Simultaneous quantification of glutathione, glutathione disulfide and glutathione-S-sulfonate in grape and wine using LC-MS/MS.

A fast, sensitive and reproducible method using LC-MS/MS for simultaneous quantification of glutathione (GSH), glutathione disulfide (GSSG) and glutathione-S-sulfonate (GSSO3H) was developed, optimised and applied in analysis of grape juice and wine samples. The results show that only GSH (10-60 mg·L-1) and GSSG (2-11 mg·L-1) are found in grape juice when SO2 is not added. GSSO3H was detected in must samples treated with SO2 but only at a low concentration (<1 mg L-1). In the wine samples, the dominant form of glutathione was GSSO3H (5-11 mg L-1), followed by GSH (0-5 mg L-1) and GSSG (0-6 mg L-1), underscoring the importance of GSSO3H quantification. GSSO3H formation in wine was correlated with the total SO2 level in the wine. We believe this is the first report on GSSO3H quantification in wine.

Implementation of an Enzyme Linked Immunosorbent Assay for the Quantification of Allergenic Egg Residues in Red Wines Using Commercially Available Antibodies.

Since the early 2000s, labeling of potentially allergenic food components to protect people who suffer from food allergies is compulsory in numerous industrialized countries. In Europe, milk and egg components used during the winemaking process must be indicated on the label since July 1, 2012. Several ELISA procedures have been developed to detect allergenic residues in wines. However, the complexity of the wine matrix can inhibit the immunoenzymatic reaction. The aim of this study was to implement an ELISA assay for the detection of ovalbumin in red wines using commercially available antibodies. The specificity of the acquired antibodies and the absence of cross reactivity were assessed by immunoblotting and ELISA. An ELISA assay with a LOD of 14.2 µg/L and a LOQ of 56.4 µg/L of ovalbumin in aqueous solution was obtained. Differences in ELISA signals were observed when analyzing various fining agents, although reproducible conformation of the antigen could be reached for the comparison of ovalbumin and Ovicolle. The differences between samples in terms of pH could be leveled but the inhibition of the ELISA signal, positively correlated to the tannin content of the wines, could not be suppressed. Thus, standard curves of ovalbumin in several wines were obtained by relative quantification. The control steps and the difficulties encountered presented in this study should be considered by anybody working toward the development of ELISA assays for the detection of allergenic residues in complex food matrices.

UDP-glucose 4-epimerase isoforms UGE2 and UGE4 cooperate in providing UDP-galactose for cell wall biosynthesis and growth of Arabidopsis thaliana.

Five Arabidopsis thaliana genes that encode UDP-glucose 4-epimerase (UGE) and represent two ancient plant UGE clades might be involved in the regulation of cell wall carbohydrate biosynthesis. We tested this hypothesis in a genome-wide reverse genetic study. Despite significant contributions of each gene to total UGE activity, none was essential for normal growth on soil. uge2 uge4 displayed dramatic general growth defects, while other mutant combinations were partially aberrant. UGE2 together with UGE3 influenced pollen development. UGE2 and UGE4 synergistically influenced cell wall galactose content, which was correlated with shoot growth. UGE2 strongly and UGE1 and UGE5 lightly supported UGE4 in influencing root growth and cell wall galactose content by affecting galactan content. By contrast, only UGE4 influenced xyloglucan galactosylation in roots. Secondary hypocotyl thickening and arabinogalactan protein carbohydrate structure in xylem parenchyma depended on the combination of UGE2 and UGE4. As opposed to cell wall galactose content, tolerance to external galactose strictly paralleled total UGE activity. We suggest a gradual recruitment of individual UGE isoforms into specific roles. UGE2 and UGE4 influence growth and cell wall carbohydrate biosynthesis throughout the plant, UGE3 is specialized for pollen development, and UGE1 and UGE5 might act in stress situations.

Distinct properties of the five UDP-D-glucose/UDP-D-galactose 4-epimerase isoforms of Arabidopsis thaliana.

Plant genomes contain genetically encoded isoforms of most nucleotide sugar interconversion enzymes. Here we show that Arabidopsis thaliana has five genes encoding functional UDP-D-glucose/UDP-D-galactose 4-epimerase (named UGE1 to UGE5). All A. thaliana UDP-d-glucose 4-epimerase isoforms are dimeric in solution, maximally active in vitro at 30-40 degrees C, and show good activity between pH 7 and pH 9. In vitro, UGE1, -3, and -5 act independently of externally added NAD+, whereas cofactor addition stimulates the activity of UGE2 and is particularly important for UGE4 activity. UGE1 and UGE3 are most efficiently inhibited by UDP. The five isoforms display kcatUDP-Gal values between 23 and 128 s(-1) and KmUDP-Gal values between 0.1 and 0.3 mm. This results in enzymatic efficiencies ranging between 97 and 890 mm(-1) s(-1) for UGE4 = UGE1 < UGE3 < UGE5 < UGE2. The KmUDP-Glc values, derived from the Haldane relationship, were 0.76 mm for UGE1, 0.56 mm for UGE4, and between 0.13 and 0.23 mm for UGE2, -3, and -5. The expression of UGE isoforms is ubiquitous and displays developmental and cell type-dependent variations. UGE1 and -3 expression patterns globally resemble enzymes involved in carbohydrate catabolism, and UGE2, -4, and -5 expression is more related to carbohydrate biosynthesis. UGE1, -2, and -4 are present in the cytoplasm, whereasUGE4 is additionally enriched close to Golgi stacks. All UGE genes tested complement the UGE4rhd1 phenotype, confer increased galactose tolerance in planta, and complement the galactose metabolization deficiency in the Saccharomyces cerevisiae gal10 mutant. We suggest that plant UGE isoforms function in different metabolic situations and that enzymatic properties, gene expression pattern, and subcellular localization contribute to the differentiation of isoform function.