Determination of the Role of Proteins in the Physical Properties of Equine Synovial Fluid

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 www.valleyvet.com/ct_detail.html?pgguid =b3a23e6a-62a1-4e9b-9700-d54971e2da5f http://oregonstate.edu/dept/biochem/ hhmiundergradresearch/2009/program.htm

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 Polymer Labs Aquagel-OH SEC Columns Wyatt Dawn EOS Hitachi HPLC Pump Hitachi DRI

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 Undigested Digested

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 Severe Trauma Fracture 5 mo. old Colt, Normal 9 mo. 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. • 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 • 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=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC. Accessed: September, 2010. • 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.

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