Nitric Oxide-Dependent Blood Flow in Patients with Sickle Cell Disease Is Reflected by the Reactive Hyperemia-Peripheral Arterial Tonometry Index
Authors: Krajewski Megan L. , Kim James A. , Mendelsohn Laurel G. , Williams Candice B. , Gladwin Mark T. , Kato Gregory J. 
Published: April 2020

Abstract
Background: Decreased nitric oxide (NO) bioavailability is increasingly recognized as an important contributor to endothelial dysfunction and vascular complications of sickle cell disease. Unlike in other states of endothelial dysfunction, where NO production is impaired but response to exogenous NO donors is preserved, the vascular dysfunction observed in sickle cell disease is characterized by a diminished response to NO donors. Our group has previously demonstrated this NO resistance state through invasive strain-gauge venous occlusion plethysmography, the gold standard for evaluating peripheral endothelial function. Reactive hyperemia-peripheral arterial tonometry (RH-PAT) (ENDO-PAT 2000, Itamar Medical) is a relatively new, non-invasive technique for assessing endothelial function that records the reactive hyperemia response by measuring finger pulse wave amplitude after occlusion of the arm. We sought to compare these two techniques and validate peripheral arterial tonometry (PAT) as a tool for assessing vascular function in the sickle cell population.
Methods: Thirteen patients with HbSS sickle cell disease have participated in this ongoing study. Subjects were recruited while in steady state and were not receiving chronic transfusions. Following an overnight fast, subjects underwent PAT. According to manufacturer specifications, RH-PAT indices ≤ 1.67 are indicative of endothelial dysfunction and those &gt; 1.67 represent normal endothelial function. The subjects subsequently underwent venous occlusion plethysmography (AI6, D.E. Hokanson, Inc). Infusions of acetylcholine (ACh) and sodium nitroprusside (SNP) were administered to measure endothelium-dependent and endothelium-independent blood flow, respectively. To further characterize the nitric oxide resistance state, plasma samples from the first eight patients were assayed for NO consumption, and nitrite, a NO metabolite.
Results: Following intra-arterial infusion of 7.5, 15, and 30 μg/min of ACh, patients with an RH-PAT index ≤ 1.67 had an increase in blood flow over baseline of 196 ± 30%, 287 ± 43%, and 338 ± 53%, respectively. This was not significantly different from the percentage change in blood flow response in patients with RH-PAT index &gt; 1.67 (281 ± 68%, 277 ± 48%, 407 ± 53%, respectively, p = 0.60). In contrast, patients with an RHPAT index ≤ 1.67 demonstrated a significantly blunted response to 0.8, 1.6, and 3.2 μg/min infusions of SNP (28 ± 7%, 45 ± 11%, and 76 ± 17% vs. 85 ± 18%, 142 ± 36%, and 240 ± 48%, respectively, p &lt; 0.001). This blunted response was also observed when comparing absolute forearm flow values (p &lt; 0.01).
Lactate dehydrogenase, an established biomarker of the NO resistance state and associated endothelial dysfunction in sickle cell disease, was found to be inversely correlated with plasma nitrite levels (Spearman r = −0.95, p = 0.001). Plasma nitrite levels were also inversely correlated with NO consumption (Pearson r = −0.80, 95% CI = −0.96 to −0.23, p = 0.016). Patients with an RH-PAT ≤ 1.67 demonstrated greater NO consumption than patients with an RH-PAT &gt; 1.67 (3.32 μM ± 0.49, n = 4 vs. 2.06 μM ± 0.18, n = 4, (mean ± SEM), p= 0.029), consistent with a state of decreased NO bioavailability. Plasma nitrite reserve in patients with sickle cell disease has not been clearly established, but these data suggest a potential relationship between decreased nitrite and NO resistance. Interestingly, decreased plasma nitrite levels have been shown to correlate with endothelial dysfunction in other patient populations.
Conclusions: In this cohort of sickle cell patients, RH-PAT was closely associated with the NO-mediated component of endothelial function. Lower RH-PAT is associated with blunted response to SNP and increased NO consumption, suggesting that RH-PAT provides a non-invasive method for assessing NO resistance in sickle cell disease. In addition, our results present preliminary support that plasma hemoglobin redirects NO metabolism away from nitrite production.