Abstract
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of all isoforms of nitric oxide synthase, the enzyme that synthesizes nitric oxide from arginine. Elevated plasma concentrations of ADMA are associated with hypertension and other risk factors for cardiovascular disease. Symmetric dimethylarginine (SDMA), a stereoisomer of ADMA that does not inhibit nitric oxide synthase, is also present in plasma in concentrations that are almost equal to ADMA concentrations. Any analytical method used for the determination of ADMA should therefore be able to discriminate between ADMA and SDMA. In this chapter a high-performance liquid chromatography (HPLC) method for the simultaneous analysis of arginine, ADMA, and SDMA is described. Solid-phase extraction is used to isolate all basic amino acids. Subsequently, amino acids are converted into relatively stable adducts by derivatization with o-phthalaldehyde reagent containing mercaptopropionic acid. Derivatives are then separated by reversed-phase HPLC using isocratic elution and fluorescence detection. The method requires only 0.05-0.2 mL of sample, allowing the analysis of plasma from small laboratory animals. Because of its high precision, this method is particularly suited to detect small concentration differences between samples, e.g., in the assessment of ADMA metabolism at the organ level by measurement of arterio-venous concentration differences.
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References
Andrew, P. J. and Mayer, B. (1999) Enzymatic function of nitric oxide synthases. Cardiovasc. Res. 43, 521–531.
Vallance, P., Leone, A., Calver, A., Collier, J., and Moncada, S. (1992) Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 339, 572–575.
Matsuoka, H., Itoh, S., Kimoto, M., et al. (1997) Asymmetrical dimethylarginine, an endogenous nitric oxide synthase inhibitor, in experimental hypertension. Hypertension 29, 242–247.
Surdacki, A., Nowicki, M., Sandmann, J., et al. (1999) Reduced urinary excretion of nitric oxide metabolites and increased plasma levels of asymmetric dimethylarginine in men with essential hypertension. J. Cardiovasc. Pharmacol. 33, 652–658.
Päivä H., Laakso, J., Laine, H., Laaksonen, R., Knuuti, J., and Raitakari, O. T. (2002) Plasma asymmetric dimethylarginine and hyperemic myocardial blood flow in young subjects with borderline hypertension or familial hypercholester-olemia. J. Am. Coll. Cardiol. 40, 1241–1247.
Kielstein, J. T., Bode-Böger, S. M., Frölich, J. C., Ritz, E., Haller, H., and Fliser, D. (2003) Asymmetric dimethylarginine, blood pressure, and renal perfusion in elderly subjects. Circulation 107, 1891–1895.
Vallance, P. (2001) Importance of asymmetrical dimethylarginine in cardiovascular risk. Lancet 358, 2096–2097.
Böger, R. H., Bode-Böger, S. M., Szuba, A., et al. (1998) Asymmetric 274 dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction. Its role in hypercholesterolemia. Circulation 98, 1842–1847.
Miyazaki, H., Matsuoka, H., Cooke, J. P., et al. (1999) Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Circulation 99,1141–1146.
Valkonen, V. P., Päivä H., Salonen, J. T., et al. (2001) Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine. Lancet 358, 2127–2128.
Zoccali, C., Bode-Böger, S. M., Mallamaci, F., et al. (2001) Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study. Lancet 358,2113–2117.
Nijveldt, R. J., Teerlink, T., van der Hoven, B., et al. (2003) Asymmetrical dimethylarginine (ADMA) in critically ill patients: high plasma ADMA concentration is an independent risk factor of ICU mortality. Clin. Nutr. 22, 23–30.
Gary, J. D. and Clarke, S. (1998) RNA and protein interactions modulated by protein arginine methylation. Prog. Nucleic Acid Res. Mol. Biol. 61, 65–131.
Leiper, J. M., Santa Maria, J., Chubb, A., et al. (1999) Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases. Biochem. J. 343, 209–214.
Ogawa, T., Kimoto, M., and Sasaoka, K. (1989) Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney. J. Biol. Chem. 264,10205–10209.
Teerlink, T., Nijveldt, R. J., de Jong, S., and van Leeuwen, P. A. (2002) Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chro-matography. Anal. Biochem. 303,131–137.
Mulder, C., Wahlund, L. O., Blomberg, M., et al. (2002) Alzheimer’s disease is not associated with altered concentrations of the nitric oxide synthase inhibitor asymmetric dimethylarginine in cerebrospinal fluid. J. Neural Transm. 109, 1203–1208.
Nijveldt, R. J., van Leeuwen, P. A., van Guldener, C., Stehouwer, C. D., Rauwerda, J. A., and Teerlink, T. (2002) Net renal extraction of asymmetrical (ADMA) and symmetrical (SDMA) dimethylarginine in fasting humans. Nephrol. Dial. Transplant. 17, 1999–2002.
Teerlink, T., Neele, S. J., de Jong, S., Netelenbos, J. C., and Stehouwer, C. D. (2003) Oestrogen replacement therapy lowers plasma levels of asymmetrical dimethylarginine in healthy postmenopausal women. Clin. Sci. 105, 67–71.
Post, M. S., Verhoeven, M. O., van der Mooren, M. J., Kenemans, P., Stehouwer, C. D. A., and Teerlink, T. (2003) Effect of hormone replacement therapy on plasma levels of the cardiovascular risk factor asymmetric dimethylarginine: a randomized, placebo-controlled 12-week study in healthy early postmenopausal women. J. Clin. Endocrinol. Metab. 88,4221–4226.
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Teerlink, T. (2005). Determination of the Endogenous Nitric Oxide Synthase Inhibitor Asymmetric Dimethylarginine in Biological Samples by HPLC. In: Fennell, J.P., Baker, A.H. (eds) Hypertension. Methods In Molecular Medicine™, vol 108. Humana Press. https://doi.org/10.1385/1-59259-850-1:263
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DOI: https://doi.org/10.1385/1-59259-850-1:263
Publisher Name: Humana Press
Print ISBN: 978-1-58829-323-7
Online ISBN: 978-1-59259-850-2
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