Eno 20 nitric oxide analyzer

Venous blood samples were drawn into BD 4 ml lithium heparin tubes and centrifuged at 4,000 rpm at room temperature for 3 minutes within 1 minute of collection. For the HTN study, blood was drawn immediately before and one hour after BRJ or placebo consumption. For the HFpEF study, blood was drawn one hour after BRJ or placebo consumption. Plasma was transferred in polypropylene microtubes containing no additives and frozen at −70°C for later analysis. Nitrite and nitrate were measured as described previously [37 (link)] using an ENO-20 nitric oxide analyzer (EICOM, San Diego, CA USA). Plasma was mixed with equal volume of 100% methanol, vortexed and centrifuged at 11,500g for 10 minutes. The supernatant was loaded into 96 well plates and nitrate and nitrate concentrations was measured using ENO-20 NOx analyzer (EICOM, San Diego, CA, USA). The nitrite and nitrate is separated via column chromatography and reacted individually with the Greiss reagent to synthesize a red diazo compound that is read at a wavelength of 540 nm by a visible light detector in the ENO-20, sample preparation described above are as per the manufacturer’s instructions. Standard nitrite and nitrate samples were made freshly each day before plasma samples were measured and used for calibration.

After 10 minutes of supine rest (~ 1 hour after the participant consumed the juice), venous blood samples were drawn into 4 ml lithium heparin tubes and centrifuged at 4,000 rpm at 20°C for 3 minutes within 1 minute of collection. Plasma was transferred in 400 μl volumes to sterile 500 μl Sarstedt polypropylene microtubes containing no additives and frozen at −70°C for later analysis. Nitrite (NO₂⁻) and nitrate were measured as described previously (42 (link)) using an ENO-20 nitric oxide analyzer (EICOM, San Diego, CA USA).

On all occasions, blood was drawn from the antecubital vein and stored in two 4 mL lithium heparin vials. Blood samples were immediately centrifuged at 1700 g for two minutes. Using a micropipette, two 0.4 mL aliquots of plasma were removed and stored in a −80 degrees Celsius freezer. Plasma was thawed, treated with equal volume of methanaol and centrifuges at 11,000 g for 10 minutes. A ENO-20 nitric oxide analyzer (EICOM, CA) was used to quantify nitrate and nitrite micromolar concentrations in plasma from both the pre-consumption and post-consumption blood draws. The intraclass correlation coefficients for the day-to-day test-retest reliability in our laboratory was 0.987 for nitrate and 0.987 for nitrite.