Suppression of NOS-mediated constitutive DAF-FM-T formation within RBCs after inhibition of NO synthesis or NO scavenging. (A) RBCs were left either untreated or pretreated with the specific NOS inhibitor L-NAME or the NO scavenger Fe[DETC]₂, washed, loaded with DAF-FM diacetate and analyzed by HPLC with fluorescence detection. (B) RBCs were treated as described in panel A and analyzed by LC-MS. The top panel shows the mass spectrum of the respective peaks at 4.2 minutes for DAF-FM and 4.4 minutes for DAF-FM-T. The bottom panels show the SRM chromatograms for DAF-FM-T, monitoring the fragmentation from 424 to 380 because of the loss of a CO₂ from the parent molecule, and DAF-FM monitoring the transition from 413 to 369. (C-D) DAF-FM-T related peak areas from 3 to 5 independent experiments with different blood donors; samples treated as in panel A. (E-F) Estimation of the DAF-FM-T quantities formed by constitutive NOS activity in RBCs. (E) Regression curve obtained by diluting DAF-FM-T standards in RBC lysates. Measured peak areas were plotted against DAF-FM-T concentrations, assuming 100% conversion of DAF-FM into DAF-FM-T. (F) Calculated DAF-FM-T equivalents (eq) formed in ∼10⁷ red cells loaded with DAF-FM diacetate and incubated for 30 minutes at room temperature. After removing the outlier (according to Tuckey, see box plot) the mean DAF-FM-T concentration in RBC was 64 ± 12nM (n = 19).