Concentrations of Total Glutathione and Cysteine in Wheat Flour as Affected by Sulfur Deficiency and Correlation to Quality Parameters JULIA REINBOLD, † MICHAEL RYCHLIK,* ,‡ STEFAN ASAM, ‡ HERBERT WIESER, † AND PETER KOEHLER † Deutsche Forschungsanstalt fu ¨r Lebensmittelchemie and Lehrstuhl fu ¨r Lebensmittelchemie der Technischen Universita ¨t Mu ¨nchen, Lichtenbergstrasse 4, D-85748 Garching, Germany A method for the simultaneous quantitation of total glutathione and total cysteine in wheat flour by a stable isotope dilution assay using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) was developed. As internal standards, L-[ 13 C 3 , 15 N]cysteine and L-γ-glutamyl-L-[ 13 C 3 , 15 N]cysteinyl-glycine were used. The method consisted of the extraction and reduction of flour with tris(2-carboxyethyl) phosphine after the addition of internal standards, protection of free thiol groups with iodoacetic acid, derivatization of free amino groups with dansyl chloride, and HPLC-MS/MS. The limits of detection and quantitation for glutathione were 0.75 nmol/g and 2.23 nmol/ g flour, respectively. For cysteine, the limits of detection and quantitation were 0.72 nmol/g and 2.12 nmol/g flour, respectively. The developed method was found to be sensitive enough for quantitation of total glutathione and cysteine levels in wheat flour. This method was then utilized to investigate the effect of sulfur (S) deficiency on the amount of total glutathione and cysteine in flour. In S-deficient wheat, the concentrations of total glutathione and cysteine were proportional to the amount of S supplied during growth. The calculation of correlations revealed that GSH and Cys concentrations influenced the rheological dough properties and the baking performance at least as much as protein parameters. Thus, the low concentration of GSH and Cys in flour from S-deficient wheat had a similar effect on the technological properties as the altered composition of gluten proteins. INTRODUCTION Since the fundamental discoveries by Justus von Liebig in the first half of the 19th century, sulfur (S) has been known as one of the major plant nutrients along with nitrogen (N), phosphorus, and potassium. However, S fertilization was not common for wheat crops until the 1980s with air pollution from traffic and industry providing enough S in the soil for wheat growth. After the 1979 Geneva Convention on Long-Range Transboundary Air Pollution (1), sulfur dioxide (SO 2 ) emissions were dramatically reduced, and as a consequence, S deficiency in soil was observed (2). Since then, S fertilization and the effect of S deficiency on growing plants, crops, and the properties of dough prepared from wheat flour with S deficiency have attracted considerable interest. Several studies (3–7) indicated that S deficiency affects the amount and the proportions of different gluten protein types in flour, while the content of albumins, globulins, and total gluten proteins were hardly influenced by S fertilization. S deficiency caused a significant increase in the amounts of S-free ω-gliadins and moderately increased the amount of S-poor high molecular weight (HMW) glutenin subunits. Conversely, S rich γ-gliadins as well as low molecular weight (LMW) glutenin subunits decreased dramatically in the case of S deficiency, while the amount of R-gliadins declined only moderately. Additionally, it has been shown that S deficiency causes low yield and poor technological properties of wheat, the latter of which results in doughs that are less extensible and more resistant to extension and in loaves of smaller volume and poorer texture (3, 5, 6, 8–13). Changes in the proportions of gluten protein fractions and types havebeenproposedtoberesponsiblefortheseimpairments(3,5,7,14). However, another possible effect of S deficiency on dough properties and baking quality has been discussed by Zhao et al. (14) through the involvement of low molecular weight (LMW) thiols present in flour such as cysteine (Cys) and glutathione (GSH). Numerous studies have demonstrated the large weaken- ing effect of these thiols on dough by SH/SS interchange reactions with gluten proteins (15). However, a certain amount of thiols in the developing dough is thought to be necessary owing to an optimal arrangement of gluten proteins (16). Thus, Zhao et al. (14) speculated that low S supply to wheat may decrease the concentration of endogenous thiols in flour resulting * To whom correspondence should be addressed. Phone: +49-8161 713153. Fax: +49-8161 714212. E-mail: michael.rychlik@wzw.tum.de. Current Address: BIOANALYTIK Weihenstephan, Alte Akademie 10, D-85350 Freising, Germany. † Deutsche Forschungsanstalt fu ¨r Lebensmittelchemie. ‡ Lehrstuhl fu ¨r Lebensmittelchemie der Technischen Universita ¨t Mu ¨nchen. 6844 J. Agric. Food Chem. 2008, 56, 6844–6850 10.1021/jf800880n CCC: $40.75  2008 American Chemical Society Published on Web 07/26/2008