanoparticles of about 20÷150 nm were synthesized by chemical co-precipitation of Fe2+ and Fe3+
in NH4OH solution and were coated with citric acid. The goal of this in vivo study was to analyze the biodistribution of
MNPs in different organs by means of electron paramagnetic resonance (EPR) and histological analysis with Prussian
blue after intravenous administration to Sprague Dawley rats. Their distribution was studied at 24 hours after
intravenous administration through the jugular vein at a dose of 15 μmol of Fe/kg. The results showed larger depositsof nanoparticles in spleen and liver and not so significant ones in kidney, pancreas and lung.
We have studied these aspects with our novel iron oxide formulation, which can be used in the research of therapeuticareas and magnetic resonance imaging (MRI) and justify further assessment.
Keywords: MNP, biodistribution, murine model

INTRODUCTION
Owing to the evolving field of bioapplications,magnetic iron oxide nanoparticles have found different
directions such as magnetic separation and biodetectionof cells, proteins, viruses etc., clinical diagnosis andtherapy (magnetic resonance imaging and magnetic fluid
hyperthermia), targeted drug delivery as well as cancer
treatment [1]. When testing and designing innovative
nanoparticles it should be taken into account importantparameters like their pharmacokinetics properties andbiodistribution characteristics. The first step in
nanoparticles retention and cellular internalization is theirpassage from the bloodstream to the tissue of interest,
followed by binding of molecular target so that it may
reach an adequate concentration of nanoparticles in thedesired tissue. However, a large percentage ofsystemically injected nanoparticles have a narrow
therapeutic index because they are quickly eliminatedfrom the bloodstream by the reticuloendothelial systemand the mononuclear phagocyte system, of liver, spleen
and bone marrow [2,3].

Also serum half-life and magnetic nanoparticles
biodistribution may be influenced by coating materials,
nanoparticle core and linker together with synthesis andpurification procedures [4].
While naked magnetic nanoparticles have ahydrophobic nature they are not stable in normal
physiological conditions and tend to form aggregates[5-7]. In water based magnetic fluids, the magnetic phase(mainly iron oxides) requires different coating materials
that give them stability and compatibility with biologicalfluids [8-11].
Coating materials used to sterically stabilize the ironoxide nanoparticles often contain carboxyl functionalgroups. These functional groups are popular because
they form stable covalent linkage between coating agentsand iron oxide surface.
To stabilize the suspension of magnetic nanoparticles
we used citric acid, which is adsorbed on the surface offreshly prepared iron oxide nanoparticles. The terminal
carboxylic functional group of citric acid, not only thatmakes the nanoparticles water dispersible, but also
provides a starting point for surface functionalization.

Received September 10th 2015. Accepted October 12th 2015. Address for correspondence: Nicu Florian Trincu, PhD, Faculty of
Pharmacy, University of Medicine and Pharmacy of Craiova, 2-4 Petru Rares Street, 200349 Craiova, Romania; phone:+40746276998, e-mail: nicu_trincu@yahoo.com

54

Fiziologia - Physiology • 2015.25.4(88)Because a suspension of magnetic nanoparticles inan artificial physiological environment might not fully
simulate different types of immune system cells or
biological system, we focused our attention on evaluating
the pharmacokinetics of nanoparticles in a in vivo
situation, using a murine model.
The aim of this research is to investigate thebiodistribution of biocompatible magnetite nanoparticles
covered with citric acid in Sprague Dawley rats, after
intravenous administration.

MATERIALS AND METHODS
1. Magnetic nanoparticles
Citric acid-coated superparamagnetic iron oxide
nanoparticles (MNPs) were obtained at the Institute ofMacromolecular Chemistry "Petru Poni" (Iasi, Romania)(Figure 1).

Fig. 1. MNPs seen at 20x with Olympus
SZ-4045ESD Microscope

The coated magnetic nanoparticles were obtained insitu by chemical co-precipitation of Fe3+ and Fe2+
solutions with NH4OH solution, at pH 9–10, followed bydispersing in a solution of de-ionized water with citric acid.
Briefly, 5.40 g of FeCl3 x 6H2O (1M) and 1.98 g of FeCl2 x
4H2O (2M), were dissolved in 25 mL HCl (2M) , and theresulted solutions were added to 90 mL of de-ionizedwater containing 3.25 g citric acid, under argon
atmosphere and vigorously stirred. Further, 20 mL of
NH4OH solution (5M) was added over the previous
mixture and stirred for 30 min until the colour shift fromorange to black indicating the formation of an iron oxide
nanoparticles suspension. The suspension was heated to
80°C and stored for 2 hours followed by centrifugation for
5 minutes at 900 × g. The supernatant was eliminatedand the coated nanoparticles were washed several times
with water and then finally dispersed in 100 ml de-ionized
Fiziologia - Physiology • 2015.25.4(88)water and sonicated for 3–5 min. The hydrodynamic
diameters are between 20–150 nm.
2. Procedures
Our studies have been conducted using a group often Sprague–Dawley male rats of 20 weeks old and withan average weight of 400g. The animals have beenraised in standard-sized cages for rats in sterile
laboratory environment. All the experiments wereapproved by Ethics Committee of the University of
Medicine and Pharmacy of Craiova, Romania, and wereconducted in accordance with the European Legislation
regarding animal rights, Directive 2010/63/EU.
The experimental models were anesthetized with an
intraperitoneal injection using a mixture of 100 mg/kg
ketamine hydrochloride (Richter, Germany) and 10 mg/kg,
xylazine hydrochloride (Interchemie, Netherlands). A
suspension of citric-acid coated magnetic nanoparticles(14 mg Fe/mL) was prepared in glycolyzed solution andinjected slowly through the jugular vein at a dose of 15
μmol of Fe/kg. Control experiments were carried out on
two rats without injecting them.
At 30 minutes after injection, blood sample werecollected from rats tail vein, to be used as reference
concentration in determining nanoparticle accumulationin organs. Animals were allowed to recover after theprocedure and were monitored to be euthanized 24
hours after MNPs administration, with 120mg/kg
pentobarbital sodium (Produlab Pharma B.V.,
Netherlands), intraperitoneally administered. Rats werethen perfused transcardially with saline for 20 min, andvarious organs were collected (liver, spleen, pancreas,kidney, brain and muscle), washed thoroughly with saline,
and were frozen at -80 °C [12]. These tissues samples
were used for determination of magnetic nanoparticles
content. Half of the samples were analyzed using
electron paramagnetic resonance (EPR) following alyophilization process [13].
The other half of the organ samples were kept
overnight in a neutral buffered formalin 10% andordinarily processed in paraffin. Sections of 3-4 µm
thickness of the right lobe of the liver and spleen sections
were sectioned axially and sections of the lung, heart andkidney were cut in the coronal plane. All sections were
stained with Prussian Blue and counterstained withhematoxylin and eosin followed by examination underlight microscopy [14,15].
RESULTS
Magnetic nanoparticles are not delivered only to theliver, but they are also transferred to other organs of therat [16]. Considering the concentration of nanoparticles inthe sample at 30 minute after MNPs administration, as
55

the reference concentration, CREF, the magnetic
nanoparticles had the following organ concentrations,
measured with EPR: Liver - (120 +/- 24) CREF; Spleen (706 +/- 141) CREF; Kidney: (3.3 +/- 1.5) CREF; Pancreas:(0.7 +/- 1.5) CREF. In brain and muscle the MNP
concentrations were approximately zero.

some deposits were noted in the periphery of thecapillary sinusoids and also in Kupfer cells and Ito cells.
Smaller deposits were found in the portal andcentrilobular spaces. As for the kidneys, MNPs wereprimarily located within the proximal convoluted tubules
and to a lesser extent in the distal convoluted tubules,
more precisely in the epithelial cells which cover thecollector tubes.

Fig. 4. MNP deposits within the spleen. Prussian Blue staining 40x

Fig. 2. EPR spectra at room temperature of different lyophilized
rat organs 24 h after MNPs administration

Fig. 5. Histological study of rats’ livers sacrificed 24 hours after the
intravenous administration of MNPs. Prussian Blue staining 40x

Fig. 3. MNP concentrations in organs 24 h after intravenous
administration

Iron nanoparticles content in the spleen and liver ishigher than in other organs studied (Figure 3), which isconsistent with previous studies [17].
Histopathological assessment confirmed all the deposits
using Prussian Blue staining (Figures 4-6). As expected,
in spleen were found nanoparticle deposits in and out of
the macrophages, especially in the red pulp. In the liver

56

Fig. 6. MNP deposits within the kidney. Prussian Blue staining 40x

Fiziologia - Physiology • 2015.25.4(88)DISCUSSION
This study had a few limitations because the numberof rats was too small for an accurate analysis of thenanoparticles distribution and second, because due totechnical challenges it could not determine the absoluteconcentration of MNPs but only a relative one.
Briley-Sæbø et al. reported that for nanoparticles witha diameter less than 40 nm, both the biodistribution andblood half-life of iron oxide particles are influenced by thematerial of the coating rather than the average particle size[18]. Our nanoparticles are coated with citric acid which
contributes to the increase of the hydrodynamic diameter.
Gu et al. evaluated the biodistribution anddegradation of PEGylated iron oxide nanocrystals inmice. The iron accumulated in the organs 24 h after
intravenous injection (5 mgFe/kg) of Feridex and PEG
phospholipid-coated IO nanocrystals, (5÷30 nm) confirm
the results obtained in this study [19].
A more ample study was conducted by Jain el al. [20]in which they studied the distribution of iron oxide
nanoparticles coated with oleic acid and pluronic in rats,
after intravenous administration. From the analysis withinductively coupled plasma-mass spectrometry (ICP-MS),
it was determined that about 55% of the injected
nanoparticles were stored in the liver at 6 h but thendeclined to 20% after 24 h and raised at about 50% at 3
weeks after nanoparticle injection. An explanation ofrising levels of iron in the liver at 3 weeks could be due tothe intraportal transport of free iron released frombiodegraded MNPs or or due to the formation of smaller
particles as a result of degradation in the spleen.
Furthermore, Gamarra et al. [21] observed thatdextran coated magnetite nanoparticles (80-150 nm)
reach maximum concentration in liver at 95 min after the
intravenous administration with the half-life of thenanoparticles in the blood of 11.6 ± 0.6 min measured by
EPR.
This study highlighted that the type of iron oxide
nanoparticles we used in intravenous administration in rats
have been stored in the major tissues of the organism.

CONCLUSIONS
The results suggest that MNPs, as characterized inour study, can be potentially used as contrast agent in
MRI imaging, cancer therapy and drug delivery,
distributing especially in spleen and liver.
A future objective of this research is to enhance thethermal ablation procedures in liver tumors through theuse of computer-controlled radio-frequency ablation (RFA)
needle combined with targeted intratumoral (MNP)
delivery and induction of tumor hyperthermia as a novel
method for ablating tumors. Although this technology
holds interesting potential for the ablation of cancer,
significant further research is needed.
Fiziologia - Physiology • 2015.25.4(88)Acknowledgments
Nicu Florian Trincu, PhD, was supported by the
project "Program of excellence in doctoral andpostdoctoral research in multidisciplinary chronic
diseases", contract number POSDRU/159/1.5/S/133377,
financed by the European Social Fund and Romanian
Government through Sectoral Operational Programme
Human Resources Development 2007-2013.
Author contributions: N.F. Trincu, J. Neamtu, T.A.
Balseanu and A. Saftoiu performed the procedures andwrote the paper. B.S. Ungureanu and I. Pirici assessed
and interpreted the pathological images and evaluatedthe EPR spectra. S.A. Buteica helped with MNPs
preparation.

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4. Emerich DF, Thanos CG. The pinpoint promise of
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5. Mahmoudi M, Simchi A, Imani M. Recent advances in surface
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6. Mornet S, Portier J, Duguet E. A method for synthesis andfunctionalization of ultrasmall superparamagnetic covalentcarriers based on maghemite and dextran. J Magn Magn Mater.
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7. Jain TK, Morales MA, Sahoo SK, Leslie-Pelecky DL,
Labhasetwar V. Iron oxide nanoparticles for sustained deliveryof anticancer agents. Mol Pharm. 2005; 2: 194-205.
8. Radbruch A, Mechtold B, Thiel A, Miltenyi S, Pflüger
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11. Lübbe AS, Alexiou C, Bergemann C. Clinical applications ofmagnetic drug targeting. J Surg Res. 2001; 95(2): 200-6.
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13. Gamarra LF, Pontuschka WM, Amaro E, et al. Kinetics ofelimination and distribution in blood and liver of biocompatible57ferrofluids based on Fe3O4 nanoparticles: An EPR and XRF
study. Mater Sci Eng C 2008; 28: 519-525.
14. Schlachter EK, Widmer HR, Bregy A, et al. Metabolic
pathway and distribution of superparamagnetic iron oxide
nanoparticles: in vivo study. Int J Nanomedicine. 2011; 6:
1793-1800.
15. Tsuchiya K, Nitta N, Sonoda A, et al. Histological study ofthe biodynamics of iron oxide nanoparticles with different
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17. Tate J, Ogden J, Strawbridge R, Pierce Z, Hoopes P.
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18. Briley-Saebo K, Bjornerud A, Grant D, Ahlstrom H, Berg T,
Kindberg GM. Hepatic cellular distribution and degradation ofiron oxide nanoparticles following single intravenous injection in
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19. Gu L, Fang RH, Sailor MJ, Park J-H. In Vivo Clearance andToxicity of Monodisperse Iron Oxide Nanocrystals. ACS Nano.
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20. Jain TK, Reddy MK, Morales MA, Leslie-Pelecky DL,
Labhasetwar V. Biodistribution, clearance, and biocompatibility of ironoxide magnetic nanoparticles in rats. Mol Pharm. 2008; 5(2): 316-27.
21. Gamarra LF, Pontuschka WM, Amaro E, et al. Kinetics ofelimination and distribution in blood and liver of biocompatible
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study. Mater Sci Eng C 2008; 28: 519-525.

BIODISTRIBUȚIA NANOPARTICULELOR DE FE3O4/ACIDCITRIC ÎNTR-UN MODEL MURIN
REZUMAT
Nanoparticulele magnetice (MNP) de oxid de fier de aproximativ 20÷150 nm au fost sintetizate prin metoda
chimică de coprecipitare a Fe2+ și Fe3+ în soluție de NH4OH, după care acestea au fost acoperite cu acid citric.
Scopul acestui studiu in vivo a fost de a analiza biodistribuția MNP la nivelul diferitelor organe, dupăadministrarea intravenoasă a MNP la șobolanii Sprague Dawley. Analiza a fost efectuată prin metoda rezonanței
paramagnetice a electronilor (EPR), urmată de analiza histologică în colorația albastru de Prusia. Studiul
distribuției MNP a fost efectuat la 24 de ore de la administrarea intravenoasă la nivelul venei jugulare, a unei
doze de 15 μmol Fe/kg. Rezultatele au arătat o mai mare aglomerare a nanoparticulelor la nivelul splinei șificatului, fără ca MNP să fie distribuite la nivelul rinichilor, pancreasului și plămânilor. Aceste aspecte au foststudiate utilizând o nouă formulare a oxidului de fier, care ar putea fi folosită în cercetări privind posibilitățile
terapeutice ale acesteia, precum și în domeniul rezonanței magnetice nucleare (RMN), în studii viitoare.
Cuvinte cheie: MNP, biodistribuție, model murin

58

Fiziologia - Physiology • 2015.25.4(88)BODY COMPOSITION ASSESSMENT IN PATIENTS WITH
CHRONIC OBSTRUCTIVE PULMONARY DISEASE
VOICA SORINA ALINA1, CRISTIAN OANCEA2, CRISAN ALEXANDRU F2,
TUDORACHE EMANUELA2, FIRA-MLADINESCU OVIDIU2, TUDORACHE VOICU2
1University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania
2Department of Pulmonology, University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania

ABSTRACT
Chronic obstructive pulmonary disease (COPD) is a preventable and treatable disease with significant extra-pulmonary
effects, which can contribute to serious complications in certain patients. The systemic component results in weightloss and muscle weakness, both of which contribute to progression of the disease and deterioration in quality of life.
Malnutrition is one of the most investigated extra-pulmonary features in COPD patients. Loss of body weight anddepletion of fat free mass (FFM) are common and severe risk factors for mortality in COPD.
Surrogate measures such as the well known Body Mass Index (BMI) give no indication of body composition, muscle
mass or nutritional state. Different methods are used for nutritional assessments beyond BMI, such as bioelectrical
impedance analysis (BIA). BIA is simple, inexpensive, quick and non-invasive technique for assessing bodycomposition and its changes over time. Given the usefulness of BIA in determining body compartments and being aquick and simple method, its use could become generalized and constitute another instrument of measurement in
COPD patients.
Key words: COPD, body composition, malnutrition, cachexia.

INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is achronic inflammatory disorder of the lung with many
extra-pulmonary effects, caused mainly by tobaccosmoking, and is characterized by progressive, persistent
airflow obstruction. There is a global increase in thenumber of patients suffering from COPD and the World
Health Organization (WHO) estimates that COPD will
become the third leading cause of death in the world until
2020. This progressive increase in the number of patientswith COPD, especially in acute stages, will create aconsiderable medico-economic burden, therefore,
preventive measure and therapies for this disorder are
being assiduously pursued [1].
Loss of body weight and depletion of fat free muscle
mass are common and serious problems in patients with
COPD indifferent of the degree of airflow limitation andare caused by a combination of several physiological andpathophysiological alterations such as systemic
inflammation, inadequate energy intake and/or increased
energy expenditure [2].
Malnutrition in COPD has been associated withsystemic inflammation, cachexia, anorexia, skeletal muscle
dysfunction, dyspnea, reduced health status, enhanced riskof exacerbations and increased mortality [3]. Consequently,
current COPD guideline recommendations by the Global
Initiative for Chronic Obstructive Lung Disease (GOLD)consider nutritional monitoring an important part of routine
evaluation of COPD patients [4].
Common definitions of malnutrition include weight lessthan 90% of predicted value as given by the Metropolitan
Insurance Company, and/or body mass index (BMI) of lessthan 18.4 kg/m2. Often the terms malnutrition and cachexia
are used interchangeably and defined by the BMI but here
are different states of low weight. In a large study ofpatients with COPD, Schols et al. distinguished threedifferent types of impaired nutrition: semi-starvation (lowBMI with normal or above normal fat-free mass [FFM]
index), muscle atrophy (low FFM index and normal or
above normal BMI) and cachexia (low BMI and low FFM
index). Schols observed that patients with muscle atrophy
and those with cachexia had similar outcomes. On theother hand, the groups with normal or above-normal BMI(semi-starvation or no impairment) had better survival.
Thus the groups with low FFM have greater mortality thanthe others, and it would appear that low FFM is a better
predictor of mortality than low BMI [2].
The prevalence of cachexia is presented to be
between 20-40% in the COPD population [5]. The

Received 11th of September 2015. Accepted 20th of October 2015. Address for correspondence: Cristian Oancea MD, PhD,
Department of Pneumology, University of Medicine and Pharmacy “Victor Babes” Timisoara, Eftimie Murgu Sq No. 2A, 300041,
Timisoara, Romania; phone: +40769221057; e-mail: oancea@umft.roFiziologia - Physiology • 2015.25.4(88)59significance of cachexia in COPD is strongly evident in
the survival curves, as shown by Schols et al. [6].
He observed that the median survival was cut almostin half by cachexia from 4 to 2 years. The differences insurvival are not due to the differences in severity of
COPD by spirometric criteria. What is not known iswhether cachexia can be prevented, or even reversed,
once developed, and how this will affect survival [2].
Other important consequence of cachexia relates to
impaired quality of life. With fatigue and muscle
weakness being even greater than for non-cachectic
COPD patients, cachexia worsens the quality of life
considerably.

also increased. Furthermore, exercise training appears toaccentuate oxidative stress in COPD. Thus, ROS and/or
RNS may play a role in cachexia as molecules capableof incurring tissue damage. However, these authors
found that cachectic patients were able to increase their
exercise capacity after training [12,13].
In this context, it should not be forgotten that ROS
can also serve as signalling molecules in the process ofadaptation to exercise. It remains a puzzle whether
exercise, both acute and repeated (training), is pro- or
maladaptive in these patients, and whether exercise has
positive or negative (or no) effect on quality of life andmortality in cachectic COPD patients.

IMPORTANT MECHANISMS OF CACHEXIA IN COPD
PATIENTS

THE IMPACT OF OBESITY ON COPD PATIENTS

A mechanism that would cause cachexia in patientswith COPD is the energy imbalance. Due to airways
obstruction, the work of breathing is increased. Sergi et
al. noted that the resting expenditure was found to bet
10% higher in COPD patients than in normal subject ofsimilar age, height and weight [7].
The oxygen cost of exercise has also been noted to
be increased than in normal subjects [8].
In addition to this concept, it is well known that there
are some fiber-type shifts in skeletal muscle in COPD
from type I to type II. Muscle fiber II uses more oxygen
per unit work output than type I, thus there is an extra
metabolic load due to oxygen-inefficient muscle usage.
Another factor is that cachexia itself leads to anorexia,
and this leads to a vicious circle, such that the moreweight lost, the less food is taken in. Aggressive
nutritional augmentation and appetite stimulation havenot solved the problem, casting some doubt on thecentrality of this mechanism [9].
Another mechanism proposed for cachexia in COPD
patients would be the disuse of the muscles that lead toatrophy. Patients with COPD are very inactive thus
extreme inactivity may play an important role in cachexia.
As with nutritional guidance, instituting a regimen of regularexercise does not cure cachexia per se, but just as withnutrition, physical activity is always encouraged [10].
Latest research has focused on systemic
inflammation to be the genesis of cachexia in COPD.
There is considerable evidence that this may be
important, but there are many gaps in present knowledge
that stand in the way of making any definitive conclusions
about the role of inflammation in developing cachexia.
The most important molecules that are receiving
attention are TNF-α, IL-1β, IL-6, C-reactive protein, andreactive oxygen species (ROS) and reactive nitrogenspecies (RNS) [11].
Oxidative stress is greater in COPD patients than incontrols, and the oxidised/reduced glutathione ratio is60

The prevalence of obesity and COPD is increasing at
remarkable rate in the Western world, and this has amajor negative health and economic ramifications [14].
The clinical consequences of obesity in COPD arediverse and include increased perceived activity related
dyspnea, decreased quality of life, sleep disorders andelevated oxygen uptake at peak exercise. Obesity in
COPD is known to be associated with reduced
self-reported daily activity levels, and this, in turn, may
predispose to increased risk for comorbidities, which
include skeletal muscle de-conditioning, cardiovasculardiseases, insulin resistance and osteoporosis. The
mechanisms of activity limitation in obese COPD patients
have only recently become the focus of systemic studies[15].
Jones and Nzeku have analyzed the impact of theabnormalities on lung volumes across the stages ofobesity and demonstrated an inverse relationshipbetween increasing BMI and decreasing end expiratory
lung volume (EELV) and expiratory reserve volume (ERV)in healthy population. Total lung capacity (TLC) declines
modestly with mild to moderate obesity [16].
Consequently, inspiratory capacity (IC) increases withhigher BMI, reflecting the relative preservation of TLC in
the presence of decreased EELV. The reduced EELV in
obesity means that airways resistance (Raw) is increased,and expiratory flow limitation may exist during restingbreathing [17,18].
The presence of diffuse small airways disease in
COPD further amplifies the before mentioned associationof increased BMI and higher air resistance in obesity.
Although much is known about the respiratory muscles in
COPD there has been no large systematic evaluation of
respiratory muscle structure and function in obese
patients with COPD [19].
Ora et al. in a physiological study of patients with
COPD and mild to moderate obesity observed that there
was no difference in respiratory muscle strength duringrest and exercise in the obese group compared with the
Fiziologia - Physiology • 2015.25.4(88)normal-weight group. The former had greater static lungrecoil and intra-abdominal pressures despite similar lungfunction impairment by forced expiratory volume in thefirst second (FEV1) criteria. The factors that predispose to
hypercapnic ventilatory failure in patients in whom COPD
and morbid obesity coexist have not been systematicallystudied [20].
Low BMI is associated with increased COPD-related
mortality, unrelated to disease severity. Recent studies
indicate that the FFM index is an even more important
determinant of prognosis in moderate to severe COPD than
BMI. Fat mass index was not predictive for prognosis inpatients with moderate to severe COPD [2,21].
Besides the presence of chronic airflow obstruction,
low-grade systemic inflammation is one of themechanisms that may be responsible for the increased
rate of cardiovascular complications in COPD [22].
The role for smoking-related systemic inflammation isevident in the pathogenesis of COPD and led to the
proposal of the term chronic systemic inflammatory
syndrome complementary to the diagnosis of COPD [23].
In a recent meta-analysis of longitudinal studies, thepresence of the metabolic syndrome was strongly
associated with cardiovascular morbidity and mortality.
Indeed, some studies suggest the presence of insulin
resistance in COPD, especially in normal weight to obese
patients and those who are hypoxaemic [24].
BODY COMPOSITION ASSESSMENT
Surrogate measures such as the well known BMI
give no indication of body composition, muscle mass or
nutritional state. Thus malnutrition that requires
intervention can exist although the individual has anormal BMI. These patients are usually not detected bysubjective global assessment of nutritional status. In
COPD patients, it has been recognized that FFM further
differentiates into body cell mass (BCM) and extra
cellular mass (ECM) rather than low BMI that should beconsidered as a critical parameter of disease severity
and prognosis [25].
Monitoring changes in FMM and fat mass (FM) can
improve our understanding of disease processes andenergy metabolism, leading to the development of more
effective nutrition and exercise intervention strategies tocounteract the loss of FMM associated with factors suchas malnutrition, injury, aging and certain diseases suchas COPD.
There are a number of sophisticated but expensive
methods that may be used to obtain reference measures ofbody composition, including magnetic resonance imagery,
neutron activation analysis and computerized tomography.
Alternatively, hydrometry, dual energy x-ray absorptiometry
and densitometry are more commonly used in researchsettings for measures of body composition.
Fiziologia - Physiology • 2015.25.4(88)Densitometry
Densitometry refers to the measurement of total
densitometry body (Db) and the estimation of bodycomposition from Db. Db is the ratio of body mass to body
volume (BV). Body volume is measured by either water
displacement or air displacement. For years, the water
displacement method, known as hydrodensitometry orhydrostatic weighing, has been considered by someexperts as a gold standard method in light of the relatively
small technical error associated with the accurate
measurement of Db (0.0015 g/cc or approximately 0.7%
BF). In order to achieve this degree of accuracy, total bodymass, underwater weight, water temperature, and residuallung volume (RV) must be measured precisely (within 0.20
kg for body mass and underwater weight, within 0.0005
degrees Celsius (°C) for water temperature, and within
100 ml for RV). The estimated technical error associatedwith the RV measurement (0.00139 g/cc) is relatively large
compared to the other three sources of error combined(0.0006 g/cc) [26].
Hydrometry
Hydrometry, or the measurement of total body water(TBW), is also limited when used singly to derivereference measures of body composition. With thismethod, the concentration of hydrogen isotopes(deuterium or tritium) in biological fluids (saliva, plasma,and urine) after equilibration is measured and used toestimate TBW [27].
This method assumes that the distribution andexchange of the isotope by the body are similar to the
distribution and exchange of water. However, due to theexchange of the isotope with nonaqueous hydrogen in
the body, TBW may be overestimated by 1 to 5%. Using
this method in conjunction with the two component
molecular model to obtain estimates of FFM, it is further
assumed that the hydration of the FFM is constant for all
individuals (~ 73% of FFM). Because TBW fluctuateswidely within and among individuals depending on age,gender, level of obesity, and disease, large errors may
result when hydrometry is used with the two component
model to derive reference measures of body composition.
Siri estimated that biological variability (2%) in thehydration of the FFB would produce a substantial error in
the estimation of body fat (2.7% BF) for the general
population [28].
Dual-energy X-ray AbsorptiometryDual-energy x-ray absorptiometry (DXA) is arelatively new technology that is gaining recognition as areference method for body composition research. Thismethod is based on three compartment model that
divides the body into total-body mineral, mineral-free lean,
and fat tissue masses. The precision of DXA in
measuring %BF is estimated to be 1.2%BF [29].

61DXA is highly reliable, and there is good agreement(~0.4%BF difference) between %BF estimates obtainedby hydrodensitometry (Db adjusted for relative total-bodymineral and TBW) and DXA [30].
In addition to obtaining estimates of relative body fat
and lean tissue mass, DXA provides segmental andregional measures of body composition. DXA is an
attractive alternative to hydrodensitometry as a reference
method because it is rapid (a total body scan takes 20
minutes), safe, requires minimal subject cooperation, and,most importantly, takes into account inter-individual
variability in bone mineral content. Also, DXA estimatesof body composition appear to be less affected byfluctuations in TBW compared to hydrodensitometry andhydrometry.
Kohrt estimated that a 5% difference in the relative
hydration of the FFB (78 vs 73% FFB) would produce<0.5 kg error in fat and FFM, suggesting that hydration
status has a relatively small effect on soft-tissue
estimates obtained via DXA [31].
The majority of the physiologists often use to assess
body composition with the methods of skinfolds,
anthropometry and bioelectrical impedance analysis(BIA). Given the choice of methods and numerous
prediction equations published in the literature, it is often
difficult for the clinician to select an appropriate method
or prediction equation that accurately assesses the bodycomposition of each patient.
Skinfold Method
The skinfold (SKF) is an indirect measure of the
thickness of subcutaneous adipose tissue at a specified
site. Most SKF equations use two or more SKF
measurements to predict either Db or %BF. The accuracy
and precision of SKF measurements is highly dependent
on technician skill, type of SKF caliper, and client factors.
It takes a great amount of time and practice to develop
skill as a SKF technician, and standardized proceduresmust be carefully followed [32].
Anthropometry
Anthropometry refers to the measurement of the sizeand proportions of the human body. Anthropometricprediction equations estimate Db, %BF, or FFM fromcombinations of body mass, standing height, skeletal
diameters, and circumference measures. Compared to
SKF measures, these anthropometric techniques are
relatively simple, inexpensive, and require less skill andtraining. The accuracy and precision of anthropometricmeasures, however, are affected by technician skill andclient factors. Circumferences are preferable to SKFs
when measuring obese clients for a number of reasons.
Regardless of size, circumferences of obese individuals
can be measured, whereas, the SKF thickness may
exceed the maximum aperture of the caliper, andcircumferences require less technician skill, and the62

difference between technicians is smaller compared to
SKF measurements [33].
Bioelectrical impedance analysis
The bioelectrical impedance analysis is a commonlyused method for estimating body composition. It
measures the impedance or resistance to a small
electrical current as it travels through the body`s waterpool. An estimate of total body water (TBW) is acquired
from which total body FFM is calculated using theassumption that 73% of the body`s FFM is water. The
resistance to current flow will be greater in individuals
with large accounts of body fat given that adipose tissue
is a poor conductor of electrical current due to its
relatively low water content [34].
This body composition assessment method is simple,
inexpensive, quick and non-invasive technique forassessing body composition and its changes over time.
BIA is largely used in clinical trial settings and there are awhole series researches regarding the theory andmethodology of several different BIA techniques.
However, there is a considerable lack of information on
the practical aspects of BIA for those primarily interested
in learning how to apply this method in practice [35].
Thus, BIA still is an underused and underestimatedtool for nutritional assessment in primary care. This canbe further explained by the fact that the costs of BIA are
currently not always refundable and that there are no
guidelines outlining the methods for assessing
malnutrition in patients with COPD [36].
In order to ensure the accuracy of the method, thepatients must strictly follow each of the BIA testing
guidelines. In addition, standardized testing proceduresmust be followed. Compared to the skinfold method BIA
analysis does not require a high degree of technical skill,
it is more comfortable and less intrusive for the client andit can be used to estimate body composition of obese
individuals [37].

BIA client guidelines:

Urinate within 30 minutes of the test
No exercise within 12 hours of the test
No eating or drinking within 4 hours of the test
No diuretic medications within 7 days of the test
No alcohol consumption within 48 hours of the test
No testing of the female clients who perceive they
are retaining water during the stage of the menstrual
cycle.
The principle of BIA is to determine the electric
impedance of an electric current passing through the body[38].
The electrical impedance (Z) consists of two
components, resistance (R) and reactance (Xc). Reactanceis a measure of body cell mass (BCM) and resistance ameasure of TBW. From the determined impedance a
number of BIA parameters can be estimated [39].
‐
‐
‐
‐
‐
‐

Fiziologia - Physiology • 2015.25.4(88)The BIA analysis determines the following
parameters [40]:‐ BCM that consists of all cells that have an effect on
metabolism (e.g. nervous system, internal organs,muscle); this analysis is the most important whenassessing the nutritional status of the patient.
‐ FFM that represents everything that is not body fat,it consists of BCM and ECM; it is decreased inelderly people and chronic diseases. The Fat massis indirectly determined from the body weight minus
FFM.
‐ TBW which is elevated in high portion of muscle
and water retention such as oedema, and isdecreased in situations such as dehydration.
‐ ECM is mainly extracellular water; it is increased due
to extracellular water retention such as oedema anddecreased due to water loss e.g. diuretics.
The BIA results are influenced by the following
factors [41]:‐ Individual characteristics (e.g. skin temperature,
race, age, sex)
‐ Medical conditions and medication that have an
impact on the fluid and electrolyte balance;
infection and cutaneous disease that may alter theelectrical transmission between electrode and skin
‐ Environmental conditions (e.g. ambient temperature)‐ Non-adherence of electrodes, use of wrong electrodes,
loosing of cable clip, interchanging of electrodes‐ High and weight (should be measured directly by
the investigator)‐ Position of the body and limbs‐ Moderate to intense level of physical activity/exercise
before BIA measurements (last exercise at least 12
hours previously)‐ Consumption of food and beverages (at least 12
hours previously, fasted state for at least 2 hours)
It has been demonstrated that BIA is a useful bodycomposition measurement in patients with COPD [42].
FFM assessed by BIA is an important factor in thesepatients in determining maximum exercise performance,along with other pulmonary function parameters [43].
Other study has described that in patients with COPD
with acute respiratory failure, BIA estimates nutritional status
better than anthropometric parameters or plasma levels ofvisceral proteins. The marked inflation of the extracellularcompartment (extra-/intracellular water volume ratio) that isshown in the BIA analysis in the acute hospitalized COPD
patients presumably leads to inaccurate anthropometricresult(e.g. overestimation of triceps skinfold thickness andfat mass, and inaccurate measurement of middle-arm
muscle circumference). In out-patients with COPD, bodycomposition estimated by BIA analysis presented good
reliability and correlation with DXA and the two methods
presented satisfactory clinical accuracy [44].
Abbatecola et al. observed in a study thatinvestigated the correlations of lean and FM on walking
Fiziologia - Physiology • 2015.25.4(88)speed and gait speed in patients with COPD that FM wasan independent determinant for slower walking speed,
while lean mass was not [45]. The importance of mobility
limitation using different walking speed distances has
been shown to be important even if not attributable tospecific disease states in older persons [46].
A community dwelling cohort study of older persons
suggested that the absolute amount of FM wasnegatively associated with physical performance, whilelean mass is not significant in absolute terms [47].
A study found that the lean-to-fat ration was
associated with greater walking distance in younger men,but not in women with COPD. The same authors
showed that higher measures of total adiposity (BMI) andcentral adiposity were related to functional limitation inboth genders [37].
Some authors examined the idea that BIA can predictclinical outcomes, such as mortality and length of
hospital stay. An issue that has received little attention iswhether the standard procedure for predicting suchoutcomes using estimates of body composition madeusing impedance and anthropometry is superior using
anthropometry (weight + height) alone or bodycomposition calculated from simple anthropometry alone.
Kyle et al. applied a BIA equation (with R, Xc, W andH as predictive variables), derived from 343 healthy
subjects whose body composition was assessed with
DXA to a wide range of hospitalized patients, including
those with cardiovascular disease and COPD. BMI wasfound to be associated with prolonged length of hospitalstay. The study also reported that both low FFM and FM
significantly predicted length of stay [48].
In a series of studies involving groups of patients with
COPD and a study of mixed groups of patients, it was
found that there were variable associations between the
FFMI (or lean/fat mass ratio), determined by whole-body
BIA and anthropometry, and clinically relevant outcome
measures, such as length of hospital stay, mortality andlung function tests. However, when FFMI was not
associated with significant relationships, BMI was also
not associated with significant relationships. When FFMI
was associated with significant relationships, BMI was
generally also associated with significant relationships.
An exception is a COPD study in which some of theoutcomes (dyspnea and lung function tests FEV1%
predicted and FEV1/forced vital capacity) weresignificantly related to FFMI and not BMI [25].

CONCLUSIONS
Bioelectrical impedance analysis is non-invasive,
relatively inexpensive, does not expose to ionizing
radiation, has very limited variations, can be performed inalmost any subject and provides a viable opportunity forevaluating body composition in COPD patients.

63

Our understanding of the mechanism underlying
malnutrition and subsequent muscle dysfunction in
COPD patients has increased significantly. In addition to
BMI, determination of FFM is crucial to adequately detectmalnutrition in patients with COPD.
There is a high prevalence of malnutrition in
hospitalized COPD patients. Given the usefulness of BIA
in determining body compartments and being a quick andsimple method, its use could become generalized andconstitute another instrument of measurement in COPD
patients. There is a relationship between FEV1 and the
distance covered in the six minutes walking test, numberof hospitalization day and readmissions and the FFM
index. Furthermore, low FFM index measured by BIA isan independent predictor of mortality in COPD patients.
Further validation of BIA is necessary to understandthe mechanisms for the changes in acute illness,extreme heights and body shape abnormalities.

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64

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Clin Nutr. 2005; 24: 133-142.

MĂSURAREA COMPOZIȚIEI CORPORALE LA PACIENȚII CU BPOC
REZUMAT
Bronhopneumopatia obstructivă cronică (BPOC) este o boală ce poate fi prevenită şi tratată, cu efecte
semnificative extrapulmonare, care pot contribui la complicaţii semnificative la anumiţi pacienţi. Componenta
sistemică rezultă în pierderea în greutatem corporală, slăbiciune musculară, amândouă putând contribui laprogresia bolii şi la scăderea calităţii vieţii. Malnutriţia este una din cele mai investigate caracteristici
extrapulmonare la pacienţii cu BPOC. Pierderea greutăţii corporale şi depleţia masei fără grăsimi sunt factori derisc comuni şi severi pentru mortalitatea în BPOC.
Măsurători surogate cum ar fi bine cunoscutul indice de masă corporală (IMC) nu dau indicaţii în privinţa
compoziţiei corporale, masei musculare sau a stării nutriţionale. Diferite metode sunt utilizate pentru evaluarea
nutriţională dincolo de IMC, cum ar fi analiza de impedanţă bioelectrică (BIA). BIA este simplă, ieftină, având otehnică rapidă şi non-invazivă de evaluare a compoziţiei corporale şi a schimbărilor în timp. Având în vedere
utilitatea BIA în determinarea diferitelor compartimente corporale şi fiind o metodă simplă şi rapidă, utilizarea
acestuia ar putea deveni generalizată şi constituie încă un instrument de măsurare a compoziţiei corporale lapacienţii cu BPOC.
Cuvinte cheie: BPOC, compoziția corporală, malnutriție, cașexie

Fiziologia - Physiology • 2015.25.4(88)65THE EFFECTS OF TWO LIPID-LOWERING DRUGSON MITOCHONDRIAL RESPIRATION IN H9C2 CELL LINE
ILEANA SCURTU1, ROXANA POPESCU2, ANDREEA PRIVISTIRESCU1,ADRIAN STURZA1,3, IOANA Z. PAVEL1, DOINA VERDEȘ2,
OANA M. DUICU1,3*, DANINA M. MUNTEAN1,3
1Department of Pathophysiology
2Department of Cellular and Molecular Biology
3Center for Translational Research and Systems Medicine, “Victor Babeș” University of Medicine and Pharmacy, Timișoara,

Romania

ABSTRACT
Pravastatin (PR), a HMG-CoA reductase inhibitor, and nicotinic acid (NA), a water-soluble vitamin B, are widelyprescribed lipid-modifying drugs. Controversy exists about the effects of these agents on mitochondrial function. The
present study was purported to characterize the acute effects of PR and NA (10 µM) on mitochondrial respiration inH9c2 cardiomyoblasts. Mitochondrial respiration of permeabilized cells was assessed by high-resolution respirometry
using the Oxygraph-2k in the presence of complex I and complex II substrates (glutamate/malate and succinate) and,of the successive addition of the electron transport chain inhibitors: rotenone, oligomycin, FCCP, and antimycin A,
respectively. Our preliminary data show an inhibitory effect on mitochondrial respiration in the presence of pravastatin,
whereas nicotinic acid stimulated respiration in H9c2 cardiomyoblasts.
Keywords: pravastatin, nicotinic acid, mitochondrial respiration, H9c2 cardiomyoblasts

INTRODUCTION
In the 21st century the persistence of a highprevalence of cardiovascular diseases worldwide isdriven by the dual burden of obesity and diabetes, bothconditions being largely associated with dys- or
hyperlipidemia. According to guidelines, statins represent
the most widely prescribed hypocholesterolemiant drugs
for both primary and secondary prevention ofcardio-metabolic disorders [1] and are currently accepted
as a safe therapy view their pleiotropic beneficial effects[2, 3]. However, statins have also been associated withskeletal muscle symptoms that may progress to toxic
myopathy [4] and require the association of non-statin
therapies in order to attain the recommended cholesterollevels [5]. Among the mechanisms proposed to underlie
statins-induced myopathy an important contributor isrepresented by the impairment of mitochondrial function[6, 7].
Pravastatin (PR), one of the firstly developedhydrophilic statins, was found already back to 1998 toaccelerate the age-dependent impairment of the electrontransport chain complex I function in rat muscle cells [8].

However, later studies reported that PR was lessmyotoxic as compared to the lipophilic statins both in a
rat skeletal muscle cell line and after in vivo
administration in rats [9, 10]. Recently, Bonifacio et al.
showed that simvastatin is toxic to the H9c2 rodent
cardiomyocyte cell line by decreasing mitochondrial
respiration, the activity of enzyme complexes I and IV of
the respiratory chain, and the ATP content [11]. Whether
PR interferes with mitochondrial function in
cardiomyocytes it is not known.
Nicotinic acid (niacin) is one of the oldest known
anti-atherosclerotic water-soluble drug, mainly indicatedfor its HDL cholesterol-elevating effect, the reduction of
lipoprotein a (whose level is not improved by statins)
along with its anti-lipolytic properties; similarly to statins,
lipid independent-beneficial effects (anti-inflammatory,
vasculo-protective, immuno-modulatory) have also been
described [12]. Interestingly, a recent study demonstrated
that NA increased the total cellular respiratory capacity infibroblast cell lines [13].
In line with the above mentioned data, the aim of thepresent study was to assess the effects of PR and NA on
mitochondrial respiration in H9c2 cardiomyoblasts.

Received 12th of September 2015. Accepted 17th of October 2015. Address for correspondence: Oana M Duicu, MD, PhD,
Department of Pathophysiology, “Victor Babeș” University of Medicine and Pharmacy of Timişoara, 14 Tudor Vladimirescu st.,300173 Timisoara, RO, Tel/Fax: +40-256-493085; e-mail: oanaduicu@umft.ro66

Fiziologia - Physiology • 2015.25.4(88)MATERIALS AND METHODS
Reagents
All chemicals were supplied by Sigma-Aldrich.
Cell culture
The rat cardiomyoblast H9c2 cell line was purchasedfrom the European Collection of Cell Cultures. Cells weregrown in Dulbecco’s modified Eagles high-glucose
medium (4.5g/l) containing 1 mM sodium pyruvate, 5 mM
HEPES, 10 % fetal bovine serum (FBS), and 100 U/ml
penicillin-streptomycin. Cells were kept in 5% CO2 at
37°C and grown for 4 days to achieve the optimum
confluence.
Experiment protocol
1. Preparation of permeabilized cell line H9C2
2 ml cell suspension (containing 1 million cells/ml) in
MIR05 buffer was added to each O-2K chamber.
Digitonin (8.1 µM final concentration) was used topermeabilize cell membranes in the respiratory chamber(5 minutes of incubation) before adding the substrates
and then the inhibitors [16-18]. Data are expressed inpmol O2 s−1mg−1.
2. Measurement of OXPHOS
Mitochondrial respiration was measured at 37°C by
high-resolution respirometry using the Oxygraph-2K(Oroboros Instruments, Austria) with 2 chambers, eachcontaining the respiration buffer MIR05 (0.5 mM EGTA, 1
g/l BSA, 3 mM Mg Cl2 6H2O, 20 mM taurine, 10 mM
KH2PO4, 110 mM sucrose, 60 mM K- lactobionate, and20 mM HEPES, pH 7.1). Respiratory rates were
measured in the presence of complex I(glutamate/malate) or complex II (succinate) substrates
according to the Substrate-Uncoupler-Inhibitor Titration(SUIT) protocol described by Gnaiger [14] and adaptedfrom Duicu et al [15]. Addition of CI/CII-substrates is a
step referred as basal respiration (the classical state 2)
whereas addition of ADP to activate oxidative
phosphorylation (OXPHOS) is referred to as the active
respiration (the classical state 3) or the OXPHOS state.
After measuring the basal and the active respiration,
oligomycin (an inhibitor of ATP synthase) was added inorder to induce the LEAK state, followed by the titrationof the classic uncoupler FCCP (0.5 μM steps) in order toobtain the maximum mitochondrial respiration or theelectron transfer system (ETS) capacity. The integrity ofthe outer mitochondrial membrane was assessed byaddition of cytochrome c. Respiratory control ratio (RCR)
was calculated as the ratio between the OXPHOS
capacity (state 3) and the LEAK flux (state 2) [16, 17].

Fiziologia - Physiology • 2015.25.4(88)Statistical Analysis
All values are presented as mean  SEM. Group
comparisons were performed by one-way analysis ofvariance (ANOVA) and Dunnett multiple comparison test
(GraphPad Prism version 5.0). Values for p < 0.05 wereconsidered statistically significant.

RESULTS AND DISCUSSION
Statin therapy, albeit being the cornerstone oftreatment in conditions associated with dys- andhyperlipidemia, has been unequivocally related tomitochondrial distress. Since mitochondria are mostly
abundant in tissues with high energy demand (such as
the myocardial tissue), cardiomyocytes should be firstly
affected. Paradoxically, opposite effects of lipophilic
statins (simvastatin, atorvastatin) on skeletal and cardiac
muscle (albeit both striated muscles) have been reported
in the literature, with mitochondrial impairment of skeletal
muscle whereas mitochondrial respiratory function in theheart was either not affected or increased [20, 21]. Withreference to pravastatin, it has also been reported thatchronic administration (40 mg/kg/day) of pravastatin for 3
weeks in mice induced marked alterations of skeletal
muscle mitochondria while the ultrastructure of the heartin the treated animals appeared normal [22].
In the present study the classic parameters of
mitochondrial respiration were assessed in order tocharacterize the acute effects of two lipid-modifying drugs,
PR and NA (10 µM) in H9c2 cardiomyoblasts. In
particular, the following respiratory parameters were
measured: State 2 (basal respiration) measured after the
addition of complex I (glutamate/malate) or complex II
substrate/inhibitor (succinate/rotenone), OXPHOS state(ADP-stimulated respiration), LEAK state, and ETS (themaximal mitochondrial respiratory capacity), respectively- whereas the RCR was calculated.
Our preliminary data show that, in cells respiring on
complex I dependent-substrates (glutamate/malate), PR(but not NA) elicited a generalized reduction in all
respiratory parameters (Fig. 1). Thus, both basal (State 2)
and active (OXPHOS) respiratory rates were significantly
decreased by PR (**p < 0.01 vs. Ctrl), leading to an
obvious decrease in the RCR (Fig. 1). Similarly, both ETS(**p < 0.01) and the LEAK state (*p < 0.05) displayed low
values when cells were incubated with PR as comparedto the non-treated controls (Fig. 1). The measurement of
ETS by progressively adding FCCP reflects how asystem reacts to an increased ATP demand. A diminishedmaximal respiration is the case of cells treated with PR,
suggests the risk for an energetic crisis.
At variance, an improved cellular respiratory capacity
was obtained when cells were incubated with the same67

concentration of NA. The most important increase wasfound for the basal respiration (***p < 0.001) but also
active and uncoupled respiration were higher than theones of the non-treated cells respiring on
glutamate/malate (**p < 0.01 vs. Ctrl) - Figure 1.

Fig. 2. Respiratory rates in cells treated with PR ad NA inpresence of Complex II-dependent substrates.
(**p < 0.01 vs Ctrl.)

CONCLUSION
Fig. 1. Respiratory rates and RCR in cells treated with PR
ad NA in presence of Complex I-dependent substrates.
(*p < 0.05, **p < 0.01, ***p < 0.001 vs Ctrl.)

Interestingly, the effects of the lipid-lowering drugs on
the active and uncoupled respiration, respectively, werecompletely very different for complex II-supported
respiration as compared to complex I. Accordingly, an
important decrease in both OXPHOS and ETS in cells
respiring on succinate (plus rotenone to prevent theretrograde flow of electrons toward complex I) wasrecorded when myoblasts were incubated with the statin(PR) while no effect was elicited in the presence of niacin(Figure 2).
It is known that NA plays an important role in theelectron transport chain via the conversion in
nicotinamide adenine dinucleotide to form the reduced
dinucleotide that in turn plays a vital role in providing the
reducing equivalents to fuel the oxidative phosphorylation[24]. We can speculate that the changes elicited by NA incomplex I-supported respiration I (Fig. 1) are related to
the fact that NADH is the substrate for Complex I (andhas no effect on Complex II-supported respiration, which
has as substrate FADH) - Fig. 2.

68

In conclusion, in H9c2 cells, our preliminary data
show an inhibitory effect of a low dose of pravastatin on
complex I-dependent mitochondrial respiration whereas
nicotinic acid exerts a stimulatory effect in the same
conditions. Moreover, PR also decreased oxidative
phosphorylation in the presence of complex II substrate
whereas NA did not interfere with succinate-supported
cellular respiration. Further experiments are required toelucidate whether these results are dose dependent andcommon for other liposoluble statins.
ACKNOWLEDGEMENTS
Research supported by the Hungarian-Romaniancrossborder cooperation project code HURO/1101/086(HURO-TWIN).

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Global Transcriptome and Nutrient-Sensing Signaling Network.
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2014; 65(8): 956-959.
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EFECTELE A DOUĂ MEDICAMENTE HIPOLOPEMIANTE ASUPRARESPIRAȚIEI MITOCONDRIALE LA NIVELUL LINIEI CELULARE H9C2
REZUMAT
Pravastatinul (PR), un inhibitor al HMG-CoA reductazei și acidul nicotinic (AN), o vitamină B hidrosolubilă, suntprescrise pe scară largă ca medicamente cu rol reglator al lipidelor circulante. Efectele acestor compuși la nivelulfuncției mitocondriale sunt controversate. Scopul prezentului studiu a fost de a caracteriza efectele acute ale PRși AN (10 µM) asupra respirației mitocondriale la nivelul cardiomioblaștilor H9c2. Respirația mitocondrială lanivelul celulelor permeabilizate a fost realizată prin tehnica respirometriei de înaltă rezoluție cu ajutorul
oxigrafului-2k (Oroboros Instr., Austria) în prezența substratelor complexului I și II ale lanțului respirator(glutamat/malat, și succinat) și respectiv, a adiției succesive a inhibitorilor clasici ai complexelor acestuia:
rotenonă, oligomicină, FCCP și antimicina A. Rezultatele preliminare arată un efect inhibitor asupra respirației
mitocondriale în cazul pravastatinului, în timp ce acidul nicotinic a stimulat respirația cardiomioblaștilor H2c9.
Cuvinte cheie: pravastatin, acid nicotinic, cardiomioblaști H2c9, respiratia mitocondrială

Fiziologia - Physiology • 2015.25.4(88)69

PULMONARY EMBOLISM: TO BE OR NOT TO BE
CASE REPORTSIMINA CRIŞAN1, ALINA-RAMONA NICOLA2, MIHAELA VALCOVICI1,2,
OANA MATEI2, MIHAI ANDREI LAZAR2, MARIAN GAŞPAR1,2,
LUCIAN PETRESCU1,2, CONSTANTIN TUDOR LUCA1,2
1“Victor Babes” University of Medicine and Pharmacy Timisoara, Romania
2Cardiovascular Diseases Institute Timisoara, Romania

ABSTRACT
Background. Pulmonary artery angiosarcoma are rare to find and diagnosis is a great challenge for the clinician,especially before surgery, due to the very unspecific signs.
Case presentation. We present the case of a 62 years old male who was admitted in hospital for evaluation of moderate
dyspnea that progressively aggravated in the last 3 months, with progressive alteration of the general status. On admission
in the Timisoara Cardiovascular Diseases Institute, in September 2014, the examination revealed an overweight patient(BMI= 27 kg/m² BM), with minor central cyanosis. The patient also presented varicose veins on both lower limbs, withmoderate peripheral edema, but without signs of post-thrombotic syndrome. Hepatomegaly 5 cm below the right costal
margins and hepatojugular reflux were noticed. On pulmonary auscultation, the patient had diminished vesicular murmur
on the level of the inferior left hemithorax, without any rales and with a spontaneous arterial oxygen saturation of 90%.
Cardiac examination showed rhythmic heart sounds, with no murmurs, with a ventricular rate of 92 beats/minute and anarterial blood pressure of 110/80 mmHg. Transthoracic echocardiography revealed a left ventricle with volumetricdimensions in normal range, conserved ejection fraction, and no kinetic abnormalities, while the right ventricle wasenlarged. Doppler continuous flow investigation showed severe secondary pulmonary hypertension. Angiocoronarographic
evaluation showed coronary arteries without significant angiographic lesions. Right heart catheterization revealed
increased pressures in the right ventricle and also in the pulmonary artery, with increased vascular pulmonary resistance,
but with no left to right shunts. First conclusion was that the patient has post-embolic secondary pulmonary hypertension,
with repeated pulmonary embolism episodes, with indication to undergo pulmonary thromboendarterectomy. The surgicalteam found, on the level of the right pulmonary artery, a large tumor mass, almost occlusive, with a friable macroscopic
aspect, a mass that infiltrated the vascular wall and was also present at the level of the left pulmonary artery, while themacroscopic aspect of the tumor mass was suggestive for a pulmonary artery sarcoma.
Conclusion. The differential diagnosis of pulmonary vascular diseases are a great challenge for the clinician. Due tothe non-specific symptoms, the first diagnosis taken into account is of pulmonary embolism, conducting tothromboendarterectomy, or even surgery. In case of tumors, the prognosis is poor and survival is of approximately one
year after surgical tumor mass removal.
Key words: tumor mass, thromboendarterectomy, pulmonary embolism, pulmonary hypertension

BACKGROUND
Pulmonary artery angiosarcoma are rare to find anddiagnosis is a great challenge for the clinician, especiallybefore surgery, due to the very unspecific signs.

CASE PRESENTATION
We present the case of a 62 years old male, admitted
for clinical evaluation in Timisoara Cardiovascular

Diseases Institute in September 2014. The patientpresented moderate dyspnea that progressively
aggravated in the last 3 months, with progressivealteration of the general status of the patient.
The patient, a former smoker until 12 years before
admission, first presented an episode of hemoptysis in
August 2013. At that time, he underwent a pulmonary
computer tomography examination, that revealed multiple
intra-arterial pulmonary thrombosis and the diagnosis of
pulmonary embolism was established (Figure 1). Since
September 2013 the patient was under anticoagulant

Received 10th of October 2015. Accepted 15th of November 2015. Address for correspondence: Nicola Alina-Ramona, MD,
Cardiovascular Diseases Institute Timisoara, Gheorghe Adam Street No. 13A, Timisora, Romania; phone: +40256-207355; fax:
+40256-207362; e-mail: nicoramo_alina@yahoo.com

70

Fiziologia - Physiology • 2015.25.4(88)treatment with acenocumarol, with an INR usually intherapeutic range (with a value between 2 and 3). However,
he presented two more hemoptysis episodes, one in
December 2013 and the last one in April 2014.

Fig. 1. Computer Tomographic acquisition showing a lacunaryimage on the level of the arterial pulmonary trunk crossing

On admission in the Timisoara Cardiovascular
Diseases Institute, in September 2014, the examination
revealed an overweight patient (BMI= 27 kg/m² BM), withminor central cyanosis. The patient also presented
varicose veins on both lower limbs, with moderate
peripheral edema, but without signs of postthrombotic
syndrome. Hepatomegaly 5 cm below the right costal
margins and hepatojugular reflux were noticed. On
pulmonary auscultation, the patient had diminishedvesicular murmur on the level of the inferior left
hemithorax, without any rales and with a spontaneousarterial oxygen saturation of 90%. Cardiac examination
showed rhythmic heart sounds, with no murmurs, with aventricular rate of 92 beats/minute and an arterial bloodpressure of 110/80 mmHg.
Biological samples were in normal range, with an INR
of 2.45 and the EKG of the patient showed a sinus rhythm,
VA=72 bpm, QRS axis+ 40 degrees, without any
repolarization abnormal features. We completed theevaluation of the patient with a transthoracic
echocardiography that revealed a left ventricle withvolumetric dimensions in normal range (end-diastolicvolume of 110 ml), with conserved ejection fraction(EF=50-55%, planimetric determination) and no kinetic
abnormalities. However, the right ventricle was enlarged,
with a diameter in parasternal long axis view of 4.6 mm.
Using Doppler continuous flow, a gradient of 78 mm Hg on
the level of the tricuspid valve was observed, with thecalculation of a systolic pressure in the pulmonary artery of
85 mmHg (severe secondary pulmonary hypertension).
Still, the systolic function of tight ventricle was not altered(TAPSE=28 mm). The inferior vena cava was dilated (7mm), with inspiratory collapse and the acceleration timemeasured while superimposing pulsed Doppler flow on thelever of the pulmonary valve was shortened (60 msec).
The evaluation of the patient continued with aperipheral lower limbs veins Doppler echocardiography
Fiziologia - Physiology • 2015.25.4(88)that showed no signs of intravascular thrombosis, with anormal venous flow. Spirometric evaluation of the patient
revealed normal parameters of respiratory function.
The next investigation was an invasive
angiocoronarographic evaluation that evidenced
coronary arteries without significant angiographic lesions.
Right heart catheterization revealed increased pressuresin the right ventricle and also in the pulmonary artery,with increased vascular pulmonary resistance, but withno left to right shunts.
At this point of the patient evaluation, taking into
consideration the clinical manifestations and also theresults of the paraclinical ones, we concluded that wewere in front of a patient with progressively alteredpostembolic secondary pulmonary hypertension, withrepeated pulmonary embolism episodes, a patient that, in
our opinion had the indication to undergo pulmonary
thromboendarterectomy.
Subsequently, September the 5th 2014, a bilateralpulmonary artery thromboendarterectomy, in profoundhypothermia was performed. To our surprise, the operatingteam found, on the level of the right pulmonary artery, alarge tumor mass, almost occlusive, with a friable
macroscopic aspect, a mass that infiltrated the vascularwall. The tumor mass was also present on the level of theleft pulmonary artery (Figure 3) and the macroscopic
aspect of the tumor mass was suggestive for a pulmonary
artery sarcoma. However, the final diagnosis was to beestablished by the histopathological examination.

Fig. 2. Right pulmonary artery mass removal

Fig. 3. Bilateral pulmonary artery tumor mass71

In postoperatory care, the evolution of the patient
was favourable, and the transthoracic ecocardiography
performed one day after surgery revealed dilated rightheart cavities but with a value of the pulmonary arterysystolic pressure of 44 mmHg.
Unfortunatelly, the result of the histopathologicalexam confirmed our suspicion, and diagnosed amalignant mesenchymal tumor proliferation (taken into
account chondrosarcoma, mixoid chondrosarcoma or
osteosarcoma. For further histological differentiation,
immunohistochemical tests would have been useful, butwere unfortunatelly unavailable.
When discharged, approximately two weeks after
surgery, the patient was on anticoagulant treatement withacenocumarol, diuretic and calcium channel blocker(Diltiazem 3x60 mg/day). He was further refered foroncological treatment and survey, and was unfortunately
lost to our follow-up.

DISCUSSION
The differential diagnosis of pulmonary vasculardiseases are a great challenge for the clinician. Out of
those, the neoplasms of arterial pulmonary trunc are veryrare diseases, with an incidence of 0,001% up to 0,03%
in the general population [1].
Due to the non-specific symptoms (dyspnoea, cough,hemoptysis, thoracic pain), the first diagnosis taken into
account is the one of pulmonary embolism [2,6].
In most cases, the diagnosis is established while
conducting further investigations previous tothromboendarterectomy, or even during surgery (as was
our case) [3].
The prognosis is poor, given the fact that, despite
their reduced metastatic proliferation, survival after

diagnosis is for only a few months, that is 2 months
without surgery and approximately one year after surgicaltumor mass removal [4,5].

CONSENT
Written informed consent was obtained from the patient
for publication of this case report and any accompanying
images. A copy of the written consent is available forreview by the Editor-in-Chief of this journal.

REFERENCES
1. Ramjee V, Lewis MM, Clements SD. High-grade,nonmyogenic pulmonary artery sarcoma: rare findings oncoronary angiography. Tex Heart Inst J. 2011; 38(1):71-3.
2. Weijmer MC, Kummer JA, Thifs LG. Case report of apatient with an intimal sarcoma of the pulmonary trunkpresenting as a pulmonary embolism. Neth J Med. 1999;
55: 80-3.
3. Matotoo A, Fedullo PF, Kapelanski D, Ilowite JS.
Pulmonary artery sarcoma, a case report of surgical cureand 5-year follow-up. Chest. 2002; 122: 745-7.
4. Mandel J, Mark EJ. Case records of the
Massachusetts General Hospital. A 69-year-old woman
with pleuritic pain and a pulmonary arterial obstruction. N
Engl J Med. 2000; 343: 493-500.
5. Parish JM, Rosenow EC 3rd, Swensen SJ, Crotty TB.
Pulmonary artery sarcoma. Clinical features. Chest. 1996;
110(6): 1480-8.
6. Hsing JM, Thakkar SG, Borden EC, Budd GT. Intimal
pulmonary artery sarcoma presenting as dyspnea: casereport. Int Semin Surg Oncol 2007; 4: 14.

EMBOLISMUL PULMONAR: A FI SAU A NU FIREZUMAT
Introducere. Angiosarcomul arterei pulmonare este o tumoră rar întâlnită și diagnosticată și reprezintă oadevărată provocare pentru clinician, în special înainte de intervenția chirurgicală, datorită semnelor extrem denespecifice ale acesteia.
Prezentarea cazului. Prezentăm cazul unui bărbat de 62 de ani, care a fost internat în spital pentru evaluarea
dispneei moderate, care s-a agravat progresiv în ultimele 3 luni, cu alterarea progresivă și a statusului general alpacientului. La internarea în Institutul de Boli Cardiovasculare Timisoara, în septembrie 2014, examenul fizic aarătat că pacientul este supraponderal (BMI= 27 kg/m² BM) și prezenta cianoză centrală minoră. Pacientul avea
de asemenea varice venoase la nivelul ambelor membre inferioare, cu edem periferic moderat, dar fără semne
de sindrom post-trombotic. Au fost remarcate de asemenea hepatomegalia la 5 cm sub nivelul marginii costale72

Fiziologia - Physiology • 2015.25.4(88)drepte și refluxul heptojugular. La auscultația plmonară, pacientul prezenta murmur vezicular diminuat la nivelul
hemitoracelui ingerior stâng, fără raluri și cu saturație arterială spontană a oxigenului de 90%. Examinarea
cardiacă a arătat zgomote cardiace ritmice, fără murmur, cu o frecvență ventriculară de 92 bătăi/min. și tensiune
arterială de 110/80 mmHg. Ecocardiografia transtoracică a arătat un ventricul stâng cu dimensiuni volumetrice înlimite normale, fracție de ejecție păstrată și fără anomalii kinetice, chiar dacă ventriculul drept era mărit.
Investigația Doppler cu flux continuu a arătat hipertensiune pulmonară secundară severă. Evaluarea
angiocoronarografică a arătat că arterele coronare nu prezentau leziuni semnificative. Cateterizarea inimii dreptea arătat presiuni crescute la nivelul ventriculului drept și arterei pulmonare, cu o creștere a rezistenței vasculare
pulmonare, dar fără existența șunturilor stânga-dreapta. Prima concluzie a fost că pacientul prezintă
hipertensiune pulmonară secundară post-embolică, cu episoade repetate de embolism pulmonar, având indicația
de trombendarterectomie pulmonară. Echipa chirurgicală a găsit la nivelul arterei pulmonare drepte o masătumorală mare, aproape ocluzivă, cu un aspect macroscopic friabil, o masă care infiltra peretele vascular și era
prezentă și la nivelul arterei pulmonare stângi, aspectul macroscopic al tumorii fiind sugestiv pentru sarcom alarterei pulmonare.
Concluzii. Diagnosticul diferențial al bolilor vasculare pulmonare reprezintă o provocare pentru clinicieni.
Datorită simptomelor nespecifice, primul diagnostic considerat este cel de embolism pulmonar, având indicație
de trombendarterectomie sau chiar de intervenție chirurgicală. In cazul tumorilor, prognosticul este rezervat, iarrata de supraviețuire este de aproximativ 1 an după îndepărtarea chirurgicală a masei tumorale.
Cuvinte cheie: masă tumorală, trombendarterectomie, embolism pulmonar, hipertensiune

Fiziologia - Physiology • 2015.25.4(88)73