Dataset Information

Nitrosylated hemoglobin levels in human venous erythrocytes correlate with vascular endothelial function measured by digital reactive hyperemia.

ABSTRACT:

Unlabelled

Impaired nitric oxide (NO)-dependent endothelial function is associated with the development of cardiovascular diseases. We hypothesized that erythrocyte levels of nitrosylated hemoglobin (HbNO-heme) may reflect vascular endothelial function in vivo. We developed a modified subtraction method using Electron Paramagnetic Resonance (EPR) spectroscopy to identify the 5-coordinate α-HbNO (HbNO) concentration in human erythrocytes and examined its correlation with endothelial function assessed by peripheral arterial tonometry (PAT). Changes in digital pulse amplitude were measured by PAT during reactive hyperemia following brachial arterial occlusion in a group of healthy volunteers (50 subjects). Erythrocyte HbNO levels were measured at baseline and at the peak of hyperemia. We digitally subtracted an individual model EPR signal of erythrocyte free radicals from the whole EPR spectrum to unmask and quantitate the HbNO EPR signals.

Results

Mean erythrocyte HbNO concentration at baseline was 219+/-12 nmol/L (n = 50). HbNO levels and reactive hyperemia (RH) indexes were higher in female (free of contraceptive pills) than male subjects. We observed a dynamic increase of HbNO levels in erythrocytes isolated at 1-2 min of post-occlusion hyperemia (120+/-8% of basal levels); post-occlusion HbNO levels were correlated with basal levels. Both basal and post-occlusion HbNO levels were significantly correlated with reactive hyperemia (RH) indexes (r = 0.58; P<0.0001 for basal HbNO).

Conclusion

The study demonstrates quantitative measurements of 5-coordinate α-HbNO in human venous erythrocytes, its dynamic physiologic regulation and correlation with endothelial function measured by tonometry during hyperemia. This opens the way to further understanding of in vivo determinants of NO bioavailability in human circulation.

SUBMITTER: Lobysheva II

PROVIDER: S-EPMC3794924 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Reduced bioavailability of nitric oxide (NO) is a major feature of endothelial dysfunction characteristic of cardiovascular and metabolic diseases but the short half-life of NO precludes its easy quantification in circulating blood for early diagnosis. In erythrocytes, NO can react with hemoglobin to form an iron-nitrosyl complex (5-coordinate-α-HbNO) directly quantifiable by Electron Paramagnetic Resonance spectroscopy (EPR) in mouse, rat and human venous blood ex vivo. However, the sources of the nitrosylating species in vivo and optimal conditions of HbNO preservation for diagnostic use in human erythrocytes are unknown. Using EPR spectroscopy, we found that HbNO stability was significantly higher under hypoxia (equivalent to venous pO2; 12.0±0.2% degradation of HbNO at 30 minutes) than at room air (47.7±0.2% degradation) in intact erythrocytes; at 20°C (15.2±0.3% degradation after 30 min versus 29.6±0.1% at 37°C) and under acidic pH (31.7±0.8% versus 62.2±0.4% degradation after 30 min at physiological pH) at 50% of haematocrit. We next examined the relative contribution of NO synthase (NOS) from the vasculature or in erythrocytes themselves as a source of nitrosylating NO. We detected a NOS activity (and eNOS expression) in human red blood cells (RBC), and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice, as measured by HbNO formation and nitrite/nitrate accumulation. NO formation was increased after inhibition of arginase but abrogated upon NOS inhibition in human RBC and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice. However, the HbNO signal from freshly drawn venous RBCs was minimally sensitive to the inhibitors ex vivo, while it was enhanced upon caveolin-1 deletion in vivo, suggesting a minor contribution of erythrocyte NOS to HbNO complex formation compared with vascular endothelial NOS or other paracrine NO sources. We conclude that HbNO formation in rodent and human venous erythrocytes is mainly influenced by vascular NO sources despite the erythrocyte NOS activity, so that its measurement by EPR could serve as a surrogate for NO-dependent endothelial function.

Project description:BackgroundImpaired perfusion indices signal potential microvascular dysfunction preceding atherosclerosis and other cardiometabolic pathologies. Post-occlusive reactive hyperemia (PORH), a vasodilatory response following a mechanically induced ischemia, is a transient increase in perfusion and can assess microvascular function. The greatest blood flow change corresponding to the first minute of hyperemia (represented by time-to-peak, hyperemic velocity, AUC within 1st min) has been shown to indicate microvascular dysfunction. However, the reproducibility of these temporal kinetic indices of the PORH response is unknown. Our aim was to examine the inter- and intra-day reproducibility and standardization of reactive hyperemia, with emphasis on the kinetic indices of PORH, using laser speckle contrast imaging (LSCI) technique.Methods and resultsSeventeen healthy adults (age = 24 ± 3 years) completed three PORH bouts over two lab visits. LSCI region of interest was a standardized 10 cm region on the dominant ventral forearm. A 5-min brachial artery occlusion period induced by inflating an arm cuff to 200 mmHg, preceded a 4-min hyperemic period. Inter- and intra-day reliability and reproducibility of cutaneous vascular conductance (LSCI flux / mean arterial pressure) were determined using intraclass correlation (ICC) and coefficient of variation (CV%). Maximal flow and area under the curve standardized to zero perfusion showed intra- and inter-day reliability (ICC > 0.70). Time to maximal flow (TMF) was not reproducible (inter-day CV = 18%). However, alternative kinetic indices such as 1-min AUC and overshoot rate-of-change (ORC), represented as a piecewise function (at 5s, 10s, 15s, and 20s into hyperemia), were reproducible (CV< 11%). Biological zero was a reliable normalization point.ConclusionPORH measured with LSCI is a reliable assessment of microvascular function. However, TMF or its derived hyperemic velocity are not recommended for longitudinal assessment. Piecewise ORC and 1-min AUC are reliable alternatives to assess the kinetic response of PORH.