Volume 23 (2015): Edizione 4 (December 2015)

The present paper is a literature review of the recent years dealing with the most important separation techniques of fatty acids in biological samples. Our aim was to make a synthesis of the analytical methods used, to note the most used ones, but also to mention other methods that are less utilized, which can have important advantages (such as less time consuming, greener reagents, etc.). Gas-chromatographic separation methods were described and compared to liquid chromatographic separations of fatty acids in different types of biological samples. In the same time, the importance of determining fatty acids profiles in biological samples was revealed, pointing out the possible implications in diagnostics of different types of disorders or remarking different profiles compared to healthy states.

Serum paraoxonase-1 (PON1) binds mainly to high density lipoproteins (HDLs) and protects low density lipoproteins (LDLs) against oxidation. While paraoxonase and arylesterase activities are traditionally assayed, lactonase activity, accounting for protection against LDL oxidation, was less investigated in obese children and adolescents. Therefore, we aimed to measure lactonase, paraoxonase and arylesterase activities, oxidized LDL (ox-LDL) and malondialdehyde (MDA) levels in obese children and adolescents.

Study population included 68 children (35 obese and 33 normal-weight). Arylesterase and paraoxonase activities were assayed spectrophotometrically. Lactonase activity, ox-LDL and MDA levels were measured using a pH-sensitive colorimetric assay, an ELISA technique and a fluorimetric method, respectively. The lipid profile was assessed by common methods.

Lactonase and arylesterase activities were decreased in the presence of obesity. MDA, but not ox-LDL levels, showed significant differences between groups. Multiple regression analysis identified a reciprocal relationship and a possible association between lactonase and arylesterase activities and obesity.

Background. Oxidative stress (OS) and inflammation are major mechanisms involved in the progression of chronic heart failure (CHF). Serum uric acid (sUA) is related to CHF severity and could represent a marker of xanthine-oxidase activation. The relationship between sUA, oxidative stress (OS) and inflammation markers was assessed in patients with moderate-severe CHF and reduced left ventricular (LV) ejection fraction (EF).

Methods. In 57 patients with stable CHF, functional NYHA class III, with EF<40%, the LV function was assessed by N-terminal of the prohormone brain natriuretic peptide (NT-proBNP) levels and echocardiographically through the EF and E/e’ ratio, a marker of LV filling pressures. The relationship between LV function, sUA, malondialdehyde (MDA), myeloperoxidase (MPO), paraoxonase 1 (PON-1) as OS markers and high sensitivity C-reactive protein (hsCRP) and interleukin 6 (IL-6) as markers of systemic inflammation was evaluated.

Results. The mean sUA level was 7.9 ± 2.2 mg/dl, and 61% of the CHF patients had hyperuricemia. CHF patients with elevated LV filling pressures (E/e’ ≥ 13) had higher sUA (8.6 ± 2.3 vs. 7.3 ± 1.4, p=0.08) and NT-proBNP levels (643±430 vs. 2531±709, p=0.003) and lower EF (29.8 ± 3.9 % vs. 36.3 ± 4.4 %, p=0.001). There was a significant correlation between sUA and IL-6 (r = 0.56, p<0.001), MDA (r= 0.49, p= 0.001), MPO (r=0.34, p=0.001) and PON-1 levels (r= −0.39, p= 0.003).

Conclusion. In CHF, hyperuricemia is associated with disease severity. High sUA levels in CHF with normal renal function may reflect increased xanthine-oxidase activity linked with chronic inflammatory response.

Coronary chronic total occlusion (CTO) is caused by organized thrombi or atherosclerotic plaque progression. The presence of a CTO is an independent predictor of mortality in patients presenting with ST-segment elevation myocardial infarction (STEMI). Platelets have a crucial role in the pathophysiology of atherosclerosis. The aim of this retrospective study was to investigate platelet indices as predictors of CTO in patients with STEMI treated with primary percutaneous coronary intervention (pPCI). A total number of 334 patients admitted for STEMI between January 2011 and December 2013 were included and divided in two groups based on the presence of CTO (48 patients in CTO+ group, 286 patients in CTO-group). Platelet count, mean platelet volume (MPV), platelet distribution width (PDW), platelet-large cell ratio (P-LCR), lymphocyte and neutrophil count determined on admission were analyzed. MPV was larger in patients with CTO compared with patients without CTO (p=0.02), as were PDW (p=0.03) and P-LCR (p=0.01). Platelet-to-lymphocyte ratio (PLT/LYM) was lower in patients with CTO: 105.2 (75.86-159.1) compared to 137 (97-188.1), p<0.01. Receiver-operator characteristic curve analysis identified an area under the curve of 0.61 (95%CI=0.57-0.67, p< 0.01) for PLT/LYM in predicting the presence of a CTO, with a cut-off value at 97.73. Lower values than this were independent predictors of a CTO in multivariate logistic regression analysis, with an Odds Ratio of 2.2 (95%CI=1.15-4.20, p=0.02). Our results support the use of platelet indices and PLT/LYM as predictors of CTO in patients presenting with STEMI.

Background: We hypothesized that an ideal heart valve replacement would be acellular valve root scaffolds seeded with autologous stem cells. To test this hypothesis, we prepared porcine acellular pulmonary valves, seeded them with autologous adipose derived stem cells (ADSCs) and implanted them in sheep and compared them to acellular valves.

Methods: Fresh porcine pulmonary valve roots were decellularized with detergents and enzymes. ADSCs were isolated from subdermal fat and injected within the acellular cusps. Valves were then implanted in an extra-anatomic pulmonary position as RV to PA shunts: Group A (n=6) consisted of acellular valves and Group B (n=6) of autologous stem cell-seeded acellular xenografts. Sheep were followed up for 6 months by echocardiography and histologic analysis was performed on explanted valves.

Results: Early evolution was favorable for both groups. All Group A animals had physiologic growth without any signs of heart failure and leaflets were found with preserved structure and mobility, lacking signs of thrombi, inflammation or calcification. Group B sheep however expressed signs of right ventricle failure starting at one month, accompanied by progressive regurgitation and right ventricle dilatation, and the leaflets were found covered with host tissue. No cells were found in any Group A or B explants.

Conclusions: Acellular stabilized xenogeneic pulmonary valves are reliable, stable, non-immunogenic, non-thrombogenic and non-calcifying scaffolds with excellent hemodynamics. Seeding these scaffolds with autologous ADSCs was not conducive to tissue regeneration. Studies aimed at understanding these novel observations and further harnessing the potential of stem cells are ongoing.

Objective: the pourpose of the study was to determine if there are any differences between placenta derived plasmatic levels of messenger RNA in normal and future preeclamptic pregnancies and if these placental transcripts can predict preeclampsia long before clinical onset

Study design: we compared plasmatic expression of two placental transcripts from 12 women who ultimately developed preeclampsia with 224 controlled subjects, at the end of the first trimester of pregnancy. After multiplse-of-the-median conversion of markers we developed a multivariate model using logistic regression to determine preeclampsia risk.

Results: we found lower multiples of the median values for both placental transcripts (mRNA corresponding to placental growth factor and pregnancy associated plasmatic protein A) in cases who ultimately developed preeclampsia and the multivariate model we obtained offered a preeclampsia detection rate of 75% at 10% false positive rate.

Conclusion: specific early changes of placenta-derived messenger RNA could be used as preeclampsia predictors.

The entry of the generic drugs on the market was an impressive development of the pharmaceutical industry and due to their lower prices also a decrease in the cost price for the treatment of patients. The difference in price (sometimes even 50%) between generics and original and different response to therapy sometimes raised serious questions related to their therapeutic equivalence. The scientific community is increasingly interested in this aspect, with studies (in vitro and on patients) demonstrated statistically significant differences in terms of differences generic / original drug. In this context, the aim of our study was to assess the in vitro cytotoxic activity of oxaliplatin (original and generic drug) on DLD-1 cell lines, HT-29, and carboplatin cytotoxic activity (and the reference molecule from Santa Cruz Biotechnology) on cell line A2780. Cell viability was evaluated using the MTT assay.

Regarding the cell line DLD-1, IC50 values of generics was lower than the original after exposure for 24 hours to oxaliplatin but after 48 hours of exposure were not statistically significant differences. HT-29 line has a higher resistance to chemotherapy compared with oxaliplatin, the IC 50 values after 48 hours of exposure are higher than those for the line DLD-1. IC50 values are confirmed by morphological analysis of cells. Regarding carboplatin were not recorded statistically significant differences between the two generic drugs tested.

Although other studies reported differences between generic and branded drugs in terms of hypersensibility reactions, adverse effects and efficacity, we cannot extrapolate our findings to the patients. Further studies on patients are neeeded for a better evaluation of the efficacity of generic vs. original drugs.

In recent years Clostridium difficile infection (CDI) has represented a serious public health issue, mainly due to the global spread of the hypervirulent strain NAP1/027/BI. The purpose of the present study was to evaluate the utility of a PCR coupled with electrospray ionization mass spectrometry (ESI-MS) commercial assay for the detection of C. difficile virulence markers. Non-duplicative C. difficile isolates from patients with CDI diagnosed in a tertiary level hospital from Bucharest were tested for toxin A, toxin B, binary toxin genes and deletion in tcdC gene using PCR/capillary gel electrophoresis and PCR/ESI-MS. The study analysed 45 non-duplicative isolates, 33 strains (73.3%) belonging to ribotype 027. The concordance between PCR/capillary gel electrophoresis and PCR/ESI-MS was 100% for toxin A gene, 97.8% for toxin B gene, 91.1% for binary toxin subunit A gene and 95.6% for binary toxin subunit B gene. The general concordance for the complete panel of markers was 88.9% but was 100% for ribotype 027 isolates. PCR/ESI-MS might be a valid method for the detection of C. difficile virulence markers, including binary toxin.

Objective: To evaluate the bacteriological features in non-struvite nephrolithiasis and in its associated urinary tract infection, and to establish the relationship between the two pathologies.

Methods: The non-struvite calculi from 132 patients were aseptically extracted by percutaneous nephrolithotomy (PNL). The midstream urine and calculi were bacteriologically and biochemically processed.

Results: Most calculi (78%) were located to renal pelvis, associated with hydronephrosis, the biochemical composition confirming the lack of struvite and revealing the predominance of calcium oxalate. The females presented significantly more colonized calculi (50%) than males (21.9%), with higher bacteriological diversity. There is a significant relation between the presence of colonized calculi and urinary tract infections, 24.2% of calculi and 25.8% of the urine samples presenting positive cultures. In 70.4% of cases, we found the same antibiotic resistance pattern between the pathogens isolated from calculi and urine, thus considering them identical strains. The Enterobacteriaceae represented the most predominant bacteria both from calculi (62.5%) and urine (63.6%), approximatively 30% being resistant to cephalosporins and over 50% resistant to fluoroquinolones, ampicillin and tetracycline. There were 3.8% of cases in which the calculi were colonized but the urine was sterile, the bacteria being sensitive to cephalosporins that are used as prophylaxis.

Conclusions: In all the cases, the same bacterial species was found both in calculi and urine, and 70.4% of them were phenotypically identical. The resistance to the second generation cephalosporins is lower than in the case of other antibiotics, which makes them the most suitable for prophylaxis in PNL.

Background: P-glycoprotein (P-gp), a drug efflux transporter, encoded by the gene MDR1 ABCB1 multidrug resistant, reduces the penetration through the brain by the AEDs. Overexpression of Pgp in blood-brain barrier in epileptic patients play an important rol in pharmacoresistance. The aim of this study was to evaluate a possible association between C1236T and G2677T ABCB1 gene polymorphisms and drug-resistant epilepsy in Romanian children.

Material and Methods: A total of 194 children with epilepsy hospitalized in the Paediatric Neurology and Psychiatry Clinic in Tirgu Mureş and 153 healthy controls were included in this study. Of the initial group, those cases that met the criteria for idiopathic and cryptogenic epilepsies (114 cases) were stratified in 31drug-resistant and 83 drug-responsive patients. The C1236T and G2677T genetic polymorphisms were assessed by RFLP-PCR (polymerase chain-restriction fragment length polymorphism) followed by gel electrophoresis.

Results: Molecular analysis of ABCB1 gene polymorphism identified 1236CT heterozygous to be highly represented in drug-responsive group, with a p value of 0.001. Also, in idiopathic epilepsy subgroups (with partial and generalized type of seizures), in case of 1236TT and CT genotypes we found highly significant differences between drug-responsive patients and those resistant to antiepileptic treatment (p-0.003 and p-0.002 respectively). No association between G2677T polymorphism and total epilepsy was found.

Conclusions: Our results show that MDR1 C1236T and G2677T polymorphisms are not associated with drug-resistant epilepsy in the study population, but 1236TT and 1236CT genotype variants and also 2677TT were found to be significantly associated with drug-responsive idiopathic epilepsy.

The study objective was to examine the clinical and hematological significance of receptor CX3CR1 and megakaryocytes in patients with aplastic anemia.

Method. 40 patients diagnosed with aplastic anemia and 10 case-control were included in the study. Were analyzed bone-marrow biopsies regarding cellularity, the presence of megakaryocytes and immunohistochemical expression of CX3CR1, CD4, CD8, CD45RO. We divided patients according to CX3CR1 intensity and the presence of megakaryocytes in 4 groups, which were analyzed comparatively. We realized the second division of patients in 4 groups, depending on the CX3CR1 intensity and cellularity of bone-marrow biopsy.

Results. Statistically significant differences between the case group and the control group were observed in terms of the percentage of CD8, CD45RO positive cells and positivity for CX3CR1. In the lot of patients with aplastic anemia, we found statistically significant differences between groups with megakaryocytes present and absent, in terms of the number of lymphocytes, platelets, hemoglobin, ESR at 1 hour, ESR at 2 hours, bone marrow cellularity.

Conclusions. CX3CR1 could be involved in the pathogenesis of aplastic anemia, influencing bone marrow cellularity. Megakaryocytes influence more hematological parameters, so we suggest using thrombopoietin receptor analogues as 1st line treatment along with the immunosuppressive treatment.