The Optimization of Synovial Fluid Protein Quantification with Hyaluronidase

Abstract

Synovial fluid (SF) is critical for joint health as it provides lubrication via high molecular
weight carbohydrates like hyaluronic acid (HA) and contains signaling molecules to respond to
injury¹. HA, known for its high viscosity, sequesters inflammatory modulating cytokines in SF,
reducing inflammation. This property of HA interferes with accurate protein quantification using
immunoassays to study joint inflammation². Various protocols exist to address HA interference
but yield inconsistent results^3,4,5. One approach treats synovial fluid with hyaluronidase for 0.5-1
hour at 37℃ or 20℃¹. However, commercially available hyaluronidases are contaminated with proteases6, and synovial fluid contains proteases⁷. Although protease inhibitors can be used,
some also inhibit hyaluronidase⁸. The research goals were to determine the optimum HA
digestion protocol, protease contamination of three hyaluronidase products, and whether
protease inhibitors interfere with hyaluronidase. The optimal digestion protocol was determined
by measuring the viscosity of high molecular weight HA after digestion at 20℃ or 37℃ for 0.5,
1, or 24 hours. Inhibition of hyaluronidase by protease inhibitors was also determined by
measuring viscosity as a proxy for HA concentration. To assess protease contamination in
commercial hyaluronidases, bovine serum albumin was treated using the HA digest protocol,
and protein was quantified using Bradford Assay and spectrophotometry. Preliminary results
indicate that hyaluronidase treatment of HA is optimal at 37℃ for 30 minutes. Protease
inhibitors did not interfere with the digestion of HA. Initial results showed contamination of
hyaluronidase, but this research is ongoing. These results aim to provide an optimized protocol
to prepare synovial fluid for immunoassays in orthopedic research.

¹ Necas, J., Bartosikova, L., Brauner, P., & Kolar, J. (2008). Hyaluronic acid (hyaluronan): A review.

Veterinary Medicine, 53(8), 397–411. Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic; Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.

² Anderson, J. R., Phelan, M. M., Rubio-Martinez, L. M., Fitzgerald, M. M., Jones, S. W., Clegg, P. D., & Peffers, M. J. (2020). Optimization of synovial fluid collection and processing for NMR metabolomics and LC-MS/MS proteomics. *Journal of Proteome Research, 19*(7), 2585-2597. https://doi.org/10.1021/acs.jproteome.0c00035

³ Keiser, H. D., & Hatcher, V. B. (1979). The Effect of Contaminant Proteases in Testicular Hyaluronidase Preparations on the Immunological Properties of Bovine Nasal Cartilage Proteoglycan. Connective Tissue Research, 6(4), 229–233. https://doi.org/10.3109/03008207909152325

⁴Jayadev, C., Rout, R., Jackson, W., Price, A., & Hulley, P. (2012, April). Synovial fluid preparation to improve immunoassay precision for biomarker research using multiplex platforms. *Osteoarthritis and Cartilage*, 20(S1), S169-S170. https://doi.org/10.1016/j.joca.2012.02.077

⁵ Keiser, H. D., & Hatcher, V. B. (1977). A Comparison of Bovine Nasal Cartilage Proteoglycan Core Protein Produced by Chondroitinase and Hyaluronidase: The Possible Role of Protease Contaminants. Connective Tissue Research, 5(3), 147–155. https://doi.org/10.3109/03008207709152265

⁶ Keiser, H. D., & Hatcher, V. B. (1977). A Comparison of Bovine Nasal Cartilage Proteoglycan Core Protein Produced by Chondroitinase and Hyaluronidase: The Possible Role of Protease Contaminants. Connective Tissue Research, 5(3), 147–155. https://doi.org/10.3109/03008207709152265

⁷ Marco Maiotti, Giovanni Monteleone, Umberto Tarantino, Giovanni F. Fasciglione, Stefano Marini, Massimiliano Coletta,Correlation between osteoarthritic cartilage damage and levels of proteinases and proteinase inhibitors in synovial fluid from the knee joint, Arthroscopy: The Journal of Arthroscopic & Related Surgery, Volume 16, Issue 5, 2000, Pages 522-526, ISSN 0749-8063, https://doi.org/10.1053/jars.2000.4632.

⁸ Mio, K., & Stern, R. (2001). Inhibitors of the hyaluronidases. Matrix Biol. 2002 Jan;21(1):31-7. doi: 10.1016/s0945-053x(01)00185-8.