Doubts concerning functional endothelial nitric oxide synthase in human erythrocytes

To the editor:

Nitric oxide (NO) is involved in the modulation of multiple physiologic functions. NO is produced from L-Arg by the catalytic action of NO synthase (NOS; EC 1.14.13.39).¹  Erythrocytes have been reported to express NOS,^2-8  an eNOS isoform.⁵  However, findings on a functional erythrocytic eNOS (eeNOS) in humans are contradictory.^2-8  Discrepancies may be because of different experimental conditions and methodological shortcomings.⁹  In consideration of the paradoxical occurrence of eeNOS in red blood cells (RBCs),⁶  which are mainly responsible for NO inactivation,⁹  we attempted to measure NOS activity in RBCs freshly collected from normal healthy humans by a fully validated, highly sensitive and specific gas chromatography-mass spectrometry (GC-MS) assay.¹⁰  Approval from the local Ethics Committee of the Hannover Medical School was obtained. Blood was drawn from the antecubital vein of healthy male and female volunteers using EDTA monovettes and processed immediately. RBCs were separated by centrifugation (800g, 4°C, 5 minutes) and used either unwashed or after repeated wash with physiologic saline. Unwashed RBCs were lyzed by freezing (30 minutes, −80°C) followed by slow defrosting in an ice bath and by rapid vortex-mixing (1 minute) with ice-cold distilled water (1:1, vol/vol).

Both in whole blood and in washed RBCs, externally added L-[guanidine-¹⁵N₂]-arginine (L-[¹⁵N₂]-Arg; Figure 1A) but not ¹⁵NO-derived [¹⁵N]nitrite and [¹⁵N]nitrate (not shown) was detected in RBCs cytosol at concentrations comparable with erythrocytic L-Arg concentrations. In washed RBCs isolated from blood of a healthy female volunteer, the peak area ratio (PAR) of m/z 47 ([¹⁵N]nitrite) to m/z 46 ([¹⁴N]nitrite) and the PAR of m/z 63 ([¹⁵N]nitrate) to m/z 62 ([¹⁴N]nitrate) measured in the RBCs cytosol did not differ between untreated and L-[¹⁵N₂]-Arg-treated RBCs (Figure 1B). These findings suggest no formation of ¹⁵NO from L-[¹⁵N₂]-Arg by native RBCs.

We did not find [¹⁵N]nitrite and [¹⁵N]nitrate above baseline levels in lyzed RBCs from freshly obtained blood of 5 healthy volunteers on incubation with L-[¹⁵N₂]-Arg (Figure 1C). In contrast, external addition of a recombinant human eNOS (heNOS) resulted in formation of [¹⁵N]nitrate indicating functional heNOS activity in lyzed RBCs. Addition of NADPH to lyzed RBCs did not further increase heNOS activity suggesting that sufficient endogenous NADPH is present in the hemolysate (Figure 1C). heNOS activity in buffer was found not to differ for H₄B concentrations between 100 and 2500nM.¹¹  That heNOS activity was measurable in lyzed RBCs suggest that H₄B is present in lyzed and native RBCs at concentrations high enough to ensure NOS activity. In addition, we found that glutathione reductase (GR), which shares with NOS the cofactors NADPH and FAD, was active in lyzed RBCs (Figure 1D). This finding suggests that other pathways are intact and functional in the lyzed RBCs used in the present study.

We used a sophisticated GC-MS assay to measure NOS activity in human RBCs. Our results suggest that human RBCs either contain inactive eeNOS or eeNOS activity is very low. The physiologic function of eeNOS in human erythrocytes is elusive and remains to be established.