Simultaneous Assessment of Endothelial Function, Nitric Oxide Synthase Activity, Nitric Oxide–Mediated Signaling, and Oxidative Stress in Individuals with and without Hypercholesterolemia

Abstract

Endothelial function is impaired in hypercholesterolemia and atherosclerosis. Based on mostly indirect evidence, this impairment is attributed to reduced synthesis or impaired biological activity of endothelium-derived nitric oxide (NO). It was the aim of this study to directly estimate and compare whole-body NO production in normo- and hypercholesterolemia by applying a nonradioactive stable isotope dilution technique in vivo. We enrolled 12 normocholesterolemic and 24 hypercholesterolemic volunteers who were all clinically healthy. To assess whole-body NO synthesis, we intravenously administered l-[guanidino-((15)N(2))]-arginine and determined the urinary excretion of (15)N-labeled nitrate, the specific end product of NO oxidation in humans, by use of gas chromatography-mass spectrometry. In addition, we measured flow-mediated vasodilation (FMD) of the brachial artery, expression of endothelial NOS (eNOS) in platelets, plasma concentration of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), and urinary excretion of 8-isoprostaglandin F(2alpha) (8-iso-PGF(2alpha)). After infusion of l-[guanidino-((15)N(2))]-arginine, cumulative excretion of (15)N-labeled-nitrate during 48 h was 40% [95% CI 15%-66%] lower in hypercholesterolemic than normocholesterolemic volunteers [mean 9.2 (SE 0.8) micromol vs 15.4 (2.3) micromol/l, P = 0.003]. FMD was on average 36% [4%-67%] lower in hypercholesterolemic than normocholesterolemic volunteers [6.3 (4.0)% vs 9.4 (4.6)%, P = 0.027]. Normalized expression of NOS protein in platelets was also significantly lower in hypercholesterolemic volunteers, whereas there were no significant differences in plasma ADMA concentration or urinary excretion of 8-iso-PGF(2alpha) between the 2 groups. This study provides direct evidence for a decreased whole body NO synthesis rate in healthy people with hypercholesterolemia.