Quantitation of Endogeneous Retinoids

描述（由申请方提供）：本研究拟开发一种LC/MS分析方法，用于区分、鉴定和定量生物组织中的内源性类维生素A。尽管视黄酸（RA）在发育、分化、视觉、生殖和免疫反应中的重要性，但不存在能够测量内源性水平的灵敏测定。具有各种检测方案的HPLC缺乏灵敏度，GC/MS需要衍生化并且不能分辨RA的几何异构体，并且LC/MS测定迄今为止尚未显著提高RA的检测限和/或尚未开发为分析有力的测定。RA的内源性水平的测量和RA将与合成RA的酶共定位的假设的调查是对RA功能的理解的极大兴趣。具体目的是（1）开发和验证生物样品中RA及其异构体的灵敏、特异性测定，（2）通过将其应用于动物组织研究来证明该测定的实用性，所述动物组织研究包括（a）测量wt和CRBP -/-小鼠中的类维生素A，（B）评估乙醇中毒对wt小鼠中全反式RA水平的影响，和（c）研究伊拉浓度是否与在小鼠脑和建立的细胞系中合成它的酶装置共定位。

DESCRIPTION (provided by applicant): This research proposes to develop an LC/MS assay to distinguish, identify, and quantitate endogeneous retinoids in biological tissues. Despite the importance of retinoic acid (RA) in development, differentiation, vision, reproduction, and immune response, no sensitive assay exists capable of measuring endogeneous levels. HPLC with various detection schemes lacks sensitivity, GC/MS requires derivitization and cannot resolve geometric isomers of RA, and LC/MS assays to date have not significantly improved detection limits of RA and/or have not been developed as an analytically vigorous assay. The measurement of endogeneous levels of RA and investigation of the hypothesis that RA will colocalize with enzymes that synthesize RA is of great interest towards the understanding of RA function. Specific aims are (1) to develop and validate a sensitive, specific assay for RA and its isomers in biological samples, (2) to demonstrate the utility of the assay by applying it to animal tissue studies consisting of (a) the measurement of retinoids in wt and CRBP -/- mice, (b) the assessment of the impact of ethanol intoxication on all-trans RA levels in wt mice, and (c) the investigation of whether IRA concentration colocalizes with the enzymatic apparatus that synthesizes it in mouse brain and established cell lines.