A MICROMETHOD FOR ASSAY OF TOTAL TOCOPHEROLS IN BLOOD SERUM

Abstract

A simple, accurate micromethod for vitamin E in blood would be a desirable addition to assay procedures now available. There appears to be no vitamin E method which requires less than 2 to 5 ml. of serum or plasma, and this amount is frequently unobtainable from small children or experimental animals. Economy of space and time is another advantage of using a micromethod. The micromethod for measuring vit,amin E in blood to be described here appears to fit these requirements. A pair of analysts can do 80 assays in a working day (and carotene is also measured in the method). The volume of blood serum or oxalated plasma which is required (0.06 ml.) is easily obtained by finger-tip puncture.. The method employs techniques previously described, whereby the Beckman spectrophotometer is adapted to measurement of optical density of very small volumes and micro pipettes of the Lang-Levy type are used (1, 2). For determining vitamin E in blood the direct measurement of its absorption peak at about 295 rnp is attractive. However, owing to the very low extinction value of tocopherol and to the presence of many interfering substances which absorb in this region, this method is not practical. Of the oxidimetric color reactions for vitamin E, only two seem to be sensitive enough for use in a blood method. One is oxidation (e.g. with silver nitrate) to give tocopherylquinone with an absorption peak at about 265 rnp; it does not seem useful for microassay because of carotene interference and because of the rigid control required to obtain precise results. The other is the Emmerie and Engel (8) color reaction with ferric chloride and a!, ar’-dipyridyl to give a red color. Since the latter reaction is precise and easy to perform, it has been used. The method is based on the macroprocedure of Quaife and Harris (4). However, the hydrogenation step, which obviates interference due to carotene, has been omitted since it was not found feasible to hydrogenate