Determination of the Role of Proteins in the Physical Properties of Equine Synovial Fluid By: Shannon Cahill-Weisser Advisor: Dr. Skip Rochefort Oregon State University School of Chemical, Biological, and Environmental Engineering HHMI Summer 2010 reboundsportsphysio.com.au/information/glucosamine

The Problem 21.6% of U.S. adults reported doctor diagnosed arthritis ( ) 8.8% of U.S. adults reported activity limitations ( ) Arthritis cost the United States $128 Billion in 2003

Synovial Fluid Viscoelastic fluid in diarthrotic joints Keeps join lubricated, absorbs shock Supplies nutrients to cartilage Largest molecules present are proteins and hyaluronic acid health/adam/19309Kneejoint.html

Equine Joint Atlas

Hyaluronic Acid (HA) Key anionic glycosaminoglycan (GAG) polymer in synovial fluid Molecular weight million Daltons Conc. range in synovial fluid 2-4 mg/mL Found in lower concentrations with lower molecular weight in diseased joints hhmiundergradresearch/2009/program.htm =b3a23e6a-62a1-4e9b-9700-d54971e2da5f

Proteins Plasma proteins such as albumin and globulin Serum albumin ~ 66 thousand Daltons, most prominent protein in synovial fluid Globulins have a wider molecular weight range Total protein conc. in synovial fluid mg/mL Studies show they may have electrostatic interactions with HA, forming protein-GAG complexes May also form aggregated protein structures

Research Plan Objectives: Characterize the interactions between synovial fluid proteins and hyaluronic acid. Hypothesis: Interaction exists that affects the rheological properties of synovial fluid. Plan: Refine method of digestion/extraction of synovial fluid proteins without removal or compromise of HA.

Size Exclusion Chromatography- Multi Angle Laser Light Scattering

Size Exclusion Columns Light Scattering Geometry media.wiley.com/CurrentProtocols/PS/ps0708/ps0708-fig full.gif

Laboratory SEC-MALLS Wyatt Dawn EOS Polymer Labs Aquagel-OH SEC Columns Hitachi DRIHitachi HPLC Pump

Digestion and Extraction Breaks proteins into amino acids Pieces removed through phenol-chloroform extraction Results:  Lower HA peak in digested sample, implying loss of HA  Run to run inconsistencies in protein Mw UndigestedDigested

Samples of Study Sample 1: RLTR, 2005,Normal  HA Elution: min  HA Mw: 2.65E+06 Da  Protein Elution: min  Protein Mw: 7.61E+04 Da Sample 2:Left Stifle, 2005, Severe Trauma Fracture  Peak 1 Elution: min  Peak 1 Mw: 7.288E+05 Da  Peak 2 Elution: min  Peak 2 Mw: 8.490E+04 Da

Elution Time Comparisons

Gel Electrophoresis Procedure Bradford Assay: optical density measurement of coomassie blue bound proteins Run on SDS- polyacylamide gels

Gel Electrophoresis 9 mo. Normal Severe Trauma Fracture 5 mo. old Colt, Normal

Future… Run gel configured for glycosaminoglycan analysis  Fluorophore-Assisted Carbohydrate Electrophoresis  Try toluidine blue and other dyes Examine synovial fluid for degraded HA pieces before and after digestion.  Can show HA sizes at lower end of range

References  Calabro, Anthony, Benavides, Maria, Tammi, Markku, Hascall, Vincent C., and Midura, Ronald J. (1999). “Microanalysis of Enzyme Digests of Hyaluronan and Chondroitin/Dermatan Sulfate by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)”. Glycobiology 2000, vol. 10 no. 3, pp  Centers for Disease Control and Prevention (2006 and 2007). “Prevalence of Doctor- Diagnosed Arthritis and Arthritis-Attributable Activity Limitation, 2003—2005”. Morbidity and Mortality Weekly Report (MMWR), 2006: 55(40); and 2007/ 56(03); 55. Available at: Accessed: August 6,  Bhusari, Amol. (2007). Computational and Experimental Investigation of the Role of Hyaluronic Acid-Protein Interactions in the Rheology of Synovial Fluid. Texas Tech University.  Kvam, Catrine, Granese Daniela, Flaibani, Antonella, Zanetti, Flavio, and Paoletti, Sergio (1993). “Purification and Characterization of Hyaluronan form Synovial Fluid”. Analytical Biochemistry 1993, 211,  Hans, Tricia, Latimer, Kenneth S., LeRoy, Bruce E., Bain, Perry J., Tarpley, Heather L., Frank, Paul M. (2004). “Synovial Fluid Findings in Degenerative Joint Disease”. Accessed: September,  Leiske, Danielle Lurisa. (2004). Molecular and Rheological Characterization of Sodium Hyaluronate (HA) and Equine Synovial Fluid. Oregon State University.  Sigma Aldrich. (2010). “Albumin from Bovine Serum”. RCH_CONCAT_PNO|BRAND_KEY&F=SPEC. Accessed: September, RCH_CONCAT_PNO|BRAND_KEY&F=SPEC  Yelin, E (PhD), Cisternas, M (MA), Foreman, A (MA), Pasta, D (MS), Murphy L (PhD), Helmick, CG (MD) (2007). “National and State Medical Expenditures and Lost Earnings Attributable to Arthritis and other Rheumatic Conditions—United States, 2003”. MMWR 2007: 56(01); 4-7. Available at: Accessed: August,

Acknowledgements Howard Hughes Medical Institute Pete and Rosalie Johnson Internship Dr. Skip Rochefort Dr. Jill Parker, OSU Veterinary School Dr. Weis, Angela Poole, and Emilie Dicks Talia Helman, Dan Foster, Karl Schilke, Marsha Lampi, Marshall Lake, Danielle Leiske