1. 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

2. The Problem 21.6% of U.S. adults reported doctor diagnosed arthritis (2003-2005) 8.8% of U.S. adults reported activity limitations (2003-2005) Arthritis cost the United States $128 Billion in 2003

3. Synovial Fluid Viscoelastic fluid in diarthrotic joints Keeps join lubricated, absorbs shock Supplies nutrients to cartilage Largest molecules present are proteins and hyaluronic acid http://www.nytimes.com/imagepages/2007/08/01/ health/adam/19309Kneejoint.html

4. Equine Joint Atlas http://oregonstate.edu/dept/biochem/hhmi/undergradresearch/2005/index.html

5. Hyaluronic Acid (HA) Key anionic glycosaminoglycan (GAG) polymer in synovial fluid Molecular weight 0.2-10 million Daltons Conc. range in synovial fluid 2-4 mg/mL Found in lower concentrations with lower molecular weight in diseased joints http://oregonstate.edu/dept/biochem/ hhmiundergradresearch/2009/program.htm www.valleyvet.com/ct_detail.html?pgguid =b3a23e6a-62a1-4e9b-9700-d54971e2da5f

6. 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 18-48 mg/mL Studies show they may have electrostatic interactions with HA, forming protein-GAG complexes May also form aggregated protein structures

7. 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.

8. Size Exclusion Chromatography- Multi Angle Laser Light Scattering http://www.ap-lab.com/images/LS_setup.gif

9. Size Exclusion Columns Light Scattering Geometry media.wiley.com/CurrentProtocols/PS/ps0708/ps0708-fig-0001-1-full.gif http://www.viscotek.com/images/Theory/separate.jpg

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

11. 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

12. Samples of Study Sample 1: RLTR, 2005,Normal  HA Elution: 33.994 min  HA Mw: 2.65E+06 Da  Protein Elution: 47.328 min  Protein Mw: 7.61E+04 Da Sample 2:Left Stifle, 2005, Severe Trauma Fracture  Peak 1 Elution: 43.921 min  Peak 1 Mw: 7.288E+05 Da  Peak 2 Elution: 47.513 min  Peak 2 Mw: 8.490E+04 Da

13. Elution Time Comparisons

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

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

16. 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

17. 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.273-281.  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); 1089-1092 and 2007/ 56(03); 55. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5540a2.htm. Accessed: August 6, 2009.http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5540a2.htm  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, 44-49.  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”. http://www.vet.uga.edu/vpp/clerk/hans/index.php. Accessed: September, 2010 http://www.vet.uga.edu/vpp/clerk/hans/index.php  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”. http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=A2153|SIGMA&N5=SEA RCH_CONCAT_PNO|BRAND_KEY&F=SPEC. Accessed: September, 2010. http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=A2153|SIGMA&N5=SEA 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: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5601a2.htm. Accessed: August, 2009.http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5601a2.htm

18. 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