Рязанский государственный медицинский университет имени академика И.П. Павлова, Рязань, Российская Федерация _____________________________________________________________________________ Актуальность. Гликопротеин-Р (Pgp, АВСВ1)-белок-транспортер, обеспечивающий защиту органов и тканей от ксенобиотиков, являющихся его субстратами, выводя их из клеток во внеклеточное пространство и биологические жидкости. Цель. Изучить влияние эстрадиола на активность Pgp in vitro на линии клеток Caco-2. Материалы и методы. Исследования выполнены на линии клеток Caco-2. Активность Pgp анализировали по транспорту его маркерного субстрата-фексофенадина в трансвеллсистеме. Концентрацию фексофенадина оценивали методом высокоэффективной жидкостной хроматографии. Количество Pgp определяли методом ИФА. Эксперимент включал следующие серии: клетки, которые преинкубировали с чистой транспортной средой без добавления каких-либо веществ (контрольная серия); влияние рифампицина в концентрации 10 мкмоль/л при преинкубировании в течение 3 сут на активность и синтез Pgp (контроль индукции); влияние эстрадиола в концентрациях 1 и 10 мкмоль/л при преинкубировании в течение 30 мин на активность и синтез Pgp; влияние эстрадиола в концентрациях 1 и 10 мкмоль/л при преинкубировании в течение 3 сут на активность и синтез Pgp. Результаты. Эстрадиол в концентрациях 1 и 10 мкМ при инкубации с клетками в течение 30 мин достоверно не влиял на активность и синтез Pgp, также как и 1 мкМ эстроген при инкубации 3 сут. В то же время эстрадиол в концентрации 10 мкМ при инкубации в течение 3 сут повышал активность и синтез белка-транспортера. Заключение. Эстрадиол в эксперименте in vitro на клетках линии Caco-2 в концентрации 10 мкМ при инкубировании в течение 3 сут повышает синтез и активность белкатранспортера гликопротеина-Р.
Analysis of the effects of retinol acetate on LPO processes in vivo revealed antioxidant effects under normal conditions and during experimental free-radical pathology (toxic hepatosis-hepatitis). The concentration inversion of antioxidant effects of retinol acetate into prooxidant effects were observed only under normal conditions.
INTRODUCTION: Human breast cancer resistance protein (BCRP, ABCG2) is a transport protein of ABC superfamily of transporters that uses ATP energy of for its work. BCRP plays an important role in the pharmacokinetics of drugs, therefore all new drugs are recommended to be tested for belonging to its substrates, inducers and inhibitors. The influence of substances on the activity of this transport protein in vitro is evaluated by a change of transmembrane transfer of its substrates, one of which being quercetin. This, in turn, requires the development and validation of a method for its quantitative analysis in an appropriate matrix. AIM: To develop a method for the quantitative determination of quercetin using high performance liquid chromatography (HPLC) with tandem mass selective detection (MS/MS) with full validation. MATERIALS AND METHODS: The method was developed on Ultimate 3000 HPLC equipped with TSQ Fortis (Thermo Fisher, USA) MS/MS detector. The conditions of chromatographic analysis were as follows: column UCT Selectra C18 4.6 mm*100 mm 5μm, 100 A, Selectra C18 Guard Cartridges SLC-18 GDC46-5UM. The analysis time was 11 min at separation temperature 35°С, flow rate 0.5 ml/min, injected sample volume 5 μl. Gradient elution regime was used: on 0th minute, the ratio of 0.1% formic acid solution and acetonitrile was 70% and 30%; 0.3 min — 30% and 70%; 4 min — 1% and 99%; 9 min — 70% and 30%. Quercetin retention time was 3.91 minutes. Detection conditions: negative ionization mode, 301 m/z → 150.9 m/z with collision energy 22 V, 301 m/z →178.9 m/z with collision energy 17 V, source fragmentation 5 V, electrospray voltage 3 000 V, CID gas pressure 1 mTorr, Sheath gas 50 Arb, Aux gas 10 Arb, Sweep gas 10 Arb, ion transfer tube temperature 300°C, vaporizer temperature 350°C. The matrix was transport medium after incubation for 3 hours with Caco-2 cells overexpressing BCRP. In this medium, the transport of quercetin through the cell monolayer was further evaluated in vitro. Precipitation of protein and isolation of quercetin from the transport medium was realized by a mixture of water and acetonitrile in a ratio of 1:1. RESULTS: The developed method was validated by the following parameters: selectivity, linearity, accuracy, precision, limit of quantification, sample transfer, matrix effect, sample stability. The analytical range of the technique was 5–500 nmol/l. In this case, the correlation coefficient was more than 0.99. The limit of detection and the lower limit of quantification of quercetin (LLQQ) were 1 and 5 nmol/l, respectively. The calculation of intra- and inter-cycle accuracy and precision showed that these parameters do not exceed 20% for the concentration corresponding to the lower limit of quantitative determination, and 15% for other concentrations. The analyte demonstrated stability in triple freeze-defreeze cycle at -80°C, in storage at -80°C for 60 days, after sample preparation and being kept in the autosampler for 24 hours. There was no sample transfer and no matrix effect. CONCLUSION: A method for the quantitative determination of quercetin using HPLC-MS/MS in a transport medium was developed and fully validated.
The constitutive androstane receptor (CAR) is a nuclear receptor that participates in the regulation of biotransformation of toxic substances and metabolic processes. The mechanisms of expression changes of CAR under conditions of oxidative stress (OS) have not been studied yet and this was the purpose of the study. OS was modeled by incubating Caco2 cells with hydrogen peroxide 10-100 μM for 72 h. The amount of CAR was determined by the Western blot, nuclear factor erythroid 2-related factor 2 (Nrf2) was evaluated by a heterogeneous enzyme immunoassay malondialdehyde (MDA), the lipid peroxidation products (LPP) was assayed by a photometric method. Incubation of cells with 10 μM and 50 μM H2O2 led to an increase in the amount of CAR and Nrf2, while incubation with 100 μM H2O2 caused their decrease. Nrf2 inhibition did not influence the CAR content under OS conditions. 10 μM MDA increased the CAR content, 100 μM MDA had no effect, while 150 μM reduced the amount of CAR.
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