Evidence of NOS activity in RBCs. (A) NOS activity was detected by enzymatic conversion of l-arginine²³  in RBC membranes derived from human blood samples (n = 4). (B-C) RBC-NOS-dependent rate of NO release was measured with 2 independent methods. (B) The oxyhemoglobin assay. Purified RBCs from venous blood were placed in a reservoir separated from circulating oxyhemoglobin by a dialysis membrane (2.5-3.0 nm [25-30 Å]), inhibiting exchange of RBCs and oxyhemoglobin. In order to measure NO release by NO-dependent conversion of oxyhemoglobin to methemoglobin, time-dependent changes in absorption between 411 nm (isosbestic point) and 401 nm (the highest difference in absorbance) were determined via difference spectrophotometric analysis.²⁴  Buffer (control), l-arginine (l-arg), and l-NMMA were applied subsequently to the system. Arrows indicate the duration of infusion of the respective agents. l-arginine led to an increase of NO release from 0.6 to 14 pmol/mL/min. Inhibition with l-NMMA reduced NO release to 1.4 pmol/mL/min. (C) The nonreductive CLD. Purified RBCs from venous blood were reconstituted with buffer (control) and placed into a reaction chamber flowed with helium (to avoid excessive foaming, antifoaming reagent was added and the Hct diluted to 0.08 [8%]). Time-dependent NO release in the presence or absence of l-arginine or l-NMMA was detected directly in the gas phase (calculated to mL whole blood per measurement period). (D-F) Changes in accumulated plasma RXNO, nitrite, and nitrate were determined in blood samples after incubation with l-arginine (stimulation) and l-NNA (inhibition). Changes were compared with control conditions (incubation with PBS). ^* indicates significant difference from control; and #, difference from l-arg, P less than .05.