Differential determination of thiamine and its phosphates, hydroxyethylthia mine and pyrithiamine, in rat brain.

Abstract

A reliable and convenient method for the fluorometric determination of free thiamine, the di- and triphosphate esters of thiamine (TDP+TTP), hydroxyethylthiamine (HET) and pyrithiamine (PTH) in individual rat brain tissue is presented. The thiamine phosphate esters, as well as HET and PTH, were extracted from the tissue by homogenizing the rat brain in cold 0.3 M HClO4. The di- plus triphosphate esters of thiamine were isolated by passing an appropriate aliquot of the extract through a small column of Amberlite CG-50, a weak cation exchange resin. The samples were then dephosphorylated, placed on a Decalso column and eluted with hot 20% KCl in 0.1 N HCl. Modifications of other methods were used to fluorometrically quantitate the thiamine, HET, and PTH in the brain tissue. There was no interference of HET or PTH in the determination of thiamine since the particular method used in this paper to oxidize thiamine to thiochrome did not cause HET or PTH to be oxidized to fluorescent compounds. There was no appreciable interference of thiamine and HET in the fluorometric determination of PTH until the ratio of μg of thiamine plus HET to μg of PTH in the assay mixture was 6/1. When the above ratio increased to 24/1, the fluorescent determination of PTH was 10% high. The fluorometric determination of HET in the presence of excess thiamine and PTH was 16 and 75% high when the ratio of μg of PTH/μg of HET in the assay solution was 20/1 and 83/1, respectively.