Quantitation of Endogeneous Retinoids

描述（由申请人提供）：本研究拟建立一种LC/MS分析方法来区分、鉴定和定量生物组织中的内源性类维甲酸。尽管维甲酸（RA）在发育、分化、视力、生殖和免疫反应中具有重要意义，但目前还没有能够测量内源性水平的敏感测定方法。各种检测方案的HPLC缺乏灵敏度，GC/MS需要衍生化，不能分辨RA的几何异构体，LC/MS检测迄今为止没有显著提高RA的检测限和/或尚未发展成为一种分析强有力的检测方法。测量RA的内源性水平和研究RA与合成RA的酶共定位的假设对理解RA的功能具有重要意义。具体目标是：(1)开发和验证生物样品中RA及其异构体的敏感、特异性分析，(2)通过将该分析应用于动物组织研究来证明该分析的实用性，该研究包括(a)测量wt和CRBP -/-小鼠中的类维生素a， (b)评估乙醇中毒对wt小鼠全反式RA水平的影响。以及(c)在小鼠脑和已建立的细胞系中，IRA浓度是否与合成它的酶装置共定位的研究。

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.