From February 2011 to February 2013, patients with primary knee osteoarthritis (Jordan et al. 2004; Sutipornpalangkul et al. 2009) were recruited to participate in the study, following approval from the institutional ethics committee. Inclusion criteria were patients with primary knee osteoarthritis and age > 55 years. Exclusion criteria were patients with rheumatoid arthritis or secondary osteoarthritis (such as post-traumatic, post-infection osteoarthritis), concomitant knee or adjacent area infection, allergy to viscoelastic medication (injection), post-steroid injection, post-knee arthroplasty, immunocompromised patient, poor nutrition, severe knee pain (from any cause in addition to primary knee osteoarthritis) with normal radiographic findings, and poor medical condition. All 24 patients were consecutively enrolled according to the inclusion and exclusion criteria. One male patient was excluded because his age was less than 45 years, leaving 23 patients in the present study. All 23 patients were consecutively enrolled according to the inclusion and exclusion criteria. Three patients were from the outpatient clinic, where they provided synovial fluid (1 mL) from arthrocentesis under sterile technique before viscoelastic injection treatment. The other 20 patients provided synovial fluid from surgery (knee arthroplasty). Surgeons retrieved synovial fluid (1 mL) after knee arthrotomy, at the beginning of the operation before bone reaming, cutting, or implantation. Our preliminary study showed that mean knee society score (KSS) (Insall et al. 1989; Asif and Choon 2005) of patients with knee arthroplasty was ~46. Therefore, we decided to divided all 23 patients into 2 groups based on pre-treatment (viscoelastic injection or knee arthroplasty) KSS (Insall et al. 1989; Asif and Choon 2005): ≤46, severe; and >46, mild-moderate. Baseline data, including Kellgren- Lawrence (KL) radiographic grade (Kellgren and Lawrence 1957), were collected from all patients. This study was approved by the ethic committee of the Faculty of Medicine, Thammasat University.
Synovial fluid analyses
Synovial fluid from each patient was centrifuged at 2330 × g (800 RPM) for 5 minutes. The supernatant was collected to assay levels of antioxidants, including α-tocopherol (vitamin E) and total glutathione (GSH), iron concentrations and lipid peroxidation (TBARs) using the following techniques.
Determination of lipid peroxidation (TBARs) levels
TBARs levels were measured fluorometrically using a spectrofluorometer (Perkin-Elmer LS55 luminescence spectrometer, Beaconsfield, UK) with excitation and emission wavelengths at 515 and 553 nm, respectively. 1,1,3,3-tetraethoxypropane was used as the standard (Asakawa and Matsushita 1980).
High performance liquid chromatography (HPLC) determination of α-tocopherol
Levels of α-tocopherol (vitamin E) in synovial fluid were determined by reverse phase HPLC following a modified method by Zaspel and Csallany (1983). Two hundred and fifty microliters of samples were sequentially extracted with methanol and hexane. After drying the hexane layer under N2 and re-dissolving with methanol, the extract was injected into the HPLC system, which consisted of a Waters 2695 (Waters, Milford, MA, US). Vitamin E levels were determined with a Jusco FP 2020 Plus fluorescence detector (Japan Spectroscopic Co. Ltd, Tokyo, Japan), with an excitation wavelength of 295 nm and emission wavelength of 370 nm. The software program, Empower Pro (Waters, Milford, MA, US), was used for data analysis. The separation was carried out on a Nova Pak C18 column (4.6 mm · 150 mm, 5 μ) with 100% methanol as the mobile phase. The flow rate was 1.0 mL/min.
Determination of GSH levels
Total GSH in synovial fluid samples was determined using the DTNB-glutathione reductase recycling method with 5, 5′ dithiobis 2-nitrobenzoic acid as the disulfide chromogen. The absorbance of the reduced chromogen was measured with a microplate reader at 405 nm (Biotex, USA) and GSH concentration was expressed as nmol/mL (Anderson 1985).
Determination of total iron levels
Total iron in synovial fluid was determined using a modification of the methods of Foy et al., and ferrozine (Sigma, St. Louis, MO, US) was used as a chromogen (Foy et al. 1967). An aliquot of 250 μL of synovial fluid was mixed with 500 μL of acid mixture (10% trichloroacetic acid, 1 mol/L hydrochloric acid), and boiled for 30 min. After cooling, the samples were centrifuged at 2330 × g for 10 min. The clear supernatant was then mixed with the chromogen (1.5 mol sodium acetate and 0.5 mmol BPT) in 1:1.5 v/v and left at room temperature for 20 min before reading the absorbance at 562 nm with a UV-visible spectrophotometer (Cintra 10e, GBC, Melbourne, VIC, Australia).
Statistical analyses
Statistical analysis was implemented using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). ANOVA was used to analyze the statistical significance of differences in the values of concentrations of vitamin E, GSH, iron or TBARs between the 2 different groups of knee osteoarthritis severity. The correlations between the KSS and the concentration of vitamin E, GSH, iron or TBARs were analyzed and interpreted via Pearson’s correlation coefficient (r). Categorical variables were analyzed using Fisher’s exact test or Chi-square test. The level of significance was identified at p < 0.05.
