Quantitation of Endogeneous Retinoids

描述(由申请人提供)：这项研究建议建立一种LC/MS分析方法来区分、鉴定和定量生物组织中的内源性维甲酸。尽管维甲酸(RA)在发育、分化、视觉、生殖和免疫反应中很重要，但目前还没有一种灵敏的方法能够测量内源性水平。不同检测方案的高效液相色谱缺乏灵敏度，GC/MS需要衍生化，不能拆分RA的几何异构体，LC/MS分析到目前为止还没有显著提高RA的检测限和/或没有发展成为一种强有力的分析方法。内源性RA水平的测定和RA与合成RA的酶的共位假说的研究对于理解RA的功能具有重要意义。具体目标是(1)建立和验证生物样品中RA及其异构体的灵敏、特异的分析方法；(2)通过将其应用于动物组织研究来证明该分析方法的实用性，这些动物组织研究包括：(A)测量wt和CRBP-/-小鼠中的维甲酸，(B)评估乙醇中毒对wt小鼠中全反式RA水平的影响，以及(C)研究IRA浓度是否与在小鼠大脑和已建立的细胞系中合成IRA的酶装置共存。

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.