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

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Abstract
The tocopherol content of the yeast, R. glutinis, was determined by spectrofluorometry and rotifer [Asplanchna sieboldi] bioassay. Comparative tocopherol measurements were made on the mushroom, A. campestris, and the alga, E. gracilis, strain Z. R. glutinis, previously reported to have a tocopherol content of < 4.75 .mu.g/g dry wt, had < 0.001 .mu.g/g dry wt tocopherol by the spectrofluorometric technique. The rotifer bioassay showed < 1.28 pg tocopherol/g dry wt yeast. The tocopherol values for A. campestris and E. gracilis, strain Z, were 0.31-0.34 and 797 .mu.g/g dry wt, 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 1 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-.alpha.-tocopherol during the extraction process. Along with rigorous solvent and glassware preparation, this technique increased the sensitivity of the spectrofluorometric assay from the previously reported 0.1 to 0.005 .mu.g/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. [Body wall outgrowth response (morphotype differentiation) in A. sieboldi was an indicator of tocopherol level.].