Oxidative stress in skin fibroblasts cultures from patients with Parkinson's disease

Twenty patients diagnosed with PD and 19 healthy age- and sex-matched controls were enrolled in this study, after informed consent. The study was approved by the Ethics Committees of the University Hospitals "Doce de Octubre" and "Príncipe de Asturias", and was conducted according to the declaration of Helsinki. Two patients received no treatment, while the other 18 were treated with antiparkinsonian drugs alone or in combination including levodopa (16 cases), dopamine agonists (14 cases), selegiline or rasagiline (8 cases), anticholinergics (3 cases), and amantadine (1 case).

The control group was composed by 19 patients evaluated in the neurology departments of the same hospitals because of tension type headache, dizziness, dorsolumbar or cervical pain, etc. The clinical data of PD patient and control groups are summarized in table 1.

The following exclusion criteria were applied both to patients and controls: A) Ethanol intake higher than 80 g/day in the last 6 months. B) Previous history of chronic hepatopathy or diseases causing malabsorption. C) Previous history of severe systemic disease. D) Atypical dietary habits (diets constituted exclusively by one type of foodstuff, such as vegetables, fruits, meat, or others, special diets because of religious reasons, etc) F) Intake of drugs which modify lipid absorption. G) Therapy with vitamin supplements in the last 6 months.

Human skin fibroblasts were obtained from the dorsal region of the upper arm of each PD patient or control. Fibroblasts from the biopsy specimens were cultured in Dulbecco's modified Eagle's medium containing penicillin (100 UI/mL), streptomycin (100 mg/dl), L-glutamine (4 mM) and supplemented with heat-inactivated fetal calf serum at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Cells were grown to confluence, harvested by trypsinization at 37°C, washed with culture medium and resuspended with phosphate buffer 20 mM, and then sonicated to obtain the cell homogenate. Care was taken not to use cultures with a passage number greater than 12.

Respiratory chain enzymes and citrate synthase activity were measured in a DU-68 spectrophotometer (Beckman), applying 35-150 μg of total protein per 1 ml test volume in every complex enzyme assay. Incubation temperatures were 30°C for NADH coenzyme Q oxidoreductase (complex I), rotenone-sensitive NADH cytochrome c reductase (complexes I and III), succinate cytochrome c reductase (complexes II and III), succinate dehydrogenase (complex II) and citrate synthase (CS), and 38°C for cytochrome c oxidase (complex IV). The activities of complexes II, I and III, II and III, IV, and CS were determined as reported elsewhere [27]. Complex I was measured by following the oxidation of NADH at 340 nm in 100 mM Tris-HCl pH 7.4, 500 mM sucrose, 2 mM EDTA, 5 mM KCN, 100 μM NADH, and 50 μM DB (2,3-dimethyl-5-decyl-6-methylbenzoquinone) [46]. The reaction was inhibited by 90% when 2 μM rotenone was present. Complex V was determined by measuring 2.5 mM ATP extinction in a mean with 50 mM Hepes-Mg buffer at pH 8.0, 0.2 mM NADH, and phosphoenol-pyruvate 2.5 mM, and then adding 5 μl of pyruvate-kinase (10 mg/ml) and 10 μL of lactate-dehydrogenase (5 mg/ml) in presence of 10 μl of antimycin A (0.2 mg/ml in 50% ethanol). The oligomycin sensitive fraction was measured by adding 10 μl of oligomycin (0.2 mg/ml in 50% ethanol).To correct for mitochondrial volume, all respiratory chain enzyme activities were normalized to the activity of CS. Protein were measured by the method of Lowry et al. [47]. Specific activities were expressed as nmol × min^-1 xmg protein^-1, and referred to the specific activities of CS to correct for mitochondrial volume. All chemicals were from Boehringer Mannheim (Boehringer Mannheim, Germany) and Sigma Chemicals (St. Louis, MO). The measures were performed three times for every sample on three different passages (most of them between passage 5 and passage 10). The standard deviation of every measure is specified in table 2. The range of linearity was defined in all our determinations in a wide range of protein concentration. So we decided to use the homogenates in order to have a total protein concentration of 35-150 micrograms per mililiter of final test volume. This range was inside the linearity to all the activities of the OXPHOS complexes.

Glutathione peroxidase specific activity was determined according to the method described by Flohé and Günzler [48] based on NADPH oxidation followed at 340 nm at 37°C.

Catalase activity was determined according to the method described by Aeby [49] based on H₂O₂ decomposition followed at 240 nm at room temperature. Catalase specific activity was determined by calculating the rate constant of a first order reaction.

Total and Mn-SOD activities were determined according to the method described by Spitz and Oberley [50] based on nitroblue tetrazolium reduction by superoxide radicals followed at 560 nm at room temperature. The Mn-SOD was distinguished from cyanide-sensitive Cu/Zn-SOD by the addition of 5 mM NaCN. Cu/Zn-SOD activity was calculated by substracting the cyanide-resistant SOD activity form the total SOD activity. One unit of SOD activity is defined as the amount of enzyme that inhibits the reaction rate by 50%.

All enzymatic activities were expressed as values normalized to total cellular protein. The measurements were performed three times for every sample.

Oxidized, reduced and total coenzyme Q₁₀ levels were determined by high performance liquid chromatography with electrochemical detection. The method used was that of Langedijk et al. [51] with some modifications. The stationary phase was a reverse phase column (HR-80 RP-C₁₈, 80 × 4,6 mm. ESA Inc). The mobile phase was prepared by dissolving 7 g of NaClO₄.H₂O in 1000 ml of methanol/propanol/HClO₄ 70%, 700.8:200:0.2 (vol/vol), and the flow rate was set at 0.8 ml/min. The programmed conditions for the electrochemical detector and the post-column valve were the same. The system was entirely controlled by a computer (Kromasystem 2000, Kontron Instruments). Injections were made in a 50 μl injection valve (Model 7161, Rheodyne, Cotaty, USA) with a 100 μl syringe from Hamilton (Bonaduz, Switzerland). The calibration method used ubiquinone as external standard. The within-run coefficients of variation for CoQ₁₀ and CoQH₂ were, respectively, 5 and 3.2%, and the day to day precisions were 9.2 and 6.3%. CoQ₁₀ recovery ranged between 88 and 93%. The measurements of CoQ₁₀ were expressed in nmol/g of protein. The measures were performed three times for every sample.

Results were expressed as mean ± SD. Statistical analysis was done by the SPSS statistical package (15.0 version). For continuous variables the Kolmogorov-Smirnoff test was used to analyze normality in the distribution. Then, the Students's two-sample t-test was used for variables that followed a normal distribution and the Mann-Whitney test was used for the rest of variables. The results of each table were corrected for multiple comparisons by the use of Bonferroni's correction. Correlation analyses were performed by using Pearson's correlation coefficient.