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"""""""".
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