Measurement of chick kidney 25-hydroxyvitamin D3-1α-hydroxylase without use of radioactive substrate

✍ Scribed by Yoko Tanaka; Hector F. DeLuca

• Publisher: Elsevier Science
• Year: 1981
• Tongue: English
• Weight: 457 KB
• Volume: 110
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(81)90118-4

✦ Synopsis

Previously, activities of enzymes that hydroxylate vitamin D and its metabolites have been determined by measurement of radioactive metabolites produced from a radioactive substrate. However, sensitive ultraviolet absorbance detectors used with high-performance liquid chromatographic systems provide a means of quantitation of the products derived from nonradioactive substrates. This coupled with the superior resolution of vitamin D metabolites by high-performance liquid chromatography has eliminated the need for radioactive substrate in the measurement of chicken kidney 25-hydroxyvitamin D,-la-hydroxylase. The method involves the incubation of chicken kidney preparations with 25hydroxyvitamin Da, extraction of the incubation mixture with chloroform/methanol, prepurification of the extract on a 0.7 x 14-cm Sephadex LH-20 column developed with a solvent system of CHCl,:hexane 65:35, application of appropriate fractions to a high-performance liquid chromatographic column (Zorbax-Sil in 10% 2-propanol in hexane), and detection of the product by ultraviolet absorbance. Recoveries of the product are determined with an internal standard of l,25-dihydroxy-[26,27-3H]vitamin Da and are 80 f 5%. Using this method the K, and V for rachitic chick kidney 25-OH-Da-lcY-hydroxylase are 7 x lo-' M and 7 x 10-r" moPZOO mg tissue/5 min, respectively.