Vitamin B6 and Pyridoxine Glucoside Content of Wheat and Wheat Flours

Cereal Chem. 73(6):770-774 Analysis of different wheats and flours for vitamin B6 using water exin PN (0.9-2.7 nmol/g). Mean values (± standard deviations) were 5.0 ± tracts and reverse-phase high-performance liquid chromatography 1.8 nmol/g for total vitamin B6; 1.7 ± 0.5 nmol/g for PN; and 3.4 ± 1.8 revealed significant variation in content of pyridoxine (PN) and pyrinmollg for PNG. American hard wheats contained significantly more doxine glucoside (PNG), a glycosidic adduct of B6 with partial human vitamin B6, mostly as PNG, compared to Canadian hard wheats. The bioavailability found in many plant foods. Other vitameric forms of same pattern was seen for Colorado hard wheats compared to others. vitamin B6 were not detected. In 22 American and Canadian wheats, Hard wheats contained more vitamin B6 than did soft wheats. Flours total vitamin B6 (the sum of PN plus PNG observed in samples) content milled from 16 of the cultivars contained =75% less B6 than did the corvaried over a threefold range of 3.4 to 11.1 nmol/g. PNG accounted for a responding wheats. significant portion of the variation (1.9-8.8 nmol/g), with less variation The U.S. Food and Nutrition Board suggested in the 1989 Recommended Daily Allowances (RDA) (National Research Council 1989) that whole-wheat foods are a good source of vitamin B6 in the American diet. However, a significant and variable fraction of vitamin B6 in plant foods, including wheat, is present as pyridoxine glucoside (5'-o-(B-D-glucopyranosyl)pyridoxine) (PNG) (Gregory and Ink 1987), which has partial bioavailability for humans (Gregory et al 1991). Many Americans consume vitamin B6 at levels significantly less than the current RDA (Kant and Block 1990). Vitamin B6 includes a group of 3-hydroxy-2-methyl-pyridine derivatives that can exist in the C-4' position as an alcohol (pyridoxine) (PN), an aldehyde (pyridoxal) (PL) and an amine (pyridoxamine) (PM). These vitameric forms can be phosphorylated or nonphosphorylated. The available data suggest that PN predominates in most plant foods (Van Schoonhoven et al 1994). However, most of the reports in the literature have used a microbiological procedure involving an acid hydrolysis step during sample extraction (Toepfer and Lehmann 1961). This procedure converts any PNG in the sample into PN (Gregory 1988). Therefore, in much of the literature about vitamin B6 in plant foods, PNG present in the food may be reported as PN. We recently reported an high-performance liquid chromatography (HPLC) method capable of measuring vitamin B6 and PNG in wheat (Sampson et al 1995). Although the method does not detect quantitatively minor forms of vitamin B6 such as polyglycosylated adducts (Gregory and Sartain 1991), this method is simple, quick and reliable, and does detect the major forms of vitamin B6 in wheat, including PN and PNG. Our initial analyses of vitamin B6 in several wheat cultivars obtained with this method showed that 36-81% of vitamin B6 in several wheat cultivars was present as PNG (Sampson et al 1995). We have now used this method to investigate the vitamin B6 content of American and Canadian wheats and wheat flours. Our objectives in this study were: 1) to evaluate the vitamin B6 content, including PNG, of various cultivars of American and Canadian wheat; 2) to determine whether differences in these 'Department of Food Science and Human Nutrition, Colorado State University Fort Collins, CO 80523. 2 Corresponding author. E-mail: [email protected]. 3 South China University of Technology, Guang Zhou, People's Republic of China. Publication no. C-1996-1007-05R. 3 1996 American Association of Cereal Chemists, Inc. vitamers occurred between American and Canadian strains and between hard vs. soft wheats; 3) to compare wheats grown in Colorado to those from other American and Canadian locations; and 4) to determine the extent of loss of these compounds associated with commercial milling. Our interest in American vs. Canadian and Colorado vs. other wheats stemmed from the importance of wheat to Colorado agriculture. MATERIALS AND METHODS Reagents Vitamin B6 standards, deoxypyridoxine, and HPLC reagents were obtained from vendors described earlier (Sampson et al 1995). Sample Preparation Seventeen hard wheats and 5 soft wheats, as well as flours milled from 16 of these wheats, were obtained from commercial mills in the United States and Canada. Information about milling, patent percentage, and origin of cultivars was obtained from the mills. Wheats were stored at -20°C in 100-ml polycarbonate bottles before analysis. Sample preparation was done under yellow fluorescent lighting. Samples (10 g) were prepared in triplicate as described previously (Sampson et al 1995) using a micro-mill (Lab Apparatus, Cleveland, OH) and homogenizer (Hi-Speed, model 45, Virtis Co., Gardiner, NY). Moisture, protein, and ash contents of the wheats and flours were determined using approved methods (AACC 1995). Protein was expressed as N x 6.25 for grain and as N x 5.7 for flours. HPLC Equipment and Method Wheats were analyzed by reverse-phase HPLC using gradient elution and fluorescence detection, as described elsewhere (Sampson et al 1995). Mobile phase A of the binary gradient contained 0.033M phosphoric acid and 0.008M 1-octanesulfonic acid at pH 2.2. Mobile phase B contained 0.033M phosphoric acid and 10% (v/v) acetonitrile at pH 2.2. Fluorescence was enhanced by postcolumn derivitization with sodium bisulfite. The binary gradient involved a linear ramp from 100% A to 100% B in 10 min, 15 min in 100% B, ramp back to 100% A in 4.5 min, and 5.5 min in 100% A. PN and PNG were quantified relative to fluorescence of the internal standard, deoxypyridoxine. Molar fluorescence of PNG was assumed to be equivalent to PN, as shown by Gregory and Ink (1987). 770 CEREAL CHEMISTRY TABLE I Identification and Proximate Composition of U.S. and Canadian Grains and Flours