Original contributionConcomitant presence of N-nitroso and S-nitroso proteins in human plasma
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 (NOx) 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.
Section snippets
Study population
Nine men and nine women with an average age of 33 ± 2 years (range 19–58 years), body weight of 70 ± 3 kg, and height of 171 ± 2 cm, gave their informed consent before participating in the study. All volunteers were in excellent general health, nonsmokers, and none were on regular medication or revealed present or past evidence of cardiovascular diseases known to affect endothelial function such as hypertension, hypercholesterolemia, heart failure, or diabetes mellitus.
Blood sampling, standards, and albumin removal
Venous blood was
Results
Using highly sensitive analytical techniques for the quantification of nitrosated species in complex biological tissues and fluids, we find physiological levels of RSNOs in human plasma to be in the nanomolar range (see Table 1). Concentrations in males did not significantly differ from those in females (p = .057). Interindividual differences appeared to be independent of the age of the participants. Triggered by the recent finding of concomitant S- and N-nitrosation products in different
Discussion
Using carefully validated chemiluminescence-based techniques for the quantification of nitroso species in biological tissues and fluids, we here demonstrate that besides SNOAlb, human plasma contains nanomolar concentrations of circulating N-nitroso proteins. To the best of our knowledge, this is the first report on the occurrence of high-molecular-weight N-nitroso compounds under physiological conditions, which demonstrates that both RSNOs and RNNOs are normal physiological constituents of the
Acknowledgements
We thank V. Specian for careful reading of the manuscript and Dr. J. Rodriguez for helpful suggestions. This work was supported in part by NIH grant R01-HL69029-01 (to M.F.) and funds from the Deutsche Forschungsgemeinschaft (SFB 1919 to M.K.; Ra 969/1-1 to T.R.).
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