The Effects of One Month Consumption of Standardized Aronia Melanocarpa Extract on Anemia in Patients on Hemodialysis: Focus on Oxidative Stress and Inflammation

1. Patients The study included 30 patients with chronic kidney failure on dialysis treatment at the Center for Nephrology and Dialysis of the Clinical Center Kragujevac. Inclusion criteria were: regular dialysis treatment for more than 3 months, 3 times a week and hemoglobin values lower than 110g / L. Exclusion criteria were: hemoglobin values lower than 80g/L, the use of antioxidant and immunosuppressive therapy, uncontrolled malignancies, proven active bleeding and presence of systemic inflammation or active infection.
2. Ethical approval The research was conducted with respect to the Helsinki Declaration on Medical Research, the approval of the Ethics Committee of the Clinical Center Kragujevac No 01-14-3039. All subjects provided written informed consent before study enrollment.
3. Used plant extract Standardized Aronia extract (SAE) is official product of pharmaceutical company Pharmanova (Belgrade, Serbia); nevertheless, procedure of extraction was done by EU-Chem company (Belgrade, Serbia). This product contains 400mg/30ml of polyphenols, while the recommended daily dosage is 30ml.
4. Blood sampling Patients included in the study consumed standardized Aronia melanocarpa extract (30ml/day) for 30 days. On the day 0, before SAE consumption, blood samples from all patients were collected for hematologic analyzes, inflammation parameters, oxidative stress parameters and antioxidant protection before the dialysis procedure. All mentioned parameters were determined again after consuming the product, ie. on the 30th day of the study.
5. Analytic Procedures Venous blood samples (2 x 4.5ml) were taken before the start of supplementation (zero day) and at the end of supplementation (30th day). Vacuum tubes with sodium citrate were used for blood sampling. The first sample was used for routine hematologic analyzes, the second sample was used for plasma and erythrocyte lysate extraction in order to determinate redox and inflammatory status.
6. Evaluation of Systemic Redox State Plasma samples were used for determination of the levels of the following pro-oxidants: superoxide anion radical (O2-), hydrogen peroxide (H2O2), nitrites (NO2-) and index of lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS), while the parameters of antioxidative defence system, such as activities of superoxide dismutase (SOD) and catalase (CAT) and level of reduced glutathione (GSH) were determined in erythrocytes lysates samples.

A) Index of lipid peroxidation (TBARS) determination The degree of lipid peroxidation in the plasma samples was estimated by measuring TBARS, using 1% thiobarbituric acid in 0.05 NaOH, which was incubated with the sample at 100°C for 15 min and measured at 530 nm. TBA extract was obtained by combining 0.8 ml sample and 0.4 ml trichloro acetic acid (TCA); afterwards, the samples were put on ice for 10 min and centrifuged for 15 min at 6000 rpm. (1).

b) Nitrite determination (NO2-)

Nitric oxide (NO) decomposes rapidly to form stable nitrite/nitrate products. The NO2- level was measured and used as an index of NO production, using Griess's reagent. For NO2 - determination in plasma 0.1 ml 3 N PCA (perchloride acid), 0.4 ml 20 mM ethylenediaminetetraacetic acid (EDTA) and 0.2 ml plasma were put on ice for 15 min, then centrifuged for 15 min at 6000 rpm. After pouring off the supernatant, 220 μl K2CO3 was added. Nitrites were measured at 550 nm. (2)

1. Superoxide anion radical determination (O2-)

Superoxide anion radical concentrations in plasma samples were measured using the NTB (Nitro Blue Tetrazolium) reagent in TRIS buffer (assay mixture). The measurement was performed at a wavelength of 530 nm. (3). 2. Hydrogen peroxide determination (H2O2)