the “glutathione potential” A new concept of glutathione quantification in wine: the “glutathione potential” Aurélie Rolanda*, Stéphane Delpecha ,Florine Cavelierb and Rémi Schneidera a Nyseos, 53 rue Claude François, Parc 2000, 34080 Montpellier, France b Institut des Biomolécules Max Mousseron - UMR 5247 CNRS-UM-ENSCM, Département des Aminoacides, Peptides et Protéines (DAPP), Université Montpellier, 34095 Montpellier cedex 05, France. *aurelie@nyseos.fr Introduction Glutathione (GSH) is widely studied in the enological field, especially for its antioxidant properties. Indeed, GSH is considered as a powerful antioxidant of wine especially for several key aroma compounds such as varietal thiols (3-mercaptohexanol and 4-methyl- 4-mercaptopentan-2-one) (1). GSH is highly reactive with either soft electrophile like o-quinones of polyphenols to form thioether bonds such as in the Grape Reaction Product (2) or with other thiols to form disulfide bonds under special oxidation conditions (3). The glutathione engaged in thioether bond is chemically stable under enological conditions and cannot be released. However, the glutathione involved in disulfide forms has to be considered with great interest since oxidoreduction potential of wine is responsible for the equilibrium between disulfides and thiols and could result in GSH release (Figure 1). The aim of this work was to developed a new analytical method able to measure both reduced GSH and all disulfide forms of GSH that is to say « the glutathione potential ». Figure 1: Possible chemical pathways of glutathione under enological conditions G-SH G-S-R’ G-S-S-R’’ Redox Addition THIOETHERS Metal chelation Cu2+ Antioxidant reserve DISULFIDES COMPLEXES R’ and R’’: alkyl groups Materials and methods Three differents wines (White wine – Manseng – Côtes de Gascogne; Rosé Wine – Grenache – Rosé de Provence; Red Wine – Gamay – Beaujolais) have been studied according to three different treatments (Figure 2) : i – control, ii – CuSO4 (10 mg/L)/GSH (50 mg/L); iii – O2/GSH (50mg/L). Oxygen consumption has been monitored over 7 days using chemoluminescence (Nomasense O2). In practice, wines were analyzed at t0 and t+7days. Samples (1 mL) were treated subsequently with tris(carboxyethyl)-phosphine and N- phenylmaleimide for 30 min at room temperature. Extract were then purified by SPE, concentrated to dryness and then analyzed by nanoChipCUBE-LC-MS/MS. The total run time was 13 min and quantification was performed using Stable Isotope Dilution Assay. Analytical performances allowed the high-throughput analysis of GSH potential at traces levels (ppb) in both grapes and wines with a high accuracy (95%) and repeatability (RSD < 10%). Figure 2: Experimental design Results and Discussion First of all, samples were characterized at t0 (Figure 3) to measure the proportion of free GSH and disulfides forms. All wines exhibited a similar repartition of glutathione forms: 20/80 respectively. However, GSH disulfides were particularly abundant in red and rosé wines. To better understand the redox equilibrium of GSH, we proposed a new analytical parameter: the GSH potential that corresponds to the sum of free GSH and disulfides forms. The GSH potential allowed measuring the « antioxidant reserve » of wine. Then, wines have been analyzed after 7 days of oxygen consumption (Figure 4): Treatment CuSO4/GSH: The addition of GSH in presence of CuSO4 destroyed the free GSH in favour of non-reversible losses Treament GSH/O2: The addition of GSH prior to the wine oxygenation modified the redox equilibrium and favoured the non-reversible losses. Figure 3: GSH potential (black arrow) in red wine after different treatments Figure 2: GSH potential of wines GSH POTENTIAL Conclusion For the first time, we proposed a new analytical measure able to characterize an « antioxidant reserve » of wine. Analytical performances showed very good accuracy, repeatability and high-throughput. The GSH potential is a potent diagnosis tool that opens avenue for future experiments on wine ageing and redox evolution or bottling effect on wine redox. Bibliography Nikolantonaki, M.; Waterhouse, A. L., A Method To Quantify Quinone Reaction Rates with Wine Relevant Nucleophiles: A Key to the Understanding of Oxidative Loss of Varietal Thiols. Journal of Agricultural and Food Chemistry, 2012, 60, 8484-8491. Cheynier, V. F.; Trousdale, E. K.; Singleton, V. L.; Salgues, M. J.; Wylde, R., Characterization of 2-S-glutathionyl caftaric acid and its hydrolysis in relation to grape wines. Journal of Agricultural and Food Chemistry, 1986, 34, 217-221. Kritzinger, E. C.; Bauer, F. F.; du Toit, W. J., Role of Glutathione in Winemaking: A Review. Journal of Agricultural and Food Chemistry, 2013, 61, 269-277. Acknowledgements We thanked the FEDER and the Languedoc-Roussillon Region for financial support. In Vino Analytica Scientia 2015 - Trento, ITALY