Determination of the Metabolism, Distribution, and Concentration of Calcitroic Acid

Abstract

Near the turn of the 20th century scientist were beginning to discover compounds thatwere essential for humans beyond the previous establish, protein, grains, and fat. This ushered in a new field of studying a class of molecules that were considered “vital amines” later shortened to vitamins. Vitamin D was the fourth of this new class of compound that was identified and over the next decades was shown to play a vital role in the homeostasis of calcium and phosphorous. The dominant active form of vitamin D is formed within the body through a series of oxidation reactions acting upon its dietary source vitamin D2 or UV light activated precursor 7-dehydrocloesteroil leading to the main active form 1,25(OH)2 D3, calcitriol. In the 1980s exploration into the activity of other vitamin D metabolites was conducted by Esvelt and it was found that the final metabolic product of the activated pathway for vitamin D, calcitroic acid, had activity in complex formation with the vitamin D receptor (VDR). Our work focuses on the implementation of new methods of separation and measurement that were not available at the time of that study to explore the endogenous localization and concentration of calcitroic acid. This work utilizes the methods of liquid chromatographic separation with tandem mass spectrometric detection. An analytical method was optimized based on the ionization and fragmentation of calcitroic acid for the detection in in vitro and in vivo studies. This method was pair with a reverse phase ultra-high performance liquid chromatographic separation (UHPLC– MS/MS). The metabolic stability of calcitroic acid was explored to determine whether it was a true end product of the metabolism of vitamin D. It was found that calcitroic acid was stabile under enzymatic oxidative conditions. The conjugation of calcitroic acid for excretion and circulation was explored using in vitro assays. Separation methods were optimized for sample extraction of calcitroic acid from mouse tissues. A strong anion exchange (SPE) method was developed for the analysis to allow for removal of interfering species during in vivo studies. The concentration of native calcitroic acid was then measured in several different mouse tissues and determined to be below the limit of detection for the developed method. Alternative forms of selectively during these studies were explored based on the derivatization of calcitroic acid using dienophile dyes. To understand the role that the VDR in inflammatory bowel disease (IBD), A outbred mouse model was optimized for the screening efficacy of compounds, including calcitroic acid. Optimization to the measurements conducted within the study, vehicle and delivering of the drugs as well as the chemical irritant and sample preparation were optimized for low variance measurement to be conducted on the reduction of colitis in mice.