ascorbic-acid and tris(2-carboxyethyl)phosphine

Cysteine is one of the most reactive amino acids. This is due to the electronegativity of sulphur atom in the side chain of thiolate group. It results in cysteine being present in several distinct redox forms inside the cell. Amongst these, reversible oxidations, S-nitrosylation and S-sulfenylation are crucial mediators of intracellular redox signalling, with known associations to health and disease. Study of their functionalities has intensified thanks to the development of various analytical strategies, with particular contribution from differential alkylation-based proteomics methods. Presented here is a critical evaluation of differential alkylation-based strategies for the analysis of S-nitrosylation and S-sulfenylation. The aim is to assess the current status and to provide insights for future directions in the dynamically evolving field of redox proteomics. To achieve that we collected 35 original research articles published since 2010 and analysed them considering the following parameters, (i) resolution of modification site, (ii) quantitative information, including correction of modification levels by protein abundance changes and determination of modification site occupancy, (iii) throughput, including the amount of starting material required for analysis. The results of this meta-analysis are the core of this review, complemented by issues related to biological models and sample preparation in redox proteomics, including conditions for free thiol blocking and labelling of target cysteine oxoforms.

Topics: Alkylation; Animals; Arsenites; Ascorbic Acid; Cysteine; Dithiothreitol; Ethylmaleimide; Eukaryotic Cells; Humans; Iodoacetamide; Models, Biological; Oxidation-Reduction; Phosphines; Proteomics; Staining and Labeling; Sulfhydryl Compounds

Ultrasensitive analytical methods are still urgent for the discovery of trace level biomarkers and the early clinical diagnosis of disease. In this work, an ultrasensitive universal sensing platform was constructed by integrating fluorescent assay with chemical-chemical redox cycling signal amplification strategy. Using Ru@SiO

Topics: Ascorbic Acid; Biomarkers; Biosensing Techniques; DNA; DNA Methylation; Limit of Detection; Manganese Compounds; Oxidation-Reduction; Oxides; Silicon Dioxide

The objective of the study was to develop a new method for the determination of the total content of vitamin C and dehydroascorbic acid in food, based on the technique of differential pulse voltammetry with the use of a boron-doped diamond electrode modified with mercury film. A comparison was made between the results obtained with the developed method and a proposed reference method based on high-performance liquid chromatography with spectrophotometric detection. The reduction of dehydroascorbic acid was performed with the use of tris(2-carboxyethyl)phosphine. The interference caused by the presence of tris(2-carboxyethyl)phosphine during the voltammetric determination of ascorbic acid was effectively eliminated through a reaction with N-ethylmaleimide. The conducted validation of the voltammetric method indicated that correct results of analysis of the total content of vitamin C and ascorbic acid were obtained. Analysis of the content of dehydroascorbic acid was imprecise due to the application of the differential method. The results of the analyses and the determined validation parameters of the developed method are characterised by a high degree of conformance with the results obtained with the chromatographic reference method, which indicates the equivalence of the two methods.

Topics: Ascorbic Acid; Chromatography, High Pressure Liquid; Dehydroascorbic Acid; Indicators and Reagents; Vitamins

Topics: Amino Acid Sequence; Ascorbic Acid; Molecular Sequence Data; Peptides; Phosphines; Sulfhydryl Compounds

Ascorbic acid (AA) has a strong anti-oxidant function evident as its ability to scavenge superoxide radicals in vitro. Moreover, AA is an essential ingredient for post-translational proline hydroxylation of collagen molecules. Dehydroascorbic acid (DHA), the oxidized form of AA, is generated from these reactions. In this study, we describe an improved method for assessing DHA in biological samples. The use of 35 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a reductant completely reduced DHA to AA after 2 h on ice in a 5% solution of metaphosphoric acid containing 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 1.5. This method enabled us to measure the DHA content in multiple tissues and plasma of 6-weeks-old mice. The percentages of DHA per total AA differed markedly among these tissues, i.e., from 0.8 to 19.5%. The lung, heart, spleen and plasma had the highest levels at more than 10% of DHA per total AA content, whereas the cerebrum, cerebellum, liver, kidney and small intestine had less than 5% of DHA per total AA content. This difference in DHA content may indicate an important disparity of oxidative stress levels among physiologic sites. Therefore, this improved method provides a useful standard for all DHA determinations.

Topics: Animals; Antioxidants; Ascorbic Acid; Clinical Laboratory Techniques; Dehydroascorbic Acid; Edetic Acid; Male; Mice; Mice, Inbred C57BL; Organ Specificity; Oxidative Stress; Phosphines; Phosphorous Acids; Reducing Agents

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.

Topics: Ascorbic Acid; Copper; Dehydroascorbic Acid; Dithiothreitol; Drug Stability; Hydrogen-Ion Concentration; Kinetics; Oxidation-Reduction; Phosphines; Phosphorous Acids; Reducing Agents; Solutions

Determination of dehydroascorbic acid in biological samples most commonly involves indirect measurement. The concentration is calculated by subtraction of the measured ascorbic acid concentration from that of total ascorbic acid analyzed after reduction of the dehydroascorbic acid present; a methodology also referred to as subtraction methods. Consequently, successful determination of dehydroascorbic acid is dependent on proper sample handling, quantitative reduction of the compound, and accurate quantification of both ascorbic acid and total ascorbic acid. In this paper, the recently introduced reductant tris[2-carboxyethyl]phosphine (TCEP) is evaluated as a reliable alternative to the commonly used reducing agent dithiothreitol (DTT). The results show that TCEP offers a more efficient reduction of dehydroascorbic acid at low pH compared to that of DTT. Moreover, while DTT maintains a reducing sample environment for less than 24 h, TCEP show complete protection from oxidation of ascorbic acid for at least 96 h following sample preparation. Removal of TCEP prior to analysis is unnecessary. A revised HPLC-EC method incorporating TCEP as reductant as well as the coanalysis of isoascorbic acid and uric acid is presented. The within- and between-day coefficients of variation for the complete assay are less than 1.5 and 3.5% for all analytes. As a whole, the method presented here is simpler and more reliable than existing methods.

Topics: Ascorbic Acid; Chromatography, High Pressure Liquid; Dehydroascorbic Acid; Dithiothreitol; Humans; Hydrogen-Ion Concentration; Models, Chemical; Oxygen; Phosphines; Reproducibility of Results; Time Factors; Uric Acid