Accessible Unlicensed Requires Authentication Published by De Gruyter June 1, 2005

Determination of asymmetric dimethylarginine (ADMA) using a novel ELISA assay

Friedrich Schulze, Reinhard Wesemann, Edzard Schwedhelm, Karsten Sydow, Jennifer Albsmeier, John P. Cooke and Rainer H. Böger

Abstract

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Elevated ADMA plasma levels have been reported in connection with diseases associated with an impaired endothelial L-arginine-NO pathway and endothelial dysfunction, such as atherosclerosis, hypercholesterolemia, chronic heart failure, diabetes mellitus, and hypertension. NO production by NOS is decreased due to elevated ADMA levels. In fact, there is increasing interest in determination of ADMA levels in samples of various origins. The aim of this work was to develop a precise and easy immunoassay in contrast to the existing methods, such as HPLC, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC)-MS. We determined cross-reactivity in our immunoassay of 1.2% for symmetric dimethylarginine and <0.02% for L-arginine. The limit of quantitation was 0.05μmol/l. We found good correlation of the values measured when we compared our assay with LC-tandem MS (n=29; r=0.984; p<0.0001). We determined ADMA levels in human serum and plasma, mouse and rat plasma, and cell culture supernatant. For human plasma we found a mean of 0.65μmol/l in healthy subjects. In the plasma of mice and rats we found mean concentrations of 1.05 and 1.09μmol/l, respectively.


Corresponding author: Prof. Dr. med. Rainer H. Böger, Arbeitsgruppe Klinische Pharmakologie, Institut für Experimentelle und Klinische Pharmakologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany Phone: +40-42803-9759, Fax: +40-42803-9757,

References

1 McDermott JR. Studies on the catabolism of Ng-methylarginine, N′g,Ng-dimethylarginine and Ng,Ng-dimethylarginine in the rabbit. Biochem J 1976; 154:179–84. Search in Google Scholar

2 Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992; 339:572–5. Search in Google Scholar

3 MacAllister RJ, Parry H, Kimoto M, Ogawa T, Russell RJ, Hodson H, et al. Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase. Br J Pharmacol 1996; 119:1533–40. Search in Google Scholar

4 Albsmeier J, Tsikas D, Frölich JC, Böger RH. Asymmetric dimethylarginine (ADMA), an explanation for the L-arginine paradox. Naunyn-Schmiedeberg Arch Pharmacol 2002; 365:SupplR14:44. Search in Google Scholar

5 Böger RH, Bode-Böger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, et al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation 1998; 98:1842–7. Search in Google Scholar

6 Cooke JP. Asymmetrical dimethylarginine: the Über marker? Circulation 2004; 109:1813–8. Search in Google Scholar

7 Zoccali C, Bode-Böger SM, Mallamaci F, Benedetto FA, Tripepi G, Malatino L, et al. Asymmetric dimethylarginine (ADMA): an endogenous inhibitor of nitric oxide synthase predicts mortality in end-stage renal disease (ESRD). Lancet 2001; 358:2113–7. Search in Google Scholar

8 Lu TM, Ding YA, Lin SJ, Lee WS, Tai HC. Plasma levels of asymmetrical dimethylarginine and adverse cardiovascular events after percutaneous coronary intervention. Eur Heart J 2003; 24:1912–9. Search in Google Scholar

9 Achan V, Broadhead M, Malaki M, Whitley G, Leiper J, MacAllister R, et al. Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol 2003; 23:1455–9. Search in Google Scholar

10 Kielstein JT, Impraim B, Simmel S, Bode-Boger SM, Tsikas D, Frolich JC, et al. Cardiovascular effects of systemic nitric oxide synthase inhibition with asymmetrical dimethylarginine in humans. Circulation 2004; 109:172–7. Search in Google Scholar

11 Bode-Böger SM, Böger RH, Kienke S, Junker W, Frölich JC. Elevated L-arginine/dimethylarginine ratio contributes to enhanced systemic NO production by dietary L-arginine in hypercholesterolemic rabbits. Biochem Biophys Res Commun 1996; 219:598–603. Search in Google Scholar

12 Chen BM, Xia LW, Zhao RQ. Determination of N(G),N(G)-dimethylarginine in human plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 1997; 692:467–71. Search in Google Scholar

13 Marra M, Bonfigli AR, Testa R, Testa I, Gambini A, Coppa G. High-performance liquid chromatographic assay of asymmetric dimethylarginine, symmetric dimethylarginine, and arginine in human plasma by derivatization with naphthalene-2,3-dicarboxaldehyde. Anal Biochem 2003; 318:13–7. Search in Google Scholar

14 Pettersson A, Uggla L, Backman V. Determination of dimethylated arginines in human plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 1997; 692:257–62. Search in Google Scholar

15 Causse E, Siri N, Arnal JF, Bayle C, Malatray P, Valdiguie P, et al. Determination of asymmetrical dimethylarginine by capillary electrophoresis-laser-induced fluorescence. J Chromatogr B Biomed Sci Appl 2000; 741:777–83. Search in Google Scholar

16 Vishwanathan K, Tackett RL, Stewart JT, Bartlett MG. Determination of arginine and methylated arginines in human plasma by liquid chromatography-tandem mass spectrometry. J Chromatogr B Biomed Sci Appl 2000; 748:157–66. Search in Google Scholar

17 Martens-Lobenhoffer J, Bode-Böger SM. Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry with very straightforward sample preparation. J Chromatogr B Anal Technol Biomed Life Sci 2003; 798:231–9. Search in Google Scholar

18 Tsikas D, Schubert B, Gutzki FM, Sandmann J, Frölich JC. Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative. J Chromatogr B Anal Technol Biomed Life Sci 2003; 798:87–99. Search in Google Scholar

19 Albsmeier J, Schwedhelm E, Schulze F, Kastner M, Böger RH. Analysis of NG,NG-dimethylarginine (asymmetric dimethylarginine, ADMA) with gas chromatography-mass spectrometry using NG,NG-[2H6]dimethylarginine as internal standard. J Chromatogr B Anal Technol Biomed Life Sci 2004; 809:59–65. Search in Google Scholar

20 Dayoub H, Achan V, Adimoolam S, Jacobi J, Stuehlinger MC, Wang BY, et al. Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis: genetic and physiological evidence. Circulation 2003; 108:3042–7. Search in Google Scholar

21 Teerlink T, Nijveldt RJ, de Jong S, van Leeuwen PA. Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography. Anal Biochem 2002; 303:131–7. Search in Google Scholar

Received: 2004-8-3
Accepted: 2004-9-10
Published Online: 2005-6-1
Published in Print: 2004-12-1

©2004 by Walter de Gruyter Berlin New York