Measurement of Arginine Metabolites: Regulators of Nitric Oxide Metabolism
UNIT 17.16
Molly S. Augustine1 and Lynette K. Rogers1,2 1
Center for Perinatal Research, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio 2 Department of Pediatrics, The Ohio State University, Columbus, Ohio
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. C 2013 by John Wiley & Sons, Inc. 58:17.16.1-17.16.9. Keywords: arginine r ADMA r HPLC r methylarginine
INTRODUCTION L-arginine is an essential amino acid that is the substrate for both arginase isoforms, leading to polyamine and proline production, which are essential for cell proliferation and wound healing (Witte and Barbul, 2003). L-Arginine is also the substrate for nitric oxide synthases (NOS), the enzymes responsible for the production of nitric oxide (NO), which is important in many physiological processes. Metabolism of L-arginine by the arginases results in the production of L-ornithine and urea, and metabolism of L-arginine by the NOS results in the production of L-citrulline and NO. Although regulation of NO production is complex, studies have identified roles for methylated arginine metabolites (Teerlink et al., 2009), including asymmetrical dimethylarginine (ADMA), monomethylL-arginine (NMMA), and symmetrical dimethylarginine (SDMA), and implicated them in many disease states.
Measurement of methylarginines by HPLC has been widely accepted for many years. However, the technique incorporated ortho-phthalaldehyde (OPA) as the fluorometric derivatizing agent (Schwedhelm, 2005). While effective, OPA is highly unstable and light sensitive, and requires immediate pre- or post-column derivatization of the sample. In developing the method described in this unit, our goal was to identify a more stable fluorometric agent that provides sufficient HPLC separation and reliable quantification of the desired compounds. We have used a new fluorometric derivatizing agent (AccQ-Fluor) that is stable for several weeks (Heresztyn et al., 2004) and yields reliable separation of L-arginine, L-citrulline, ADMA, NMMA, SDMA, proline, and L-ornithine. The Basic Protocol describes the use of this method for plasma samples, whereas the Alternate Protocol applies the method to solid tissue and urine samples, which require preparation using solid-phase extraction cation-exchange columns. Oxidative Stress Current Protocols in Toxicology 17.16.1-17.16.9, November 2013 Published online November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471140856.tx1716s58 C 2013 John Wiley & Sons, Inc. Copyright
17.16.1 Supplement 58
BASIC PROTOCOL
MEASUREMENT OF ARGININE METABOLITES IN PLASMA This protocol describes preparation of samples derivatized with Waters AccQFluor Reagent (6-aminoquinolyl-N-hydrozysuccinimidyl carbamate), a stable fluorescent tag developed for amino acid analysis. It also provides directions for preparing standards and carrying out chromatographic separation of the fluorescent metabolites.
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 Prepare plasma samples 1. Thaw frozen plasma samples on ice. 2. Dispense 20-μl aliquots of plasma into 1.5-ml microcentrifuge tubes (three replicates per sample). 3. Spike with internal standard, e.g., 10 μl of 100 μM L-homoarginine in AccQ-Fluor buffer. 4. Add ice-cold ethanol at a ratio of 1:1.25 plasma/ethanol (v/v), e.g., 20 μl sample and 25 μl ethanol. Acetonitrile has been used in place of ethanol with equal results.
5. Vortex three times for 10 sec each time. Measurement of Methylarginines
6. Incubate 10 min on ice.
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Table 17.16.1 Preparation of Calibration Standards
Standard concentration (μM)
Stock concentration (μM)
Stock volume (μl)
Internal standarda (μl)
AccQ-Fluor buffer (μl)
AccQ-Fluor tag (μl)
0
0
0
10
70
20
0.1
5
2
10
68
20
0.5
5
10
10
60
20
1
5
20
10
50
20
2
5
40
10
30
20
5
50
10
10
60
20
10
50
20
10
50
20
20
50
40
10
30
20
a 100
μM L-homoarginine in AccQ-Fluor buffer.
7. Centrifuge 5 min at 10,621 × g, 4ºC. After centrifuging a pellet, containing the precipitated proteins, should form.
8. Collect the supernatant by aspiration, and transfer it to another tube. This supernatant contains the metabolites of interest.
9. Repeat steps 4 to 8 with the pellet, eliminating step 6. This second addition of ethanol is used to rinse the precipitate and sample tube to increase recovery. After the second rinse, a second pellet will not be observed; this step may be eliminated if adequate recovery (>75% of the internal standard) is achieved without it.
10. Pool the two supernatants, and dry under a stream of nitrogen gas, without heat. A crust often forms as the sample dries; a quick vortex of the sample will break this up to expedite drying time.
Derivatize metabolites and standards 11. Reconstitute the dried sample in 80 μl of AccQ-Fluor buffer and 20 μl of AccQFluor tag. Vortex three times for 10 sec each time. If necessary, centrifuge briefly to bring sample to bottom of the tube.
12. Heat 10 min at 55ºC. 13. Transfer the samples into sterile 0.22-μm microfiltration tubes and centrifuge 5 min at 10,621 × g, 25ºC, to remove any residual particulates. 14. Transfer the filtered samples to HPLC autosampler injection vials containing 200-μl inserts. 15. Prepare calibration standards (final concentrations in the first column of Table 17.16.1) as follows:
a. Prepare individual solutions of 1 mg/ml in 0.01 M HCl for L-arginine, L-citrulline, proline, L-ornithine, ADMA, SDMA, and NMMA. b. Make mixed standard stock solutions of 50 μM and 5 μM I by diluting all of the individual stock solutions together in AccQ-Fluor buffer. c. Prepare an internal standard stock solution of 100 μM L-homoarginine in AccQ-Fluor buffer. d. Create calibration standards by combining the volumes of stock, internal standard, AccQ-Fluor buffer, and AccQ-Fluor tag indicated in Table 17.16.1. Current Protocols in Toxicology
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17.16.3 Supplement 58
The linear range studied is 0.05 to 20 μM. Peaks are identified by retention time and by coinjection with standards.
16. After preparing the standards, heat 10 min at 55ºC for derivatization.
Analyze samples 17. Maintain an Agilent Zorbax C18-SB reverse-phase column (3 × 250 mm, 3.5-μm particle size) at 34ºC with a flow rate of 0.5 ml/min. Also use a 4.6 × 12-mm, 5-μm particle size C18-SB guard column (see Critical Parameters). Carry out Chromatographic separation using mobile phases A and B, with a gradient elution as follows: 0 to 3 min isocratic at 5% B 3 to 15 min linear to 8% B 15 to 35 min to 20% B on a gradient curve of +3 35 to 55 min linear to 60% B 7 min 100% B organic flush 10 min 10% B column re-equilibration. 18. Inject calibration standards intermixed with the injected samples used for quantitation, and monitor fluorescence at an excitation wavelength of 250 nm and an emission wavelength of 395 nm. 19. Generate a calibration curve by plotting the concentration of the standard versus the area of the peak, divided by the area of the internal standard peak. This can be accomplished with either the HPLC software or Microsoft Excel.
20. Determine the concentration of the unknown by using the formula generated by the calibration curve. ALTERNATE PROTOCOL
MEASUREMENT OF ARGININE METABOLITES IN OTHER SAMPLE MATRICES Measurement of arginine metabolites in cell media (100- to 500-μl aliquots) and cell protein lysates (20-μl aliquots) is carried out identically to plasma measurement. Depending on the amount of L-arginine present in cell media, dilutions are often required to accurately measure L-arginine and L-citrulline because the L-arginine peak often overwhelms the L-citrulline peak and must be brought within scale. More complex matrices, such as urine or tissues, require more extensive preparation before carrying out measurements, using solid-phase extraction cation-exchange columns. These methods are described below.
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
Measurement of Methylarginines
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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For tissue 1a. Homogenize 200 mg of tissue over liquid nitrogen in 1 ml lysis buffer, and transfer it to a 1.5-ml microcentrifuge tube. 2a. Centrifuge 15 min at 10,621 × g, 4ºC, and aspirate the supernatant. 3a. Combine 100 μl of the superanatant with 10 μl of 100 μM L-homoarginine and 900 μl of PBS. 4a. Condition the Oasis MCX SPE columns with 2 ml 50:40:10 (v/v/v) methanol/ water/ammonia. 5a. Condition the columns with 2 ml PBS. 6a. Load the diluted sample, and discard the eluent. 7a. Wash the column with 2 ml 0.1 M hydrochloric acid, and then with 2 ml methanol. 8a. Elute the sample from column with 1 ml of 50:40:10 (v/v/v) methanol/water/ ammonia. 9a. Dry the samples under a stream of nitrogen at 60ºC. 10a. Follow steps 11 to 14 in the Basic Protocol to form the AccQ-Fluor derivative for analysis as in the succeeding steps in the Basic Protocol.
For urine 1b. Thaw urine samples on ice. 2b. Dispense 200-μl aliquots of urine in 5-ml glass centrifuge tubes. 3b. Spike the samples with an internal standard, e.g., 10 μl of 100 μM L-homoarginine. 4b. Add 800 μl PBS. 5b. Vortex three times for 10 sec each time. 6b. Centrifuge 10 min at 1301 × g, 4ºC, and collect the supernatant. 7b. Condition the column with 2 ml methanol/water/ammonia (50/40/10) and then with 2 ml PBS. 8b. Load the sample. 9b. Wash the column with 2 ml of 0.1 M hydrochloric acid and then with 2 ml of methanol. 10b. Elute the sample from the column with 1 ml methanol/water/ammonia (50:40:10). 11b. Dry the samples under a stream of nitrogen at 60ºC. 12b. Follow steps 11 to 14 in the Basic Protocol to form the AccQ-Fluor derivative for sample analysis as in the succeeding steps in the Basic Protocol.
REAGENTS AND SOLUTIONS Use biological grade TYPE 1, 18 MΩ resistivity water or equivalent for all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Lysis buffer Weigh the following into a 100-ml graduated cylinder: 0.88 g NaCl (reagent grade, Fisher; final concentration 150 mM) (continued)
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0.0452 g EDTA (Sigma-Aldrich; final concentration 1 mM) 0.0380 g EGTA (Sigma-Aldrich; final concentration 1 mM) 0.0368 g sodium orthovanadate, 90% (Sigma-Aldrich; final concentration 2 mM) Add 1 ml Triton X-100 (electrophoresis grade, Fisher; final concentration 1% v/v) and 2 ml of 1 M Trizma-HCl, pH 7.4 (see recipe; final concentration 20 mM). Fill the cylinder to volume with water. Store up to 3 months at 4ºC.
Mobile phase A Weigh 13.61 g of sodium acetate tetrahydrate (136.08 g/mol, HPLC grade; final concentration 100 mM; Fisher), and rinse into a 1-liter graduated cylinder. Weigh 3.5 g of citric acid (anhydrous, ACS certified; final concentration 15 mM; Fisher), and rinse into the same graduated cylinder. Fill to 990 liters with deionized water. Adjust the pH to 5 with 1 M citric acid (19.2 g/100 ml). Fill the cylinder to 1 liter with deionized water, and pass through a 0.22-μm cellulose filter (GSWP, Millipore). Store up to 3 months at room temperature. Mobile phase B For acetonitrile/methanol/5% water 1:1 (v/v), combine 475 ml acetonitrile (HPLC grade; Fisher), 475 ml methanol (HPLC grade; Fisher), and 50 ml water. Store up to 3 months at room temperature. Phosphate-buffered saline (PBS), pH 7.4 Weigh the following into a 1-liter graduated cylinder: 8.00 g NaCl (reagent grade; Fisher) 1.12 g Na2 HPO4 (ACS certified; Fisher) 0.20 g KH2 PO (ACS certified; Fisher) 0.20 g KCl (reagent grade; Sigma-Aldrich) Fill the cylinder to 990 ml with water, adjust the pH to 7.4 with ammonium hydroxide (trace metal grade; Fisher) or 11.65 N hydrochloric acid (ACS reagent grade; Fisher). Fill the cylinder to 1 liter with water. Store up to 3 months at room temperature.
Trizma-HCl, 1 M, pH 7.4 Weigh 15.76 g Trizma hydrochloride (reagent grade; Sigma-Aldrich) into a 1-liter graduated cylinder. Fill to 990 ml with water. Adjust the pH to 7.4 with ammonium hydroxide (trace metal grade; Fisher) or 11.65 N hydrochloric acid (ACS reagent grade; Fisher). Fill the cylinder to 1 liter with water. Store up to 3 months at room temperature. COMMENTARY Background Information L-arginine is an essential amino acid that is
Measurement of Methylarginines
necessary for ammonia detoxification. It is the substrate for both arginase isoforms, leading to polyamine and proline production, as well as for nitric oxide synthases (NOS), the enzymes responsible for the production of nitric oxide (NO). Polyamines and proline are essential for cell proliferation and wound healing (Witte and Barbul, 2003). Metabolism of L-arginine by the arginases results in the production of L-ornithine and urea, and metabolism of Larginine by the NOS results in the production
of L-citrulline and NO. NO plays very important roles in physiology, e.g., protection from infection, smooth muscle relaxation, mucosal blood flow, and maintenance of mucosal integrity and barrier function. The regulation of NO production is multifaceted, but studies have identified fundamental roles for methylated arginine metabolites, which are endogenous regulators of NO (Teerlink et al., 2009). Asymmetrical dimethylarginine (ADMA), monomethyl-Larginine (NMMA), and symmetrical dimethylarginine (SDMA) are forms of methylated
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arginine found in measureable quantities in eukaryotes (Zakrzewicz and Eickelberg, 2009). ADMA and NMMA are asymmetrical methylarginines that directly inhibit NOS enzymes, whereas SDMA inhibits the cellular import of arginine by interfering with cationic amino acid transporters. ADMA is present in higher concentrations than NMMA or SDMA and is considered a significant biological regulator of NOS activity. ADMA is eliminated by renal excretion (10% to 30%) and through metabolism by dimethylarginine dimethylaminohydrolases (DDAH) into L-citrulline and dimethylamine (Leiper et al., 1999). ADMA is synthesized when arginine residues in proteins are methylated by protein arginine methyltransferases (PRMT; Wilcken et al., 2007). De novo synthesis of methylarginines has not been identified; therefore, biologically available ADMA, NMMA, and SDMA are the result of protein degradation and release. Thus, intracellular levels of methylarginines are regulated by PRMT activity, protein turnover, and DDAH activity (Wilcken et al., 2007). Increasing evidence in the literature indicates that ADMA is associated with a wide range of human diseases (Richir et al., 2007; Kiechl et al., 2009; Teerlink et al., 2009; Chen et al., 2012). Much of the research has implicated elevated ADMA levels with cardiovascular disease, including hypertension and hypercholesterolemia (Kiechl et al., 2009; Chen et al., 2012). However, other associations between ADMA and pathology have been reported in preeclampsia (Boger et al., 2010), depression (Selley, 2004), heart failure (Visser et al., 2010), pulmonary hypertension (Shao et al., 2012), polycystic kidney disease (Schwedhelm and Boger, 2011), and other disorders (Cua et al., 2011; El-Shanshory et al., 2013). Therapeutic strategies have focused on manipulation of DDAH to indirectly target ADMA levels (Zakrzewicz and Eickelberg, 2009). Measurement of methylarginines is challenging and encompasses complex sample preparation and derivatization. Most methods use HPLC separation of fluorescently derivatized samples. The most common fluorescent methods measure orthophthalaldehyde derivatives, which are highly unstable and require immediate analysis (Zhang and Kaye, 2004). As technology has progressed, several LC-MS/MS methods have been developed; however, attaining separation of chemically similar compounds with exact or very similar masses, while using a mobile
phase that is compatible for mass spectrometry, has been challenging (Schwedhelm et al., 2005). ELISA-based assays have also been developed but have proven problematic because of the lack of antibody specificity for very similar compounds (Schwedhelm, 2005). We have developed a revised HPLC method which relies on ion-pairing chromatography and fluorescence detection, using the AccQFluor reagent. In developing this method, our goal was to identify a more stable fluorometric agent that provides sufficient HPLC separation and reliable quantification of the desired compounds. The AccQ-Fluor reagent was tested and found to yield consistent derivatization to form a reasonably stable adduct (up to 1 week with no loss) with appropriate chromatographic properties.
Critical Parameters and Troubleshooting The sample preparation for this analysis is simple and straightforward; however, the HPLC separation can be complicated. Some considerations that greatly affect this include the following: Filtering the samples before injecting reduces blockages and over-pressuring of the HPLC system. Using a column heater to regulate temperature greatly reducing retention time shifts between samples, particularly because the queued batches run over a large period of time. Having a guard column to help retain impurities to protect the chromatographic column, and replacing the guard column. This often is enough to regain peak separation and a quiet baseline. Diluting the samples (sometimes required) for baseline separation between L-arginine and L-citrulline, especially in cases where the Larginine peak is particularly large, e.g., in enriched cell media. A large interference peak overlaying Lhomoarginine, NMMA, and ADMA, completely preventing quantitation (observed in human plasma samples). A possible correlation has been made between this interfering peak and samples from patients exposed to opiate drugs.
Anticipated Results If samples are obtained and processed carefully, we are able to obtain reliable quantification of the metabolites described. Figure 17.16.1 illustrates the chromatographic separation of a mixed standard in an average run. Figure 17.16.2 illustrates the same chromatography in a plasma sample. Because the
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Supplement 58
220
220 200
180
180
160
160
140
140
120
120
100
100
80
80 ornithine
60
SDMA ADMA
40 arginine citrulline
20 0
60 40
NMMA
20
proline
0
homoarginine
⫺20
⫺20 0
2
4
Figure 17.16.1
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Time (min)
Chromatographic separation of standards using the methods described in this unit.
150
150
140
140
130
130
120
120
110
ornithine
arginine
100 Absorbance
Absorbance
200
110 100 90
90
80
80 citrulline
70
70 60
60
50
50 proline
40
Absorbance
Absorbance
homoarginine
40 30
30 homoarginine
20
NMMA
20
ADMA
10
SDMA
10
0
0 0
2
Figure 17.16.2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Time (min)
Chromatographic separation of a plasma sample.
AccQ-Fluor reagent derivatizes amine groups (primary and secondary), many other peaks are visible within this particular chromatographic separation and are likely to be free amino acids and polyamines.
Time Considerations Measurement of Methylarginines
Depending on the volume of the starting sample, thawing times from −80ºC on ice are 15 min for 100 μl to 1 hr for 1.5 ml. Tissue homogenization takes 2 min per sam-
ple. The time-determining factor for completing the protein precipitation and derivatization of the samples is the evaporation under a stream of nitrogen, which varies depending on the volume recovered. For plasma, the drying time is generally 30 min; for cell matrices, it is considerably longer, ranging up to and beyond 1 hr. Overall, 24 samples (the number of ports on our nitrogen evaporator) can easily be processed in one work day; 48 samples, processed in two batches, are still attainable in one work
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day. The HPLC gradient method is lengthy, at 73 min per injection. The AccQ-Fluor derivative is stable up to 1 week, allowing samples to be batched and queued for analysis.
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. 170:859863. El-Shanshory, M., Badraia, I., Donia, A., Abd ElHameed, 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 205:261265. 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. 7:275285. 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-2nonenal and asymmetric dimethylarginine concentrations and decreased nitric oxide concentrations in the plasma of patients with major depression. J. Affect Disord. 80:249256. 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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UNIT 17.16
Molly S. Augustine1 and Lynette K. Rogers1,2 1
Center for Perinatal Research, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio 2 Department of Pediatrics, The Ohio State University, Columbus, Ohio
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. C 2013 by John Wiley & Sons, Inc. 58:17.16.1-17.16.9. Keywords: arginine r ADMA r HPLC r methylarginine
INTRODUCTION L-arginine is an essential amino acid that is the substrate for both arginase isoforms, leading to polyamine and proline production, which are essential for cell proliferation and wound healing (Witte and Barbul, 2003). L-Arginine is also the substrate for nitric oxide synthases (NOS), the enzymes responsible for the production of nitric oxide (NO), which is important in many physiological processes. Metabolism of L-arginine by the arginases results in the production of L-ornithine and urea, and metabolism of L-arginine by the NOS results in the production of L-citrulline and NO. Although regulation of NO production is complex, studies have identified roles for methylated arginine metabolites (Teerlink et al., 2009), including asymmetrical dimethylarginine (ADMA), monomethylL-arginine (NMMA), and symmetrical dimethylarginine (SDMA), and implicated them in many disease states.
Measurement of methylarginines by HPLC has been widely accepted for many years. However, the technique incorporated ortho-phthalaldehyde (OPA) as the fluorometric derivatizing agent (Schwedhelm, 2005). While effective, OPA is highly unstable and light sensitive, and requires immediate pre- or post-column derivatization of the sample. In developing the method described in this unit, our goal was to identify a more stable fluorometric agent that provides sufficient HPLC separation and reliable quantification of the desired compounds. We have used a new fluorometric derivatizing agent (AccQ-Fluor) that is stable for several weeks (Heresztyn et al., 2004) and yields reliable separation of L-arginine, L-citrulline, ADMA, NMMA, SDMA, proline, and L-ornithine. The Basic Protocol describes the use of this method for plasma samples, whereas the Alternate Protocol applies the method to solid tissue and urine samples, which require preparation using solid-phase extraction cation-exchange columns. Oxidative Stress Current Protocols in Toxicology 17.16.1-17.16.9, November 2013 Published online November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471140856.tx1716s58 C 2013 John Wiley & Sons, Inc. Copyright
17.16.1 Supplement 58
BASIC PROTOCOL
MEASUREMENT OF ARGININE METABOLITES IN PLASMA This protocol describes preparation of samples derivatized with Waters AccQFluor Reagent (6-aminoquinolyl-N-hydrozysuccinimidyl carbamate), a stable fluorescent tag developed for amino acid analysis. It also provides directions for preparing standards and carrying out chromatographic separation of the fluorescent metabolites.
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 Prepare plasma samples 1. Thaw frozen plasma samples on ice. 2. Dispense 20-μl aliquots of plasma into 1.5-ml microcentrifuge tubes (three replicates per sample). 3. Spike with internal standard, e.g., 10 μl of 100 μM L-homoarginine in AccQ-Fluor buffer. 4. Add ice-cold ethanol at a ratio of 1:1.25 plasma/ethanol (v/v), e.g., 20 μl sample and 25 μl ethanol. Acetonitrile has been used in place of ethanol with equal results.
5. Vortex three times for 10 sec each time. Measurement of Methylarginines
6. Incubate 10 min on ice.
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Table 17.16.1 Preparation of Calibration Standards
Standard concentration (μM)
Stock concentration (μM)
Stock volume (μl)
Internal standarda (μl)
AccQ-Fluor buffer (μl)
AccQ-Fluor tag (μl)
0
0
0
10
70
20
0.1
5
2
10
68
20
0.5
5
10
10
60
20
1
5
20
10
50
20
2
5
40
10
30
20
5
50
10
10
60
20
10
50
20
10
50
20
20
50
40
10
30
20
a 100
μM L-homoarginine in AccQ-Fluor buffer.
7. Centrifuge 5 min at 10,621 × g, 4ºC. After centrifuging a pellet, containing the precipitated proteins, should form.
8. Collect the supernatant by aspiration, and transfer it to another tube. This supernatant contains the metabolites of interest.
9. Repeat steps 4 to 8 with the pellet, eliminating step 6. This second addition of ethanol is used to rinse the precipitate and sample tube to increase recovery. After the second rinse, a second pellet will not be observed; this step may be eliminated if adequate recovery (>75% of the internal standard) is achieved without it.
10. Pool the two supernatants, and dry under a stream of nitrogen gas, without heat. A crust often forms as the sample dries; a quick vortex of the sample will break this up to expedite drying time.
Derivatize metabolites and standards 11. Reconstitute the dried sample in 80 μl of AccQ-Fluor buffer and 20 μl of AccQFluor tag. Vortex three times for 10 sec each time. If necessary, centrifuge briefly to bring sample to bottom of the tube.
12. Heat 10 min at 55ºC. 13. Transfer the samples into sterile 0.22-μm microfiltration tubes and centrifuge 5 min at 10,621 × g, 25ºC, to remove any residual particulates. 14. Transfer the filtered samples to HPLC autosampler injection vials containing 200-μl inserts. 15. Prepare calibration standards (final concentrations in the first column of Table 17.16.1) as follows:
a. Prepare individual solutions of 1 mg/ml in 0.01 M HCl for L-arginine, L-citrulline, proline, L-ornithine, ADMA, SDMA, and NMMA. b. Make mixed standard stock solutions of 50 μM and 5 μM I by diluting all of the individual stock solutions together in AccQ-Fluor buffer. c. Prepare an internal standard stock solution of 100 μM L-homoarginine in AccQ-Fluor buffer. d. Create calibration standards by combining the volumes of stock, internal standard, AccQ-Fluor buffer, and AccQ-Fluor tag indicated in Table 17.16.1. Current Protocols in Toxicology
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The linear range studied is 0.05 to 20 μM. Peaks are identified by retention time and by coinjection with standards.
16. After preparing the standards, heat 10 min at 55ºC for derivatization.
Analyze samples 17. Maintain an Agilent Zorbax C18-SB reverse-phase column (3 × 250 mm, 3.5-μm particle size) at 34ºC with a flow rate of 0.5 ml/min. Also use a 4.6 × 12-mm, 5-μm particle size C18-SB guard column (see Critical Parameters). Carry out Chromatographic separation using mobile phases A and B, with a gradient elution as follows: 0 to 3 min isocratic at 5% B 3 to 15 min linear to 8% B 15 to 35 min to 20% B on a gradient curve of +3 35 to 55 min linear to 60% B 7 min 100% B organic flush 10 min 10% B column re-equilibration. 18. Inject calibration standards intermixed with the injected samples used for quantitation, and monitor fluorescence at an excitation wavelength of 250 nm and an emission wavelength of 395 nm. 19. Generate a calibration curve by plotting the concentration of the standard versus the area of the peak, divided by the area of the internal standard peak. This can be accomplished with either the HPLC software or Microsoft Excel.
20. Determine the concentration of the unknown by using the formula generated by the calibration curve. ALTERNATE PROTOCOL
MEASUREMENT OF ARGININE METABOLITES IN OTHER SAMPLE MATRICES Measurement of arginine metabolites in cell media (100- to 500-μl aliquots) and cell protein lysates (20-μl aliquots) is carried out identically to plasma measurement. Depending on the amount of L-arginine present in cell media, dilutions are often required to accurately measure L-arginine and L-citrulline because the L-arginine peak often overwhelms the L-citrulline peak and must be brought within scale. More complex matrices, such as urine or tissues, require more extensive preparation before carrying out measurements, using solid-phase extraction cation-exchange columns. These methods are described below.
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
Measurement of Methylarginines
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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For tissue 1a. Homogenize 200 mg of tissue over liquid nitrogen in 1 ml lysis buffer, and transfer it to a 1.5-ml microcentrifuge tube. 2a. Centrifuge 15 min at 10,621 × g, 4ºC, and aspirate the supernatant. 3a. Combine 100 μl of the superanatant with 10 μl of 100 μM L-homoarginine and 900 μl of PBS. 4a. Condition the Oasis MCX SPE columns with 2 ml 50:40:10 (v/v/v) methanol/ water/ammonia. 5a. Condition the columns with 2 ml PBS. 6a. Load the diluted sample, and discard the eluent. 7a. Wash the column with 2 ml 0.1 M hydrochloric acid, and then with 2 ml methanol. 8a. Elute the sample from column with 1 ml of 50:40:10 (v/v/v) methanol/water/ ammonia. 9a. Dry the samples under a stream of nitrogen at 60ºC. 10a. Follow steps 11 to 14 in the Basic Protocol to form the AccQ-Fluor derivative for analysis as in the succeeding steps in the Basic Protocol.
For urine 1b. Thaw urine samples on ice. 2b. Dispense 200-μl aliquots of urine in 5-ml glass centrifuge tubes. 3b. Spike the samples with an internal standard, e.g., 10 μl of 100 μM L-homoarginine. 4b. Add 800 μl PBS. 5b. Vortex three times for 10 sec each time. 6b. Centrifuge 10 min at 1301 × g, 4ºC, and collect the supernatant. 7b. Condition the column with 2 ml methanol/water/ammonia (50/40/10) and then with 2 ml PBS. 8b. Load the sample. 9b. Wash the column with 2 ml of 0.1 M hydrochloric acid and then with 2 ml of methanol. 10b. Elute the sample from the column with 1 ml methanol/water/ammonia (50:40:10). 11b. Dry the samples under a stream of nitrogen at 60ºC. 12b. Follow steps 11 to 14 in the Basic Protocol to form the AccQ-Fluor derivative for sample analysis as in the succeeding steps in the Basic Protocol.
REAGENTS AND SOLUTIONS Use biological grade TYPE 1, 18 MΩ resistivity water or equivalent for all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.
Lysis buffer Weigh the following into a 100-ml graduated cylinder: 0.88 g NaCl (reagent grade, Fisher; final concentration 150 mM) (continued)
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0.0452 g EDTA (Sigma-Aldrich; final concentration 1 mM) 0.0380 g EGTA (Sigma-Aldrich; final concentration 1 mM) 0.0368 g sodium orthovanadate, 90% (Sigma-Aldrich; final concentration 2 mM) Add 1 ml Triton X-100 (electrophoresis grade, Fisher; final concentration 1% v/v) and 2 ml of 1 M Trizma-HCl, pH 7.4 (see recipe; final concentration 20 mM). Fill the cylinder to volume with water. Store up to 3 months at 4ºC.
Mobile phase A Weigh 13.61 g of sodium acetate tetrahydrate (136.08 g/mol, HPLC grade; final concentration 100 mM; Fisher), and rinse into a 1-liter graduated cylinder. Weigh 3.5 g of citric acid (anhydrous, ACS certified; final concentration 15 mM; Fisher), and rinse into the same graduated cylinder. Fill to 990 liters with deionized water. Adjust the pH to 5 with 1 M citric acid (19.2 g/100 ml). Fill the cylinder to 1 liter with deionized water, and pass through a 0.22-μm cellulose filter (GSWP, Millipore). Store up to 3 months at room temperature. Mobile phase B For acetonitrile/methanol/5% water 1:1 (v/v), combine 475 ml acetonitrile (HPLC grade; Fisher), 475 ml methanol (HPLC grade; Fisher), and 50 ml water. Store up to 3 months at room temperature. Phosphate-buffered saline (PBS), pH 7.4 Weigh the following into a 1-liter graduated cylinder: 8.00 g NaCl (reagent grade; Fisher) 1.12 g Na2 HPO4 (ACS certified; Fisher) 0.20 g KH2 PO (ACS certified; Fisher) 0.20 g KCl (reagent grade; Sigma-Aldrich) Fill the cylinder to 990 ml with water, adjust the pH to 7.4 with ammonium hydroxide (trace metal grade; Fisher) or 11.65 N hydrochloric acid (ACS reagent grade; Fisher). Fill the cylinder to 1 liter with water. Store up to 3 months at room temperature.
Trizma-HCl, 1 M, pH 7.4 Weigh 15.76 g Trizma hydrochloride (reagent grade; Sigma-Aldrich) into a 1-liter graduated cylinder. Fill to 990 ml with water. Adjust the pH to 7.4 with ammonium hydroxide (trace metal grade; Fisher) or 11.65 N hydrochloric acid (ACS reagent grade; Fisher). Fill the cylinder to 1 liter with water. Store up to 3 months at room temperature. COMMENTARY Background Information L-arginine is an essential amino acid that is
Measurement of Methylarginines
necessary for ammonia detoxification. It is the substrate for both arginase isoforms, leading to polyamine and proline production, as well as for nitric oxide synthases (NOS), the enzymes responsible for the production of nitric oxide (NO). Polyamines and proline are essential for cell proliferation and wound healing (Witte and Barbul, 2003). Metabolism of L-arginine by the arginases results in the production of L-ornithine and urea, and metabolism of Larginine by the NOS results in the production
of L-citrulline and NO. NO plays very important roles in physiology, e.g., protection from infection, smooth muscle relaxation, mucosal blood flow, and maintenance of mucosal integrity and barrier function. The regulation of NO production is multifaceted, but studies have identified fundamental roles for methylated arginine metabolites, which are endogenous regulators of NO (Teerlink et al., 2009). Asymmetrical dimethylarginine (ADMA), monomethyl-Larginine (NMMA), and symmetrical dimethylarginine (SDMA) are forms of methylated
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arginine found in measureable quantities in eukaryotes (Zakrzewicz and Eickelberg, 2009). ADMA and NMMA are asymmetrical methylarginines that directly inhibit NOS enzymes, whereas SDMA inhibits the cellular import of arginine by interfering with cationic amino acid transporters. ADMA is present in higher concentrations than NMMA or SDMA and is considered a significant biological regulator of NOS activity. ADMA is eliminated by renal excretion (10% to 30%) and through metabolism by dimethylarginine dimethylaminohydrolases (DDAH) into L-citrulline and dimethylamine (Leiper et al., 1999). ADMA is synthesized when arginine residues in proteins are methylated by protein arginine methyltransferases (PRMT; Wilcken et al., 2007). De novo synthesis of methylarginines has not been identified; therefore, biologically available ADMA, NMMA, and SDMA are the result of protein degradation and release. Thus, intracellular levels of methylarginines are regulated by PRMT activity, protein turnover, and DDAH activity (Wilcken et al., 2007). Increasing evidence in the literature indicates that ADMA is associated with a wide range of human diseases (Richir et al., 2007; Kiechl et al., 2009; Teerlink et al., 2009; Chen et al., 2012). Much of the research has implicated elevated ADMA levels with cardiovascular disease, including hypertension and hypercholesterolemia (Kiechl et al., 2009; Chen et al., 2012). However, other associations between ADMA and pathology have been reported in preeclampsia (Boger et al., 2010), depression (Selley, 2004), heart failure (Visser et al., 2010), pulmonary hypertension (Shao et al., 2012), polycystic kidney disease (Schwedhelm and Boger, 2011), and other disorders (Cua et al., 2011; El-Shanshory et al., 2013). Therapeutic strategies have focused on manipulation of DDAH to indirectly target ADMA levels (Zakrzewicz and Eickelberg, 2009). Measurement of methylarginines is challenging and encompasses complex sample preparation and derivatization. Most methods use HPLC separation of fluorescently derivatized samples. The most common fluorescent methods measure orthophthalaldehyde derivatives, which are highly unstable and require immediate analysis (Zhang and Kaye, 2004). As technology has progressed, several LC-MS/MS methods have been developed; however, attaining separation of chemically similar compounds with exact or very similar masses, while using a mobile
phase that is compatible for mass spectrometry, has been challenging (Schwedhelm et al., 2005). ELISA-based assays have also been developed but have proven problematic because of the lack of antibody specificity for very similar compounds (Schwedhelm, 2005). We have developed a revised HPLC method which relies on ion-pairing chromatography and fluorescence detection, using the AccQFluor reagent. In developing this method, our goal was to identify a more stable fluorometric agent that provides sufficient HPLC separation and reliable quantification of the desired compounds. The AccQ-Fluor reagent was tested and found to yield consistent derivatization to form a reasonably stable adduct (up to 1 week with no loss) with appropriate chromatographic properties.
Critical Parameters and Troubleshooting The sample preparation for this analysis is simple and straightforward; however, the HPLC separation can be complicated. Some considerations that greatly affect this include the following: Filtering the samples before injecting reduces blockages and over-pressuring of the HPLC system. Using a column heater to regulate temperature greatly reducing retention time shifts between samples, particularly because the queued batches run over a large period of time. Having a guard column to help retain impurities to protect the chromatographic column, and replacing the guard column. This often is enough to regain peak separation and a quiet baseline. Diluting the samples (sometimes required) for baseline separation between L-arginine and L-citrulline, especially in cases where the Larginine peak is particularly large, e.g., in enriched cell media. A large interference peak overlaying Lhomoarginine, NMMA, and ADMA, completely preventing quantitation (observed in human plasma samples). A possible correlation has been made between this interfering peak and samples from patients exposed to opiate drugs.
Anticipated Results If samples are obtained and processed carefully, we are able to obtain reliable quantification of the metabolites described. Figure 17.16.1 illustrates the chromatographic separation of a mixed standard in an average run. Figure 17.16.2 illustrates the same chromatography in a plasma sample. Because the
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220
220 200
180
180
160
160
140
140
120
120
100
100
80
80 ornithine
60
SDMA ADMA
40 arginine citrulline
20 0
60 40
NMMA
20
proline
0
homoarginine
⫺20
⫺20 0
2
4
Figure 17.16.1
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Time (min)
Chromatographic separation of standards using the methods described in this unit.
150
150
140
140
130
130
120
120
110
ornithine
arginine
100 Absorbance
Absorbance
200
110 100 90
90
80
80 citrulline
70
70 60
60
50
50 proline
40
Absorbance
Absorbance
homoarginine
40 30
30 homoarginine
20
NMMA
20
ADMA
10
SDMA
10
0
0 0
2
Figure 17.16.2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Time (min)
Chromatographic separation of a plasma sample.
AccQ-Fluor reagent derivatizes amine groups (primary and secondary), many other peaks are visible within this particular chromatographic separation and are likely to be free amino acids and polyamines.
Time Considerations Measurement of Methylarginines
Depending on the volume of the starting sample, thawing times from −80ºC on ice are 15 min for 100 μl to 1 hr for 1.5 ml. Tissue homogenization takes 2 min per sam-
ple. The time-determining factor for completing the protein precipitation and derivatization of the samples is the evaporation under a stream of nitrogen, which varies depending on the volume recovered. For plasma, the drying time is generally 30 min; for cell matrices, it is considerably longer, ranging up to and beyond 1 hr. Overall, 24 samples (the number of ports on our nitrogen evaporator) can easily be processed in one work day; 48 samples, processed in two batches, are still attainable in one work
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day. The HPLC gradient method is lengthy, at 73 min per injection. The AccQ-Fluor derivative is stable up to 1 week, allowing samples to be batched and queued for analysis.
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. 170:859863. El-Shanshory, M., Badraia, I., Donia, A., Abd ElHameed, 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 205:261265. 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. 7:275285. 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-2nonenal and asymmetric dimethylarginine concentrations and decreased nitric oxide concentrations in the plasma of patients with major depression. J. Affect Disord. 80:249256. 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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