Abstract
This chapter demonstrates the in vitro formation of several metabolites of 25-hydroxyvitamin D 3 (25- OHD 3 ), which can be logically placed on a single metabolic pathway, using the isolated perfused rat kidney. Each metabolite can be identified by a combination of high-performance liquid chromatography (HPLC), ultraviolet (UV) spectrophotometric, and mass spectrometric techniques. The enzymes involved in the pathway are present only in the vitamin D-repleted animal, are stimulated by vitamin D intoxication, and probably play some role in excretion of the potentially toxic molecule, 25-OHD 3 . The study of the pathway of nonradioactive 25-OHD 3 metabolism in preparative sized samples requires a different approach that involves three steps from bulk extract to pure material suitable for mass spectrometry, and a third step back on Zorbax-SIL with the initial solvent to confirm purity. Such a scheme separates most of the metabolites of 25-OHD 3 based upon their hydroxyl content while avoiding the column overloading problems encountered with large lipid contents of bulk perfusate extracts. The chapter also discusses the diode-array spectrophotometric detection of metabolites of 25-OHD 3 and identification of metabolites of pathway. The metabolite 24,25-(OH) 2 D 3 has been known as an in vivo and in vitro product of 25-OHD 3 metabolism. Using 24,25-(OH) 2 D 3 as a substrate for the perfused rat kidney it is shown that the three metabolites described in the chapter appear in the perfusate within 2-4 hr. If a vitamin D-depleted donor rat is used, no such metabolites are formed from 24,25-(OH) 2 D 3 until 4-6 hr has elapsed. Use of 24-oxo-25-OHD 3 as substrate in the vitamin D-repleted kidney results in synthesis of 24-oxo-23,25-(OH) 2 D 3 and 24,25,26,27-tetranor-23-OHD 3 but not 24,25-(OH) 2 D 3 . Use of 23(S),25- (OH) 2 D 3 gives only 25-OHD 3 -26,23-lactone. Use of 24-oxo-23,25-(OH) 2 D 3 as substrate in the vitamin D-repleted kidney results in synthesis of only 24,25,26,27-tetranor-23-OHD 3 .
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