Béla Kiss

Investigation into the role of Cu/Zn-SOD delivery system on its antioxidant and antiinflammatory activity in rat model of peritonitis

BACKGROUNDnThe current study evaluated the role of delivery system (solution, conventional liposomes and PEG-ylated liposomes) on superoxide dismutase (SOD) antioxidant and antiinflammatory properties in a rat model of lipopolysaccharide (LPS)-induced peritonitis.nnnMETHODSnFifty male albino rats (Wistar-Bratislava) were divided into five groups (n=10). Control group received saline and the other four groups received intraperitoneal injections of LPS (5mg/kg). Among the LPS-injected groups, one was LPS control group and the other three groups received the endotoxin injection 30min after receiving the same dose of SOD (500U/kg, ip) in different delivery systems: saline solution (SOD-S), conventional liposomes (SOD-L) or PEG-ylated liposomes (SOD-PL). The animals were euthanized 6h after LPS injection, blood samples were collected and acute phase response (total and differential leukocytes count; tumor necrosis factor α), antioxidants (total antioxidants; reduced glutathione), oxidative stress (total oxidants; lipid peroxidation) and nitrosative stress (nitric oxide metabolites; nitrotyrosine) were evaluated.nnnRESULTSnIntraperitoneal administration of LPS to rats induced a marked inflammatory and oxidative response in plasma. On the other hand, all SOD formulations had protective effect against endotoxin-induced inflammation and oxidative/nitrosative stress, but PEG-ylated liposomes had the most significant activity. Thus, SOD-PL administration significantly reduced the effects of LPS on bone marrow acute phase response, the oxidative status and production of nitric oxide metabolites, while increasing the markers of antioxidant response in a significant manner.nnnCONCLUSIONnSOD supplementation interferes both with inflammatory and oxidative pathways involved in LPS-induced acute inflammation, PEG-ylated liposomal formulation being of choice among the tested delivery systems.

A Rapid Method for Determination of Resveratrol in Wines by HPLC-MS

Abstract A new, sensitive, high throughput liquid chromatography coupled with mass spectrometry (LC-MS/MS) method for quantification of trans- andcis-resveratrol in wine samples was elaborated. During method development the influence of multiple factors on the ionization efficiency of resveratrol was studied. These parameters included ion source (APCI and ESI, both being operated in positive and negative ionization mode), percentage of aqueous and organic phase, type of organic solvent, and concentration of additives. The optimum conditions were as follows: a mobile phase consisting of a mixture of 1 mM ammonium acetate/acetonitrile (73/27, v/v) with a flow rate of 1 mL/min, MS/MS detection with an APCI interface, operated in negative ionization mode. The monitored ion transition was m/z 227→184.7. Calibration curves were generated for both isomers of resveratrol in the range of 10.47–837.86 ng/mL (trans isomer) and 9.12 − 730.14 ng/mL (cis isomer), respectively. The developed method has been successfully applied to the analysis and comparison of trans- and cis-resveratrol content of 20 different Romanian wine samples.

Influence of Genista Tinctoria L or Methylparaben on Subchronic Toxicity of Bisphenol A in Rats

OBJECTIVEnTo evaluate the influence of an extract of Genista tinctoria L. herba (GT) or methylparaben (MP) on histopathological changes and 2 biomarkers of oxidative stress in rats subchronicly exposed to bisphenol A (BPA).nnnMETHODSnAdult female Wistar rats were orally exposed for 90 d to BPA (50 mg/kg), BPA+GT (35 mg isoflavones/kg) or BPA+MP (250 mg/kg). Plasma and tissue samples were taken from liver, kidney, thyroid, uterus, ovary, and mammary gland after 30, 60, and 90 d of exposure respectively. Lipid peroxidation and in vivo hydroxyl radical production were evaluated by histological analysis along with malondialdehyde and 2,3-dihydroxybenzoic acid detection.nnnRESULTSnThe severity of histopathological changes in liver and kidneys was lower after GT treatment than after BPA or BPA+MP treatment. A minimal thyroid receptor antagonist effect was only observed after BPA+MP treatment. The abnormal folliculogenesis increased in a time-dependent manner, and the number of corpus luteum decreased. No significant histological alterations were found in the uterus. The mammary gland displayed specific estrogen stimulation changes at all periods. Both MP and GT revealed antioxidant properties reducing lipid peroxidation and BPA-induced hydroxyl radical generation.nnnCONCLUSIONnGT L. extract ameliorates the toxic effects of BPA and is proved to have antioxidant potential and antitoxic effect. MP has antioxidant properties, but has either no effect or exacerbates the BPA-induced histopathological changes.

Rapid high-performance liquid chromatography–tandem mass spectrometry method for determination of pentoxifylline and its active metabolites M1 and M5 in human plasma and its application in bioavailability study

A new rapid, sensitive and selective liquid chromatography coupled with mass spectrometry method was developed and validated for the simultaneous quantification of pentoxifylline (PTX) and two major active metabolites in human plasma (M1 and M5). After a deproteinization step, chromatographic separation of the selected analytes was performed on a RP-C18 column. The detection of target compounds was in multiple reaction monitoring mode using an ion trap mass spectrometer equipped with an electrospray ion source. The method was validated and proved to be linear, accurate and precise over the range 5.08-406.14, 10.08-806.40 and 20.15-1611.60 ng/mL in case of PTX, M1 and M5, respectively. The major advantages of this method are the small sample volume, simple sample processing technique, the high sensitivity and the very good selectivity guaranteed by the MS/MS (in case of PTX) or MS/MS/MS (in case of M1 and M5) detection. The validated method has been successfully applied to a bioequivalence study.

A rapid UPLC–MS/MS method for simultaneous determination of flunitrazepam, 7-aminoflunitrazepam, methadone and EDDP in human, rat and rabbit plasma

A simple, high-throughput, sensitive LC-ESI-MS/MS method is presented for the simultaneous determination of methadone (MET), flunitrazepam (FNZ) and their major metabolites, EDDP (2-ethilidene-1,5-dimethyl-3,3-diphenylpyrrolidone) and 7-aminoflunitrazepam (7-AFNZ), respectively, in human, rat and rabbit plasma. The isolation of the selected compounds involved a liquid-liquid extraction with ethyl acetate at a basic pH. Good chromatographic separation was achieved on a HSS T3 column (1.8 μm particle size), with a 3 min gradient elution using a mixture of acetonitrile with 0.1% formic acid (solvent A) and 5mM ammonium acetate (solvent B) as the mobile phase. The tandem mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with ionization of the analytes in positive mode. The assay was fully validated according to current acceptance criteria for bioanalytical methods validation. It was proved to be linear in the range of 0.5-250 ng/mL, with adequate accuracy and precision over this range. Based on accuracy and CV% values the LOQ and ULOQ values were set at 0.509 ng/mL and 2036 ng/mL for MET, 0.520 ng/mL and 2080 ng/mL for EDDP, 0.524 ng/mL and 2096 ng/mL for FNZ and 0.528 ng/mL and 2114 ng/mL for 7-AFNZ, respectively. The method was tested for potential matrix effects, without observing significant ion suppression. The investigated compounds stability was examined in plasma at room temperature and after three freeze-thaw cycles and in the final extract when maintained at 4 °C in the autosampler. Potential stability issues were observed only for FNZ at room temperature. The method was successfully applied to quantify the selected compounds in human, rat and rabbit plasma samples, after exposure to FNZ or simultaneous exposure to FNZ and MET.

Individual and combined in vitro (anti)androgenic effects of certain food additives and cosmetic preservatives.

The individual and combined (binary mixtures) (anti)androgenic effect of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) was evaluated using the MDA-kb2 cell line. Exposing these cells to AR agonists results in the expression of the reporter gene (encoding for luciferase) and luminescence can be measured in order to monitor the activity of the reporter protein. In case of the evaluation of the anti-androgenic effect, the individual test compounds or binary mixtures were tested in the presence of a fixed concentration of a strong AR agonist (1000 pM 5-alpha-dihydrotestosterone; DHT). Cell viability was assessed using a resazurin based assay. For PG, this is the first report in the literature concerning its (anti)androgenic activity. In case of both individual and mixture testing none of the compounds or binary combinations showed androgenic activity. When tested in the presence of DHT, BuPB, BHA and BHT proved to be weak anti-androgens and this was confirmed during the evaluation of binary mixtures (BuPB+BHA, BuPB+BHT and BHA+BHT). Besides performing the in vitro testing of the binary combinations, two mathematical models (dose addition and response addition) were evaluated in terms of accuracy of prediction of the anti-androgenic effect of the selected binary mixtures. The dose addition model guaranteed a good correlation between the experimental and predicted data. However, no estimation was possible in case of mixtures containing PG, due to the lack of effect of the compound in case of the individual testing.

Determination of Flunitrazepam in Human Plasma and Urine by HPLC with Mass Spectrometry Detection

Abstract A new, sensitive, and selective liquid chromatography coupled with mass spectrometry (LC/MS/MS) method for quantification of flunitrazepam in human plasma and urine was validated. The detection of flunitrazepam was in multiple reaction monitoring mode using an ion trap mass spectrometer equipped with an atmospheric pressure chemical ionisation ion source. The method was validated and proved to be linear, accurate, and precise over the range of 0.7–49.4 ng/mL in plasma samples and 0.5–33 ng/mL in urine. This is the first reported method for analysis of flunitrazepam in human plasma and urine that uses protein precipitation for plasma/direct injection for urine as a sample processing procedure. The total run time of the analytical method is less than 2 minutes. Another advantage of the method, besides its simplicity, is the very good recovery of the analyte. The validated LC/MS/MS method has been successfully applied to a pharmacotoxicological study of flunitrazepam.

ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS OF FLUNITRAZEPAM AND 7-AMINOFLUNITRAZEPAM IN HUMAN PLASMA

A high-throughput ultra-performance liquid chromatography (UPLC) method with UV detection was elaborated and validated for the simultaneous quantification of flunitrazepam and its major metabolite, 7-aminoflunitrazepam, in human plasma. After a simple liquid-liquid extraction with ethyl acetate, chromatographic separation was performed on a BEH C18 chromatographic column with a binary mixture of acetonitrile/10 mM potassium monohydrogen phosphate (pH = 8.5) as the mobile phase, under gradient elution (flow rate = 0.275 mL/min). The total run time of the chromatographic analysis was 4.2 min. UV detection was performed at 314 nm for flunitrazepam and at 242 nm for 7-aminoflunitrazepam. To our knowledge, this is the first reported UPLC-PDA method for the quantification of flunitrazepam and 7-aminoflunitrazepam in human plasma. The assay was applied successfully for the analysis of the selected two analytes in real human plasma samples obtained from a healthy volunteer, after ingestion of a single dose of flunitrazepam.

Estrogenic/antiestrogenic activity of selected selective serotonin reuptake inhibitors

Background and aims Selective serotonin reuptake inhibitors (SSRIs) are one of the most prescribed classes of psychotropics. Even though the SSRI class consists of 6 molecules (citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine and sertraline), only fluoxetine was intensively studied for endocrine disruptive effects, while the other SSRIs received less attention. This study was designed to evaluate the estrogenic/antiestrogenic effect of fluoxetine, sertraline and paroxetine. Methods The in vitro (anti)estrogenic activity was assessed using a firefly luciferase reporter construct in the T47D-KBluc breast cancer cell line. These cells express nuclear estrogen receptors that can activate the transcription of the luciferase reporter gene upon binding of estrogen receptor agonists. Results All three compounds were found to interact with the estrogen receptor. Fluoxetine had dual properties, weak estrogenic at lower concentrations and antiestrogenic effect at higher concentrations. Sertraline shared the same properties with fluoxetine, but also increased the estradiol-mediated transcriptional activity. Paroxetine presented only one type of effect, the ability to increase the estradiol-mediated transcriptional activity. Conclusions Overall, our results indicate a possible interaction of SSRIs with the estrogen receptor. As SSRIs are being used by all categories of population, including pregnant women or children, establishing whether they can affect the endocrine mediated mechanisms should be a priority.

A HIGH-THROUGHPUT UPLC-MS/MS FOR THE SIMULTANEOUS ANALYSIS OF SIX PHYTOESTROGENS FROM GENISTA TINCTORIA EXTRACTS

A new rapid, sensitive UPLC method coupled with ESI-MS/MS detection was developed and validated for the simultaneous quantification of six phytoestrogens (daidzin, genistin, genistein, daidzein, formononetin, and coumestrol) from Genista tinctoria L. extracts. An adequate chromatographic separation was achieved with gradient elution and a mobile phase consisting of a mixture of 0.1% acetic acid/methanol (70/30, v/v). The total run time was of 5.5 min. The detection of target compounds was performed in multiple reaction monitoring mode. The method was validated and proved to be linear, accurate, and precise over the range of 10–800 ng/mL. The LLOQ values estimated based on the signal to noise ratio were of 5 ng/mL for each analyte, except for daidzein (LLOQ = 10.78 ng/mL). The elaborated assay shows shorter run time, lower solvent consumption, and higher sensitivity than most of the existing LC methods described in the literature. The method was used to quantify the selected phytoestrogens in Genista tinctoria herba or flos extracts. The major compounds found were genistein and its glycosidic form genistin. The method proved to be adequate for the characterization of plant extracts from the point of view of their phytoestrogen content, a group of active compounds with great therapeutic potential.