ANALYSIS OF TOCOPHEROL IN RHODOTORULA GLUTINIS, AGARICUS CAMPESTRIS, AND EUGLENA GRACILIS USING SPECTROFLUOROMETRY AND ROTIFER BIOASSAY

Abstract

The tocopherol content of the yeast, Rhodotorula glutinis, was determined by spectrofluorometry and rotifer bioassay. Comparative tocopherol measurements were made on the mushroom, Agaricus campestris, and the alga, Euglena gracilis, strain Z. R. glutinis, previously reported to have a tocopherol content of <4.75μg/g dry weight, was shown to have <0.001μg/g dry weight tocopherol by the spectrofluorometric technique. The rotifer bioassay showed that there was <1.28pg tocopherol/g dry weight yeast. The tocopherol values for A. campestris and E. gracilis, strain Z, were 0.31-0.34 and 797μg/g dry weight, respectively, as determined by spectrofluorometry. These values are somewhat lower than previously reported determinations, possibly because of the greater specificity of the spectrofluorometric method. A slightly modified spectrofluorometric procedure for the analysis of tocopherol is presented; homogenization of tissue samples, lyophilization, lipid extraction with acetone, and one purification step precede spectrofluorometric determination. Use of a cold acetone fractional crystallization procedure for lipid purification removed 99.27% of the originally extracted lipid material. The average recovery of introduced tocopherol was 97.6%, indicating only minor loss of d-α-tocopherol during the extraction process. Along with rigorous solvent and glassware preparation, this technique has increased the sensitivity of the spectrofluorometric assay from the previously reported 0.01 to 0.005μg/ml tocopherol. Specificity of tocopherol fluorescence was determined by introducing known amounts of tocopherol into samples, examination of excitation and fluorescent spectra, and adding FeCl₃.