Determination of OA Pathology by Biomarker Dating

描述（申请人提供）： 目前不可能在单个时间点基于单个生物标志物可靠地区分个体OA患者。然而，我们的工作和其他人的工作已经确定了几个生物标志物与OA，包括血清软骨寡聚基质蛋白（COMP），血清透明质酸，和各种表位的II型胶原蛋白，仅举几例。在这项研究中，我们提出了一种新的策略，这是一个完善目前的OA生物标志物的方法，其目标是提高目前的OA生物标志物的预测能力。这种改进是基于测量体液中发现的选定软骨大分子，特别是II型胶原蛋白和聚集蛋白聚糖中D-天冬氨酸的分数。氨基酸在天然蛋白质中以L-构型光学异构体存在。L-异构体通过自发过程（外消旋化）转化为生物学上不常见的D-异构体，该过程取决于时间、温度和较小程度的pH值。虽然一般来说，外消旋化是一个非常缓慢的过程，但天冬氨酸是“最快”外消旋化的氨基酸之一;这使得它能够在不更新或周转率慢的蛋白质中检测到。天冬氨酸的外消旋也可在两种软骨大分子中检测到，其中已对其进行了研究，胶原蛋白和聚集蛋白聚糖。这些软骨大分子中D-天冬氨酸的定量揭示了存在不同的分子库，其具有不同的周转率，胶原蛋白为100-400年，聚集蛋白聚糖为3-25年。我们建议，D-Asp的馏分中的片段来自这些大分子存在于血清，尿液和滑液将反映软骨大分子的最古老的池caterion的程度。我们将使用已建立的HPLC方法并开发基于ELISA的方法来测量OA和非OA受试者血清、尿液和滑液中选定软骨大分子中D-Asp的分数水平。我们假设，与目前可用的OA生物标志物相比，体液中最古老的II型胶原和聚集蛋白聚糖片段的定量将更好地区分OA受试者与非OA受试者。我们期望当前生物标志物技术的这种改进产生对分解代谢过程（具有高D-Asp含量的高生物标志物水平）与合成代谢过程（由于高转换状态而具有相对低的D-Asp含量的高生物标志物水平）对OA受试者中生物标志物水平的贡献的有价值的见解。我们将量化软骨大分子中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".