Original Contribution THE EFFECT OF DIETARY NITRATE ON SALIVARY, PLASMA, AND URINARY NITRATE METABOLISM IN HUMANS ANANTH S. PANNALA,* ALI R. MANI, † JEREMY P. E. SPENCER,* VERNON SKINNER, ‡ K. RICHARD BRUCKDORFER, ‡ KEVIN P. MOORE, † and CATHERINE A. RICE-EVANS* *Centre For Age-Related Diseases, GKT School of Biomedical Sciences, King’s College London, London, UK; † Centre For Hepatology, and ‡ Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School, UCL, London, UK (Received 4 September 2002; Revised 13 November 2002; Accepted 15 November 2002) Abstract—Dietary nitrate is metabolized to nitrite by bacterial flora on the posterior surface of the tongue leading to increased salivary nitrite concentrations. In the acidic environment of the stomach, nitrite forms nitrous acid, a potent nitrating/nitrosating agent. The aim of this study was to examine the pharmacokinetics of dietary nitrate in relation to the formation of salivary, plasma, and urinary nitrite and nitrate in healthy subjects. A secondary aim was to determine whether dietary nitrate increases the formation of protein-bound 3-nitrotyrosine in plasma, and if dietary nitrate improves platelet function. The pharmacokinetic profile of urinary nitrate excretion indicates total clearance of consumed nitrate in a 24 h period. While urinary, salivary, and plasma nitrate concentrations increased between 4- and 7-fold, a significant increase in nitrite was only detected in saliva (7-fold). High dietary nitrate consumption does not cause a significant acute change in plasma concentrations of 3-nitrotyrosine or in platelet function. © 2003 Elsevier Science Inc. Keywords—Nitrate, Nitrite, Pharmacokinetics, 3-Nitrotyrosine, Platelet function, Free radicals INTRODUCTION Studies that assess nitric oxide (NO) metabolism and the formation of reactive nitrogen species (RNS) in vivo usually rely on the measurement of nitrite, nitrate, and 3-nitrotyrosine in biological fluids. Although it is widely recognized that dietary nitrate can affect plasma and urinary nitrate concentrations, there are very little data on the pharmacokinetics of ingested nitrate, which is con- verted to nitrite in saliva, and its impact on the formation of these biomarkers. Nitrite when swallowed, under the acidic conditions of the stomach (pH 1–2), is converted into nitrous acid and other RNS (Fig. 1) [1]. Since dietary nitrate can be converted to nitrite in the saliva and hence to nitrous acid in the stomach, there have been concerns regarding the potentially harmful effects of dietary nitrate/nitrite in man [1–5]. RNS derived from acidic nitrite can cause nitrosative stress leading to the nitration of aromatic compounds such as tyrosine, de-amination of DNA bases, and N- nitrosation of amines [6 –9]. A putative mechanism of tyrosine nitration is via the formation of RNS from nitrite in acidic conditions (Fig. 1) [9 –11], conditions akin to the human gastric milieu and to some extent in ischemic tissues [12]. RNS such as N 2 O 3 , formed from NO and NO 2 , have been shown to react with thiols to form S-nitrosothiols [13,14], which could have longer half-lives and greater stability than NO, while exerting similar biological ac- tivity [15–17]. Large amounts of NO are reported to be generated in the gastric lumen after oral ingestion of inorganic nitrate [18 –20], suggesting a possible physio- logical role of nitrate in the diet. Enterosalivary circula- tion of ingested nitrate may lead to continuous produc- tion of NO in the gastric lumen, which could in theory exert beneficial effects such as vasodilatation [21,22] and decreased platelet aggregation [15,23,24]. Platelet adhe- sion and aggregation are important events in the patho- genesis of vascular occlusion disease. NO, organic and inorganic nitrates, and S-nitrosothiols have been shown Address correspondence to: Professor Catherine Rice-Evans, Centre for Age-Related Diseases, GKT School of Biomedical Sciences, King’s College London, St. Thomas’s Street, London SE1 9RT, UK; Tel: +44 (20) 7848-6141; Fax: +44 (20) 7848-6143; E-Mail: catherine.rice- evans@kcl.ac.uk. Free Radical Biology & Medicine, Vol. 34, No. 5, pp. 576 –584, 2003 Copyright © 2003 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter doi:10.1016/S0891-5849(02)01353-9 576