Determination of OA Pathology by Biomarker Dating

描述(由申请人提供)： 目前还不可能在单个时间点根据单个生物标记物可靠地区分单个骨性关节炎患者。然而，我们和其他人的工作已经确定了几个与OA相关的生物标志物，包括血清软骨低聚基质蛋白(COMP)、血清透明质酸和II型胶原的各种表位，仅举几例。在这项资助中，我们提出了一种新的策略，这是对现有的OA生物标记物方法的改进，目的是改善现有的OA生物标记物的预测能力。这种改进是基于测量体液中发现的选定软骨大分子中D-天冬氨酸的比例，特别是II型胶原和聚集素。氨基酸以L构型光学异构体的形式存在于天然蛋白质中。L异构体通过自发过程(消旋化)转化为生物上不常见的D-异构体，该过程依赖于时间、温度和较小程度的pH。虽然消旋化通常是一个非常缓慢的过程，但天冬氨酸是消旋化最快的氨基酸之一；这使得它能够在没有更新或周转率较慢的蛋白质中检测到。天冬氨酸的消旋作用也在两种已被研究过的软骨大分子--胶原和聚天冬氨酸中被检测到。对这些软骨大分子中D-天冬氨酸的定量检测表明，这些大分子中存在着不同的分子池，其周转率不同，胶原蛋白的周转率为100-400年，聚集素的周转率为3-25年。我们认为，从这些大分子衍生出来的碎片中D-Asp的比例将反映最古老的软骨大分子池的分解代谢程度。我们将使用已建立的高效液相色谱方法和建立基于ELISA的方法来检测骨性关节炎和非骨性关节炎受试者血清、尿液和滑液中选定的软骨大分子中D-天冬氨酸的含量。我们假设，对体液中最古老的II型胶原和聚集素片段的量化将比目前可用的骨关节炎生物标志物更好地区分骨关节炎患者和非骨关节炎患者。我们期望这种对当前生物标记物技术的改进将对分解代谢过程(高生物标记物水平与高D-Asp含量)与合成代谢过程(由于高周转状态而高生物标记物水平，但D-Asp含量相对较低)对OA受试者的生物标记物水平的贡献提供有价值的见解。我们把测定软骨大分子中D-天冬氨酸含量的技术称为“生物标记物测年”。

DESCRIPTION(Provided by Applicant): It is currently impossible to reliably distinguish an individual OA patient on the basis of a single biomarker at a single timepoint. Nevertheless, our work and that of others has identified several biomarkers associated with OA, including serum cartilage oligomeric matrix protein (COMP), serum hyaluronan, and various epitopes of type II collagen, to name a few. In this grant, we propose a novel strategy that is a refinement of current OA biomarker methods with the goal of improving upon the predictive capability of current OA biomarkers. This refinement is based upon measuring the fraction of D-aspartate in select cartilage macromolecules found in body fluids, in particular, type II collagen, and aggrecan. Amino acids exist in native proteins as the L-configurational optical isomer. The L-isomer is converted to the biologically uncommon D-isomer by a spontaneous process (racemization) that is dependent on time, temperature, and to a lesser extent pH. Although in general, racemization is a very slow process, aspartate is one of the 'fastest' racemizing amino acids; this enables its detection in proteins that are not renewed or have a slow turnover rate. Racemization of aspartate is also detectable in the two cartilage macromolecules in which it has been studied, collagen and aggrecan. The quantification of D-aspartate in these cartilage macromolecules has revealed the presence of distinct pools of molecules with different turnover rates ranging from 100-400 years for collagen and from 3-25 years for aggrecan. We propose that the fraction of D-Asp in the fragments derived from these macromolecules present in the serum, urine and synovial fluid will reflect the degree of catabolism of the oldest pool of cartilage macromolecules. We will use established HPLC methods and develop ELISA based methods to measure the fractional levels of D-Asp in select cartilage macromolecules in serum, urine and synovial fluid from OA and non-OA subjects. We hypothesize that quantification of the oldest type II collagen and aggrecan fragments in body fluids will better discriminate an OA subject from a non-OA subject than is possible with currently available OA biomarkers. We expect this refinement of current biomarker technology to yield valuable insights into the contribution of catabolic processes (high biomarker level with a high D-Asp content) versus anabolic processes (high biomarker level due to high turnover state but with a relatively low D-Asp content) to the level of a biomarker in an OA subject. We refer to the technique of quantifying the D-aspartate content of cartilage macromolecules as "biomarker dating".