Determinación de arginina y sus metabolitos en plasma

ANALYTICAL BIOCHEMISTRY
Analytical Biochemistry 326 (2004) 87–92 www.elsevier.com/locate/yabio

Simultaneous determination of arginine and seven metabolites in plasma by reversed-phase liquid chromatography with a time-controlled ortho-phthaldialdehyde precolumn derivatization
Wei-zheng Zhang and David M. Kaye*
Wynn Department of Metabolic Cardiology, Baker Heart Research Institute,Melbourne Vic. 8008, Australia Received 26 September 2003

Abstract In an attempt to simultaneously detect molecules generated through the metabolism of L -arginine, a high-performance liquid chromatography method with on-line time-controlled preinjection reaction of ortho-phthaldialdehyde derivatization was developed. Plasma concentrations of citrulline, N G -hydroxy-L -arginine, N G -monomethyl-L-arginine, asymmetric N G , N G -dimethyl-L -arginine, 0 symmetric N G , N G -dimethyl-L -arginine, ornithine, and agmatine were analyzed within 35 min, using only 20 ll of sample, pretreated by a simple cold ethanol cleanup procedure. Plasma samples of 35 healthy human volunteers were analyzed and results were comparable to other published data. All detection parameters of the method demonstrate thatit is a reliable and efficient means for the comprehensive determination of arginine and its metabolites, making this approach suitable for routine clinical applications. Ó 2003 Elsevier Inc. All rights reserved.
Keywords: Arginine; Metabolites; HPLC; Plasma; Determination

Arginine serves as a precursor for generating nitric oxide, urea, creatine, agmatine, and other essential biologicalsubstances. Apart from the synthesis of protein in vivo, arginine and its metabolites participate in many facets of biology, including cellular proliferation, vasodilation, neurotransmission, calcium release, and immunity (review in [1,2]). Furthermore, alterations in the metabolism of arginine have also recently been implicated in disease(s) other than those typically considered ‘‘inborn errors ofmetabolism’’ [3]. Arginine can be converted to citrulline, NOHA1 [4], and ornithine [5] through various pathways, including the nitric oxide pathway and the urea cycle. Citrulline is associated not only with endogenous synthesis of arginine [6] but also as a product of the NO pathway catalysed by NOS through NOHA as an intermediate. Ornithine and
Corresponding author. Fax: +61-3-9276-2495. E-mailaddress: d.kaye@alfred.org.au (D.M. Kaye). 1 Abbreviation used: NOHA, N G -hydroxy-L -arginine; NMMA, N G monomethyl-L -arginine; ADMA, asymmetric N G , N G -dimethyl-L 0 arginine; SDMA, symmetric N G , N G -dimethyl-L -arginine; OPA, ortho-phthaldialdehyde; NO, nitric oxide; NOS, nitric oxide synthase; SPE, solid-phase extraction. 0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rightsreserved. doi:10.1016/j.ab.2003.11.006
*

agmatine, on the other hand, can be generated under the ureagenesis [5] and the decarboxylation of arginine [7], respectively. NMMA, ADMA, and SDMA can be formed in vivo by modification with methyl group(s) on arginine. To fully characterize the roles of arginine and its metabolites in disease physiology, it is necessary to assess multiple factors, including(a) the transport systems for arginine and some of its metabolites, (b) the enzymes involved in the pathways, and (c) the half-life of those compounds in specific compartments. Many of the arginine by-products and arginine itself participate in regulation of arginine pathways to cope with pathophysiological conditions. However, detailed knowledge of arginine metabolism and its regulation is stilllimited [1]. This, in part, could be due to lack of a comprehensive determination method needed for measurement of arginine and its key metabolites simultaneously. High-performance liquid chromatography (HPLC) has been the most used technique for determination of arginine and its metabolites. Combined analysis of arginine, agmatine, citrulline, and ornithine [8]; arginine, ADMA and SDMA [9–11] and...