Competitive protein-binding radioassay of thiamine in selected biological materials

Abstract

A principle of competitive protein-binding radioassay is developed for thiamine determination in some biological samples. Thiamine in an assay sample competes with radiolabelled thiamine for Sepharose-immobilized buckwheat-seed thiamine-binding protein. A blank sample is prepared by destruction of theiamine in hot alkaline solution. Model studies show that the radioassay works in thiamine concentration range of 1–10 μM, in samples of moderate ionic strenght (up to 0.25 M NaCl) and is specific for thiamine in the presence of up tp 5-fold molar excess of thiamine phosphates. Thiamina phosphates can be determined but after hydrolysis with a suitable phosphatase enzyme (Taka-Diastase). Using this method, thiamine contents are successfully determined: (i) in spinach juice, directly, (ii) in cow's milk, after deproteinization, and (iii) in human urine, after desalting. Both the precision (C.V. less than 15%) and the recovery of thiamine supplements (82–100%, depending on thiamine pre-extraction method) are reasonable. Results of thiamine radioassay show a good correlation with control determinations by the standard thiochrome method.