Characterization of the metabolic pathway of 1,25-dihydroxy-16-ene vitamin D3 in rat kidney by on-line high performance liquid chromatography-electrospray tandem mass spectrometry.
1,25-Dihydroxy-16-ene vitamin D3 is a synthetic analog of 1,25-dihydroxyvitamin D3, the most physiologically active metabolite of vitamin D3. The renal metabolism of 1,25-dihydroxy-16-ene vitamin D3 had been studied previously using a perfused rat kidney system [Reddy et al., Bioorg Med Chem Lett 3: 1879-1884, 1993], and its C-24 oxidative metabolic pathway had been found to be different from that of 1,25-dihydroxyvitamin D3 by HPLC. To further delineate the differences between the C-24 oxidative metabolic pathways of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxy-16-ene vitamin D3 in this present study we investigated the C-24 oxidation pathway of 1,25-dihydroxy-16-ene vitamin D3 using a novel detection approach based on on-line capillary liquid chromatography coupled to electrospray tandem mass spectrometry. Two types of tandem mass spectrometric detection were employed to characterize the metabolites in the kidney perfusate: (a) the preliminary screening of metabolites by parent scan, which led to the tentative discovery of the production of 1,23,25-trihydroxy-24-oxo-16-ene vitamin D3, a new metabolite of 1,25-dihydroxy-16-ene vitamin D3, and (b) the pharmacokinetic studies of the substrate, 1,25-dihydroxy-16-ene vitamin D3 and its metabolites by multiple reaction monitoring. In the latter, the mass spectrometric sensitivity for quantification was found to be about 20-fold better than UV detection. The current work concluded that the C-24 oxidative metabolic pathway of 1,25-dihydroxy-16-ene vitamin D3 closely mimicked that of its natural counterpart. Furthermore, the use of mass spectrometry permitted the clearance rate of the starting substrate to be studied at a more physiological level (ng/mL or submicromolar level), which had not been possible previously by HPLC-UV detection.
Yeung
B
B
Department of Chemistry, Northeastern University, Boston, MA 02115, USA.
Vouros
P
P
Siu-Caldera
M L
ML
Reddy
G S
GS
D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007668:Kidney / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D014230:Triazoles / Q000737:chemistry
D014230:Triazoles; C087630:1,25-dihydroxy-16-ene-vitamin D3; C053540:4-phenyl-1,2,4-triazoline-3,5-dione; D002117:Calcitriol