Measurement of Arginine Metabolites: Regulators of Nitric Oxide Metabolism

Molly S. Augustine1, Lynette K. Rogers2

1 The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, 2 The Ohio State University, Columbus, Ohio
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.16
DOI:  10.1002/0471140856.tx1716s58
Online Posting Date:  November, 2013
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Abstract

Arginine is the substrate for nitric oxide synthases (NOS), and arginine availability regulates the production of nitric oxide. Through the activity of methyltransferases, arginine can be methylated to form monomethylarginine (NMMA), asymmetrical dimethylarginine (ADMA), and symmetrical dimethylarginine (SDMA). NMMA and ADMA directly inhibit NOS, whereas SDMA inhibits the cellular import of arginine through the cationic amino acid transporter. Increased levels of methylarginine compounds have been associated with many diseases including atherosclerosis, renal failure, pulmonary hypertension, and preeclampsia. Previous HPLC methods to measure these molecules rely on derivatization with ortho‐phthalaldehyde, which is unstable and requires immediate pre‐ or post‐column reactions. We have identified a new fluorometric agent that is stable for at least 1 week and provides chromatographic properties that facilitate separation of these chemically similar compounds by reverse phase chromatography. Curr. Protoc. Toxicol. 58:17.16.1‐17.16.9. © 2013 by John Wiley & Sons, Inc.

Keywords: arginine; ADMA; HPLC; methylarginine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Arginine Metabolites in Plasma
  • Alternate Protocol 1: Measurement of Arginine Metabolites in Other Sample Matrices
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Arginine Metabolites in Plasma

  Materials
  • Plasma samples, frozen at −80°C
  • Internal standard, e.g., 100 μM L‐homoarginine (Sigma‐Aldrich) in AccQ‐Fluor buffer (Waters)
  • Ethanol, 200 proof, HPLC/spectrophotometric grade (Sigma‐Aldrich)
  • Nitrogen gas
  • AccQ‐Fluor reagent kit (Waters), containing AccQ‐Fluor buffer and AccQ‐Fluor tag (store desiccated at 4ºC)
  • L‐arginine, L‐citrulline, proline, L‐ornithine, ADMA, SDMA, and NMMA
  • 0.1 M HCl (ACS reagent grade; Fisher)
  • Mobile phase A (see recipe)
  • Mobile phase B (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Vortex
  • Refrigerated centrifuge
  • 0.22‐μm, 47‐mm GSWP filter microfiltration tubes, sterile (Ultrafree, GV Durapore; Millipore)
  • HPLC autosampler injection vials with 200‐μl inserts
  • 3 × 250–mm, 3.5‐μm particle size C18‐SB reverse‐phase column (Zorbax, Agilent)
  • 4.6 × 12–mm, 5‐μm particle size C18‐SB guard column (Zorbax, Agilaent)
  • Column heater (Timberline)
  • HPLC system (Shimadzu), including:
    • System controller SCL‐10AVP
    • Solvent Delivery Module LC‐10ATVP
    • Low‐pressure gradient flow control valve FCV‐10ALVP
    • Degasser unit DGU‐14A
    • Auto injector SIL‐10ADVP
  • Spectrofluorometric detector RF‐10AXL

Alternate Protocol 1: Measurement of Arginine Metabolites in Other Sample Matrices

  Additional Materials (also see Basic Protocol)
  • ∼200 mg of tissue sample of interest or 200‐μl urine sample, frozen at −80ºC
  • Liquid nitrogen
  • Lysis buffer (see recipe)
  • Phosphate‐buffered saline, pH 7.4 (PBS; see recipe)
  • 50:40:10 (v/v/v) methanol (HPLC grade, Fisher)/water/ammonia (7 N in methanol, Sigma‐Aldrich)
  • Methanol
  • 7 × 95‐mm electric sawtooth homogenizer (e.g., PowerGen, Fisher)
  • 3‐cc (60‐mg) Oasis MCX SPE solid‐phase extraction cation‐exchange columns (Waters)
  • 5‐ml glass centrifuge tubes
  • 60°C heating block
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Figures

Videos

Literature Cited

Literature Cited
  Boger, R.H., Diemert, A., Schwedhelm, E., Luneburg, N., Maas, R., and Hecher, K. 2010. The role of nitric oxide synthase inhibition by asymmetric dimethylarginine in the pathophysiology of preeclampsia. Gynecol. Obstet. Invest. 69:1‐13.
  Chen, X.M., Hu, C.P., Li, Y.J., and Jiang, J.L. 2012. Cardiovascular risk in autoimmune disorders: Role of asymmetric dimethylarginine. Eur. J. Pharmacol. 696:5‐11.
  Cua, C.L., Rogers, L.K., Chicoine, L.G., Augustine, M., Jin, Y., Nash, P.L., and Nelin, L.D. 2011. Down syndrome patients with pulmonary hypertension have elevated plasma levels of asymmetric dimethylarginine. Eur. J. Pediatr. 170:859‐863.
  El‐Shanshory, M., Badraia, I., Donia, A., Abd El‐Hameed, F., and Mabrouk, M. 2013. Asymmetric dimethylarginine levels in children with sickle cell disease and its correlation to tricuspid regurgitant jet velocity. Eur. J. Haematol. 91:55‐61.
  Heresztyn, T., Worthley, M.I., and Horowitz, J.D. 2004. Determination of L‐arginine and NG, NG‐ and NG, NG'‐dimethyl‐L‐arginine in plasma by liquid chromatography as AccQ‐Fluor fluorescent derivatives. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 805:325‐329.
  Kiechl, S., Lee, T., Santer, P., Thompson, G., Tsimikas, S., Egger, G., Holt, D.W., Willeit, J., Xu, Q., and Mayr, M. 2009. Asymmetric and symmetric dimethylarginines are of similar predictive value for cardiovascular risk in the general population. Atherosclerosis 205:261‐265.
  Leiper, J.M., Santa Maria, J., Chubb, A., MacAllister, R.J., Charles, I.G., Whitley, G.S., and Vallance, P. 1999. Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases. Biochem. J. 343:209‐214.
  Richir, M.C., Siroen, M.P., van Elburg, R.M., Fetter, W.P., Quik, F., Nijveldt, R.J., Heij, H.A., Smit, B.J., Teerlink, T., and van Leeuwen, P.A. 2007. Low plasma concentrations of arginine and asymmetric dimethylarginine in premature infants with necrotizing enterocolitis. Br. J. Nutr. 97:906‐911.
  Schwedhelm, E. 2005. Quantification of ADMA: Analytical approaches. Vasc. Med. 10:S89‐S95.
  Schwedhelm, E. and Boger, R.H. 2011. The role of asymmetric and symmetric dimethylarginines in renal disease. Nat. Rev. Nephrol. 7:275‐285.
  Schwedhelm, E., Tan‐Andresen, J., Maas, R., Riederer, U., Schulze, F., and Boger, R.H. 2005. Liquid chromatography‐tandem mass spectrometry method for the analysis of asymmetric dimethylarginine in human plasma. Clin. Chem. 51:1268‐1271.
  Selley, M.L. 2004. Increased (E)‐4‐hydroxy‐2‐nonenal and asymmetric dimethylarginine concentrations and decreased nitric oxide concentrations in the plasma of patients with major depression. J. Affect Disord. 80:249‐256.
  Shao, Z., Wang, Z., Shrestha, K., Thakur, A., Borowski, A.G., Sweet, W., Thomas, J.D., Moravec, C.S., Hazen, S.L., and Tang, W.H. 2012. Pulmonary hypertension associated with advanced systolic heart failure: Dysregulated arginine metabolism and importance of compensatory dimethylarginine dimethylaminohydrolase‐1. J. Am. Coll. Cardiol. 59:1150‐1158.
  Teerlink, T., Luo, Z., Palm, F., and Wilcox, C.S. 2009. Cellular ADMA: Regulation and action. Pharmacol. Res. 60:448‐460.
  Visser, M., Paulus, W.J., Vermeulen, M.A., Richir, M.C., Davids, M., Wisselink, W., de Mol, B.A., and van Leeuwen, P.A. 2010. The role of asymmetric dimethylarginine and arginine in the failing heart and its vasculature. Eur. J. Heart Fail. 12:1274‐1281.
  Wilcken, D.E., Sim, A.S., Wang, J., and Wang, X.L. 2007. Asymmetric dimethylarginine (ADMA) in vascular, renal and hepatic disease and the regulatory role of L‐arginine on its metabolism. Mol. Genet. Metab. 91:309‐317.
  Witte, M.B. and Barbul, A. 2003. Arginine physiology and its implication for wound healing. Wound Repair Regen. 11:419‐423.
  Zakrzewicz, D. and Eickelberg, O. 2009. From arginine methylation to ADMA: A novel mechanism with therapeutic potential in chronic lung diseases. BMC Pulm. Med. 9:5.
  Zhang, W.Z. and Kaye, D.M. 2004. Simultaneous determination of arginine and seven metabolites in plasma by reversed‐phase liquid chromatography with a time‐controlled ortho‐phthaldialdehyde precolumn derivatization. Anal. Biochem. 326:87‐92.
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