Category Archives: Original Research

LC-MS Method for Determining Amiodarone and Desethylaminodarone in Rat Plasma Used in Endogenous Overdosing Conditions Following Lipolysis

George Jîtcă, Bianca-Eugenia Ősz, Szende Vancea, Amalia Miklos, Amelia Tero-Vescan

University of Medicine and Pharmacy of Tîrgu Mureș, Romania

DOI: 10.2478/amma-2018-0016

Objective: The purpose of this study was to develop a LC-MS method to determine amiodarone (AMI) and its major metabolite desethylamiodarone (DEA) from rat plasma released from the adipose tissue of AMI treated rats subjected to a weight gain/weight loss cycle.
Methods: Separation of the compounds was performed on a Kinetex 2.6 μm C18 100 x 4.6 mm column under isocratic conditions using a mixture of acetonitrile: 0.1% formic acid 65:35 at a flow rate of 0.5 ml/min. Detection of the analyte was performed by electrospray positive ionization, the monitored ions being 135 m/z from 646 for AMI and 135 m/z of 618 for DEA. Analytes were extracted after plasma protein precipitation with methanol.
Results: The developed method presented specificity and linearity on the concentration range of 25-2500 ng/ml plasma for AMI and 2.5-1250 ng/ml plasma for DEA and the precision and accuracy of the method at all of quality control concentration levels including LLOQ were according to official guidelines for validating analytical methods.
Conclusions: A sensitive and accurate LC-MS method has been developed with a much lower LLOQ than literature data to detect the plasma concentration differences of the studied analytes that result from forced lipolysis and mobilization from the adipose tissue.

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Characterization of Inclusion Complexes between Miconazole and Different Cyclodextrin Derivatives

Hajnal Kelemen1, Gabriel Hancu1, Serban Andrei Gâz-Florea2, Eniko Nemes-Nagy3, Lajos Attila Papp1, Eleonora Mircia4

1. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy, Tîrgu Mureş, Romania
2. Department of Organic Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy, Tîrgu Mureş, Romania
3. Department of Biochemistry, Faculty of Pharmacy, University of Medicine and Pharmacy, Tîrgu Mureş, Romania
4. Department of Pharmaceutical Industry, Faculty of Pharmacy, University of Medicine and Pharmacy, Tîrgu Mureş, Romania

DOI: 10.2478/amma-2018-0012

Objective: Miconazole, an imidazole antifungal derivative, is a very hydrophobic compound, a major drawback in obtaining topical pharmaceutical formulations with optimal bioavailability. Cyclodextrins (CDs) may increase local drug delivery by enhancing the drug release and/or permeation. The aim of the study is the characterization of inclusion complexes between miconazole and different CD derivatives.
Methods: Several CD derivatives were tested in the experiments. The binary systems between miconazole and different CDs were prepared in 1:1 molar ratios by physical-mixture and kneading methods. Differential scanning calorimetry (DSC) and Fourier transformed-infrared spectroscopy (FT-IR) methods were used to characterize solid state interactions between miconazole and CDs in their binary systems.
Results: The FT-IR analysis suggests the formation of a new solid phase, indicating a molecular interaction between the components. The DSC analysis sustains the hypothesis of formation of partial inclusion complexes between miconazole nitrate and CD. Conclusion: The thermic behaviour of the complexes depends both on the preparation method and the composition of the products.

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Phenolic Content from Medicinal Plants and their Products Used in Veterinary Medicine

Erzsebet Varga1, Ibolya Schmidt1, Beáta Szövérfi1, Mădălina Daniela Pop1, Hajnal Kelemen2, Anita Tóth3

1. University of Medicine and Pharmacy of Tîrgu Mures,Faculty of Pharmacy, Romania
2. University of Medicine and Pharmacy of Tîrgu Mures, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Romania
3. Semmelweis University, Department of Pharmacognosy, Budapest, Hungary

DOI: 10.2478/amma-2018-0013

Objective: The aim of the study was to determine the content of polyphenols and flavonoids from sixteen selected medicinal plants from the spontaneous Romanian flora and fifteen tinctures obtained with propylene glycol.
Methods: The polyphenols were determined by the Folin-Ciocalteu method while the flavonoids by using a colorimetric method from the 10th edition of the Romanian Pharmacopoeia. The antioxidant activities of the most common nine medicinal plants and fifteen tinctures were determined by DPPH and ABTS methods.
Results: The results highlighted that the phenolic compounds and flavonoids have contributed to their antioxidant activities and the medicinal plants and tinctures included in the study are rich sources of natural antioxidants.
Conclusions: There are a wide variety of extraction methods for the determination of phenolics and flavonoids. The study confirms a correlation between phenolic and flavonoid contents obtained by using the DPPH and ABTS tests.

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Optimization of a Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells

Georgiana Mihaela Şerban, Ion Bogdan Mănescu, Doina Ramona Manu, Minodora Dobreanu

University of Medicine and Pharmacy of Tîrgu Mureş, Romania

DOI: 10.2478/amma-2018-0011

Objective: Peripheral blood mononuclear cells (PBMC) are extremely important in the body’s immune response. Their isolation represents a major step in many immunological experiments. In this two phase study, we aimed to establish an optimum protocol for PBMC isolation by density-gradient centrifugation.
Methods: During Phase-1, we compared two commercially available PBMC isolation protocols, Stemcell Technologies (ST) and Miltenyi Biotec (MB), in terms of PBMC recovery and purity. Twelve blood samples were assigned to each protocol. Each sample was divided in three subsamples of 1ml, 2ml and 3ml in order to assess the influence of blood sample volume on isolation performance. During Phase-2, a hybrid protocol was similarly tested, processing six blood samples. Additionally, we performed a flow cytometric analysis using an Annexin-V/Propidium-Iodide viability staining protocol.
Results: Phase-1 results showed that, for all subsample volumes, ST had superior PBMC recovery (mean values: 56%, 80% and 87%, respectively) compared to MB (mean values: 39%, 54% and 43%, respectively). However, platelet removal was significantly higher for MB (mean value of 96.8%) than for ST (mean value of 75.2%). Regarding granulocyte/erythrocyte contamination, both protocols performed similarly, yielding high purity PBMC (mean values: 97.3% for ST and 95.8% for MB). During Phase-2, our hybrid protocol yielded comparable results to MB, with an average viability of 89.4% for lymphocytes and 16.9% for monocytes.
Conclusions: ST yields higher cell recovery rates and MB excels at platelet removal, while the hybrid protocol is highly similar to MB. Both cell recovery and viability increase with blood sample volume.

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Aquaporin 2 Expression in Human Fetal Kidney Development

Gergő Ráduly, Zsuzsánna Pap, Loránd Dénes, Annamária Szántó, Zoltán Pávai

Department of Anatomy and Embryology, University of Medicine and Pharmacy of Tîrgu Mureş

DOI: 10.2478/amma-2018-0010

Introduction: The metanephrogenic zone, renal cortex and renal pyramids develop into their final form by week 13. The metanephric kidney produces large quantities of diluted urine in order to maintain volumes of amniotic fluid. Aquaporins are transmembrane protein channels that enable water transport through biological membranes. Aquaporin 2 (AQP2) is a water channel found in the supranuclear region and apical area of the cell membrane of the kidneys collecting tubule cells. Its main function is reabsorption of water through vasopressin stimulation.
Materials and methods: Immunohistochemistry was used to study fetal renal tissue of 34 post-mortem fetuses of 9 weeks to 24 weeks gestational age.
Results: AQP2 expression is present in connecting tubules and collecting tubules during the targeted time period. From week 9 to 12, the expression is cytoplasmic. From week 13 to 20 the enhancement of expression in the apical cell membrane occurs with the advancement of fetal age. At the end of the studied period, from week 21 to 24, both cytoplasmic and apical expression were observed. In animal studies AQP2 expression has an increasing trend during development. In contradiction with these results, other authors described low AQP2 levels in the human fetal kidney.
Conclusions: This study helps to understand the amniotic fluid’s homeostasis during pregnancy. In the beginning of the fetal period AQP2 protein is present in the cytoplasm of epithelial cells of the collecting duct and distal connecting duct. During the fetal period, AQP2 expression in collecting ducts becomes more enhanced in the apical membrane of the cells.

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MTHFR – Ala222Val Effects on Metabolic Syndrome Progression

Katalin Csép, Eszter Szigeti, Krisztina Szalman

University of Medicine and Pharmacy Tirgu Mures, Romania

DOI: 10.2478/amma-2018-0009

Objective: Methylene-tetrahydrofolate reductase (MTHFR) is involved in adapting metabolism to environmental challenges by various mechanisms, including the control of gene expression by epigenetic and post-translational changes of transcription factors. Though a metabolic syndrome candidate gene, association studies of its common polymorphism rs1801133 (MTHFR-Ala222Val) remain inconclusive with important ethnic differences, and the effect on disease progression was not addressed.
Methods: 307 middle-aged metabolic syndrome patients in a central Romanian hospital setting were investigated metabolically, and genotyped by PCR-RFLP. Disease progression was assessed by the age of onset of metabolic components, as well as development of non-alcoholic fatty liver disease and atherosclerotic complications.
Results: The minor allele frequency of rs1801133 was 30.13%. Metabolic parameters showed no statistically significant differences according to genotype, but variant carriers developed dysglycemia and dyslipidemia earlier (53.28±10.8 vs 59.44±9.31 years, p<0.05 and 58.57±11.31 vs 64.72±10.6 years, p<0.1).While the polymorphism did not influence hepatic complications, an inverse association was found for manifest atherosclerosis (OR=0.49, p=0.006, 95%CI:0.29-0.81), which may be folate-status dependent, and needs further investigations. Simultaneous analysis with transcription factor polymorphisms (rs1801282, rs8192678) showed that the more protective genotypes were present the later metabolic disturbances developed, and in the presence of the other two variants the apparent protective cardiovascular effect disappeared.
Conclusions: The common functional polymorphism rs1801133 may influence metabolic syndrome progression, the age of onset of components and development of atherosclerotic complications. Besides simple additive effects, complex mitigating and aggravating variant interactions may exist, and the protective or predisposing outcome may depend on modifiable environmental factors.

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The Value of Perioperative ECG Recordings in the Cardiovascular Monitoring and Risk Assessment of Asymptomatic Patients with Total Hip Arthroplasty

Attila Frigy1, István Szabó-Györke2, Krisztina Csomor2, András Czédula3, Sándor-György Zuh4, István Gergely4

1. Department of Internal Medicine IV, University of Medicine and Pharmacy of Tirgu Mures, Romania
2. University of Medicine and Pharmacy of Tirgu Mures, Romania
3. Clinic of Orthopedics and Traumatology, Clinical County Hospital Mures, Tirgu Mures, Romania
4. Department of Orthopedics I, University of Medicine and Pharmacy of Tirgu Mures, Romania

DOI: 10.2478/amma-2018-0008

Aim: To evaluate the value of a more closed perioperative ECG follow-up in the investigation and forecasting of cardiovascular events during and after hospital admission.
Methods: We included in the study 30 asymptomatic patients from cardiac point of view (14 women, 16 men, mean age 63 years), who underwent total hip arthroplasty. ECGs were performed on the day before surgery, on the day of surgery, after the intervention, and on the 3rd day. We looked for the correlations between ECG changes and clinical events.
Results: The commonest postoperative new ECG changes were QTc prolongation (14 patients – 46,6%) and sinus tachycardia (8 patients – 26,6%). There were no major cardiac events during hospitalization, and we found possible correlations in two cases between perioperative ST-T changes and postdischarge clinical events: one exitus and one case of ischemic heart disease.
Conclusions: Our data do not support the efficacy of a more closed, routine ECG follow-up in the studied, low risk patient population, but new perioperative ST-T changes always have to be taken into account.

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Mediators of Inflammation as a Link Between Diabetes Mellitus and Periodontal Breakdown

Adriana Monea, Lia Yero Eremie, Csilla Bukhari, Gabriela Beresescu, Carmen Biris

University of medicine and pharmacy Tirgu-Mures, Romania

DOI: 10.2478/amma-2018-0005

Our objective was to investigate immunological changes that occur in saliva of subjects with type 2 diabetes mellitus (T2DM) without signs of periodontal disease and to establish if salivary inflammatory cytokines are a possible link between diabetes mellitus and periodontal breakdown. Material and methods. Twenty T2DM subjects with no periodontal disease and twenty healthy controls were registered for the present study. TNF-α and IL-6 level from saliva and serum were measured. Periodontal tissue samples were histologically examined.

Results: TNF-α and IL-6 levels were higher in T2DM subjects compared to controls, with an extremely significant difference in saliva (p<0.001). Significant inflammation, affecting both epithelial and connective tissues was present in periodontal biopsies. Conclusions: The subjects showed an increased TNF-α and IL-6 levels, both in serum and -mostly in -saliva of diabetics without signs of periodontal disease, confirming the hypothesis of immunological implication, as a correlation between periodontal disease incidence and diabetes mellitus. Histologic alterations, suggesting a local inflammatory state, were present in periodontal tissue of diabetics, confirming the above hypothesis. The study reveals that saliva analysis is a quite efficient method in testing the periodontal breakdown progression in the subjects with T2DM.

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Sciatic Nerve Regeneration in Wistar Albino Rats Evaluated by in vivo Conductivity and in vitro 1H NMR Relaxometry

Anamaria Victoria Bumbu1, Radu Fechete2, Marcel Perian3, Bogdan Septimiu Bumbu3, Klara Brinzaniuc3

1. Spitalul Clinic Judetean De Urgenta Oradea, Romania
2. Technical University of Cluj-Napoca, Romania
3. University of Medicine and Pharmacy Tîrgu Mures, Romania

DOI: 10.2478/amma-2018-0004

Objective: The aim of this study was to evaluate and quantify functional and structural nerve regeneration after reconstruction using either direct suture or silicon graft.
Methods: Thirty-two adult Wistar Albino rats were divided in two equal groups. The left sciatic nerve was cross-sectioned and reconstructed using either direct suture (DS group) or a silicone graft (SG group). At 4, 6, 8 and 10 weeks two rats were randomly chosen from each group for in vivo measurement of nerve electric conductivity and subsequently sacrificed together with other two rats from the same group for in vitro 1H NMR relaxometry measurements. The T2 distributions were assigned to 1H located in different pools corresponding to the nerve structure.
Results: In the injured nerve we observed a significant increase in the stimulation threshold and a decrease in conduction velocity when compared with the healthy nerve in both groups. Whereas the conduction velocity increased progressively from 4 to 10 weeks in the DS group, the opposite evolution was observed in the SG group. In both groups, the first two peaks corresponding to water bound to collagen and epineurium had smaller transverse relaxation times in the injured nerves, while there was no change in the peaks corresponding to perineurium and free water between healthy and injured nerves.
Conclusions: Significant differences were observed between direct suture and nerve graft reconstructions from both a functional and structural point of view. In the case of direct suture reconstruction, the nerve was functionally healed at 10 weeks after injury.

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Clinical and Therapeutic Trial for the Efficacy of Narrow Band – UVB Phototherapy versus Systemic Therapy in Moderate and Severe Atopic Dermatitis of the Adult

Gyula Laszlo Fekete, László Fekete

University of Medicine and Pharmacy Tirgu Mures, Romania

DOI: 10.2478/amma-2018-0003

Objectives: The aim of this clinical and therapy study was to evaluate the efficacy of NB-UVB phototherapy versus systemic therapy in moderate-to-severe atopic dermatitis of the adult.
Material and methods: The subjects of the study were divided into two groups of 25 adult patients with moderate and severe atopic dermatitis according to the inclusion criteria. The first group of 25 patients were treated with systemic corticosteroids while the second group of 25 patients were treated with NB-UVB phototherapy. At the end of the study, after all the data were centralized, we performed a statistical analysis of the results, comparing the two groups as well as the efficacy of the different therapies.
Results: In group I the clinical efficacy of the systemic corticosteroid treatment was achieved, on average, at 4 weeks in patients with moderate atopic dermatitis and at 6 weeks in patients with severe atopic dermatitis. In group II the clinical effecacy of NB-UVB phototherapy was achieved, on average, at 6 weeks for patients with moderate atopic dermatitis and at 8 weeks for those with the severe form. In both groups, the total IgE serum levels were elevated at the beginning, and they became normal throughout the clinical improvement. Remarkable therapy-related side effects were found in the first study group.
Conclusion: We conclude that NB-UVB phototherapy had similar efficacy in treating moderate-to-severe atopic dermatitis with minimal side effects compared to systemic corticosteroid therapy.

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