1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species (ROS) such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene (DBB). In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which DPBF reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion, and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is DBB. Only under the action of H₂O₂ 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis, and cyclic voltammetry (CV). A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196–3.941 mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8 μM, respectively. The kinetics of the reaction between DBBF and H₂O₂ was also studied.

Aim: (1) To describe the whole blood content of thiamine diphosphate (TDP), a biologically active form of vitamin B1 in end-stage kidney disease patients treated with hemodialysis (HD); (2) to establish the impact of a single HD procedure on TDP blood concentrations; and (3) to describe potential explanatory variables influencing TDP dialysis related losses, including dialysis prescription, vitamin B1 dietary intake and supplementation. Methods: Single-center, cross-sectional study in 50 clinically stable maintenance HD patients. The assessment of whole blood TDP with the High Performance Liquid Chromatography method, before and after a single, middle-week dialysis session and analysis of clinical and laboratory parameters potentially influencing TDP status Results: We report a significant difference in TDP levels before and after HD sessions - 42.5 (95% CI 38.7-46.2) μg/L and 23.6 (95% CI 18.9-28.2) μg/L, respectively (p = 0.000). The magnitude of intradialytic TDP changes is highly variable among individuals and is negatively associated only with the body weight of the patients (p < 0.013). Vitamin B1 dietary intake and supplementation do not influence whole blood TDP and dialysis-related loss of TDP. Conclusions: TDP, a bioactive compound of vitamin B1, is substantially lost during the HD procedure, and the magnitude of its loss is associated with the patient's body weight but it is not influenced by vitamin B1 dietary intake and standard supplementation dose.

This paper reports on dihydroxycoumarins as fluorescent probes suitable for the detection and determination of the nitroxide radical, namely 4-amino-TEMPO. Since 4-amino-TEMPO is used as a spin label for the detection of various radicals and damage caused by these species, its determination under physiological conditions might help us to understand the mechanism of the oxidative stress. Among different coumarins studied, only dihydroxy-substituted derivatives show high sensitivity, specificity, and selectivity for the nitroxide radical. In this assay, dihydroxy-substituted coumarins under the action of 4-amino-TEMPO show a very fast and significant increase in fluorescence intensity and lifetime. Among them 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement (up to 40 times), with an estimated limit of detection equal to 16.7 nM—a significantly lower value when compared with UV-Vis or electron paramagnetic resonance (EPR) spectroscopy. The method is characterized by an easy procedure of sample preparation and very short time of analysis. The mechanism of the interaction between 6,7-dihydroxycoumarin and 4-amino-TEMPO has been examined with the use of a series of complementary techniques, such as steady-state and time-resolved fluorescence spectroscopy, UV-Vis spectroscopy, electron paramagnetic resonance spectroscopy, potentiometric titration, and high-performance liquid chromatography. It has been proven that the only route of the reaction in the system studied is a proton transfer from the molecule of esculetin to the amino group of the nitroxide. Biological studies performed on prostate cancer cells, breast cancer cells, and normal skin fibroblasts revealed significant anticancer properties of 6,7-dihydroxycoumarin, which caused a considerable decrease in the viability and number of cancer cells, and affected their morphology, contrary to normal fibroblasts. Furthermore, the experiment performed on prostate cancer cells showed that fluorescence emission of esculetin is closely related to intracellular pH—the higher pH, the higher observed fluorescence intensity (in accordance with a chemical experiment). On the other hand, the studies performed in different pH levels revealed that when pH of the solution increases, the observed fluorescence intensity enhancement under the action of 4-amino-TEMPO decreases (better sensing properties of esculetin towards the nitroxide in lower pH).

Probiotics are live microorganisms that provide health benefits when administered in adequate amounts. Due to the increasing popularity of probiotic supplements, concerns have arisen regarding their quality, microbial composition, and safety. This study aimed to evaluate the quantitative and qualitative characteristics of the selected probiotics available on the Polish market, including both dietary supplements and foods for special medical purposes, and to compare the obtained results with the information provided on the product labels. Fifteen commercial probiotic products were analysed. Viable microorganism counts were determined using the traditional culture-based plate count method and by flow cytometry for selected products. Species identification was performed using MALDI-TOF MS and qPCR, whereas microbiological purity testing was conducted to confirm the absence of pathogenic bacteria. Significant differences were observed between the declared and experimentally determined numbers of viable microorganisms. Only a few products maintained bacterial counts consistent with label claims, while most contained considerably low viable cells. Flow cytometry revealed higher viable cell counts than plate counting, indicating the presence of viable but non-culturable bacteria. The declared species composition of the strains was mostly confirmed, although in several cases, undeclared probiotic microorganisms were identified. All tested products were free from pathogens. The study indicates significant discrepancies in the quality of probiotic supplements available on the Polish market. From a consumer perspective, these findings highlight the importance of verifying probiotic quality and suggest that not all commercial products may guarantee the full range of claimed health benefits. The implementation of standardised analytical procedures and enhanced quality control measures is therefore essential to ensure the product safety, strain authenticity, and reliability of health-related claims.