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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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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Basic Protocol 1: Measurement of Arginine Metabolites in Plasma

  • 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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Literature Cited

Literature Cited
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  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.
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  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.
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