Synthesis of a reagent for fluorescence-labeling of vitamin D and its use in assaying vitamin D metabolites.
The fluorogenic dienophile 1,2,4-triazoline-3,5-dione with a highly fluorescent quinoxalinone group at the 4-position (DMEQ-TAD) was synthesized and exploited as a reagent to assay vitamin D metabolites. 25-Hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3, and 24(R),25-dihydroxyvitamin D3 reacted quantitatively with DMEQ-TAD when the two substrates were mixed in dichloromethane at room temperature to yield the corresponding 6,19-cycloadduct. The reaction was very fast so that 1 alpha,25-dihydroxyvitamin D3 at a concentration as low as 10(-8) M could be quantitatively labeled with the fluorescent reagent within 30 min at room temperature. With this reagent, down to 10 fmol of vitamin D metabolites could be quantified linearly. The detection limit of the labeled vitamin D using high-performance liquid chromatography was usually about 1 fmol. Thus, it was shown in a model system that the fluorometric method using the new reagent (DMEQ-TAD) can be applied to the assay of the three major vitamin D metabolites in 1 ml of plasma. This is the first practical fluorometric method for assaying the active vitamin D metabolite.
Shimizu
M
M
Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, Japan.
Kamachi
S
S
Nishii
Y
Y
Yamada
S
S
D015650:24,25-Dihydroxyvitamin D 3 / Q000032:analysis*; D002112:Calcifediol / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism; D005470:Fluorometry; D011810:Quinoxalines / Q000138:chemical synthesis* / Q000378:metabolism; D014230:Triazoles / Q000138:chemical synthesis* / Q000378:metabolism; D014807:Vitamin D / Q000378:metabolism*
D005456:Fluorescent Dyes; D011810:Quinoxalines; D014230:Triazoles; C070204:4-(2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl)-1,2,4-triazoline-3,5-dione; D014807:Vitamin D; D015650:24,25-Dihydroxyvitamin D 3; D002112:Calcifediol