Competitive protein-binding radioassay of thiamine in selected biological materials.
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 thiamine in hot alkaline solution. Model studies show that the radioassay works in thiamine concentration range of 1-10 microM, in samples of moderate ionic strength (up to 0.25 M NaCl) and is specific for thiamine in the presence of up to 5-fold molar excess of thiamine phosphates. Thiamine phosphates can also 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.
Jagiellonian University, Institute of Molecular Biology, Kraków, Poland.
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D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D006801:Humans; D008892:Milk / Q000737:chemistry*; D010936:Plant Extracts / Q000032:analysis*; D011485:Protein Binding; D011869:Radioligand Assay; D013831:Thiamine / Q000032:analysis* / Q000652:urine; D014675:Vegetables / Q000737:chemistry*
D010936:Plant Extracts; D013831:Thiamine