Measurement of Arginine Metabolites: Regulators of Nitric Oxide Metabolism

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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

Figure 17.16.1 Chromatographic separation of standards using the methods described in this unit.

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Figure 17.16.2 Chromatographic separation of a plasma sample.

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Videos

Literature Cited

Literature Cited
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