Figure 1.
Combination of finger plethysmography (peripheral arterial tonometry, EndoPAT) with venous blood sampling from the same arm for EPR measurements of HbNO level. Changes of pulse amplitude, recorded by EndoPAT probes at baseline, during arterial occlusion (with a brachial cuff), and during reactive hyperemia (after cuff deflation) at the tip of the index finger of the ischemic arm (B) and contralateral finger of control arm (not undergoing reactive hyperemia) (A). Blood sampling for HbNO EPR assay was done at baseline (sample 1) before EndoPAT signal recording and at 1 (sample 2) and 2 (sample 3) minutes post-deflation of the cuff (post-occlusive hyperemia).
Figure 2.
Measurements of HbNO in human RBCs by a modified EPR subtraction procedure.
(A) Typical EPR spectra of free radicals in RBCs left for 45 min. in open air at room temperature: the individual model spectrum of free radicals in RBCs (lowest spectrum, a–b) was obtained after subtraction (subtraction I) of the EPR spectrum of RBCs treated with ascorbic acid (AA, 10 mmol/L, for the last 15 minutes) (b), from the EPR spectrum of RBC treated similarly with solvent (a). (B) Measurements of HbNO level in RBCs isolated from venous blood by EPR spectroscopy using subtraction (subtraction II) of individual free radical signal. The high field components of the EPR spectrum of RBCs frozen immediately after sampling (upper spectrum, a′) are shown at diapason 3250–3350 Gauss. The final EPR spectrum (a′–b′) shows the hf components of gz HbNO signal after subtraction of the model EPR spectrum of free radicals (b′), divided by the correspondent proportional scale factor (gain = 6.23×104); a typical EPR spectrum of RBCs, frozen after incubation with NO-donor (Dea-NONOate, 50 µmol/L) under low O2 level (gain = 0.62×104) is shown for comparison (d). The peak-to-peak amplitude of the 1st hf component (A(I)) of the triplet hf structure of the EPR spectrum, attributed to 5-coordinate α-HbNO, was used for quantitation.
Table 1.
Clinical and biological characteristics of the study subjects.
Figure 3.
Quantification of Hb-NO from human venous erythrocytes.
(A) Typical EPR spectra of RBCs, frozen after blood centrifugation (a), and after 45 minutes of exposure to air (b) (diapason: 3100–3400 Gauss). (B) Final EPR spectrum of RBCs (c, gain = 6.2×104), after subtraction of the model spectrum of free radicals (b), divided by the correspondent proportional scale factor, from the EPR spectrum of the initial sample frozen immediately (a). Typical EPR spectra of RBCs, frozen after incubation under low O2 level with the NO-donor, Dea-NONOate (50 µmol/L; c, gain = 3.1×104). A peak-to- peak amplitude of hf component, A(I), of the HbNO triplet structure attributed to 5-coordinate α-HbNO was used for quantitation.
Figure 4.
Hb-NO levels correlate with endothelial function in normal subjects.
(A) Linear regression analysis between basal HbNO level in RBCs isolated from venous blood and FRHI calculated as described in Methods (r = 0.58, P<0.0001). (B) Analysis of HbNO levels in RBCs isolated from venous blood of subjects distributed in different tertiles of FRHI as measured in the same arm; one-way analysis of variance (ANOVA) shows significant difference of mean values between groups (P = 0.0002).
Figure 5.
Hb-NO levels correlate with reactive hyperemia both in males and females.
Linear regression analysis between basal HbNO level (nmol/L) in RBCs isolated from venous blood of male (A) and female (B) subjects and FRHI calculated as described in Methods (r = 0.57; P<0.005; n = 23 and r = 0.55; P<0.005; n = 27 respectively).
Figure 6.
Post-occlusive Hb-NO correlates with resting Hb-NO and reactive hyperemia.
Linear regression analysis between HbNO level in RBCs isolated from brachial vein during the post-occlusion (PO) period (maximum HbNO value at 1 or 2 minutes PO) and basal HbNO level (A; r = 0.53; n = 46; P = 0.0001) or FRHI (calculated as described in Methods) (B; r = 0.4; n = 46; P = 0.004). The line represents the least-square regression line.