title: Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration.
abstract: N(G) ,N(G) -dimethyl-l-arginine (asymmetric dimethylarginine, ADMA),N(G) -monomethyl-l-arginine (l-NMMA) and N(G) ,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.
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, Maryland, USA.|Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, UK.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Alkaitis
authors.affiliation: Research Technologies Branch, National Institute of Allergy and Infectious Diseases, Rockville, Maryland, USA.
authors.forename: Glenn
authors.initials: G
authors.lastname: Nardone
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, Maryland, USA.
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Chertow
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, Maryland, USA.
authors.forename: Hans C
authors.initials: HC
authors.lastname: Ackerman
mesh_terms: D000818:Animals; D001120:Arginine / Q000008:administration & dosage* / Q000097:blood*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015203:Reproducibility of Results
chemical_list: D001120:Arginine
keywords: ADMA; Arginine; L-NMMA; Nitric Oxide Synthase; SDMA; Vascular Homeostasis