Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration.

Matthew S Alkaitis,Glenn Nardone,Hans C Ackerman,Jessica H Chertow

doi:10.1002/bmc.3548

Abstract

NG,NG‐dimethyl‐l‐arginine (asymmetric dimethylarginine, ADMA),NG‐monomethyl‐l‐arginine (l‐NMMA) and NG,N G’‐dimethyl‐l‐arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l‐NMMA inhibit nitric oxide synthase by competing with l‐arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high‐performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l‐NMMA and SDMA. Our assay requires 12 μL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed‐mode cation‐exchange columns, using synthetic monoethyl‐l‐arginine as an internal standard. Metabolites were derivatized with ortho‐phthaldialdeyhde and 3‐mercaptopropionic acid, separated by reverse‐phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter‐day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l‐NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples. © 2015 The Authors Biomedical Chromatography published by John Wiley & Sons, Ltd.

Highlights

Protein-incorporated arginine residues may be methylated posttranslationally by protein arginine methyltransferases (PRMTs; Kakimoto and Akazawa, 1970; Lee et al, 1977)
To support further investigation of methylarginine accumulation in diseases characterized by endothelial dysfunction, we developed a high-performance liquid chromatography (HPLC) assay to quantify arginine, asymmetric dimethylarginine (ADMA), L-NMMA and symmetric dimethylarginine (SDMA) in a single analytical run
Peak identities and retention times were determined by separate analysis of individual arginine, L-NMMA, homoarginine, ADMA, SDMA and MEA standards (Fig. 1)

Summary

Introduction

Protein-incorporated arginine residues may be methylated posttranslationally by protein arginine methyltransferases (PRMTs; Kakimoto and Akazawa, 1970; Lee et al, 1977). ADMA is metabolized to dimethylamine and citrulline by dimethylarginine dimethylaminohydrolase (DDAH; Achan et al, 2003) or cleared by renal excretion (Vallance et al, 1992). Nitric oxide (NO) production by nitric oxide synthase (NOS) requires L-arginine as a substrate and is inhibited by ADMA and L-NMMA (Cardounel et al, 2007). ADMA, L-NMMA and SDMA inhibit cellular uptake of arginine, which could limit NOS substrate availability (Closs et al, 1997). ADMA infusion decreases forearm blood flow, decreases plasma cGMP, increases mean arterial blood pressure and increases systemic vascular resistance (Vallance et al, 1992; Achan et al, 2003; Kielstein et al, 2004). Elevated plasma ADMA increases risk of complications and death in sickle-cell disease

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