Original Contribution Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans Petra Kleinbongard f,1 , Andre ´ Dejam b,f,1 , Thomas Lauer f , Thomas Jax a , Stefan Kerber a , Putrika Gharini f , Jan Balzer f , Rainer B. Zotz c , Ru ¨ diger E. Scharf c , Reinhart Willers d , Alan N. Schechter b , Martin Feelisch e , Malte Kelm f, * a Division of Cardiology, Pulmonary Diseases, and Angiology, Department of Medicine, Heinrich Heine University, D-40225 Du ¨ sseldorf, Germany b Molecular Medicine Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA c Institute of Hemostasis and Transfusion Medicine, Heinrich Heine University, D-40225 Du ¨ sseldorf, Germany d Department of Statistical Computations, Heinrich Heine University, D-40225 Du ¨ sseldorf, Germany e Whitaker Cardiovascular Research Institute, Boston University School of Medicine, Boston, MA 02118, USA f Medical Clinic I, University Hospital RWTH Aachen, D-52074 Aachen, Germany Received 16 May 2005; revised 31 July 2005; accepted 17 August 2005 Available online 10 November 2005 Abstract A reduced nitric oxide availability is a hallmark of endothelial dysfunction occurring early in atherosclerosis. Recently, we have shown that plasma nitrite mirrors acute changes in endothelial nitric oxide synthase activity in various mammals, including humans. Here, we examined the hypothesis that plasma nitrite levels are reduced in humans with endothelial dysfunction and the decrease is correlated with increasing numbers of cardiovascular risk factors (RF). Plasma nitrite concentrations were quantified by flow-injection analysis. The coefficient of variation for repeated measurements of plasma nitrite was <8%, and heart rate and blood pressure at the time of blood sampling had no significant effect on nitrite values measured (n = 10). Baseline levels of plasma nitrite followed a normal distribution in each group studied and decreased progressively with increasing numbers of cardiovascular risk factors (n = 351, p < 0.001): 351 T 13 (0 RF), 261 T 10 (1 RF), 253 T 11 (2 RF), 222 T 18 (3 RF), and 171 T 29 nmol/L (4 RF). Intima media thickness (IMT) and flow-mediated dilation (FMD) were determined via ultrasound. Plasma nitrite and FMD levels were lower, whereas IMT was greater in individuals with endothelial dysfunction (n = 12) compared to healthy volunteers (n = 12). Nitrite correlated significantly with FMD (r = 0.56, p < 0.001) and inversely with IMT (r = À0.49, p < 0.01). Plasma nitrite levels are reliably measurable in humans, indicate endothelial dysfunction, and correlate with cardiovascular risk factors. Future studies are necessary to identify the prognostic relevance of plasma nitrite determination in patients suffering from cardiovascular disease. D 2005 Elsevier Inc. All rights reserved. Keyword: Free radicals Hyperlipidemia, arterial hypertension, diabetes, smoking, and aging are major risk factors for atherogenesis and manifestation of cardiovascular events [1]. Endothelial dys- function represents a key event in early atherosclerosis. Decreased bioactivity of endothelium-derived nitric oxide (NO), because of either impaired synthesis or exaggerated breakdown, is a major feature of endothelial dysfunction [2]. Although direct biochemical evidence for reduced NO avail- ability has been obtained in various experimental models, this approach has been impossible in patients so far. Instead, surrogates of NO bioactivity have been introduced in clinical settings, testing vasomotor function such as measurement of acetylcholine-induced or flow-mediated dilation in the coro- nary and systemic circulation. Endothelium-derived NO either undergoes oxidation or participates in nitrosylation and nitrosation reactions once released into the vascular lumen. The NO-related compounds produced differ greatly in biological activity, concentration, and stability as well as compartmentalization between plasma and blood cells. The relatively low steady-state concentrations 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.08.025 * Corresponding author. Fax: +49 241 8082545. E-mail address: mkelm@ukaachen.de (M. Kelm). 1 These authors contributed equally to this work. Free Radical Biology & Medicine 40 (2006) 295 – 302 www.elsevier.com/locate/freeradbiomed