Vesna Kuntić

The endogenous opioid system in human alcoholics : molecular adaptations in brain areas involved in cognitive control of addiction

The endogenous opioid system (EOS) plays a critical role in addictive processes. Molecular dysregulations in this system may be specific for different stages of addiction cycle and neurocircuitries involved and therefore may differentially contribute to the initiation and maintenance of addiction. Here we evaluated whether the EOS is altered in brain areas involved in cognitive control of addiction including the dorsolateral prefrontal cortex (dl‐PFC), orbitofrontal cortex (OFC) and hippocampus in human alcohol‐dependent subjects. Levels of EOS mRNAs were measured by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR), and levels of dynorphins by radioimmunoassay (RIA) in post‐mortem specimens obtained from 14 alcoholics and 14 controls. Prodynorphin mRNA and dynorphins in dl‐PFC, κ‐opioid receptor mRNA in OFC and dynorphins in hippocampus were up‐regulated in alcoholics. No significant changes in expression of proenkephalin, and µ‐ and δ‐opioid receptors were evident; pro‐opiomelanocortin mRNA levels were below the detection limit. Activation of the κ‐opioid receptor by up‐regulated dynorphins in alcoholics may underlie in part neurocognitive dysfunctions relevant for addiction and disrupted inhibitory control.

Effects of rutin and hesperidin and their Al(III) and Cu(II) complexes on in vitro plasma coagulation assays.

Two flavonoids, rutin and hesperidin, were investigated in vitro for anticoagulant activity through coagulation tests: activated partial thromboplastin time (aPTT), prothrombin time (PT) and thrombin time (TT). Only an ethanolic solution of rutin at the concentration of 830 µM prolonged aPTT, while TT and PT were unaffected. In order to evaluate whether the prolongation of aPTT was due to the decrease of coagulation factors, the experiment with deficient plasma was performed, showing the effects on factors VIII and IX. Since pharmacological activity of flavonoids is believed to increase when they are coordinated with metal ions, complexes of these flavonoids with Al(III) and Cu(II) ions were also tested. The results showed that complexes significantly prolonged aPTT and had no effects on PT and TT. Assay with deficient plasma (plasma having the investigated factor at less then 1%) revealed that complexes could bind to the coagulation factors, what may lead to a non-specific inhibition and aPTT prolongation. An effort was made to correlate stability of complexes with their anticoagulant properties.

Radioprotectors – the Evergreen Topic

To protect organisms from ionizing radiation (IR), and to reduce morbidity or mortality, various agents, called radioprotectors, have been utilized. Because radiation‐induced cellular damage is attributed primarily to the harmful effects of free radicals, molecules with radical‐scavenging properties are particularly promising as radioprotectors. Early development of such agents focused on thiol synthetic compounds, known as WR protectors, but only amifostine (WR‐2721) has been used in clinical trials as an officially approved radioprotector. Besides thiol compounds, various compounds with different chemical structure were investigated, but an ideal radioprotector has not been found yet. Plants and natural products have been evaluated as promising sources of radioprotectors because of their low toxicity, although they exhibit an inferior protection level compared to synthetic thiol compounds. Active plant constituents seem to exert the radioprotection through antioxidant and free radical‐scavenging activities. Our research established that plants containing polyphenolic compounds (raspberry, blueberry, strawberry, grape, etc.) exhibit antioxidative activities and protect genetic material from IR.

Spectrophotometric Investigation of the Pd(II)-Quercetin Complex in 50% Ethanol

Summary. Composition and stability constant of the Pd(II)-quercetin complex were determined by suitable spectrophotometric methods and pH-metric measurements in 50% Ethanol. It was found that the Pd2+ ion and quercetin form a 1:1 complex in which Pd2+ is linked to quercetin through the carbonyl and the 3-hydroxyl group. The stability constant log β1 ranged from 6.05 at pH=5.00 to 4.96 at pH=6.50. The conditions for the spectrophotometric determination of quercetin by means of complex formation were investigated. Beer’s law was obeyed up to 5.00×10−5 M quercetin.Zusammenfassung. Zusammensetzung und Stabilitätskonstante des Pd(II)-Quercetin-Komplexes in 50% Ethanol wurden mittels geeigneter spektrophotometrischer Methoden und pH-metrischer Messungen bestimmt. Es wurde festgestellt, daβ Pd2+ und Quercetin einen 1:1-Komplex bilden, in dem das Pd-Ion über seine Carbonyl- und 3-Hydroxylgruppe an Quercetin gebunden ist. Die Stabilitätskonstante log β1 bewegte sich im Bereich von 6.05 (pH=5.00) bis 4.96 (pH=6.50). Die Bedingungen für eine spektrophotometrische Bestimmung von Quercetin mittels Komplexierung wurden untersucht. Das Beersche Gesetz wird bis zu 5.00×10−5 M Quercetin befolgt.

Diverse Elements in Herbal Tea Products Consumed in Serbia Using Inductively Coupled Plasma Mass Spectrometry

Nine elements (Fe, Cu, Mn, Zn, As, Cd, Sn, Hg, and Pb) were determined in 8 teas and 25 herbal teas widely consumed in Serbia for medical purposes. Green, black, peppermint, chamomile, cranberry, sage, rosehip, uva, senna, marigold, and fennel teas were investigated. Microwave-assisted acid digestion was used for all of the samples, and the element contents were determined by inductively coupled plasma mass spectrometry. From all of the determined microelements, the highest content was that of Mn, 2912.8 and 2541.8 mg/kg of dry matter in the black and green tea, respectively. The contents of toxic elements (As, Cd, and Pb) were below the maximum permissible levels, except for one brand of peppermint tea in which the level of Cd was 8.61 mg/kg, much higher than the allowed 0.3 mg/kg.

An Improved HPLC Method with the Aid of a Chemometric Protocol: Simultaneous Determination of Atorvastatin and Its Metabolites in Plasma

The aim of the present study was to optimize a chromatographic method for the analysis of atorvastatin (acid and lactone forms), ortho- and para-hydroxyatorvastatin by using an experimental design approach. Optimization experiments were conducted through a process of screening and optimization. The purpose of a screening design is to identify the factors that have significant effects on the selected chromatographic responses, and for this purpose a full 23 factorial design was used. The location of the true optimum was established by applying Derringer’s desirability function, which provides simultaneously optimization of all seven responses. The ranges of the independent variables used for the optimization were content of acetonitrile in mobile phase (60–70%), temperature of column (30–40 °C) and flow rate (0.8–1.2 mL min−1). The influences of these independent variables were evaluated for the output responses: retention time of first peak (p-hydroxyatorvastatin) and of last peak (atorvastatin, lactone form), symmetries of all four peaks and relative retention time of p-hydroxyatorvastatin. The primary goal of this investigation was establishing a new simple and sensitive method that could be used in analysis of biological samples. The method was validated and successfully applied for determination of atorvastatin (acid and lactone forms) and its metabolites in plasma.

Direct UV Spectrophotometry and HPLC Determination of Triton X-100 in Split Virus Influenza Vaccine.

One of the most commonly used surfactants in the production of split virus influenza vaccine is nonionic surfactant Triton X-100. After splitting of the virus is accomplished, Triton X-100 is removed from the vaccine by subsequent production steps. Because of toxicity of Triton X-100, which remains in the vaccine in residual amounts, a sufficiently sensitive method for its detection and quantification needs to be defined. Two methods for determination of Triton X-100 residuals were developed: the UV-spectrophotometry and HPLC methods. For both methods, preparation of vaccine samples and removal of proteins and virus particles were crucial: samples were treated with methanol (1:1) and then centrifuged at 25 000 × g for 30 min. After such treatment, the majority of vaccine components that interfered in the UV region were removed, and diluted samples could be directly measured. The chromatographic system included C18 column, step methanol gradient, and detection at 225 nm with a single peak of Triton X-100 at 12.6 min. Both methods were validated and gave satisfactory results for accuracy, precision, specificity, linearity, and robustness. LOQ was slightly lower for the HPLC method. Hence, it was shown that both methods are suitable for analysis of residual amounts of Triton X-100, with the advantages of the UV method being its simplicity and availability in most laboratories.

Surface-Enhanced Raman Scattering (SERS) Biochemical Applications

Surface-enhanced Raman scattering (SERS) is achieved when an analyte is adsorbed onto or in close proximity to a prepared roughened metal surface (primarily gold or silver) in a process which greatly enhances the Raman emission. SERS provides high sensitivity, selectivity, and specificity to the field of biomolecule detection and identification, and a range of diseases can be detected. Of particular interest is the development of SERS tags for cancer detection, as well as SERS-labeled antibodies in SERS immunoassays for protein localization in blood plasma/serum or tissue. The suitability of SERS for biochemical diagnostics and future implementation in a clinical practice is promising.