Stable isotope dilution assay for liquid chromatography–tandem mass spectrometric determination of l-homoarginine in human plasma
Introduction
l-Homoarginine is a cationic amino acid of which the carbon chain is by one CH2 group longer than in l-arginine. It is putatively formed by the transamination of l-lysine by the enzyme l-arginine:glycine-amidinotransferase (AGAT, EC 2.1.4.1) [1], [2]. Given its structural similarity to l-arginine, l-homoarginine can interact and interfere with l-arginine metabolism and signalling. l-Homoarginine can serve as a substrate and competitive inhibitor of nitric oxide synthases (NOS) [3], the enzymes which generate nitric oxide (NO) from l-arginine [4], [5], [6]. NO plays an important role in the vascular system, it inhibits vascular inflammation, prevents adhesion of immune cells and aggregation of platelets and acts as vasodilator. Furthermore NO maintains smooth muscle cells in a nonproliferative quiescent state [4], [6]. Thus, competition of l-homoarginine with l-arginine for binding to the NOS’ active site may interfere with endothelial function. To date, reports of l-homoarginine plasma concentrations in health and disease and its relation to human pathophysiology are sparse. Valtonen et al. reported increased l-homoarginine plasma concentrations during the second and third trimester of pregnancy [7]. There are several metabolic diseases such as hyperornithinaemia, hypercitrullinaemia and hyperammoniaemia in which l-homoarginine and l-homocitrulline excretion into the urine is increased [8]. A recent study by März et al. suggested that low l-homoarginine plasma concentrations are associated with cardiovascular and all-cause mortality in patients subjected to coronary angiography or hemodialysis [9].
Analytical methods for the measurement of arginine and arginine derivatives using gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometry (MS) have been reviewed elsewhere [10], [11], [12], [13], [14]. LC–tandem MS-based methods for the quantitative determination of asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and l-arginine have been widely applied in clinical studies and have elucidated the detrimental role of ADMA and SDMA in human pathophysiology [15]. The LC–tandem MS method described herein allows the quantification of l-homoarginine in human plasma. A stable isotope labeled homolog of l-homoarginine was used as internal standard for l-homoarginine. With the ability to avoid the need of solid phase extraction, this LC–tandem MS-based method is suitable to analyse l-homoarginine in large clinical trials and may be applicable for clinical laboratory routine.
Section snippets
Chemicals and materials
l-Arginine hydrochloride reference standard was purchased from US Pharmacy (Rockville, MD, USA). l-Homoarginine hydrochloride, l-N-monomehtylarginine (l-NMMA), acetate, and Nɛ-acetyllysine were obtained from Sigma–Aldrich (Steinheim, Germany). Aqueous stock solutions of l-homoarginine, l-NMMA and Nɛ-acetyllysine were made by weighing authentic material supplied by the manufacturer. l-[13C6]-Lysine dihydrochloride (l-[1,2,3,4,5,6-13C6]-lysine, 97–99 atom% 13C) was purchased from Euriso-top
Plasma dialysis
Method calibration and validation were performed in dialysed human plasma. The determination of l-homoarginine before and after the dialysis demonstrated efficient removal of >99% of the analytes. The chromatograms depicted in Fig. 1 show the concentrations of l-homoarginine prior (A) and after (B) plasma dialysis.
LC–tandem MS of the butyl ester derivatives of l-homoarginine and its congeners l-NMMA and Nɛ-acetyllysine
The parent ions formed from ESI+ of the butyl esters of l-homoarginine, its congeners l-NMMA and Nɛ-acetyllysine as well as the stable-isotope labeled l-homoarginine represent the
Discussion
The LC–MS/MS method described herein permits the determination of l-homoarginine in human plasma. Up to now, l-homoarginine was commonly used as internal standard for quantifying the methylated arginines ADMA and SDMA [17]. Knowledge about the endogenous occurrence of l-homoarginine complicated the use of l-homoarginine as internal standard for arginine derivatives in biological samples [18]. The presented method uses the in-house synthesized stable-isotope labeled internal standard l-[13C6
Acknowledgements
We thank Mariola Kastner and Daniel Auge for their excellent technical assistance and we gratefully acknowledge the Cusanuswerk for providing personal funding to DA. This study was supported by grants from the Deutsche Forschungsgemeinschaft (Bo 1431/4-1) and from the Deutsche Stiftung für Herzsforschung (F/12/08).
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