Assessment of redox status parameters
The frozen knee tissue was pulverized with a mortar and pestle and
resuspended in nine volumes of cold 0.1 mol/L phosphate buffer (pH=7.4)
containing 1.15% KCl. After the homogenization process, samples were
centrifuged in a cooling centrifuge at 800×g for 10 min, and then at
9500×g for 20 min to obtain a post-mitochondrial supernatant which was
used for redox status analysis. We measured pro-oxidative parameters
(total pro-oxidant status, TOS, and superoxide anion,
O2•-), prooxidant-antioxidant balance
(PAB) and parameters indicating the severity of oxidative damage in the
protein and lipid structure of the diseased knee, respectively (advanced
oxidation protein products, AOPP, and malondialdehyde, MDA, one of the
final products of polyunsaturated fatty acids peroxidation) [24].
Total oxidant status (TOS) was measured by a colorimetric method
optimised by Erel based on the ability of oxidants to oxidise the
ferrous ion-o-dianisidine complex to ferric ion, which forms a coloured
complex with xylenol orange in an acidic medium; colour intensity is
proportional to the total amount of oxidant molecules present in the
sample. Superoxide anion radical
(O2•-) content was measured as
nitrobluetetrazolium reduction rate according to Auclair and Voisin. In
the prooxidant-antioxidant balance (PAB) method, 3,3’,
5,5’-tetramethylbenzidine was used as a chromogen. The concentration of
advanced oxidation protein products (AOPP) was determined by measuring
the absorbance of the complex formed during the reaction with glacial
acetic acid with potassium-iodide. The concentration of malondialdehyde
(MDA) was measured by the TBARS (thiobarbituric acid reactive substance)
assay. Protein concentration in knee tissue was determined by the method
described by Bradford, using bovine serum albumin as a standard.
Biochemical parameters in knee tissue were normalized per gram of
protein in the sample. TOS, O2•- and
AOPP were determined with an ILAB 300 Plus analyzer (Instrumentation
Laboratory, Milan, Italy), whereas the PAB and MDA concentrations were
measured with a continuous spectrophotometer (Pharmacia LKB, Cambridge,
UK) [24].