Raman spectroscopy of monosodium urate crystals: A novel tool for non-invasive gout diagnosis

Gout is the most common form of inflammatory arthritis. It results in the deposition of monosodium urate crystals (MSU) at the surface of a joint or the articular cartilage. The gold standard for gout diagnosis is synovial fluid (SF) analysis which requires aspiration of the fluid and subsequent analysis by polarized light microscopy (PLM). This has poor reproducibility, is invasive and requires trained personal to perform the analysis. Raman spectroscopy (RS) has the potential to be a non-invasive diagnostic tool that can detect the presence of MSU crystals. The purpose of the research was to determine whether Raman spectroscopy applied onto the surface of a joint could detect MSU crystals through the skin. Two clinical studies were conducted, entitled the Pilot study (Pilot) and the sons of gout study (SOG). Pilot (n=20) considered ten clinically diagnosed gout sufferers and ten clinically diagnosed Osteoarthritis (OA) patients that acted as control. SOG (n=25) considered nine asymptomatic patients where gout was confirmed by clinicians at time of experiment by ultrasound, and 16 Non-Gouty patients, which did not show any signs of gout. An algorithm was implemented in Matlab® 2016 that removed background florescence, performed denoising and identified the presence or absence of MSU peaks. The comparisons were made against Raman peaks that are known to relate to MSU according to the literature. Three peak combinations, entitled C1, C2 and C3 were evaluated based on their resulting sensitivities and specificities for both studies. C1 was chosen as it provided the highest sensitivity for both studies. Pilot was found to have a sensitivity and specificity of 0.8 and 0.7, respectively. SOG had a sensitivity and specificity of 1 and 0.5, respectively. The results indicated that RS diagnosis is able to achieve good to high sensitivity comparable to other gout detection techniques but a moderate to good specificity. The results also show that RS is fully capable of detecting MSU crystals in-vivo, but results in a high number of false positives, 2 for Pilot and 7 for SOG. However, the false positives in Pilot may be attributed to the control subjects suffering from osteoarthritis, which can be a precursor of gout, and the false positives in the SOG study may be a result of ultrasound (US) being used as the confirmatory diagnostic technique that RS is being compared to. US is known to have sensitivity as low as 0.22 and is operator-dependent. Larger population studies are needed to confirm the ability of RS as a diagnostic tool for detecting gout.

Raman spectroscopy of monosodium urate crystals: A novel tool for non-invasive gout diagnosis

Gout is the most common form of inflammatory arthritis. It results in the deposition of monosodium urate crystals (MSU) at the surface of a joint or the articular cartilage. The gold standard for gout diagnosis is synovial fluid (SF) analysis which requires aspiration of the fluid and subsequent analysis by polarized light microscopy (PLM). This has poor reproducibility, is invasive and requires trained personal to perform the analysis. Raman spectroscopy (RS) has the potential to be a non-invasive diagnostic tool that can detect the presence of MSU crystals. The purpose of the research was to determine whether Raman spectroscopy applied onto the surface of a joint could detect MSU crystals through the skin. Two clinical studies were conducted, entitled the Pilot study (Pilot) and the sons of gout study (SOG). Pilot (n=20) considered ten clinically diagnosed gout sufferers and ten clinically diagnosed Osteoarthritis (OA) patients that acted as control. SOG (n=25) considered nine asymptomatic patients where gout was confirmed by clinicians at time of experiment by ultrasound, and 16 Non-Gouty patients, which did not show any signs of gout. An algorithm was implemented in Matlab® 2016 that removed background florescence, performed denoising and identified the presence or absence of MSU peaks. The comparisons were made against Raman peaks that are known to relate to MSU according to the literature. Three peak combinations, entitled C1, C2 and C3 were evaluated based on their resulting sensitivities and specificities for both studies. C1 was chosen as it provided the highest sensitivity for both studies. Pilot was found to have a sensitivity and specificity of 0.8 and 0.7, respectively. SOG had a sensitivity and specificity of 1 and 0.5, respectively. The results indicated that RS diagnosis is able to achieve good to high sensitivity comparable to other gout detection techniques but a moderate to good specificity. The results also show that RS is fully capable of detecting MSU crystals in-vivo, but results in a high number of false positives, 2 for Pilot and 7 for SOG. However, the false positives in Pilot may be attributed to the control subjects suffering from osteoarthritis, which can be a precursor of gout, and the false positives in the SOG study may be a result of ultrasound (US) being used as the confirmatory diagnostic technique that RS is being compared to. US is known to have sensitivity as low as 0.22 and is operator-dependent. Larger population studies are needed to confirm the ability of RS as a diagnostic tool for detecting gout.

POS0133 MONOSODIUM URATE CRYSTALS REDUCE HUMAN LIGAMENT CELLS VIABILITY THROUGH INCREASE OF ROS PRODUCTION

Background:Ligament destruction is a frequent complication of gout and is strongly associated with tophi. Ligament fibroblasts are important cellular mediators of ligament remodeling. None of study has paid attention to the effects of monosodium urate (MSU) crystals on ligament fibroblasts.Objectives:The study aims to investigate the effects and mechanism of MSU crystals on ligament fibroblasts.Methods:MSU crystals were added to human ligament fibroblasts(HLFs) cultures or primary ligament cells cultures. Cell counting kit-8 (CCK-8) assay, cell migration assay, Annexin V-FITC/PI assay were conducted. Reactive Oxygen Species(ROS) was tested by ROS Assay Kit.Results:The higher concentrations of MSU crystals (0.5-1mg/mL) reduced the viability of HLFs or primary ligament cells after 24 h as assessed by CCK8 assays, with a further reduction in viability observed at the 48 h time point. When observed under light microscopy, HLFs cultured with MSU crystals (0.5mg/mL) appeared unhealthy with fewer cells present. The cell migration ability of HLFs was decreased significantly on MSU crystals (0.5mg/mL). According to the result of Annexin V-FITC/PI assay, the survival rate of HLFs on MSU crystals (0.5mg/mL) was lower than that of 0.25mg/ml and 0 mg/ml at 72h. ROS assay results showed that the production of ROS increased as the concentrations of MSU crystals increased.Conclusion:MSU crystals inhibit human ligament cells viability through the increase of ROS production. It may contribute to disordered ligament remodeling in gout patients with ligament destruction.References:[1]Ashika Chhana, et al. Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state. Arthritis Res Ther. 2018, 20(1): 208.Figure 1.MSU crystals reduce human ligament fibroblasts and primary human ligament cells viability over time. A: CCK-8 assay; B: Observation of HLFs morphology; C: Annexin V-FITC/PI assay.Disclosure of Interests:None declared

STUDY OF EFFICACY OF PIPPALI VARDHAMANAK FOR TREATMENT OF VATARAKTA WITH SPECIAL REFERENCE TO GOUT

Introduction- Vatarakta is an illness where both Vata and Rakta are afflicted by distinct etiological factors. Vata- rakta is also possible when Gati of Vata is hindered by morbid Kapha Dosha. In modern science Gout shows sim- ilar characteristics like Vatarakta. It is occurred by accumulation of urate crystals in tissues. It starts from small joints to major joints. Pippali Vardhamanak is advised by Aacharya Sushruta for treatment of Vatarakta and other Vata-Kaphaj Vyadhi. In this VardhamanMatra of Pippali is given as said by Aacharya. Pippali has anti- inflammatory, rejuvenating properties and also act as Vata-Kapha Shamak. Aim- To evaluate therapeutic efficacy of Pippali Vardhamanak in Vatarakta with special reference to Gout. Objectives- To study therapeutic efficacy of Pippali Vardhamanak to reduce the symptoms of Vatarakta with special reference to Gout. Materials and Methods- The present study was conducted in 30 patients having clas- sical symptoms of Vatarakta. The patients were given Pippali Vardhamanak for 15 days. Pippali has Katu Ras, Laghu-Teekshna Snigdha Guna, Madhur Vipaka and Anshna Veerya, due to these proper- ties it acts as Kaphashamak, Vatashamak and Raktashama, Strotoshodhana. Piperine inhibits monosodium urate crystals. Conclusion- Pippali Vardhamanak gives significant relief in Vatarakta with special reference to Gout. Keywords: Vatarakta, Gout, Pippali, Pippali Vardhamanak

Detection of intracellular monosodium urate crystals in gout synovial fluid using optical diffraction tomography

Optical diffraction tomography (ODT) enables imaging of unlabeled intracellular components by measuring the three-dimensional (3D) refractive index (RI). We aimed to detect intracellular monosodium urate (MSU) crystals in synovial leukocytes derived from gout patients using ODT. The 3D RI values of the synthetic MSU crystals, measured by ODT, ranged between 1.383 and 1.440. After adding synthetic MSU crystals to a macrophage, RI tomograms were reconstructed using ODT, and the reconstructed RI tomograms discerned intracellular and extracellular MSU crystals. We observed unlabeled synthetic MSU crystal entry into the cytoplasm of a macrophage through time-lapse imaging. Furthermore, using gout patient-derived synovial leukocytes, we successfully obtained RI tomogram images of intracellular MSU crystals. The 3D RI identification of MSU crystals was verified with birefringence through polarization-sensitive ODT measurements. Together, our results provide evidence that this novel ODT can identify birefringent MSU crystals in synovial leukocytes of patients with gout.

Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

How macrophages are programmed to respond to monosodium urate crystals (MSUc) is incompletely understood partly due to the use of a toll-like receptor-induced priming step. Here, using genome wide transcriptomic analysis and biochemical assays we demonstrate that MSUc alone induces an in vitro metabolic and inflammatory transcriptional program in both human and murine macrophages markedly distinct from that induced by LPS. Genes uniquely up-regulated in response to MSUc belonged to lipids, glycolysis, and transport of small molecules via SLC transporters pathways. Sera from individuals and mice with acute gouty arthritis provided further evidence for this metabolic rewiring. This distinct macrophage activation may explain the initiating mechanisms in acute gout flares and is regulated through JUN binding to the promoter of target genes through activation of JNK (but not by P38) in a process that is independent of inflammasome activation. Finally, pharmacological JNK inhibition limited MSUc-induced inflammation in animal models of acute gouty inflammation.