Subjects with acute ACL injury have a high risk of developing osteoarthritis (OA), even after ACL reconstruction. It is hypothesized that biochemical and inflammatory cascades caused by the injury may contribute to the post-traumatic OA development. This study aims to develop novel NMR techniques for characterizing metabolic and inflammatory processes as well as relaxation and diffusion properties of synovial fluids after ACL injury in order to identify novel NMR biomarkers associated to joint inflammation and degeneration.Synovial fluid samples were collected from eight patients (18 – 44 yo, 5M/3F) with acute ACL-injuries during ACL reconstruction, and one patient with OA (47 yo, M). The HR-MAS NMR spectra were acquired on a Varian INOVA 500 MHz spectrometer with a consistent spin rate of 2250 kHz[1]. The sequences included 1D CPMG spectrum (Fig. 1, TE = 144 ms), followed by 2D sequences for the measurements of relaxation times and diffusion coefficients: inverse recovery for T1, CPMG spin-echo for T2, spin-locking for T1ρ, and pulsed field gradient stimulated echo (PFG-STE) for D, respectively. Saturation pulses were applied for solvent suppression. By gradually changing the acquisition parameters (Table 1), a set of spectra with peak intensities associated to the parameters are collected. To solve the multiple relaxation and diffusion components and to avoid prejudice and the requirement of prior knowledge when extracting relaxation time and diffusion coefficient information from data, inverse Laplace transform (ILT) with an iterative regularization algorithm[2] is used for point-by-point data inversion. Inspired by the diffusion-ordered spectroscopy (DOSY)[3] technique, we also generate 2D relaxation spectra after data processing (Fig. 2). Spectra for metabolites (small molecules) and macromolecules were reconstructed separately by summing the components of longer and shorter relaxation times separately during reconstruction. The ratio of macromolecule was calculated by the ratio of total macromolecule intensity divided by the sum of total metabolite intensity and total macromolecule intensity. The reproducibility of the techniques were tested by scan-rescanning 99.9% D2O samples, and the coefficients of variation (CV) were calculated.The 1D CPMG spectrum (Fig. 1) demonstrates major components presented in SF. Among the peaks, lactate has been reported to correlate with level of inflammation, and N-acetyl and glycine (and alanine) represent breakdown products from proteoglycan and collagen, respectively[4]. The 2D NMR spectra (Fig. 2) successfully resolved relaxation time and diffusion coefficient distributions for SF. T2 and T1ρ projection along chemical shift direction (red curves in Fig. 2) showed distinct fast and slow relaxing components, while T1 and diffusion projection only showed one dominating component. Fig. 3 illustrated 1D and 2D T2 spectra. Using our novel reconstruction algorithm, the 2D T2-reconstructed spectrum reliably separated spectrum from metabolites (Fig. 3d) and macromolecules (Fig. 3e), and better quantified the metabolite levels especially for the CH3 lipids (0.92 ppm) and the N-acetyl group (2.04 ppm) resonance (resonances arising from the chondroitin sulfate side changes of proteoglycan). While in comparison, the traditional 1D CPMG spectrum (Fig. 3a, TE = 144 ms) failed to recover correct intensities for the two peaks due to different local T2 modulation. Using this technique, we quantified the ratio of macromolecule (Fig. 3e) signal intensity and found notable reduction in the OA SF specimen (67.4%) compared to ACL (80.1% ± 3.7%), suggesting more severe breakdown of hyaluronon in synovial fluids of OA knee as compared to ACL-injured knees. Table 2 summarizes the scan/rescan CVs of relaxation time and diffusion coefficient quantification in 99.9% D2O samples, indicating excellent reproducibility of the technique.We have developed 1D and 2D NMR protocols for comprehensive profiling metabolic, relaxation and diffusion properties of synovial fluids in ACL-injured knees. We are currently applying these techniques in more samples collected from ACL-injured knees and will correlate NMR measures with longitudinal quantitative MRI measures of cartilage degeneration to identify novel NMR markers that predicts the joint degeneration.