Fluorimetric determination of histidine by exploiting its inhibitory effect on the oxidation of thiamine by cobalt-containing Prussian Blue nanocubes.

A fluorometric assay for histidine (His) is described. It is based on the inhibitory effect of His on nanocubes consisting of cobalt-containing Prussian Blue analog (CoFe NCbs), which have a strong oxidation effect on thiamine (THI) in the presence of NaOH. THI is nonfluorescent but the oxidized form (thiochrome; ThC) has a strong blue fluorescence, with excitation/emission maxima at 370/445 nm. His inhibits the oxidation effect of the CoFe NCbs due to the strong interaction between its imidazole side chain and the amino groups of the CoFe NCbs. This method is fast and has good sensitivity and selectivity. The lower detection limit is 14.3 nM of His, the linear range extends from 0.05 to 2.5 µM, and the relative standard deviation is calculated to be 1.5%. The method was successfully employed to quantify His in spiked serum samples. Graphical abstractSchematic representation of cobalt-containing Prussian Blue nanocubes (CoFe NCbs)-thiamine (THI)-based fluorometric assay for Histine (His). His inhibits the generation of thiochrome (ThC; the oxidized form of THI). The detection limit is 14.3 nM with the linear range of 0.05-2.5 µM.

Fluorometric determination of sulfide ions via its inhibitory effect on the oxidation of thiamine by Cu(II) ions.

A fluorometric assay is described for sulfide ions determination. It is based on the finding that the oxidation of the non-fluorescent substrate thiamine (TH) by Cu(II) in basic solution to form fluorescent thiochrome is inhibited by sulfide ions. This results in a decrease in fluorescence intensity which is proportional to the concentration of sulfide ions. Under the optimized conditions, the decrease in fluorescence, best measured at excitation/emission wavelengths of 370/440 nm, decreases linearly in the 0.03 to 2.5 µM sulfide ions concentration range. The detection limit is 20 nM. The method shows excellent selectivity over many potentially interfering ions and has been successfully applied to the determination of sulfide ions in spiked tap water, lake water and the synthetic wastewater samples. The method is time-saving and environmentally friendly, and in our perception shows a great potential in water quality inspection and environmental monitoring. Graphical abstract A fluorescent assay for sulfide ions detection is proposed based on its inhibitory effect on the oxidation of thiamine by Cu(II) ions.

Thiamine antivitamins--an opportunity of therapy of fungal infections caused by Malassezia pachydermatis and Candida albicans.

Severe skin diseases and systemic fungaemia are caused by Malassezia pachydermatis and Candida albicans respectively. Antifungal therapies are less effective because of chronic character of infections and high percentage of relapses. Therefore, there is a great need to develop new strategies of antifungal therapies. We previously found that oxythiamine decreases proliferation of yeast (Saccharomyces cerevisiae), therefore we suggest that thiamine antivitamins can be considered as antifungal agents. The aim of this study was the comparison of thiamine antivitamins (oxythiamine, amprolium, thiochrome, tetrahydrothiamine and tetrahydrooxythiamine) inhibitory effect on the growth rate and energetic metabolism efficiency in non-pathogenic S. cerevisiae and two potentially pathogenic species M. pachydermatis and C. albicans. Investigated species were cultured on a Sabouraud medium supplemented with trace elements in the presence (40 mg l(-1)) or absence of each tested antivitamins to estimate their influence on growth rate, enzyme activity and kinetic parameters of pyruvate decarboxylase and malate dehydrogenase of each tested species. Oxythiamine was the only antivitamin with antifungal potential. M. pachydermatis and S. cerevisiae were the most sensitive, whereas C. albicans was the least sensitive to oxythiamine action. Oxythiamine can be considered as supportive agent in superficial mycoses treatment, especially those caused by species from the genus Malassezia.

A smartphone-based ratiometric fluoroprobe based on blue-red dual-emission signals of thiochrome and copper nanoclusters for sensitive assay of metam-sodium in cucumbers.

It is of great importance to develop the highly efficient fluorescence strategy for rapid/sensitive detection of metam-sodium (MES) in evaluating its residual safety, especially in fresh vegetables. Herein, we prepared an organic fluorophore (thiochrome, TC) and glutathione-capped copper nanoclusters (GSH-CuNCs), and their combination (TC/GSH-CuNCs) was sucessfully employed as a ratiometric fluoroprobe by means of the blue-red dual emission. The fluorescence intensities (FIs) of TC decreased upon the addition of GSH-CuNCs via the fluorescence resonance energy transfer (FRET) process. When fortified at the constant levels of GSH-CuNCs and TC, MES substantially reduced the FIs of GSH-CuNCs, while this was not the case in the FIs of TC except for the prominent red-shift of ∼30 nm. Compared to the previous fluoroprobes, the TC/GSH-CuNCs based fluoroprobe supplied wider linear range of 0.2-500 µM, lower detection limit (60 nM), and satisfactory fortification recoveries (80-107%) for MES in the cucumber samples. Based on the fluorescence quenching phenomenon, a smartphone application was used to output RGB values of the captured images for the colored solution. The smartphone-based ratiometric sensor could be utilized for the visual fluorescent quantitation of MES by virtue of the R/B values in cucumbers, which gave linear range (1-200 µM) and LOD (0.3 µM). By means of blue-red dual-emission fluorescence, the smartphone-based fluoroprobe provides a cost-effective, portable and reliable avenue for the on-site, rapid and sensitive assay of MES's residues in complex vegetable samples.

A simple, sensitive, and specific method for the extraction and determination of thiamine and thiamine phosphate esters in fresh yeast biomass.

Thiamine is an essential vitamin for most living organisms, of which yeasts are a rich nutritional source. In this study we developed a thiamine extraction and determination method to detect thiamine in fresh yeast biomass. The thiamine determination method combines the derivatization of thiamine to a highly fluorescent product, with chromatographic separation (HPLC) and fluorescence detection. The method specifically detects free thiamine (T), thiamine phosphate (TP), and thiamine pyrophosphate (TPP). It has a high sensitivity of 2 ng/ml for TPP and TP, and 1 ng/ml for T, excellent instrumental repeatability, and low day-to-day variation in retention time of the different phosphate forms. We demonstrated the robustness of the method by proving that the fluorescence signals of the derivatised samples are stable for at least 82 h after derivatization, and by showing that the final pH of the samples does not influence the fluorescent response. In addition, we developed and validated a thiamine extraction method consisting of beads beating the fresh yeast biomass in 0.1 M HCl using a lysing matrix composed of 0.1 mm silica spheres. The performance of this method was compared to extraction via heat treatment at 95 °C for 30 min, and a combination of beads beating and heat treatment carried out in different order. We demonstrated that thiamine extraction via beads beating is the only method that prevents the biologically active form thiamine pyrophosphate to be degraded to thiamine phosphate, therefore, the extraction method developed and described in this study is preferred when the different thiamine vitamers need to be detected in their actual proportions. The combination of the extraction via beads beating, the conversion of all vitamers to the thiochrome derivatives, and the separation of these compounds on the reversed phase HPLC with a fluorescence detector, yielded a sensitive, specific, repeatable, and robust method for extraction and determination of vitamin B1 in fresh yeast biomass.

Photolysis of thiochrome in aqueous solution: A kinetic study.

The photolysis of thiochrome (THC), an oxidation product of thiamine (vitamin B1) (THE), used for its fluorimetric assay, has been studied in the pH range 7.0-12.0. THC undergoes photooxidation to oxodihydrothiochrome (ODTHC) which is oxidized to a non-fluorescent compound (OP1) on UV irradiation. The kinetics of the consecutive first-order reactions: THC→k1ODTHC→k2OP1, has been evaluated and the values of first-order rate constants, k1 (0.58-4.20 × 10-5, s-1) and k2 (0.05-2.03 × 10-5, s-1), at pH 7.0-12.0 have been determined. The rates of degradation of THC and ODTHC are enhanced with pH and the second-order rate constants k1' and k2' for the OH- ion-catalyzed reaction are in the range of 0.002-58.3 M-1 s-1. The quantum yields of the photolysis of THC and ODTHC in the pH range 7.0-12.0 have been determined. THC, ODTHC and OP1 have been identified by chromatographic, spectrometric and fluorimetric methods. THC and ODTHC have similar fluorescence characteristics and emit at 450 and 445 nm, respectively. THC, ODTHC and OP1 with distinct absorption maxima (370, 344 and 290 nm, respectively) have been determined by a newly developed and validated multicomponent spectrometric method during the photolysis reactions. The on-line formation of THC by the photooxidation of THE may lead to the degradation of THC and give erroneous results in the fluorimetric assay of THE. A scheme for the photolysis reactions of THC in aqueous solution is presented.

Thiamin analysis in red wine by fluorescence reverse phase-HPLC.

The derivatization of thiamin vitamers to their respective thiochrome by ferricyanide to facilitate fluorescence detection following separation by HPLC provides a powerful analytical tool. However the polyphenolic compounds in red wine readily interact with ferricyanide, reducing the effectiveness of ferricyanide oxidation in the derivatization of thiamin. We describe a method to facilitate the removal of polyphenolic compounds that interfere with the ferricyanide derivatization of thiamin. Polyvinylpolypyrrolidone afforded the total removal of phenolic compounds from red wines and allowed a spike recovery of thiamin vitamers (101% for thiamin; 104% for TMP; and 100% for TDP) in a wide range of red wines. This research found that Merlot styles of red wine contained the highest concentration of total thiamin (29.01 ng/mL) while Pinot Noir wines contained the lowest total concentration (8.27 ng/mL).

[Extracting and study of biochemical properties of thiamine pyrophosphokinase from non-malignant and tumor tissue of myometrium].

The method of extraction and purification of thiamine pyrophosphokinase from non-malignant and tumor tissue of myometrium has been elaborated. Kinetic characteristics of T-kinase from non-malignant and tumor tissue of women myometrium have been studied. It has been shown, that malignization of myometrium is accompanied by a decrease in affinity of thiamine pyrophosphokinase from tumor to thiamine and by an increase in sensitivity of the enzyme from tumor to thiochrome.