Concomitant presence of N-nitroso and S-nitroso proteins in human plasma

Abstract

Nitric oxide (NO)-mediated nitrosation reactions are involved in cell signaling and pathology. Recent efforts have focused on elucidating the role of S-nitrosothiols (RSNO) in different biological systems, including human plasma, where they are believed to represent a transport and buffer system that controls intercellular NO exchange. Although RSNOs have been implicated in cardiovascular disease processes, it is yet unclear what their true physiological concentration is, whether a change in plasma concentration is causally related to the underlying pathology or purely epiphenomenological, and to what extent other nitrosyl adducts may be formed under the same conditions. Therefore, using gas phase chemiluminescence and liquid chromatography we sought to quantify the basal plasma levels of NO-related metabolites in 18 healthy volunteers. We find that in addition to the oxidative products of NO metabolism, nitrite (0.20 ± 0.02 μmol/l nitrite) and nitrate (14.4 ± 1.7 μmol/l), on average human plasma contains an approximately 5-fold higher concentration of N-nitroso species (32.3 ± 5.0 nmol/l) than RSNOs (7.2 ± 1.1 nmol/l). Both N- and S-nitroso moieties appear to be associated with the albumin fraction. This is the first report on the constitutive presence of a high-molecular-weight N-nitroso compound in the human circulation, raising the question as to its origin and potential physiological role. Our findings may not only have important implications for the transport of NO in vivo, but also for cardiovascular disease diagnostics and the risk assessment of nitrosamine-related carcinogenesis in man.

Introduction

The continuous production and release of endothelial nitric oxide (NO) plays an important role in vascular homeostasis by regulating blood vessel tone and inhibiting smooth muscle cell proliferation, blood cell adhesion, and lipid oxidation [1]. Since NO is released not only abluminally to exert its effects on cells of the vascular wall, but also into the vessel lumen, a significant part of the NO produced by the endothelium is believed to come into direct contact with blood. Given the extremely short half-life of NO in blood and its rapid reaction with hemoglobin in vitro, the fate of this fraction of NO is thought to be dictated largely by its interaction with red blood cells [2]. In addition to hemoglobin, however, NO is likely to interact with a number of other molecular targets in blood following reaction with molecular oxygen or reactive oxygen species to form nitrogen dioxide (NO₂), dinitrogen trioxide (N₂O₃), or peroxynitrite (ONOO⁻). Unlike NO, these species are nitrosating agents that can react with aromatic compounds, amines, alcohols and thiols to form C-, N-, O-, and S-nitroso species [3]. Earlier investigations in biological systems 4, 5 demonstrated that products of NO oxidation (NO_x) rapidly react with thiols to form S-nitrosothiols (RSNOs). As a result, RSNOs have been the major focus of most recent studies, and numerous pathways have been proposed to be under regulatory control by S-nitrosation [6]. The biological relevance of covalent attachment of an NO moiety to the sulfhydryl group of a plasma protein is exemplified by the occurrence of S-nitrosoalbumin (SNOAlb) in human plasma [4]. Albumin is the principal plasma protein and fulfills a plethora of different functions, including the maintenance of colloid osmotic pressure, acid/base buffering, and antioxidative actions, as well as binding and transport of bilirubin, hormones, fatty acids, metals, and other endogenous ligands [7]. Human albumin contains a single free sulfhydryl group in Cys-34, which is responsible for much of its antioxidative and ligand-binding properties. Apart from a change in its binding characteristics following reaction with NO 8, 9 little information is available as to whether any of the other physiological functions of albumin are altered upon nitrosation of its free SH-group. What is clear, however, is that nitrosation of albumin confers NO⁺ (nitrosonium), NO⁻ (nitroxyl), and NO-donating properties to the molecule. Transnitrosation reactions, i.e., the transfer of a NO⁺-equivalent from one molecule to another, are a common feature of all RSNOs and may account for the inhibition of cysteine-dependent enzymes by SNOAlb [10]. Whereas reaction with glutathione and ascorbate stimulates NO release from SNOAlb [11], spontaneous decomposition to NO is rather slow, which is compatible with the notion that SNOAlb acts as a buffer and transport system for NO 4, 12. In spite of the fact that the physiological concentration of this circulating NO-adduct is the subject of much debate 13, 14, a number of recent clinical investigations have indirectly implicated the involvement of RSNOs in disease processes 15, 16, 17, 18. However, whether these species represent reporter molecules suitable to monitor the progression of illnesses associated with an increased or decreased NO production, play a protective role in vivo, or rather represent bioinactive detoxification products is far from being clear. Similarly little is known regarding the potential role of other nitrosative protein modifications that lead to formation of, e.g., N-nitroso compounds (RNNOs). Whether endogenous RNNOs of high molecular weight exist in humans is hitherto unknown.