Uptake of L-[¹⁵N₂]-Arg in RBCs and assessment of NO synthase and glutathione reductase activity in intact and lyzed human RBCs. Enrichment of L-[¹⁵N₂]-Arg (A), [¹⁵N]nitrite, [¹⁵N]nitrate and L-[¹⁵N₂]-Arg (B) in RBCs cytosol after incubation of L-[¹⁵N₂]-Arg with whole blood and washed RBCs of 2 healthy volunteers (A,B); NOS activity in lyzed human RBCs in the absence and in the presence of externally added heNOS (C); glutathione reductase (GR) activity in lyzed human RBCs (D). (A) Peak area ratio (PAR) of m/z 588 to m/z 586 after incubation of whole blood and washed RBCs with L-[guanidine-¹⁵N₂]-arginine (L-[¹⁵N₂]-Arg; 98% at both ¹⁵N atoms; Cambridge Isotope Labs). L-[¹⁵N₂]-Arg (10mM with respect to the blood volume) was incubated for 20 minutes at 37°C. In case of washed RBCs, the original blood plasma volume was replaced by the same volume of physiologic saline or physiologic saline that contained glucose (1mM). The blood used in this experiment was donated by a healthy, 27-years old male volunteer. (B) PAR of m/z 47 to m/z 46 (for [¹⁵N]nitrite), m/z 63 to m/z 62 (for [¹⁵N]nitrate) and m/z 588 to m/z 586 (for L-[¹⁵N₂]-Arg) on incubation of washed RBCs with L-[¹⁵N₂]-Arg (0.4mM with respect to the blood volume) for 20 minutes at 37°C. The original blood plasma volume was replaced by physiologic saline that contained glucose (1mM). Blood used in this experiment was donated by a healthy, 28-years old female volunteer. Note the logarithmic scale on the y axis. (C) PAR of m/z 63 to m/z 62 measured in lyzed RBCs isolated from EDTA blood donated by 5 healthy volunteers (aged 28–56 years; 4 females). Lyzed RBCs were incubated with L-[¹⁵N₂]-Arg (5mM) in the absence (open symbol) or in the presence (closed symbol) of externally added recombinant heNOS (50 μg/mL; ALEXIS). Incubations were performed at 37°C. Data are shown as mean ± SEM. Asterisks indicate statistically significant difference between incubation time 0 minutes and incubation time 10 minutes (P = .029), incubation time 20 minutes (P = .021) and incubation time 30 minutes (P = .016). (D) Decrease in the concentration of externally added NADPH (100μM) in lyzed RBCs diluted with phosphate buffered saline (1:200, vol/vol) after addition of the substrate GSSG (1mM). NADPH was measured spectrophotometrically by recording absorbance at 340 nm.