Application of the slope ratio assay technique to the estimation of thiamine activity of thiamine analogues

Abstract

Experiments were conducted to determine the antithiamine activity of two thiamine antagonists, dimethialium (3-[2-methyl-4-amino-5-pyrimidinyl)-methyl]-4, 5-dimethyl thiazolium nitrate) and amprolium (1-[(4-amino-2-n-propyl-5-pyrimidinyl)-methyl]-2-picolinium chloride hydrochloride) by symmetrical 5-point slope ratio assay. Thiamine activity of vitamine supplement for formula feed stored at two different temperature were also determind by 3-point slope ratio assay.After two preliminary experiments, corn-soy type diet with 5mg of thiamine•HNO3 and either 1.2g of dimethialium or 3g of amprolium per kg of diet was found to be satisfactory for the basal diet. Either growth rate or feed efficiency (gain/feed) of 10-day-old White Leghorn male chick was depressed linearly by increasing dietary levels of dimethialium up to 2.0g/kg and of amprolium up to 5.0g/kg or by decreasing dietary levels of thiamine•HNO3 to 3mg/kg of the basal diet. The linear relation ship was observed already after 7 days on the test diets. On the most deficient diet, some of the 10-day-old chicks showed slight deficiency symptoms of thiamine after 14 days, so that the experimental period was decided to be 10 to 14 days.Combined variance analysis of two 5-point slope ratio assays on dimethialium revealed that 179mg of dimethialium counterbalanced to 1mg of thiamine•HNO3 on growth rate of chicks with 95% fiducial interval between 253 to 105mg.Combined variance analysis of two 5-point slope ratio assays on amprolium revealed that 666mg of amprolium counterbalanced to 1mg of thiamine•HNO3 on growth rate of chicks with 95% fiducial interval between 848 to 484mg. Antithiamine activity of dimethialium was 3.7 times as high as that of amprolium.No significant difference of thiamine activity was observed by 3-point slope ratio assay between vitamine supplement stored at -4°C and 30°C for 5 months. The activity corresponding to 1.47 and 1.45mg of thiamine per g of supplement, respectively.