BEETROOT JUICE INTAKE in POSTMENOPAUSAL HYPERTENSIVE WOMEN

The evaluation of the blood pressure (BP) reactivity was carried out under mental stress, through the electronic version of Stroop protocol (3 minutes). For the test, the volunteers sited in front of a computer screen with a headset and listened to a voice speaking names of different colors and with images changing every 2 seconds. Each image consisted of a word forming the name of a color, with the word's letters color being different from the color's name, and with the bottom of the word with another color different from the color of the word and the color's name formed by the word. The volunteers should speak as quickly as possible the name of the color formed by the letters in the image. Before the test, baseline BP were measure once after resting in siting position during 15 minutes. During the test, the volunteers BP was measured every minute, through the auscultatory method.

Resting R-R intervals will be recorded for 15 min in the seated position using an HR monitor (POLAR® RS800cx; Polar Electro Oy, Finland; sampling frequency = 1000 Hz) with spontaneous breathing. Data will be downloaded to a computer using an infrared interface with specific software (POLAR PRO TRAINER5®, Polar Electro, Kempele, Finland). Heart rate variability (HRV) analysis was performed using KUBIOS HRV 3.0. Prior to the analysis, the signal will be visually inspected and filtered, and a range of 5 min interval with few artifacts will be selected near the end of the recording for analysis. The resulting R-R intervals will be analyzed in the time domain, in the frequency domain using spectral analysis (Fast Fourier Transform), and non-linearly through the Poincaré diagram.

Superoxide Dismutase (SOD) activity will be assessed by inhibition of pyrogallol autoxidation. Samples will be mixed with 50 mmol L Tris-HCl buffer (pH 8.2) containing 1 mmol L EDTA to deactivate metal-dependent enzymes, 80 U mL 1 catalase, and 24 mmol L 1 pyrogallol, and the kinetic assay will be monitored for 10 min at 420 nm using an analytical curve constructed with SOD as a standard.

Catalase (CAT) activity will be based on the decomposition of hydrogen peroxide. Samples will be mixed with a 10 mmol_L^-1 potassium phosphate buffer (pH 7.0 containing 0.2% hydrogen peroxide. Hydrogen peroxide decomposition will be monitored at 240 nm for 10 min.

To measure glutathione peroxidase (GPx) activity, plasma will be incubated with GPx buffer (100 mM potassium phosphate containing 1 mM EDTA, pH 7.7), sodium azide (40 mM), glutathione (GSH) (diluted in 5% metaphosphoric acid), glutathione reductase (GR) (diluted in GPx buffer), NADPH (diluted with 5% sodium bicarbonate), and tert-butyl hydroperoxide (0.5 mM). The reduction in NADPH concentration will be assessed for 10 min in a spectrophotometer at 340 nm.

The total antioxidant capacity due to iron reducing antioxidant power (FRAP) will be determined by the ability of the antioxidants present in the blood samples to reduce Fe3+ to Fe2+, which is chelated by TPTZ (2,4,6-tris(2-pyridyl)-s-triazine) and forms the Fe2+ TPTZ complex. This complex will be quantified in a spectrophotometer at 593 nm, and the antioxidant activity will be determined by means of an analytical curve, constructed with trolox as a standard.

Plasma proteins will be precipitated with metaphosphoric acid (MPA) and centrifuged at 7000 g for 10 min. The supernatant will be collected and mixed with 100 mM sodium phosphate buffer (pH 8.0) containing 5 mM EDTA and 15 μL ortho-phthaldialdehyde (1 mg/mL in methanol, HPLC grade, 99.9%). The solution will be incubated in the dark at room temperature for 15 min. Fluorescence will be read at 350 nm (excitation) and 420 nm (emission). Glutathione (GSH) concentrations of the sample will be calculated using a GSH standard curve (0.001-0.1 mM)

Human cytokines (IL-8, IL-1β, IL-6, TNF, IL-10 and IL-12p70) in serum samples will be measured by flow cytometry technique (FACSCantoII BD™, San Jose, CA, USA) using a human inflammatory cytokine kit (BD™ Cytometric Bead Array (CBA), BD Biosciences, San Jose, CA, USA) according to the manufacturer\'s instructions.